A new approach to determining derivatization degree and its use for the investigation of silylation of methyltestosterone in nano-/microgram amounts

A new approach to establishing the yield of derivatization reactions of difficult-to-derivatize steroids is proposed on the basis of the comparison of the areas of chromatographic peaks between the native form of the analyte and its derivative. The influence of material to be derivatized, i.e., methyltestosterone, in an amount of 5 and 500 ng, and the time of reaction, 5–120 min, on the degree of conversion is studied. Methyltestosterone is derivatized at 80°C in a mixture of pyridine and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane. Prior to the injection of samples into the gas chromatograph, pyridine and BSTFA are replaced with an inert organic solvent, methyl tert-butyl ether. The degree of methyltestosterone derivatization is shown statistically independent of the amount of the steroid processed. The performance of the reaction for 1 h at 80°C ensures reaction yield of around 90%.