Clenbuterol in the horse: confirmation and quantitation of serum clenbuterol by LC-MS-MS after oral and intratracheal administration

Clenbuterol is a beta2 agonist/antagonist bronchodilator, and its identification in post-race samples may lead to sanctions. The objective of this study was to develop a specific and highly sensitive serum quantitation method for clenbuterol that would allow effective regulatory control of this agent in horses. Therefore, clenbuterol-d9 was synthesized for use as an internal standard, an automated solid-phase extraction method was developed, and both were used in conjunction with a multiple reaction monitoring liquid chromatography-tandem mass spectrometry (LC-MS-MS) method to allow unequivocal identification and quantitation of clenbuterol in 2 mL of serum at concentrations as low as 10 pg/mL. Five horses were dosed with oral clenbuterol (0.8 microg/kg, BID) for 10 days, and serum was collected for 14 days thereafter. Serum clenbuterol showed mean trough concentrations of approximately 150 pg/mL. After the last dose on day 10, serum clenbuterol reached a peak of approximately 500 pg/mL and then declined with a half-life of approximately 7 h. Serum clenbuterol declined to 30 and 10 pg/mL at 48 and 72 h after dosing, respectively. By 96 h after dosing, the concentration was below 4 pg/mL, the limit of detection for this method. Compared with previous results obtained in parallel urinary experiments, the serum-based approach was more reliable and satisfactory for regulation of the use of clenbuterol. Clenbuterol (90 microg) was also administered intratracheally to five horses. Peak serum concentrations of approximately 230 pg/mL were detected 10 min after administration, dropping to approximately 50 pg/mL within 30 min and declining much more slowly thereafter. These observations suggest that intratracheal administration of clenbuterol shortly before race time can be detected with this serum test. Traditionally, equine drug testing has been dependent on urine testing because of the small volume of serum samples and the low concentrations of drugs found therein. Using LC-MS-MS testing, it is now possible to unequivocally identify and quantitate low concentrations (10 pg/mL) of drugs in serum. Based on the utility of this approach, the speed with which new tests can be developed, and the confidence with which the findings can be applied in the forensic situation, this approach offers considerable scientific and regulatory advantages over more traditional urine testing approaches.