Development of
analytical methods for
counterfeit drugs
investigation
Nadia Muleri
Università di Roma “La Sapienza”
Facoltà di Farmacia e Medicina
Istituto Superiore di Sanità
January 2012
Advisors
Internal:
External:
prof. Alhaique
dr. Valvo
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Summary
1.
INTRODUCTION .................................................................................................................................. 4
1.1 AIM OF THE THESIS ............................................................................................................................. 8
2.
MATERIALS AND METHODS ............................................................................................................... 9
2.1 MATERIALS.......................................................................................................................................... 9
2.2 METHODS ......................................................................................................................................... 11
3.
DEVELOPMENT OF ANALITICAL METHOD FOR COUNTERFEIT DRUGS ANALYSIS ............................ 13
3.1 COLORIMETER .................................................................................................................................. 16
3.1.1 Training Stage ............................................................................................................................... 19
3.1.2 Testing Stage ................................................................................................................................ 25
3.1.3 Ruggedness................................................................................................................................... 30
3.2 SOLID STATE TECHNIQUES: IR SPECRTOSCOPY AND THERMOANALYSIS ......................................... 32
3.2.1 IR ................................................................................................................................................... 32
3.2.2 DSC and TGA ................................................................................................................................. 46
3.2.3 Case studies .................................................................................................................................. 56
3.2.4 Detection of solid state properties by thermoanalysis ................................................................ 69
3.2.5 Results on summary ..................................................................................................................... 78
3.3 STABILITY STUDIES ............................................................................................................................ 79
3.4 DISSOLUTION .................................................................................................................................... 88
3.4.1 Development of a discriminating dissolution method ................................................................. 91
3.4.2 Dissolution test medium validation ............................................................................................. 94
3.4.3 Dissolution of original and illegal drugs ....................................................................................... 95
3.5 THE ANALOGUES............................................................................................................................. 111
3.5.1 The analogues in the literature .................................................................................................. 112
3.5.2 Detection of analogues by LC_MS: development of a new method.......................................... 123
3.5.3 Characterization of synthesized references by thermoanalysis and IR ..................................... 124
3.5.4 Detection of analogue molecules in a pharmaceutical product by solid state analyses ........... 131
3.6 NMR ................................................................................................................................................ 137
3.6.1 Validation of a quantitative NMR method for the analysis of counterfeit drugs ...................... 141
3.6.1.1 Materials and method for validation ...................................................................................... 141
3.6.1.2 Instrumental parameters ........................................................................................................ 143
3.6.1.3 Validation with Acyclovir ......................................................................................................... 144
3.6.1.4 Validation with Maleic acid ..................................................................................................... 147
3.6.1.5 Validation with Di-Nitro Benzoic Acid ..................................................................................... 150
3.6.1.6 Ruggedness.............................................................................................................................. 152
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
3.6.1.7 Recovery experiments ............................................................................................................. 154
3.6.1.8 Method applicability ................................................................................................................ 154
4.
CONCLUSIONS .................................................................................................................................156
5.
REFERENCES ....................................................................................................................................158
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
1.
INTRODUCTION
Medical product counterfeiting is a more and more widespread crime. Not only it damages the
patented brand which is imitated, but most of all it causes dangerous hazard for the public health.
According to World Health Organization (WHO) definition, “a counterfeit medicine is one which is
deliberately and fraudulently mislabeled with respect to identity and/or source. Counterfeiting can
apply to both branded and generic products and counterfeit products may include products with the
correct ingredients or with the wrong ingredients, without active ingredients, with insufficient active
ingredients or with fake packaging".
Counterfeit drugs are never equivalent to originals in safety, efficacy and quality because neither the
composition nor the declared therapeutic efficacy is guaranteed.
Since counterfeit drug manufacturers do not respect Good Manufacturing Practices (GMP), the quality
of raw materials is completely unknown; toxic impurities, heavy metals, residual solvents could be
present and dosage uniformity is not guaranteed. Counterfeit drugs are not stored and distributed
according to the international quality standard regulations that all pharmaceutical manufacturers must
respect.
Fig.1 : The manufacture of illegal
drugs is not subject to any control
thus inappropriate equipment
can be employed, like this dirty
coating pan.
In this regard the resounding case of counterfeit heparin is illustrative [1-2]. This drug is a very common
anticoagulant. Some batches of raw material for injectable preparations, manufactured in China,
contained a contaminant, that is oversulfated chondroitin sulfate (OSCS). This contaminant caused very
strong adverse reactions and the death of many people. The counterfeit heparin met the quality tests of
the Pharmacopoeias in the same way as the original one, thus it was necessary to set up an ad hoc
Nuclear Magnetic Resonance (NMR) method to distinguish between them. To face the emergency the
new method was included in the European Pharmacopoeia (Ph.Eur.).
Further dangers can arise from bad packaging that may chemically or physically interact with the
medicine and cause its deterioration.
Sealing defects may lead to degradation of the active substance and generation of toxic impurities and
dangerous contaminants. The same risks occur when expired drugs are re-labeled and put on the illegal
market with a fake expiry date.
There are different kinds of counterfeit drugs; some of them are almost identical to the original while
others use fantasy brand but declare therapeutic effects typical of the original drug. Some others,
classified as “illegal drugs”, are generic medicines that are legal in some countries where intellectual
property is not recognized (like China and India) but illegally sold also in countries where they are not
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
authorized and where the required quality standards are higher (e.g. Kamagra is a legal generic drug of
Viagra in India) [1].
Recently a new phenomenon related to pharmaceutical counterfeiting has arisen : the circulation of
fake food supplements and herbal products[3-4-5]. These products are declared to be made of natural
origin substances, minerals and vitamins, without any synthetic active substance. On the contrary some
of them were reported to contain pharmaceutical active substances or even new synthetic analogue
molecules. These analogues are active molecules structurally very similar to known synthetic active
substances [6] but neither toxicological studies nor clinical trials have been carried out on them, thus
they can be very dangerous.
In some counterfeit drugs the active substance is present in the correct amount, but cases where it is
absent, under-dosed or overdosed are very frequent. Sometimes then the declared active substance is
substituted with a cheaper one with similar activity.
The lack of the active substance or its underdosage can cause the failure of the therapy and, in case of
antibiotics, the development of resistant strains. On the other hand both the over-dosage and the
substitution of the active substance can cause high risks, related to the possible increase of side effects
and the onset of intolerances and pharmaceutical interactions.
The RIVM, the Dutch Institute for Public Health and the Environment proposes the following counterfeit
drug classification to group these products according to the health risk assessment for patients [7].
 Counterfeit drugs: falsifications that are look-a-likes of the genuine medicine, having the correct
shape, color, and embossing.
 Imitation drugs: illegal drug products that do not look like the genuine medicine but their name or
claim strongly suggest the therapeutic effect of the original medicine.
In each of these groups, samples are further sorted depending on the type and the amount of active
substance:
 Professional: contains the Active Pharmaceutical Ingredient (API) within 90-110% of
declared amount; no other APIs are present
 Non-professional: contains API outside 90-110% of declared amount; no other APIs are
present
 Mixed: contains the correct API and another known API
 Fraudulent: contains other known APIs
 Analog: contains other unapproved APIs
The highest risks are related to Mixed, Fraudulent and Analog classes, since possible toxic effects,
unexpected side effects and pharmaceutical interactions can arise.
Excipients contained in counterfeit drugs are frequently different from those used in the original
formulation, thus leading to a possible different bioavailability; on one hand a delay in the active
substance absorption may occur causing therapeutic inefficacy, because the API therapeutic
concentration is not reached in the site of action. On the other hand an acceleration in the API
absorption may amplify side effects.
The presence of extraneous materials and even toxic components has also been observed. E.g. the
presence of ethylene glycol instead of glycerin has been detected in some cough syrups [1-2]. In many
tablets the presence of building materials, like concrete and chalk, has also been observed [8-9].
The extent of pharmaceutical counterfeiting phenomenon cannot be accurately quantified because of
its underground nature. However evaluations by WHO (World Health Organization, United Nations
Health Care Authority) and FDA (Food and Drug Administration, the U.S. Department of Health and
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Human Services) point out that counterfeit drugs business fluctuates between 1% and 10% of the total
pharmaceutical business. This estimation strongly increases in developing countries, where a proper
regulation for drug control is missing and pharmaceutical counterfeiting is not prosecuted by law. On
the other hand in Europe this estimation is reduced to about 1%, in particular through illegal channels.
Counterfeiting mainly involves medicines that offer high profits and are easy to sell on the illegal
distribution channels.
In developing countries this phenomenon involves antibiotics, vaccines and other lifesaving drugs.
In developed countries the main target of counterfeiters are the lifestyle products even though lately
also some counterfeit lifesaving drugs, as antibiotics, are becoming quite common. The lifestyle
products are medicines for the improvement of the quality of life, like impotence treatment drugs,
anorectic drugs, steroids, glucocorticoids [10], psychotropic drugs.
However, counterfeit drug manufacturing varies according to market changes and focuses on
emergency fields where supplying problems may arise (i.e. vaccines during the swine flu crisis).
The main circulation way of counterfeit drugs occurs through illegal channels, first of all the internet
trade, especially in countries, like Italy, where there is a traceability system that follows medicines from
manufacture to selling or disposal. This system, operating through an optical sticker present on the
packaging of medicines, prevents the intrusion of counterfeit drugs in the legal channel [1-2].
Several factors contribute to increase the circulation of counterfeit drugs, particularly the market
globalization and the internet trade. In many countries the internet trade of drugs is illegal. Nevertheless
patients could be tempted to buy drugs online because prices are low, no medical prescription is
generally needed and privacy is protected. The phenomenon is increasing also because in many
countries a regulation that prosecutes pharmaceutical counterfeiting as a crime against the public
health is missing.
The European Union is working to build a common regulation against the pharmaceutical crime and
harmonize the pharmaceutical trade in Europe [1]. Since 2008 a revision work on Directive 2001/83/CE
has started to establish new responsibility for importers and distributors of raw materials and drugs, to
assure the quality and to trace the source of active substances, to raise the required standards of GMP
and Good Distribution Practices (GDP), to employ a traceability system.
The revision proposes also some regulation about the internet trade. The revision will set a qualification
system for the on-line pharmacies; the legal on-line pharmacies will have to respect official quality
standards and will be easily distinguishable from the illegal ones.
In the same direction but by different instruments, the Council of Europe (an international organization
aimed to promote the democratic development, the human rights, the rule of law and cultural cooperation) is working on an agreement, named Medicrime, to introduce the principle of pharmaceutical
crime in the penal system of the 47 participant states [1].
Production, distribution, promotion of counterfeit drugs will be treated as a crime, thus the new
regulation will allow to promptly seizure on-transit products and to shut down the suspicious sites. In
this way the prosecution of pharmaceutical counterfeiting will be proportional to the health risk posed.
Alert signals will warn about unauthorized on-line pharmacies and there will be sanctions also for
patients purchasing these drugs.
The WHO is the Institution that first promoted the fight against pharmaceutical counterfeiting. During
the WHO International Conference "Combating Counterfeit drugs”, held in Rome in 2006, the
International Medical Products Anti-Counterfeiting Taskforce (IMPACT) was established. IMPACT is a
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
coalition in which international authorities (like WHO, Council of Europe, European Commission,
Interpol), governmental authorities (like Medicines Agencies, Customs, Police), private organizations
(like worldwide and European industrial federations, pharmacists, patients, providers ) are involved [1].
By international, national and regional approaches, IMPACT promotes the international cooperation. It
plans to improve the knowledge of the pharmaceutical counterfeiting phenomenon, achieve a
regulation adequate to the health risks it poses, implement surveillance measures, improve the
circulation of information, develop scientific and technical expertise to detect counterfeit drugs and to
authenticate originals, improve information to patients.
In Italy a national taskforce, IMPACT Italia, representing the Italian link of the international team, has
been established in 2007. The Italian Medicines Agency (AIFA), the Italian National Institute of Health
(ISS), the Ministry of Health, the Customs Agency, the Ministry of Economic Development, the Ministry
of Interior, the Carabinieri NAS Police Force, are involved in this coalition. The cooperative approach of
this taskforce makes the strategies more effective and prevents from the waste of resources and
competence conflicts [11-12].
IMPACT Italia works to monitor illegal channels and to develop informatics instrument to be used on the
field, like the logos data bank for authorized drugs. It also deals with laboratory analyses on suspicious
drugs, officers training and patients information. Information to patients is one of the main goals of
IMPACT Italia as it is crucial to combat pharmaceutical counterfeiting. In fact, a misinformed patient can
be tempted to purchase drugs on the illegal channels underestimating the serious danger to his health.
Recently even very young internet users have been involved in counterfeit medicines purchases. Illegal
advertisement, spam, hidden links have popularized the wrong belief that some drugs, especially those
for erectile dysfunctions treatment, are "leisure drugs"; thus these medicines can be used in a totally
inappropriate and dangerous way [1] as evidenced by some seizures where these counterfeit drugs
were confiscated together with cocaine.
The easy access to medicines by internet, without a medical prescription, can induce to unaware
consumption of drugs. As reported by an AIFA study in 2009, there are different kinds of e-pharmacies
[1-13]. Legal pharmacies are those authorized in some European countries (like United Kingdom and
Germany) and inspected by certifying organizations. They commonly handle original drugs but often
exploit the national regulation to sell medicines even in other countries, like Italy, where on-line
pharmacies are not legal.
Then there are fake pharmacies, aimed only to computer frauds and digital identity theft with phishing
scam. Generally these sites are quickly shut down by government but their structure is immediately
recycled to build new sites.
Finally there are illegal pharmacies that put into circulation counterfeit and illegal drugs mostly
delivered by mail. The medicines they deliver do not have a uniform quality since they stock up products
without respecting any regulation. Their only interest is making high profits. Thus these “pharmacies”
supply the cheapest products (fake generics, drugs that imitate original brands, expired drugs, of illegal
manufacture (in particular from Asia), depending on what is available from time to time on the illegal
market.
It has been reported that often the same organization is head of many illegal on-line pharmacy sites,
apparently not linked one each other. In this way a customer, noticing the same structure in many sites,
is induced to believe that this kind of system is made by trustworthy and serious company [1].
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
These on-line pharmacies are the main circulation channel of counterfeit drugs in Italy and, since their
web sites are in an extraterritorial space, it is difficult to apply the national regulations against
pharmaceutical counterfeiting. Also border controls are not always effective since it is not possible to
check every delivery. Thus an international cooperation among all the involved authorities is crucial for
an effective global strategy against pharmaceutical counterfeiting.
1.1 AIM OF THE THESIS
This work was carried out at the Istituto Superiore di Sanità (ISS), that is the Italian National Institute of
Health and belongs to the European Network of the Official Medicines Control Laboratories (OMCLs).
The ISS is a founder member of IMPACT Italia and the analyses of suspicious counterfeit drugs are
performed in the Section "Quality of Chemical Drugs - Anti-counterfeiting Unit" [14].
Suspicious counterfeit samples come from seizures by Carabinieri NAS (a special police force working on
health matters), by Customs and even from purchases on the internet. On WHO’s initiative a study on
best seller medicines from internet market has begun, thus drugs for erectile disease, anorectic drugs,
anabolics are purchased from on-line pharmacies and analyzed not only to detect the counterfeits but
also to highlight the health risks arising from the use of these products.
To reach this goal a research work to develop new analytical methods for the analysis of counterfeit
drugs has been undertaken: methods for a rapid screening, easily exportable in sites where goods
transit (like Customs) and usable even by untrained personnel; but even much more in depth methods
to acquire information as complete as possible about counterfeit drugs and health risks that they can
cause.
Once aware of these risks it is possible to implement a defense strategy, to alert people about the real
dangers of counterfeit drugs and to state the adequate legal actions against this crime.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
2.
MATERIALS AND METHODS
2.1 MATERIALS
The active substances Sildenafil citrate, Tadalafil, Vardenafil Chlorohydrate Thrihydrate were
provided respectively by Pfizer, Eli Lilly and Bayer.
The excipients and the materials analyzed to develop the solid state method by Infrared spectroscopy
(IR), Differential scanning calorimetry (DSC) and Thermogravimetric analysis (TGA) are listed:
 in table 1 the excipient that are present in the formulation of Viagra, Cialis and Levitra
 in table 2 other common pharmaceutical excipients
 in table 3 materials from food industry and building industry
The excipients employed for this study were provided by pharmaceutical manufacturing industries.
Materials from food industry were purchased in a common supermarket. Materials from building
industry were purchased in hardware stores.
Table 1:
Name
Function
Medicine
Sildenafil citrate
Active substance
Viagra
Tadalafil
Active substance
Cialis
Vardenafil Chlorohydrate
Active substance
Levitra
Adsorbent, suspending, diluent, disintegrating
Viagra, Cialis, Levitra
Thrihydrate
Microcrystalline cellulose
agent
Anhydrous Calcium Hydrogen
Diluent agent
Viagra
Anhydrous Lactose
Diluent agent
Viagra
Monohydrate Lactose
Diluent agent
Viagra, Cialis
Magnesium stearate
Lubricating agent
Viagra, Cialis, Levitra
Sodium croscarmellose
Coating, disintegrating, binder, stabilizing ,
Viagra, Cialis
Phosphate
suspending, thickening, adsorbent agent
Hydroxypropylmetylcellulose
Coating, modulating, binder, stabilizing ,
Viagra, Cialis
suspending, thickening agent
Hydroxypropylcellulose
Coating, emulsifying, stabilizing, suspending,
Cialis
binder, thickening agent
Crospovidon
Disintegrating agent
Levitra
Talc
Diluent agent
Cialis
colloidal Silica (aerosil)
Adsorbent, glidant, suspending, disintegrating,
Levitra
thickening agent
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Table 2
Name
Function
Boric acid
Acidifying, preservative agent
Anhydrous citric acid
Acidifying, antioxidant, tampon, flavoring agent
Monohydrate citric acid
Acidifying, antioxidant, tampon, flavoring agent
Stearic acid
Emulsifying, solubilizer, lubricating agent
Corn starch
Glidant, diluent, disintegrating, binder agent
Calcium carbonate
Diluent, therapeutic agent
Anhydrous sodium carbonate
Acidity corrector
Carnauba wax
Coating agent
Microcrystalline cellulose
Adsorbent, suspending, diluent, disintegrating agent
Sodium citrate
Alkalinizing, tampon, emulsifying agent
Dextrates
Diluent agent
Emcompress (Dihydrate
Diluent agent
calcium hydrogen phosphate)
Explotab
Disintegrating agent
Tragacanth gum
Suspending, thickening agent
Arabic gum
Emulsifying, stabilizing, suspending, binder, thickening agent
Hydroxyethylcellulose
Coating, emulsifying, dispersant, disintegrating, binder, thickening
agent
Hydroxyprophylcellulose
Coating, emulsifying, stabilizing, suspending, binder, thickening agent
Anhydrous lactose
Diluent agent
Monohydrate lactose
Diluent agent
Magnesium stearate
Lubricating agent
Maltodextrin
Coating, binder, thickening agent
Mannitol
Sweetening, diluent, osmotic agent
Methylcellulose
Coating, emulsifying, dispersant, disintegrating, binder, thickening
agent
Polivinyl-pirrolidon
Disintegrating, dissolvent, dispersant, binder agent
Sucrose
Granular, coating, sweetening, suspending, diluent, thickening agent
Amorphous silica
Adsorbent, glidant, suspending, disintegrating, thickening agent
Sodium dodecyl sulfate (SDS)
Anionic surfactant, detergent, emulsifying, carrier, lubricating,
wetting agent
Sodium stearate
Wetting agent, jelling, emulsifying agent
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Talc
Diluent agent
Xantam gum
Stabilizing, suspending, thickening agent
Table 3
Name
Probable function
White concrete
Low cost diluent or pollution agent in dirty mixer
Coconut flour
Diluent agent
Sugar cane
Low cost replacement of sucrose and lactose
Potato stark
Binder agent, low cost replacement of corn stark
Gypsum - Chalk
Low cost diluent, binder agent
Counterfeit and illegal drugs were obtained from seizures of Carabinieri NAS, Customs, Guardia di
Finanza. In addition other suspicious samples were purchased on the internet.
Original Viagra®, Cialis® and Levitra® were mostly bought from Italian pharmacies; about a 10% of the
samples were bought from online UK pharmacies.
Sildenafil citrate raw material: La Fenice Smilax
DMSO d6 99.8% Euriso top
All the other reagent were of analytical grade.
2.2 METHODS
FTIR
FTIR spectra were obtained directly on untreated powder by means of an ATR (attenuated total
reflection) sampling system (Golden Gate-Specac, England) coupled with a Perkin Elmer FTIR System
2000 spectrometer (Perkin Elmer, USA). Spectra were recorded at room temperature from 4000 to 370
cm−1 on a Perkin Elmer System 2000 spectrometer. For each sample 16 scans were collected at a
resolution of 4 cm−1.
DSC
DSC curves were recorded using a Perkin Elmer DSC 7 instrument. Approximately 1-2 mg of powder was
weighed into a DSC pan. The DSC profiles were recorded at 10°C min−1, under nitrogen flux, from 25°C to
about 250°C or earlier when the first decomposition occurred. The experiments were conducted using
closed pans with a hole made by the Perkin Elmer’s Accupik system. The DSC temperature scale was
calibrated using extrapolated onset temperatures of the fusion endotherms of indium and lead pure
standards (Perkin Elmer), heated at the same rates used for the samples.
TGA
Thermogravimetric curves were recorded with a Perkin Elmer Pyris1 TGA. The TGA thermograms were
recorded at 10°C min−1, under nitrogen flux, from 25°C (or 15°C) to about 500°C. Approximately 3.5mg
of substance was weighed into a TGA pan (only 2mg were weighted when analyzing pure substances like
active ingredients or excipient reference standards). The experiments were conducted using closed pans
with a cover hole made by the Perkin Elmer’s Accupik system. The cooling accessory C6 chiller
(PerkinElmer) allowed starting from 15°C. A temperature calibration of the thermogravimetric apparatus
was performed measuring the magnetic transition temperature of two standards, alumel and nickel
(supplied by Perkin Elmer).
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
The colorimeter
The colorimeter was the eye-one model by X-rite (Regensdorf , Switzerland).
The Gretag Macbeth i1Match monitor profiling software was employed. It permitted to export data in
Microsoft Excel format as reflectance vs wavelength and this data format was employed.
The colorimeter covers the whole visible spectrum, from 380 to 730 nm in 10 nm steps (i.e. it scans 36
points). The calibration of the colorimeter was performed on a white tile of barium sulfate.
The GTI Mini Matcher (GTI Graphic Technology, Inc., 211 Dupont Avenue, Newburgh, NY –
www.gtilite.com) was employed to obtain a calibrated and reproducible type of lighting.
Dissolution test
Both Apparata I and II (basket and paddle) of the European Pharmacopoeia were tested and finally the
apparatus I (basket method) was chosen, employing 900 ml of dissolution medium at a temperature of
37±0.5 °C and a rotational speed of 50 rpm.
The dissolution system was a AT7 Smart Sotax Dissolutor equipped with a linear 7- cell transporter with
a Perkin Elmer lambda 25 Spectrophotometer hyphenated. The software employed was WinSOTAX.
Released percentages of the active ingredient were automatically measured in a time interval
dependent on the release rate (usually from 2 to 15 minutes) at the maximum absorption wavelength of
sildenafil that is 292nm. The flowcell pathlength was 5 or 10 mm depending on the dosage of the tablet.
Filtration of aqueous samples was performed on-line on 0.7µm Whatman GF/C.
The intrinsic dissolution was conducted with the rotating disk apparatus, as the dissolution was
performed by the Apparatus I. The holder was immersed, with the pellet side down, into the dissolution
vessel. Pellets were prepared compressing about 70 mg of sildenafil citrate in a Perkin-Elmer hydraulic
press using an 8mm punch and die set. The linear portion of each dissolution profile, i.e., before
depletion of the disc and alteration of its surface area, was used to derive the intrinsic dissolution rate
(IDR).
Composition of dissolution media:
Medium pH 4,5 Phosphate buffer solution, pH 4.5
Medium pH 2 HCl
Medium pH 6.8 Phosphate buffer solution
Potassium dihydrogen phosphate
Hydrochloric Acid
Phosphoric acid
Stability studies:
Stability studies were conducted in a Instrument srl Bernareggio PID SYSTEM
HPLC-Diode Array (DAD)
The chromatographic apparatus consisted of an HPLC system Series 1100 equipped with an automatic
injector and a photo-diode array detector (Agilent Technologies Deutschland GmbH, Waldbronn,
Germany). The chromatographic column was a Symmetry C18, 150mm×3.9mm i.d., 5µm particle size
(CPS Analytical) thermostated at 30°C. The detection wavelength was 290 nm and the injection volume
was 10µl.
Mobile phase: 580ml of triethylamine 0.05M adjusted to pH3 with Phosphoric acid, 250 ml methanol,
170 ml acetonitrile.
A System Suitability was performed according to the reference method.
NMR
NMR Bruker 700 MHz
NMR Bruker 400 MHz
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
3.
DEVELOPMENT OF ANALITICAL METHOD FOR COUNTERFEIT DRUGS ANALYSIS
In the laboratory of “Quality of Chemical Drugs - Anti-counterfeiting Unit” of The Istituto
Superiore di Sanità, the analysis of suspicious drugs begins with visual inspection. Tablet dimensions,
shape and color, primary and secondary packaging features, in comparison with the original, in some
cases can be useful to ascertain if a medicine is counterfeited. Nevertheless it can occur that original
drugs sold in different countries have different secondary packaging. Expiry date, correspondence
between blister and box batch numbers, leaflet (in particular the leaflet language: if it is not Italian, the
medicine is illegally sold in Italy), presence of manufacturing company logo and hologram (identification
marks that complicate counterfeit manufacture) are very important details for detecting counterfeit
drugs.
Subsequently HPLC analysis is performed, commonly with DAD-UV detector, to acquire information
about the active product ingredient (API) in the sample. The API is the most important component of a
medicine, being related to the pharmacological effects. By a comparison with a certified standard of the
declared active ingredient, this is first indentified by the retention time and next quantified through a
calibration curve.
Cases where the API is totally absent are not infrequent, since it is the most expensive component [1].
The API underdosage is very frequent too, as it has been observed during this work. It causes the
therapy inefficacy and its consequences vary depending on the kind of drugs.
On the other hand over-dosage cases have also been detected where the API was present in a quantity
larger than declared or, in some cases, in quantity larger than the highest authorized dosage. This
implies many risks related to the increase of the side effects since the highest dosage of API is
authorized following a risk-benefit assessment.
Over-dosage occurs for different reasons; drug manufacturing not in respect of good manufacturing
practices can cause a non uniform production, thus some tablets may be under-dosed and some other
overdosed.
On the other hand, manufacturing overdosed drugs could be the purpose of forgers; to ensure a large
business it is important that customers are very pleased with the efficacy of the medicine, in particular
in the start up phase. Thus unscrupulous forgers may employ bigger amount of API neglecting to the
increase in side effects occurrence.
Another hypothesis that follows some observations is that the over-dosage may be necessary to balance
the effects of an inadequate formulation that, for instance, causes a too slow release (such a case was
encountered in this work and has been reported in following paragraphs).
Another kind of manipulation is the substitution of the declared API with a totally different one which
gives the impression of being effective, like paracetamol in place of antiviral [1].
For instance many cases of substitution of Tadalafil, the API of Cialis ®, with Sildenafil, (the API of
Viagra®) were detected; Sildenafil has the same therapeutic indication as Tadalafil, that is the treatment
of the erectile dysfunction, but is less expensive; therefore the manufacturer’s profit increases.
This substitution can result in both inefficacy of the medication, as Tadalafil and Sildenafil have a
different duration of action, and risks for the patient because the higher dosage of Sildenafil compared
to Tadalafil can result in more pronounced side effects.
Indeed, even very similar active substances may differ in duration of the pharmaceutical action, side
effects, and may cause different pharmacological interactions and intolerance reactions.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Thus, even with a similar API, the substitution of the active substance hides considerable risks for the
health of patients.
The contemporary presence of two active substances, one of which in a very little amount, was also
observed. This is caused, probably, by the use of dirty production pipelines, since the illegal production
is neither bound to GMP respect nor to any kind of guarantee controls; dirty pipelines can cause
contamination phenomena, very dangerous because of possible pharmacological interactions.
In addition to API by HPLC equipment it is possible to detect API impurities; these are side products, not
totally removed, of the synthesis process of the API, or substances deriving from normal degradation
phenomena but that are intensified when drugs are not well preserved. The presence of a significant
amount of this impurities denotes a low quality of the drug since they can be toxic. Hence it is
convenient to verify that these impurities are of very small amount, otherwise it is necessary to identify
them, for example by a HPLC-MS, to know their actual toxicity.
The investigation about the active substance is fundamental and provides the first evaluation about a
suspicious sample and its dangerousness since it examines the most important component.
However it is very important to analyze a suspicious sample in a more complete way to totally evaluate
the risks for the patients’ health. Hence it is necessary to verify the absence of residual solvents or heavy
metals that could abound in a drug not manufactured in respect to GMP.
And it is also very significant to investigate about the total composition of a sample, and even about
excipients, to verify the absence of low quality ingredients, non-pharmaceutical ingredients or toxic
components.
On one hand even if good quality pharmaceutical excipients different from those of the original
formulation are employed some consequences can arise. As a matter of fact the excipients can
modulate the dissolution of the active substance from a tablet and affect the bioavailability of the drug;
for this reason during the development of a new drug, formulation studies are conducted to find an
adequate formulation to the therapeutic purpose. A counterfeit drug, if has a different formulation,
could be not adequate for the therapeutic indication. In addition an inadequate formulation can cause
stability problems, accelerating the degradation phenomena of the active substance and causing the
production of toxic impurities.
On the other hand the usage of low quality ingredients, non pharmaceutical ingredients and above all
toxic components is much more dangerous.
The use of non-pharmaceutical excipients was denounced in the report of INVIMA (Instituto Nacional de
Vigilancia de Medicamentos y Alimentos) in 2004; indeed in Colombia 38 illegal laboratories which
employed boric acid, cement, floor wax, talcum powder in illegal drugs manufacturing were discovered
[15].
Excipients cannot be analyzed by HPLC-DAD since the most of them are insoluble in aqueous and
organic solvents and do not absorb in the UV range.
Instead it is convenient to employ solid state techniques by which it is possible to analyze directly the
sample without dissolving it.
From scientific literature [16-17-18-19-20-21], it results that excipients are successfully investigated by
NIR and Raman techniques, applied directly on powder or on the whole tablet, depending on the
sampling system.
Near Infrared Spectroscopy provides an indicative spectrum of the total composition of the tablet. The
spectrum is not easy to read and it is not possible to link spectrum regions to single contributes of the
14
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
components. But after collecting a significant number of reference spectra and after processing the data
by a statistical analysis (by chemometric software) a NIR spectrum provides very complete information
about correspondence between the analyzed sample and the original drug and highlights possible
resemblance among counterfeit samples, i.e. when they have the same formulation.
