1-3
SPECIFIC QUALITY PROBLEMS
WITH ANTIBIOTICS AND ANTITUBERCULOTICS
Pr A. NICOLAS, PhD
Head of the French Laboratories and Controls Directorate
AFSSAPS
Interregional Seminar for Quality Control Laboratories involved in WHO Prequalification Programme and/or participating in
respective sampling and testing projects, Nairobi, Kenya, 23-25 September 2009
ANTIBIOTICS AND
ANTITUBERCULOTICS
 Classification
 Origin
 Drug quality reports
 Fermentation products
 Impact on the composition of a monograph
 Antituberculosis dosage forms
 Conclusion
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
ANTIBIOTICS - CLASSIFICATION
 Very diverse class of compounds
 Generally grouped according chemical structures
– Aminoglysides
– Cephalosporins and related beta lactams
– Glycopeptides
– Macrolides
– Penicillins
– Quinolones
– Sulphonamides and diaminopyrimidines
– Tetracyclines
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
ANTITUBERCULOSIS - CLASSIFICATION
 Miscellaneous group of antibacterials
 Include
– Cycloserine
– Ethambutol
– Isoniazid
– Pyrazinamide
– Rifampicin
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
WHO List of Prequalified Medicinal
Products
 Prequalified antituberculosis products (june 2009)
– Cycloserine
– Ethambutol
–
–
–
–
Isoniazid
Ethionamide
Pyrazinamide
Ethambutol + Isoniazid
– Ethambutol + Isoniazid + Rifampicin
– Ethambutol + Isoniazid + Pyrazinamide + Rifampicin
– Isoniazid + Rifampicin
– Isoniazid + Pyrazinamide + Rifampicin
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
Drug Quality Reports Affecting
USAID-assisted Countries (1)
 USP Drug Quality and Information Program
– Updated june 2009
– Repository of publicly-available information to raise awereness
of the problem of counterfeit and substandard medicines
– More often cited drugs : antimalarials, antiretrovirals
– But also : antibiotics, antituberculosis products
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
Drug Quality Reports Affecting
USAID-assisted Countries (2)
 Ghana
– 13/08/2004 Counterfaiting of Ampicillin (root crop cassava)
 Nigeria
– 29/05/2008
• Augmentin (amoxicillin/clavulanic acid)
• Ampicillin
– 15/05/2009
• Amoxicillin, ciprofloxacin,
erythromycin
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ampicillin,
Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
cloxacillin,
Drug Quality Reports Affecting
USAID-assisted Countries (3)
 Sierra Leone
– 17/06 2008 : chloraphecol, tetracycline
– 9/06/2008 : amoxicillin, ampicillin
 South Africa
– 27/05/2009
• Antib-4 (pyrazinamide, ethambutol, isoniazid,
rifampicin)
• Ebsar (isoniazid + rifampicin)
 Tanzania
– 5/10/2008 : antibiotics
 Uganda
– 1/10/2005 : cloxacillin
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
ORIGIN
 These active substances could be obtained by :
– synthetic route
– semi-synthetic route derived from fermentation product by
process involving at least cleavage and/or formation of covalent
bonds followed by extraction/purification steps
– fermentation
 The origin impacts the purity profile, choice of the analytical methods
and their performance
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
REFERENCES : ICH and Ph. Eur.
 Guideline ICH Q3A(R2) « Impurity in New Drug Substances »
 Ph. Eur. Chapter 5.10 «Control of Impurities in substances for
pharmaceutical use»
 Ph. Eur. General monograph (2034) «Substances for pharmaceutical
use» do not cover « fermentation products and semi-synthetic
products derived therefrom » for application of reporting,
identification and qualification thresholds
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
THRESHOLDS FOR ORGANIC IMPURITIES
IN ACTIVE SUBSTANCES (General case)
Maximum
Reporting
Identification Qualification
daily dose
threshold
threshold
threshold
Human use or
human and
veterinary use
≤ 2 g/day
> 0.05 per cent
> 0.10 per cent
> 0.15 per cent
Human use or
human and
veterinary use
> 2 g/day
> 0.03 per cent
> 0.05 per cent
> 0.05 per cent
Veterinary use
Not applicable > 0.10 per cent
only
> 0.20 per cent
> 0.50 per cent
Use
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
PRODUCTS OF FERMENTATION (1)
 Relevant monograph from Ph. Eur. « Products of
fermentation (1468) »
This monograph applies to :
• products obtained by semi-synthesis from a product of
fermentation and those obtained by biocatalytic transformation
• whole broth concentrates or raw fermentation products
A complex class of compounds with many analytical challenges
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
PRODUCTS OF FERMENTATION (2)
 Fermentation processes are biosynthetic pathways consisting of
parallel reactions
– Some antibiotics are mixtures of structurally related substances
– Not always clear which of these substances contribute to activity
(active or impurity ?)
