Quantitation of 17beta-nandrolone metabolites in boar and horse urine by gas chromatography-mass spectrometry

A method to quantify metabolites of 17beta-nandrolone (17betaN) in boar and horse urine has been optimized and validated. Metabolites excreted in free form were extracted at pH 9.5 with tert-butylmethylether. The aqueous phases were applied to Sep Pak C18 cartridges and conjugated steroids were eluted with methanol. After evaporation to dryness, either enzymatic hydrolysis with beta-glucuronidase from Escherichia coli or solvolysis with a mixture of ethylacetate:methanol:concentrated sulphuric acid were applied to the extract. Deconjugated steroids were then extracted at alkaline pH with tert-butylmethylether. The dried organic extracts were derivatized with MSTFA:NH4I:2-mercaptoethanol to obtain the TMS derivatives, and were subjected to analysis by gas chromatography mass spectrometry (GC/MS). The procedure was validated in boar and horse urine for the following metabolites: norandrosterone, noretiocholanolone, norepiandrosterone, 5beta-estran-3alpha, 17beta-diol, 5alpha-estran-3beta, 17beta-diol, 5alpha-estran-3beta, 17alpha-diol, 17alpha-nandrolone, 17betaN, 5(10)-estrene-3alpha, 17alpha-diol, 17alpha-estradiol and 17beta-estradiol in the different metabolic fractions. Extraction recoveries were higher than 90% for all analytes in the free fraction, and better than 80% in the glucuronide and sulphate fractions, except for 17alpha-estradiol in the glucuronide fraction (74%), and 5alpha-estran-3beta, 17alpha-diol and 17betaN in the sulphate fraction (close to 70%). Limits of quantitation ranged from 0.05 to 2.1 ng mL(-1) in the free fraction, from 0.3 to 1.7 ng mL(-1) in the glucuronide fraction, and from 0.2 to 2.6 ng mL(-1) in the sulphate fraction. Intra- and inter-assay values for precision, measured as relative standard deviation, and accuracy, measured as relative standard error, were below 15% for most of the analytes and below 25%, for the rest of analytes. The method was applied to the analysis of urine samples collected after administration of 17betaN laureate to boars and horses, and its suitability for the quantitation of the metabolites in the three fractions has been demonstrated.