title: Alternative methodology for the analysis of progesterone, testosterone, and epi-testosterone in bovine liver and veal muscle.
abstract: 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 microg/kg. Homogenized samples were pretreated with methanol to denature endogenous enzymes. Following removal of methanol, samples were treated overnight with Helix pomatia beta-glucuronidase to deconjugate glucuronide conjugates. Alkali digestion of the samples in KOH solutions was done under shaking at 37 degrees 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 microg/kg) and low (0.3 microg/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.
authors.affiliation: Canadian Food Inspection Agency, Saskatoon Laboratory, Centre for Veterinary Drug Residues, 116 Veterinary Rd, Saskatoon, SK, Canada S7N 2R3. rfedeniuk@inspection.gc.ca
authors.forename: Rick W
authors.initials: RW
authors.lastname: Fedeniuk
authors.forename: Michelle
authors.initials: M
authors.lastname: West
authors.forename: Ron
authors.initials: R
authors.lastname: Gedir
authors.forename: Massey
authors.initials: M
authors.lastname: Mizuno
authors.forename: Constance
authors.initials: C
authors.lastname: Neiser
authors.forename: James D
authors.initials: JD
authors.lastname: MacNeil
mesh_terms: D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D004845:Epitestosterone / Q000032:analysis*; D005966:Glucuronidase / Q000737:chemistry; D006868:Hydrolysis; D007202:Indicators and Reagents; D008099:Liver / Q000737:chemistry*; D013058:Mass Spectrometry; D008460:Meat / Q000032:analysis*; D011374:Progesterone / Q000032:analysis*; D011489:Protein Denaturation; D012015:Reference Standards; D015203:Reproducibility of Results; D012997:Solvents; D013739:Testosterone / Q000032:analysis*
chemical_list: D007202:Indicators and Reagents; D012997:Solvents; D013739:Testosterone; D004845:Epitestosterone; D011374:Progesterone; D005966:Glucuronidase