Keywords: Elemental impurity, Toxic elements, X-ray fluorescence, energy dispersive Xray
fluorescence, Method validation.
Introduction:
1 Principles of fluorescence spectroscopy
1. The light intensity from the excitation source is a function of wavelength. Even if the intensity
of the exciting light is monitored via the beam splitter shown in Figure 2.1, and corrected
by division, the response of the reference solution or detector may be dependent upon
wavelength. (Ferreira et al., 1991, Lakowicz, 1999)
2. The transmission efficiency of the excitation monochromators is a function of wavelength.
(Ferreira et al., 1991)
3. The optical density of the sample may exceed the linear range, which is about 0.1 absorbance
units, depending upon sample geometry. (Andersen and Mortensen, 2008, Bacia et al., 2014)
2 Instrumentation:
2.1 Light sources
We now describe the individual components of a spectrofluorometer. The general
characteristics of these components are considered along with the reason for choosing specific
parts. Understanding the characteristics of these components allows one to understand the
capabilities and limitations of spectrofluorometers. We will first consider
light sources. (Bacia et al., 2006, Beć et al., 2020, Becker et al., 2007)
1.
2.
3.
4.
5.
6.
7.
8.
Arc and Incandescent Xenon Lamps.
Pulsed Xenon Lamps.
High-Pressure Mercury (Hg) Lamps.
Xe–Hg Arc Lamps.
Quartz–Tungsten Halogen (QTH) Lamps.
Low-Pressure Hg and Hg–Ar Lamps.
LED Light Sources.
Laser Diodes.(Braeckmans et al., 2010)
2.2 Monochromators
Monochromators are used to disperse polychromatic or white light into various colours or
wavelengths. This dispersion can be accomplished using prisms or diffraction gratings. The
monochromators in most spectrofluorometers use diffraction gratings rather than prisms. The
performance specifications of a monochromator include dispersion, efficiency, and stray light
levels. Dispersion is usually given in nm/mm. The slit width is sometimes expressed in mm,
which requires knowledge of the dispersion.(Kirby, 1971, Wampler and Kutz, 1988) A
monochromator for fluorescence spectroscopy should have low stray light levels to avoid
problems due to scattered or stray light.
By stray light we mean light transmitted by the monochromator at wavelengths outside the
chosen wavelength and bandpass.(Lakowicz, 1999, Sharma, 1981)
2.2.1 Optical filters
2.2.1.1 Coloured filters:
While spectrofluorometers have monochromators for wavelength selection, it is often
important to use optical filters in addition to monochromators. Optical filters are used to
compensate for the less-than-ideal behaviour of monochromators. Also, when the spectral
properties of a fluorophore are known, maximum sensitivity is often obtained using filters
rather than monochromators. (Parker and Barnes, 1957, Parker and Rees, 1960)
2.2.1.2 Thin-Film Filters
A wide variety of coloured-glass filters are available, but the transmission curves are not
customized for any given application. During the past ten years, there have been significant
advances in the design of thin-film optical filters.14 Almost any desired transmission curve can
be obtained. Filters are now being designed for specific applications, rather than
choosing the colored-glass filter that best suits an application. Long-pass filters are an example
of this type filter. These filters have a sharp cut on the transmission above 325 nm or 488 nm,
which are wavelengths available from a helium–cadmium or argon ion laser, respectively.
(Erdogan et al., 2004) The transmission above the cut-on wavelength is close to 100% to
provide maximum sensitivity. Neutral-Density Filters Neutral-density filters are used to
attenuate light equally at all wavelengths. They are typically composed of sheets of glass or
quartz coated with a metal to obtain the desired optical density. Quartz transmits in the UV and
is preferred unless no experiments will be done using wavelengths below 360 nm. Neutraldensity filters are described by their optical density, and can typically be obtained in increments
of 0.1, up to optical densities of 4. It is often necessary to adjust or match the intensity of two
signals, which is conveniently accomplished using neutral-density filters.(Coskun et al., 2010)
2.3 Photomultiplier tubes (detectors):
Almost all fluorometers use photomultiplier tubes (PMTs) as detectors, and it is important
to understand their capabilities and limitations. A PMT is best regarded as a current source.
