Determination of Steroids in Bovine Serum: Validation of a Reliable LC-MS/MS Method and In Vivo Studies with Boldenone Undecylenate and Testosterone Propionate.

Serum analysis has received much attention in regulatory analysis of food-producing animals, especially for anabolic steroids. The possibility of confirming the parent drugs with minimum metabolization enables the detection of intact steroid esters, whose identification represents unequivocal proof of drug administration. This work involved the development and validation of a quantitative LC-MS/MS method to determine 30 steroids and steroid esters in bovine serum. Sensitivity was improved using microwave-assisted chemical derivatization with methoxyamine hydrochloride. The validation was successfully conducted in accordance with the Decision 657/2002/EC guidelines. An in vivo experiment was performed on 12 crossbred steers in which two commercial formulations containing boldenone undecylenate and testosterone propionate were administrated via intramuscular injections. The samples were collected over a period of 120 days, in which both intact esters were identified within 11 days postadministration. 17β-Boldenone was observed after 92 days for 2 steers and 56 days for the other animals. The applicability of a cut-off level to the ratio between 17β-testosterone and epitestosterone was evaluated in an attempt to differentiate testosterone abuse from endogenous production. It could be observed that a calculated ratio above this level is strong evidence of drug administration, although a high false-negative rate was obtained.