Development and validation of a liquid chromatography-electrospray tandem mass spectrometry method for mebendazole and its metabolites hydroxymebendazole and aminomebendazole in sheep liver

Abstract

A quantitative liquid chromatography-electrospray tandem mass spectrometry method for the determination of mebendazole and its hydrolysed and reduced metabolites in sheep liver has been developed and validated. The benzimidazole substances were extracted with ethyl acetate after the sample mixture had been made alkaline. The HPLC separation was performed on a reversed-phase C₁₈ column with gradient elution using a mobile phase consisting of water containing 0.1% formic acid and acetonitrile. The analytes were detected after atmospheric pressure electrospray ionization on a tandem quadrupole mass spectrometer in MS-MS mode. The components were measured by the MS-MS transitions of the molecular ion to the two most abundant daughter ions. The detection limits are lower than 1 μg kg⁻¹. For this application, the validation limit was set at 50 μg kg⁻¹. The examined validation parameters were in accordance with the permitted tolerances ranges stipulated in the proposed new European validation criteria for residue surveillance. For the three analytes, the overall recovery was higher than 90%. The RSD for the repeatability ranged from 5 to 11%. The range for the within-laboratory reproducibility was between 2 and 17%. The decision limits for mebendazole, the hydrolysed and the reduced metabolite were 56.6, 61.8 and 64.2 μg kg⁻¹, respectively. The detection capabilities for these substances were 60.0, 86.1 and 90.9 μg kg⁻¹, respectively.