Pitfalls and solution for simultaneous determination of estrone and 17α-ethinylestradiol by gas chromatography-mass spectrometry after derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide

A procedure for the simultaneous determination of both natural and synthetic estrogenic steroids (estrone and 17α-ethynylestradiol) in water is described. Analytes were derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) + trimethylchlorosilane (TMCS) in pyridine solution and determined by GC-MS. The effects of solvent, temperature, and reaction time on the derivatization of EE2 were investigated in order to prevent the formation of undesired multiple derivatization products and conversion of trimethylsilyl (TMS) derivatives of EE2 formed to their respective El derivatives reported in previous studies. The identity and mechanism of formation of multiple derivatives have been discussed. A new silylation mixture (BSTFA:TMCS:pyridine = 49.5:05:50, v/v/v) was employed to overcome these pitfalls and generate a single product of di-TMS derivative of EE2. In the presence of pyridine, variations in reaction temperature (25-80 °C) and time (30 min versus 60 min) did not significantly affect the derivatization efficiency and stability of the di-TMS derivative of EE2 formed. A good linearity of response in the concentration range from 1.0 to 800 μg/mL estrogens with a coefficient R2 > 0.996 at 50 °C for quantitative analysis was also achieved.