DETERMINATION OF ENDOGENOUS URACIL AND DIHYDROURACIL IN HUMAN PLASMA BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

AIM To develop a sensitive and specific HPLC/MS/MS method for the determination of endogenous uracil and its metabolite dihydrouracil in human plasma. This method will be useful to estimate the activity of dihydropyrimidine dehydrogenase (DPD), a rate-limiting enzyme of fluoropyrimidine chemotherapy drug, which would help investigate individual variation of DPD and direct to adjust clinical dosage of pyrimidine in chemotherapy. METHODS Plasma samples (200 μL) were extracted with 5 mL ethyl acetate-isopropanol (85∶15) followed by adding 150 mg ammonium sulfate and 100 μL internal standard (bromouracil, 400 ng·mL^-1). After 1 min vortex-mixing and 20 min shaking, the plasma protein was denatured and adhered to the wall of the extraction tube. The supernanant was transferred into another glass tube, and evaporated at 45℃ for 15 min under a stream of nitrogen. The residue was dissolved in 100 μL 10% methanol and transferred into autosampler-tubes. The solution (20 μL) was automatically injected into HPLC/MS/MS. The analytes were separated on Discovery Amide C₁₆ column with water as the mobile phase, then ionized in the gas-auxiliary electrospray ionization source and uracil (MRM m/z 111.0 → 41.9) as well as dihydrouracil (MRM m/z 113.0 → 42.0) was detected by the triple quadrupole mass analyzer. The peak area of uracil, dihydrouracil and the internal standard was recorded for quantitation. RESULTS The detection selectivity of uracil and dihydrouracil was excellent because of the use of high performance HPLC instrument. The limits of quantitation of the method for uracil and dihydrouracil were 0.5 ng·mL^-1 and 5 ng·mL^-1, the linear ranges were 0.5-100 ng·mL^-1 and 5-1 000 ng·mL^-1, respectively. Extraction efficiencies were all over 80% for the two analytes. The RSDs were less than 8.0% and the average recoveries were 98.0% and 100.5% for uracil and dihydrouracil, respectively. CONCLUSION The method is specific, sensitive and accurate, it is suitable for the determination of endogenous uracil and dihydrouracil in plasma and other biological samples. The concentration ratios of dihydrouracil to uracil would provide some data for the investigation of individual variation in DPD activity.