Nieuwe ontwikkelingen in de behandeling van de complicaties van HIV-infectie : 4. HIV-infectie en AIDS bij vrouwen

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Rapid, sensitive and specific derivatization methods with 9-(hydroxymethyl)anthracene for the fluorimetric detection of carboxylic acids prior to reversed-phase high-performance liquid chromatographic separation

Three derivatization procedures are described for the pre-column fluorescence labelling of carboxylic acids. The methods are based on esterification with 9-(hydroxymethyl)anthracene. The carboxylic acid function is activated with 2-bromo-l-methylpyridinium iodide, N,N′-carbonyldiimidazole or N-ethyl-N′-(3-dimethylanopropyl)carbodiimide hydrochloride, respectively. Benzoic acid was completely converted into the corresponding ester with all three methods. About 100 fmol of the acid could be detected after high-performance liquid chromatographic analysis of the derivative. The methods are well suited for the analysis of carboxylic acids in plasma

Development of an on-line size exclusion chromatographic - reversed-phase liquid chromatographic two-dimensional system for the quantitative determination of peptides with concentration prior to reversed-phase liquid chromatographic separation

Complex samples, containing endogenous peptides require analytical methods with high sensitivity and selectivity to enable reliable analysis of these compounds in biological samples. In a number of cases, the selectivity of mono-dimensional separation systems is not sufficient. In order to improve the separation efficiency two- or multi-dimensional systems can be the solution. The present study aims at the development and validation of an on-line two-dimensional separation system, using size exclusion chromatography (SEC) and reversed-phase liquid chromatography (RP-LC), to quantitate structurally related peptides in the presence of large proteins such as albumin in contrast with the available coupled-column systems which are focussed on the qualitative determination of peptides. As model peptides a number of enkephalins have been chosen. Albumin was added to mimict a real biological sample. If the SEC dimension, the RP-LC dimension and the interface have been chosen properly, the entire peak, eluting from the SEC column and containing the enkephalins, can be trapped in a loop after complete separation from albumin. Subsequent separation of the enkephalins is achieved in the RP-LC dimension. Detection is performed using UV absorbance. The procedure is validated with respect to recovery, linearity and intra- and interday precision. The lower limit of quantitation (LOQ) is equal for all enkephalins studied and determined to be 5 ng at a signal-to-noise ratio of 5. Based on the injection volume of 5 μl in the first dimension a concentration LOQ of 1000 ng/ml has been established. Although the developed on-line coupled two-dimensional separation system is suited for the quantitation of various enkephalins in the presence of albumin with a satisfactory linearity, precision and recovery, the method lacks still the sensitivity to allow measurements of endogenous enkephalins in a biological matrix. More sensitive detection methods or miniaturization of the system are being tested currently. The value of the present study, however, is that it demonstrates the potency of the two-dimensional system for quantitative purposes in peptide profiling in order to establish the normal peptide profiles of healthy biosystems and to study quantitative modifications of these profiles in case of diseases. © 2001 Elsevier Science B.V. All rights reserved

On-line multidimensional liquid chromatography and capillary electrophoresis systems for peptides and proteins

Peptides and proteins are gaining increasing attention in biosciences and, consequently, in analysis. This overview highlights the different approaches to couple on-line various separation techniques for the determination of proteins and peptides. The first section discusses the liquid chromatography (LC)-LC coupling, the second one reviews the on-line LC-capillary electrophoresis (CE) coupled systems and the third section summarizes the strategies for on-line CE-CE. The advantages, disadvantages, most relevant difficulties and particular systems for on-line coupling are discussed. Special attention is paid to the interface between the two dimensions. Applications are summarized in tables and a few typical examples are discussed. Many multidimensional separation methods are available, and it is demonstrated that peptide and protein mapping, or quantitation of proteins or peptides in various samples (aqueous solutions, cells, plasma) require different coupled systems. For mapping a semi-quantitative detection is often sufficient, while comprehensiveness is very important. For quantitation of a certain peptide or protein at a low concentration level a validated method should be used, while a heart-cut transport of the first dimension to the second one can offer sufficient selectivity. The combination with mass spectrometry as part of the total system is stressed and illustrated. © 2004 Published by Elsevier B.V

Nieuwe ontwikkelingen in de behandeling van de complicaties van HIV-infectie : 3.Pneumocystis carinii-pneumonie

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Pre-, on- and post-column derivatization in capillary electrophoresis.

This survey gives a short overview of the various reagents and procedures that can be used for pre-, post- and on-column derivatization in capillary electrophoresis. First there is an introduction about capillary electrophoresis as an analytical technique; this is followed by a discussion of the pros and cons of the various modes of derivatization and a comparison with liquid chromatography. In the following paragraphs the reagents for a number of functional groups are discussed. The emphasis is on derivatization of the amine group. Most of the information on the reagents and derivatization procedures is listed in tables together with information on the detection mode, analytes, sensitivity and samples. In addition to the amino group, information is given on labeling of aldehyde, keto, carboxyl, hydroxyl and sulfhydryl groups