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

A method to quantify metabolites of 17 beta-nandrolone (17 beta N) 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 Cis 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, 5 beta-estran-3 alpha, 17 beta-diol, 5 alpha-estran-3 beta, 17 beta-diol, 5 alpha-estran-3 beta, 17 alpha-diol, 17 alpha-nandrolone, 17 beta N, 5(10)-estrene-3 alpha, 17 alpha-diol, 17 alpha-estradiol and 17 beta-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 17 beta-estradiol in the glucuronide fraction (74%), and 5 alpha-estran-3 beta, 17 alpha-diol and 17 beta N 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 17 beta N laureate to boars and horses, and its suitability for the quantitation of the metabolites in the three fractions has been demonstrated. (c) 2006 Elsevier B.V. All rights reserved.