CONFIRMATION OF ANABOLIC HORMONE RESIDUES IN BOVINE BLOOD BY MICRO-HPLC-ION SPRAY TANDEM MASS-SPECTROMETRY

Sensitive, specific analytical methods for the determination of anabol ics in biological matrices are essential to control the illegal use of these substances in food-producing animals. Programs of residue contr ol are performed annually in Italy for the determination of endogenous sex hormones (17 beta-estradiol, progesterone, testosterone) for whic h 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 radioimmunoassay (RIA), due to relatively low levels of these substances in plasma/serum. In this st udy, we report a new method based on tandem mass spectrometry with on- line micro-high performance liquid chromatography (micro-HPLC-MS-MS) u sing an atmospheric pressure ionization (API) source and an ion spray (IS) interface for the specific direct detection of natural (progester one and testosterone), and synthetic (17 beta 19-nortestosterone and m edroxyprogesterone) hormone residues in bovine serum. 17-Methyltestost erone was used as the internal standard. Analytes were extracted with acetate buffer, purified on C18 Solid Phase Extraction (SPE) cartridge and separated on a reverse phase C18 micro-HPLC column (300 mm x 1 mm , 5 mu m), using acetonitrile-water, 80:20 (v/v) containing 2 mM ammon ium acetate as the mobile phase, at a flow rate of 10 mu l/min. When a nabolic hormones were ionized in the IS interface operating in the pos itive ion mode, only the protonated molecules, [M+H](+), were generate d, without evidence of any fragmentation. These served as precursor io ns for collision induced dissociation (CID) and diagnostic daughter io ns for each analyte were identified in order to carry out analysis by micro-HPLC-MS-MS in the selected reaction monitoring (SRM) mode. For t he 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 8 3% for 17 beta-19-nortestosterone, testosterone, and 17-methyltestoste rone, and 72% for medroxyprogesterone, and progesterone. The micro-HPL C-MS-MS method for the determination of anabolic hormones in bovine bl ood requires no sample derivatization, minimal sample preparation, and provides a sensitive, selective, rapid alternative to the existing pu rification, separation, and detection techniques. At present this very sensitive method is being successfully applied to measure bovine seru m concentrations of natural hormones, such as testosterone and progest erone, in order to then confirm any illegal administration of these su bstances.