(Studholme and Blau, 1994). The current is proportional to the light intensity. A PMT responds
to individual photons, and the pulses can be detected as an average signal or counted as
individual photons Photon Counting versus Analog Detection of Fluorescence A PMT is capable
of detecting individual photons. Each photoelectron results in a burst of 105 to 106 electrons,
which can be detected as individual pulses at the anode. Hence, PMTs can be operated as
photon counters or can be used in the analog mode in which the average photocurrent is
measured. Note that we are considering steady-state measurements. (Hungerford et al., 1996)
(Gifford and Bigliardi, 1988).
3 Determination of active ingredients in drugs:
3.1 Dapoxetine hydrochloride (DAP) and avanafil (AVA):
By the year of (2020) Mohamed and his research group determined (DPA) and (AVA), The
procedure is based on measurement of native fluorescence intensity of both drugs at λEm 337
nm and 370 nm using λEx 290 nm and 314 nm for DAP and AVA in methanol respectively. A
simple, rapid, sensitive and cost-effective spectrofluorimetric technique was developed for the
determination of DAP and AVA. In this study, the native and synchronous spectrofluorimetric
method was used for simultaneous analysis of two drugs in finished product and biological fluids
and used in uniformity of dosage units with good results. The proposed method is fast. Finally,
the developed method can be used in the clinical analysis of DAP and AVA and in quality
control labs. (Mohamed et al., 2020) (Shah et al., 2017, Patel and Kothari, 2016) (Öztürk Er et
al., 2019)
3.2 Acetylsalicylic acid (ASA), paracetamol and caffeine:
Alves and Poppi worked on determination of Acetylsalicylic acid (ASA), paracetamol and
caffeine (2000) using solid-phase molecular fluorescence and second order multivariate
calibration. This methodology is applicable even in the presence of unknown interferences and
with spectral overlap of the components in the mixture. Parallel factor analysis (PARAFAC) was
used for model development, whose effectiveness was demonstrated by analysis of variance
(ANOVA). Errors below 10% were obtained for all compounds using an external validation set.
Benefits of the new procedures not included in the reference methods such as low cost, no
need of sample preparation, simple and fast analysis using fluorescence spectrometer and no
generation of waste, make this method very attractive, allowing for the simultaneous
determination of compounds with good reproducibility and accuracy. Benefits, such as, low
cost, no need for extensive sample preparation, fast analysis with no generation of waste (other
than the sample itself), make this method an attractive alternative, allowing the simultaneous
determination of compounds with intense spectral overlap. The model was validated by
analysis of variance (ANOVA). (Alves and Poppi, 2009, Moreira et al., 2005a, McKemy et al.,
2000)
3.3 Paracetamol:
Paracetamol is determined by fluorescence spectroscopy in (2005) by Moreira et al.
Fluorescence intensity is linearly dependent on PA concentration within the 100–400 mg g−1
range. The analytical frequency is 200 h−1. Detection and quantification limits were estimated
within the 13.0–16.7 and 43.1–55.7 mg g−1 ranges for samples with different ingredient
proportions. The results demonstrate the potentiality of the proposed method for PA
determination in solid matrices. Precise results, in agreement with those obtained by the British
Pharmacopoeia reference method, were obtained. The procedure is simple, rapid and nondestructive The method, however, is dependent on the ingredients and powdering, being
necessary a previous knowledge of the ingredients of the sample, and a good powdering
system. Circumvented these points, a simple reading of the fluorescence intensity in the solid
sample makes an easy and fast determination possible. (Moreira et al., 2005b). Circumvented
these points, a simple reading of the fluorescence intensity in the solid sample makes an easy
and fast determination possible. (Moreira, 2005).
3.4 Antiepileptic drugs:
By the year of (2020) Zeid and his friends performed an estimation of active materias in
some antiepileptic drugs, a facile, sensitive, and precise lab-on-a-chip electrophoretic method
coupled with light-emitting diode induced fluorescence (LED-IF) detection was developed to
assay three antiepileptic drugs, namely, vigabatrin, pregabalin, and gabapentin, in
pharmaceutical formulations. The analytes were derivatized offline for the first time with
fluorescine-5-isothiocyanate (FITC) to yield highly fluorescent derivatives with λex/em of
490/520 nm. The FITC-labelled analytes were injected, separated, and quantitated by a
microfluidic electrophoresis device using fluorescence detection. The labelled analytes were
monitored using a blue LED-IF system. HDM- β-CD acted as a selective host for the studied
antiepileptic drugs, rendering a high separation efficiency. Methylcellulose was used as an
efficient dynamic coating polymer to prevent the labelled drugs frombeing adsorbed on the
inner surfaces of the poly (methylmethacrylate) microchannels. (Zeid et al., 2020, Castro and
Manz, 2015).
