Interference by metabolites and the corresponding troubleshooting during the LC-MS/MS bioanalysis of G004, a...

The quantitative determination of drugs in bio-samples may be interfered by the drug-related metabolites during the high-throughput LC-MS/MS analysis. When quantifying bromine or chlorine containing compounds, the ⁸¹Br/³⁷Cl isotopic forms of their mono-hydroxylated metabolites after in-source dehydration could produce ions which are isobaric with the precursor ions of the parent compounds at the ⁷⁹Br/³⁵Cl isotopic form. In this report, we described the identification of an interfering hydroxylated metabolite of G004, a novel bromine-containing hypoglycemic agent, during LC-MS/MS analysis of plasma samples. Several different MRM transitions were tested and evaluated to minimize the metabolite influence on the quantification of G004. Furthermore, the standard addition method using incurred samples was used to evaluate the matrix effect caused by the interfering metabolite. The lower limit of quantitation of the established method was 0.2 ng/ml, which was 10 times lower than the existing one. The method was successfully applied to investigate the single-dosing pharmacokinetic profile of G004 in beagle dogs. The above results indicated that when quantifying chlorine or bromine containing compounds, evaluation of the interference from mono-hydroxylation or dehydrogenation metabolites should be undertaken, and if such metabolites existed, their impact on quantification of the parent compounds could be eliminated by the proper selection of the MRM transitions.