Development and validation of UPLC-MS/MS method for 5-Fluorouracil quantification in dried blood spot

Area of Science:

• Pharmacology
• Analytical Chemistry
• Biochemistry

Background:

• 5-Fluorouracil (5-FU) is a critical antimetabolite chemotherapy agent.
• Therapeutic drug monitoring of 5-FU is essential due to its narrow therapeutic index and susceptibility to genetic variations.
• Dried Blood Spot (DBS) sampling offers a less invasive alternative for drug monitoring, enhancing sample stability.

Purpose of the Study:

• To develop and validate a robust analytical method for quantifying 5-Fluorouracil levels in Dried Blood Spots.
• To establish a reliable method for therapeutic drug monitoring of 5-FU in cancer patients.

Main Methods:

• Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was employed for analysis.
• Sample preparation involved DBS extraction using 2-Propanol: ethyl acetate (16:84).
• Analysis utilized a C18 column with negative electrospray ionization and Multiple Reaction Monitoring (MRM).

Main Results:

• The method achieved a lower limit of quantification of 0.1 μg/mL.
• A linear calibration curve was established over the range of 0.1 μg/mL to 60 μg/mL.
• The developed method demonstrated full compliance with FDA and EMA validation requirements.

Conclusions:

• The UHPLC-MS/MS method provides accurate and precise quantification of 5-Fluorouracil in DBS samples.
• This validated method supports effective therapeutic drug monitoring, improving patient outcomes in cancer treatment.
• DBS-based monitoring of 5-FU offers advantages in patient comfort and sample stability.