2.3. Standard preparation stock solutions

5-FU and 5-Bromouracil (1 mg/mL): 10 mg of pure powder of 5-FU was dissolved in 5 mL of Milli Q water and final volume was raised to 10 mL q.s, aliquoted and stored at −20 °C.

At the time of analysis, stock solutions were diluted with water to create spike-in working stock solutions containing 2, 5, 10, 20, 50, 100, and 200 μg/mL of 5-FU. To generate calibration standards, 950 μL of drug-free plasma were mixed with 50 μL of each spike-in from the working stock solution. This yielded the following concentrations: 0.10, 0.25, 0.50, 1.00, 2.50, 5.00, and 10.00 μg/mL. Comparably, four distinct concentrations (lower limit of quantification) of quality control samples were produced independently in blank plasma for 5-FU: LLOQ, 0.10 μg/mL; low-quality controls, 1 μg/mL; medium-quality controls, 2.5 μg/mL; and high-quality controls, 5 μg/mL. Samples of spiked plasma were all kept at −20 °C. The internal standard (I.S.) stock solution was made with a concentration of 1 mg/mL in water. I.S. working stock was further diluted with water and kept at −20 °C. Supplementary Materials and Procedures contain more information about preparing stocks.