Quantification of clenbuterol at trace level in human urine by ultra-high pressure liquid chromatography–tandem mass spectrometry

Clenbuterol is a β2 agonist agent with anabolic properties given by the increase in the muscular mass in parallel to the decrease of the body fat. For this reason, the use of clenbuterol is forbidden by the World Anti-Doping Agency (WADA) in the practice of sport. This compound is of particular interest for anti-doping authorities and WADA-accredited laboratories due to the recent reporting of risk of unintentional doping following the eating of meat contaminated with traces of clenbuterol in some countries. In this work, the development and the validation of an ultra-high pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC–ESI-MS/MS) method for the quantification of clenbuterol in human urine is described. The analyte was extracted from urine samples by liquid–liquid extraction (LLE) in basic conditions using tert butyl-methyl ether (TBME) and analyzed by UHPLC–MS/MS with a linear gradient of acetonitrile in 9 min only. The simple and rapid method presented here was validated in compliance with authority guidelines and showed a limit of quantification at 5 pg/mL and a linearity range from 5 pg/mL to 300 pg/mL. Good trueness (85.8–105%), repeatability (5.7–10.6% RSD) and intermediate precision (5.9–14.9% RSD) results were obtained. The method was then applied to real samples from eighteen volunteers collecting urines after single oral doses administration (1, 5 and 10 μg) of clenbuterol-enriched yogurts.