Current Pharmaceutical Analysis - Volume 2, Issue 1, 2006

In this review, the analytical applications of carbon paste electrodes for pharmaceutical analysis are summarized, and future prospects examined, especially in connection with chemically and biologically modified carbon paste electrodes.

Capillary electrophoresis, as an analytical tool for drugs, offers several advantages for pharmaceuticals and clinical applications; however, it suffers from poor detection limits. Concentration on the capillary (stacking) improves greatly this problem and is very easy to perform. One of the simple and practical methods to perform stacking is dissolving the sample in organic solvents and injecting a large volume of sample on the capillary. This leads to concentration of the sample 10-30 folds directly on the capillary, removes the excess of proteins found in biological fluids and overcomes the deleterious effects of salts. The stacking can be performed in both the hydrodynamic and electroinjection. This stacking brings the detection limits of the CE closer to that of the HPLC. The mechanism, practical applications, different factors, and optimum conditions for this type of stacking are reviewed and discussed.

A method for determining the concentration of cefepime, a cephem anti-microbial agent, by direct serum injection using micellar electrokinetic capillary chromatography is developed and its validation of the assay is studied. A borate buffer (100 mM; pH 8.0) containing sodium dodecyl sulfate (80 mM) is used as a run buffer. Ultraviolet detection is carried out at 260 nm. The migration time of cefepime is about 5.2 min. The limit of detection is 0.5 mg/l (S/N=3). The RSD's of intra-day and inter-day precisions are 1.57-4.08 % (6.8-94.2 mg/l) and 1.47-2.75 % (10.2-81.5 mg/l), respectively, and the recovery rate is 94-108%. 13 other cephem anti-microbial agents are measured under the analytical conditions of this method and the results show that cefatrizine, whose migration time coincides with that of cefepime, interferes with the determination of cefepime. This method is characterized by the ability of allowing analysis by the direct injection of serum samples of micro-quantities into the capillary.

The neccessity to derivatize polar analytes prior to separation often disqualifies gas chromatography (GC) as a method of choice in the field of biomedical/pharmaceutical analysis. Laborious and lengthy protocols for treating compounds prior to the analysis were discouraging. Only few derivatization approaches were well-established over decades, primarily silylations. To its assets belongs universality and efficacy, to shortcomings necessity for dry residue and prolonged reaction time, often under heating. Similarly, the next field-proven esterification-acylation two-step procedures suffered from the same pre-requisites. Current investigations in the field of derivatization turned attention to chemical reactions proceeding in aqueous environment and obviating multiple reaction steps and heating. Application of alkyl chloroformates (RCF), under conditions discovered more than a decade ago, met such criteria. Instantaneous conversion of hydrophilic compounds to organophilic ones became often an integral part of sample preparation procedures with negligible time and costs required. This review attempts to bring forward some of the most important studies on RCF-mediated derivatizations in the last decade and to figure out general utility of the approach in analyzing polar organic compounds by GC, with particular attention to polyfunctional organic acids, especially amino acids (AAs).

Amyloidosis refers to a heterogeneous group of disorders characterized by the extracellular deposition of amyloid proteins in various tissues of the body. About 25 different amyloid proteins originating from presumably normal precursor proteins have been described so far. Identification of the chemical nature of amyloid is necessary in clinical practice, since both prognosis and treatment regimens are different in the various amyloidoses. This review is aimed at summarizing the new biochemical micro-techniques for precise typing of amyloid proteins in diagnostic biopsy specimens. The reported micro-techniques vary with respect to the state of the amyloid-bearing tissue, i.e., unfixed-frozen or formalin-fixed, the purification strategy (chromatographic or electrophoretic) and the analytical method used (ELISA, Western blotting, amino acid sequencing, mass spectroscopy). The utility and advantages of the described protocols are discussed with regard to tissue state, sample size, amyloid content, simplicity of procedures and possible applicability in diagnostic laboratories.