– Separation not always achievable
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
PRODUCTS OF FERMENTATION (3)
 Biosynthetic pathways difficult to control (variation from batch to
batch)
– Different strains of same species may lead to different
impurity profiles
– Large differences in media components in fermentation may
lead to different impurity profiles
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MONOGRAPH – DEFINITION (1)
 Substances obtained by synthetic or semi-synthetic route
correspond to a major known compound
 Substances obtained by fermentation are often but not always
defined as mixtures
 The name of the strain is indicated
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MONOGRAPH – DEFINITION (2)
 Synthetic : ciprofloxacin
1-Cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4dihydroquinoline-3-carboxylic acid.
 Semisynthetic : rifampicin
2S,12Z,14E,16S,17S,18R,19R,20R,21S,22R,23S,24E)5,6,9,17,19-Pentahydroxy-23-methoxy-2,4,12,16,18,20,22heptamethyl-8-[[(4-methylpiperazin-1-yl)imino]methyl]-1,11-dioxo1,2-dihydro-2,7-(epoxypentadeca[1,11,13]trienimino)naphto[2,1b]furan-21-yl acetate
Semisynthetic antibiotic obtained from rifamycin SV.
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
MONOGRAPH – DEFINITION (3)
 Fermentation leading to a known major compound : josamycin
Josamycin is a macrolide antibiotic produced by certain strains of
Streptomyces narbonensis var. josamyceticus var. nova, or
obtained by any other means. The main component is……
 Fermentation leading to a mixture : gentamicin
Mixture of the sulphates of antimicrobial substances produced by
Micromonospora purpurea, the main components being
gentamicins C1, C1a, C2, C2a and C2b.
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
MONOGRAPH - IDENTIFICATION
 Synthetic and semisynthetic
– Identification by IR spectroscopy (2.2.24)
 Mixtures obtained by fermentation
– IR if structures of the individual compounds are very similar :
erythromycin
– Identification by examination of the chromatograms obtained
in the test for composition
• Gentamicin : the chromatogram obtained with the test solution shows
5 principal peaks having the same retention times as the 5 principal
peaks in the chromatogram obtained with reference solution (gentamicin
sulphate CRS).
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
MONOGRAPH - TESTS
 Composition
– Used to specify complex mixture such as some fermented
polypeptides (tyrothricin) or aminoglycosides (gentamicin)
 Related substances
– ICH impurity limits/thresholds cannot be applied because of
wide variety in purity and complexity
– Not possible to establish one fixed set of thresholds for
identification and qualification
– Specifications should be derived on a case by case basis
considering batch results of various suppliers
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MONOGRAPH - TESTS
COMPOSITION
In the case of mixture a « composition test » is described
 Composition. Liquid chromatography (2.2.29): use the normalisation
procedure taking into account only the peaks due to gentamicins
C1, C1a, C2, C2a and C2b; use the chromatogram supplied with
gentamicin sulphate CRS to identify the corresponding peaks.
 Limits
– gentamicin C1 : 20.0 per cent to 40.0 per cent,
– gentamicin C1a : 10.0 per cent to 30.0 per cent,
– sum of gentamicins C2, C2a, and C2b: 40.0 per cent to
60.0 per cent
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
COMPOSITION – GENTAMICIN (1)
 Known structures of 5 components
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COMPOSITION – GENTAMICIN (2)
J. Pharm. Biomed. Analysis, 2007, 45, 257-262
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MONOGRAPH - TESTS
RELATED SUBSTANCES (1)
 Same method as for composition but different specifications
 Content in impurities can be very high and chemical structures are
not always known
 Gentamicin
Limits (for related substances eluting before gentamicin C1a):
– any impurity: maximum 3.0 per cent
– total: maximum 10.0 per cent
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MONOGRAPH - TESTS
RELATED SUBSTANCES (3)
 Threshold for « any unspecified impurity » in current
antibiotics monographs from Ph. Eur.
