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

Abstract

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.

Broad screening and identification of β-agonists in feed and animal body fluid and tissues using ultra-high performance liquid chromatography-quadrupole-orbitrap high resolution mass spectrometry combined with spectra library search

“…These methods include liquid chromatography (Chang, Chou, & Hwang, 2005;Meng et al, 2012;Turberg, Macy, Lewis, & Coleman, 1994), gas chromatography (Bocca, Fiori, Cartoni, & Brambilla, 2003), liquid chromatography tandem mass spectrometry (LC-MS/MS) (Shishani, Chai, Jamokha, Aznar, & Hoffman, 2003;Wu, Liu, & Peng, 2014), gas chromatography-mass spectrometry (Montrade, Lebizec, Monteau, & Andre, 1995;Wang, Li, Zhang, & Shen, 2006), enzyme-linked immunoassay (Hahnau & Julicher, 1996;Harkins, Woods, Lehner, Fisher, & Tobin, 2001;Pinheiro et al, 2008), colloidal gold test strip (Khamta, Pattarawarapan, Nangola, & Tayapiwatana, 2009;Zhang et al, 2009), surface plasmon resonance immunosensors (Traynor, Crooks, Bowers, & Elliott, 2003), and electrochemical immunosensors (He, Wang, Zhang, & Yang, 2009). Table 1 Chemical formula, accurate mass, retention time and characteristic fragment ions of 32 b-agonists.…”

Determination of beta‐agonists in swine hair by μFIA and chemiluminescence

“…Urine and plasma have been proposed as substrates for the detection of residues of ␤-agonists in animals [17,18,32], however, ␤-agonists cannot be detected in urine later than 11 days after sample collection, and in plasma it is even undetectable 4 days after the last application [33,34]. Therefore, the traceability, which is important for legal accounting process, is poor for the samples in these two forms.…”

An Automated Immunoassay Based on Magnetic Microspheres for the Determination of Clenbuterol in Food

“…Numerous methods for the determination of β 2 -agonists in pharmaceuticals and biological fluids have been reported that include gas chromatography -mass spectrometry, [3][4][5][6][7][8] high-performance liquid chromatography, [9] ultra-high performance liquid chromatography -tandem mass spectrometry, [10] and liquid chromatography -tandem mass spectrometry. [11] These methods may determine the analyte in the sample, but are expensive and time-consuming for routine analysis.…”