Clenbuterol Determination in Calf Hair by UPLC-MS-MS: Case Report of a Fraudulent Use for Cattle Growth

Abstract

A method for clenbuterol determination in hair has been developed. Hair specimens collected from two calves were decontaminated using hot water followed by methylene chloride. Hair was cut into small pieces, and 100 mg was incubated in 1 mL 0.1M hydrochloric acid overnight at 45°C in the presence of 1 ng acebutolol used as internal standard. After neutralization with 1 mL 0.1M NaOH, 2 mL of bicarbonate buffer (pH 8.6) were added and the preparation was then purified using solid-phase extraction with an Isolute C₁₈ column. Methanolic eluent was evaporated to dryness and the residue was reconstituted with 50 µL methanol. A 5-µL portion was injected onto an ACQUITY^™ UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm) and separation was achieved using a gradient of acetonitrile and formate buffer delivered at a flow rate of 0.6 mL/min. Detection was done on a Waters^® Micromass^® Quattro Micro™ API triple-quadrupole mass spectrometer. Ionization was achieved using electrospray in positive mode. Clenbuterol was identified by two transitions (m/z 277.1 > 203.2 and m/z 277.1 > 132.1). Quantitation was performed with the most intensive transition (m/z 277.1 > 203.2) versus the internal standard monitored using the transition (m/z 337.3 > 116.1). When compared with gas chromatography methods that are generally used for the determination of β-adrenergics, the major advantages of this method were the sensitivity, a shorter run time, and the absence of a derivatization step. The analysis of two hair samples from calves suspected of drug administration showed low clenbuterol concentrations at 3.6 and 4.8 pg/mg.