–
–
–
–
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Ciprofloxacine : 0.1 %
Cefradine : 0.25 %
Amoxicillin trihydrate : 1 %
Gentamicin sulphate : 3.0 %
Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
MONOGRAPH ASSAY – MICROBIOLOGICAL
TITRATION
 Microbiological assay of antibiotics (2.7.2)
– Two methods
• A. Diffusion method (19 antibiotics)
• B. Turbidimetric method (14 antibiotics)
– Indication of the reagents, micro-organisms, pH, temperature
– Chapter 5.3. Statistical analysis of results of biological assays
and tests
– Use of CombiStats www.edqm.eu
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
MONOGRAPH - ASSAY
HPLC (1)
 ASSAY – HPLC (2.2.29)
– For synthetic and semi-synthetic drugs
– For mixtures obtained by fermentation if similar activity of the
individual compounds
– Possibilty of sums of active substances
 HPLC/UV
– For chromophoric compounds (general case)
– Or after derivatization (amikacine)
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
MONOGRAPH - ASSAY
HPLC (2)
 Erythromycin
– Content:
• sum of the contents of erythromycin A, erythromycin B and
erythromycin C: 93.0 per cent to 102.0 per cent (anhydrous
substance),
• erythromycin B: maximum 5.0 per cent,
• erythromycin C: maximum 5.0 per cent.
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
MONOGRAPH - ASSAY
HPLC (3)
 Other detection modes (aminosides)
– HPLC/PAD (pulsed amperometric detection), Ph. Eur and US,
Ph. Int.
• Difficult to handle, poor precision
• Noisy, high disregard limit (1.0 % for framycetine and
neomycine sulphate)
– HPLC/ELSD
• Gentamicin sulphate (Chinese Ph.)
• Simultaneous determination of content in sulphates (in
place of titration)
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
MONOGRAPH - STORAGE
 General conditions : in airtight container
 Specific conditions
– Protected from light: tiamuline
– Multiple conditions
• In an airtight container, at a temperature of 2 °C to 8 °C :
potassium clavulanate
• Under nitrogen in an airtight container, protected from light,
at a temperature not exceeding 25 °C : rifampicin
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MONOGRAPH - TRANSPARENCY LIST
(IMPURITIES)
 List of specified and other detectable impurities (for definition
see Ph. Eur. General monograph 2034)
 All cases
– Absence to a very long list
• Nystatin, Josamycin, tyrothricin : no list
• Tiamuline : 6 specified impurities, 12 other detectable
impurities
• GRAHEK R and ZUPANCIC-KRALJ
J. Pharm. Biomed. Anal. XXX(2009)xxx in press
Identification of gentamicin impurities by LC tandem
mass spectrometry : 17 impurities
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ANTITUBERCULOSIS DOSAGE FORMS
 Ph. Int., 1st Suppl : 6 new monographs including 2, 3 and 4
component fixed-dose preparations
–
–
–
–
–
–
Doxycycline capsules
Isoniazid and ethambutol hydrochloride tablets
Rifampicin capsules and tablets
Rifampicin and isoniazid tablets
Rifampicin, isoniazid and pyrizinamide tablets
Rifampicin, isoniazid, pyrizinamide and
hydrochloride tablets
ethambutol
 Introduction of dissolution test in monographs for tablets and
capsules : doxycycline, isoniazid
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
4 COMPONENTS ANTITUBERCULOSIS
TABLETS
 Rifampicin (150 mg), isoniazid (75 mg), pyrizinamide (400 mg) and
ethambutol hydrochloride (275 mg) tablets
 Rifampicin : semisynthetic antibiotic obtained from rifamycin SV.
 Isoniazid, pyrizinamide
synthetic compounds
and
ethambutol
 Monographs in Ph. Eur., USP, Ph. Int., Chinese
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hydrochloride
:
ETHAMBUTOL, HCl
Ph. Eur.
Content
99.0 – 101.0 %
Titrimetry
Impurities HPLC 215 nm
Imp B : 1.0 %
USP
98.0 – 100.5 % 98.0 – 100.5 %
Titrimetry
Titrimetry
Total : 1.0 %
GC (2.4.24)
Fluorimetry
TLC : 1.0 %
Imp D : 5 ppm
(1-2 dichloroethane)
TLC Imp A : 1.0%
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Chinese
Min 98.5 %
Titrimetry
Aminobutanol Aminobutanol Aminobutanol
Derivatization
by
fluorescamine
Other : 0.10 %
Ph. Int.
385/485 nm :
1.0 %
Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
TLC : 1.0 %
ISONIAZID
Content
Ph. Eur.
USP
Ph. Int.
Chinese
99.0 – 101.0 %
98.0 – 102.0 %
98.0 – 101.0 %
Min 99.0 %
Titrimetry
HPLC
Titrimetry
Titrimetry
Impurities TLC
Hydrazine : 0.05 %
NONE
TLC
Hydrazine: 0.02 % Hydrazine: 0.02 %
Other : 0.2 %
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TLC
Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
PYRAZINAMIDE
Content
Ph. Eur.
