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In the present paper we report the LC-MS/MS determination of residues of 12 anabolic steroids in bovine serum, as an expansion of our work protocols for steroids determination in biological matrices. Steroids analyzed included alpha-zearalanol, beta-zearalanol, alpha-trenbolone, beta-trenbolone, methyl-testosterone, alpha-estradiol, beta-estradiol, ethynylestradiol, alpha-boldenone, beta-boldenone, alpha-nortestosterone and beta-nortestosterone. Following protein precipitation, serum samples were cleaned up by solid-phase extraction using Oasis HLB and Amino cartridges. Atmospheric pressure chemical ionization (APCI) in both positive and negative ionization modes was used and mass spectrometry detection was carried out in multiple reaction monitoring mode following two or (in most cases) three product ions per precursor ion. The method was validated in accordance with the Commission Decision 2002/657/EC. The decision limit (CC alpha) values obtained, ranged from 0.01 to 0.07 ng/ml and the detection capability (CC beta) values obtained ranged from 0.02 to 0.12 ng/ml. The recoveries ranged from 70.2% to 118.2%. The developed method is suitable for routine and confirmatory purposes such as control of illegal use in livestock production. (C) 2010 Elsevier B.V. All rights reserved.