Quantification of allantoin and other metabolites of the purine degradation pathway in human plasma samples using a newly developed HILIC‐LC‐MS/MS method

Abstract The development of a simple HILIC‐LC‐MS/MS method to quantify the plasma levels of allantoin, inosine, hypoxanthine, and adenosine, using stripped plasma for the bioanalytical method validation, was the purpose of this study. Chromatographic separation conducted using an XBridge BEH Amide column (2.1 × 150 mm, 3.5 μm) was achieved under gradient elution with two mobile phases: 0.1% formic acid–ACN (5:95) and 0.1% formic acid–ACN (50:50). Multiple reaction monitoring MS detection was performed using a triple quadrupole. The method validation experiments were performed according to the European Medicines Agency and the U.S. Food and Drug Administration guidelines. The lower LOQ was 50 nM, 5 nM, 20 nM, and 2 nM for allantoin, inosine, hypoxanthine, and adenosine, respectively. The recovery was repeatable and stable. The intraday precision ranged from 1.6% to 6.5%, while the interday precision ranged from 3.4% to 58.7%. Therefore, it is necessary to make a matrix‐matched calibration curve each day to overcome this issue. Since the quality control samples’ stability did not always comply with the guidelines, the samples need to be analyzed soon after collection.