Raman spectroscopy as well provides valued information about total composition of an unknown drug,
about both active substance and excipients. A Raman spectrum is simpler to read than a NIR one, and it
is possible to search for peculiar bands of functional groups of components. Also this technique, needs a
data bank with reference spectra of excipients and active substances that may be present in an
unknown sample.
However these effective techniques are expensive (above all Raman spectroscopy equipment) and
require well trained users.
The X-Ray instrument is even more expensive and complicated but it offers a high sensitivity and high
capability to investigate counterfeit drugs composition [22-23].
15
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
3.1 COLORIMETER
The aim of this thesis was to develop new techniques to investigate counterfeit and illegal drugs.
Some of these techniques are cheap, some other are more expensive being also more exhaustive; by the
application of different methods of analysis it is possible to obtain different and more complement
information about an illegal sample that is totally unknown.
A preliminary screening technique by a colorimeter was validated; it is very cheap, simple and even
portable, thus it can be used by even not qualified personnel. This method can be very useful in places
where there are lots of suspicious samples in transit, like customs. By this technique it is possible to
reduce the amount of suspicious samples to send to a specialized laboratory for a more exhaustive
analysis.
In particular this method can examine the suspicious counterfeit samples which imitate a registered
brand and look like original drugs.
By this technique the external coating of a tablet or a peculiar area of the secondary packaging is
investigated measuring accurately the color of the surfaces [24]. As a matter of fact the color of tablets
and of some areas of external boxes are distinctive of a particular drug; thus it can be hypothesized that
a counterfeit drug cannot imitate it accurately.
This measure of color don’t provide any information about neither the composition of the tablets nor
the health risks of a counterfeit drug; nonetheless it can be very useful as preliminary screening.
Actually this method operates similarly as a visual inspection, but by an instrument; this instrument is
usually more accurate than the human eye in detecting minor color differences. Moreover while a visual
inspection is often prone to subjective judgment, an objective instrumental measure can assure greater
reproducibility and most importantly provide an electronic data bank. This would spare inspectors from
carrying with them the original samples for comparison.
Fig.2 : The colorimeter is portable: it can be connected to a computer through a standard USB port.
The colorimeter employed was the eye-one model by X-rite (Regensdorf, Switzerland).
A first typical entry-level use of this kind of colorimeter is to calibrate pc monitors and video-projectors
or to monitor-to-printer match, a process that ensures photographicers that the color they are seeing
on the PC monitor will be the same on their printed output.
This instrument is very inexpensive (it costs about 1000 euros) and indeed portable: it is very small and
it can be connected to every personal computer through a standard USB port, through which it is
powered without the need for a dedicated battery.
The Gretag Macbeth i1Match monitor profiling software was employed. It permitted to have a
description of each scanned color in terms of many different scales. Most importantly it permitted to
export data in Microsoft Excel format as reflectance vs wavelength. This data format was employed
during this study in every calculation concerned.
16
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
The colorimeter covers the whole visible spectrum, from 380 to 730 nm in 10 nm steps (i.e. it scans 36
points).
Fig. 3: Reflectance spectra obtained from tablets of Viagra, Levitra and Cialis.
Reflectance spectrum acquisition is performed placing the ocular of the probe upon the surface to be
scanned, making it adhere perfectly in order to avoid interferences from the ambient light.
Fig. 4 : On the left, the scanning of Cialis 20 mg secondary packaging. On the right, the scanning of a tablet.
The scanning requires not more than a couple of seconds. Usually for each measure 10 color scans are
acquired and the results mediated to have more reproducible data. Every ten scans a calibration of the
colorimeter is performed on a white barium sulfate tile.
Since the response to ambient light conditions was unknown prior to this study, all the analyses for the
method validation were performed in homogeneous conditions putting the scanner under a GTI Mini
Matcher (GTI Graphic Technology, Inc., 211 Dupont Avenue, Newburgh, NY – www.gtilite.com) which is
a booth that provides a calibrated and reproducible type of lighting (daylight, office, incandescent
illumination or ultra-light; in particular daylight was employed for the validation).
By this method the color of the tablets and of the external boxes of suspicious samples are compared to
those of the original drug. A “wavelength distance pattern recognition” is executed to compare the
reflectance spectra in all the considered wavelengths.
To correctly make this comparison the normal variability of color, thus of reflectance values, within
original tablets and boxes must be considered. There are some small color differences among different
batches of the same original brand. In addition also the method imprecision, that arises from differences
in the ambient light and in sampling by operators must be evaluated.
If this normal variability within original samples is not correctly evaluated, for instance an original
sample manufactured in a different batch may be erroneously considered counterfeit.
17
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
For this reason a large number of original tablets and external boxes manufactured in different batches
was tested with this method by different operators for each examined brand. Subsequently all data
collected were statistically analyzed to obtain the original statistical population reflectance data for the
examined brand drug. In this way both the normal variability within original samples and the method
imprecision was evaluated.
After this elaboration, the reflectance spectrum of a suspicious drug can be compared to the original
population reflectance data to verify if the analysis of the color classify it as original or not.
This method was applied both to tablets and to external boxes of three different original drugs, Viagra®
from Pfizer, Cialis® from Lilly and Levitra® from Bayer. These drugs are those authorized in Italy for the
treatment of erectile dysfunction. The examined dosage were the following:
 Viagra Pfizer : 100 mg, 50 mg, 25 mg
 Cialis Lilly : 20 mg, 10 mg
 Levitra Bayer : 20 mg, 10 mg
Fig. 5 : On the left, reflectance spectra of tablets are depicted; on the right reflectance spectra of the packages.
This pharmaceutical class was chosen because it is strongly involved in pharmaceutical counterfeiting.
Indeed Viagra and Cialis are the most counterfeited drugs in Italy. In addition the tablets of these brands
have peculiar color (Viagra tablet is blue, Cialis tablet is dark yellow, Levitra tablet is salmon yellow). It is
important that tablets are colored because this method is neither discriminating not reliable when white
tablets are tested. In such cases only the external boxes can be analyzed.
For external boxes, the dark blue area was tested in the Viagra packaging, the dark green one in Cialis,
the dark violet in Levitra.
Fig. 6 : Packaging and tablets of Viagra, Cialis, Levitra.
The validation of this method was executed in two different steps, called the Training stage and the
Testing stage.
18
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
3.1.1 Training Stage
The Training stage is the preliminary work of analysis of a representative amount of original samples.
These data are subsequently processed to build the statistical population of original drugs for a certain
brand.
This population is defined through a mean reflectance spectrum (that is the Grand Mean of all the data
collected for the original samples, calculated for each wavelength) and a range of acceptability, obtained
from the standard deviation. (see fig.7)
0.8
Cialis tablets 20mg
0.75
0.7
reflectance
0.65
0.6
0.55
0.5
0.45
0.4
0.35
0.3
530
580
630
wavelenght (nm)
680
730
Fig 7 : The green line reports the average reflectance spectrum for original Cialis tablets. The red lines delimit the
statistical population of originals: outwards there are samples which are considered suspicious.
The different dosages of the same brand were considered as different statistical groups. Tablets from
different dosage usually have different size, thus they may have different color because of a different
coating depth; also boxes may come from different manufacturing line.
It is very important that the training set, that is the set of original samples examined during the training
stage, is representative of the normal variability within original samples and that new samples are
added regularly to update the training set.
Ten to twenty batches were analyzed for each dosage. Batches production date spanning 5 years were
chosen to maximize heterogeneity. Original Viagra, Cialis and Levitra were mostly bought from
Italian pharmacies; about a 10% of the samples were bought from online UK pharmacies in order to
widen the variability among batches.
Tablets were scanned on the less carved side (which for Viagra and Levitra is the one indicating the
brand, while for Cialis is the one not carved at all) to avoid introducing a source of heterogeneity.
For those tablets of irregular shape the operators were instructed to make the scan always on the same
spot.
The building and updating of this library of original drugs is the slow step of this technique. But once the
library is in place, the whole analytical procedure is very fast and it is not necessary anymore to dispose
of original samples for the comparison.
The validation was conducted in the following way: for every dosage of each drug considered three
operators made ten replicates in two different days on each batch available. (see fig.8)
While the operator was acquiring the spectra, he could immediately evaluate if he had committed any
blunder (ambient light can ruin the acquisition if the ocular is not perfectly in place) and replace it with a
19
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
new one, since the colors obtained in all the ten replicates were displayed on the PC monitor at the
same time.
......
Batch 1
Operator 1
Day 1
Replicate
1
…
Operator 2
Operator 1
Operator 3
Day 2
Day 1
Replicate
10
Replicate
1
- Mean for each Batch:
𝑋̅1
- Std.deviation for each Batch :
𝑠1
- Grand Mean on all batches :
…
......
......
Operator 2
Operator 3
Day 2
Replicate
10
𝑋̅𝑛
𝑠𝑛
𝑋̿ = 𝑚𝑒𝑎𝑛 (𝑋̅1 , …, 𝑋̅𝑛 )
- Total method precision (for the specific dosage considered): Sdosage
- Total standard deviation
Batch n
(e.g. Scialis_10 mg, Scialis_20 mg, and so on): calculated by pooling s1, s2, …, sn
STOT: The standard deviation made on all the data collected for the specific dosage: it comprises both method imprecision and
batch-to-batch variability.
Fig. 8 : a scheme of the experimental design is reported together with the statistical data collected for each drug
and dosage.
Before processing the data the Cochran test and the Grubbs test were performed to highlight the
possible presence of anomalous data. The Cochran test was used to evaluate if the data from each
operator in each day was consistent or not, by the comparison of the variances. Then the Grubbs test
was applied on every data sets and on each sets of means to remove anomalous data.
2
𝑠𝑚𝑎𝑥
𝐶=
∑𝑗 𝑠𝑗2
𝐺 = (𝑥𝑖 − 𝑥̅ )
Subsequently the data were processed by two different statistical approaches: Nested ANOVA and
ANOVA One Way [25-26-27].
NESTED ANOVA
A three-factor nested ANOVA experiment was conducted, the three factors being operators, days (thus
environmental variability) and replicates. By this statistical elaboration the method imprecision was
estimated and it was evaluated which of the three factors contributed to it the most. Subsequently the
method imprecision was compared to the batch to batch variability.
20
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
This procedure permitted to know separately for each batch both the method precision and its various
components, namely inter-operator precision, inter-day precision and repeatability. Subsequently
pooling the method precisions of each batch the average method precision was obtained for the
examined dosage.
Then a Grand Mean and a standard deviation at each wavelength i were calculated on all the spectra
collected. The grand mean was the one from Fig. 3 and the standard deviation was the total standard
deviation from all the measurements collected during the training stage. This total standard deviation of
course comprised both method imprecision and batch to batch heterogeneity (see Fig. 3).
ONE WAY ANOVA
Apart from the nested one also a crossed layout of data was designed to carry out a One-way ANOVA.
This in order to evaluate whether the method imprecision was significant compared to the batch to
batch heterogeneity. In particular a matrix was built of n columns (one for each batch) by six rows (the
data collected in two days by three operators; the ten replicates made each day by every operator were
averaged to simplify calculations thus and in the matrix there is 𝑥̅ ) (see Table 4).
Table 4
Operator 1 – Day
1
Operator 1 – Day
2
Operator 2 – Day
1
Operator 2 – Day
2
Operator 3 – Day
1
Operator 3 – Day
2
Batch 1
Batch 2
…
Batch k
𝑥̅111
𝑥̅112
…
𝑥̅11𝑘
…
…
…
…
…
…
…
…
…
…
…
…
…
…
…
…
𝑥̅321
𝑥̅322
…
𝑥̅32𝑘
To evaluate if batch to batch variability was significant in comparison to method imprecision an F test
was then carried out: the ratio between the batch to batch mean square and the residual mean square
was calculated and compared to the tabulated F for k-1 and 6∙k-k degrees of freedom (see for example
[28] for a more detailed discussion about the significance of variance sources in ANOVA).
On the whole the following evidence were collected:
 batch to batch heterogeneity accounted for almost the entire variability of the training set for all the
packages and most of the tablets. This was not true for those tablets showing a markedly convex
shape: Cialis 20mg and 10mg and in a less extent Viagra 100mg. In these specific cases the method
imprecision (which is the combination of inter replica, inter day and inter operator variability) was
greater or comparable (in the case of Viagra) to batch to batch heterogeneity. This means that the
colorimeter is sufficiently precise when the surface to be analyzed is flat (as in packages) or not very
convex (as in many tablets), but its precision diminishes considerably with the convexity of the
sample surface.
21
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Fig. 9 : the maximum absolute values of standard deviation components from ANOVA experiments due to batch-tobatch heterogeneity (in light grey) and method imprecision (dark grey) are reported. On the left, tablet values are
reported; on the right, packaging values are reported. Only the wavelength ranges of the specific colour were
considered, i.e. for tablets: 560-620nm for Cialis (yellow-orange), 400-490nm for Viagra (azure), 560-620nm for
Levitra (yellow-orange); for packages: 490-560nm for Cialis (green), 400-510nm for Viagra (blue), 400-530 for
Levitra (violet).
 In Cialis (and in a less extent in Viagra) batch to batch variability proved slightly larger for packages
then for tablets. This shows that the physico-chemical process of tablet filming is more controlled
and reproducible then the process of printing on the cartons; the mixture of coloring agents used for
the tablet filming is standardized with a higher degree of accuracy in comparison to boxes color,
which is instead apparently more prone to variation from one lot to another.
 The nested ANOVA experiment showed that the three factors investigated contribute to a different
extent to the global precision of the method (not reported in fig. 4): the replicates contribution (the
short term repeatability of a single operator) is less significant than that from either operator or day.
Operator and day variability are similar, although the first one is smaller for packages. The reason for
this resides in the role of the operator being minimal in the scanning of boxes, because their surface
is flat. On the contrary a more pronounced operator to operator variability was noticed for tablets,
because in this case the operator ability in handling the tablet played a major role. As expected this
resulted particularly true for Cialis 20 mg, because of its marked convexity.
Results from this statistical processing is presented in a graphical way:
The area between the two red lines represents the statistical population of originals for the examined
brand. The red lines are obtained from the Grand Mean (the mean value of all the data, depicted by the
green line) minus / plus the standard deviation calculated for each wavelength. In this way the normal
variability within original samples due to variability among batches and method imprecision, is
evaluated.
22
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Cialis tablets 20mg
0.8
0.75
0.7
0.65
0.6
0.55
0.5
0.45
0.4
0.35
0.3
reflectance
reflectance
Cialis tablets 10mg
530
580
630
680
wavelenght (nm)
0.8
0.75
0.7
0.65
0.6
0.55
0.5
0.45
0.4
0.35
0.3
530
730
0.25
0.25
0.2
0.2
0.15
0.15
0.1
0.05
0.1
0.05
0
480
530
580
630
680
0
730
480
wavelenght (nm)
Viagra tablets 25mg
0.8
0.8
0.7
0.7
0.6
0.6
0.5
0.4
580
wavelenght (nm)
680
Viagra tablets 50mg
reflectance
reflectance
630
680
wavelenght (nm)
Cialis packages 20mg
reflectance
reflectance
Cialis packages 10mg
580
0.3
0.5
0.4
0.3
0.2
380
480
580
wavelenght (nm)
0.2
680
380
23
480
580
wavelenght (nm)
680
730
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Viagra tablets 100mg
Viagra packages 100mg
0.8
0.7
reflectance
reflectance
0.6
0.5
0.4
0.3
0.2
380
480
580
0.2
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
380
680
Viagra packages 25mg
680
Viagra packages 50mg
reflectance
reflectance
380
580
wavelenght (nm)
wavelenght (nm)
0.2
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
480
wavelenght (nm)
480
580
0.2
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
380
680
Levitra tablets 10mg
480
580
wavelenght (nm)
680
Levitra tablets 20mg
0.9
0.8
0.8
reflectance
0.9
reflectance
0.7
0.7
0.6
0.6
0.5
0.5
0.4
0.4
0.3
0.3
530
580
630
680
730
530
wavelenght (nm)
24
580
630
wavelenght (nm)
680
730
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Levitra packages 20mg
0.2
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
0.2
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
reflectance
reflectance
Levitra packages 10mg
530
580
630
wavelenght (nm)
680
530
730
580
630
680
730
wavelenght (nm)
Fig 10 : Results from the statistical processing is presented in a graphical way for each type of original samples. The
green line reports the average reflectance spectrum. The red lines delimit the statistical population of originals.
3.1.2 Testing Stage
After completing the preliminary work on the training stage, the testing stage began as the method was
applied to suspicious samples.
To investigate if a suspicious sample was genuine or counterfeit a wavelength distance pattern
recognition was conducted; considering all the reflectance values collected at 36 wavelengths by the
colorimeter, it was evaluated if this sample was part of the population of the originals or not. After
acquiring the reflectance spectrum of the suspicious sample, its residual spectrum made of 36
normalized deviation 𝑍𝑖 , one for each wavelength, was calculated according to equation:
𝑍𝑖 =
(𝑥𝑖 − 𝑥̅𝑖 )
𝑠𝑖
Where 𝑥̅𝑖 is the Grand Mean at each wavelength i from the training set of genuine samples, 𝑥𝑖 is the
reflectance of the suspicious sample at each wavelength i and 𝑠𝑖 the standard deviation of the training
set at each wavelength i.
A sample was considered genuine when all its 𝑍𝑖 were ≤ of the tabulated 𝑍𝑐𝑟𝑖𝑡 , the critical value of 𝑍
from the two-sided Student’s distribution at 95% confidence level (i.e. 1.96); in this case it is not
necessary to carry on further analyses with more in-depth techniques. Thus in a plot where 𝑍𝑖 values are
on y-axis and wavelengths on x-axis, all y points of an original sample are under the 𝑍𝑐𝑟𝑖𝑡 value (1.96).
When one or more 𝑍𝑖 resulted > 𝑍𝑖 two cases were distinguished based on the 𝑍𝑖 failing the test being
or not related to wavelengths in the range of the specific color of the sample (for example this range is
560-620 nm for the yellow-orange color in Cialis and Levitra, or 490-560 nm for the green in the carton
box of Cialis etc):
 when the 𝑍𝑖 > 𝑍𝑐𝑟𝑖𝑡 fell in the range of the specific color, the sample was considered a counterfeit;
 when the 𝑍𝑖 > 𝑍𝑐𝑟𝑖𝑡 fell outside this range, the sample was considered dubious.
This distinction had the sole purpose of better studying the colorimeter performances: for future in-thefield uses of the colorimeter inspectors should be instructed to bring the sample to the specialized
laboratory in any case.
25
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
In wavelength distance pattern recognition methods generally larger tolerances are allowed compared
to the 95% chosen in this study: often 𝑍𝑐𝑟𝑖𝑡 up to a value of 6 are used, depending on the number of
trials in the set and of wavelengths considered [29].
Anyway it should be noted that wavelength distance pattern recognition method are normally used in
quality control of pharmaceutical industry, by instruments like Near Infrared spectrometers, to ascertain
if a sample manufactured fulfils the set quality standard; in such case the risk is that of erroneously
discarding a good sample. In the specific case of counterfeiting detection, instead, the major danger lies
in a bad sample being labeled as genuine. Furthermore this approach is a technique for a preliminary
screening. Thus the need for a more cautious choice of 𝑍𝑐𝑟𝑖𝑡 (see [29] for a detailed description of this
matter).
Suspicious samples were collected from police seizures or purchased from online pharmacies. Most of
them didn’t have any secondary package so only the results obtained with tablets will be discussed.
A total of 58 samples were collected containing 2 to 30 tablets each.
Approximately a 45% was constituted by Viagra, another 45% by Cialis and 10% by Levitra. All the
samples underwent a preliminary visual inspection by an attentive comparison with the originals:
 Group 1: 14 samples were sorted as quite different in color from the corresponding original
 Group 2: 21 as faintly different
 Group 3: 23 as extremely similar or indistinguishable.
All the samples were analyzed with the method described. After the colorimetric analysis they were also
analyzed with HPLC/UV and IR spectroscopy for confirmation.
All the sample resulted counterfeited apart from 6 samples in the “extremely similar” group, which were
in fact genuine.
The colorimeter correctly classified as counterfeit with no need for further investigations all the tablets
from group one and two. Furthermore from group 3 the colorimeter could correctly identify the
counterfeit medicines (15 samples) and the original ones (8 samples), agreeing with HPLC and IR
analysis. Only one extremely good looking Cialis fake tablet was classified as dubious and deserving
further analysis according to the approach over described, instead of directly counterfeit.
This sample and other illustrative cases are reported.
26
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Fig. 11 : an original 100 mg Viagra tablet and a tablet almost indistinguishable from it are presented together with
their spectra (on the left) and the Z test results (on the right) for every wavelength. The calculated Z value is much
greater than the critical one for most of the wavelengths so the tablet failed to pass the test and was correctly
identified as counterfeit.
Fig. 12 : an original 20 mg Cialis tablet and a tablet indistinguishable from it are presented together with their
spectra (on the left) and the Z test results (on the right) for every wavelength. Only a few Z values trespassing the
Zcrit cut-off, and they lies outside the 560-620nm range, which is the range specific of the yellow/orange colour of
Cialis. So the sample still failed to pass the test but was classified as deserving further investigations by other
techniques.
27
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
COUNTERFEIT
ORIGINAL
0.75
0.65
0.55
0.45
0.35
0.25
0.15
380
480
580
Z
9
8
7
6
5
4
3
2
1
0
Original
Samples Grand
Mean
Suspect Sample
0.85
Zcrit
95%
380
680
480
580
680
Fig. 13 : This counterfeit sample was quite different in colour from the original. Its spectrum together with that of
the original (on the left) and the Z test results (on the right) are reported. The calculated Z value is much greater
than the critical one for most of the wavelengths so the tablet failed to pass the test and was correctly identified as
counterfeit.
SUSPICIUOS
Original Samples
Grand Mean
0.85
0.75
Suspect Sample
0.65
ORIGINAL
2.5
2
1.5
0.55
0.45
Z
Zcrit 95%
1
0.35
0.5
0.25
0.15
0
380
480
580
680
380
480
580
680
Fig. 14 : This suspicious sample looked exactly the same as the original. Its reflectance spectrum overlapped with
the original (on the left); the calculated Z value was smaller than the critical one for all the wavelengths (on the
right) thus the sample was correctly identified as original. This evidence was successively confirmed by HPLC and IR.
28
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
COUNTERFEIT
ORIGINAL
7
0.9
suspicious
sample mean
0.8
0.7
0.5
Z crit 95%
5
Original
samples Grand
Mean
0.6
Z
6
4
0.4
3
0.3
2
0.2
1
0.1
0
0
380
480
580
380
680
480
580
680
Fig. 15 : This sample looked very similar to the original but the colorimeter highlighted some differences, as
pointed out by the Z test (on the right.) This sample was correctly identified as counterfeit.
COUNTERFEIT
1.00
ORIGINAL
20.00
0.90
0.80
0.70
0.60
suspicious
sample
18.00
Original Sample
Grand Mean
14.00
16.00
10.00
0.40
8.00
0.30
6.00
0.20
4.00
0.10
2.00
0.00
0.00
480
Z crit 95%
12.00
0.50
380
Z
580
680
380
480
580
680
Fig. 16 : This sample was nearly indistinguishable from the original but the colorimeter correctly classified it as a
counterfeit as subsequently proved by other techniques.
29
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Some tests were also made to check the ability of the method to correctly identify genuine materials. A
variety of genuine samples were tested. About a 25% of packages and a 15% of tablets were wrongly
identified as counterfeit at the probability level chosen. Moreover some packages spectra from Viagra
original samples bought in this second stage resulted quite different from the library spectra. The
probable explanation for this is that Viagra packages color is not as much reproducible as the tablets
one is. This seemingly prove that for Viagra package (but it may also be true for other packages not
tested here) there is a frequent change in color from the manufacturer that may compromise the
possibility to use effectively the colorimeter. Levitra and Cialis package color resulted instead more
reproducible. However it is very important to take care to constantly update the spectral library
especially for packages. Moreover package testing may result somehow more tricky then tablets
because European market allows foreign packaging for each EU country. So in principle a database
should better be made of samples from as many countries as possible.
Also stickers that cannot be removed may be present on the package place assigned for the
measurement. In this case of course the inspector has to check the sole tablet.
3.1.3 Ruggedness
Ruggedness studies were conducted on both packaging and tablets of all the three drugs at every
dosage. The parameters deemed interesting were ambient light, temperature and sampling. Each one
was investigated at two levels as in the Plackett-Burman experimental design [30]. Analyses were
carried out respectively: in a dark room or with all lights turned on under the Mini Matcher booth
described in 2.2; at 20 °C and at 30°C; laying the tablet on a plane surface or holding it in the palm of the
hand (this of course was not tested for packaging).
Table 5
Experiment n°
Ambient Light
Temperature
Sampling
Result
1
+
+
+
Y1
2
+
Y2
3
+
Y3
4
+
Y4
 External light: - = light turned off ; + = light turned on
 Sampling: + = sample firmly kept in the palm of the hand ; - = sample leant on a plane surface
 Temperature: + = 30°C ; - = 20 °C
Reflectance spectra obtained in all these conditions were compared following the scheme:
Ambient Light = [(Y1 +Y3) - (Y2 +Y4)] /2
Temperature = [(Y1 +Y2) - (Y3 +Y4)] /2
Sampling
= [(Y1 +Y4) - (Y2 +Y3)] /2
Then a Student’s t is calculated for each parameter
t = /s
where s is the total Standard Deviation calculated during the training stage.
The t calculated for each parameter is compared to tcritical at 95% probability level, for infinite degrees of
freedom, to verify if that parameter has a significant influence on the measure or not.
30
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Ruggedness experiments confirmed what was hinted by precision experiment about tablets handling.
The only parameter that may have a perceptible influence on the final result is in fact the way in which
the sample is handled, especially for Cialis tablets. This confirms the importance of a basic training of the
operator: to obtain a reliable result he has to let the scanner adhere as perfectly as possible to the
tablet surface in order to avoid ambient light to interfere with the analysis and most importantly he has
to hold the tablet firmly in order to avoid wobbling, which could caused a certain amount of variability
among repetitions.
Temperature and ambient light, instead, didn’t show any influence on the analysis at least in the
investigated range.
In conclusion the method validation proved that the colorimeter is sufficiently precise for most kind of
tablets and packages. In case of very convex tablet, like Cialis, the moderate imprecision due to its shape
may lead to less thorough responses when extremely accurate counterfeits are analyzed. At the
moment the small number of extremely accurate Cialis counterfeits tested does not allow for conclusive
statements on this issue. In the meantime the colorimeter remains a very promising choice as a support
for in the field inspectors that have to decide whether to collect a sample for the laboratory or not and
for those developing countries that cannot afford high-tech analytical devices.
31
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
3.2 SOLID STATE TECHNIQUES: IR SPECRTOSCOPY AND THERMOANALYSIS
3.2.1 IR
Deepening the investigation on counterfeit medicines, Infrared spectroscopy was applied. A
Golden Gate Reflectance FT-IR equipped with a diamond ATR sampling system was employed.
Tablet external coating was mechanically removed; subsequently the sample was ground and analyzed.
By this technique it is possible to look into a tablet examining its total composition; as a matter of fact IR
spectrum can provide information on both the API and the excipients. Nevertheless FT-IR is a very fast
technique considering that no further sample preparation is needed and spectrum acquisition requires
only a few seconds.
By an accurate comparison between the IR spectrum of a suspicious sample and that of the original drug
it is possible to determine if they have the same composition or not, thus it permits to ascertain
definitively if the investigated sample is counterfeit or not. In this way IR spectroscopy is employed for a
qualitative analysis of the whole composition of a tablet, thus as a fingerprinting technique.
Also comparisons among counterfeit samples can be made to obtain additional information. I.e. if
different counterfeit drugs have very similar composition it could be hypothesized that they are
manufactured in the same industry.
Fig.17 : Perkin Elmer FTIR
System 2000 with ATR
Golden Gate sampling
system was employed.
Based on such bases a study on the possible application of IR spectroscopy to recognize the single
contributes of all the components of an suspicious drug was conducted.
For this purpose a data bank was built analyzing separately, as reference standards, both active
substances and excipients of the investigated original drugs.
In particular Viagra  Pfizer, Cialis Ely Lilly, Levitra Bayer were examined.
In addition many other excipients have been screened, in particular the most common and cheap, and
also some substances from food industry that could be used in illegal drug manufacturing to substitute
the more expensive pharmaceutical excipients (like sucrose, coconut flour etc.). But even inedible
materials, (like materials from construction industry) have been analyzed because their use in
manufacturing of counterfeits has been reported [8-9-10].
