UPLC-MS/MS determination of steroid hormones via a novel reaction based on derivatisation by a cyclic-organophosphate

Abstract

A cyclic-organophosphate, specifically 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane-2-oxide, was used to derivatise the hydroxyl group at the C3 position of selected steroid hormones to analyse the derivatives using UPLC-MS/MS (ultra-performance liquid chromatography-tandem mass spectrometry). Reactions were performed in an anhydrous pyridine environment in the presence of AlCl3 at 50 °C. The developed reaction is suitable for analytical chemistry applications and was validated by analysis of selected contraceptive drugs. The sensitivity of the method depends on hormone tested and the limit of detection ranges from 130 pg/mL for β-estradiol to 240 pg/mL for estriol. The estimated efficiency of derivatisation reactions varies in the range from 77.5 to 95.7%, and depends upon the hormone undergoing derivatisation. The method's recovery rate for the lowest concentration tested (800 pg/mL) is 88.1–96.3%. The method exhibits linearity in the 390 pg/mL to 2.5 μg/mL range, with R2 = 0.997. The developed steroid hormone derivatisation reaction was validated experimentally using UHPLC-QTOF-MS (ultra-high performance liquid chromatography quadrupole time of flight mass spectrometry) and NMR (nuclear magnetic resonance) spectroscopy. These studies show that the developed derivatisation reaction provides a precise and repeatable determination of selected steroid hormones in contraceptive drugs. At n = 10, CV (Coefficient of Variation) did not exceed 7%, which is a very good result compared with other analytical methods.