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Invited Review www.jpbsonline.org

The role of assay methods in characterizing the

quality of bulk pharmaceuticals
Wieslawa Misiuk

Department of Biology ABSTRACT

and Chemistry, University
This study presents the role of assay methods in characterizing the quality of bulk substances in pharmaceutical
of Bialystok, Hurtowa 1,
Bialystok, Poland
analysis. High-performance liquid chromatography (HPLC) is the most remarkable development and the
technique has become very significant in the quality control of bulk drugs and pharmaceutical formulations,
Address for correspondence: even at the pharmacopoeial level. Development of HPLC and other chtromatographic techniques, coupled with
Dr. Wieslawa Misiuk, mass spectrometry, is also useful in the determination of drugs and their metabolites in biological samples.
E-mail: wiesmisi@uwb. The role of electrophoretic, spectroscopic, and other methods in pharmaceutical analysis are discussed here.
edu.pl
There are separate sections devoted to microscopy techniques that are useful in the pharmaceutical field, as
also the regulatory aspects of drug analysis, with emphasis on questions related to validation.
Received : 01-05-10
Review completed : 04-05-10
Accepted : 21-05-10
DOI: 10.4103/0975-7406.67007
J Pharm Bioall Sci 2010;2:88-92 KEY WORDS: Chromatography, drug, pharmaceutical formulation, microscopy, spectroscopy, validation

I n the field of drug analysis, the analytical investigation

of bulk drug materials, the intermediates in their
synthesis, products of drug research, drug formulations,
Application of Chromatographic Techniques

Rapid development of analytical methodology in pharmaceutical

impurities and degradation products, and biological
samples containing the drugs and their metabolites is a very
important area of research. The aim of this study is to obtain
data that can contribute to high quality, maximal efficacy,
and safety of drug therapy, as also maximum economy during
drug production.
and biomedical analyses has led to various forms of high-
performance liquid chromatography (HPLC) becoming
undoubtedly the most important methods.[29-31] The theoretical
and practical foundations for this method were laid down at the
end of 1960s and the beginning of 1970s. The latter decade was
the period that saw a rapid spread of this technique.

From the point of view of public health, the safety, efficacy,
and economy of drug therapy are extremely important issues.
Not only Financial, but also political aspects of the problems
are also real in the European and World community. For
that reason, pharmaceutical and biomedical analyses are
among the most important branches of applied analytical
chemistry.[1-8]
The importance of drug impurity and stability-related issues
has also been characterized by a number of books and articles
devoted to this subject.[9-12] The determination of drugs and
metabolites in biological samples,[13-17] with particular attention
to toxicological and forensic analysis, [18-25] requires special
techniques and a special manner of thinking, as reflected by
many books and articles on these issues. Drug discovery requires
a solid analytical background, with a great variety of methods
to be used.[26-28] Innumerable drug-related chapters have also
appeared in general analytical books and special issues of
scientific journals.
In pharmaceutical analysis, the HPLC method shares its
importance with various techniques. HPLC has been used
to solve no less than 50% of the problems, leaving the other
50% to about 15 other chromatographic, spectroscopic, and
other methods, about 10% to gas chromatography (GC), 5%
to thin-layer chromatography (TLC), 10% to ultraviolet (UV)
spectrophotometry, and the rest to electroanalytical methods.
The contribution of HPLC in drug analysis has further increased
in this long period. For example, in 1983, a UV detector was
applied almost exclusively, leaving a little share to refractive
index, fluorimetric, and electrochemical detection, but in 2005,
a mass spectrometer was applied as a detector in about one-
third of the analysis. HPLC coupled with mass spectrometry,
HPLC/MS (MS) or LC/MS (MS) due to high sensitivity and
selectivity, have become the predominant method in bioassays
and pharmacokinetic and metabolic studies, as well as in
the structure elucidation of drug impurities and degradation
products.[32-34] A new development in the field of HPLC/MS has
been the introduction of column packing with ultrafine particles

