Quantification of uracil and dihydrouracil plasma levels

A volume of 4 ml of heparinized whole blood was centrifuged at 1500 g for 10 min at 4°C. Isolated plasma was stored at −20°C until further analysis. DHU and U were quantified in plasma using mass spectrometry 31. After defrosting, a volume of 20 μl of internal standard working solution containing 1,3‐U‐¹⁵N₂ and 5,6‐DHU‐¹³C₄,¹⁵N₂ was added to 300 μl plasma. Protein precipitation was performed using 900 μl of MeOH and acetonitrile (1:1, v/v). Samples were vortex‐mixed for 10 s, shaken for 10 min at 1250 rpm (Labinco, Breda, The Netherlands) and centrifuged at 14 000 g for 10 min. Clear supernatants were dried under a stream of nitrogen at 40°C and reconstituted in 100 μl 0.1% formic acid in water. U and DHU plasma levels were determined using an ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) system. Chromatographic separation was performed on an Acquity UPLC® HSS T3 (150 × 2.1 mm ID, particle size 1.8 μm; Waters, Milford, MA, USA) column. Mobile phases consisted of 0.1% formic acid in UPLC‐grade water (eluent A) and 0.1% formic acid in UPLC‐grade acetonitrile (eluent B) at a flow rate of 0.3 ml min⁻¹. The following gradient was used: 0% B at 0–3.0 min, 0–90% B at 3.0–3.2 min, 90% B at 3.2–3.7 min, 0% B at 3.7–5 min. A Qtrap 5500 triple quadrupole mass spectrometer (AB Sciex, Framingham, MA, USA) was operated in the negative mode for quantification of U and in the positive mode for quantification of DHU. Selected mass transitions for U and DHU were m/z 110.9 ➔ m/z 42.0 and m/z 114.9 ➔ m/z 55.0. Validated concentration ranges for U and DHU were 1–100 ng ml⁻¹ and 10–1000 ng ml⁻¹, respectively.