Alternative methodology for the analysis of progesterone, testosterone, and epi-testosterone in bovine liver and veal muscle.

Research has shown that traditional solvent extraction procedures used for the analysis of endogenous steroids often give inconsistent recoveries and results. However, a single-laboratory validation of a liquid chromatography/tandem mass specrometry method using 2 product ions per transition for progesterone, testosterone, and epi-testosterone in bovine liver and veal muscle showed accuracy and precision to within 23% at concentrations ranging from 0.5 to 2.0 μg/kg. Homogenized samples were pretreated with methanol to denature endogenous enzymes. Following removal of methanol, samples were treated overnight with Helix pomatia β-glucuronidase to deconjugate glucuronide conjugates. Alkali digestion of the samples in KOH solutions was done under shaking at 37°C for 30 min. The digestate was extracted with methyl tert-butyl ether, and the extracts were cleaned by partitioning between acetonitrile-hexane, followed by solid-phase extraction cleanup on silica cartridges. In bovine liver, average recoveries exceeded 54% for all analytes, and the within-run assay coefficients of variations were <6 and 13% for high (2.0 μg/kg) and low (0.3 μg/kg) analyte concentrations, respectively. In veal muscle, average recoveries exceeded 60%, and the analysis of blind spikes gave accuracy estimates of over 85%, with coefficients of variation (CVs) <15% for all analytes. The CVs for the multiple reaction monitoring ion ratios for all compounds were <22% for all validation data. The method meets the requirements for confirmatory methods as outlined in 2002/657/EC. An analyst is capable of processing up to 20 samples within 5 days.