Long-term storage stability of uracil in plasma

5-fluorouracil (5-FU) is widely used in cancer treatment. Both 5-FU and uracil are metabolized by dihydropyrimidine dehydrogenase (DPD); therefore, the European Medicines Agency (EMA) recommends measuring plasma uracil (P-uracil) and/or DPYD genotyping before treatment initiation. EMA classifies partial DPD deficiency as P-uracil ≥ 16 µg/L and <150 µg/L and complete deficiency as P-uracil ≥ 150 µg/L, why accurate measurement is crucial. This study investigated the three-year stability of P-uracil at −80 °C storage in seventy-eight patient samples collected during routine work-up before 5-FU initiation in 2022. Following initial analysis, samples were stored at −80 °C and reanalyzed in December 2025. P-uracil was measured using an in-house LC-MS/MS method. Seventy-six patients were included in the statistical analysis. Median P-uracil was 15.7 µg/L µg/L (range 6.2–69.0 µg/L) in 2022 and 15.0 µg/L (7.0–98.7 µg/L) in 2025, with no significant systematic difference (p = 0.241). The median percentage difference (PD%) was −2.2% (range −55.1% to +80.1%). A PD% exceeding ±15% was observed in 34 samples across all P-uracil levels: 53.3%, 50.0%, and 36.8% of samples with P-uracil <10, 10 to <16, and ≥16 µg/L, respectively. Samples with P-uracil ≥16 µg/L showed the widest PD% range (−55.1% to 80.1%). In nine patients, variability led to crossing the clinical threshold of 16 µg/L (absolute P-uracil change of 3.43–11.45 µg/L). In conclusion, no systematic change in P-uracil was observed during storage at −80 °C for up to three years; however, variability between repeated measurements was observed at individual level.