Some of the IR spectra, collected for the data bank, are now presented:
32
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
IR spectra of active substances:
SILDENAFIL CITRATE (API of Viagra)
92,9
90
85
80
75
70
65
%T
60
55
50
45
40
35
30
28,3
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
400,0
1600
1400
1200
1000
800
600
400,0
1400
1200
1000
800
600
400,0
cm- 1
TADALAFIL (API of Cialis)
92,9
92
90
88
86
84
82
80
78
76
74
72
70
%T
68
66
64
62
60
58
56
54
52
50
48
45,2
4000,0
3600
3200
2800
2400
2000
1800
cm- 1
VARDENAFIL CHLOROHYDRATE THRIHYDRATE (API of Levitra)
92,9
90
88
86
84
82
80
78
76
74
72
70
68
%T
66
64
62
60
58
56
54
52
50
48
46
44
42
39,3
4000,0
3600
3200
2800
2400
2000
1800
1600
cm- 1
33
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
IR spectra of excipients and common materials :
ANHYDROUS CITRIC ACID
BORIC ACID
96,7
95
99,8
95
90
90
85
85
80
80
75
75
70
65
70
60
65
55
%T 60
%T
50
55
45
40
50
35
45
30
40
25
20
35
15
30
10
25,8
4000,0
7,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
400,0
4249,0
4000
3600
3200
2800
2400
2000
cm-1
STEARIC ACID
1800
cm-1
1600
1400
1200
1000
800
600
400,0
SODIUM CITRATE
112,9
100,9
112
110
95
108
90
106
85
104
80
102
100
75
98
70
96
%T
%T 65
94
60
92
90
55
88
50
86
45
84
40
82
80
35
78,0
31,1
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
400,0
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
400,0
1000
800
600
400,0
cm-1
cm-1
MICROCRYSTALLINE CELLULOSE
CORNFLOUR
95,9
96,7
95
90
90
85
85
80
80
75
75
70
70
65
60
65
%T
%T
55
60
50
55
45
50
40
45
35
30
40
25
35
18,7
31,9
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
400,0
4000,0
3600
3200
2800
2400
2000
1800
1600
cm-1
cm-1
34
1400
1200
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
SODIUM CROSCARMELLOSE
POVIDONE – CROSPOVIDONE
96,6
94
92
90
88
86
84
82
80
78
76
74
72
70
%T
68
%T
66
64
62
60
58
56
54
52
50
48
46
44
42
40,0
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
400,0
4000,0
cm-1
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
400,0
cm-1
METHYLCELLULOSE - HYDROXYETHYLCELLULOSE – HYDROXYPROPYL CELLULOSE - HYDROXYPROPYL
METHYLCELLULOSE
%T
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
cm-1
GLUCOSE
SUCROSE
35
800
600
410,0
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
99,0
97,1
95
95
90
90
85
85
80
80
75
70
75
65
70
60
65
%T
%T
55
60
50
55
45
50
40
45
35
40
30
35
25
20,9
4249,0
30,2
4000
3600
3200
2800
2400
2000
1800
cm-1
1600
1400
1200
1000
800
600
400,0
4249,0
4000
3600
3200
2800
2400
2000
1800
cm-1
1600
1400
1200
1000
800
ANHYDROUS LACTOSE – MONOHYDRATE LACTOSE
%T
4000,0
3600
3200
2800
2400
2000
1800
1600
cm-1
36
1400
1200
1000
800
600
400,0
600
400,0
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
MANNITOL
MALTODEXTRIN
138,2
202,9
200
135
190
130
180
125
170
120
160
115
150
110
140
105
130
100
%T 120
%T
95
110
90
100
85
90
80
80
75
70
70
60
65
50
60
58,3
41,9
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
400,0
4000,0
cm-1
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
405,0
cm-1
DEXTRATES
EXPLOTAB (SODIUM STAARCH GLYCOLATE)
98,6
98
95,8
97
90
96
95
85
94
93
80
92
75
91
90
70
89
88
65
87
86
60
85
%T
%T
84
55
83
50
82
81
45
80
79
40
78
77
35
76
30
75
74
25
73
72
71,2
19,1
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
4000,0
400,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
402,0
cm-1
cm-1
EMCOMPRESS
ANHYDROUS CALCIUM HYDORGEN PHOSPHATE
95,0
97,3
96
94
90
92
90
85
88
86
80
84
82
75
80
78
70
76
74
65
72
70
60
68
%T 66
%T
55
64
62
50
60
58
45
56
54
40
52
50
35
48
46
30
44
42
25
40
20,2
36,3
38
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
400,0
4000,0
3600
3200
2800
2400
2000
1800
1600
cm-1
cm-1
37
1400
1200
1000
800
600
400,0
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
COLLOIDAL SILICA – AMORPHOUS SILICA
TALC
100,4
95
90
85
80
75
70
65
60
55
50
%T
%T
45
40
35
30
25
20
15
10
5
0
-3,4
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
4000,0
400,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
400,0
800
600
400,0
cm-1
cm-1
MAGNESIUM STEARATE
SODIUM LAURYL SULFATE
97,5
97
200,0
96
190
95
180
94
93
170
92
91
160
90
150
89
88
140
87
86
130
85
120
84
%T
83
%T 110
82
81
100
80
90
79
78
80
77
76
70
75
60
74
73
50
72
71
40
70
30
26,2
68,5
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
4000,0
400,0
POLISORBATE 80
94,4
90
85
80
75
70
65
60
55
%T
50
45
40
35
30
25
20
14,7
4000,0
3600
3200
2800
2400
2000
1800
3600
3200
2800
2400
2000
1800
1600
cm-1
cm-1
1600
1400
1200
1000
800
600
400,0
cm-1
38
1400
1200
1000
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
XANTAN GUM – TRAGACANTH
96,7
95
90
85
80
75
70
65
60
%T
55
50
45
40
35
30
25
20
17,4
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
400,0
cm-1
GUM ARABIC
CARNAUBA WAX
96,8
96
106,2
94
100
92
95
90
90
88
85
86
80
84
75
82
70
80
%T
%T
65
78
60
76
55
74
72
50
70
45
68
40
66
35
64
30
27,6
62,1
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
400,0
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
405,0
cm-1
cm-1
COCONUT FLOUR
POTATO STARCH – CORN STARCH
96,6
101,5
100
94
98
92
96
90
94
88
92
86
90
84
88
82
86
80
84
78
82
76
80
74
78
72
76
70
74
68
%T 72
%T 66
70
64
68
62
66
60
64
58
62
56
60
54
58
52
56
50
54
48
52
46
50
44
48
42
40
46
37,3
43,5
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
400,0
4000,0
3600
3200
2800
2400
2000
1800
1600
cm-1
cm-1
39
1400
1200
1000
800
600
400,0
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
SODIUM BICARBONATE
ANHYDROUS SODIUM CARBONATE
102,1
96,1
94
100
92
98
90
96
88
94
86
84
92
82
90
80
88
78
86
76
84
74
82
72
%T
%T
70
80
68
78
66
76
64
74
62
72
60
70
58
56
68
54
66
52
64
50
62
60,6
47,5
4249,0
4000
3600
3200
2800
2400
2000
1800
cm-1
1600
1400
1200
1000
800
600
4000,0
400,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
400,0
cm-1
CALCIUM CARBONATE
MAGNESIUM CARBONATE
101,6
100
101,7
100
95
95
90
90
85
85
80
80
75
75
70
70
65
65
60
%T
%T 55
50
60
55
45
50
40
45
35
40
30
35
25
30
20
15
25
22,3
11,5
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
400,0
4000,0
3600
3200
2800
2400
2000
1800
ANHYDROUS SODIUM SULFATE
100,8
98
96
94
92
90
88
86
84
82
80
78
%T
76
74
72
70
68
66
64
62
60
58
55,2
4249,0
4000
3600
3200
2800
2400
2000
1600
cm-1
cm-1
1800
cm-1
40
1600
1400
1200
1000
800
600
400,0
1400
1200
1000
800
600
400,0
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
IR spectra of materials from building industry:
HYDRATE LIME
CEMENT
98,7
97,0
95
94
92
90
90
88
86
85
84
82
80
80
78
75
76
74
70
72
70
%T
%T 65
68
66
60
64
62
55
60
58
50
56
54
52
45
50
40
48
35
33,5
42,3
46
44
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
400,0
4249,0
4000
3600
3200
2800
2400
2000
1800
cm-1
cm-1
WHITE CEMENT
1600
1400
1200
1000
800
600
400,0
CHALK
197,0
102,7
100
180
160
95
140
90
120
85
100
80
80
60
75
40
70
20
65
%T 0
%T
60
-20
-40
55
-60
50
-80
45
-100
-120
40
-140
35
-160
30
-180
-192,0
25,9
4249,0
4000
3600
3200
2800
2400
2000
1800
cm-1
1600
1400
1200
1000
800
600
400,0
4000,0
102,35
102,0
101,5
101,0
100,5
100,0
99,5
99,0
98,5
98,0
%T
97,5
97,0
96,5
96,0
95,5
95,0
94,5
94,0
93,60
3600
3200
2800
2400
2000
1800
3200
2800
2400
2000
1800
1600
cm-1
STUCCO
4000,0
3600
1600
1400
1200
1000
800
600
420,0
cm-1
41
1400
1200
1000
800
600
400,0
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
The following approach was employed to detect the components of a suspicious drug from its IR
spectrum:
 First, the peculiar bands of every reference substance of the IR data bank were identified. Each
substance has a unique IR spectrum; some bands of the spectrum are peculiar of a particular
substance.
 Second, the presence of these peculiar bands was searched in the IR spectrum of suspicious drugs. In
this way it was detected which substances, among those of the data bank, were present in the
investigated drug product.
First of all this approach was tested on original tablets, of Viagra, Cialis and Levitra, whose qualitative
composition is precisely known.
 Viagra: API : sildenafil citrate
Excipients : microcrystalline cellulose, anhydrous calcium hydrogen phosphate, sodium
croscarmellose, magnesium stearate
 Cialis:
API : Tadalafil
Excipients : microcrystalline cellulose, sodium croscarmellose, magnesium stearate, sodium
dodecyl sulfate (SDS), monohydrate lactose, hydroxypropylcellulose.
 Levitra: API : Vardenafil chlorhydrate trihydrate
Excipients : crospovidon, magnesium stearate, microcrystalline cellulose, anhydrous
colloidal silica.
For Viagra all the authorized dosage strengths have the same qualitative and quantitative composition,
while in Cialis and Levitra the active substance/excipients ratio is different for each dosage, thus they
have been analyzed separately.
The IR spectra of the brand drug products Viagra, Cialis an Levitra are presented below:
42
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
BRAND VIAGRA
96,6
96
94
92
90
88
86
Sildenafil
Mg stearate
84
82
%T 80
78
76
74
Sildenafil
72
70
68
Cellulose
66
64,1
4249,0
4000
3600
3200
2800
2400
2000
1800
cm-1
1600
1400
1200
1000
800
600
400,0
600
400,0
BRAND CIALIS
95,9
90
85
80
75
Tadalafil
70
Mg stearate/SDS
65
%T
60
55
50
Lactose
45
40
Cellulose
35
30
24,3
4249,0
4000
3600
3200
2800
2400
2000
1800
cm-1
1600
1400
1200
1000
800
BRAND LEVITRA
95,5
94
92
90
88
86
84
82
Vardenafil
80
78
76
74
Mg stearate
72
70
68
66
Vardenafil
%T 64
62
Vardenafil
60
58
56
Cellulose
54
52
50
48
46
44
42
Cellulose
40
38
36
33,7
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
400,0
cm- 1
Fig 18 : IR spectra of brand Viagra, Cialis and Levitra. The contributes form each components of the drug products
are highlighted with different colors.
43
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
With this approach it is possible to detect the presence of the three active substances (Sildenafil,
Tadalafil and Vardenafil) and the presence of the main excipients: microcrystalline cellulose, lactose,
magnesium stearate, that actually is contained in a very little amount but which alchilic chain gives a
very strong and peculiar signal. In very elaborate formulations like that of Cialis, that contains a large
number of excipients, it was not possible to point out all components, in particular those contained in a
little amount, also because the predominant presence of lactose covers the other signals. After all it is
reasonable to suppose that big amounts of cheap raw materials are commonly used in counterfeit
manufacturing, rather than a large assortment of technological and expensive excipients, like for
example hydroxypropylcellulose, that operate in modulating the API release.
In the range 3000 – 2800 cm-1 both magnesium stearate and sodium dodecyl sulfate (SDS) give a
characteristic signal due to C-H bond stretching of the alchilic chain that is easily detected in drug
products; thus from this band the presence either of magnesium stearate or SDS is pointed out.
Afterwards this approach was applied to illegal drugs and its capability was stressed: i.e. mannitol was
detected in many chewable sildenafil tablets (like Kamagra and Filagra, illegal drugs). Mannitol is
commonly used as sweetening agent in chewable tablets but a formulation containing mannitol and
sildenafil has never been approved in Italy.
%T
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
400,0
cm-1
Fig. 19 : Comparison between the IR spectra of a chewable Kamagra sample (black line) and that of mannitol (blue
line). The peculiar peaks of mannitol, circled in orange, are evident in Kamagra spectrum.
Often lactose was detected together with sildenafil while in the original Viagra formulation lactose is
absent. Lactose employed in pharmaceutical industry must be controlled for TSE (Transmissible
spongiform encephalopathy) contamination and it is quite unlikely that these tests are done by
counterfeiters [31]. Moreover lactose, not labeled, may cause allergic reaction.
44
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
%T
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
400,0
cm-1
Fig. 20 : Comparison between the IR spectra of a sample named Hindgra (black line) and that of monohydrate
lactose (blue line). The peculiar peaks of lactose, circled in yellow, are evident in Hindgra spectra.
In another formulation tragacanth gum was detected by the sole IR.
%T
4000,0
3600
3200
2800
2400
2000
1800
cm-1
1600
1400
1200
1000
800
600
500,0
Fig. 21 : Comparison between the IR spectra of a counterfeit Viagra (black line) and that of Tragacanth gum (blue
line). The peculiar peaks of Tragacanth gum, circled in sky blue, are evident in the counterfeit Viagra.
Studies to apply IR spectroscopy for quantitative analysis were also carried out, in particular to quantify
the active substances. Nevertheless it was not possible to develop a reliable and rapid quantification
method because the diamond ATR sampling system does not allow to have a reliable thickness of the
analyzed substance. For this reason just an approximate indication about the amount of API in respect
to the other components in the product can be obtained.
45
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
The blind spot of this application in qualitative analysis of counterfeit drugs is that signals from different
components can overlap, thus a very abundant component can mask the presence of the others.
3.2.2 DSC and TGA
To support IR, two thermoanalysis techniques on solid state samples were employed, Differential
Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA) [32-33-34-35].
These techniques are rapid and the sample preparation is very simple: neither extraction nor dissolution
in any solvent is necessary; only the external coating of tablets must be removed and the tablet must be
ground as for IR, thus the same sample preparation can be employed both for IR and for thermal
analyses. The instrumental run takes about 20-40 minutes, depending on the investigated temperature
range and the temperature scan rate; DSC and TGA can operate simultaneously. A single run is not very
expensive because there is no consumption of materials apart from the aluminium pan. These aspects
are very important because the amount of samples that a control laboratory have to screen is increasing
at a very fast pace these days.
Fig. 22 : on the left: the Perkin Elmer DSC 7 employed to acquire DSC thermograms. On the right: Perkin Elmer
Pyris1 TGA employed for TGA analyses.
Differential scanning calorimetry is a technique used to study the thermal transitions that samples
undergo on heating. Thermal transitions are changes in the structure of a material, such as melting,
crystallisation and glass transition.
In DSC the sample (which is put in an aluminium pan) and an inert reference (an empty identical pan)
are heated separately by two heaters at a predefined rate (measured in °C/min). A computer control,
connected to sample and reference, assures that the two pans remain at the same temperature
throughout the entire experiment. When the sample reaches the temperature at which it undergoes
some thermal transition, its heater will have to put out an amount of heat that is different from the one
of the reference. This difference in heat is measured and reported in a plot: this means that there will be
a specific signal for each thermal transition. In particular a DSC thermogram is a plot in which
temperature is reported on the x-axis and the difference in heat output of the two heaters on the y-axis.
In this way the heat absorbed/released by the sample is plotted against temperature, as reported in
figure 23.
46
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Fig. 23 : Schematic representation of thermal phenomena detectable by DSC. Tg: glass transition temperature; Tc:
crystallization temperature; Tm: melting temperature.
Endothermic transitions in which the sample absorbs heat, like melting, produce a positive peak, while
exothermic transitions in which the sample gives off heat because it goes into a more stable state, like
crystallisation, will produce a negative peak.
But also other phenomena like polymorphic transitions, in which the sample changes its crystalline
structure, and loss of water or solvents can be detected by a DSC.
Water and solvent losses produce wide endothermic peaks when the solvent is weakly bound to the
substance (e.g. when it is adsorbed on the surface) while loss of water of crystallisation shows a sharp
peak in the thermogram.
Commonly DSC is used to investigate the physico-chemical properties of a pure substance. For example
in pharmaceutical analysis it is used to establish thermal behaviour of active substances: onset
temperature of fusion, glass transition, crystallization, desolvation, polymorphic transitions etc.
However DSC can be successfully employed to analyse mixtures of substances, such as pharmaceutical
products, as each ingredient in the mixture shows its own set of signals that are specific to that
substance at that temperature.
For this reason this technique has been employed in the analysis of counterfeit/illegal pharmaceutical
products.
For a correct interpretation a data bank of reference substances is needed: the signals in the
thermogram of an unknown mixture are identified by matching them with those of the various
reference substances that can possibly be present in the sample under investigation.
For the building of the data bank, the analysis of all the reference substances must be conducted at the
same operative conditions that will be used in the screening of counterfeits, i.e. the same kind of pan
and the same temperature scan speed. This is because the temperature at which thermal phenomena
happen is strictly related to these aspects, especially to the scan speed.
When analysing mixtures a problem can arise from the overlap of signals from different substances.
However the temperature range investigated was in general sufficiently wide to avoid such overlapping
of signals.
47
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Analyses were conducted from ambient temperature to 200 - 250°C, the limit being the decomposition
of one or more of the substances in the mixture. To improve resolution the amount of sample analyzed
was quite small (1 - 1,5 mg) so that the peaks were not too large, but still maintaining sufficient
sensitivity even for substances present at 10% or even less. Furthermore closed pans were used: in this
way signals become much sharper because the gradual equilibration of the sample condition with the
ambient that occurs in an open pan is avoided. Anyway since in many thermal phenomena some
materials are released from the sample, i.e. aqueous vapour during a water loss phenomenon, a hole is
made in the pan cover to let such materials freely expand in the ambient without compromising the
very structure of the pan.
Thermogram resolution is also dependent on the temperature scan speed. Analyses were conducted
operating at 10°C/min to reach the best compromise possible between time of analysis and resolution.
The actual limit of the DSC analysis of mixture resides in the fact that when one of the ingredient
decomposes, it generates very intense signals that mask every phenomenon happening at higher
temperatures. So the temperature at which the first decomposition happens (often about at 220°C, that
is the temperature at which celluloses char and lactose decomposes) is the maximum temperature that
can be investigated by DSC.
For this reason much more information are obtained using simultaneously DSC and TGA.
A thermo gravimetric analyser is basically a very sensitive microbalance (with a digit of a tenth of
microgram) in which the sample is weighted inside a furnace, where the temperature can be varied
according to a specific heating program. It measures the changes in sample weight that occur upon
heating. Such changes may be due for example to water or solvent loss, or to decompositions. Thus
through TGA it is possible to quantify the water content of a sample.
In a TGA plot the weight of the sample is constantly measured and reported on the y-axis against
temperature.
Fig. 24 : TG analysis of sodium citrate. Weight loss and its derivative versus temperature are showed. Various
thermal phenomena occurring at different temperature, marked with arrows, are shown as peaks in the thermal
profiles. Dashed line: weight; bold line: derivative weight; A: loss of water, B: decomposition.
A TGA plot is generally made by a series of steps representing the various sample weight losses as a
function of temperature. To have a better representation of these phenomena, the derivative weight
loss is reported on the y-axis and the various signals will be peaks instead of steps.
48
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Since in TGA the signals are generated by weight losses, TGA cannot detect fusions or phase transitions
like DSC does. However it is better suited than DSC for detecting decompositions: when a substance
decomposes it loses weight in neat, consecutive steps, which are clearly represented in a TGA
thermogram. Decompositions in TGA do not mask the subsequent signals like they do in DSC and, most
importantly, substances that decompose without melting, which are quite elusive in DSC, are clearly
detected by TGA. So it can be stated that DSC and TGA can be regarded as complementary.
As TGA was used together with DSC, the same temperature program (10 °C/min) was employed for both
of them, in order to compare the plots generated by the two techniques. In this way the various thermal
phenomena will be detected at the same temperatures and the two thermograms, obtained by DSC and
TGA respectively, can be superimposed to have a single and complete representation of the thermal
behavior of a compound (see figure 25). The sample is put inside an aluminium pan with the cover
holed, identical to that used in DSC. A flux of nitrogen is used to avoid oxidation of samples and to help
carrying away the vapours produced during the analysis.
Fig. 25 : Thermal analysis of sucrose. Various thermal phenomena, marked with arrows are shown. Fusion (A) is
detectable only with a DSC analysis; decompositions (B, C) are detected at the same temperature in both analysis;
higher temperature decompositions are detectable only with TGA analysis, since earlier decompositions usually
mask all subsequent phenomena in a DSC analysis. Dashed/dotted line: weight; bold line: derivative weight; dotted
line: heat flow; A: fusion, B: decomposition; C: decomposition; D: decomposition.
In TGA the thermogram of a mixture can be considered the sum of the signals (weight losses in this case)
of each components. Thus also TGA can be useful to find out the exact composition of an unknown
pharmaceutical product such as counterfeits and illegal medicines.
The temperature range that can be scanned by TGA in the analysis of mixtures is wider than that of DSC
because TGA is informative even after one ingredient decomposes. Usually a TGA works from ambient
temperature to 500 °C, beyond this temperature the aluminum of the pan melts.
49
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
For the analysis of mixtures an amount of powder between 3 and 4 mg was weighed. Greater amounts
would result in an undesirable loss of resolution, because weigh losses steps would became wider and
overlap each other.
The ability of thermoanalysis in helping to detect which components are present in a suspicious
medicine depends on the thermal behaviour of the active substances and excipients and on the
specificity of their thermal profiles. Hence, similarly to the approach developed with FT-IR, also for
thermal analyses a data bank of active substances and excipients, analyzed separately as reference
standards, was built.
The API usually are easily detected in DSC by their melting peaks. From the presence (or the absence) of
a peculiar melting peak it is possible to determine the presence (or absence) of the declared API. In the
same way the substitution with another ingredient is detectable by the presence of its own melting
peak. Nevertheless some API are not detectable by DSC because they don’t melt in the DSC temperature
range, or decompose without melting, or their melting peak is masked by the decomposition of some
excipient; in such cases TGA thermogram may be helpful, if the investigated API shows specific
decomposition signals.
However, most of the excipients undergo thermal transitions or weight losses in the temperature range
investigated by DSC and TGA so their presence in a suspect drug sample can be identified with success
by detecting their peculiar signals.
Most excipients contain water, adsorbed on the surface or crystallisation water, many show melting.
Practically all of them decompose, often in multiple steps, with a profile that is unique of each one. In
figure 26 the DSC and TGA thermograms of some common excipients but also cheaper materials from
food industry or from construction industry are depicted. These examples constitute part of a more
extensive database.
50
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Fig. 26 : A collection of thermograms of various substances. Different substances that may be found in counterfeit
drugs (pharmaceutical or alimentary excipients or other substances reported to be used in counterfeit
preparations) were analyzed both by TGA and by DSC to obtain their thermograms. Here the data for some of the
most common substances are reported, showing that different substances have signals at different temperatures
and thus are distinguishable if present in the same mixture. For an easier comparison of the thermograms of
different substances, only the interval between 50 and 400°C is reported. Only the derivate of the TGA thermogram
is reported. Vertical heat flow and derivative weight % scales are not reported. Bold lines: derivative weight; dotted
lines: heath flow.
51
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
In figure 27A is reported the case of an illegal drug that was declared to be a generic of Cialis, reporting
Tadalafil to be the API, both on the box and in the leaflet. The DSC scans unequivocally uncovered the
fraudulent presence of Sildenafil. A confirmation of this can be obtained by TGA: as showed by figure
27B, the decomposition pattern of Sildenafil is clearly present in the thermogram of the drug.
Fig. 27 : Detection of the substitution of the API. Thermal analysis of a counterfeit drug claiming to contain
Tadalafil and an original Cialis are reported. In A a comparison between DSC analyses is showed, in B a comparison
between TGA analyses. Since both the original and the counterfeit drugs have the same peaks at the same
temperatures, we can assume that the original drug and the counterfeit one have very similar composition.
However, in the thermal analysis of the counterfeit drug there are additional peaks, marked with arrows, that are
not present in the thermograms of the original drug. These peaks correspond to the ones detected analysing
Sildenafil (in particular for DSC, the endothermic peak between 190 and 200°C is the melting of Sildenafil). For
easier interpretation of the data, only the derivate of the TGA thermogram is reported. A: DSC analysis. Bold line:
counterfeit drug; dotted line: original drug. B: TGA analysis. Bold line: counterfeit drug; dotted line: original drug.
52
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Therefore comparing the thermogram of a suspicious drug to that of the corresponding original it is
possible to determine if it is a counterfeit or not; as a matter of fact if the two thermograms are
different then the drug must be a counterfeit (see figure 28) because it contains one or more substances
different from the original drug or it does not contain one or more ingredients of the original.
Fig. 28 : Direct comparison of the thermograms of original and counterfeit drugs. In A comparison between DSC
analysis of an original Cialis and a counterfeit one is showed; in B comparison between TGA analysis of an original
Viagra and a counterfeit one. The thermograms are easily distinguishable, demonstrating that the analyzed
counterfeit drugs have different compositions than those of the original drugs. For easier visualization, DSC
thermograms were shifted across the heat flow scale. For an easier interpretation of the data for TGA, only the
derivate thermogram is reported. A: DSC analysis. Bold line: original drug; dotted line: counterfeit drug. B: TGA
analysis. Bold line: original drug; dotted line: counterfeit drug.
By these techniques it is possible to identify which ingredients have been employed in the formulation
of an unknown sample, if they are different from those of the original and if there are unhealthy
ingredients.
In cases where the active substance and excipients are the same as the original drug then, it may still be
useful to determine differences in the quality of raw material, evidenced by minor solid state differences
53
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
due for example to different polymorphic forms or hydration or crystalline habit; thus this approach
may help to discover if low quality ingredients have been employed, detecting counterfeiting even when
the fake drug is very well made.
Actually the better results on unknown drugs investigation are obtained when the three solid state
techniques, FT-IR, DSC and TGA, are used together and the information derived from each data bank
matched each other.
By DSC and TGA it is possible to have complete information about the thermal phenomena of the
components of an illegal drug. But FT-IR can provide precious additional information.
The combined use of these three techniques is a very effective solid state approach to detect the
composition of an unknown medicine.
Indeed these solid state techniques should not be regarded as alternatives, but instead as
complementary to gather a more comprehensive set of information about the suspect sample. As a
matter of fact, on one hand the FT-IR signals of the major components of a drug may cover those of the
minor ones; sometimes signals from two or more substances overlap; many inorganic substances are
invisible, since they lack the covalent bounds necessary to generate IR signals. In all these cases thermal
analysis can be of help: a minor component of a mixture could be detected by DSC or TGA if its signals,
even if small compared to those of the more abundant components, fall in a signal-free temperature
region. In addition many substances invisible in spectroscopy are instead clearly visible in DSC and TGA,
because for example they lose water at a specific temperature.
On the other hand not all the substances present in a mixture can be detected by thermal analysis;
some substances don’t give any signal in thermoanalysis because they don’t contain water and don’t
melt. In addition thermal signals may be covered by decomposition phenomena of the other
components of the mixture. In such cases IR is the sole source of information.
Anyway, even employing this solid state approach by FT-IR, DSC and TGA, if a counterfeit sample is very
similar to the original it could be tricky to recognize the drug as a counterfeit because there may be just
very small differences. On the other hand some minor differences in the DSC and TGA thermograms
may arise even from normal batch to batch variability among originals.
For this reason these solid state techniques, although quite inexpensive and fast, need skilled and
experienced operators to be effective.
The information on mixtures collected by these techniques are merely qualitative. So to detect an
underdosage or over-dosage of an API in a counterfeit drug other techniques such as HPLC are needed.
Nonetheless when the quantity of one ingredient in the investigated drug differs significantly from the
original formulation, its specific DSC and TGA signals will be correspondingly very different in intensity.
So a rough statement on possible over or underdosage of the API could be made also with thermal
analysis.
In figure 29 the DSC thermograms of three mixtures containing Sildenafil and lactose in different ratios
is reported.
54
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Fig. 29 : Influence of the relative concentration of two substances on DSC thermal response. The DSC analysis of
three different mixtures of lactose monohydrate and Sildenafil citrate generate different thermograms. For easier
visualization, DSC thermograms were shifted across the heat flow scale. Bold line: 3-1 mass/mass ratio mixture of
monohydrate lactose/Sildenafil citrate; dotted line: line: 1-1 mass/mass ratio mixture of monohydrate lactose Sildenafil citrate; dashed line: 1-9 mass/mass ratio mixture of monohydrate lactose - Sildenafil citrate; black
arrows: thermal phenomena related to monohydrate lactose; white arrows: Sildenafil fusion/decomposition.
It can be noticed that upon lowering the amount of Sildenafil the height of its fusion peak (190 – 200°C)
diminishes accordingly. In particular when lactose is the prevalent component of the mixture (bold line),
two peaks for lactose (one at 145°C showing a loss of water and one at 210°C due to fusion and
decomposition) are present, and one peak for Sildenafil (at 195°C due to fusion/decomposition). When
the two substances are present at a similar concentration (dotted line) the same peaks are presents at
the same temperatures, but the peak of Sildenafil citrate is noticeably higher and wider. When Sildenafil
citrate is present in a superabundant amount (dashed line), the lactose peak at 210°C is no longer visible
and the peak at 145°C become much smaller. Thus DSC, while not being a quantitative technique, shows
different responses to different amounts of substances and so can provide information about the
relative concentration of substances in a mixture.
The thermal analyses can also provide another kind of valuable information, about interaction problems
among components of a illegal drug. Very often counterfeit drugs have a different formulation from the
original one, containing different ingredients. But if these ingredient are not accurately chosen active
substance/excipient interaction problems can arise.
Generally a DSC and TGA thermogram of a well formulated pharmaceutical product is the sum of the
signals of each components of the mixture. Yet a different evidence occurs if two or more substances in
a mixture interact with each other when the right temperature is reached. In this case the thermogram
won’t be the sum of the signals of the two substances because, as a consequence of the interaction
between them, the original signals will change either in position or in shape.
55
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
This ability of DSC to detect interaction between substances in a mixture is commonly used by
pharmaceutical industry during formulation studies [36–37]: inert excipients that do not interact with
the active ingredient are generally chosen, because interactions may have an unpredictable and
unwanted influence on the release of the active ingredient from the matrix, bringing bioavailability
problems, and can also lower the stability of the product.
Also in the analysis of counterfeit and illegal products this DSC ability can be very useful, because it
allows highlighting the possible presence of API/excipient or excipient/excipient interaction that could
involve an incorrect formulation and bring risks for patients.
3.2.3 Case studies
Some case studies will now be presented:
A case of substitution of one excipient with a cheaper one
This sample from India, sold by an online pharmacy, is named Tadalafil. Its name, together with the
tablet shape and colour, which have no resemblance with the original brand drug, Cialis, (on the right in
the picture) clearly suggests that the manufacturer had no intention to deceive customers into believing
that this was the original drug. So this is not a counterfeit, but an illegal “generic” version of Cialis. Of
course there is no authorisation for example in USA or Europe to produce and market generics of this
brand (since its patent is not yet expired) so this sample was sold illegally.
Comparing IR spectrum, DSC and TGA thermogram of this drug with those of Cialis, it can be noted that:
 In IR spectrum some more peaks, in addition to those of original Cialis, were detected.
 In DSC the signals of lactose at about 140°C and 220°C (respectively its water loss and its first
decomposition step) are less pronounced
 Both in DSC and in TGA there is a quite prominent peak at about 190°C
Checking the thermograms and the spectra in the database, this additional signals could be assigned to
the Emcompress (Dihydrate Calcium Hydrogen Phosphate), a pharmaceutical excipient that is not
present in original formulation of Cialis. In particular the additional signals in DSC and TGA arose from
the water loss of this excipient.
Thus the manufacturer used a minor amount of lactose and substituted the rest of it with Emcompress,
which has a similar function, but is considerably cheaper. This kind of formulation for a drug containing
Tadalafil was never studied, so it is not known if it is suitable to assure good stability and correct release
of the active substance.
