Measurement of endogenous uracil and dihydrouracil in plasma and urine of normal subjects by liquid chromatography–tandem mass spectrometry

A sensitive and specific HPLC–MS–MS method was developed for the determination of endogenous uracil (Ura) and its metabolite dihydrouracil (UH2) in human plasma and urine samples. Plasma samples were extracted with ethyl acetate–isopropanol (85:15, v/v) following added ammonium sulfate, and then separated on a Discovery Amide C16 column with 3% methanol solution as the mobile phase; urine samples were just centrifuged at 2500 g for detection. Quantitation was carried out by LC–MS–MS in the multiple reaction monitoring (MRM) mode. The limits of quantitation of the method for Ura and UH2 were 0.5 and 5 ng ml−1 (for plasma), and 50 and 100 ng ml−1 (for urine), respectively. This method can be useful to evaluate the activity of dihydropyrimidine dehydrogenase (DPD), a rate-limiting enzyme of the chemotherapy drug fluoropyrimidine, which will be helpful in investigating subject variation of DPD and adjusting clinical dosage in pyrimidine chemotherapy.