Quantitative radio‐HPLC analysis
dryness
Radio‐HPLC analysis was conducted on selected urine, fecal, and plasma samples on a 1200 HPLC system (Agilent Technologies, Palo Alto, CA). Radio‐HPLC conditions are described in Data Urine and feces samples were injected onto the HPLC and the column eluate from each sample was fractionated into a set of four LumaPlate‐96 microplates at 12 s/well. The mobile phase solvent in microplate wells was evaporated to dryness. The dried plates were sealed and counted on a TopCount scintillation counter for 5 or 10 min/well. The counting data were processed in Laura v4.1 to generate radiochromatograms.Plasma samples were analyzed in a similar manner to that of the urine and feces samples except that significant steps were taken to maximize radioactive signal intensity as follows: (1) each plasma extract was injected up to 8 times, (2) the resulting column eluate from each injection was cumulatively collected into the same set of microplates, and (3) after the last injection, the dried microplates were counted for 30 min/well.Each peak on a radiochromatogram was calculated as a percentage of the total count detected and expressed as nanogram equivalents of daprodustat per gram of plasma or the percentage of the dose recovered in urine and feces (all corrected for the overall recovery of extraction and reconstitution). A Topcount peak height of 10× standard deviation of background counts was defined as the lower limit of quantification (LLQ).[To deconvolute and estimate concentrations of M2 and M33, which coeluted in the major radio peak, the concentration values of M3, M4, and M6 determined using a validated LC/MS bioanalytical method