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(< 2 um), enabling short columns (5 cm or less) to be used, and
rapid analyses (e.g., 5 minutes or even less than 1 minute) to
be carried out by UPLC, for example, ultra performance liquid
chromatography.
In the compendial analysis of small organic molecules, the
breakthrough of HPLC was also extremely rapid. In the twenty-
ninth United States Pharmacopoeia,[35] HPLC was applied to
the assay of bulk drug materials of this type in about 45% of
the monographs. This share was somewhat higher than that of
the non-selective ones, but with titration methods that were
less time-consuming, leaving only about 10% to other methods,
mainly the similar, non-selective UV-Vis spectrophotometry.
HPLC and TLC were used almost exclusively, with almost equal
shares for the purity control of bulk drug materials and the related
compounds test. Even more spectacular was the propagation
of HPLC in the assay of pharmaceutical formulations, which
needed specific methods indicating stability. No other method
had spread so rapidly in pharmaceutical analysis.
Among other chromatographic methods the important
application field of modern TLC is the separation of the
components of complex mixtures, for example, impurities
and degradation products of drug materials and extracts of
medicinal plants. The speed and the resolution could be greatly
improved by the introduction of special techniques, such as
high-performance thin-layer chromatography (HPTLC), using
ultra thin layers and coatings with ultrafine particles or over
pressured-layer chromatography (OPLC). The development of
densitometers enables classical TLC and the latter techniques
could be successfully used as tools for the quantitative analysis
of complex mixtures.
The introduction and rapid spread of HPLC and HPLC/MS
decreased the importance of GC and GC/MS in pharmaceutical
analysis.[36] Nevertheless, these are still important techniques
in many fields of drug analysis, where the analytes are volatile
and thermally stable. In the past 15 to 20 years a new field
application is being used for the determination of residual
solvents in drugs. Almost always the headspace technique is used
to fulfill the demanding requirements, that is, determination
of solvents at the 10-ppm level; and down to the ppm level in
the case of carcinogenic or genotoxic solvents.
Application of Capillary Electrophoresis
Since the introduction of the commercially available
instruments capillary electrophoresis (CE), related methods
such as micellar electrokinetic chromatography (MEKC),
microemulsion electrokinetic chromatography (MEEKC), and
capillary electrochromatography (CEC),[37-39] have attracted
great interest in pharmaceutical analysis as possible alternatives
or amendments to HPLC.
This share is an underestimation, as there are researchers
specializing in CE analysis. It is an overestimation because,
despite CE having several advantages such as a flat flow profile
that results in an extremely high column efficiency, due to its
J Pharm Bioall Sci April-June 2010 Vol 2 Issue 2 89 
Misiuk: Role of assay method in bulk drugs 
limitations with regard to its general applicability, it does not yet
seem to be a real rival to HPLC in the practice of compendial-
industrial pharmaceutical analysis. CE is already an official
method in USP XXIX,[35] but its contribution to the monographs
is still negligibly low. However, CE is already an inevitable tool
in the analysis of proteins and other biopolymers, particularly
with respect to miniaturization, which leads to chip-based
bioanalytical chemistry. Of the new techniques mentioned
earlier, CEC will certainly have a bright future.
The separation and quantification of enantiomeric mixtures
are among the greatest challenges of the past years in
pharmaceutical and biomedical analysis. [40-45] The main
problems to be solved are, to determine the enantiomeric purity
of drugs being used in therapy as pure enantiomers and the
simultaneous determination of the components of race-mates
in the biological samples. The latter type of enantiomeric
separation has been successfully adapted to CE.[45] The present
situation can be characterized by the spread of this technique
and the continuous development and commercialization of new
types of chiral HPLC columns.
Application of Spectroscopy
In pharmaceutical and biomedical analysis, the development
of nuclear magnetic resonance (NMR) and mass spectrometry
(MS), along a road paved with Nobel Prizes, has also been
successfully exploited. The dramatic decrease in the demanding
requirements for sample size and the solution of the difficult
problems of interfacing these techniques with chromatographic
(and electrophoretic) separation methods have greatly expanded
