Use of non-specific and specific interactions in the analysis of testosterone and related compounds
By addebook • Jun 28th, 2008 • Category: Chemistry •
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Use of non-specific and specific interactions in the analysis of testosterone and related compounds by capillary electromigration techniques
by Lotta K. Amundsen
ISBN 978-951-38-7085-0 (soft back ed.)
ISSN 1235-0621 (soft back ed.)
Copyright © VTT Technical Research Centre of Finland 2008
Page: 114
Determination of testosterone and related compounds in body fluids is of utmost
importance in doping control and the diagnosis of many diseases. Capillary
electromigration techniques are a relatively new approach for steroid research.
Owing to their electrical neutrality, however, separation of steroids by capillary
electromigration techniques requires the use of charged electrolyte additives that
interact with the steroids either specifically or non-specifically. The analysis of
testosterone and related steroids by non-specific micellar electrokinetic
chromatography (MEKC) was investigated in this study. The partial filling (PF)
technique was employed, being suitable for detection by both ultraviolet
spectrophotometry (UV) and electrospray ionization mass spectrometry (ESI-MS).
Efficient, quantitative PF-MEKCUV methods for steroid standards were
developed through the use of optimized pseudostationary phases comprising
surfactants and cyclodextrins. PF-MEKCUV proved to be a more sensitive,
efficient and repeatable method for the steroids than PF-MEKCESI-MS. We
discovered that in PF-MEKC analyses of electrically neutral steroids, ESI-MS
interfacing sets significant limitations not only on the chemistry affecting the
ionization and detection processes, but also on the separation. The new PF-MEKCUV
method was successfully employed in the determination of testosterone in male
urine samples after microscale immunoaffinity solid-phase extraction (IA-SPE).
The IA-SPE method, relying on specific interactions between testosterone and a
recombinant anti-testosterone Fab fragment, is the first such method described
for testosterone. Finally, new data for interactions between steroids and human
and bovine serum albumins were obtained through the use of affinity capillary
electrophoresis. A new algorithm for the calculation of association constants
between proteins and neutral ligands is introduced.
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