Duodenal bile
AMS
COLD
To assess the drug‐related material (DRM) arising from the radiolabeled intravenous dose (TP1), individual bile strings were extracted with either acetonitrile or water in two separate extraction procedures. An aliquot of 500 μL from each acetonitrile or water extract was pipetted into a scintillation vial and mixed with 15 mL ScintiLogic U cocktail. An equal volume of the opposite solvent to which the extraction was conducted (i.e., either water or acetonitrile) was also added.All scintillation vials were stored in the dark at approximately 4°C for at least 24 h, briefly mixed, and counted for 60 minutes on a low‐level LSC to determine radioactivity content.Based on the levels of extracted radioactivity, equal volume aliquots of 100 μL from three subjects were pooled and spiked with 1200 μL of a cold standard mix solution in water to create a single diluted pool of bile string extracts at approximately 15 DPM/mL for HPLC followed by analysis by accelerator mass spectrometry (AMS). Preparation of the cold standard mix solution is described in Data A concurrent inline UV‐chromatogram was obtained before fractions were collected for analysis by AMS so that the retention times for the radioactive peaks for daprodustat and the six metabolites (M2–6 and M13) for which authentic standards were available could be verified.To assess DRM arising from the nonlabeled oral dose, the remaining bile extracts (pooled and individual) were prepared into a single sample as described below, for metabolite identification by liquid chromatography–tandem mass spectrometry (LC–MS