Application of two-dimensional high-performance liquid chromatography in metabolism studies of ivermectin

Two-dimensional high-performance liquid chromatography (HPLC) of alternate reversed-phase and normal-phase columns was used in the purification, quantification and identification of submicrogram quantities of drug residue and metabolites in tissues from animals dosed with ivermectin. In reverse isotope dilution assay of the parent drug, two-dimensional HPLC assured constant specific activity of the drug isolates. For identification of metabolites from liver tissue, HPLC in two dimensions not only facilitated the purification, but also provided information (capacity factor k') in both systems for the elimination of possible metabolite structures in the course of compound identification. These approaches exemplified by the ivermectin studies should be generally applicable in analyses of complex biological samples in which quantity is frequently a limiting factor.