56
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
%T
4000,0
3600
3200
2800
2400
2000
1600
1800
1400
1200
1000
800
600
400,0
cm-1
Fig. 30 : Comparison between the IR spectra of brand Cialis (black line), Emcompress - calcium hydrogen phosphate
bihydrate (blue line), and counterfeit Cialis (red line). The peculiar peaks of emcompress are circled in orange: they
are evident in the counterfeit drug; on the contrary they are lacking in the spectra of brand Cialis.
57
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Fig. 31 : DSC (A) and TGA (B) analyses of an illegal drug and of an original Cialis are reported. In the thermograms
of the counterfeit drug an anomalous peak is present, marked with black arrows, proving the presence of
Emcompress. Furthermore, in the DSC thermogram, signals of lactose (white arrows) are less pronounced than
those in the original drug. For easier visualization of the thermograms, DSC thermograms were shifted across the
heat flow scale and only derivative weight is shown for TGA. Bold line: counterfeit drug; dotted line: original drug;
black arrow: emcompress; white arrow: lactose monohydrate.
58
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
A case of an undeclared excipient
This sample is an imitation of Viagra, illegally sold in Europe. It is currently manufactured in India and
sold by various online pharmacies. Even if this drug is not strictly a counterfeit because it has a different
name (Filagra) from the original brand, looking at its diamond shape and its blue colour, the
resemblance with original Viagra (at the right side of figure 32) seems obvious.
Fig. 32 : DSC (A) and TGA (B) analyses of an illegal drug and of an original Viagra are reported. In the thermograms
of the illegal drug are presents anomalous peaks, marked with arrows, proving the presence of lactose
monohydrate. For easier visualization, DSC thermograms were shifted across the heat flow scale and only
derivative weight is shown for TGA. Bold line: counterfeit drug; dotted line: original drug; black arrow: lactose
monohydrate.
59
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
%T
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
400,0
cm-1
Fig. 33 : Comparison between the IR spectra of brand Viagra (black line), monohydrate Lactose (blue line), and
counterfeit Viagra (red line). The peculiar peaks of lactose are circled in orange: they are evident in the counterfeit
drug; on the contrary they are lacking in the spectra of brand Viagra.
From the observation of its DSC and TGA profiles, the presence of some very intense signals can be
immediately noticed. By a quick comparison with the thermograms of the database they are
immediately matched to signals from lactose monohydrate: in particular the water loss at 140°C, the
first decomposition step at 220°C and the second decomposition step at over 300°C. Also in IR spectrum
the presence of lactose is evident from some peculiar peaks.
The fusion and decomposition of the API Sildenafil citrate (190 – 200°C) shows very faint signals here
because lactose signals are so intense that they partly hide them. This means that lactose is by far the
most abundant component of the mixture, actually the sole component apart from Sildenafil itself and
cellulose, whose water loss signal is visible between ambient temperature and 100°C both in DSC and
TGA plots. As it was the case for the use of Emcompress reported in the previous example, the use of
lactose in a formulation containing Sildenafil has never been studied, thus there are no proofs about its
safety as there is no data to support that it is stable and that it provides a suitable bioavailability. But in
this case there is another risk for the customers: the presence of lactose, should be declared on both the
leaflet and the packaging, because it may result in intolerances for many patients.
A cross contamination case
I-Pramil is a counterfeit of Pramil, a generic medicine containing Sildenafil that is legally sold in
Paraguay. This is a special kind of counterfeiting, since the counterfeited drug in this case is not the
original brand Viagra, but a generic of it, since it is probably more requested by Paraguayan customers.
In both TGA and DSC thermograms (see figure 34) the presence of Sildenafil is indicated by the
decomposition and fusion signals respectively (190-200°C). In IR spectrum as well the bands of Sildenafil
Citrate are evident. The presence of cellulose then is shown by the wide signal between ambient
60
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
temperature and 100°C in both TGA and DSC thermograms. In DSC thermogram it can be also noticed
the very faint signal of Magnesium Stearate at about 105 °C, which is detected also by Infrared
Spectroscopy and, most interestingly, another more pronounced peak at 150°C. This is the signal of the
water loss of lactose. The presence of lactose in such low quantity is probably due to a crosscontamination or a carry-over from a previous production campaign of the plant. This demonstrates
how counterfeit manufacture neglect the correct cleaning of their production equipment, as well as all
the GMP, and alerts on how unreliable the content of an illegal medicine can be.
Fig. 34 : DSC (A) and TGA (B) analyses of a illegal drug and of an original Viagra are reported. In the
thermograms of the illegal drug are presents very small unexpected peaks, marked with arrows, proving
the presence of traces of lactose monohydrate. For easier visualization, DSC thermograms were shifted
across the heat flow scale and only derivative weight is shown for TGA. Bold line: counterfeit drug;
dotted line: original drug; black arrow: lactose monohydrate.
61
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
%T
Sildenafil
Mg stearate
Sildenafil
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
400,0
cm-1
Fig. 35 : Comparison between the IR spectra of brand Viagra (black line) and I-Pramil (blue line). The peculiar bands
of Sildenafil (circled in green) and that of magnesium stearate (circled in red) prove their presence in both Viagra
and I-Pramil.
It is also interesting to note how sensitive the DSC is: the presence of lactose could not be detected by
Infrared Spectroscopy, probably because it was masked by the very intense signals of Sildenafil, which is
present in the mixture in a greater ratio than in the original. To confirm the presence of lactose it was
necessary to recur to Nuclear Magnetic Resonance (NMR) analysis after solvent extraction. DSC can be a
very sensitive instrument and can detect even traces of materials that normally go unnoticed by the
investigation with other techniques.
Illegal medicine with wrong formulation: presence of an excipient that could be not inert
Kamagra is an illegal imitation of Viagra manufactured in India and is also available in chewable tablets.
Also this sample, like Filagra, even though it is not strictly a counterfeit, is misleading since its diamond
like shape hint to the original brand.
Comparing IR spectrum of a Kamagra chewable tablet to that of Viagra some differences can be pointed
out. Searching in the data bank, the additional peaks were identified as the contribute of an unexpected
excipient, that is mannitol, often used as sweetening agent in chewable tablets.
Also in the DSC thermograms of Kamagra it was possible to detect the fusion peaks of Mannitol, beside
signals of Cellulose, Magnesium Stearate and Sildenafil (see figure 36).
Actually both Mannitol and Sildenafil citrate DSC signals are shifted in temperature respect to what
expected from the data bank; furthermore they are not as sharp as they should be, thus actually the two
signals seems almost to coalesce. The shift in temperature was so marked that to confirm the nature of
these signals also a support from another instrument, like NMR, was needed. Such a marked shift
indicates that Mannitol could be not completely inert in this formulation, but it may interact with
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Sildenafil citrate. So the two substances may be incompatible and their interaction could possibly cause
detrimental effect on the tablets stability and on the bioavailability of the API. This problem was
subsequently deepened by stability studies, as after described.
Thus thermo-analysis can be a precious instrument for revealing incorrect formulations of illegal
medicines.
Fig. 36 : DSC (A) and TGA (B) analyses of a counterfeit/illegal drug and of an original Viagra are reported. In the
DSC thermogram of the counterfeit drug there is an anomalous peak, marked with a white arrow, due to the
presence of mannitol. Moreover, both in DSC and TGA, signals of Sildenafil citrate (black arrows) are different in
position and shape from the original drug thermograms. This may be due to an interaction between mannitol and
Sildenafil. For easier visualization, DSC thermograms were shifted across the heat flow scale and only derivative
weight is shown for TGA. Bold line: counterfeit drug; dotted line: original drug; black arrow: Sildenafil citrate; white
arrow: mannitol.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
%T
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
400,0
cm-1
Fig. 37 : Comparison between the IR spectra of Kamagra (black line) and Mannitol (blue line). The peculiar bands of
mannitol (circled in violet) are present in the IR spectra of Kamagra.
A formulation containing an harmful excipient and API substitution
The reported case is about a practice that is quite common among illegal medicines: the use of chalk as
excipient. This sample is a counterfeit of Cialis that was bought from an online pharmacy.
The DSC and TGA thermograms are remarkably different from the ones of the original Cialis (see figure
38). It is evident the presence of Sildenafil, in particular from its fusion DSC peak, so this is clearly a case
of substitution of API. But what is most interesting in both DSC and TGA thermograms is the double-step
signal at about 140°C. Also in IR spectrum anomalous bands can be noted, beside the peculiar band of
Sildenafil citrate. Searching this anomalous signals and bands in DSC, TGA and IR data bank, it was
possible to match them with those of Calcium Sulfate Dihydrate. This substance is commonly known as
gypsum or blackboard chalk. This material, from building industry, was employed in place of lactose,
that is the main excipient of the original drug, because it is much cheaper.
Chalk, as afterwards will be described, was probably employed also for its disaggregating effect on the
tablet.
The presence of chalk may cause many adverse effects, from simple constipation and intestinal
disorders, to anaemia, weight loss, intestinal obstructions, intestinal perforation and life threatening
haemorrhage.
Therefore again the solid state approach was very useful in pointing out a danger coming from the
excipient composition.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Fig. 38 : DSC (A) and TGA (B) analyses of a counterfeit drug and of an original Cialis are reported. In the
thermograms of the counterfeit drug there is a peaks not present in the original drug, marked with a white arrow,
indicating the presence of Sildenafil citrate instead of Tadalafil. Moreover, the peaks marked with black arrows are
different from the original drug peaks. These double-step signals are generated by dehydratation of Calcium Sulfate
Dihydrate, so it indicates the presence of chalk instead of lactose monohydrate. For easier visualization DSC
thermograms were shifted across the heat flow scale and only derivative weight is shown for TGA. Bold line:
counterfeit drug; dotted line: original drug; black arrow: chalk; white arrow: Sildenafil citrate.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Sildenafil
%T
Sildenafil
4000,0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
cm-1
Fig. 39 : Comparison between the IR spectra of brand Viagra (black line), chalk (blue line), and counterfeit Cialis (red
line). The peculiar band of Sildenafil citrate (circled in blue) and those of chalk (circled in black) are present in the IR
spectrum of counterfeit Cialis.
Another counterfeit drug containing chalk
This sample was a counterfeit of Viagra, looking also very similar to the original, both in tablet
appearance and in packaging.
In this case TGA was particularly informative. In TGA thermogram the double peak of water loss at about
130-150°C is that of calcium sulfate dihydrate, thus in the formulation of this counterfeit the chalk is
employed. Then, near to the decomposition peak of sildenafil citrate (at about 190°C) another sharp
peak is visible. Matching this peak with the data bank, this peak was identified as the water loss of the
Emcompress (dihydrate calcium hydrogen phosphate).
In the original formulation of Viagra, instead, anhydrous calcium hydrogen phosphate is present and its
thermogram is very different. It can be also noted that the broadened peak, due to decomposition of
66
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
cellulose, in the TGA of the counterfeit is at lower temperature (about 310°C instead of 330°C). This
probably arise from an excipient substitution, that is the use of corn starch in place of microcrystalline
cellulose employed in the original formulation. After all, as counterfeit drug manufactures don’t follow
any regulation, probably they employ the raw materials that are more easily found in the market from
time to time, without caring of maintaining a uniform composition of the products.
Also from DSC thermogram the chalk is detectable, while the presence of the Emcompress is much less
evident because of the near fusion/decomposition peak of sildenafil citrate.
From IR spectrum as well the chalk is detectable while the signals of emcompress are much more
elusive.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Fig. 40 : DSC (A) and TGA (B) analyses of a counterfeit drug and of an original Viagra are reported. In the
thermograms of the counterfeit drug there are some peaks not present in the original drug, marked with a white
arrow, indicating the presence of chalk and Emcompress. Moreover, the peaks marked with black arrows are
different from the original drug peaks: probably it arises from an excipient substitution (corn starch in place of
microcrystalline cellulose). For easier visualization DSC thermograms were shifted across the heat flow scale and
only derivative weight is shown for TGA. Bold line: counterfeit drug; dotted line: original drug; black arrow: corn
starch; white arrow: chalk; gray arrows: emcompress.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
%T
4000,0
3600
3200
2800
2400
2000
1600
1800
1400
1200
1000
800
600
400,0
cm-1
Fig. 41 : Comparison between the IR spectra of brand Viagra (black line), counterfeit Viagra (blue line). The peculiar
bands of chalk (circled in gray) are present in the IR spectrum of counterfeit Viagra, while are lacking in brand
Viagra spectrum.
3.2.4 Detection of solid state properties by thermoanalysis
Finally the thermoanalysis shown capability in detecting even small differences in the solid state of a
drug product.
A different manufacturing process, a wrong storage, the use of poor quality starting materials may be
noticed by thermal analyses. These differences appear for instance when one active ingredient or one
excipient is in a different polymorphic form, or in a different hydration state, or even when, despite
having the same polymorphic form, it is in a different crystalline habit.
For this reason in building data banks as much samples as possible for each excipient, differing in origin
and quality, were analysed. In this way it was intended to detect the possible presence of different solid
state forms and quality problems of the excipients (i.e. hydration, ageing and degradation problems,
changing in polymorphic/pseudopolimorphic form).
From this point of view some excipients were interesting:
 Magnesium Stearate, that is present in many formulation as lubricating agent. This excipient can be
detected in a drug product both by IR, as it has a peculiar double and sharp band at 3000 cm-1, and
by DSC, where is identified by an endothermic signal at 80°C. It is detectable even if it is present in a
very little amount (about 3-5% m/m).
The most interesting DSC thermograms, among those obtained from different batches of Magnesium
Stearate analyzed as reference standard, are depicted in figure 42.
Magnesium Stearate have an interesting DSC thermogram; in the first sample of this excipient
(depicted by the black line) there is a quite sharp endothermic phenomenon at about 120°C due to
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
both water loss and fusion. Subsequently there is an exothermic peak due to a crystallization
process.
In the thermogram of the second sample (red line), before the water loss phenomenon, is also visible
another smaller endothermic signal, again due to water loss but at a lower temperature. In this case
there must be a different distribution of the water in the crystal and the first small signal is caused by
the evaporation of weakly bound water.
In the other two cases (green and blue line) the crystal must be much less regular because there are
two broadened endothermic phenomena. The water must be bound very weakly, as the water loss
begins at low temperature.
Thereby from the first to the last sample the quality of this excipient seems to decrease. Actually this
variability may come from a different composition of the mixture commonly defined magnesium
stearate: for European Pharmacopeia (Ph.Eur) it is a mixture made mainly of magnesium stearate
with variable amount of magnesium palmitate and magnesium oleate. But more probably these
differences are due to ageing phenomena of this excipient.
Fig. 42 : Comparison between DSC thermograms of different samples of magnesium stearate.
Also the TGA thermograms point out some differences among these samples (not reported in figure),
while FT-IR spectra don’t evidence anything of this features.
 Lactose Monohydrate is a common diluent agent and is the prevalent ingredient of Cialis. The DSC
thermogram of lactose is described in figure 43, depicted in black: there is an endothermic signal due
to dehydratation at about 140°C and a fusion peak at 220°C followed by decomposition phenomena.
However two of the monohydrate lactose samples analyzed had a DSC thermogram very different
(depicted in red, in figure 43), even though their IR and NMR spectra were identical to the other.
It was hypothesized that it followed the partial conversion of the α lactose to the β form after the
water loss [32]; this would explain why two distinct fusion peak are visible.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Again DSC highlights that the examined excipient is of lower quality.
Fig. 43 : Comparison between DSC thermograms of two samples of lactose monohydrate. DSC highlights differences
in the solid state form that were not detected by IR.
 Crospovidon is a disaggregating agent present in Levitra. European Pharmacopeia sets the maximum
water content for this excipient at 5% m/m, as it is very hygroscopic; some crospovidon samples
were stored in a close container but without any other particular care. So when analyzed by TGA,
these samples were discovered to contain about 15% m/m of water. Thus this ingredient needs to be
stored in appropriate conditions to maintain its quality.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Fig. 45 : Comparison between DSC and TGA thermograms of two samples of crospovidon. The sample described by
the red line contained a correct amount of water according to Ph.Eur., while that one depicted with the black line
had a too high water content. This evidence was clear both form DSC and TGA thermograms, but only by TGA it
was possible to determinate in a quantitative way the water content.
These differences in the solid state of substances are easily detectable by DSC and TGA when these
substances are analyzed alone; when a mixture is analyzed, highlighting differences in the solid state of
one of the components can be much trickier. So this matter deserves a more thorough investigation.
Solid state techniques pointed out interesting problems of polymorphism (actually pseudopolymorphism) in some Diclofenac sodium salt tablets.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Reactin is a generic Indian drug containing Diclofenac sodium salt, manufactured by Cipla that is an
Indian pharmaceutical industry. It is not authorized in Italy thus here it is illegally sold. Tablets were
analyzed by DSC, TGA and IR and were compared to the API reference standard and to the
corresponding Diclofenac sodium salt tablets legally sold in Italy.
Fig. 46 : Comparison between DSC thermograms of Reactin (black line), Diclofenac free acid (yellow line),
Anhydrous Diclofenac sodium salt (green line). The peak highlighted by the red arrow points out the presence of a
certain amount of Diclofenac free acid in Reactin drug product.
From DSC profile an anomalous peak is visible at about 180°C. Anhydrous diclofenac sodium salt doesn’t
give any signal in DSC in the investigated temperature range, while diclofenac free acid gives a neat
sharp fusion peak at about 175°C (see figure 46).
The presence of a small peak at about 180°C in the DSC of Reactin highlights that the active substance is
partly in a different chemical form, that is the free acid form, whose fusion signal is visible. The small
temperature shift between the fusion peak of diclofenac free acid alone and that in Reactin is explicable
considering that in a mixture the thermal phenomena are much more complex and can influence each
other.
Diclofenac free acid is less stable than diclofenac sodium salt, and it is less bioavailable since it is less
soluble in aqueous phase [38].
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Fig. 47 : Comparison between TGA thermograms of Reactin (black line), Diclofenac free acid (yellow line),
Anhydrous Diclofenac sodium salt (green line), Hydrate Diclofenac sodium salt (orange line). In the TGA
thermogram of Reactin a contribution from Hydrate Diclofenac sodium salt (pointed out by blue arrows) and
another one from Diclofenac free acid (pointed out by red arrows) are visible.
The TGA analysis clearly confirms that the active substance is partly in free acid form (see figure 47); but
it also highlights that another part of it is in hydrated form. Hydrate Diclofenac sodium, that is
thermodynamically more stable and exists in different polymorphic forms, is less bioavailable because is
less water soluble [38].
The simultaneous presence of different Diclofenac forms, anhydrous Diclofenac sodium salt, hydrate
Diclofenac sodium salt, Diclofenac sodium free acid, proves that in counterfeit drug manufacturing often
low quality materials are employed.
Furthermore it is very difficult to manufacture tablets with a correct amount of active substance if this is
made of 3 different chemical forms in a variable amount, since it will be impossible to weight it
accurately.
FT-It spectrum confirms the presence of Diclofenac free acid in Reactin (see figure 48).
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
%T
4000,0
3600
3200
2800
2400
2000
1600
1800
1400
1200
1000
800
600
370,0
cm-1
Fig. 48 : Comparison between the IR spectra of Anhydrous Diclofenac sodium salt (red line), Hydrate Diclofenac
sodium salt (green line), Reactin (black line), Diclofenac free acid (blue line). The presence of Diclofenac free acid in
Reactin drug product is pointed out by the small the band at 3300 cm-1, circled in yellow.
Afterwards the reference drug, that is the corresponding Diclofenac sodium salt drug product legally
sold in Italy, was analyzed.
Comparing its DSC profile to that of hydrate Diclofenac sodium salt and of anhydrous Diclofenac sodium
salt it resulted that also in the legal drug a big amount of hydrate active substance was present (see
figure 49). Instead the authorized formulation should contain only the anhydrous Diclofenac sodium
salt.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Fig. 49 : Comparison between DSC thermograms of legal Diclofenac sodium drug (black line), anhydrous Diclofenac
sodium salt (green line), hydrate Diclofenac sodium salt (orange line). The DSC thermogram of the legal Diclofenac
sodium drug stresses the presence of hydrate Diclofenac sodium salt (see the area circled in yellow).
TGA analysis confirms this evidence and the same does IR spectrum through small but significant
peculiars bands.
Fig. 50 : Comparison between TGA thermograms of legal Diclofenac sodium drug (black line), anhydrous Diclofenac
sodium salt (green line), hydrate Diclofenac sodium salt (orange line). The DSC thermogram of the legal Diclofenac
sodium drug stresses the presence of hydrate Diclofenac sodium salt (see the area circled in yellow).
76
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
%T
4000,0
3600
3200
2800
2400
2000
1600
1800
1400
1200
1000
800
600
370,0
cm-1
Fig. 51 : Comparison between the IR spectra of legal Diclofenac sodium drug product (black line), anhydrous
Diclofenac sodium salt (red line), hydrate Diclofenac sodium salt (green line). The doublet at 1600 cm-1 is very
indicative: in the anhydrous form the band on the left is longer, in the hydrate form, instead, that on the right. Since
in the legal product these two band are of the same length, the anhydrous form and the hydrate one are both
present. Also the bands at 1300 cm-1 and 750 cm-1 are very informative because are reliably different for the
anhydrous and hydrate forms. These bands confirm the presence of both the anhydrous form and the hydrate one
in the Diclofenac sodium original drug.
The hydration of the active substance can arise from the use of a low quality raw material or from poor
preservation; but moisture could be absorbed during the processing or from the excipients.
On the purpose to avoid these problems GMP had been introduced: imposing strict regulations and high
level quality standards, their respect permits to guarantee adequate quality, safety and efficacy for
medicines.
Also dissolution test was performed both on Reactin and on the legal drug. It highlights that the
dissolution of the legal drug tablets is slower than the specification, accordingly to what resulted from
solid state analyses, that is the presence of the hydrate Diclofenac sodium salt, less water soluble.
The dissolution of Reactin, then, is even slower and this can be explained through the major problems
on the quality of the active substance pointed out by DSC, TGA and IR.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
3.2.5 Results on summary
By this solid state approach, that employs FT-IR, DSC and TGA, a large number of suspicious samples
were analyzed, in particular drugs for the treatment of the erectile dysfunction that are the most
counterfeited.
The major part of these samples resulted illegal or counterfeit drugs. Very often their formulation was
different from that of the original drug.
In particular about excipients it resulted:
1. Frequently some excipients employed in the formulation of the original drug are substituted with
other less expensive ingredients, to obtain more profit. This to detriment of the quality of the drug.
These different formulations were not developed, thus it is not known their performance in terms of
bioavalilability or stability of the active substance.
2. In some illegal formulations the DSC pointed out possible active substance/excipient interaction
problems. In particular it was noted that sildenafil and mannitol could interact since their fusion
peaks change in temperature and shape when they are in mixture. A similar behavior was observed
in sildenafil/lactose monohydrate mixture.
Since DSC is referred in the scientific literature as a technique employed during formulation studies
for a new drug to verify the absence of interaction among components [36-38], these evidences
were alarming.
Interactions among the ingredients of a drug may produce toxic compounds and bring a dangerous
variability.
3. The illegal market provides innovative pharmaceutical form, like chewable tablets, oral gels, also
because it bypasses every preliminary study imposed by regulatory authorities. Thus there are no
guarantees about the safety and the effectiveness of these new pharmaceutical forms.
4. The use of non pharmaceutical materials but even of toxic substances, like building materials, was
detected. These materials are much less expensive than pharmaceutical excipients, thus the profit is
increased.
In manufacturing of counterfeit drug even the use of commonly used in medicine excipients can
bring risks because they are not of pharmaceutical grade. For example lactose, commonly employed
in counterfeit formulation, must be tested for TSE contamination [31]; talc, that is an anticaking,
glidant, diluent and lubricant agent, must be tested for possible asbestos contamination [39].
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
3.3 STABILITY STUDIES
As DSC highlighted possible interaction problems in some counterfeit formulations different from
that of the original drug, stability studies were carried to deepen this matter.
An inadequate formulation may alter the bioavailability of the active substance; moreover it can
decrease the stability of a drug and cause production of toxic substances.
To study the effect of suspicious formulations on the stability of an active substance, some mixtures of
active substance and excipients were prepared and stored at 40°C and 75% of relative humidity, that are
accelerated stability conditions. The degradation impurities were observed every month, till the 6th
month.
In particular possible interaction problems between the active substance sildenafil citrate and the
excipients lactose and mannitol were studied, thus mixtures containing sildenafil, lactose and mannitol
were prepared beside reference mixtures for comparison.
The following sample, some binary mixtures and some in house products, were prepared and stored in
accelerated stability conditions.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Table 6
Sample
Raw material
Sildenafil Citrate
Brand Viagra
In house product Viagra
like
Binary mixture Sildenafil
Citrate– Monohydrate
Lactose, 1,5: 1 m/m ratio
Binary mixture Sildenafil
Citrate – Anhydrous
Calcium Hydrogen
Phosphate, 1,5: 1 m/m
ratio
In house product with
lactose, 1,5:1 m/m ratio
Binary mixture Sildenafil
Citrate– Monohydrate
Lactose, 1:3 m/m ratio
Binary mixture Sildenafil
Citrate – Anhydrous
Calcium Hydrogen
Phosphate, 1:3 m/m ratio
In house product with
lactose, 1:3 m/m ratio
Abbreviation
M
V
P. V
Sildenafil citrate
Viagra Tablets ground after removal of the external
coating
Known amount of the following ingredients were
blended: sildenafil citrate, microcrystalline cellulose,
anhydrous calcium hydrogen phosphate,
croscarmellose, magnesium stearate
M. S/L
Sildenafil Citrate/Lactose, 1,5:1 w/w ratio
M. S/P
Sildenafil Citrate /Anhydrous Calcium Hydrogen
Phosphate, 1,5:1 w/w ratio
P. S/L
It is made of sildenafil citrate, cellulose (about 50% of
the whole), lactose monohydrate, croscarmellose,
magnesium stearate; Sildenafil Citrate/Monohydrate
Lactose rate is about 1,5:1 w/w
M. L/S
Sildenafil Citrate/Monohydrate Lactose, 1: 3 w/w ratio
M. P/S
Sildenafil Citrate /Anhydrous Calcium Hydrogen
Phosphate, 1: 3 w/w ratio
P. L/S
It is made of sildenafil citrate, cellulose, lactose
monohydrate, croscarmellose, magnesium stearate;
Sildenafil Citrate/Monohydrate Lactose rate is about 1:3
w/w
Binary mixture Sildenafil
citrate – Mannitol, 1:3
m/m ratio
M. M/S
In house product with
Mannitol, 1:3 m/m ratio
P. M/S
In house product made of
excipients
Ingredient
P. E
Sildenafil Citrate/Mannitol, 1:3 w/w ratio
It is made of sildenafil citrate, mannitol, cellulose (about
5%), magnesium stearate; Sildenafil Citrate/Mannitol
rate is about 1:3 w/w
In this mixture there is not active substance but only
excipients: mannitol, cellulose, lactose, anhydrous
calcium hydrogen phosphate, croscarmellose,
magnesium stearate
Through these studies it was researched if the suspected interaction between sildenafil/mannitol and
sildenafil/lactose, highlighted by DSC, increased degradation impurities of the active substance.
The in house products composition was chosen to imitate the composition of some counterfeit drugs
analyzed in this laboratory.
Reference products were also necessary for comparison.
Binary mixtures were prepared to directly investigate the subjects of the suspected interaction; these
were compared to binary mixtures of sildenafil citrate/anhydrous calcium hydrogen phosphate; actually
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
this excipient, that is present in the original Viagra formulation, often is not detected in counterfeits
drugs, thus it was hypothesized that other excipients are used instead of it.
Every month a little amount of these samples were drawn and analyzed by HPLC-DAD, using a reference
method for impurities of sildenafil citrate:
 Chromatographic column Symmetry C18 150x3.9 mm, 5 μm.
 Mobile phase: 580 ml 0.05M triethylammine solution (pH3 by phosphoric acid), 250 ml methanol,
170 ml acetonitrile.
 Flow rate 1ml/min
 Detector 290 nm
 Injection volume 20µl
Accordingly all samples were analyzed seven times, at the starting point and every month till the 6th,
during the storage in accelerated stability conditions.
At starting point, after 3 months and after 6 months of storage, also DSC and TGA of the samples were
performed.
Results of the HPLC impurities analyses are presented graphically. For an easier representation only the
starting point and the sixth month results are depicted, as they are the most revealing to display
humidity and temperature effects on degradation impurities production.
Impurities were identified by the relative retention time (respect to that of sildenafil), and quantified as
percentage area.
The retention times are on abscissas axis, the areas on ordinates.
In the same graphic all the binary mixture are compared at the starting point and at the 6th month. In
another graphic then all the “in house products” are compared. Thus comparisons were made among
samples of similar composition (active substance and one excipient or active substance and a mixture of
excipients).
Every sample is indicated with an abbreviation (that is explained in the scheme) and with a different
color.
Every line imitates the chromatogram and in particular the impurities profile of each sample. Every point
in the graphic corresponds to a certain impurity, with its relative retention time and its area. But the
graphic differs from the chromatogram because only each impurity’s area and the relative retention
time is visualized, not its peak profile. Furthermore the active substance signal is absent to better
visualize the much smaller impurities signals.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Binary Mixtures - Starting point
M S/L
M S/P
M L/S
M P/S
M M/S
0.300
Area (%)
0.250
0.200
0.150
0.100
0.050
0.000
0.20
0.40
0.60
0.80
1.00
1.20
1.40
Relative retention time
1.60
1.80
2.00
2.20
Fig. 52 : Impurity profiles of Binary Mixtures at the starting point of the study. The abbreviations of the mixtures are
the same of Table 6. In the graphic every impurity is reported as a point: its abscissa corresponds to the retention
time, its ordinate to the area. All the points of the same mixture are joined by a line, that has a different colour for
each mixture.
Binary Mixtures - 6° mounth
M. S/L
M S/P
M. L/S
M P/S
M. M/S
0.300
0.250
Area (%)
0.200
0.150
0.100
0.050
0.000
0.20
0.70
1.20
1.70
Relative retention time
2.20
Fig. 53 : Impurity profiles of Binary Mixtures after 6 months in accelerated stability conditions. The abbreviations of
the mixtures are the same of Table 6. In the graphic every impurity is reported as a point: its abscissa corresponds
to the retention time, its ordinate to the area. All the points of the same mixture are joined by a line, that has a
different colour for each mixture.
At the starting point (see figure 52) the impurities profile of all the binary mixtures is the same and
corresponds to that of the raw material sildenafil citrate used for samples manufacture. These
impurities are synthesis impurities. The same observation, as after described, is applied for “in house
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
products”: at the starting point all of them have the same impurities. Thus it results that the operation
of mixing the active substance with various excipients actually didn’t affect the impurities profile.
