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 by gas chromatography–mass spectrometry

• Published in: Journal of Chromatography A 2006-03, Vol.1108 (1), p.121-128
• Main Authors: Shareef, Ali, Angove, Michael J., Wells, John D.
• Format: Article
• Language: English
• Subjects: Analysis; Biological and medical sciences; Estrone; Ethinylestradiol; GC–MS; General pharmacology; Medical sciences; Pharmacology. Drug treatments; Silylation
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2005.12.098

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.