Abstract

Identification of polar metabolites of drug candidates during development is often challenging. Several prominent polar metabolites of 2-amino-1-(2-(4-fluorophenyl)-3-((4-fluorophenyl)amino)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)ethanone ([¹⁴C]KAF156), an antimalarial agent, were detected in rat urine from an absorption, distribution, metabolism, and excretion study but could not be characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) because of low ionization efficiency. In such instances, a strategy often 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 serve 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]KAF156-1 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]KAF156-1 (specific activity 3.65 μCi/mg) and [¹³C₆]KAF156-1 with a mass ratio of [¹²C]/[¹³C₆] as 0.9 and the mass difference as 6.0202. By 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]KAF156-2 were synthesized to allow the detection of any putative polar metabolites that 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.