Development of achiral and chiral 2D HPLC methods for analysis of albendazole metabolites in microsomal fractions using multivariate analysis for the in vitro metabolism

• Development of multidimensional methods for the analysis of drugs in microsomes.
• Development of the in vitro metabolism of ABZ using multivariate design.
• Direct injection of microsomes by means of RAM columns for sample clean-up.
• On line injection plus multivariate analysis: less organic solvents consumption.
• Application of a chiral multidimensional method for the in vitro metabolism of ABZ.

In this work, the development of two multidimensional liquid chromatography methods coupled to a fluorescence detector is described for direct analysis of microsomal fractions obtained from rat livers. The chiral multidimensional method was then applied for the optimization of the in vitro metabolism of albendazole by experimental design. Albendazole was selected as a model drug because of its anthelmintics properties and recent potential for cancer treatment. The development of two fully automated achiral–chiral and chiral–chiral high performance liquid chromatography (HPLC) methods for the determination of albendazole (ABZ) and its metabolites albendazole sulphoxide (ABZ-SO), albendazole sulphone (ABZ-SO2) and albendazole 2-aminosulphone (ABZ-SO2NH2) in microsomal fractions are described. These methods involve the use of a phenyl (RAM-phenyl-BSA) or octyl (RAM-C8-BSA) restricted access media bovine serum albumin column for the sample clean-up, followed by an achiral phenyl column (15.0 × 0.46 cm I.D.) or a chiral amylose tris(3,5-dimethylphenylcarbamate) column (15.0 × 0.46 cm I.D.). The chiral 2D HPLC method was applied to the development of a compromise condition for the in vitro metabolism of ABZ by means of experimental design involving multivariate analysis.