their field of application. In addition to the offline applications
that are still widely used, online HPLC/MS, HPLC/ NMR,
HPLC/NMR/MS, and other hyphenated methods, are becoming
leading methods, for example, the structure elucidation of drug
impurities, degradation products, metabolites, and bioactive
components in natural products.[46-48] Due to its high sensitivity
and selectivity, HPLC/MS(MS) has become the predominant
method, even in the quantitation of these minor components
(e.g., in pharmacokinetic and bioequivalence studies).
UV spectroscopy[49] is observable due to the availability of diode-
array detectors attached to HPLC and TLC densitometers,
both suitable for obtaining good-quality spectra, which are
often useful; in identifying impurities for example. As for
the quantitative analytical application of this technique,
approximately 10% of the share in pharmacopoeias for the assay
of bulk drug materials and pharmaceutical formulations is very
slowly decreasing.
Multiwavelength / chemometric measurements were interesting
and successful research areas at the beginning of the 30-year
period. At present, these can be considered to be fairly important
routine methods.
In modern pharmaceutical and biomedical analysis the most
important application of fluorimetry is as detectors attached
to HPLC or related techniques. In particular, laser-induced
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 Misiuk: Role of assay method in bulk drugs
fluorimetry based on native or derivatization-based fluorescence
enables very sensitive determinations to be carried out.
The most important field of application of infrared (IR)[50]
and near-infrared (NIR) spectroscopy[51] is the identification of
drugs. IR has greatly decreased (almost completely eliminated)
the importance of the classical color tests, while NIR is a
method of increasing importance in the in-process control of
manufacturing pharmaceutical formulations. IR and Raman
spectroscopy, together with solid-phase NMR, X-ray diffraction,
and thermal methods are the up-to-date methods in solid-phase
characterization,[52] which is of great importance in developing
pharmaceutical formulations, with optimal bioavailability.
In recent times FTIR spectroscopy has been coupled with
ATR. FTIR spectroscopic imaging in ATR (attenuated Total
Reflection) mode is a powerful tool for studying biomedical
samples and dissolution of pharmaceutical formulations and
drug release. One of the key advantages of ATR-FTIR imaging
is that is requires minimal or no sample preparation prior
to spectral measurements. Consequently, this approach is
particularly suitable to measure substances with strong infrared
absorption such as water. The application of ATR-FTIR imaging
also allows for the characterization of biomedical materials in
tissue engineering. Also the quantitative information about the
spatial distribution of chemical components on pharmaceutical
tablets in contact with water, as a function of time, provides an
important basis for building new mathematical models for the
optimization of controlled drug delivery. ATR-FTIR imaging is
suitable for imaging of realistic tablets in contact with aqueous
solutions because of the shallow penetration of the evanescent
wave into the sample.
In pharmacopoeias, for the study of toxic metal impurities,
the classical sulfide and other limit tests are still widely used.
At present, the rapidly increasing importance of the much
more selective and sensitive atomic spectroscopic methods
can be observed, such as, graphite furnace atomic absorption
spectrometry (GF-AAS), inductively coupled plasma atomic
emission spectrometry (ICP/AES), and mass spectrometry
(1CP/ MS).
Others
The classical titrations non-selective method is still widely
used in compendial analysis for the assay of bulk drug
materials. Even in the USP, where the breakthrough of HPLC
has been much faster, more than 40% of the low molecular
weight organic compounds are determined by aqueous or
non-aqueous titration. Other electroanalytical methods have
always been only modestly important in pharmaceutical
analysis. Classical polarographic methods using toxic mercury
electrodes are being driven out from practice and replaced by
new electrodes, for example, glassy carbon electrodes modified
with carbon nanotubes, which provide highly sensitive analyses.
Another field where remarkable results have been obtained
is the development of ion-specific and molecule-specific
sensors. Flow-injection analysis with various detectors such as
spectroscopic, electroanalytical chemiluminescence is often
 90 J Pharm Bioall Sci April-June 2010 Vol 2 Issue 2
used in the analysis of drug formulations.[53] All antibiotics, 30
years ago, were determined using microbiological methods.[54]
