Clenbuterol in the Horse: Confirmation and Quantitation of Serum Clenbuterol by LC-MS-MS after Oral and Intratracheal Administration*

Abstract

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.

Detection, pharmacokinetics and cardiac effects following administration of clenbuterol to exercised horses

“…Following cessation of administration, the half-life of elimination of clenbuterol in the present study was 10.4 ± 4.4 h. This is in agreement with the half-life calculated in previous studies (10.4 [9] and 7 h [11]). Clenbuterol fell below detectable levels in plasma by 2-7 days post administration of the final dose in all horses studied for the low-dose regimen.…”

“…Clenbuterol fell below detectable levels in plasma by 2–7 days post administration of the final dose in all horses studied for the low‐dose regimen. This is similar to previous reports whereby clenbuterol was nondetectable in plasma by 72 h , 96 h and 7 days following administration of the final dose. Soma and colleagues reported that the drug was below the LOQ (13 pg/ml) in 3 of 6 horses studied by 96 h post administration (the final time point investigated) and nondetectable in the other 3.…”

The β‐agonist clenbuterol in mane and tail hair of horses

“…Clenbuterol in hair of horses was first described in a conference report (Popot et al 2000). The determination of clenbuterol in the tail or mane hair of horses will not replace blood or urine analysis, which are well established methods for detection of recent drug use (Lehner et al 2001;Harkins et al 2001). However, hair analysis enables identification of application and duration of application outside the time range covered by blood and/or urine analysis.…”

“…Random tests for illegal drug application have been carried out but have rarely yielded positive results, possibly because treatment was discontinued early enough to avoid urine or blood detection. The ß-agonist clenbuterol cannot be detected in horse urine later than 11 days after withdrawal, and in blood it is undetectable 4 days after the last application (Hagedorn et al 1995;Harkins et al 2001). It was our hypothesis that detection in mane or tail hair would be possible over an extended period, as shown in rat hair (Adam et al 1994), poultry feathers (Malucelli et al 1994), cattle (Gaillard et al 1997;Fente et al 1999) and human hair (Gleixner et al 1996).…”

Quantitative detection of salmeterol after inhalation in equine urine by liquid chromatography/tandem mass spectrometry

“…A recent study on derivatisation of β‐agonists with different agents and catalysts has shown that MSTFA (without catalyst) is the best choice for the derivatisation of salmeterol 10. Recently, a number of LC/MS n methods for the detection of β‐agonists, other than salmeterol, in bovine urine,11 bovine liver12 and equine serum13 have been described.…”