4 Total reflection X-ray fluorescence (TXRF) to determine trace
inorganic impurities in drugs:
4.1 Palladium (Pd) in paracetamol:
Palladium (Pd) has been determined in paracetamol in (1996) by (Shaw et al. in general X-ray
sources capable of accessing Pd-K lines were found to be most effective in determination of Pd
in APIs.
It is clear that the most important factor for the usage of TXRF as a screening platform for
analysis of pharmaceutical drug substances is the instrument configuration and the X-ray
source in particular. Analysis of Pd in pharmaceutical drug substances has interference
limitations when Pd-L lines are used for this analysis, but are alleviated when Pd-K lines are
used. (Shaw et al., 2012, Lamblin et al., 2010, Wagner et al., 1996)
4.2 Determination of (Cd, Pb, As, Hg, Co, V, Ni) in solid oral tablets:
Six inorganic trace metals contaminants has been demonstrated by Klopper in oral solid
dosage drug products (OSD products) by the year of 2017 within a defined matrix range.
Method development and
validation focused on class 1 (Cd, Pb, As, Hg) and class 2A (Co, V, Ni) elements, as defined by
ICH guideline Q3D. In order to limit validation activities, a novel cluster approach was applied,
based on matrix properties. (Klopper, 2017, Marguí et al., 2009) This included comprehensive
characterization of method performance parameters for exemplary pharmaceutical matrices and
demonstration of LOQ independence from matrix effects by using a set of limit samples
representing typical matrix variations of OSD products. The methodology can be used as a limit
test for class 1 and class 2A elements and is fully compliant with method validation
requirements according to the European Pharmacopeia. (Sauer et al., 2020, D'Elia et al., 2020).
4.3 Gold and platinum metallodrugs:
Antosz et al in 2012 used TXRF to detect gold and platinum in metalodrugs. The observed
recovery rates of their study are in good general agreement with recent reports on the use of
TXRF for the analysis of pharmaceutical compositions. (Antosz et al., 2012, De La Calle et al.,
2012, Shaw et al., 2012) In these reports various metals were investigated mainly in the ppm
range. Their study thus indicates that samples can be analyzed with comparable accuracy and
precision also in the ppb concentration range. It can be expected that after optimization of
sample preparation procedures or further technical improvements a substantially further
improved analyte recovery will be reached. (Meyer et al., 2012)
4.4 Nickel and Manganese Contaminants in Pharmaceutical Iron Supplements:
Cardoso et al. investigated the capability of energy dispersive X-ray fluorescence (EDXF)
spectrometry in a triaxial geometry apparatus as a fast and nondestructive determination
method of both dominant and contaminant elements in pharmaceutical iron supplements. The
following iron supplements brands with their respective active ingredients were analyzed:
Neutrofer fo´ lico (iron gylcinate), Anemifer (iron(II) sulfate monohydrate), Noripurum (iron(III)-
hydroxide polymaltose complex), Sulferbel (iron(II) sulfate monohydrate), and Combiron Fo´
lico (carbonyl iron). (Balarajan et al., 2011, Al-Omari et al., 2016) We have applied the EDXRF in
triaxial geometry technique for the quantification of both the dominant and contaminant metal
elements. Manganese and nickel were measured as important contaminants of these
supplements even though the prescriptions do not warn of their presence. (Cardoso et al.,
2017, Beard et al., 2003, Sims et al., 2011)
4.5 Bromine traces in sodium diclofenac:
Raggi et al. tried a fast method for bromine detection in solid sodium diclofenac samples,
The method proposed based on the use of XRF spectroscopy on solid samples, appears suitable
in terms of feasibility and sensitivity for determination of bromine in commercial samples of
sodium diclofenac, advantages of this procedure are ease and speed of detection the possibility
to utilize samples without special pretreatments or solution preparation, and the long term
stability of pellets, both of standards and of samples, which allows long delays between
calibration and analysis. Another advantage lies in the fact that XRF is an elemental technique,
capable of detecting bromine in whatever form it is present without requiring any hypothesis
on the nature of the molecule containing it. On the negative side one might mention the fact
that the homogenization of standards is fairly laborious, especially for low bromine levels and
the fact that the method, although very promising, still suffers from the “matrix effect”. (Raggi
et al., 1990, Raggi et al., 1991)
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