A liquid chromatography-tandem mass spectrometry method for the simultaneous quantitation of endogenous uracil (U) and dihydrouracil (UH2) was developed and tested in a Brazilian population of patients with gastrointestinal cancer previously exposed to 5-fluorouracil (5FU).
The analytes were extracted by a liquid–liquid method using 5-clorouracil as internal standard. The separation was performed on a reversed-phase XTerra C18 column with a mobile phase composed of methanol and aqueous 0.1% ammonium hydroxide (15:85). Mass spectrometry detection was carried out using negative electrospray ionization and selected reaction monitoring. Bovine serum albumin was employed as an alternative matrix to prepare the calibration standards, aiming to avoid the measurement of physiologic U and UH2. Calibration curves were constructed over the range of 5–200 ng/mL for U and 10–500 ng/mL for UH2.
The mean RSD values in the intrarun precision were 6.5% and 10.0% and in the interrun precision were 7.8% and 9.0% for U and UH2, respectively. The mean accuracy values were within the range of 90%–110% for both analytes. The analytes were stable in plasma under different conditions of temperature and time. The validated method was successfully applied to determine the plasma concentrations of U and UH2 in patients with gastrointestinal cancer (n = 32) previously treated with 5FU and for whom clinical toxicity was well documented. U concentrations varied from 21.8 to 56.6 ng/mL, whereas UH2 concentrations varied from 57.7 to 271.5 ng/mL. UH2/U ratio ranged from 1.56 to 6.18.
The method has proved to provide a quick, reliable, and reproducible quantitation of the plasma concentrations of U and its metabolite UH2. The UH2/U ratios did not discriminate patients previously exposed to 5FU with and without severe toxicities, possibly due to the small sample. Further studies in a larger population are desirable.