Multiresidue HPLC method to measure benzimidazole anthelmintics in plasma and egg from laying hens. Evaluation of albendazole metabolites residue profiles

A method to quantify residual concentrations of benzimidazole (BZD) anthelmintic drugs in plasma and total egg from laying hens was developed and validated. The analytical method allowed detection of ten (10) BZD molecules by HPLC with UV detection: albendazole (ABZ) and its metabolites, albendazole sulphoxide (ABZSO), albendazole sulphone (ABZSO₂) and amino albendazole sulphone (NH₂-ABZSO₂); fenbendazole (FBZ) and its metabolites, fenbendazole sulphoxide (FBZSO) and fenbendazole sulphone (FBZSO₂); and flubendazole (FLBZ) and its reduced (FLBZ-R) and hydrolysed (FLBZ-H) metabolites. Analytes were separated on a C₁₈ reversed-phase column using a mobile phase compose of acetonitrile and ammonium acetate buffer (0.025M, pH 5) at a flow rate of 1.2mLmin⁻¹. The total run time for the method was 25min. The egg and plasma extraction processes were based on liquid–liquid extraction obtaining recoveries between 68.9% and 98.3% (egg) and from 71.4% to 100% (plasma). The linearity of the method was demonstrated with correlation coefficients ranging between 0.995 and 0.999 (egg) and between 0.993 and 0.997 (plasma). The method exhibited a high degree of intra- and inter-day precision and accuracy demonstrated by the low CV (⩽14.1% for egg, and ⩽7.79% for plasma) and relative error (RE) (from 0.628% to 8.30% for egg and between 2.89% and 9.08% for plasma). The LOQ were low and accurate enough for the application of the method to study the plasma pharmacokinetic disposition of ABZ metabolites and their residual concentrations in eggs following an oral administration of ABZ to laying hens. ABZ parent drug and its ABZSO and ABZSO₂ metabolites were measured in plasma. The ABZSO and ABZSO₂ plasma concentrations were much higher than that of the parent drug, with Cₘₐₓ values of 2.22 and 1.50μg/mL, respectively. The AUC values were 21.02 and 23.08μgh/mL for ABZSO and ABZSO₂. Residual ABZSO and ABZSO₂ concentrations were also measured in eggs. The assessment of the plasma disposition kinetics and egg residues after ABZ administration to laying hens is presented as an application of the developed analytical methodology.