Comparing the binary mixtures at the sixth month (see figure 53), it is interesting to note that
sildenafil/lactose (M S/L blue, M L/S green) and sildenafil/mannitol mixture (S M/S azure) have not a
worst profile than the reference samples (sildenafil/anhydrous calcium hydrogen phosphate) both in
number of impurities and in amount.
The DSC and TGA analyses didn’t highlight any modification of the samples during the storage (see
figure 54 and 55).
Fig. 54 : Comparison among DSC thermograms of Binary Mixtures.
Binary mixtures Sildenafil Citrate/anhydrous calcium hydrogen phosphate 1,5:1 - black line; binary mixtures
anhydrous calcium hydrogen phosphate/Sildenafil Citrate 3:1 – black line; binary mixture Sildenafil citrate/lactose
monohydrate 1,5:1 - blue line; binary Mixtures Lactose/Sildenafil citrate 3:1 - green line; binary Mixtures
Mannitol/Sildenafil citrate 3:1 - red line.
The anhydrous calcium hydrogen phosphate doesn’t show any thermal phenomena and it doesn’t give
DSC and TGA peaks. Thus the thermograms of the binary mixtures sildenafil/anhydrous calcium
hydrogen phosphate highlight only the melting and decomposition of sildenafil citrate.
The two sildenafil/lactose mixtures have different DSC thermograms having different composition. In M
S/L mixture, where sildenafil is present in bigger amount, is clearly visible the melting peak of sildenafil,
with a little shoulder due to the melting and the decomposition of lactose; the dehydratation peak of
lactose is detectable but it is small. In M L/S mixture instead, where lactose is more abundant than
sildenafil, signals from lactose are predominant even though the melting peak of sildenafil is still
detectable.
In the DSC of the mannitol/sildenafil mixture, the mannitol melting peak is clear, followed by the less
marked signal of sildenafil, that is at lower temperature than when sildenafil citrate is analyzed alone.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Fig. 55 : Comparison among TGA thermograms of Binary Mixtures.
Binary mixtures Sildenafil Citrate/anhydrous calcium hydrogen phosphate 1,5:1 - black line; binary mixtures
anhydrous calcium hydrogen phosphate/Sildenafil Citrate 3:1 – black line; binary mixture Sildenafil citrate/lactose
monohydrate 1,5:1 - blue line; binary Mixtures Lactose/Sildenafil citrate 3:1 - green line; binary Mixtures
Mannitol/Sildenafil citrate 3:1 - red line.
From TGA similar information can be acquired.
In the binary mixture Sildenafil citrate/anhydrous calcium hydrogen phosphate only the sildenafil citrate
decomposition is detectable.
In the TGA thermogram of the two Sildenafil/lactose mixtures, the most abundant component has the
strongest decomposition signal. These signals shape is different respect to those when sildenafil and
lactose are analyzed alone.
In the mannitol/sildenafil mixture the decomposition signal of mannitol is clear while the signals from
sildenafil are much less marked and altered in shape.
In house Products - Starting Point
0.250
V
0.200
P. V
0.150
P. S/L
0.100
P. L/S
0.050
P. M/S
0.000
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
Fig. 56 : Impurity profiles of “in house products” at the starting point of the study. The abbreviations of the mixtures
are the same of Table 6. In the graphic every impurity is reported as a point: its abscissa corresponds to the
retention time, its ordinate to the area. All the points of the same mixture are joined by a line, that has a different
colour for each mixture.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
In house Products - 6° mounth
0.250
V
P. V
P. S/L
0.200
P. L/S
P. M/S
0.150
0.100
0.050
0.000
0.20
0.70
1.20
1.70
2.20
Fig. 57 : Impurity profiles of “in house products” after 6 months in accelerated stability conditions. The
abbreviations of the mixtures are the same of Table 6. In the graphic every impurity is reported as a point: its
abscissa corresponds to the retention time, its ordinate to the area. All the points of the same mixture are joined by
a line, that has a different colour for each mixture.
As well, observing the “in house products”, also compared to brand Viagra, meaningful phenomena in
lactose (P. S/L green, P L/S violet) and mannitol (P. M/S azure) mixtures are not highlighted (see figure
57).
The P. S/L mixture, with lactose, have a big amount of impurities, in some cases more than the “in house
Viagra” but still similar to those of brand Viagra; yet, increasing the amount of lactose present in the
mixture (P. L/S), impurities become smaller.
Also mannitol, instead of increasing the degradation phenomena, seems to improve the impurities
profile.
In conclusion the suspected interaction between the active substance sildenafil citrate and the
excipients mannitol and lactose, highlighted by DSC, didn’t give any evidence of increasing the
degradation impurities.
As a consequence it can be hypothesized that the thermal phenomena of sildenafil citrate, in presence
of mannitol or lactose, change in temperature and shape not because of a chemical or physical
interaction but rather because of the influence from the thermal phenomena of these excipients. As
decomposition is a exothermic phenomenon, the decomposition of mannitol and lactose releases
energy that is available for the near substances. To melt and decompose sildenafil needs to reach a
certain temperature when it has enough energy. But this temperature may decrease if there is energy
available for sildenafil from the decomposition phenomena of other components. Also the widening of
the melting peak of sildenafil may be explained: the energy from thermal phenomena of other
components may diffuse in a not uniform way causing a less uniform melting of sildenafil.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Fig. 58 : Comparison among DSC thermograms of “in house products”.
In house product Viagra alike (P. V) - black line; in house product with lactose 1,5:1 m/m (P. S/L) - blue line; in house
product with lactose 1:3 m/m (P. L/S) - green line; in house product with mannitol 1:3 m/m (P. M/S) - red line.
The comparison among DSC and TGA thermograms of “in house products” is depicted in figures 58 and
59.
The DSC of the “in house product” with anhydrous calcium hydrogen phosphate/Sildenafil citrate
highlights the presence of sildenafil, cellulose and magnesium stearate.
The DSC of the “in house products” containing sildenafil and lactose, as well as the binary mixtures,
stresses the signals from the most abundant component between sildenafil and lactose.
The DSC of “in house product” with mannitol and sildenafil is very similar to the corresponding binary
mixture, but is visible the contribute of magnesium stearate.
Fig. 59 : Comparison among TGA thermograms of “in house products”.
In house product Viagra alike (P. V) - black line; in house product with lactose 1,5:1 m/m (P. S/L) - blue line; in house
product with lactose 1:3 m/m (P. L/S) - green line; in house product with mannitol 1:3 m/m (P. M/S) - red line.
The TGA of the in house product “Viagra alike”, that is identical to that of the original Viagra, points out
the presence of the active substance and of cellulose; the cellulose is detectable from the broaden
water loss signal at room temperature and the decomposition signal at 350°C that partly covers the
sildenafil decomposition.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
A strong decomposition signal of cellulose is visible also in the in house product P S/L that contains
about 50% w/w of cellulose while it is absent in P L/S where cellulose is 5% w/w.
In the P M/S the decomposition signal of mannitol is different from that of the binary mixture
sildenafil/mannitol because the cellulose contributes with its decomposition.
The impurities profile of the raw material sildenafil citrate remains unchanged during the storage in
accelerated stability conditions; these impurities are the synthesis impurities detectable even at the
starting point.
The blank, that is the mixture of the sole excipients without active substance, didn’t give meaningful
signals as expected, thus there are no interferences by excipients.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
3.4 DISSOLUTION
Dissolution test is employed for various applications [40].
It is often employed in quality control of scale-up and production batches, to ensure batch-to-batch
consistency and consistency with the pivotal clinical trial batches.
In Biopharmaceutic Classification System (BCS) the active substances are classified in four different
classes according to their aqueous solubility and intestinal permeability; for immediate release drugs,
BCS-based biowaiver can be applied when the active substance is in class I (High Solubility – High
Permeability) or III (High Solubility – Low Permeability), and some conditions about excipients are
respected [41].
Furthermore the dissolution test is normally used as bioequivalence surrogate to demonstrate similarity
between different formulations of the same active substance, e.g. for marketing authorization of new
generic drugs. [42]
The release profile of the active substance in different operating conditions can provide valuable
information to choice a formulation adequate to the therapeutic purpose.
Formulation is very important for the therapeutic effect; e.g. when an active substance is not very
soluble in water, excipients can promote its solubilization.
During formulation studies the physico-chemical properties of the active substance are evaluated. A
powder must be mixed with lubricant and glidant agents to be pressed. In this way frictions and
adherence to the metal press decrease.
Also binders are useful for wet granulation. But these kinds of excipients reduce drug wettability, thus
delay the drug dissolution; e.g. lubricant agents produce a hydrophobic film around particles.
To circumvent this issue disintegrating agents are added to increase the contact surface between
powder and dissolution medium.
To reach the appropriate mass, diluents agents are also added, in particular to the low dosage strengths.
Commonly coloring agents, waxes and matting agents are also employed to improve the appearance of
a tablet and simplify its identification by patients.
In some cases flavoring and sweetening agents are added to improve the taste of the drug.
An adequate formulation guarantees stability of the active substance and an appropriate bioavailability
of the drug.
If a formulation is not adequate, chemical interactions between active substance and excipients may
arise, through covalent or non covalent bounds. Furthermore API/excipient interactions may lead to
physical transformations, like amorphization of a crystalline active substance, and alterations of
technological properties like solubility, wettability, porosity, hardness. Accordingly, the API
bioavailability can be modified.
Dissolution test can be profitably applied for analysis of counterfeit and illegal drugs because it can
discriminate among different formulations highlighting differences both in composition and in
manufacturing technology.
The in vitro dissolution test was applied on counterfeit and illegal drugs to obtain more in-depth
information about the specific tablet analyzed. In particular the dissolution test permitted to study the
API release from the tablets and compare it to that in the corresponding original drug. As the API release
determined by in vitro dissolution is partly related to bioavailability, such a study permitted to
investigate the issue of bioavailability of counterfeit drugs.
Indeed the inadequate release of the active substance is one of the dangers coming from counterfeit
drugs. On one hand a faster release of the API may cause a too fast absorption and increase side effects.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
On the other hand a slower release may prevent the active substance from reaching the optimal
concentration, causing the delay of therapeutic effect or even complete ineffectiveness. Thus a patient
may be induced to arbitrarily increase the dose, with possible dangerous accumulation effects.
In an illegal drug a different API release in respect to the original one can follow from the use of a
different compression force during the manufacturing, or a different particle size of raw materials or a
different formulation. Excipients can strongly affect bioavailability, in particular the extent and the rate
of the absorption of the active substance.
The analysis of counterfeit drugs by dissolution test can be performed with two different purposes.
First of all a discriminating dissolution test is a further instrument to highlight if a suspicious drug is
counterfeit or not by comparison to the original API release profile.
Some studies on antimalarial drugs demonstrated the effectiveness of dissolution test in detection of
counterfeit tablets [43-44-45].
Second if a counterfeit or illegal drug contains the same active substance but in a different formulation
from the original, this test can determine the release rate of API from tablets and highlight possible
dangers arising from an inadequate release.
In this case the same criteria of the similarity studies on generics drugs can be employed, performing the
test at different pH.
Thus a discriminating dissolution test on counterfeit and illegal drugs can provide valuable information,
different from those obtainable by other techniques, in particular about formulation characteristics,
quality and quantity of excipients, technology of manufacturing process.
Many elements affect the release profile of API in a dissolution test [40].
Enviromental factors during dissolution:
 Intensity of agitation
 Concentration gradient (i.e. the difference in concentration between the solubility of API in
dissolution medium and its average concentration in bulk fluid)
 Composition of dissolution medium; e.g. pH, ionic strength, viscosity, surface tension of dissolution
medium are all important factors
 Temperature of dissolution medium
Then there are factors related to the physico-chemical properties of the drug:
Factor affecting solubility
 Polymorophism
 Amorphous state and solvation
 Free acid, free base or salt form
 Complexation, solid solution and eutectics
 Particle size
 Surfactants
Factors affecting surface area available for dissolution
 Particle size
 Tablet shape
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
 porosity
Factors related to the composition and the method of manufacture:
 Amount and type of diluents and filler and other adjuvants, such as neutral salt
 Type of tablet manufacture employed
 Granule size and size distribution
 Amount and type of disintegrant and method incorporating it
 Amount and type of surfactant and method incorporating it
 Compression force and speed of compression
Finally there are enviromental factors involved with dosage form:
 Humidity during manufacture
 Storage condition for dosage form
 Age of dosage form
Since dissolution test is affected by many different elements, a strict interpretation of results is not
possible.
Fig. 60 Test dissolution equipment
A dissolution test is performed on six tablets that are simultaneously investigated, each one in a
different vessel where a stated volume of dissolution medium is contained. The temperature, regulated
by a thermostat, usually is 37°C to simulate the human body conditions.
There are two main stirring systems:
 paddles, that mix the dissolution medium all around the tablet. The tablet is on the bottom of the
vessel (figure 61 on the left);
 baskets, made of a holed cylindrical rotating container. The tablet is placed inside this container (the
figure 61 on the right).
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Fig. 61 : The two main stirring systems for dissolution test are Paddles (depicted on the left) and Baskets (on the
right).
Different rotating speed can be employed: the faster the rotating system, the faster the release of API
from the tablet.
In automated instruments at fixed time intervals an amount of dissolution medium is taken from each
vessel and the concentration of the API is detected by a spectrophotometric measure; in manual
instruments the sampling of dissolution medium is made manually.
The dissolution profile of the drug is registered and it is graphically reported. Dissolution percentage
(that is the amount of dissolved active substance in respect to the declared API content) is reported on y
axis, time of dissolution on x axis.
3.4.1 Development of a discriminating dissolution method
The dissolution test finalized to the investigation of counterfeit and illegal drugs was applied to drugs for
the treatment of erectile dysfunction, in particular those with sildenafil citrate as active ingredient.
A discriminating dissolution method, to highlight differences in release of the sildenafil citrate from
counterfeit tablets in respect to the original Viagra tablets was developed.
In future possible application of the same dissolution test to other drugs for the treatment of erectile
dysfunction (i.e. Cialis and Levitra) will be investigated. As a matter of fact the substitution of the
declared active substance with a cheaper one with similar pharmaceutical effect is often detected; thus
it would be convenient to use the same dissolution test for the analysis of all the drugs of the same
therapeutic class.
Viagra Pfizer® is an immediate release oral dosage form. The active ingredient is sildenafil citrate, whose
solubility is pH dependent. Being a weak base, its solubility decreases going to basic pH.
Commonly for immediate release tablets the dissolution test is performed in acidic dissolution media to
simulate the stomach environment where the tablets dissolve.
Sildenafil citrate is freely soluble in acidic pH (pH 1= 0.1M HCl), slightly soluble at pH 4.5, practically
insoluble at pH 6.8 and 7.5.
A preexisting dissolution method, for batch-to-batch consistency quality control, operates at pH 2, with
HCl.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Instead for the purpose of this study the dissolution test was performed in low solubility condition for
the active substance, thus in more basic media, to have a slower dissolution. In such conditions
differences in formulation and manufacturing technology were better higlighted.
Two different dissolution media were examined: pH 4.5 by phosphate buffer, pH 6.8 by phosphate
buffer.
Three standard solutions 0.1 mg/ml of sildenafil free base (about 14 mg of sildenafil citrate in 100ml),
were prepared at pH 4.5 and pH 6.8 dissolution media. The UV spectra were acquired to detect the
wavelength corresponding to the maximum of absorbance, that is 292 nm. Thus spectrophotometric
measurements to evaluate sildenafil concentration in dissolution medium were acquired at this
wavelength.
The molar extinction coefficient at 292nm was calculated. Repeatability and Linearity of
spectrophotometric measures in the concentration range of interest (that is 0.1 mg/ml – 0.003 mg/ml
with cuvettes of 10 mm, thus the range 200% - 6.25% of the final concentration for dissolution of 50 mg
Sildenafil tablets) were evaluated.
The 5 mm optical path cuvettes were employed for 100 mg Sildenafil tablets and the 10 mm optical path
cuvettes for 25 mg and 50 mg Sildenafil tablets.
After this preliminary analyses, investigations to develop the better operative conditions for the
dissolution test were performed. These investigations were carried out with original tablets.
First the Ph.Eur. apparatus II, the paddle stirring system, was used.
120
PADDLE - Viagra 50 mg
Method development
100
Dissolution (%)
Medium : Phosphate Buffer
Medium Volume : 900 ml
80
Apparatus : PADDLE
60
Rotation Speed : 50 RPM
pH 4,5
40
Wavelength UV Detector : 292nm
Filter : 1.2 µm GF Whatman
pH 6,8
20
Temperature : 37°C
0
0
10
20
30
40
50
60
t (min)
Fig. 62 : Dissolution of brand Viagra tablets in the operative conditions described in the table on the right, both at
pH 4,5 (blue line) and 6,8 (red line). The percentage of dissolution is reported versus time.
As observed from the results reported in the graphic (see figure 62), the release is quite fast at both pH
4.5 and pH 6.8; indeed after only 10-20 minutes the dissolution is complete. However at pH 6.8 the was
plateau reached at 70% because of the low solubility of the active substance.
Moreover using the paddle system a coning effect was observed: some insoluble powder settled over
the tablet, hindering the further release of API.
For this reason the paddle system was considered not appropriate and the Ph.Eur. apparatus I, the
basket system, was tried. The following operating conditions were tested:
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
160
BASKET - Viagra 50 mg
Method development
140
Medium : Phosphate Buffer
Dissolution (%)
120
Medium Volume : 900 ml
50 RPM - pH 4,5
100
Apparatus : BASKET
50 RPM - pH 6.8
80
Wavelength UV Detector : 292nm
75 RPM - pH 6.8
60
Filter : 1.2 µm GF Whatman
40
Temperature : 37°C
20
0
0
50
100
150
200
t (min)
Fig. 63 : Dissolution of brand Viagra tablets in the operative conditions described in the table on the right, at pH 4,5
with a rotation speed of 50 RPM (blue line), at pH 6,8 and 50 RPM (red line) and pH 6,8 and 75 RPM (green line).
The percentage of dissolution is reported versus time.
pH 6.8 medium was discarded because the low solubility of the active substance determines a release
much lower than 100% (see figure 63); even increasing the rotation speed the API release doesn’t
improve meaningfully. In this conditions the investigation range of dissolution is too limited, so this is
not convenient parameter investigation.
The release profile observed at pH 4.5, with rotation speed of 50 RPM (Revolutions per minute), is much
more interesting. The dissolution is complete and quite slow, thus these operating conditions would be
appropriate to highlight formulation differences.
However the observed release of almost 140% is anomalous and it can’t arise from an over-dosage as
the analyzed tablets were original. Thus it was attributed to the use of not appropriate filters. Indeed
the use of low selective filters may not retain some fine particles of insoluble excipients which could
interfere with the spectrophotometric measure.
To evaluate this problem the concentration of sildenafil in the dissolution medium was measured by UV
analysis, after filtration with filters of different porosity: GF Whatman 1.2, 0.7, 0.45 µm. It resulted that
1,2 µm filters are not able to retain all insoluble particles, while both 0.7 and 0.4 µm filters are
appropriate. GF Whatman 0.7 µm filters were employed, as they can be used with automated system.
Using 0.45 µm filters the manual sampling of the dissolution medium for spectrophometric analysis is
necessary because these filters generate a very high pressure, too strong for the automated systems.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
3.4.2 Dissolution test medium validation
The test on 50 mg Viagra tablets at pH 4.5 was repeated with the chosen filters. It was verified that the
filters were appropriate. In addition different rotation speeds were tested (see figure 64).
BASKET - Filtri 0.7µm - Viagra 50 mg
100
Method development
Medium : Phosphate Buffer pH 4.5
Dissolution (%)
80
Medium Volume : 900 ml
60
75 RPM
40
50 RPM
20
Apparatus : BASKET
Wavelength UV Detector : 292nm
Filter : 0.7 µm GF Whatman
0
0
50
100
150
200
250
300
Temperature : 37°C
t (min)
Fig. 64 : Dissolution of brand Viagra tablets in the operative conditions described in the table on the right, both at
50 RPM as rotation speed (red line), and at 75 RPM (blue line). The percentage of dissolution is reported versus
time.
It resulted that a rotation speed of 50 RPM was more convenient because the dissolution is still
complete but slower, thus a wider investigation field is obtained.
After developing the operating conditions of work, further verifications were conducted:
 The selectivity of the method was tested. It was verified that the excipients present in the
formulation of Viagra don’t interfere with Sildenafil UV absorption at 292nm. Only the coloring agent
indigotine E132, employed in the coating film, slightly absorbs in the range of interest and causes a
weak azure color of the dissolution medium. However it was evaluated that this interference is
below 2%, thus unimportant for this method.
 It was verified that the dissolution of all the marketed dosage strengths (25mg, 50mg, 100mg of
sildenafil) was conducted in sink conditions. To respect the sink conditions, the concentration of API
in the dissolution medium should be never higher than 1/3 of its solubility limit concentration. This
condition is necessary to simulate what happens in vivo. In vivo there is a strong API concentration
gradient between the matrix of the tablet and the aqueous environment of the stomach; indeed the
dissolved API is quickly absorbed through the membranes, facilitating further dissolution of the
active substance from the tablet.
A solution of 0.338 mg/ml of sildenafil (that is three times more concentrated than the 900ml
medium after complete dissolution of a 100mg sildenafil tablet) was prepared and the complete
solubility of the API in this solution was verified by spectrophotometric analysis. In particular the
original solution was progressively diluted and it was verified the linearity of the spectrophometric
response at 292nm.
 The intrinsic dissolution test was performed on the raw material sildenafil citrate to study the
dissolution of the sole active substance in the chosen conditions. It was conducted with the rotating
disk apparatus, as the dissolution was performed by the Apparatus I (baskets). The intrinsic
dissolution was subsequently employed for comparison with the dissolution profile of the
investigated drugs. From this comparison it was possible to evaluate in what measure the dissolution
rate is affected by active substance solubility and in what measure by excipients and formulation.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
3.4.3 Dissolution of original and illegal drugs
Original, counterfeit and illegal drugs with Sildenafil citrate were tested by the dissolution method
developed.
In addition the release of the active substance was evaluated with a pre-existing dissolution method for
Sildenafil citrate. This method employs classical dissolution conditions for immediate release dosage
forms, that are acidic pH to simulate the stomach environment where tablets dissolve. This method has
a specification: after 30 minutes the release must be higher than the 85% of the declared dosage.
New Dissolution Method pH 4.5
Dissolution Method pH 2
Medium : Phosphate Buffer pH 4.5
Medium : HCl 0.01 M pH 2.0
Medium Volume : 900 ml
Medium Volume : 900 ml
Apparatus : BASKET
Apparatus : BASKET
Rotation speed : 50 RPM
Rotation speed : 100 RPM
Wavelength UV Detector : 292nm
Wavelength UV Detector : 292nm
Filter : 0.7 µm GF Whatman
Filter : 0.7 µm GF Whatman
Temperature : 37°C
Temperature : 37°C
Specification: 85% of release after 30 min
Testing counterfeit and illegal drugs with two different dissolution methods, at different pH, more
information about the API release were obtained and hypotheses about their possible bioavalability
respect to the original drug were made. Actually, to operate in the same way as for comparison
between generics drugs, dissolution should be tested at 3 different pH and many tablets (commonly 12)
should be analyzed. Unfortunately for the analysis of counterfeit drugs with sildenafil citrate it was not
possible to test the dissolution at 3 different pH because of the low solubility of sildenafil at pH higher
than 4.5. Moreover only 6 or 3 tablets were tested because suspicious samples commonly contain just
few tablets.
Hence this was not a similarity study; however hypotheses and general considerations from comparison
between original and illegal drugs will be presented.
Where it was possible each dissolution test was performed on 6 tablets. Each point in the graphics is the
average of 6 different data; an error bar, obtained by the standard deviation among these data, is
associated to each point.
The dissolution profile of illegal drugs is compared to the reference, that is the original drugs with the
same amount of Sildenafil citrate.
The illegal drugs analyzed were:
1. Filagra
2. Sildeanfil Soft
3. AX
4. Counterfeit Cialis 20mg
5. Counterfeit Viagra 50 mg
6. Counterfeit Viagra 100 mg
7. I-Pramil
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
1.
FILAGRA :
Fig. 65 : On the left: a Filagra tablet compared to a brand Viagra one (on the right in the picture). On the right :
Filagra blisters.
Through a purchase on the internet two distinct samples named Filagra, manufactured in India, were
obtained and analyzed. The primary packaging reported the indication: “Sildenafil Citrate Tablets,
equivalent to Sildenafil 50 mg”. The tablets had a blue coating film, darker than the original Viagra;
tablets diamond shape strongly resembled the original, but the size was bigger.
The active substance was identified and quantified by HPLC analysis and the results complied with the
declared dosage. Solid state techniques, IR, DSC and TGA, highlighted that the two Filagra samples had
the same composition. In particular cellulose, magnesium stearate and lactose in addition to the API
sildenafil citrate were detected. Lactose was in such big amount to cover the decomposition peak of
Sildenafil citrate in TGA thermogram.
After removing the external coating film, the presence of residues of the same coating was observed
inside the bulk of Filagra tablets. It was hypothesized that this phenomenon arose from these tablets
being obtained from the rough chipping of other tablets for re-use drugs trafficking.
Dissolution test were performed on both Filagra samples with the new method (see figure 66); the test
confirmed that the formulation and probably the manufacturing line of the two Filagra samples were
the same as their dissolution profiles are almost identical.
Dissolution profile of Filagra was slower than that of Viagra, but it reached a plateau of 100% as well.
The slower release of Filagra probably was due not to the presence of lactose. Lactose in wide
concentration ranges, as reported by literature[46-47], usually increases the dissolution rate of slightly
soluble active substances. On the contrary this evidence can arise from a different production
technology, a greater compression force, a different granulometry of raw material in respect to those of
the original drug. Otherwise it can arise from the absence of disintegrating agents in formulation of
Filagra, like sodium croscarmellose that, instead, is present in the formulation of Viagra. This
disintegrating agent has a macroscopic effect on dissolution of Viagra tablets: when Viagra tablets come
into contact with dissolution medium, they bloat increasing the exchange surface with the medium and
detaching the external film in a very short time.
96
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Filagra
100
New Dissolution Method pH 4.5
80
Medium : Phosphate Buffer pH 4.5
Dissolution (%)
Medium Volume : 900 ml
Apparatus : BASKET
60
Rotation speed : 50 RPM
brand Viagra 50 mg
40
Wavelength UV Detector : 292nm
Filter : 0.7 µm GF Whatman
Filagra 1
Temperature : 37°C
20
Filagra 2
0
0
50
100
150
200
250
300
350
400
t (min)
Fig. 66 : Dissolution of two different samples of Filagra tablets (red and green lines) compared to that of brand
Viagra (blue line), in the operative conditions described in the table on the right. The percentage of dissolution is
reported versus time.
Filagra
80
New Dissolution Method pH 4.5
70
Medium : Phosphate Buffer pH 4.5
60
Medium Volume : 900 ml
Apparatus : BASKET
Dissolution (%)
50
Rotation speed : 50 RPM
40
Wavelength UV Detector : 292nm
30
brand Viagra 50 mg
20
Temperature : 37°C
Filagra 1
10
Filter : 0.7 µm GF Whatman
Filagra 2
0
0
5
10
15
20
t (min)
25
30
35
40
Fig. 67 : A particular of the first few minutes of the dissolutions depicted in figure 66 are now presented.
Observing thoroughly the first few minutes of dissolution (figure 67) it can be noted that Filagra has got
a longer lag phase than Viagra. This is due to differences in the coating film, in both thickness and
composition, i.e. a greater amount of thickening agent or hydrophobic components.
97
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Filagra
Dissolution Method pH 2
100
Medium : HCl 0.01 M pH 2.0
Dissolution (%)
80
Medium Volume : 900 ml
60
Apparatus : BASKET
brand Viagra 50 mg
Rotation speed : 100 RPM
40
Filagra
Wavelength UV Detector : 292nm
20
Filter : 0.7 µm GF Whatman
0
Temperature : 37°C
0
10
20
t (min)
30
40
50
Specification: 85% of release after 30 min
Fig. 68 : Dissolution of Filagra tablets (red line) compared to that of brand Viagra (blue line), in the operative
conditions described in the table on the right (the pre-existent method). The percentage of dissolution is reported
versus time.
The dissolution test was performed also with the pre-existent method (figure 68). In these conditions
the dissolution is much faster than in the other method because Sildenafil citrate is much more soluble
at pH 2 than at pH 4.5. Furthermore also the rotation speed is faster (100 RPM instead of 50).
In this conditions the dissolution profile of Filagra and that of Viagra can’t be distinguished each other.
Thus the new dissolution method results more discriminating and more effective in detecting
formulation differences.
2.
SILDEANFIL SOFT
Fig. 69 : On the left: a Sildenafil Soft blister. On the right : a Sildenafil soft tablet.
Also Sildenafil Soft was received through a purchase on the internet. The secondary packaging was
absent but a leaflet in English and an analytical certificate with the specification of the product were
attached.
For these reasons it was hypothesized that this drug is a generic drug approved in the state where it is
manufactured, that is India, where the intellectual property is not recognized.
Tablets were contained in a blister where the indication “Sildenafil Citrate Soft Tablet 50 mg” was
reported. Tablets were white, apparently without external film, with diamond shape, and minty smell.
HPLC analysis confirmed the presence of Sildenafil in the declared amount. The solid state techniques
highlighted the presence of cellulose, magnesium stearate and lactose in small amount in respect to the
active substance.
98
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Sildenafil Soft
100
New Dissolution Method pH 4.5
Dissolution (%)
80
Medium : Phosphate Buffer pH 4.5
Medium Volume : 900 ml
60
Apparatus : BASKET
Sildenafil Soft
Rotation speed : 50 RPM
40
Wavelength UV Detector : 292nm
brand Viagra 50 mg
Filter : 0.7 µm GF Whatman
20
Temperature : 37°C
0
0
50
100
150
200
t (min)
250
300
350
Fig. 70 : Dissolution of Sildenafil Soft tablets (red line) compared to that of brand Viagra (blue line), in the operative
conditions described in the table on the right. The percentage of dissolution is reported versus time.
The dissolution profile of Sildenafil Soft with the new dissolution method was very similar to that of
Viagra even though the formulation is different (see figure 70). As a matter of fact in Sildenafil Soft,
unlike Viagra, lactose was present and an external coating was absent. But these differences may
balance each other to give altogether a similar API release to that of the original.
However, observing the first few minutes of dissolution, differences in release profile shape can be
detected (see figure 71). The linear release in Sildenafil Soft and the absence of a lag phase arise from
the lack of an external coating.
Sildenafil Soft
90
New Dissolution Method pH 4.5
80
Medium : Phosphate Buffer pH 4.5
Dissolution (%)
70
Medium Volume : 900 ml
60
50
Apparatus : BASKET
40
Rotation speed : 50 RPM
Sildenafil Soft
30
20
Wavelength UV Detector : 292nm
Filter : 0.7 µm GF Whatman
brand Viagra 50 mg
10
Temperature : 37°C
0
0
10
20
30
40
50
t (min)
Fig. 71 : A particular of the first few minutes of the dissolutions depicted in figure 70 are now presented.
