Quantitative Method for the Determination of 17-Oxosteroid Fractions by Thin-Layer Chromatography

Abstract

17-oxosteroid fractions were determined quantitatively by thin-layer chromatography. A small glass "extractor" was constructed for the rapid extraction of hormones from a silica plate with minimal losses. This apparatus is generally applicable for extracting material from thin-layer plates. The hydrolysis and final Zimmermann reaction were performed in the same way as in the procedure for total 17-oxosteroids. Every fraction was identified on the plate by a coloured reaction, extracted, and the Zimmermann reaction was carried out in the tube in the classical way. The method was proved for accuracy and reproducibility. The sources of steroid metabolites differ according to sex and pregnancy; therefore values for total 17-oxosteroids may very often be in a normal range in spite of great disturbances in the metabolism of these hormones. An attempt was therefore made at the beginning of this work to obtain more precise information on the composition of the 17-oxosteroid fractions and to find a method for the rapid and reproducible separation of individual hormones. Thin-layer chromatography was soon found to be suitable for this purpose with its advantages of simplicity and speed of separation. A good separation of hormones on the plates was achieved (2) and it is possible to separate the following fractions: dehydroepiandrosterone, androsterone, aetiocholanolone, 11-oxoandrosterone, 11-jS-hydroxyandrosterone, 11-oxoaetiocholanolone and ll-/?-hydroxyaetiocholanolone. However, the quantitative determination was much more difficult. The reason for this was the insufficient and uncertain detection of spots (2), a high degree of coloured background and the instability of the colour on the plate (3), or the relatively extended extraction procedure (4). In this paper, for the separation of the fractions on the Chromatographie plate, the technique of BETTER and coworkers (1) was applied while an attempt was made to find a simple and rapid method for the identification and quantitative determination. Procedure