Utilization of stable isotope labeling to facilitate the identification of polar metabolites of KAF156, an antimalarial agent

Identification of polar metabolites of drug candidates during development is often challenging. Several prominent polar metabolites of [14C]KAF156, an antimalarial agent, were detected in rat urine from an ADME study, but could not be characterized by LC-MS/MS due to low ionization efficiency. In such instances a strategy chosen by investigators is to use a radiolabeled compound with high specific activity, having an isotopic mass ratio (i.e., [12C]/[14C]) and mass difference that serves as the basis for a mass filter using accurate mass spectrometry. Unfortunately, [14C]KAF156-1 was uniformly labeled (n=1-6) with the mass ratio of ~0.1. This ratio was insufficient to be useful as a mass filter despite the high specific activity (120 μCi/mg). At this stage in development, stable isotope labeled [13C6]KAF156 was available as the internal standard for the quantification of KAF156. We were thus able to design an oral dose, as a mixture of [14C]KAF156 (specific activity 3.65 μCi/mg) and [13C6]KAF156 with a mass ratio of [12C]/[13C6] as 0.9 and the mass difference as 6.0202. Using this mass filter strategy, four polar metabolites were successfully identified in rat urine. Subsequently, using a similar dual labeling approach, [14C]KAF156-2 and [13C2]KAF156 were synthesized to allow the detection of any putative polar metabolites which may have lost labeling during biotransformations using the previous [14C]KAF156-1. Three polar metabolites were thereby identified and M43, a less polar metabolite, was proposed as the key intermediate metabolite leading to the formation of a total of seven polar metabolites. Overall this dual labeling approach proved practical and valuable for the identification of polar metabolites by LC-MS/MS.