An improved approach to determining the yield of derivatization reaction and its application to the investigation of the silylation of some anabolic steroids

An approach to determining the yield of derivatization reaction is based on a comparison of chromatographic peak areas of the derivative and native (underivatizied) compound. In contrast to the previous publication [J. Anal. Chem., 2011, vol. 66, no. 12, pp. 1186–1189], ratio of the sensitivity coefficients of the derivative and native forms of the analyte was calculated using only experimental data obtained upon varying the derivatization conditions (solution containing equal amounts of underivatized compound and a respective derivative was analyzed previously). The approach was used to investigate the influence of the reaction time and the type of an external action on the yield of the derivatization (silylation) reaction for some anabolic steroids (methyltestosterone, methandienone, oxandrolone and oral-turinabol) containing a hindered tertiary hydroxyl group at C17. The amounts of the derivatized steroids were equal to about 20–60 ng (depending on the component). Steroids were derivatized with a mixture of pyridine and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane. The derivatization reaction was carried out for 15, 30 or 45 min under conventional heating, sonication at room temperature, and sonication at elevated temperature.