On the contrary with the pre-existent method, at pH 2, the dissolution test highlights a different release
of Sildenafil soft respect to that of the original (see figure 72). Thus the pre-existent method in this case
seems to better detect the formulation differences.
Actually after 30 minutes the dissolution both of Sildenafil Soft and Viagra are complete and both of
them fulfill the method specification.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Sildenafil Soft
Dissolution Method pH 2
100
Medium : HCl 0.01 M pH 2.0
80
Dissolution (%)
Medium Volume : 900 ml
Apparatus : BASKET
60
Rotation speed : 100 RPM
Sildenafil Soft
40
Wavelength UV Detector : 292nm
Brand Viagra 50
mg
20
Filter : 0.7 µm GF Whatman
Temperature : 37°C
Specification: 85% of release after 30 min
0
0
10
20
30
t (min)
40
50
60
Fig. 72 : Dissolution of Sildenafil Soft tablets (red line) compared to that of brand Viagra (blue line), in the operative
conditions described in the table on the right (the pre-existent method). The percentage of dissolution is reported
versus time.
3.
AX
Fig. 73 : On the left: the tablets of the sample AX didn’t have any secondary packaging; on the right: comparison
between a tablet of AX (above) and that of brand Viagra (below).
This sample was made of azure tablets, apparently without external coating. The inscription “AX” was
impressed on one side, “100 mg” on the other side.
The HPLC analysis detected in each tablet approximately an amount of 160 mg of Sildenafil, that is much
more than the higher authorized strength: a such strong over-dosage can be very dangerous. The
development studies on Viagra, indeed, pointed out that an API dosage over 100 mg increased adverse
effects (headache, flush, visual disorders, blood pressure disorders) without improving pharmaceutical
effectiveness. If such amount of Sildenafil citrate released quickly, the consume of the product AX would
be very dangerous.
The solid state analyses on AX detected the presence of cellulose and lactose; in spite of the overdosage, the active substance/excipients ratio was similar to that in the original drug. The presence of
the coloring agent both on surface and in bulk of the tablets was noted.
The dissolution test with the preexistent method, which simulates the stomach environment, showed
that the release of API in the sample AX was actually very slow (see figure 74). The plateau was not
reached even after 3 hours; thus in vivo the active substance probably doesn’t reach such high blood
concentration to produce hazardous effects.
Accordingly this sample was completely off-specification: such a slow API release is not adequate to an
immediate release dosage form.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
AX
100
Dissolution Method pH 2
Medium : HCl 0.01 M pH 2.0
Dissolution (%)
80
Medium Volume : 900 ml
Apparatus : BASKET
60
Rotation speed : 100 RPM
Wavelength UV Detector : 292nm
40
brand Viagra 100 mg
Filter : 0.7 µm GF Whatman
Temperature : 37°C
20
Specification: 85% of release after 30 min
AX
0
0
20
40
60
80
100
120
140
160
180
t (min)
Fig. 74 : Dissolution of AX tablets (red line) compared to that of brand Viagra (blue line), in the operative conditions
described in the table on the right (the pre-existent method). The percentage of dissolution is reported versus time.
Also with the new dissolution method, at pH 4.5, the API release of the sample AX was very slow (see
figure 75). The plateau wasn’t reached even after 12 hours.
At the end of the test the dissolution medium resulted colored in blue, because of the solubilization of
the coloring agent present in a big amount inside the tablet.
The presence of big amounts of a soluble coloring agent can contribute to explain the very slow release
of the active substance in AX; as reported in literature [48], soluble coloring agents diminish the
solubility of active substances in the dissolution medium through salting out effect, thus they reduce the
release rate of API.
AX
New Dissolution Method pH 4.5
160
Medium : Phosphate Buffer pH 4.5
140
Medium Volume : 900 ml
Dissolution (%)
120
Apparatus : BASKET
Rotation speed : 50 RPM
100
Wavelength UV Detector : 292nm
80
brand Viagra 100 mg
60
Temperature : 37°C
AX
40
Filter : 0.7 µm GF Whatman
20
0
0
100
200
300
400
t (min)
500
600
700
Fig. 75 : Dissolution of AX tablets (red line) compared to that of brand Viagra (blue line), in the operative conditions
described in the table on the right. The percentage of dissolution is reported versus time.
Observing thoroughly the first few minutes of dissolution (see figure 76), it can be noted in AX the
absence of a lag phase, thus the tablets of AX are not coated.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
AX
50
New Dissolution Method pH 4.5
Dissolution (%)
40
Medium : Phosphate Buffer pH 4.5
Medium Volume : 900 ml
30
Apparatus : BASKET
Rotation speed : 50 RPM
20
Wavelength UV Detector : 292nm
10
brand Viagra 100 mg
AX
Filter : 0.7 µm GF Whatman
Temperature : 37°C
0
0
10
20
30
40
t (min)
Fig. 76 : A particular of the first few minutes of the dissolutions depicted in figure 75 are now presented.
Subsequently a comparison between the dissolution profiles of the sample AX and Sildenafil Soft were
made. Indeed as the DSC thermograms of these two samples were very similar, their composition was
similar too (see figure 77). In this way it was verified if the dissolution test was able to stress some
differences.
Fig. 77 : Comparison between DSC thermograms of Sildenafil Soft (black line) and AX (red line).
AX - Sildenafil Soft
COMPARISON
Concentration (mg/L)
70
New Dissolution Method pH 4.5
60
Medium : Phosphate Buffer pH 4.5
50
Medium Volume : 900 ml
Apparatus : BASKET
40
Rotation speed : 50 RPM
30
Wavelength UV Detector : 292nm
AX 100 mg
20
R² = 0.9992
Filter : 0.7 µm GF Whatman
Sildeanfil Soft 50 mg
10
Temperature : 37°C
0
0
20
40
60
80
100
120
140
t (min)
Fig. 78 : Comparison between dissolution profile of AX (red line) and Sildenafil Soft (green line). The concentration
of sildenafil in dissolution medium is reported versus time.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
In the graphic the concentration of Sildenafil citrate is reported on the ordinates instead of the
percentage of dissolution (see figure 78). This is to make a comparison between two sample, Sildenafil
Soft and AX, which contain a different amount of active substance.
The dissolution test points out that these two samples have different release profiles, hence in this case
it can stress differences that are not detectable by other techniques. These differences can arise from
differences in production technology, compression force during the manufacture, granulometry of the
raw material, but also from the presence of a big amount of coloring agent in AX.
Then it can be noted that AX seems to release the API linearly in the examined time interval. This is also
confirmed by the linear regression curve overlapped. For this reason the dissolution profile of AX was
compared to the intrinsic dissolution of the active substance Sildenafil citrate.
The intrinsic dissolution of sildenafil citrate was studied as the release from a disk of pressed raw
material in the first few minutes of dissolution, when this release is still linear. As a matter of fact at the
beginning of analysis, when there is a strong concentration gradient between the area close to the disk
and the bulk of the dissolution medium, and the surface of disk is still smooth, the dissolution is only
dependant from the solubility of raw material; thus the dissolution is linear and its slope is related to the
solubility of API.
Since also the release of the sample AX was linear, the first few minutes of this dissolution were
compared to those of the intrinsic dissolution of sildenafil (see figure 79). In this way it was evaluated if
for the sample AX a contribution to the release rate of API arises from the excipients or, on the contrary,
its dissolution simply depends on the solubility of sildenafil
AX - Viagra - Intrinsic Dissolution
COMPARISON
0.025
Sildenafil citrate Intrinsic dissolution
0.02
New Dissolution Method pH 4.5
Medium : Phosphate Buffer pH 4.5
Conc (mg/ml)
Brand Viagra 100 mg
0.015
Medium Volume : 900 ml
AX
Apparatus : BASKET
0.01
Rotation speed : 50 RPM
Wavelength UV Detector : 292nm
0.005
Filter : 0.7 µm GF Whatman
Temperature : 37°C
0
0
2
4
6
8
10
t (min)
Fig. 79 : Comparison among the first few minutes of dissolution of brand Viagra (blue line), those of AX (red line)
and the Sildenafil citrate Intrinsic dissolution (green line). The concentration of sildenafil in dissolution medium is
reported versus time.
The dissolution profile of AX is very similar to the intrinsic dissolution profile of sildenafil: as a matter of
fact they have the same slope, even though with different intercepts. Hence, it can be hypothesized that
the excipients in AX don’t cooperate in facilitating the API release. In Viagra, instead, the role of the
excipients is very important as they facilitate significantly the release of the active substance, as proved
by a much faster dissolution than the intrinsic dissolution. For this reason the formulation of Viagra is
adequate to an immediate release dosage form.
What results is that the sample AX is very anomalous. Doubts remain about the strong over-dosage as
the active substance is the most expensive component of the drug. It can follows a manufacture which
doesn’t fulfill the GMP and thus doesn’t guarantee the uniformity of dosage.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
However it can be also hypothesize that the over-dosage is a consequence of the inadequate
formulation which doesn’t produce an appropriate release. An additional amount of the active
substance could have been arbitrarily employed by counterfeiters to obtain the therapeutic effect. For
counterfeiting market indeed it is particularly important that patients are satisfied to increase the
business.
It may be hypothesized also the manufacturers intended to produce not an immediate release but an
extended release dosage form to lengthen the duration of the effects. But no evidences have been
found on this hypothesis.
4.
5.
COUNTERFEIT CIALIS 20mg
COUNTERFEIT VIAGRA 50 mg
Fig. 80 : On the left: a counterfeit tablet of Cialis (on the left) compared to the original one. On the right: a
counterfeit tablet of Viagra (on the bottom) compared to the original one
Two samples purchased on the same internet website, one counterfeit of Cialis 20mg and one
counterfeit of Viagra 50 mg, were analyzed.
The active substance of original Cialis is Tadalafil, which interacts with a site of the receptor
phosphodiesterase 5 different from that of Sildenafil.
The tablets were delivered in a paper bag, without neither a blister nor a secondary packaging. They
looked very similar to the originals.
The solid state techniques (IR, DSC, TGA) were particularly informative for these samples: they
highlighted that the two samples had the same qualitative composition and, in particular, both of them
contained sildenafil citrate as active substance. In addition the presence of magnesium stearate,
cellulose, and another unknown component in a big amount were detected. This anomalous component
was successively identified, through the data bank; it was chalk, that is calcium sulfate dihydrate, a
product commonly employed as building material and absolutely inappropriate as pharmaceutical
excipient.
The HPLC analysis confirmed the presence of Sildenafil in both samples, thus in the counterfeit Cialis the
declared active substance, tadalafil, had been substituted with Sildenafil. The actual dosage of API was
sildenafil 50 mg in the counterfeit Cialis and sildenafil 100 mg in the counterfeit Viagra (that was
declared 50mg).
The chalk makes tablets extremely hard to grind, thus it was hypothesized that the release of the API
would be very slow.
On the contrary, as it is reported in the plot, the dissolution of these tablets was very fast.
104
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Counterfeit Cialis
New Dissolution Method pH 4.5
100
Dissolution (%)
Medium : Phosphate Buffer pH 4.5
80
Medium Volume : 900 ml
60
Rotation speed : 50 RPM
Apparatus : BASKET
counterfeit Cialis (50 mg Sildenafil Citrate)
Wavelength UV Detector : 292nm
40
Filter : 0.7 µm GF Whatman
brand Viagra 50 mg
20
Temperature : 37°C
0
0
50
100
150
200
250
300
350
t (min)
Fig. 81 : Dissolution of counterfeit Cialis (red line) compared to that of brand Viagra (blue line), in the operative
conditions described in the table on the right. Indeed in this counterfeit Cialis sample, the active substance of
Viagra is present. The percentage of dissolution is reported versus time.
Counterfeit Viagra
100
New Dissolution Method pH 4.5
Medium : Phosphate Buffer pH 4.5
Dissolution (%)
80
Medium Volume : 900 ml
60
Apparatus : BASKET
counterfeit Viagra 50 mg (100 mg sildenafil citrate)
40
Rotation speed : 50 RPM
Wavelength UV Detector : 292nm
brand Viagra 100 mg
Filter : 0.7 µm GF Whatman
20
Temperature : 37°C
0
0
50
100
150
200
250
300
350
400
t (min)
Fig. 82 : Dissolution of counterfeit Viagra (red line) compared to that of brand Viagra (blue line), in the operative
conditions described in the table on the right. The percentage of dissolution is reported versus time.
The dissolution profile of the two counterfeit samples is almost the same (see figure 81 – 82), in
accordance with what stressed by solid state analyses: that the composition of the two samples is
identical.
Observing thoroughly the beginning of the dissolution (see figure 83), the same sigmoidal profile can be
noted for both samples. This evidence, connected to the very fast API release, induces to hypothesize
that chalk, present in a big amount in these two formulations, has a disaggregating effect.
105
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Counterfeit Viagra
Counterfeit Cialis
100
100
80
Dissolution (%)
Dissolution (%)
80
60
40
counterfeit Cialis (50
mg Sildenafil Citrate)
60
40
counterfeit Viagra 50 mg
(100 mg sildenafil citrate)
20
20
brand Viagra 50 mg
brand Viagra 100 mg
0
0
0
20
40
60
t (min)
80
100
0
120
20
40
60
80
100
120
t (min)
Fig. 83 : On the left: a particular of the first few minutes of the dissolutions depicted in figure 81 are presented.
On the right: a particular of the first few minutes of the dissolutions depicted in figure 82 are presented
The counterfeit Cialis has a long lag phase but it is not possible to make consideration about this
because both coating and shape of Cialis tablets are completely different from those of brand Viagra
employed for the dissolution test comparison (as the API contained in counterfeit Cialis was Sildenafil in
place of tadalafil).
While the new dissolution method can stress how different these counterfeit samples are respect to
original Viagra, the pre-existent method at pH2 is not able to highlight all these differences (see figure
84 - 85).
Counterfeit Cialis
Dissolution Method pH 2
Medium : HCl 0.01 M pH 2.0
Dissolution (%)
100
Medium Volume : 900 ml
Apparatus : BASKET
80
counterfeit Cialis (50 mg Sildenafil Citrate)
Rotation speed : 100 RPM
60
Wavelength UV Detector : 292nm
brand Viagra 50 mg
40
Filter : 0.7 µm GF Whatman
Temperature : 37°C
20
Specification: 85% of release after 30 min
0
0
10
20
30
40
50
60
70
t (min)
Fig. 84 : Dissolution of counterfeit Cialis (red line) compared to that of brand Viagra (blue line), in the operative
conditions described in the table on the right (the pre-existent method). Indeed in this counterfeit Cialis sample, the
active substance of Viagra is present. The percentage of dissolution is reported versus time.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Counterfeit Viagra
Dissolution Method pH 2
Medium : HCl 0.01 M pH 2.0
100
Medium Volume : 900 ml
80
Apparatus : BASKET
Dissolution (%)
brand Viagra 100 mg
Rotation speed : 100 RPM
60
Wavelength UV Detector : 292nm
counterfeit Viagra 50 mg (100 mg
Sildenafil Citrate)
40
Filter : 0.7 µm GF Whatman
Temperature : 37°C
Specification: 85% of release after 30 min
20
0
0
10
20
30
t (min)
40
50
Fig. 85 : Dissolution of counterfeit Viagra (red line) compared to that of brand Viagra (blue line), in the operative
conditions described in the table on the right (the pre-existent method). The percentage of dissolution is reported
versus time.
In these conditions, indeed, for both original and counterfeit tablets the dissolution is complete after
only 10 minutes. With the pre-existent method the dissolution test doesn’t provide any indication about
the presence in the formulations of an anomalous component, like chalk. At pH 4.5, the dissolution of
the counterfeit samples is much faster than the originals; thus an unknown sample containing chalk
would be easily detected by this method.
Hence also in this case the new method results more discriminating and more effective in detecting
differences in the formulation.
6.
COUNTERFEIT VIAGRA 100 mg
Fig. 86 : On the left: comparison between secondary packages of a counterfeit Viagra sample (on the left) and an
original one. On the right: comparison the between the counterfeit Viagra tablet (on the bottom) and the original
one.
The tablets of this sample were packaged in a blister. A secondary packaging was also present, similar to
that of original Viagra but with different colors. The secondary packaging of original drugs however can
be different from country to country, thus differences in the secondary packaging could not be
informative about counterfeiting.
Thermal analyses and IR detected sildenafil citrate, magnesium stearate, cellulose, and chalk. By HPLC
the amount of Sildenafil citrate was quantified at 60 mg, hence under-dosed compared to the declared
100 mg, but the tablets showed a non uniformity of dosage.
107
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Containing 60 mg of sildenafil, this counterfeit sample was compared both to Viagra 100 mg and to
Viagra50 mg (see figure 87). In the plot the concentration of sildenafil, in mg/L, is reported on the
ordinates.
Counterfeit Viagra 100 mg
100.0
80.0
Concentration (mg/L)
New Dissolution Method pH 4.5
Medium : Phosphate Buffer pH 4.5
60.0
Medium Volume : 900 ml
40.0
Rotation speed : 50 RPM
Apparatus : BASKET
Wavelength UV Detector : 292nm
counterfeit Viagra (60 mg Sildenafil Citrate)
20.0
Filter : 0.7 µm GF Whatman
brand Viagra 100 mg
Temperature : 37°C
brand Viagra 50 mg
0.0
0
20
40
60
80
t (min)
100
120
140
160
Fig. 87 : Dissolution of counterfeit Viagra (red line) compared to that of brand Viagra 100mg (blue line), and to
brand Viagra 50mg, in the operative conditions described in the table on the right. Indeed, in this counterfeit
Viagra the API content was about 60mg. The concentration of sildenafil in dissolution medium is reported versus
time.
Also in this case the disaggregating effect of chalk is evident. Indeed at pH 4.5 the API release in
counterfeit tablets containing chalk is much faster than that of the originals.
Counterfeit Viagra 100 mg
80.0
counterfeit Viagra (60 mg Sildenafil Citrate)
Concentration (mg/L)
70.0
60.0
brand Viagra 100 mg
New Dissolution Method pH 4.5
brand Viagra 50 mg
Medium : Phosphate Buffer pH 4.5
Medium Volume : 900 ml
50.0
Apparatus : BASKET
40.0
Rotation speed : 50 RPM
30.0
Wavelength UV Detector : 292nm
20.0
Filter : 0.7 µm GF Whatman
10.0
Temperature : 37°C
0.0
0
10
20
t (min)
30
40
50
Fig. 88 : A particular of the first few minutes of the dissolutions depicted in figure 87 are presented.
In the first few minutes of dissolution (see figure 88) a lag phase very similar to that of Viagra is
highlighted. Thus probably the composition and the thickness of the external coating is similar too.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
7.
I-PRAMIL
Fig. 89 : On the left: I-Pramil blisters. On the right : an I-Pramil tablet.
Some information about I-Pramil has already been reported in the thermoanalysis section. It is a
counterfeit that imitates Pramil, a legal generic drug in the South America.
The tablets were coated with a blue film; they were packaged in a low-quality blister, where the
indication “Sildenafil 50 mg” was reported, while the secondary packaging was lacking. These tablets
were very small (they weighed about 130 mg while a Viagra 50 mg tablet weighs about 310 mg). The
HPLC analysis confirmed the presence of the declared active substance in the correct amount, thus in IPramil the API/excipient ratio is higher than in Viagra. Thermal analyses detected the presence of
cellulose and magnesium stearate.
The dissolution test at pH 4.5 points out that I-Pramil has a different release from Viagra, faster in spite
of a longer lag phase (see figure 90). Hence the new dissolution method can distinguish between IPramil and Viagra.
I-Pramil
100
New Dissolution Method pH 4.5
Dissolution (%)
80
Medium : Phosphate Buffer pH 4.5
Medium Volume : 900 ml
60
Apparatus : BASKET
Rotation speed : 50 RPM
40
brand Viagra 50 mg
20
Wavelength UV Detector : 292nm
Filter : 0.7 µm GF Whatman
I-Pramil
Temperature : 37°C
0
0
10
20
30
40
50
60
70
80
90
t (min)
Fig. 90 : Dissolution of I-Pramil tablets (red line) compared to that of brand Viagra (blue line), in the operative
conditions described in the table on the right. The percentage of dissolution is reported versus time.
The faster release probably is due to a low compression force applied during the manufacturing.
109
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
I-Pramil
100
Dissolution Method pH 2
Medium : HCl 0.01 M pH 2.0
Dissolution (%)
80
Medium Volume : 900 ml
Apparatus : BASKET
60
brand Viagra 50 mg
40
Rotation speed : 100 RPM
Wavelength UV Detector : 292nm
I-Pramil
Filter : 0.7 µm GF Whatman
20
Temperature : 37°C
Specification: 85% of release after 30 min
0
0
5
10
15
20
t (min)
25
30
35
40
Fig. 91 : Dissolution of Sildenafil Soft tablets (red line) compared to that of brand Viagra (blue line), in the operative
conditions described in the table on the right (the pre-existent method). The percentage of dissolution is reported
versus time.
Operating at pH2, no differences can be noticed between the dissolution of Viagra and I-Pramil (see
figure 91) despite there were significant differences, on the shape of the tablets, their weights,
composition, coating and technology of production.
In conclusion the new developed method resulted more discriminating than the pre-existent one in
most of the examined cases.
The dissolution test, performed in discriminating conditions, is another effective instrument to detect
counterfeit drugs from the original ones and, more significantly, to acquire valuable information about
the release rate of the active substance and the formulation of unknown samples.
In future possible application of the same dissolution test to other drugs for the treatment of erectile
dysfunction (i.e. Cialis and Levitra) will be investigated. As a matter of fact the substitution of the
declared active substance with a cheaper one with similar pharmaceutical effect is often detected; thus
it would be convenient to use the same dissolution test for the analysis of all the drugs of the same
therapeutic class.
110
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
3.5 THE ANALOGUES
In addition to the risks described so far, a new alerting danger has arisen, that is the proliferation
of fake herbal products and food dietary supplements. These products claim to contain only vitamins
and natural herbs, thus not synthetic active substances. The common belief that herbal supplements
don’t have neither side effects nor interactions with other drugs has increased the market of this kind of
products. Thus also counterfeiters have begun to be interested in this profitable business.
In some herbal products and food dietary supplements, in particular those recommended for the
treatment of erectile dysfunction and obesity, synthetic active substances were detected. In some cases
they were the same active substances as the legal medicines but not declared on the label: in this way a
patient takes a drug without being aware of it and not evaluating possible side effects and
pharmacological interactions. For example the erectile dysfunction often arises in patients with
cardiovascular diseases, that need to take some medicines, like nitroglycerine, that have dangerous
interactions with the active substances of Viagra, Cialis e Levitra.
But the greatest danger arises from the presence, in some fake herbal products, of illegal active
substances (e.g. sibutramine, not authorized in Italy after a risk/benefit assessment) and the analogues,
that are new and unknown active substances.
The analogues are molecules structurally very similar to known active substances, but with some
structural modifications in sites that are not crucial for the activity. Hence these analogues usually have
a similar activity to the active substances they imitate, but actually they are new and unknown
substances, not authorized by the competent authority. Preclinical and clinical studies were not
conducted on these new molecules, thus neither their efficacy nor their safety are known. Even though
these molecules are very similar to approved active substances, their efficacy, selectivity, absorption,
distribution, metabolism, excretion can be completely different. They can have different toxicity,
adverse effects and interactions with other drugs.
The analogues most frequently detected are those of Sildenafil, Vardenafil and Tadalafil.
In some cases permanent damages after a single administration were reported [49]. It was reported, for
examples, that the Piperidino Vardenafil, an analogue of Vardenafil, is metabolized more slowly than
Vardenafil and remains active longer. As it is more lipophilic, it can enter into the hematoencephalic
barrier and cause brain damages.
Acetildenafil, an analogue of Sildenafil, is much less selective toward PDE 6, to which visual disorders are
associated.
Amino-tadalafil, an analogue of Tadalafil, is a very reactive hydrazone and can cause a permanent
inhibition of the PDE-5 enzyme [7].
These substances are illegally employed in fake herbal products and dietary supplements production
because they usually have a similar pharmaceutical effect as the approved active substances but are
much more difficult to detect by chemical analyses. As a matter of fact the analogues have different
retention times in chromatographic analyses respect to the approved API they imitate, and reference
standards are not available.
Very complex analytical techniques are usually necessary to detect the analogues, also because the
matrix of these samples is very complex since there are also many herbal substances.
Furthermore the use of analogues instead of the approved API is increased for the belief that in this way
the intellectual property rights are not breached, thus that there can’t be prosecution for counterfeiting.
111
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Herbal products and food dietary supplements can be in different dosage form. Some of them are
granulates contained in sachets, other are capsule with heterogeneous content, pressed or not, other
are gels.
3.5.1 The analogues in the literature
To study this new phenomenon, the health risks it can cause and the possible actions against it, a
literature search was done to report all the analogues that had been detected in herbal products and
food dietary supplements. These are reported in figures 92 – 93 – 94 – 95.
This research needs to be frequently updated because new molecules are continuously synthesized and
employed in the illegal market.
Many articles reported the detection of these analogues, above in products for the erectile dysfunction
treatment. About 40 different active molecules were described [50-51-52-53-54-55].
A significant amount of analogues were reported only in Asiatic scientific literature in particular in that
of North Korea; the Asian Authorities indeed seem to be more informed about the development of the
illegal pharmaceutical market. After all in Asia the most of counterfeit drugs, spread all over the world,
are manufactured.
Many of these 40 molecules are analogues of Sildenafil; in particular some of these are nearly identical
to Sildenafil, like homosildenafil, that has only one more –CH2 group on the piperazinic nitrogen; hence
in chromatographic analyses homosildenafil has a similar retention time to Sildenafil. Other analogues
instead have a different functional group, like hydroxyhomosildenafil, where a –OH group gives a major
affinity for hydrophilic phases.
Some of these molecules have an acetyl group instead of the sulfonyl group, other a thioketone instead
of the ketone, other various substituents on the piparazinic nitrogen.
Many analogues of Vardenafil were also reported, with similar structural modifications to those
reported for Sildenafil. Vardenafil and Sildenafil indeed are very similar molecules (they only differ in the
position of nitrogen atoms in the condensed heterocyclic aromatic rings; in addition they have different
aliphatic side chains on the piperazine).
Tadalafil instead is a completely different molecule and only few analogues were reported. In addition
to the over mentioned aminotadalafil, other few analogues with a different chain on the piperazinic
nitrogen have been detected.
In this survey also structurally different molecules, that are being tested for the erectile dysfunction
disease, were considered, like udenafil, mirodenafil, avanafil, benzamidenafil [56] because, being active
molecules, they may be employed in the illegal market.
The analogues of PDE-5 inhibitors reported in scientific literature are now presented:
112
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
O
N
Sildenafil
HN
O
O
N
S
N
N
N
O
Homosildenafil
N
HN
O
O
O
N
S
N
N
N
O
O
N
HN
O
O
N
Hydroxyhomosildenafil
S
N
N
N
O
HO
O
N
Acetildenafil
N
O
HN
N
or hongdenafil
N
N
O
O
Noracetildenafil
N
or Desmethylacetildenafil
N
O
HN
N
N
or Demthylhongdenafil
N
O
O
N
HN
Dimethylacetildenafil
O
HN
N
N
N
113
O
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
O
Desmethyldenafil
N
HN
O
O
N
S
N
N
HN
O
O
N
Piperidino Sildenafil
HN
O
O
N
or Norneosildenafil
S
N
N
O
O
N
O
Piperidino Acetildenafil
HN
N
N
or Piperidino hongdenafil
N
O
O
N
NH
O
O
Methisosildenafil
N
S
or Aildenafil
N
or dimethylsildenafil
N
NH
O
O
HO
Hydroxyacetildenafil
N
O
N
HN
N
or Hydroxyhongdenafil
N
N
O
114
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
S
N
Thiosildenafil
HN
O
O
N
or SildenafilThione
S
N
or sulfosildenafil
N
N
O
S
N
Thiohomosildenafil
HN
O
O
N
or sulfohomosildenafil
S
N
N
N
O
Thiohydroxyhomosildenafil
N
HN
O
O
S
N
S
or Hydroxythiohomosildenafil
N
N
N
HO
S
O
Thiomethisosildenafil
N
or Thioaildenafil
N
S
or dimetisildenafilthione
or sulfoaildenafil or
HN
O
O
N
N
HN
O
dimethylthiosildenafil
O
O
O
N
HN
N
Benzylsildenafil
S
N
N
N
O
115
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
O
N
HN
O
O
N
Isobutylsildenafil
S
N
N
N
O
O
N
HN
O
O
N
Cyclopentafil
S
N
N
N
O
O
N
O
HN
N
Carbodenafil
N
N
N
O
O
N
N
O
HN
N
Oxohongdenafil
N
O
N
O
Cinnamydenafil
N
O
O
N
HN
N
N
N
O
116
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
O
N
HN
Nitrodenafil
N
O2N
N
O
O
N
OH
HN
Hydroxychlorodenafil
N
Cl
N
O
O
N
O
HN
Chlorodenafil
N
Cl
N
O
O
N
N
N
S
O
N
nitroso prodenafil
N
O
O
N
S
N
N
HN
O
O
O
N
Lodenafil
Carbonate
NH
N
N
O
O O
O O
S
S
N
N
O
N
N
O
117
O
N
HN
N
N
O
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Fig. 92 : Sildenafil and its analogues reported in scientific literature.
O
HN
O
O
Vardenafil
N
S
N
N
N
N
O
O
Piperidenafil
or Piperidino Vardenafil
HN
O
O
N
or Pseudovardenafil
S
N
N
N
O
Acetil Vardenafil
N
O
O
HN
N
N
N
N
O
Acetil piperidino
vardenafil
O
O
HN
N
N
N
N
O
Hydroxyvardenafil
O
O
O
HN
N
S
N
N
N
N
O
HO
118
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
O
N-desmethylvardenafil
HN
O
O
N
S
N
N
N
HN
O
O
O
HN
N
Norneovardenafil
N
HO
N
O
O
HN
N
Desulfovardenafil
N
N
O
Fig. 93 : Vardenafil and its analogues reported in scientific literature.
119
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
O
N
N
O
Tadalafil
N
H
O
O
O
NH2
N
N
O
Aminotadalafil
N
H
O
O
O
N
N
O
Ethyl Tadalafil
N
H
O
120
O
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
O
O
Cl
N
O
CloropreTadalafil
N
H
O
O
O
N
N
O
N
H
OctylnorTadalafil
O
Fig. 94 : Tadalafil and its analogues reported in scientific literature.
