Growth promotant abuse in meat-producing animals: Liquid chromatography-electrospray ionization tandem mass spectrometry for determination of β2-adrenergic agonist clenbuterol in pig urine

Abstract

Clenbuterol, a β 2-adrenergic agonist, has been found to be applied illegally in meat-producing animals to modulate growth. For residue surveillance of clenbuterol in meat-producing animals and meat, it is essential to choose appropriate matrices as well as the high degree of sensitivity derived from spectrometric analysis of suspect tissues and body fluids. The aim of the study was to evaluate the suitability of urine as a matrix for clenbuterol residue determination throughout and after its subchronic repeated administration at a growth-promoting dose to food-producing pigs, using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) as a quantification and confirmation method. Mass spectral acquisition was done in multiple reaction monitoring (MRM) mode to provide a high degree of sensitivity, archieving a limit of detection and quantification at 0.11 and 0.30 μ g/kg, respectively. The mean recovery of the analyte fortified at 0.5 μ g/kg was 88.0% with acceptable inter- and intra-day relative standard deviations in a range of 7.2-9.3%. Clenbuterol was identified by two transitions (m/z 277>259 and m/z 277>203), and quantification was performed at the most intensive transition (m/z 277>203). Study results showed the mean clenbuterol concentrations in pig urine samples to oscillate widely during the 28-day treatment period. Maximal clenbuterol concentrations were achieved in urine on day 21 (96.40p ; 45.13 ng/mL) and then decreased to 45.78p ; 8.47 ng/mL on the last day of dosing period. After a 7-day withdrawal period, clenbuterol concentration in urine (0.45p ; 0.05 ng/mL) decreased below 0.5 ppb as currently accepted maximal residue level (MRL) for clenbuterol in liver as a regulated matrix for control clenbuterol abuse. Although the concentration of clenbuterol in pig urine several times exceeded MRL throughout the treatment period, suggesting acceptability of this matrix for determination of clenbuterol residues during fattening period, the significant depletion recorded during the first seven days of withdrawal showed the urine to be of limited value as a matrix in the control of clenbuterol abuse.