Confirmatory method for the determination of nandrolone and trenbolone in urine samples using immunoaffinity cleanup and liquid chromatography–tandem mass spectrometry

A confirmatory method for the simultaneous determination of nandrolone (α and β) and trenbolone (α and β) in urine samples by liquid chromatography electrospray mass spectrometry (LC–MS-MS) was developed. After an enzymatic deconjugation, the urine was subjected to a one-step cleanup on a commercially available immunoaffinity chromatography cartridge. The analytes were detected by liquid chromatography–positive ion electrospray tandem mass spectrometry using deuterium labelled internal standards. The analytical procedure was applicable to bovine and swine urine samples. The procedure was validated as a quantitative confirmatory method according to the Commission Decision 2002/657/EC criteria. The results obtained showed that the method was suitable for statutory residues testing regarding the following performance characteristics: instrumental linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit (CCα), detection capability (CCβ) and ruggedness. The decision limits (CCα) obtained, were between 0.54 and 0.60 μg L−1; the recovery was above 64% for all the analytes. Repeatability was between 1.6% and 5.7% and within-laboratory reproducibility between 1.6% and 6.0% for all the steroids.