Harkins J D, Woods W E, Lehner A F, Fisher M, Tobin T
Maxwell H. Gluck Equine Research Center and the Department of Veterinary Science, University of Kentucky, KY 40506-0099, USA.
J Vet Pharmacol Ther. 2001 Feb;24(1):7-14. doi: 10.1046/j.1365-2885.2001.00300.x.
Clenbuterol is a beta2 agonist/antagonist bronchodilator marketed as Ventipulmin and is the only member of this group of drugs approved by the US Food and Drug Administration (FDA) for use in horses. Clenbuterol is a class 3 drug in the Association of Racing Commissioners International (ARCI) classification system; therefore, its identification in postrace samples may lead to sanctions. Recently, the sensitivity of postrace testing for clenbuterol has been substantially increased. The objective of this study was to determine the 'detection times' for clenbuterol after administration of an oral clinical dose (0.8 g/kg, b.i.d.) of Ventipulmin syrup. Five horses received oral clenbuterol (0.8 g/kg, b.i.d.) for 10 days, and urine concentrations of clenbuterol were determined by an enhanced enzyme-linked immunoabsorbent assay (ELISA) test and gas chromatography/mass spectrometric (GC/MS) analysis by two different methods for 30 days after administration. Twenty-four hours after the last administration, urine concentrations of apparent clenbuterol, as measured by ELISA, averaged about 500 ng/mL, dropping to about 1 ng/mL by day 5 posttreatment. However, there was a later transient increase in the mean concentrations of apparent clenbuterol in urine, peaking at 7 ng/mL on day 10 postadministration. The urine samples were also analysed using mass spectral quantification of both the trimethylsilyl (TMS) and methane boronic acid (MBA) derivatives of clenbuterol. Analysis using the TMS method showed that, at 24 h after the last administration, the mean concentration of recovered clenbuterol was about 22 ng/mL. Thereafter, clenbuterol concentrations fell below the limit of detection of the TMS-method by day 5 after administration but became transiently detectable again at day 10, with a mean concentration of about 1 ng/mL. Derivatization with MBA offers significant advantages over TMS for the mass spectral detection of clenbuterol, primarily because MBA derivatization yields a high molecular weight base peak of 243 m/z, which is ideal for quantitative purposes. Therefore, mass spectral analyses of selected urine samples, including the transient peak on day 10, were repeated using MBA derivatization, and comparable results were obtained. The results show that clenbuterol was undetectable in horse urine by day 5 after administration. However, an unexpected secondary peak of clenbuterol was observed at day 10 after administration that averaged approximately 1 ng/mL. Because of this secondary peak, the detection time for clenbuterol (0.8 g/kg, b.i.d. x 10 days) is at least 11 days if the threshold for detection is set at 1 ng/mL.
克仑特罗是一种β2激动剂/拮抗剂支气管扩张剂，商品名为Ventipulmin，是美国食品药品监督管理局（FDA）批准用于马匹的该类药物中的唯一成员。在国际赛马委员协会（ARCI）的分类系统中，克仑特罗属于3类药物；因此，在赛后样本中检测到它可能会导致制裁。最近，赛后克仑特罗检测的灵敏度大幅提高。本研究的目的是确定口服临床剂量（0.8μg/kg，每日两次）的Ventipulmin糖浆后克仑特罗的“检测时间”。五匹马连续10天口服克仑特罗（0.8μg/kg，每日两次），给药后30天内通过两种不同方法，即增强酶联免疫吸附测定（ELISA）试验和气相色谱/质谱（GC/MS）分析，测定尿液中克仑特罗的浓度。最后一次给药24小时后，ELISA测定的表观克仑特罗尿液浓度平均约为500ng/mL；治疗后第5天降至约1ng/mL。然而，随后尿液中表观克仑特罗的平均浓度出现短暂升高，给药后第10天达到峰值7ng/mL。还使用克仑特罗的三甲基硅烷基（TMS）和甲烷硼酸（MBA）衍生物的质谱定量分析尿液样本。使用TMS方法分析表明，最后一次给药后24小时，回收的克仑特罗平均浓度约为22ng/mL。此后，给药后第5天克仑特罗浓度降至TMS方法的检测限以下，但在第10天再次短暂可检测到，平均浓度约为1ng/mL。对于克仑特罗的质谱检测，MBA衍生化比TMS具有显著优势，主要是因为MBA衍生化产生的243m/z的高分子量基峰非常适合定量分析。因此，使用MBA衍生化对选定的尿液样本（包括第10天的短暂峰值）进行质谱分析，得到了可比的结果。结果表明，给药后第5天在马尿液中检测不到克仑特罗。然而，给药后第10天观察到克仑特罗出现意外的二次峰值，平均约为1ng/mL。由于这个二次峰值，如果检测阈值设定为1ng/mL，克仑特罗（0.8μg/kg，每日两次×10天）至少11天内仍可被检测到。
