LC-MS/MS METHOD FOR THE DETERMINATION OF DIAZOLIC ANTHELMINTIC DRUG LEVELS FROM SHEEP AND HUMAN PLASMA FOR USE IN PHARMACOKINETIC AND BIOAVAILABILITY STUDIES

Abstract

ABSTRACT. A new high-throughput, inexpensive and selective LC-MS method for determining fenbendazole, albendazole and albendazole sulfoxide from human and ovine plasma was developed and validated in accordance with current guidelines in bioanalysis. Analytes (fenbendazole, albendazole, albendazole sulfoxide) and internal standard (fluconazole) were separated on a Gemini NX-C18 analytical column in reversed phase chromatography in gradient elution using mobile phase composed of acetonitrile and aquenous 0.2% formic acid with a flow rate of 0.6 mL/min. After positive electrospray ionization analytes were detected in the mass spectrometer in selected reaction monitoring mode, monitoring fragment ion m/z 268.05 from m/z 300.08 for fenbendazole, ion m/z 234.07 from m/z 266.09 for albendazole, ion m/z 240.04 from m/z 282.09 for albendazole sulfoxide and ion m/z 220.06 from m/z 307.60 for fluconazole. Sample preparation was performed using protein precipitation. Validation of the analytical method was performed with respect to selectivity, stability, linearity (r>0.9901), precision (RSD<12.9%) and accuracy (bias<12.7%) over the concentration ranges of 5-250 ng/mL for each analyte (lower limit of quantification was 5 ng/mL for all analytes). The analytical method is simple, versatile and suitable for bioanalysis of these azole anthelmintic drugs from human and ovine samples, and applicable in pharmacokinetic studies involving fenbendazole and albendazole.