Quantitative LC–MS/MS method for the sensitive and simultaneous determination of natural hormones in bovine serum

We have developed a liquid chromatography tandem mass spectrometric (LC–MS/MS) method for the simultaneous quantitative analysis of several free forms of steroid hormones in bovine serum [pregnenolone (P5), progesterone (P4), 17hydroxyP5, 17hydroxyP4, testosterone (T), dehydroepiandrosterone (DHEA), androstenedione (A), estrone (E1), 2, 4 and 16 hydroxyE1, 2 and 4 methoxyE1]. Deuterated analogs were used as internal standards. Serum proteins were eliminated with acetonitrile. Oxime derivatives of steroids were extracted with tert-butylmethylether and analyzed in positive MRM mode. Methodology was validated in accordance with the European Commission Decision 2002/657/EC. Performance characteristics permit the use of this methodology for steroid determination in animal serum samples.