Confirmation of hormones in animal serum by liquid chromatography/tandem mass spectrometry according to european commission decision 2002/657

Abstract

A novel and rapid method was developed and validated for the confirmation of endogenous and synthetic hormones in animal serum using LC/MS/MS. Detection of 17 β-estradiol and β-testosterone below the respective European Union-recommended levels of 0.1 and 0.5 μg/L was achieved, as was a required performance level of 0.1 μg/L for 17 a-estradiol and 0.5 μg/L for 17 a-testosterone, medroxyprogesterone17-acetate, and progesterone. The method was established with dilution of serum followed by ionexchange SPE, LC separation and MS detection with electrospray ionization, selected reaction monitoring, and positive/negative switching. Two characteristic transitions were monitored for each analyte. The method was applied to bovine, ovine, porcine, equine, and avian samples and validated according to European Commission Decision 2002/657/EC and accepted for ISO/IEC 17025:2005 accreditation. An extended calibration curve allows naturally occurring levels of endogenous hormones to be quantified. Recoveries ranged from 97.3% for 17 a-testosterone to 102.0% for 17 a-estradiol. The decision limit CCa ranged from 0.02 ug/L for 17 a- and β-estradiol to 0.12 μg/L for progesterone. Detection capability CCβ ranged from 0.03 μg/L for 17 a-estradiol to 0.20 μg/L for progesterone.