121
O
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
O
N
HN
O
O
N
Udeanafil
S
N
H
N
O
N
N
HO
NH
Thioquinapiperifil
H
N
N
S
N
N
O
N
O
HN
N
Gendenafil
N
O
OH
N
N
N
O
N
H
Avanafil
Cl
N
H
N
O
N
122
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
O
N
HN
O
O
N
Mirodenafil
S
N
N
N
O
HO
HO
NH
O
Benzamidenafil
N
H
or Xanthoanthrafil
OCH3
NO2
OCH3
Fig. 95 : New active molecules with a pharmaceutical activity similar to that of Sildenafil.
3.5.2 Detection of analogues by LC_MS: development of a new method
The chemical analysis of herbal products and dietary supplements, that are suspected to contain
synthetic active substances, is very tricky and advanced equipment is needed. The complexity of the
matrix of these samples brings many interferences problems. In addition usually reference standards for
analogues are not available. Furthermore many new synthetic active molecules were reported in
literature, but it is also possible that counterfeits employ other newer molecules.
The HPLC-MS2 is a very useful instrument for the analogues detection, as proved by many scientific
works about this application. It is very complex and expensive but often it allows to detect analogues.
To employ HPLC-MS2, first the sample must be extracted with an organic solvent, e.g. methanol; hence
in the organic solvent all the soluble substances from the sample are dissolved. Afterwards these are
separated by chromatography and analyzed by the mass-mass detector; this detector not only provides
the mass of each substance but it also allows to fragment them. From these fragments very important
information can be obtained. As a matter of fact, since the analogues are structurally similar to
authorized known molecules, it is highly probable that some of the fragments they originate are the
same. In particular the literature reports some fragments produced by fragmentation both of the
original API and of many analogues [6]. As a consequence, when the masses of these fragments are
detected related to a substance with a different retention time from the original API, it can alert about
the possible presence of an analogues of the original API.
123
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
On this purpose in this laboratory a HPLC-MS2 Q-TOF, with one quadrupole and one time of flight
analyzer, able to detect accurate masses (with 4 decimal digits) and to fragment the analyzed molecules,
was purchased. The accurate mass data and the detected isotopic abundances can provide meaningful
indications about the probable molecular formula of the examined substance. In addition other valuable
information can be obtained by the fragmentation of the molecule in appropriate conditions, in
particular searching for the suspicious fragments.
A synthesis laboratory of the Istituto Superiore di Sanità, that cooperate in this project, synthesized 15
common analogues of Sildenafil to use as reference standard and a HPLC-MS2 method to separate and
detect them is currently under development.
As a matter of fact it is important to have an appropriate chromatographic method to separate the
analogues because, being structurally similar, they could have similar retention times. Separating these
molecules by chromatography is also necessary because the simultaneous presence of more analogues,
extremely dangerous for patients, was also reported [57].
A HPLC-MS2 study on analogues is also important to examine how these molecules ionize. Indeed a
molecule, to be detected by the mass analyzer, must be ionized in the HPLC mobile phase as it registers
the mass/charge ratio. Some molecules can easily accept one proton getting positive ions; in this case
formic acid is added to the mobile phase to provide protons. If instead the investigated molecule easily
loses one proton becoming a negative ion, a formiate salt is added to the mobile phase.
Some other molecules can neither accept nor lose one proton because of they are neither base nor acid,
thus they can’t be detected by the mass analyzer.
Working on reference standards of some analogues, it is possible to investigate if the HPLC-MS2
technique is able to detect these molecules and to set the appropriate instrumental conditions for the
analysis.
3.5.3 Characterization of synthesized references by thermoanalysis and IR
To employ the analogues that had been synthesized as reference standards for a HPLC-MS2 method, it
was necessary to characterize them both by HPLC-DAD analysis to detect impurities and GC analysis to
detect residual solvents. The solid state techniques, IR, DSC and TGA, were also applied.
By TGA the presence of water and crystallization solvents can be noticed, as well as the decomposition
profile of the substance. By DSC the melting point can be detected. By IR the characteristic peaks and
the fingerprint of the substance can be registered.
Furthermore it was also evaluated if the solid state techniques were able to distinguish among these
analogues for the presence of peculiar signals.
The analogue molecules examined were not citrate salts, like the authorized API Sildenafil citrate, but
free bases. In fact there are no evidence that the analogues employed in the illegal manufacturing are
citrate salts and the free base form is easier to obtain.
Pirdenafil, Mordenafil and Bradenafil are new analogues, not reported in literature, and synthesized by
the synthesis laboratory of the Istituto Superiore di Sanità.
124
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Their molecular structures are:
O
O
N
N
O
O
O
HN
O
HN
N
N
S
S
N
N
N
N
O
O
PIRDENAFIL
BRADENAFIL
O
MORDENAFIL
N
O
O
HN
N
S
N
N
O
O
Fig. 96 : Molecular structures new analogues, not reported in literature, and synthesized by the synthesis
laboratory of the Istituto Superiore di Sanità: Pirdenafil, Bradenafil, Mordenafil.
The DSC thermograms of the analogues that had been synthesized as reference standards are now
presented:
HYDROXYHOMOSILDENAFIL
DIMETHYL SILDENAFIL
125
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
PIPERIDINO SILDENAFIL
PIRDENAFIL
NORACETILDENAFIL
HOMOSILDENAFIL
THIOHYDROXYHOMOSILDENAFIL
DIMETHYLACETILDENAFIL
126
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
BRADENAFIL
MORDENAFIL
The DSC of Bradenafil points out two distinct endothermic peculiar signals. The first endothermic signal
can be attributed to a solid-solid transition to a different polymorphic form, that subsequently melts,
generating the second endothermic signal.
In the majority of these samples the presence neither of water nor of other solvents is highlighted; only
in thiohydroxyhomosildenafil and dimethylacetildenafil slightly bound solvents, that evaporated at low
temperature, were detected.
The melting temperatures of each product, registered as onset temperature, are reported in the table:
Table 7
Molecule
Melting Point
HYDROXYHOMOSILDENAFIL
195.0
DIMETHYL SILDENAFIL
205.2
PIPERIDINO SILDENAFIL
193.8
PIRDENAFIL
193.4
NORACETILDENAFIL
163.1
HOMOSILDENAFIL
199.9
THIOHYDROXYHOMOSILDENAFIL
191,1
DIMETHYLACETILDENAFIL
161,3
BRADENAFIL
137.4
MORDENAFIL
183.4
From these data it results that some of these products, like Mordenafil and Bradenafil, have peculiar
melting points thus they could be detected by a DSC analysis. Hydroxyhomosildenafil, Piperidino
Sildenafil and Pirdenafil instead have very similar melting points and may be mistaken by DSC. The
melting point of Thiohydorxyhomosildenafil is a little lower and also distinguishable. Also Homosildenafil
and Dimethylsildenafil can be distinguished because they have higher melting points than the others.
Dimethylacetildenafil and Noracetildenafil have lower and very similar melting points, hence they can be
mistaken.
The TGA thermograms of these analogues are the following:
127
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
HYDROXYHOMOSILDENAFIL
DIMETILSILDENAFIL
PIPERIDINO SILDENAFIL
NORACETILDENAFIL
PIRDENAFIL
HOMOSILDENAFIL
128
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
TIOIDROSSIHOMOSILDENAFIL
DIMETILACETILDENAFIL
BRADENAFIL
MORDENAFIL
The FT-IR of these products are the following:
108,4
106,0
105
1933,6
100
100
3479,1
1929,8
3107,1
3100,3
95
95
1193,2
2769,0
90
90
2940,4
2871,2
2809,1
85
1599,4
845,8
2860,2
632,9
1532,4
1413,9
85
874,5
1206,2
842,6
1537,6
80
2937,6
3302,5
1557,2
1578,8
75
1489,1
855,2
900,3
1372,8
80
1299,5
2965,1
70
75
%T
1456,1
1390,2
65
1099,1
1081,0
1110,1
60
55
817,1
1395,5
1283,2
65
762,4
1063,8
651,6
1103,0
60
870,6
1457,9
753,5
779,0
1146,3
1132,9
1348,2
1315,0
55
40
741,2
618,0
1247,7
50
35
1084,5
963,7
1494,0
689,7
942,6
927,8
1246,2
50
1348,6
1335,8
1583,8
1560,3
3310,5
70
1034,3
1030,2
45
1601,5
%T
819,2
775,5
1330,4
1277,5
1130,1
1166,9
1159,6
1167,6
45
30
803,7
930,0
733,8
687,8
767,9
1016,7
1688,8
1144,6
25,9
4000,0
41,5
3600
3200
2800
2400
2000
1800
cm-1
1600
1400
1200
1000
800
600,0
4000,0
HYDROXYHOMOSILDENAFIL
3600
3200
2800
2400
2000
DIMETILSILDENAFIL
129
1800
cm-1
1600
1400
1200
1000
800
650,0
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
106,4
105
106,5
105
1920,7
100
1922,9
100
95
95
90
3324,3
970,0
90
1414,2
1414,0
867,1
85
85
2983,9
2935,9
2954,7
2877,7
2868,8
80
883,2
80
1287,6
883,7
1284,4
1602,4
2936,1
1602,2
1560,2
1215,1
3283,9
75
1560,3
75
1534,1
1066,4
%T
840,3
%T
1363,6
3290,4
70
1578,6
1579,3
70
908,7
1534,6
1267,9
1267,6
863,3
1112,3
1201,6
1488,3
1447,6
65
65
929,5
1322,4
1390,0
1389,7
1076,9
60
1325,7
60
1087,8
1487,8
1463,8
711,5
749,0
780,0
1461,7
55
1101,8
1126,2
1051,4
55
779,9
690,6
1242,6
1102,5
771,1
1242,6
50
50
1026,9
810,2
748,2
1026,4
811,6
690,0
1148,4
45
45
1167,4
1706,6
927,9
1338,4
3600
3200
2800
2400
2000
1800
cm-1
1600
1337,3
1400
1200
1000
800
1014,2
4000,0
650,0
767,3
1161,4
40
38,4
39,2
4000,0
1126,4
1072,1
729,1
1705,9
3600
PIPERIDINO SILDENAFIL
3200
2800
2400
2000
1800
cm-1
1600
1400
1200
1000
800
650,0
PIRDENAFIL
105,8
105,1
100
100
95
95
90
90
85
85
835,4
2869,8
2800,9
2927,0
2956,3
80
3289,4
80
1355,2
1344,0
75
1578,5
70
1373,0
1393,2
1280,7
%T
2925,8
2956,1
75
701,6
1342,8
795,8
1536,0
1193,8
872,5
65
904,0
956,7
3288,4
70
926,6
1194,1
1577,8
992,2
966,1
926,2
1393,3
1280,6
%T 65
807,0
1561,6
2802,4
2868,9
796,0
955,9
966,3
992,0 904,0
1008,7
1535,7
806,4
701,1
1372,5
1300,4
871,8
1492,4
1317,2
60
1124,8
1490,1
60
1602,1
1123,0
1602,5
1454,1
1221,7
682,3
654,8
55
55
1300,4
1561,3
729,8
1454,7
1031,6
1317,0
1152,0
1079,9
50
1222,6
50
1032,4
1157,4
1079,7
1244,6
681,9
654,4
45
45
1243,8
1263,0
746,6
40
774,5
40
1263,1
35
35
746,8
773,9
1684,8
30
30,2
4000,0
1680,8
27,4
3600
3200
2800
2400
2000
1800
cm-1
1600
1400
1200
1000
800
650,0
4000,0
NORACETILDENAFIL
3600
3200
2800
2400
2000
1800
cm-1
1600
1400
1200
1000
800
650,0
HOMOSILDENAFIL
105,7
105,2
100
100
1739,0
95
95
90
90
85
85
2802,2
937,7
80
2803,3
2868,9
2925,5
80
2869,3
2926,3
904,5
2956,6
75
75
795,9
1342,3
903,8
2956,1
1343,5
712,7
795,8
3287,5
957,0
3287,7
1194,1
70
1536,4
1578,0
%T
1393,3
1372,5
992,4
966,2
955,6
70
1280,4
806,7
701,5
1536,0
1578,1
1393,6
1281,0
%T
926,5
992,2 926,4
966,2
1193,8
701,1
806,4
65
872,3
1372,1
65
1490,3
60
1123,4
1602,5
1122,7
871,8
1490,5
729,8
1300,6
60
1561,6
1454,8
55
1602,6
1561,3
55
1317,3
1300,3
1454,7
1316,9
729,9
1222,9
1157,7
50
1032,4
1157,8
50
682,1
654,6
1032,8
1222,6
1079,9
1080,0
681,6
654,6
1243,9
45
1244,2
45
40
1263,3
1263,2
774,2
40
747,0
35
774,0
746,6
1682,8
35
1680,8
28,8
33,1
4000,0
3600
3200
2800
2400
2000
1800
cm-1
1600
1400
1200
1000
800
650,0
TIOIDROSSIHOMOSILDENAFIL
4000,0
3600
3200
2800
2400
2000
1800
cm-1
DIMETILACETILDENAFIL
130
1600
1400
1200
1000
800
650,0
Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
107,0
97,2
105
95
1775,6
100
90
3107,8
95
85
90
80
2934,4
2963,2
2863,9
85
2960,51
75
1411,5
796,7
886,1
80
1600,37
1576,99
1529,55
3288,43
70
1532,8
75
899,8
850,8
1307,4
3300,4
736,73
1296,13
65
1601,5
1583,6
1558,6
70
%T
1268,50
%T
750,85
1388,06
60
65
1329,8
1392,3
1454,4
813,57
1463,47
1280,3
1489,8
1078,42
1099,38
1125,63
778,1
820,9
1084,1
60
55
1257,7
765,06
1199,22
1245,49
650,10
55
50
1022,3
1334,64
688,57
679,36
1016,19 930,76
761,5
50
688,8
45
1246,1
1145,79
1697,09
927,2
45
779,95
1101,2
1069,5
40
1348,6
40
716,56
35
35
1112,6
1165,8
1151,8
939,6
1686,7
727,3
32,3
30,9
4000,0
740,5
3600
3200
2800
2400
2000
1800
cm-1
1600
1400
1200
1000
800
650,0
BRADENAFIL
4000,0
3600
3200
2800
2400
2000
1800
cm-1
1600
1400
1200
1000
800
650,0
MORDENAFIL
By IR spectra the substance can be identified by the shift of some peculiar bands, like the one at 3300
cm-1, 3000 cm-1, 1700 cm-1. However detecting these shifts is much more difficult when drug products,
that contain also excipients, are analyzed instead of pure substances.
3.5.4 Detection of analogue molecules in a pharmaceutical product by solid state analyses
Some in house reformulated mixtures containing one analogue and some excipients were prepared to
test the ability of the solid state techniques in detecting which analogue was present.
REFORMULATED N°1: this mixture contains bradenafil, cellulose, magnesium stearate, emcompress
(bihydrate calcium hydrogen phosphate), chalk (often detected as component of counterfeit drugs).
1599,55
1389,42
3285,71
1244,71
1197,13
1460,79
1696,69
1333,92
814,53
778,85
927,53
%T
1599,55
3285,71
1389,42
814,53
1197,13
1460,79
778,85
1696,69
1244,71
927,53
1333,92
4000,0
3600
3200
2800
2400
2000
1800
cm-1
1600
1400
1200
1000
800
650,0
Fig. 97 : Comparison between the IR spectrum of the Reformulated N°1 (black line) and that of bradenafil (red line).
Some peculiar peaks of bradenafil, circled in blue, are visible in the spectrum of the Reformulated sample.
In the IR spectrum of the mixture some peculiar bands from bradenafil are detected (see figure 97), thus
IR seems to be able to provide some information for the detection of this analogue in a mixture.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Fig. 98 : On the left: DSC thermogram of the Reformulated N°1; on the right: DSC thermogram of bradenafil.
From the comparison between the reformulated 1 (see figure 98, on the left) and bradenafil (see figure
98, on the right) it results that also DSC is able to detect this analogue in a mixture. In spite of the
broadened endothermal signals of water loss due to the presence both of chalk and emcompress, the
two signals of bradenafil are still evident.
Fig. 99 : Comparison between the TGA thermogram of the Reformulated N°1 (red line) and that of bradenafil (black
line). The dotted lines report the TGA results, the solid lines the TGA derivatives.
Also the TGA can stress the presence of bradenafil (see figure 99): the reformulated mixture
thermogram shows a shoulder near to the decomposition signal of the cellulose, that corresponds to the
decomposition of bradenafil.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
REFORMULATED N°2 : dimethylsildenafil, cellulose, lactose, magnesium stearate, chalk.
1492,51
2964,28
1456,82
1688,76
1246,69
3309,52
802,64
929,51
766,96
%T
1014,75
2964,28
1492,51
3309,52
1456,82
766,96
1688,76
1246,69
1014,75
802,64
929,51
4000,0
3600
3200
2800
2400
2000
1800
cm-1
1600
1400
1200
1000
800
650,0
Fig. 100 : Comparison between the IR spectrum of the Reformulated N°2 (red line) and that of dimethylsildenafil
(black line). Some peculiar peaks of dimethylsildenafil, circled in blue, are visible in the spectrum of the
Reformulated sample.
From comparison between the IR spectrum of the reformulated mixture and that of dimethylsildenafil
significant common bands can be detected (see figure 100).
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Fig. 101 : On the top: DSC thermogram of the Reformulated N°2; below, on the left: DSC thermogram of
dimethylsildenafil; below, on the right: DSC thermogram of homosildenafil.
Unlike what was observed for the reformulated 1, in this case the DSC is not able to univocally identify
which of the examined analogues is present in the mixture because both dimethylsildenafil and
homosildenafil have a melting point at about 200°C (see figure 101).
Fig. 102 : Comparison between the TGA thermogram of the Reformulated N°2 (red line) and that of
dimethylsildenafil (black line). The dotted lines report the TGA results, the solid lines the TGA derivatives.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Neither the TGA analysis is very informative because the decomposition signal of dimethylsildenafil is
covered by that of the cellulose (see figure 102).
REFORMULATED N°3 : mordenafil, cellulose, lactose, magnesium stearate, chalk.
820,48
1248,67
1347,79
1686,78
939,42
1165,41
%T
1111,89
820,48
939,42
1248,67
1686,78
1111,89
1347,79
4000,0
3600
3200
2800
2400
2000
1800
cm-1
1600
1400
1165,41
1200
1000
800
650,0
Fig. 103 : Comparison between the IR spectrum of the Reformulated N°3 (black line) and that of mordenafil (blue
line). Some peculiar peaks of mordenafil, circled in red, are visible in the spectrum of the Reformulated sample.
In the IR spectrum of the reformulated mixture peculiar bands from mordenafil can be detected (see
figure 103).
Fig. 104 : On the left: DSC thermogram of the Reformulated N°3; on the right: DSC thermogram of mordenafil.
The DSC in this case is able to detect which analogue is present because mordenafil has a peculiar
melting point, at 184°C (see figure 104). The melting peak of mordenafil remains clear even inside the
mixture.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Fig. 105 : Comparison between the TGA thermogram of the Reformulated N°3 (red line) and that of mordenafil
(black line). The dotted lines report the TGA results, the solid lines the TGA derivatives.
Also the TGA can slightly notice the presence of an analogue in the mixture, through the shoulder on the
decomposition signal of the cellulose, that corresponds to decomposition of mordenafil (see figure 105).
In conclusion it results that the solid state techniques, IR, DSC and TGA, can help in detecting analogues
in a pharmaceutical product but they should be supported by more complex techniques to support the
results. Moreover, to employ these solid state techniques, reference standards of the analogue
molecules are needed, while often they are not available.
Furthermore the analogues are mostly employed in herbal products and food dietary supplements
where the matrix is usually too complex to allow an analysis by solid state techniques.
In summary, the new danger arisen from the use of the analogues is being faced with two different
strategy. On one hand as many analogues as possible are being synthesized to have reference standards
necessary for the application of some analytical techniques; on the other hand new methods, able to
detect the presence of these substances also without having the reference standards, are being
developed. One of these methods employ the Nuclear Magnetic Resonance (NMR).
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
3.6 NMR
NMR can be very useful for the analysis of unknown active substances and in general of illegal
and counterfeit drugs. The nuclear magnetic resonance is an expensive and complex equipment, hence
it can be used only by experts but it can provide very valuable information.
During this PhD work NMR was used for different purposes:
1- During the development of the solid state approach, by IR, DSC and TGA, NMR was used as
reference method. The information obtained by these solid state techniques on counterfeit drugs
were verified by a qualitative NMR analysis. To be analyzed by NMR, a sample must be dissolved in a
deuterated solvent; hence a suspicious drug was first extracted by methanol and, after the
evaporation of the solvent under nitrogen stream, the solute was re-dissolved in deuterated
methanol (MeOD) and analyzed by NMR. Of course only the methanol soluble ingredients could be
detected. Actually many excipients are totally insoluble in any solvent; however some other, like
lactose, magnesium stearate, sodium dodecyl sulfate, mannitol, sucrose, hydroxypropylcellulose are
soluble in methanol, thus their presence can be detected by NMR, researching in the spectrum of a
suspicious sample the peaks from these excipients. Furthermore many active substances, in
particular Sildenafil, Vardenafil and Tadalafil, are soluble in methanol, hence they are detectable by
NMR. The NMR spectra of these active substances are now reported:
Sildenafil citrate in MeOD
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Tadalafil in MeOD
Vardenafil in MeOD
2- NMR can provide very precious information on the chemical structure of unknown substances, for
instance illegal active substances present in some herbal products and food dietary supplements.
Before the NMR analysis it is necessary to properly treat the sample; to avoid interferences and
NMR peaks overlapping, indeed, the methanolic extract shouldn’t be directly analyzed since the
matrix of these samples is very complex; preliminary the extract should be purified by
chromatography isolating the suspicious ingredient in the fraction of interest, usually by a
semipreparative C18 column. After that, the solvent is removed by nitrogen stream, avoiding to heat
the sample because it could induce degradation of molecules; then the solute is dissolved in a
deuterated solvent, like MeOD or DMSO d6, and finally analyzed by NMR.
Even the simple proton spectrum can provide valuable information about the chemical structure of
an unknown substance. Even very similar molecules, like the analogues, show different NMR
spectra. As an example the spectra of some sildenafil analogues are reported:
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Acetildenafil in deuterated DMSO
Hydroxyacetildenafil in deuterated DMSO
Piperidino Sildenafil in deuterated DMSO
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Thiosildenafil in DMSO in deuterated DMSO
If a reference standard is available, the monodimensional spectrum of protons can be sufficient to
univocally identify the investigated substance.
If on the contrary a reference standard for comparison is not available, more complex NMR
experiments can be performed, as NMR of C13 and bidimensional experiments, like COSY
(Correlation Spectroscopy, which highlights the correlation among coupled protons) and
heterocorrelated experiments, which allow to trace which protons and carbons are bonded. In this
way more information about the chemical structure of the unknown molecule can be obtained.
An approach which uses both NMR and HPLC-MS2 is expensive and complex but allows to
investigate in a reliable way about the possible presence of illegal unknown active substances in
very complex samples like herbal products and food dietary supplements.
3- NMR can also provide quantitative determinations. NMR is an absolute technique since in the same
spectrum the area of each peak, also arising from different components of a mixture, only depends
on the number of nuclei that generate that peak.
For this reason, using an internal standard it is possible to quantify a substance without the need of
building a calibration curve and even without having its reference standard.
𝑛𝑥 𝐼𝑥 𝑁𝑥
= ×
𝑛𝑆 𝐼𝑆 𝑁𝑆
𝑛
The molar ratio between the investigated substance and the internal standard, 𝑥 , can be obtained
𝑛𝑠
if the area of one peak of the substance, 𝐼𝑥 , the number of protons in the molecule whic produces
that peak, 𝑁𝑥 , the area of one peak of the internal standard, 𝐼𝑆 , and the number of protons in the
internal standard molecule which produces it, 𝑁𝑆 , are known.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
3.6.1 Validation of a quantitative NMR method for the analysis of counterfeit drugs
The capability of this application is very remarkable and during this PhD work this technique was applied
to the analysis of counterfeit drugs.
On one hand it can provide an alternative method to HPLC for quantitative determinations of the API in
suspicious drugs. The HPLC analysis requires quite long time as the column must be conditioned and
large solvents consumption.
Furthermore, as the number of suspicious samples is increasing more and more, it can happen that all
HPLCs in a laboratory are engaged. Hence if an NMR instrument is available it can be employed to
quantify the API in place of HPLCs. The equipment of NMR is much more expensive than that of HPLC;
however it is faster and a very small amount of solvent is necessary (even if deuterated).
On the other hand the most interesting capability of NMR as quantitative method is that a reference
standard of the investigated substances is not necessary, but just a generic internal standard. For this
reason the quantitative NMR could be the only way to quantify new active substances, like analogues,
whose reference standard are not available. Indeed, if an analogue is identified, for example by HPLCMS2, it can be quantified by NMR even without having a reference standard of this new molecule.
Furthermore quantitative NMR is valuable to quantify active substances that are not visible in UV, hence
not be analyzable by HPLC-DAD.
To employ quantitative NMR for investigation on counterfeit drugs, a quantitative NMR method for the
analysis of Sildenafil Citrate was validated following the ICH guideline “Note For Guidance On Validation
Of Analytical Procedures: Text And Methodology”[58].
The following parameters were evaluated:
 Specificity
 Linearity - Range
 Trueness
 Precision: repeatability and intermediate precision
 Detection Limit (LOD)
 Quantitation Limit (LOQ)
 Ruggedness
As NMR is an absolute technique, this validation is still effective for quantitative determination even of
other substances, after ascertaining that some critical conditions are respected. In particular the NMR
signals employed for quantification should be free from interferences, both the one of the internal
standard and that of the investigated substance; furthermore they should be freely soluble in the
chosen deuterated solvent.
3.6.1.1 Materials and method for validation
An NMR Bruker 400 MHz instrument was employed for the method validation.
The working standard was a raw material Sildenafil citrate that had been previously analyzed by HPLC
for comparison to a Sildenafil reference standard.
The quantitative determination of Sildenafil citrate in the samples was carried out comparing it to an
internal standard.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Three different substances were tested as internal standards:
1. Acyclovir
2. Maleic acid
3. Di-nitro Benzoic acid
The NMR signals of the internal standard should not interfere with the signals of the investigated
substance. Indeed if these signals overlapped, their integrals would be distorted.
To employ NMR for quantitative determination the necessary condition is that at least one signal of
both the investigated substance and the internal standard is free from interferences, thus usable for the
quantification. Interferences can arise from the other soluble substances present in the drug product,
but the investigated substance and the internal standard could also interfere each other. A good
internal standard gives just few NMR signals, in the best cases just one, preferably in a region of the
spectrum where usually there are no signals.
The solvent employed for validation was DMSO d6, as many active substances are freely soluble in it.
Furthermore DMSO d6 is very viscous, hence the operative condition for a uniform magnetic field were
easily set. In addition increasing the temperature of the NMR probe, signals in DMSO d6 get very sharp
thus interference problems decrease. For this reason the temperature of the probe was set at 30°C, that
is high enough to have sharp NMR signals without deteriorating the molecules.
Since deuterated solvents are very expensive, the samples for validation were prepared in small
volumes, i.e. 1-2ml. As a consequence the measure of the volume was particularly critical. In addition it
was necessary to weigh very small amounts of powder to obtain samples with the appropriate
concentration and this procedure introduces a significant error.
The samples for validation were prepared weighing both the raw material sildenafil citrate and the
internal standard in a 1 or 2 ml volumetric flask.
In addition recovery tests were performed on “in house” reformulated mixtures of sildenafil citrate and
excipients to simulate the analysis of drug products.
Through the following formula, from the area of the sildenafil signal respect to that of the internal
standard signal, considering the protons number of each signal, the concentration of the internal
standard normalized for molecular weights (as concentrations are expressed as mg/ml, not in molarity)
it is possible to obtain the concentration of sildenafil citrate. Comparing this concentration to the actual
concentration of sildenafil citrate, evaluating the titre of this working standard by HPLC analysis, the
recovery the NMR method is obtained [59].
𝐶𝑆𝑖𝑙𝑑𝑒𝑛𝑎𝑓𝑖𝑙 =
𝐴𝑆𝑖𝑙𝑑𝑒𝑛𝑎𝑓𝑖𝑙 𝑁𝑆𝑖𝑙𝑑𝑒𝑛𝑎𝑓𝑖𝑙 𝑀𝑊𝑆𝑖𝑙𝑑𝑒𝑛𝑎𝑓𝑖𝑙
∙
∙
∙𝐶
∙ 𝑇%𝑅𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒
𝐴𝑅𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒 𝑁𝑅𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒 𝑀𝑊𝑅𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒 𝑅𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒
𝑅𝑒𝑐𝑜𝑣𝑒𝑟𝑦% =
𝐶𝑆𝑖𝑙𝑑𝑒𝑛𝑎𝑓𝑖𝑙
𝐶𝐴𝑐𝑡𝑢𝑎𝑙 ∙ 𝑇%𝑆𝑖𝑙𝑑𝑒𝑛𝑎𝑓𝑖𝑙
Where:
A= area of NMR signals considered for quantitative determination
MW = Molecular weight (in g/mol)
C = Concentration (in mg/ml or g/l)
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
N = Number of protons of the chosen signal (N=3 for the 1.3ppm signal of Sildenafil, N=2 for the 5,5ppm
signal of Acyclovir, N=2 for the 6ppm signal of Maleic Acid, N=3 for 9 ppm signal of Di-nitro benzoic acid)
T%Reference = titre expressed as a percentage of purity of the internal standard
T%Sildenafil = titre expressed as a percentage of purity of the working standard by HPLC-DAD analysis (for
comparison to a certified standard)
CSildenafil = the calculated concentration of Sildenafil expressed as mg/ml
C Actual = the actual concentration of Sildenafil expressed in mg/ml
3.6.1.2 Instrumental parameters
During the validation different sets of operative conditions were tested to optimize both trueness and
precision and develop a method as reliable as a HPLC one.
Some instrumental parameters were set with particular care as they affect the signal areas [60-61-6263-64]. In particular:
 Both the 30° and the 90° impulse were tested and finally the 30° was chosen. Indeed a 30° impulse
provides more accurate results. Also from literature it results the use of a 30° impulse for quantitative
NMR.
The duration of a 30° impulse is evaluated with an experiments called ZG490 on the Brucker Avance
equipment. By this experiment for each sample the duration of a 90° impulse is calculated and from it,
dividing by 3, the duration of a 30° impulse is obtained. A ZG490 experiment applies four times a 90°
impulse thus, when the correct value of duration of the 90°impulse is set, the NMR spectrum
disappears.
The duration of the 30° impulse resulted 3.08 µs for all the samples.
The energy of this impulse is fixed at 2dB.
 The Relaxation time, T1, is a very critical parameter for quantitative NMR analyses. T1 is the time
employed by the z component of magnetization to return at the 63% of its equilibrium value. If a too
short T1 is set, the integrals values of the NMR signals will be not correct.
The relaxation delay (called D1 on the Brucker Avance equipment), that is the delay between an
impulse and the following one, is calculated from T1. In particular D1 is usually set as 5 times T1 [BP].
