Current Pharmaceutical Analysis - Volume 1, Issue 1, 2005

This inaugural issue of “Current Pharmaceutical Analysis” presents comprehensive reviews written by eminent experts on a number of important topics including in high-throughput bioanalytical LC-MS/MS methods, identification techniques in proteomics using new analytical and bioinformatic strategies, solidphase microextraction techniques, fluorescent chiral derivatisation reagents, role of cytokines in pharmaceutical and histopathological analysis, and antagonists of endothelin receptors for drug development and physicochemical profiling by capillary electrophoresis. It is hoped that 7ldquo;Current Pharmaceutical Analysis” will soon become an indispensable journal for researchers in this field.

Swift growth in the use of LC-MS/MS for the analysis of drugs in biological matrices has been compelled by the need for timely and high-quality data at many stages in drug discovery and development process: from high throughput screening of drug candidates and rapid data generation for pre-clinical studies to almost 'real-time' analysis of clinical samples. Prompt and rational method development, validation, and transfer play a pivotal role in achieving the goals of “faster, better, and cheaper” for pharmacokinetic studies since this could easily account for more than 50% of the time and labor resources for a moderate-sized project. Strategy for rational method development, validation and transfer has been largely kept as institutional knowledge but rarely appeared in literature. In this review article, strategies for developing and validating robust high throughput LC-MS/MS methods will be critically reviewed and discussed. Automated sample preparation, fast chromatography, minimization of matrix effects, and strategy of narrowing the gap between validation and incurred sample analysis are just a few topics covered in this review. Other interesting approaches for improving method efficiency and ruggedness such as direct injection SPE and liquid/liquid extracts as well as multiplexing of LC columns will also be discussed. Potential pitfalls during method development and validation are pointed out. At the end, the question “how fast is fast enough and how fast is too fast?” will be answered after considering all aspects of the method development and validation.

Nitric oxide (NO) is a gaseous radical molecule. In human organism NO is produced in various cells from Larginine by the catalytical action of NO synthases (NOS). The L-arginine/NO pathway powerfully contributes to maintain multiple physiological functions, including vascular tone, platelet function and neurotransmission. The metabolic fate of NO is very complex due to the participation of numerous compounds resulting from the ability of NO to react practically with any biomolecule to produce biologically active metabolites (e.g. S-nitrosothiols) and biologically inactive metabolites (e.g. nitrate). The concentration in biological fluids and tissues of members of the L-arginine / NO family is of particular interest, as it may characterize the status of this pathway in health and disease as well as to monitor the progress of pharmacological interventions. Thus, measurement of the NO metabolites nitrate and nitrite is suitable to assess NO synthesis in vivo. On the other hand, measurement of the circulating NOS inhibitor asymmetric dimethylarginine (ADMA) was found to reliably identify pathological conditions associated with NO-related endothelial dysfunction. Among the various analytical methods currently available for the analysis of the L-arginine / NO family, mass spectrometry (MS)-based approaches such as gas chromatography-mass spectrometry (e.g. GC-MS / MS) and liquid chromatographymass spectrometry (e.g. LC-MS / MS) emerged indispensable analytical tools for the reliable quantitative analysis of the whole NO family. The present article discusses the currently available analytical methods especially emphasizing the importance of the MS technology to the NO field of research.

Completion of the Human Genome Project enabled a better understanding of biological functions of organisms. However, these studies still provide a limited insight into the cellular processes. Nowadays, a comprehensive analysis and characterization of all expressed proteins, called proteomics, is the point of the interest. One of the important issues in proteomics is finding of analytical and bioinformatic strategies allowing unambiguous protein identification based on the searching of the peptide sequence databases. Some examples of bioinformatic strategies for analytical data processing obtained with the use of separation techniques and mass spectrometry analysis are given to demonstrate their usefulness in proteomics. First, the application of learning algorithms for the reliable evaluation of MS/MS spectra of peptides, which were separated and processed with reversed-phase liquid chromatography-tandem mass spectrometry is discussed. Detailed considerations of the use of artificial neural networks analysis to classify automatically peptide MS/MS spectra is provided and analyzed in the aspect of utility of another learning algorithms. Moreover, the usefulness of predictions of the reversed-phase liquid chromatography retention times of peptides in proteomic research is reported. In that case, quantitative structure-retention relationships (QSRR) analysis is considered in the view of the other approaches used in this field. Finally, the contribution of analytical information from the pI-based separation methods is considered as the additional source of peptide database matching constraint.

The physicochemical properties of pharmaceuticals such as acid dissociation constant (pK), octanol-water partition coefficient (logP), protein binding constant, inclusion complex constant with cyclodextrin (CD), and selfassociation are very important in drug design, candidate selection, and drug delivery. Capillary electrophoresis (CE) is a simple, versatile, automated, and powerful separation technique and widely applied in physicochemical profiling for pharmaceuticals. It has advantages over traditional potentiometric, spectrophotometric, chromatographic, and other methods, as CE requires very small amounts of sample and can measure compounds with impurities and low aqueous solubility. Principles and applications of CE in profiling various physicochemical properties will be reviewed.

