By-products of steroid synthesis: a cause of interferences in thin-layer chromatography residue analysis

Abstract

Since the late 1980s all of the laboratories involved in high-performance thin-layer chromatography (HPTLC) control of hormonal residues in kidney fat, have occasionally detected a green fluorescent spot with similar R_F values and colour to those observed for methyltestosterone (MT). This spot (product) could lead to false positive results for MT and was thus named ‘le faux méthyl’(the false methyl) by a French-speaking colleague. All of the samples with a false methyl spot also contained a relatively high concentration of progesterone. Differentiation of this product from methyltestosterone can be performed in three ways: firstly, extra HPTLC on reversed-phase plates, secondly, extra purification of the extract with HPLC prior to HPTLC and thirdly, gas chromatography–mass spectrometry. This interference was identified as 20β-hydroxyprogesterone, a by-product of progesterone. The problem of the false methyl was not only linked with the TLC characteristics of MT but also to the progesterone used as standard. Some laboratories used an analytical-reagent grade standard and others used commercial progesterone powders as standards (e.g., obtained in crude form from pharmaceutical companies). The commercial-grade progesterones showed two spots in comparison with the analytical standard that showed just one spot. As the false methyl was observed not only in kidney fat and meat samples, but also in illegal hormone cocktails, it was concluded that we had detected a by-product of an illegally used ‘natural progesterone’.