USP
Ph. Int.
Chinese
99.0 – 100.5 %
99.0 – 101.0 %
98.5 – 101.0 %
Min 99.0 %
Titrimetry
Titrimetry
Titrimetry
Titrimetry
Impurities TLC
None
None
Any : 0.2 %
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
TLC
Any : 0.2 %
RIFAMPICIN
Content
Impurities
Ph. Eur.
USP
Ph. Int.
Chinese
97.0 – 102.0 %
95.0 – 103.0 %
97.0 – 102.0 %
Min 93.0 %
Absorbance 475 nm
HPLC 254 nm
Absorbance 475 nm
HPLC 254 nm
HPLC 254 nm
HPLC 254 nm
Rifampicinquinone
: 1.5 %
Rifampicinquinone Rifampicinquinone Rifampicinquino
: 1.5 %
ne : 1.5 %
: 1.5 %
Other : 1.0 %
Other : 1.0 %
Total : 3.5 %
Total : 3.5 %
TLC
3-formyl-rifamycin
SV : 0.5 %
Other : 1.0 %
HPLC 254 nm
Rifampicin N Oxide: 0.5 %
3-formylrifamycin SV :
0.5 %
Total other:3.0%
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
4 COMPONENTS ANTITUBERCULOSIS
TABLETS – Ph. Int. (1)
 Rifampicin (150 mg), isoniazid (75 mg), pyrizinamide (400 mg) and
ethambutol hydrochloride (275 mg) tablets
 Ph. Int. (4th Edition, 1st supplement 2008)
 Identification
– Tests A and B (retention time/HPLC for assay)
or
– Test C : 2 TLC systems (different stationary phases)
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
4 COMPONENTS ANTITUBERCULOSIS
TABLETS – Ph. Int. (2)
 Impurities
– Rifampicin-related substances (RP-HPLC-UV)
• Addition product : 3-formylrifamycin SV (specified impurity
of rifampicin limited to 0.5% in the API) and isoniazid : 5.0 %
• Rifampicin quinone : 4.0 %
• Any other impurity : 1.5 %
• Total : 10.0 %
• Disregard limit : 0.1 %
– Impurities in API
• 3-formylrifamycin SV : 0.5 %
• Rifampicin quinone : 1.5 %
• Any other impurity : 1.0 %
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
4 COMPONENTS ANTITUBERCULOSIS
TABLETS - Ph. Int. (3)
 No dissolution test
 Assay
– First RP-HPLC-UV system for isoniazid, pyrizinamide and
ethambutol hydrochloride
– Second RP-HPLC-UV system for rifampicin
– Specifications : 90.0 to 110.0 % of the amounts stated on the
label
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
4 COMPONENTS ANTITUBERCULOSIS
TABLETS - USP
 Identification
By retention time (2 HPLC systems)
 Dissolution
Not less than 75 % of the labeled amounts of each active
substance in 45 min
 Impurities
No test
 Assay
2 HPLC systems : content 90.0 to 110.0 % in each
component
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ANTIBIOTICS – COUNTERFAITING (1)
 Screening of some of the most conterfeited antibiotics in a single
chromatographic run
Gaudiano M.C. et coll, J. Pharm. Biomed. Anal., 2008, 48, 303-309
RP-HPLC method, elution by gradient, detection 230 nm
Ampicillin, amoxicillin + clavulanic acid, doxycycline, cloxacillin,
chloramphenicol
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
ANTIBIOTICS – COUNTERFAITING (2)
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009
MACROLIDES – COUNTERFAITING (1)
 Fast chemical identification system for screening of counterfeit
drugs of macrolide antibiotics
CHANG-QIN HU et coll.,
J. Pharm. Biomed. Anal., 2006, 540, 68-74
– Two color reactions + 2 TLC
– Sreening of 10 macrolides
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MACROLIDES – COUNTERFAITING (2)
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CONCLUSION
 Products from fermentation (antibiotics/antituberculosis) are a
challenge for analysts and for the authorities
– Complex mixtures
– All compounds not well known (impurity profile)
– Specific analytical methods (HPLC/amperometry)
– Content determined by activity assay
 Products obtained by synthetic routes
– Obey to ICHQ3A (R2)
– Large place for classical HPLC methods coupled to UV detection
– Impurity profile well known
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Sampling and testing for Quality Control Laboratories, Nairobi, September 2009