Determination of rosiglitazone and 5-hydroxy rosiglitazone in rat plasma using LC–HRMS by direct and indirect quantitative analysis: a new approach for metabolite quantification

Abstract

Background: With recent advances in mass spectrometry instrumentation, HRMS is of increasing interest for quantitative bioanalysis due to its high sensitivity, rapid acquisition of full scan data, and advanced software for metabolite identification. In particular, there is strong interest in use of HRMS for simultaneous quantification of parent drug and metabolites without authentic metabolite standard materials. Materials & methods: Rosiglitazone and 5-hydroxy rosiglitazone in rat plasma were analyzed using LC–Q-TOF by both direct and indirect quantitative analysis. Direct quantitative analysis used an authentic metabolite standard (5-hydroxy rosiglitazone). Indirect quantitative analysis firstly used the parent drug (rosiglitazone) calibration curve to provide a semiquantitative measure of metabolite concentration. A correction factor was then applied to the original data to re-calculate the 5-hydroxy rosiglitazone metabolite concentration. Results: The ratio of the calibration curve slope of rosiglitazone to that of 5-hydroxy rosiglitazone was determined to be 2.09 ± 0.28 using different batches of mobile phases and columns. The correction factor 2.09 was then used to correct for the 5-hydroxy rosiglitazone concentrations found from the semiquantitative results using the rosiglitazone calibration standard curve. The concentrations of 5-hydroxy rosiglitazone found by direct and indirect quantitative analysis were highly comparable (within ±8%). Conclusion: Indirect quantitative analysis provides an alternative approach for metabolite quantification for discovery PK studies.