Using acyclovir as internal standard D1 was 8s. With maleic acid it was 15s; with di-nitro benzoic acid it
was 35s.
 The spectral width was 22 ppm. Indeed it is necessary that all the signals of interest are in the 80% of
the central region of the spectrum. Furthermore the center of the spectrum (O1P) should not fall in
correspondence of significant signals because they could be distorted.
𝑠
 The signal to noise ratio ( 𝑛 ) for an NMR quantitative determination should be over 150. The
signal/noise improves increasing the number of scans (as
𝑠
𝑛
= 𝑘√𝑎 , where k is a constant value and a
is the number of scans) and increasing the concentration of the samples.
Usually from 1 to 32 scans are necessary to obtain a good signal to noise ratio for samples in a
concentration range of 2-30 mg/ml of sildenafil citrate.
Also the magnetic field strength affects the signal/noise; e.g. working with a 700 MHz instrument, that
is much more sensitive than a 400 MHz one, fewer NMR scans are necessary.
 Each spectrum was acquired with 32 K points.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Every sample was analyzed three times and the results were averaged, to avoid possible instrumental
errors.
The processing parameters were:
 An exponential apodization function, with line broadening 0.3, was applied to improve the signal to
noise ratio, even though it produces a slight loss in resolution.
 The Zero filling was applied on processing files, to double the number of points.
 The baseline correction function of the software Mestre (Whittaker Smoother)nwas employed before
peaks integration.
3.6.1.3 Validation with Acyclovir
The first validation was performed using a certified acyclovir raw material as internal standard.
Acyclovir, that is a pharmaceutical active substance, was employed as internal standard because it has a
very simple NMR spectrum. For quantitative determinations the singlet signal of acyclovir at 5.5 ppm
was considered. For sildenafil instead the triplet at 1,3 ppm was employed. Both these signals are free
from interferences.
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Acyclovir in deuterated DMSO
Sildenafil Citrate in deuterated DMSO
Fig. 106 : NMR spectrum of Acyclovir and that of Sildenafil citrate in deuterated DMSO. The green arrows highlight
the signals of sildenafil and of acyclovir that are employed for quantitative determination.
TRUENESS
Samples were prepared as follows: 5 mg of internal standard acyclovir and 4mg, 5mg and 6mg of
sildenafil citrate (respectively for the 80%, 100%, 120%) were weighed in 1 ml flask and subsequently
made up to the mark with DMSO d6. All samples were prepared twice.
80% x 2 independent samples
100% x 2 independent samples
120% x 2 independent samples
Truness – Recovery%
100.7
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
PRECISION
Intermediate precision was evaluated by two operators operating in different days:
OPERATOR 1 DAY 1
6 independent samples
OPERATOR 2 DAY 2
6 independent samples
Samples were prepared as follows: about 5 mg of both internal standard acyclovir and sildenafil citrate
were weighed in 1ml flask and subsequently made up to the mark with DMSO d6.
The Grubbs and Dixon tests were applied to detect outliers and one data was rejected.
Repeatability for each operator was calculated as standard deviation of each set of six measures.
Intermediate precision was calculated as standard deviation of the 12 measures, by two operators in 2
different days.
Repeatability
0.8%
Intermediate precision
2.3%
Results are expressed as percentage of the theoretical amount of sildenafil present in the sample.
The reported repeatability is the standard deviation pooled between the two sets of 6 experiments.
Intermediate precision is the standard deviation calculated on the 12 experiments.
LINEARITY
The linearity of the method was tested in a concentration range of sildenafil between 10% and 200% of
the reference concentration (5 mg/ml), while the concentration of acyclovir is fixed at 5mg/ml.
Samples were prepared as usual, weighing acyclovir and sildenafil citrate in 1 ml flask. Only for the 10%
level a 2ml flask was employed to increase the amount of sildenafil to weight in order to avoid excessive
weight errors.
For each concentration level two independent samples were prepared and results were pooled.
12
caclculated concentration (mg/ml)
Linearity
10
8
6
4
y = 0.9984x + 0.03
R² = 0.9998
2
0
0
5
10
actual concentration (mg/ml)
Fig. 107 : Linearity of the method in the range 10% and 200%. The linearity results from the linear regression and
correlation coefficient. In the graphic the calculated sildenafil concentration, obtained from NMR, is reported versus
the actual concentration.
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LOD E LOQ CON ACICLOVIR
The limit of detection and the limit of quantitation are calculated from linearity data by the following
equations:
𝐿𝑂𝐷 =
3.3𝜎
𝑆
𝐿𝑂𝑄 =
10𝜎
𝑆
Where:
S is the slope of the regression line from Linearity studies.
σ is carried out from the residual standard deviation of the regression line.
LOD
LOQ
0.18 mg/ml
0.56 mg/ml
RECOVERY AND SELECTIVITY
The recovery of this method was tested on a reformulated sample containing sildenafil citrate. This
mixture was composed of mannitol, lactose, magnesium stearate, corn starch, croscarmellose,
microcrystalline cellulose and the active substance sildenafil citrate working standard.
In this way, in addition to the recovery, the selectivity of the method was tested. In particular it was
investigated if the presence of common excipients could interfere with the analysis by overlapping their
NMR signals with those from sildenafil.
After mixing the reformulated sample, an aliquot of the mixture was extracted by methanol. Two
different extraction procedures were tested on two aliquots of the mixture: the first consisted in two
subsequent extractions with a small volume of methanol, that was 5ml; the second in four subsequent
extractions of 5ml of methanol.
After the addition of methanol, the sample was magnetically stirred, put in ultrasonic bath, centrifuged
to separate the supernatant from the insoluble residue. While the supernatant, in which the active
substance is dissolved, was taken with a pipette and dried under nitrogen flux, the insoluble residue was
extracted again. All the supernatants from following extraction of the same aliquot of reformulated
were collected together, dried and then dissolved in 1 ml of DMSOd6 to be analyzed.
Recovery was about 100% for both the extraction procedures, thus it resulted that two following
extractions with 5ml of methanol were adequate for a quantitative determination of a sample.
Furthermore NMR spectrum highlighted that the excipients in the mixture, both those insoluble in
methanol and DMSO and the soluble ones, didn’t give any interferences problem. The NMR signals of
the excipients didn’t overlap the sildenafil triplet employed for quantification. Thus this method resulted
selective and usable for quantitative determination of sildenafil in counterfeit drugs, as an alternative to
HPLC.
3.6.1.4 Validation with Maleic acid
Since interesting results were obtained for the validation with acyclovir, a new validation was performed
employing a common internal standard for NMR, that is maleic acid. Maleic acid gives only one NMR
signal at about 6ppm.
The procedure for samples preparation was modified to improve the intermediate precision of the
method. Instead of weighing very small amounts (5mg) of both sildenafil and internal standard, only
sildenafil citrate was weighed in a small amount (10mg). On the contrary the internal standard was
introduced together with the solvent DMSOd6 to reduce the weight error.
In practice a stock solution was prepared weighing about 100mg of maleic acid in 50ml of DMSOd6.
Thus 1ml of this stock solution contained an exactly known amount of internal standard (about 2 mg) .
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PRECISION
OPERATOR 1 DAY 1
6 independent samples
OPERATOR 2 DAY 2
6 independent samples
Repeatability
Intermediate precision
1.8%
2.0%
Two operators in two days performed 6 analysis each for a total of 12 analysis. In every analysis the
operators weighed 10mg of Sildenafil Citrate and diluted it in 1ml of a 2mg/ml solution of Maleic acid in
deuterated DMSO. The same solution was employed for all the 12 experiments. Outliers were
determined by Dixon and Grubbs tests. One was detected.
TRUENESS AND LINEARITY
After the measures to determine precision, with the same stock solution additional samples were
prepared to obtain both trueness and linearity of the method. 12 independent samples were prepared
at the following concentration levels:
10% x 2 independent samples
40% x 2 independent samples
80% x 2 independent samples
120% x 2 independent samples
160% x 2 independent samples
200% x 2 independent samples
Each sample was prepared diluting 2 to 20mg of Sildenafil Citrate in 1 ml ( 2ml for the 10% point in
linearity experiments) of the stock solution.
Truness – Recovery%
96.5%
caclculated concentration (mg/ml)
Linearity
25
20
15
10
y = 1.0286x - 0.5355
R² = 0.9942
5
0
0
5
10
15
20
25
actual concentration (mg/ml)
Fig. 108 : Linearity of the method in the range 10% and 200%. The linearity results from the linear regression and
correlation coefficient. In the graphic the calculated sildenafil concentration, obtained from NMR, is reported versus
the actual concentration.
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While precision results, obtained with a stock solution freshly prepared, were quite good, these
trueness and linearity results are not satisfactory.
Modifying the preparation procedure of the samples, as over described, actually the trueness and
linearity of the method got worse. In particular the intercept of the regression line of linearity
highlighted as the method underestimated the amount of sildenafil in the sample.
This evidence can be explained by the following considerations.
Adding the internal standard together with the solvent DMSOd6, on one hand the error from weighing a
very small amount of internal standard is reduced, but on the other hand a bigger error from a very
tricky volume measure is introduced, as DMSO is very viscous and adheres to the flask’s wall. This error
on the measure of volume strongly affects the amount of internal standard in the sample, thus the
result of analysis. On the contrary, if both sildenafil and internal standard are directly weighed in the
volumetric flask, the error on the measure of volume affects very weakly the results of analysis as it is
not bounded to the amount of the internal standard.
For this reason additional measures to test the linearity of the method were performed preparing the
sample by weighing directly both sildenafil and the maleic acid, thus without employing the stock
solution. However results were still not accurate.
Further tests were performed doubling the amount of both Sildenafil and Maleic Acid for each
concentration level, because this problems could arise from weight errors. Also tests with a double
volume of DMSOd6 were conducted to evaluate if this low accuracy could arise from volume errors but
in all cases no improvements were obtained.
Finally the low accuracy was ascribed to an interference problem between the NMR signal of water
dissolved in DMSOd6 and the that of maleic acid.
It is known that DMSO is very hygroscopic thus it absorbs water. The Maleic Acid signal at 6ppm was
affected by a slight increase in area due to the tail of the strong signal of water, that in acidic solution is
broadened. This in turn caused a consistent inaccuracy in the quantitative determination of the
Sildenafil signal.
Actually this interference was not clearly evident, but since the signal of water is much bigger than that
of maleic acid, even a very small tail that falls beneath the internal standard signal alters its exact
integration.
Thus the problems arose from the DMSO tendency of adsorbing water from the ambient. As a matter of
fact the major problems were encountered when stock solution not freshly prepared anymore was
employed, even if stored in desiccators.
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Maleic acid
Tail of
water
signal
Fig. 109 : A particular of the NMR spectrum of Sildenafil citrate with maleic acid as internal standard. The tail of the
signal of dissolved water (highlighted by the red arrow) alters the peak area of the signal of maleic acid employed
for quantitative determination (green arrow), causing inaccurate results.
This evidence was confirmed by stability studies on the stock solution of maleic acid in DMSOd6. The
stability of the stock solution was tested employing another reference standard, that is Di-Nitro Benzoic
acid. After just 15 days the dosage of maleic acid turned from 100% to 104% of the theoretical value
because of the increased dissolved water.
As DMSO is so hygroscopic, it is expensive and quite difficult to totally avoid the presence of dissolved
water: it would be necessary to employ expensive disposable phial of anhydrous DMSO d6. For this
reason it was preferred to employ a different internal standard, less susceptible to interferences from
the water signal. In particular Di-Nitro Benzoic acid was employed.
3.6.1.5 Validation with Di-Nitro Benzoic Acid
Di-Nitro Benzoic acid has two NMR signals at about 9ppm, thus enough far away from the water signal
(at about 5ppm) to not be affected by its tail. Furthermore these signals at 9ppm don’t interfere with
those of Sildenafil, thus Di-Nitro Benzoic acid resulted suitable as internal standard.
To employ Di-Nitro Benzoic acid a concentration of 10mg/ml of internal standard can’t be exceeded as
at higher concentration level its solubilization in DMSO is not complete.
Based on such basis the validation of the method was repeated employing Di-Nitro Benzoic acid as
internal standard. No Stock Solution was prepared, instead for each sample both sildenafil citrate and
Di-Nitro Benzoic acid were weighed and diluted with DMSO d6 in a 2ml volumetric flask.
The amounts of sildenafil and Di-Nitro Benzoic acid were such to have a comparable molar
concentration for both of them. Indeed in this way the error arisen from a possible inaccurate measure
of volume would minimize. Furthermore the integration of NMR peaks of similar area is easier.
To minimize errors due to weighing very small amounts of powder, the 100% concentration level was
raised to 40mg of Sildenafil (the volume of DMSO was set at 2ml). Approximately 14mg of Di-Nitro
Benzoic acid were weighed for each sample.
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PRECISION
OPERATOR 1 DAY 1
3 independent samples
OPERATOR 2 DAY 2
3 independent samples
OPERATOR 3 DAY 3
3 independent samples
Intermediate precision was evaluated on 9 measures of samples on 100% concentration level by 3
different operators in 3 different days.
OPERATOR 1 DAY 1
6 independent samples
Repeatability was calculated on 6 measures of samples on 100% concentration level by the same
operator in the same day.
Repeatability
0.26%
Intermediate precision
0.40%
This intermediate precision is very satisfactory as it is quite better than that commonly obtained from
HPLC. As a matter of fact reproducibility, that is generally quite similar to intermediate precision, of a
generic HPLC method is about 2% for pure substances (according to the Horwitz equation).
TRUENESS AND LINEARITY
With the following experimental design, trueness and linearity were determined.
OPERATOR 1 DAY 1
10% - 40% - 80% - 100% - 120% - 160% - 200%
OPERATOR 2 DAY 2
10% - 40% - 80% - 100% - 120% - 160% - 200%
Two operators performed 7 analyses each in two different days. Thus 14 samples were analyzed, 2 for
each concentration level from 10% to 200%.
Truness – Recovery%
99.35%
The gap between the obtained recovery value, 99.35%, and the theoretical value of 100% didn’t result
significant by the t-student test considering the intermediate precision values of the method.
For the linearity curve the average values from 2 samples of the same concentration level were
employed for each point.
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45
Linearity
calculated concentration (mg/2ml)
40
35
30
25
y = 0,9883x + 0,0277
R² = 0,9999
20
15
10
5
0
0
10
20
30
40
actual concentration (mg/2ml)
Fig. 110 : Linearity of the method in the range 10% and 200%. The linearity results from the linear regression and
correlation coefficient. In the graphic the calculated sildenafil concentration, obtained from NMR, is reported versus
the actual concentration.
The linearity in the range 10-200% is excellent. The correlation coefficient of the regression line is
R2=0.9999, the angular coefficient is near to 1 and the intercept near to zero.
LOD E LOQ
LOD and LOQ values were obtained from the linearity regression line as over described about the
validation with acyclovir (see 3.6.1.3).
LOD
LOQ
0.29 mg/ml
0.88 mg/ml
Thus these are detection and quantitation limits for the analysis by NMR. However it must be
considered that a further limitation arises from the difficulty of weighing very little amount of powder.
Thus it is not possible to measure accurately very low concentrations unless a bigger volume of DMSO
d6 is employed.
3.6.1.6 Ruggedness
Experiments were carried out to investigate how a deliberate change in some of the analytical
parameters could affect the results of the analysis.
In particular 7 parameters were studied at two levels, as reported in the table. As suggested by the
Plackett-Burman experimental design, 8 experiments were performed [30].
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Table 8
PARAMETER :
Number of scans
Temperature NMR
Probe (°C)
Internal standard
quantity (mg)
Relaxation delay D1
(s)
Angle of impulse P1
(µs)
Software of
integration
Room Temperature
**(°C)
EXPERIMENT N°
3
4
1
2
5
6
7
8
4
4
4
4
1
1
1
1
30
30
25
25
30
30
25
25
14
7
14
7
14
7
14
7
40
40
20
20
20
20
40
40
4.62*
3.08
4.62
3.08
3.08
4.62
3.08
4.62
Top Spin
Mestre
Mestre
Top
Spin
Top
Spin
Mestre
Mestre
Top
Spin
30
25
25
30
25
30
30
25
*P1 4.62 µs corresponds to an impulse of 45° angle while 3.08 µs to a 30° angle.
** this is the temperature of the room where samples were prepared.
The investigated parameters were:
1. Number of NMR scans. It is related to signal to noise ratio. It was investigated if it is necessary to
extend the time of analysis increasing the number of scans to improve the signal/noise.
2. Temperature of NMR probe. At 25°C in DMSO NMR signals are quite large; on the contrary at 30° the
viscosity of DMSO decreases thus NMR peaks become much sharper.
3. The amount of internal standard (di-nitro benzoic acid). It permits to have an idea of how important
is to have equimolarity between sample and reference and how critical is to weigh less then 10mg.
4. Relaxation Delay. It was investigated how critical was to employ a correct delay between a scan and
the following one.
5. The angle of the impulse. While usually a 30° impulse is employed, also 45° impulse was tested to
investigate if an inaccurate measure of this angle could affect the analysis.
6. The software used to process data. Two different software were employed for integration: Top Spin,
which allows a very accurate integration of peaks; Mestre, that disposes of an effective algorithm for
baseline correction but provide a less precise integration.
7. Temperature of the room where samples were prepared. Some samples were prepared at 25°C,
some other at 30°C. The relative humidity was as low as possible in both cases (about 40%) to avoid
an excessive hydration of DMSO d6.
The effect of a parameter change was measured as a difference between the sum of the four Titre%
determined at the first level and the four Titre% determined at the second. For instance for Number of
scans:
𝐷𝑆/𝑁 =
𝑇%𝑒𝑥𝑝1 + 𝑇%𝑒𝑥𝑝2 + 𝑇%𝑒𝑥𝑝3 + 𝑇%𝑒𝑥𝑝4 𝑇%𝑒𝑥𝑝5 + 𝑇%𝑒𝑥𝑝6 + 𝑇%𝑒𝑥𝑝7 + 𝑇%𝑒𝑥𝑝8
−
4
4
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
To determine if this difference is statistically significant and in turn the method is or not robust toward
the change in that parameter, a t test was conducted comparing the tabulated t for the appropriate
degrees of freedom with the calculated one:
𝑡=
√𝑛 ∙ |𝐷𝑆/𝑁 |
√2 ∙ 𝑠
Where n is the number of experiments carried out at each level (n=4 for the design given) and s is the
repeatability of the method. The confidence level was set at 95% and the degrees of freedom were 5,
since 6 experiments were carried out to determine repeatability.
From the ruggedness study it resulted that only two of the examined parameters are critical for the
analysis: the number of scans and the Relaxation delay.
The number of scans determines the signal to noise ratio. With just one scan the signal/noise is low and
the spectrum is noisy. For this reason the integration is less accurate. Thus it is convenient to collect
more scans of a spectrum, even extending the time of analysis, until a signal/noise of 300 is obtained.
The Relaxation delay is another critical parameter. Indeed the spectrum acquired with a shorter D1 than
the correct one gave wrong results. As a matter of fact if this delay between a scan and the following
one is not long enough, the z component of magnetization of some protons cannot relax completely;
thus the next signal they emit is incomplete, with consequent errors for the integration.
3.6.1.7 Recovery experiments
Two different recovery experiments were performed to evaluate if the validated method can be applied
also on drug products.
One test was conducted with the method of addition; to a counterfeit drug, Filagra, that contained
100mg of Sildenafil (as verified by HPLC analysis), 20 mg of raw material Sildenafil citrate were added.
One aliquot of the sample was analyzed before the addition of the raw material, another one after the
addition. Each aliquot was extracted twice with 5 ml of methanol, the supernatant was dried under
nitrogen flux and subsequently dissolved in DMSO d6.
This test was performed employing maleic acid as internal standard, dissolved in the stock solution of
DMSO d6. This experiment was considered still valid because the stock solution of maleic acid was
freshly prepared and hydration problems had not yet arisen.
Recovery value for the addition of raw material sildenafil citrate was 96.5%, that is not significantly
different from 100% according to the Student test.
Another recovery test was performed with Di-Nitro Benzoic acid as internal standard on a reformulated
mixture. A mixture containing sildenafil citrate, mannitol, lactose, magnesium stearate, corn starch,
croscarmellose, microcrystalline cellulose, was prepared and an aliquot was extracted with methanol
and analyzed. It was verified that there were no interferences among the NMR signals of sildenafil, dinitro benzoic acid and the excipients. A recovery of 99.5% was obtained.
According to these results, the validated method has proved applicable to quantitative determination of
sildenafil citrate in counterfeit and illegal samples.
3.6.1.8 Method applicability
The developed method was validated for quantitative determination of sildenafil citrate employing dinitro benzoic acid as internal standard in deuterated DMSO. However the precision, trueness, linearity
data are still effective for quantitative determination even of other substances. As a matter of fact NMR
is an absolute technique and the area of all peaks in the same NMR spectrum depends only on the
number of nuclei which generated that signal.
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Nevertheless to apply this method and its precision data to other substances it is necessary to verify that
some critical conditions are respected, as highlighted during the validation.
First selectivity must be tested: the NMR signal of the internal standard and at least one of the signals of
the investigated active substance must be free from interferences. As a matter of fact peaks of internal
standard and of active substance may either overlap each other or be interfered by other substances
dissolved in the sample, like excipients, water, residual solvents, impurities.
Hence it is necessary to detect one NMR signal of the investigated substance that is free from
interferences. Selectivity can be tested comparing the spectrum of the analyzed sample (before adding
the internal standard) to that of the internal standard in order to verify if there are some overlapping
signals. In this way a suitable peak of the active substance can be detected, which falls far enough from
signals of internal standard and water.
To eliminate the suspect that in correspondence of the chosen signal of the active substance there is
some other signals (e.g. from excipients), a comparison among the integrals of all the signals from the
active substance can be informative.
If the internal standard interferes with the investigated substance, a different internal standard can be
employed for quantification by NMR. E.g. maleic acid can be employed, as it has only one signal at
6ppm, that is an area usually devoid of NMR signals. To use maleic acid the hydration of DMSOd6 should
be avoided as dissolved water can interfere; disposable phials can be employed.
On the other hand the investigated substance should be freely soluble in DMSO; actually this solvent
was chosen because most of pharmaceutical active substances are soluble in it. If, on the contrary, the
active substance is not enough soluble in DMSO, another deuterated solvent can be employ for a
quantitative determination by NMR, but in this case a new validation would be necessary.
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4.
CONCLUSIONS
The pharmaceutical counterfeiting is a rapidly expanding phenomenon even in our society and it
is a concrete threat for public health.
For this reason authorities are investing resources to protect people from this danger.
On one hand information campaigns are conducted to illustrate risky behavior and avoid people to run
into illegal drugs, more or less unconsciously.
On the other hand authorities are promoting a research work to develop new techniques and analytical
methods for counterfeit drugs detection.
During this PhD work new methods for the analysis of counterfeit drugs have been developed; some of
these methods are fast, simple and cheap, some other are expensive, time consuming but more
exhaustive.
As a matter of fact the authorities for public health protection can pose two different questions to
specialized laboratories:
 Is the examined drug counterfeit or not?
 What risks for human health does the examined counterfeit or illegal drug pose?
Actually it is usually quite simple to answer to the first question: often an accurate visual inspection can
provide useful information. This should be supported by simple and rapid instrumental techniques to
provide reliable and final results even when suspicious samples look very similar to the originals.
For this purpose during this PhD work the Colorimeter method was developed. Through an accurate
measure of the colour of tablets and secondary packaging it can point out if the examined sample is
counterfeit or not. A comparison between the reflectance spectra, in the visible wavelengths, of the
suspicious sample and that of a data bank of original drugs is made, considering both the method
imprecision and the normal variability among originals, that had been determined by a statistical
analysis.
This method was applied on a certain number of suspicious drugs and resulted promising. Furthermore
it requires a portable equipment; thus it can be employed directly in places where there is a great
circulation of suspicious samples, like customs, and even by untrained personnel, as it is very simple.
Another rapid instrumental method developed was a solid state approach which employs IR, DSC and
TGA in combination. It allows to detect if a suspicious sample is original or counterfeit. Moreover, after
building data banks with the possible ingredients of a drug, it can provide information about the
composition of an unknown sample. In particular with IR the characteristic bands of each components
are searched; with DSC, melting peaks and water loss phenomena are detected, with TGA the
decomposition profile of components arising from a heating program.
This method was applied to the analysis of suspicious samples and provided a very remarkable
contribution to identify counterfeit drugs and to obtain information about its composition, also
detecting inappropriate and dangerous ingredients like chalk.
On the contrary the second question requires a more in depth investigation. To discover the real risk
arising from the use of illegal medicines it is necessary to employ several techniques, as each one
provides its own contribution. Usually the more exhaustive the method, the more expensive it is.
On this issue, during this work the dissolution test was employed, to investigate the risks arising from an
inappropriate release of the active substance in counterfeit and illegal drugs.
A discriminating dissolution method to obtain the release profile of Sildenafil citrate (the API of Viagra
Pfizer) from illegal and counterfeit tablets was developed. This method highlighted differences between
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
original and illegal drugs providing valuable information about formulation and technology of
production.
To fight a new great danger arising from the use of the analogues, that are unknown active substance
very similar to approved API, a HPLC-MS2 method is currently under development. These unknown
active substances are employed above all in fake herbal products and food dietary supplements, hence
the sample matrix is very complex. This advanced equipment is necessary to separate and univocally
identify these dangerous molecules, distinguishing even among very similar structures. In addition, as
the analogues are often new molecules, reference standards for comparison are not available.
Also NMR technique was employed for counterfeit drugs investigation. In particular a quantitative NMR
method was validated for determination of Sildenafil citrate content in drugs products by comparison
with an internal standard, as alternative to HPLC analysis. Since NMR is an absolute technique, this
method can be employed in the same way for quantitative determination of any other active
substances, soluble in the used solvent (DMSO d6), with at least one NMR signal free from interferences
(neither form the internal standard, nor from excipients). For this reason this method is particularly
important for quantitative determination of active substance not detectable by HPLC-DAD (because they
don’t absorb ultraviolet wavelengths) and above all active substances whose reference standard is not
available, like analogues.
In conclusion, from the study of the complex phenomenon of pharmaceutical counterfeiting it resulted
that a research work to follow its evolutions and new way of circulation is needed. As the matter of fact,
beside having advanced techniques and various analytical methods, it is necessary to know which kind
of products to examine and what kind of dangerous components to search.
Thus it results that a specialized laboratory should operate at two different levels: on one hand it has to
rapidly screen the increasing suspicious samples, to detect counterfeit drugs, allow the seizure of these
products and legal prosecutions. On the other hand it has to perform more in depth analyses on some
carefully selected samples, to evaluate the real risks and the further evolution of this phenomenon, also
to update the data for information campaigns.
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5.
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Durante questo dottorato sono state prodotte le seguenti pubblicazioni:
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


Rodomonte AL, Gaudiano MC, Antoniella E, Lucente D, Crusco V, Bartolomei M, Bertocchi P, Manna
L, Valvo L, Alhaique F, Muleri N., Counterfeit drugs detection by measurement of tablets and
secondary packaging colour. J Pharm Biomed Anal. 2010 Oct 10;53(2):215-20. Epub 2010.
Gaudiano MC, Lucente D, Antoniella E, Bertocchi P, Muleri N, Manna L, Bartolomei M, Alimonti S,
Valvo L, Rodomonte AL, “For export only" medicines come back to Europe: a RP-LC method for the
screening of six glucocorticoids in illegal and counterfeit anti-inflammatory and lightening creams. J
Pharm Biomed Anal. 2010 Oct 10;53(2):158-164. Epub 2010 May 6.
Chapter “Thermal analysis in the study of counterfeit medicines” del libro “Identification and
Analysis of Counterfeit and Substandard Pharmaceuticals” Edited by Perry G. Wang, Albert I.
Wertheimer. ILM Publications.
M.C. Gaudiano, L. Manna, P. Bertocchi, M. Bartolomei, A.L. Rodomonte, E.Antoniella, S. Alimonti, L.
Romanini, L. Rufini, N. Muleri, B. Gallinella, M.Mirra, S. Lucattini, M. Di Gregorio, L. Fucili, L. Valvo,
Lotta alla contraffazione farmaceutica: le attività dell’Istituto Superiore di Sanità - Rapporti ISTISAN
10/20. Istituto Superiore di Sanità 2010.
Il poster “Pharmaceutical counterfeit and illegal generic drugs by solid state analysis” è stato presentato
all’International Meeting “Recent Developments in Pharmaceutical Analysis” (Milano 9-12, Settembre
2009)
Partecipazione alle attività del dottorato, corsi e seminari:
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
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Advanced Analytical Methologies in drug preformulation/formulation and Quality control (Rimini
Campus branch 13-16 Giugno 2010)
"Il processo di Drug Discovery nell'industria farmaceutica" Dr. Daniele Donati, (Nerviano Medical
Sciences), 10 Giugno 2010
“Best Practicies to combact counterfeiting medicines and protect public health”(Roma, Comando dei
carabinieri per la Tutela della Salute, 19-20 maggio 2010)
“Tecnologia Innovativa che rivoluzionerà la Cromatografia liquida” Phenomenex (24 Settembre
2009)
“Fluorescent ion probes as tools for the detection of intracellular ions” Prof. Katerinopoulos (15
Settembre 2009)
“Soluzioni Waters dedicate alla Bioanalisi” (18 Giugno 2009)
“Sintesi “Diversity Oriented” di Composti Naturali e di Composti ad Attività Antitumorale” Prof.
Passarella (7 Maggio 2009)
Partecipazione a congressi:

Recent Developments in Pharmaceutical Analysis (Milano 9-12 Settembre 2009)
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Development of analytical methods for counterfeit drugs investigation – Nadia Muleri
Ringraziamenti
Desidero ringraziare tutte le persone che mi hanno permesso di vivere quest’esperienza, per me tanto
bella e formativa.
Ringrazio l’Agenzia delle Dogane per aver reso possibile lo svolgimento di questo Dottorato di Ricerca,
nella speranza che la formazione acquisita possa risultare un vantaggioso investimento.
Ringrazio la Dr. Valvo e il Prof. Alhaique, per avermi accolto nel loro gruppo di ricerca, e avermi seguito
in questi 3 anni con tanta cura e costanza.
Ringrazio di cuore tutto il gruppo del reparto “Qualità dei farmaci di origine chimica - Unità
anticontraffazione” dell’Istituto Superiore di Sanità: Andrea Luca Rodomonte, Paola Bertocchi, Livia
Manna, Monica Bartolomei, Eleonora Antoniella, Maria Cristina Gaudiano, Laura Romanini, Leandro
Rufini, Stefano Alimonti, Gabriele Civitelli. Mi hanno aiutato a crescere professionalmente e
personalmente, e mi hanno fatto sempre sentire accolta. Grazie per tutti i momenti di lavoro e di
allegria che abbiamo vissuto insieme!
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