In modern pharmacopoeias, in the majority of cases, these are
replaced by much more selective and informative methods,
mainly HPLC. Although the importance of immunoassays has
decreased in the recent years, they are still often used in the
determination of some bioactive compounds in the biological
samples. Radioimmunoassay has been greatly superseded by
various enzyme-immunoassay methods.
Application of Microscopy Techniques
A useful instrument for the structural and morphological study
of novel films, nanoparticles, hydrogels, matrices, and porous
scaffolds[55,56] is offered by modern microscopy tools, such as,
atomic force microscopy (AFM), scanning electron microscopy
(SEM), and confocal Raman microscopy (CRM).[57,58] The
imaging tools are important for analyzing the surface of soft
organic materials, as understanding these surfaces may help to
shed light on the interactions that occur between crystals and
their surrounding environment.
Drug release is the result of a complex interplay between the
drug, its carrier, and the release environment. Study of the
surface structure and morphology of pharmaceutical substances
contributes to an understanding of surface activity and is of
critical importance to the pharmaceutical industry. Modern
microscopies are tools that are applicable for the collection of
topographic data and morphological investigation of different
applicable pharmaceutical materials.
Future Trends
Globalization of the drug market and the sharpening concurrence
among the drug companies has caused pharmaceutical
analysis to become one of the battle­fields in the struggle. The
importance of issues related to drug safety has greatly increased
and this has led to the continual increase of demands with
regard to securing the quality of drugs, and often over securing
the safety of drug therapy.[59-62]
It became necessary to harmonize the demands and
analytical strategies. The first step was the establishment in
the European Pharmacopoeia, of which the Sixth Edition is
now official.[46] This became the basis of the national phar­
macopoeias of the member states of the European Union.
The next step was the formation of ICH (International
Conference on Harmonization), which was done with the aim
of harmonizing the efforts of registration agencies, principal
pharmacopoeias (Ph. Eur., USP, and Japanese Pharma­
copoeia), and pharmaceutical manufacturers’ organiza­tions,
to improve the quality of drugs and the safety and efficacy of
drug therapy. The guidelines issued by ICH are authoritative
worldwide with respect to drug quality issues. It has to be
noted that requirements with regard to the quality of drugs
and drug formulations in the drug market are, in practice,
much greater than those prescribed in the pharmacopoeias
and ICH guide­lines.
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The greater change in the pharmacopoeias in the past years
has been the increasing importance of purity tests. At the
beginning only a very limited number of monographs contained
tests related to impurities. Thanks to the development of
TLC and HPLC, at present, an overwhelming majority of the
monographs on bulk drugs, and in a fairly high propor­tion of
those on formulations, contain these tests. The impurity profile
has become the most informative indi­cator of the quality of bulk
drug materials. At the same time, the importance of assaying
bulk drugs has decreased considerably; moreover, there are
opinions that even this importance is questionable.
The tendencies toward global­i zation and harmonization
mentioned earlier and the necessity of increasing the safety
of drug therapy, have prompted the vali­dation of analytical
methods to the fore­front; moreover, it has become one of the
most important issues in contemporary drug analysis. However,
some negative tendencies are also apparent:
• A fully validated method meeting the requirements of
various guidelines needs much more analytical data than
would be strictly necessary
• Many drug analysts, espe­cially among the young generation,
feel that the essence of pharmaceutical analysis is the mass
produc­tion and handling of data, rather than problem
solving
• The way of thinking is changing, with many people, mainly
outside the circles of drug analysts, believing that possession
of up-to-date, automated / computerized instruments and
validated methods automatically give good and reliable
results. Pharmaceutical analysis is an important field of
activity in the interest of suf­fering mankind, through
increasing the safety of drug therapy.
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Source of Support: Nil, Conflict of Interest: None declared.

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