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Thin‐layer chromatography—the forgotten alternative for the quantitative determination of steroids
Author(s) -
Golf S. W.,
Graef V.,
Schiller J. Th.,
Hischer H.,
Funk W.
Publication year - 1987
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.1130020503
Subject(s) - chemistry , chromatography , derivatization , thin layer chromatography , steroid , detection limit , high performance liquid chromatography , hormone , biochemistry
We have developed a high performance thin layer chromatography (HPTLC) system for quantitative determination of androgens, corticosteroids, mineralocorticoids and gestagens on silicagel KG‐60 HPTLC‐plates with different solvent systems. A complete separation of androgens, gestagens and metabolites was achieved with dichlormethane/cyclohexane/acetone (70:25:5). Corticosteroids, mineralocorticoids and their derivatives were completely separated with diethylether/isooctane/isopropanol (70:25:5). The quantitative in situ fluorescence determination was carried out after post‐chromatographic derivatization with cinnamic aldehyde, 4‐dimethylaminobenzaldehyde and sulfuric acid. The sensitivity of detection was found between 500 pg and 1 ng per spot. The steroid metabolism as catalysed by rat liver microsomal oxidoreductases was measured by these procedures, and was compared with determination of steroids by gas chromatography (GC). According to HPTLC, steroids were reduced by NADPH‐5α‐reductase (EC 1.3.1.4) in the order progesterone > testosterone > aldosterone > cortisol > corticosterone. The enzyme activities as measured by HPTLC agree well with those obtained by GC ( r = 0.94). When turnover of enzyme assays, speed of determination, detection limit, application to labile steroids and costs of steroid determination are considered, all points speak in favour of HPTLC.