Simultaneous LC–UV–MS–MS Analysis of Nine Pivotal Metabolites in Human Serum: Application to Studies of Impaired Glucose Tolerance

A rapid, sensitive, and selective LC–UV–MS–MS method for simultaneous quantification of uric acid, creatinine, xanthine, creatine, hypoxanthine, adenosine, inosine, thymidine, and uridine in serum from patients with impaired glucose tolerance (IGT) has been developed and validated. After precipitation of protein in the serum with methanol samples were evaporated with a stream of nitrogen then reconstituted with aqueous ammonia (0.002 mol L^?1). Chromatographic separation was performed on a C₁₈ column with methanol–buffer (8 mmol L^?1 ammonium acetate adjusted to pH 6.1 with glacial acetic acid) as mobile phase at a flow rate of 0.8 mL min^?1. UV and MS–MS detection were used to quantify the metabolites on the basis of different concentrations and detector response. Linearity was excellent, with r ² no <0.998. Recovery, and intra-day and inter-day relative standard deviation (RSD) were >85, <10, and <10%, respectively. This reliable bioanalytical method enables evaluation of the levels of purines and pyrimidines in serum for IGT studies and diagnosis.