Quantitation of uridine and L-dihydroorotic acid in human plasma by LC-MS/MS using a surrogate matrix approach

Uridine and L-dihydroorotate (DHO) are important intermediates of de novo as well as salvage pathways for the biosynthesis of pyrimidines, which are the building blocks of nucleic acids - DNA and RNA. These metabolites are known to be significant biomarkers of pyrimidine synthesis during the development of DHODH inhibitor drugs for treatment of several cancers and immunological disorders. Here we are reporting a validated LC-MS/MS assay for the quantitation of uridine and DHO in K₂EDTA human plasma. Due to presence of endogenous uridine and DHO in the biological matrix, a surrogate matrix approach with bovine serum albumin (BSA) solution was used. Human plasma samples were spiked with stable isotope labeled internal standards, processed by protein precipitation, and analyzed using LC-MS/MS. Parallelism was successfully demonstrated between human plasma (the authentic matrix) and BSA (the surrogate matrix). The linear analytical ranges of the assay were set at 30.0-30,000 ng/mL for uridine and 3.00-3,000 ng/mL for DHO. This validated LC-MS/MS method demonstrated excellent accuracy and precision. The overall accuracy was between 91.9 % and 106 %, and the inter-assay precision (%CV) were less than 4.2 % for uridine in human plasma. The overall accuracy was between 92.8 % and 106 %, and the inter-assay precision (%CV) were less than 7.2 % for DHO in human plasma. Uridine and DHO were found to be stable in human plasma for at least 24 h at room temperature, 579 days when stored at -20 °C, 334 days when stored at -70 °C, and after five freeze/thaw cycles. The assay has been successfully applied to human plasma samples to support clinical studies. Novel Aspect: A surrogate matrix approach to quantify endogenous uridine and DHO concentrations in human plasma.