Chloramphenicol (CAP) is a broad-spectrum antibiotic, effective against a wide range of microorganisms, and has widely been used since the 1950's to treat food-producing animals. However, CAP has been rapidly associated to serious toxic effects, especially bone marrow depression, particularly severe when it assumes the form of the doseindependent and fatal aplastic anaemia. The well-known risk of irreversible bone marrow disorders and the absence of safe residue levels has determined European Union (EU) prohibition for veterinary use, in 1994, and no maximum residue limit (MRL) has been established for this antibiotic. Despite this legal ban, CAP has recently been found in several animal-derived foods, mainly aquaculture products. In order to prevent illegal use, it is important to develop analytical methodologies that provide performance limits capable of detecting and quantifying residual levels of this antibiotic. In this paper, the authors present a review of the most relevant procedures for sampling, pre-treatment, extraction/purification and determination of CAP residues in different food matrixes, published in the literature for the past decade. A special focus will be given to gas chromatography - mass spectrometry (GC-MS) and liquid chromatography - tandem mass spectrometry (LC-MS/MS), as they represent the most modern and reliable analytical methodologies for determination of CAP residues.

The complete spectrum of active compounds of Hypericum perforatum (St. John's wort) has not yet been fully elucidated, but some naphthodianthrones (hypericin and pseudohypericin), phloroglucinols (hyperforin) and flavonoids (primarily quercetin glycosides) are thought to be essential for the antidepressant activity of its extracts. Some of these compounds modulate the expression of the P-glycoprotein transporter or of cytochrome P450 enzymes or both although, again, their real role in clinically relevant interactions involving the extracts and conventional drugs is still not clear. Analysis of main components in body fluids and tissues has thus become of interest in studies to identify and characterize those accounting for the primary and secondary pharmacological effects, which can help in providing safe and effective dosing regimens with the appropriate extracts. Selective procedures such as high-performance liquid chromatography with fluorimetric, electrochemical, ultraviolet or photodiode detection or coupled with mass spectrometry, depending on the constituent, have been frequently used. This review provides an overview of these methods, and the related sample pretreatment procedures. The focus is also on their sensitivity and precision and consequently their reliability for pharmacokinetic studies in relation to the blood and, when animal data are available, brain concentrations of the main active components of Hypericum perforatum extracts.

F2-isoprostanes (F2-isoPs) represent a new family of biomarkers for oxidative stress generated by free radical attack of membrane-bounded arachidonic acid. Esterified F2-isoPs can be found in tissue or plasma lipids whereas the free form F2-isoPs, hydrolyzed by phospholipase, is mainly present in body fluids. The extent of systematic damage due to oxidative stress within the body can be assessed by the determination of plasma or urine F2-isoPs. The determination of F2-isoPs in clinical practice is not often used due to the complexity to extract the compounds from their biologic matrixes before the analysis step. In most of published protocols, extraction procedure is critical and timeconsuming, requiring successive chromatographic steps. Moreover, some of these procedures lead to a substantial loss of target compounds. In order to improve sample preparation steps and final recovery, others methods have been developed and optimized. For detection of F2-isoPs, two main analytical approaches have been adopted. The first one involves immunological methods and the second approach is based on chromatographic separation and detection by mass spectrometry. A large amount of works has been done in the field of isoprostane analysis, but until now, no standardized method seems to emerge. Indeed, described methodologies differ either in the sample preparation steps or in the detection techniques or both. In the present review, the most commonly used methods are presented and compared in terms of extraction, purification, and analysis of F2-isoPs, taking into account the various origins of biological samples.

The concept, molecular self-assembly, especially considering molecule-molecule interaction as an informationprocessing phenomenon, have a profoundly novel effect on thoughts and efforts related to Medicine and Pharmacology. This new style of thinking is still too novice to be used solely and independently for explanation of disease mechanisms and appropriate treatment strategies. However it calls for a range of new researches based on new predictions about disease mechanisms (especially Autoimmune diseases, Endocrinopathies, and Neoplasms) and relevant treatment strategies (superstructural drugs).

Immortalized cell lines that retain differentiated functions are required to study tissue functions at cellular and molecular levels. Transgenic animals, mice and rats, harboring temperature-sensitive simian virus 40 large T-antigen have been found to be very useful for establishing conditionally immortalized cell lines from tissues that have proved difficult to culture in vitro. Thus far we have succeeded in establishing many kinds of conditionally immortalized cell lines with differentiated functions from the transgenic animals. Novel DNA microarray technologies allow the simultaneous measurement of changes in expression of many hundreds or many thousands of genes. Using established cell lines and DNA microarrays, we have identified many genes that are differentially expressed in the process of cell differentiation and cell death. In this review, we would like to introduce the characteristics of established gastrointestinal and testicular cell lines and discuss possible applications of these cell lines to differential gene expression analysis by DNA microarray technology.