Determination of unlabeled and14C-radiolabeled drug candidates in biological fluids using LC-MS-MS—Issues and challenges

Abstract

The ability of MS-MS to monitor single isotopic species of an analyte may be disadvantageous in cases where the determination of the total concentration of all isotopic species of the analyte is required. This is the case, for example, when a14C-radiolabeled drug is administered to animals or human subjects in typical ADME studies. A number of issues and challenges connected with the determination of total and individual concentrations of12C-and14C-labeled drug in plasma using LC-MS-MS in support of human ADME studies are illustrated using as an example compound l (MK-677), a growth hormone secretagogue, recently evaluated in our laboratories. Due to a significant contribution (>60%) of the unlabeled12C-species to the MS-MS response at the14C-channel, the contributions from12C-I had to be subtracted from the MS-MS response at the14C-channel in order to determine individual concentrations of14C-I. The determination of individual concentrations of14C-I and12C-I was desired to assess the absence of an isotope effect in the disposition of isotopically labeled species. The assay in human plasma was validated in the concentration range of 0.1 to 100 ng mL−1 for the total concentration of14C-I and12C-I, and in the range of 0.0965 to 96.5 ng mL−1 for the unlabeled12C-I. For the14C-labeledI alone, the assay was validated in the concentration range of 0.175 to 3.5 ng mL−1 that was equivalent to the 5 to 100 ng mL−1 range of labeled14C-I used in the study. The strategies for the analysis of biological samples following administration of14C-labeled drugs using LC-MS-MS and the details of assay validation are presented.