Confirmatory analysis of 17β-boldenone, 17α-boldenone and androsta-1,4-diene-3,17-dione in bovine urine by liquid chromatography–tandem mass spectrometry

A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for confirmatory analysis of 17β-boldenone (17β-BOL), 17α-boldenone (17α-BOL) and androsta-1,4-diene-3,17-dione (ADD) in bovine urine was developed. [2H2]17β-Testosterone (17β-T-d2) was used as the internal standard. Sample preparation involved enzymatic hydrolysis and purification on a C18 solid-phase extraction column. Chromatographic separation of the analytes was obtained using an RP-C18 HPLC column. LC–MS–MS detection was carried out with an atmospheric pressure chemical ionisation (APCI) source equipped with a heated nebulizer (HN) interface operating in the positive ion mode. For unambiguous hormone confirmation, three analyte precursor–product ion combinations were monitored during multiple-reaction monitoring (MRM) LC–MS–MS analysis. Overall recovery (%), repeatability (relative standard deviations, RSD, %) and within-laboratory reproducibility (RSD, %) ranged from 92.2 to 97.7%, from 6.50 to 2.94% and from 13.50 to 5.04%, respectively, for all analytes. The limit of quantification in bovine urine was 0.20 ng ml−1 for 17β-BOL and ADD and 0.50 ng ml−1 for 17α-BOL. The validated method was successfully applied for determination of 17β-BOL, 17α-BOL and ADD in a large number of bovine urine samples collected within the national Official Residue Control Program.