Clenbuterol in the horse: urinary concentrations determined by ELISA and GC/MS after clinical doses

Clenbuterol is a beta (2) agonist/antagonist bronchodilator marketed as Ven tipulmin(R) and is the only member of this group of drugs approved by the U S Food and Drug Administration (FDA) for use in horses. Clenbuterol is a cl ass 3 drug in the Association of Racing Commissioners International (ARCI) classification system; therefore, its identification in postrace samples ma y lead to sanctions. Recently, the sensitivity of postrace testing for clen buterol has been substantially increased. The objective of this study was t o determine the 'detection times' for clenbuterol after administration of a n oral clinical dose (0.8 g/kg, b.i.d.) of Ventipulmin syrup. Five horses r eceived oral clenbuterol (0.8 g/kg, b.i.d.) for 10 days, and urine concentr ations of clenbuterol were determined by an enhanced enzyme-linked immunoab sorbent assay (ELISA) test and gas chromatography/mass spectrometric (GC/MS ) analysis by two different methods for 30 days after administration. Twent y-four hours after the last administration, urine concentrations of apparen t 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, peaki ng at 7 ng/mL on day 10 postadministration. The urine samples were also ana lysed using mass spectral quantification of both the trimethylsilyl (TMS) a nd methane boronic acid (MBA) derivatives of clenbuterol. Analysis using th e TMS method showed that, at 24 h after the last administration, the mean c oncentration of recovered clenbuterol was about 22 ng/mL. Thereafter, clenb uterol concentrations fell below the limit of detection of the TMS-method b y day 5 after administration but became transiently detectable again at day 10, with a mean concentration of about 1 ng/mL. Derivatization with MBA of fers significant advantages over TMS for the mass spectral detection of cle nbuterol, primarily because MBA derivatization yields a high molecular weig ht base peak of 243 m/z, which is ideal for quantitative purposes. Therefor e, mass spectral analyses of selected urine samples, including the transien t peak on day 10, were repeated using MBA derivatization, and comparable re sults 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 avera ged similar to1 ng/mL. Because of this secondary peak, the detection time f or clenbuterol (0.8 g/kg, b.i.d. x 10 days) is at least 11 days if the thre shold for detection is set.at 1 ng/mL.