Metabolic signatures of metabolites of the purine degradation pathway in human plasma using HILIC UHPLC–HRMS

Abstract

The metabolic disorders in the purine degradation pathway have proven to be closely associated with several human diseases. However, the etiology is not yet fully understood. Profile assay of purine intermediates and uric acid involved in the metabolic pathway can provide additional insight into the nature and severity of related diseases. Purine metabolites are endogenous chemicals with high hydrophilicity, polarity, and similar structures, thus there is a great need for a specific method to quantify them directly in biological fluids with a short running time. Herein, eight purine degradation pathway metabolites, including xanthine, hypoxanthine, guanine, xanthosine, inosine, guanosine, adenosine and uric acid, in human plasma were quantitatively measured using hydrophilic interaction chromatography-tandem high-resolution mass spectrometry (HILIC-HRMS) in a short running time of 10 min. The method was systematically validated for specificity, linearity of the calibration curve, the limit of detection, the limit of quantification, the lower limit of quantification, precision, accuracy, extraction recovery, matrix effect, and stability. The results showed that the method was linear (R² > 0.99), accurate (the intra- and inter-day recoveries of all analytes ranged from 90.0 % to 110.0 %), and precise (the intra- and inter-day precisions were less than 6.7 % and 8.9 %, respectively) with the lower limits of quantification ranging from 3 to 10,000 ng/mL. The extraction recoveries and matrix effects were repeatable and stable. All the analytes were stable in the autosampler and could be subject to three freeze-thaw cycles. The developed method was ultimately applied to 100 plasma specimens from healthy individuals. The results showed that the concentrations of different purine metabolites varied dramatically in plasma specimens. Diet and body mass index (BMI) were the most significant factors determining purine levels, followed by drinking and sex. Age, smoking and bedtime showed a very weak correlation with purine metabolism. The findings of the present work reveal the characteristics of purine metabolism in human plasma under non-pathological conditions. The results also highlight the factors that can cause changes in purine metabolism, which are useful in developing effective treatment strategies for metabolic disorders of purines, particularly for those caused by lifestyle factors.