Confirmatory analysis of acetylgestagens in plasma using liquid chromatography-tandem mass spectrometry

A confirmatory method has been developed and validated for the determination of chlormadinone acetate (CMA), megestrol acetate (NIGA), melengestrol acetate (MLA) and medroxyprogesterone acetate (MPA) in bovine and porcine plasma. Analytes are extracted from plasma samples using matrix-assisted liquid-liquid extraction (LLE) on Extrelut NT columns followed by C18 solid-phase extraction (SPE).

Analytes were analysed using liquid chromatography-tandem mass spectrometry (I-C-MS/MS), and quantification was performed using matrix-matched calibration standards in combination with deuterated internal standards. In accordance with Commission Decision 2002/657/EC, two ion transitions were monitored for each analyte.

Decision limits (CC alpha) were estimated by analysing 20 blank plasma samples and ranged from 0.1 to 0.2 ng mL(-1). Detection capabilities (CC beta) were estimated using 20 plasma samples fortified at 0.5 ng mL(-1) and were <0.5 ng mL(-1). In the range 0.5-2 ng mL(-1), the mean intra-laboratory reproducibility of the analytes ranged from 6 to 18% (%R.S.D.).

Analytes were shown to be stable in fortified plasma samples for > 8 months when stored at -20 degrees C.