Analysis of 5-Fluorouracil in Plasma for Clinical Research

Significance of the Topic

5-fluorouracil (5-FU) is widely used in oncology for its ability to inhibit DNA and RNA synthesis. Individual variability in drug metabolism necessitates a sensitive and selective analytical approach to support clinical research in pharmacokinetics and pharmacodynamics.

Objectives and Study Overview

This study aimed to develop and validate a rapid, robust UPLC-MS/MS method for quantifying 5-FU in human plasma using minimal sample volumes and a stable isotope-labeled internal standard.

Methodology and Instrumentation

A liquid-liquid extraction protocol was applied to 50 µL plasma samples spiked with 5-FU-13C15N2 as an internal standard. Samples were extracted with ethyl acetate containing formic acid, centrifuged, evaporated, and reconstituted in aqueous formic acid.
The chromatographic separation employed an ACQUITY UPLC HSS PFP column (2.1×100 mm, 1.8 µm) at 35 °C with a 3-minute gradient of water/0.1% formic acid (98–2% to 2–98% over 2.1 minutes) and acetonitrile at 0.4 mL/min. Detection was performed on a Xevo TQD mass spectrometer operating in negative electrospray ionization mode using MRM transitions (5-FU m/z 129→41.9 quantifier, 129→86 qualifier; ISTD m/z 132→43.9). Data acquisition and processing were managed using MassLynx v4.1 with TargetLynx.

Main Results and Discussion

5-FU eluted at ~1.18 minutes, clearly resolved from isobaric interferences. No carryover was observed up to 10,000 ng/mL. The method demonstrated a lower limit of quantification at 7.5 ng/mL (<20% RSD, <15% bias). Calibration was linear from 14 to 2600 ng/mL. Intra- and inter-day precision across QC levels (40, 350, 750, 1500 ng/mL) yielded total RSD ≤9.0%. Matrix effects (0.46–0.75) were effectively compensated by the internal standard (matrix factor ~0.96–1.02). Recovery studies with endogenous and exogenous interferents showed recoveries within 90.5–110.6%, meeting acceptance criteria (85–115%).

Benefits and Practical Applications

• High analytical selectivity and sensitivity for low-volume plasma samples.
• Rapid analysis time (3-minute runtime, 3.5-minute cycle).
• Wide dynamic range (20–2000 ng/mL) and robust quantification.
• Effective suppression of matrix and carryover effects.
• Suitable for pharmacokinetic and pharmacodynamic clinical research.

Future Trends and Opportunities

Emerging developments may include greater automation, miniaturized or microsampling techniques, multiplexed assays for concurrent drug monitoring, integration with high-resolution mass spectrometry, and personalized dosing strategies based on real-time analytical feedback.

Conclusion

The described UPLC-MS/MS method provides a fast, accurate, and reproducible tool for quantifying 5-fluorouracil in plasma. Its performance characteristics make it well suited for clinical research applications in oncology pharmacology.

Reference

1. Fakih MG et al. A Phase I, Pharmacokinetic and Pharmacodynamic Study of Vorinostat with 5-Fluorouracil and Leucovorin in Refractory Solid Tumors. Clin Cancer Res. 2010 Jul 15;16(14):3786–3794.