The role of assay methods in characterizing the quality of bulk pharmaceuticals

Abstract

This study presents the role of assay methods in characterizing the quality of bulk substances in pharmaceutical 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, even at the pharmacopoeial level. Development of HPLC and other chtromatographic techniques, coupled with mass spectrometry, is also useful in the determination of drugs and their metabolites in biological samples. The role of electrophoretic, spectroscopic, and other methods in pharmaceutical analysis are discussed here. 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.

In the field of drug analysis, the analytical investigation of bulk drug materials, the intermediates in their synthesis, products of drug research, drug formulations, 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.

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.

Application of Chromatographic Techniques

Rapid development of analytical methodology in pharmaceutical 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.

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 (< 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 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 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 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 battle-fields 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’ organiza-tions, 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.

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 propor-tion of those on formulations, contain these tests. The impurity profile has become the most informative indi-cator 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 global-ization and harmonization mentioned earlier and the necessity of increasing the safety of drug therapy, have prompted the vali-dation of analytical methods to the fore-front; 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, espe-cially among the young generation, feel that the essence of pharmaceutical analysis is the mass produc-tion 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 suf-fering mankind, through increasing the safety of drug therapy.