Analysis of purines and pyrimidines across biospecimens: influence of storage temperature and duration

Purines and pyrimidines are key cellular components for the development, proliferation, and repair of cells and tissues, whose circulating levels are associated with various diseases. Biospecimen processing delays can significantly induce hypoxanthine and inosine levels, among other changes, in blood. We developed a liquid chromatography tandem mass-spectrometry method to quantify metabolites of the purine and pyrimidine pathways and investigated the impact of delayed sample processing on single donor plasma and dried blood spots (DBS). Plasma hypoxanthine increased above the normal reference range to an average 20-fold increase across individual donors when whole blood was kept refrigerated 64 h prior to processing. The same trend was observed in dried blood spots (DBS) prepared from refrigerated whole blood. Plasma inosine also increased above the reference range after a 48-hour delay in processing refrigerated whole blood. In contrast, hypoxanthine remained stable in DBS for up to 48 h, regardless of the card storage temperature. Therefore, DBS is a feasible alternative specimen type to measure hypoxanthine if frozen shipping, delayed centrifugation, or storage interval is a concern. Understanding the impact of specimen handling conditions is essential for successful biomarker discovery, especially when new biomarkers are to be introduced into the clinical laboratory.