Indirect resolution of chiral molecules, based upon pre-column derivatization and diastereomer formation using benzofurazan bearing chiral labeling reagents, by high-performance liquid chromatography are described in this minireview. The synthesis, characteristics and application of the fluorescent chiral derivatization reagents for various functional groups, i.e. amine (NBD-PyNCS, DBD-PyNCS, DBD-β-Pro, DBD-hydroxyproline), carboxyl (NBD-APy, DBD-APy, ABD-APy), carbonyl (NBD-ProCZ, DBD-ProCZ), hydroxyl (NBD-Pro-COCl, DBD-Pro-COCl) and thiol, etc., are including in the text.

Sample preparation is essential for isolating desired components from complex matrices and greatly influences their reliable and accurate analysis. Solid-phase microextraction (SPME) is a new and effective sample preparation technique. Fibers and capillary tubes coated with an appropriate stationary phase are usually used for SPME, but alternative microextraction techniques, including solid-phase dynamic extraction using an internal coated needle, microextraction in a packed syringe and stir-bar-sorptive extraction using a coated magnetic stir bar, have been developed recently. These techniques, in combination with gas chromatography (GC), GC-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC), LC-MS or capillary electrophoresis, can be used for analysis for complex mixtures. These microextraction techniques save preparation time, as well as solvent purchase and disposal costs. This review summarizes recent advances in SPME and related microextraction techniques and their applications in pharmaceutical and biomedical analysis.

Eosinophils are major effector cells for the development of severe allergic conjunctival diseases (ACD), such as vernal keratoconjunctivitis. Recruitment of eosinophils into the conjunctiva is mediated by chemokines and cytokines. In this review, using an animal model for ACD (experimental immune-mediated blepharoconjunctivitis, EC), the roles of cytokines for eosinophil infiltration into the conjunctiva are presented. Using cytokine knockout mice, importance of endogenous IL-4 for eosinophil infiltration was confirmed. In contrast, endogenous IFN-γ was identified to be inhibitory for eosinophil infiltration. When EC was induced by transfer of in vitro-stimulated antigen (Ag)-primed lymphocytes, then the addition of IL-4 to the culture augmented eosinophil infiltration. Systemic administration of IFN-γ suppressed EC only when IFN-γ was injected during the induction phase. Concerning cytokines in the conjunctiva, expression of Th1 cytokines increased timedependently, whereas that of Th2 cytokines peaked at 12 hours after Ag challenge. In accord with the data, macrophage infiltration increased in a time-dependent manner, while eosinophil infiltration peaked at 12 hours. Subconjunctival injection of IL-4, as well as eotaxin, induced eosinophil infiltration into the conjunctiva. This IL- 4-induced eosinophil infiltration was inhibited by co-injection of IFN-γ. Taken altogether, both systemic and local IL-4 is important for eosinophil infiltration into the conjunctiva and is regulated by IFN-γ.

In 1988 Yanagisawa described endothelins, a new class of vasoconstrictor agents produced by endothelial cells. Further biological effects of these peptides have subsequently been demonstrated, for example, induction of cell proliferation and fibrosis. Two types of endothelin receptors have been described: ET are responsible for endothelininduced vasoconstriction whereas ET induce endothelial cells to release nitric oxide (NO) and prostacyclin. Many antagonists of endothelin receptors have been synthesized and evaluated in animal models and in humans. Satisfactory results have been obtained in animal models of arterial hypertension, pulmonary hypertension, stroke and heart failure but clinical trials have failed to demonstrate that these drugs have a beneficial effect in the treatment of heart failure and a dose-dependent reversible hepatic toxicity has been observed. However, the efficacy of bosentan, a mixed antagonist of endothelin receptors, in the treatment of primary pulmonary hypertension has been demonstrated and the drug is now marketed worldwide for this condition. Further studies are ongoing to evaluate other clinical applications of these drugs. Recently it has been reported that atrasentan, a selective ET antagonist, delayed the progression of hormone-refractory prostate cancer in humans. This review describes the results of the studies performed in animals and in humans and their potential future clinical applications.

In a non-chiral environment, the enantiomers of a racemate possess the same physico-chemical properties but in the biological systems they possess different activities. One of the enantiomers may be more toxic or carcinogenic, and, therefore, the present data available on the toxicity and carcinogenesis of the racemic mixtures of these chiral pollutants are not reliable and need modification in terms of the enantioselective toxicity and carcinogenesis. It is essential to explore the enantioselective toxicity and carcinogenesis due to the different enantiomers of the chiral pollutants. The knowledge of the stereoselective metabolisms of the chiral pollutants may be useful for the treatment of cancer and other diseases. The enantioselective toxicity and carcinogenesis due to the chiral pesticides, pollutants and some drugs have been discussed in this review article.