Current Pharmaceutical Analysis - Volume 2, Issue 4, 2006

Proteoglycans are ubiquitous biomolecules in the body located in the extracellular matrix, on the cell surface and also within the cells. They contain at least one glycosaminoglycan (GAG) chain covalently attached to a core protein and may also present N- or O-linked glycans. The high structural diversity and distribution relate to the various biological functions of proteoglycans. In recent years, new members have enlarged the proteoglycan family and advances in molecular biology and glycobiology contributed to elucidate more of the biological functions of proteoglycans. In order to study the structure of a proteoglycan molecule and relate it to its function (or dysfunction), its isolation and purification from cell culture or tissue extracts is necessary. Next to the widely used anion exchange chromatographic methods, techniques based on lectin affinity chromatography have created new possibilities to increase the degree of purity. Introduction of electrospray ionization (ESI) and matrix-assisted laser desorption-ionization (MALDI) sources, together with tandem mass spectrometry (MS/MS or MSn), mark a further important step towards the structural analysis of glycosaminoglycans. The aim of this review is to present the most recent advances in proteoglycan purification and analysis.

Radiopharmaceutical chemistry has addressed the issue of biomolecular chemistry, and radiopharmaceuticals are unique in their ability to monitor receptor binding sites and enzymes. The future of diagnostic and therapeutic nuclear medicine, to study the in vivo metabolism, is focused on the use of radiolabeled protein fragments, peptide structures and DNA chains. These radiolabeled molecules represent a substantial change in the paradigms of the pharmaceutical development by employing, as a source of pharmaceuticals, capabilities of our own body, instead of considering it as a simple test-tube where strange molecules interact. Research on biomolecules complexed to radioactive metals, like Tc- 99m, Re-188, Lu-177 and Y-90, that do not alter the molecular specificity, is a topic of world interest in radiopharmaceutical investigations. Some perspectives and achievements on the preparation and analysis of main diagnostic and therapeutic radiopharmaceuticals of the third generation are presented.

This review article underlines the importance of stable isotope dilution mass spectrometry analysis of endogenous and synthetic corticosteroids for the pharmacokinetic and in vivo metabolic investigations in humans. The multi-labeled corticosteroids with stable isotopes such as [1,2,4,19-13C4]cortisol, [1,2,3,4-13C4]cortisol, [1,1,19,19,19- 2H5]cortisol, [9,11,12,12-2H4]cortisol, etc. have been used not only as analytical internal standard for the mass spectrometry but as tracer in the pharmacokinetic and metabolic studies in human in vivo. Stable isotope dilution analysis of cortisol metabolites (tetrahydrocortisol (THF), allo-tetrahydrocortisol (allo-THF), tetrahydrocortisone (THE), and 6β - hydroxycortisol (6β-OHF)) and synthetic corticosteroids (prednisolone, prednisone, budesonide, fluticasone, etc.) is also the subject of this review. In the gas chromatographic mass spectrometric (GC-MS) analysis of corticosteroids, it is usual to employ derivatives in which some or all of the original functional groups are protected. Thermal stabilization of corticosteroids in the analysis has been achieved most successfully by formation of methoxime-trimethylsilyl (MO-TMS) derivatives. Bismethylenedioxy-pentafluoropropionyl (BMD-PFP) derivatives have been developed to simultaneously measure cortisol, cortisone, and their tetrahydrocorticoid metabolites (THF, allo-THF, and THE) and/or prednisolone and prednisone in plasma and urine. Stable isotope tracer methodology has been applied to the measurements of cortisol production rate, determination of human urinary cortisol metabolites, and assessment of in vivo activities of 11β- hydroxysteroid dehydrogenase (11β-HSD) in humans. This methodology also has been used for the validity of endogenous cortisol 6β-hydroxylation clearance as a new index for phenotyping the in vivo cytochrome P450 3A (CYP3A) in humans.

Research into the oxidation of lipoprotein has yielded many insights into the underlying process of the development of atherosclerosis. Oxidative modification of low density lipoprotein (LDL) has been suggested as an initial step in the pathogenesis of atherosclerosis. However, up until now, investigations of antioxidants have mostly focused on three main dietary antioxidant vitamins (β-carotene, vitamin C, and vitamin E) and some synthetic compounds. Among those antioxidants, probucol, a synthetic compound, has been shown to be an extremely potent and effective antioxidant in preventing the formation of atherosclerosis in both in vitro and in vivo studies. The present review focuses on commonly used analytical methods for measuring the antioxidant potency and outlines the critical steps on how to evaluate and design a potent antioxidant agent that can be used for the intervention of atherosclerosis. We concluded that an antioxidant should first be targeted and incorporated into human LDL. Second, the candidate compound should possess high bioavailability. The rationale and strategy for the analytical procedures are discussed in this report.

Sample preparation represents a major challenge and a very important step in the development and application of an analytical method. It is now widely accepted that sample preparation is the most time-consuming and often the costliest step in an analytical process. In addition it is the most labour-intensive and the most error prone. In the post genome era new analytical technologies are sought to address the upcoming needs: proteomics, metabolomics, holistic analytical approaches, high-throughput screening, biomarker discovery and drug discovery. Sample preparation is still in certain cases one of the bottlenecks of the data generation. However new exciting technologies and developments are continuously emerging. The present review will try to discuss critically the status and the limitations of existing methods and highlight the potential and the advantages of selected novel technologies for sample preparation and analyte detection. Emphasis is put on sample preparation methods that are used in bioanalysis in combination with separation techniques. The review will try to highlight trends in automation, derivatisation, microextraction and the implementation of molecular recognition mechanisms in bioanalytical separations. The latter includes molecular imprinting, affinity chromatography and biospecific analyte detection.

Process Analytical Technologies (PAT) are used to provide timely analysis of critical quality parameters with the end goal of improving final product quality as well as reducing manufacturing costs, thereby significantly benefiting the Pharmaceutical Industry. PAT involves mostly on-line or in-line testing, which can be an invasive or non-invasive process that analyzes the sample while it is part of the process stream. There is no sample preparation in the testing, thus saving time and avoiding possible errors in sample preparations. PAT uses vendor specifications based on science and common sense to exhibit, ‘fit for purpose ’ rather than conventional USP paradigm, ‘one specification fits all,’ which is based upon documentation but lacking in scientific logic. The current FDA guidance is to adapt its traditional regulatory scrutiny to PAT advancement. The Pharmaceutical Industry is using innovative PAT methods to improve the logical basis for establishing regulatory specifications, promoting continuous improvement and improving manufacturing. Multivariate tools are used in PAT for design, data acquisition and analysis. These tools are used in conjunction with statistical design of experiments, response surface methodologies, and process simulation and pattern recognition under the control of various knowledge management systems. For example, NIR Spectroscopy is used in drug product production at almost each step of tablet manufacturing from raw materials control to content uniformity analysis of the dosage unit. The spectral data set is usually multivariate in nature. This is due not only to the different ranges and peaks in a spectrum, which are influenced by the nature of the product but also due to several interlinked factors that influence the appearance of the spectrum. When NIR is used in PAT, the materials are blended to uniformity rather than a fixed time. This type of advance control gives better batch-to-batch consistency and better product quality, which eliminate reworks/rejects, and there are no Out Of Specification (OOS) reports to file under PAT. The product is “engineered for Quality”, as opposed to “tested to Quality”.