Application of UPLC–MS/MS for separation and quantification of 3α-Hydroxy Tibolone and comparative bioavailability of two Tibolone formulations in healthy volunteers

A novel, fast, sensitive and robust method based on ultra-performance liquid chromatography coupled to atmospheric pressure electrospray ionization tandem mass spectrometry (UPLC–ESI-MS/MS) has been developed to separate two Tibolone stereoisomers i.e., 3α-Hydroxy Tibolone and 3β-Hydroxy Tibolone and to quantify 3α-Hydroxy Tibolone using p-toulenesulfonyl isocyanate (PTSI) as a derivatizing reagent in human plasma. 3α-Hydroxy Tibolone-13CD3 was used as an internal standard (IS). The analyte and IS were extracted from human plasma by liquid–liquid extraction using ethyl acetate. Extracted samples were analyzed by UPLC–ESI-MS/MS. Chromatography was performed using binary gradient on UPLC analytical column. A linear calibration curve over the range of 0.100–35.000 ng/mL was obtained and lower limit of quantification (LLOQ) was 0.100 ng/mL demonstrating acceptable accuracy and precision. This method was successfully applied to a pharmacokinetic study in order to compare a test Tibolone 2.5 mg formulation vs. a reference 2.5 mg Tibolone tablet formulation in 50 post-menopausal/surgical menopause female human volunteers under fasting conditions. It is concluded that test formulation of Tibolone is bioequivalent to reference formulation of Tibolone.