LC–MS/MS determination of betamethasone and its phosphate and acetate esters in human plasma after sample stabilization

Two specific liquid chromatography–mass spectrometric (LC–MS/MS) assays were developed and validated for the determination of betamethasone (BET), and its acetate (BA) and phosphate (BP) esters. The plasma and the blood used for the development and validation of these two methods were previously stabilized. Liquid–liquid extraction techniques were used after the addition of prednisolone as internal standard (IS). Samples were chromatographed using C8 column, while mass detection was carried out by electrospray ionization in the positive mode (ESI+). The method was proved linear over a working range 0.50–50.00 ng/ml for BET (r2 > 0.99), while BA linear range was 1.0–20.0 ng/ml (r2 > 0.99). Sensitivity was determined as 0.50 ng/ml for BET and 1.00 ng/ml for BA. Betamethasone phosphate LC–MS/MS method involved solid phase extraction after the addition of prednisolone phosphate as (IS). Separation was carried out using C18 column, while detection was by ESI+. The method showed good linearity over the working range 2.0–200.0 ng/ml (r2 > 099). Both methods were applied to determine BET, BA and BP in plasma samples obtained for pharmacokinetics studies in human.