Optimization of silylation using N-methyl-N-(trimethylsilyl)-trifluoroacetamide, N,O-bis-(trimethylsilyl)-trifluoroacetamide and N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide for the determination of the estrogens estrone and 17α-ethinylestradiol

This paper reports an improved silylation procedure for simultaneous determination of the steroid hormones 17α-ethinylestradiol (EE2) and estrone (E1) using gas chromatography–mass spectrometry (GC–MS). This follows a re-assessment of some of the popular silylation procedures using N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), N-O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) and N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA), which lead to the formation of trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS) derivatives. Silylation of EE2 using MSTFA or BSTFA + 1% TMCS in ethyl acetate, acetonitrile and dichloromethane solvents produced multiple peaks corresponding to TMS-E1, and 3-mono-TMS-EE2 and/or 3,17-di-TMS-EE2 in variable proportions depending on the solvent used. When pyridine or dimethyl formamide solvents were used in the silylation of EE2 under the same reaction conditions, only 3,17-di-TMS-EE2 derivative was formed. Derivatization using MTBSTFA reagents using ethyl acetate, acetonitrile, dichloromethane, pyridine and dimethyl formamide resulted in almost 100% conversion of mono-TBS-EE2 to the TBS-E1. Therefore, typical methods used in some previous GC–MS determinations of E1 and EE2 in environmental water and/or sediment samples are subject to speculation. However, we can confirm that any of the TMS reagents can be used with either pyridine or dimethyl formamide under suitable reaction conditions.