Confirmation of anabolic hormone residues in bovine blood by micro‐HPLC–ion spray–tandem mass spectrometry

Abstract

AbstractSensitive, specific analytical methods for the determination of an‐abolics in biological matrices are essential to control the illegal use of these substances in food‐producing animals. Programs of residue control are performed annually in Italy for the determination of endogenous sex hormones (17β‐estradiol, progesterone, testoster‐one) for which maximum physiological levels have been established. At present the methods used in the Italian programs to determine natural hormones in bovine blood are based on the sensitive radio‐immunoassay (RIA), due to relatively low levels of these substances in plasma/serum. In this study, we report a new method based on tandem mass spectrometry with on‐line micro‐high performance liquid chromatography (micro‐HPLC‐MS‐MS) using an atmos‐pheric pressure ionization (API) source and an ion spray (IS) interface for the specific direct detection of natural (progesterone and testosterone), and synthetic (17β19‐nortestosterone and me‐droxyprogesterone) hormone residues in bovine serum. 17‐Methyl‐testosterone was used as the internal standard. Analytes were extracted with acetate buffer, purified on C18 Solid Phase Extrac‐tion (SPE) cartridge and separated on a reverse phase C18 micro‐HPLC column (300 mm × 1 mm, 5 μm), using acetonitrile‐water, 80:20 (v/v) containing 2mM ammonium acetate as the mobile phase, at a flow rate of 10 μl/min. When anabolic hormones were ionized in the IS interface operating in the positive ion mode, only the protonated molecules, [M+H]+, were generated, without evidence of any fragmentation. These served as precursor ions for collision induced dissociation (CID) and Diagnostic daughter ions for each analyte were identified in order to carry out analysis by micro‐HPLC‐MS‐MS in the selected reaction monitoring (SRM) mode. For the analytes in question, the response of the mass detector was related linearly to the quantity of each analyte injected between 10 and 300 pg, in the SRM mode. The limit of detection, based on a 3:1 signal‐to‐noise ratio, is 6–7 pg for the hormones. Recoveries were higher than 83% for 17β‐19‐nortestosterone, testosterone, and 17‐methyltestosterone, and 72% for the medroxyprogesterone, and progesterone. The micro‐HPLC‐MS‐MS method for the determi‐nation of anabolic hormones in bovine blood requires no sample derivatization, minimal sample preparation, and provides a sensi‐tive, selective, rapid alternative to the existing purification, separa‐tion, and detection techniques. At present this very sensitive method is being successfully applied to measure bovine serum concentra‐tions of natural hormones, such as testosterone and progesterone, in order to then confirm any illegal administration of these sub‐stances.