Determination of anabolic steroids in muscle tissue by liquid chromatography-tandem mass spectrometry

A specific and sensitive method based on liquid chromatography-tandem mass spectrometry using atmospheric pressure chemical ionization (LC-APCI-MS/MS) has been developed for the determination of four anabolic steroids [trenbolone, methylboldenone, methyltestosterone, and norethandrolone] in bovine muscle. Methyltestosterone- d 3 was used as internal standard. The procedure involved enzymatic hydrolysis, extraction with tert-butyl methyl ether, defattening, and final cleanup with solid-phase extraction with Oasis HLB cartridges. The analytes were analyzed by reversed-phase LC-MS/MS, acquiring two diagnostic product ions from the chosen precursor [M + H] (+) for the unambiguous confirmation of hormones. The method was validated according to the European Commission Decision 2002/657/EC for the detection and confirmation of residues in products of animal origin. The limits of detection (LOD) and limits of quantitation (LOQ) were found to be 0.3 ng/g and 1.0 ng/g, respectively. The accuracy and precision have been determined, with recoveries ranging from 83% to 104% and the CV factor not exceeding the value of 7%. The decision limits CCalpha were calculated and ranged from 0.05 to 0.15 ng/g while the detection capabilities CCbeta ranged from 0.09 to 0.25 ng/g. The method proved to be sensitive and reliable and thus renders an appropriate means for residue analysis studies.