Optimizing cancer patient care with a robust assay for 5-fluorouracil quantification and in-vitro stability in human blood for therapeutic drug monitoring

• Published in: Practical laboratory medicine 2024-05, Vol.40, p.e00415, Article e00415
• Main Authors: Gurjar, Murari, Priyan, K. Ambedkar, Asia, Priyanka, Kumar, Uday, Shukla, Kajal, Mishra, Bal Krishna, Kapoor, Akhil, Gavel, Pratibha
• Format: Article
• Language: English
• Subjects: Colorectal cancer; DPD inactivator; Method development; Personalized therapy; UV-Detection
• ISSN: 2352-5517; 2352-5517
• DOI: 10.1016/j.plabm.2024.e00415

The plasma concentration of 5-Fluorouracil (5-FU) is affected by numerous factors, thereby limiting its efficacy. The current therapeutic regimen's doses based on body surface area (BSA) are linked to increased toxicity and sometimes inadequate drug exposure. The study aims to develop an in-vitro assay to monitor 5-Fluorouracil's therapeutic efficacy in cancer patients' blood samples, focusing on pharmacokinetics to improve therapy precision. Drug levels were determined from standards, quality controls, and experimental samples using protein precipitation, liquid-liquid extraction, and separation using a C18 analytical column with an isocratic program. In EXP-1A, the mean concentration of 5-Fluorouracil was 1.15 μg/ml; in EXP-1B, it was 1.16 μg/ml, while in EXP-1C, the mean concentration was 0.9 μg/ml. The percentage difference in mean 5-Fluorouracil concentration between the experiment sample containing a DPD inactivator and EXP-1C (without a DPD inactivator) was 21.5 % higher for EXP-1A and 0.68 % higher for EXP-1B. In the second phase of the experiment, the overall stability of 5-Fluorouracil in samples containing a DPD inactivator was 24.5 % superior compared to samples without a DPD inactivator. A modified extraction technique has been developed to accurately measure 5-Flourouracil concentration in blood, preserving its stability and concentration by adding a DPD inactivator. [Display omitted] •Highly sensitive UHPLC method to determine the stability and concentration of 5-FU in plasma.•Extraction technique to determine the concentration of 5-Flourouracil in blood.•Stability is crucial for understanding the drug's performance in Therapeutic Drug Monitoring practices during clinical use and is invaluable for both clinical and pre-clinical studies.•The accurate measurements of 5-FU in the blood are highly influenced in the pre-analytical stage of the samples with presence of DPD enzyme that can be effectively suppressed with the help of specific DPD inactivator like gimeracil. Novelty. This study represents the initial in-vitro trial assessing the impact of Gimeracil, a specific DPD inactivator, on the stability of the 5-FU drug. It investigates how varying concentrations of the DPD inactivator and different volumes of whole blood affect the stability of the 5-FU drug. Additionally, it aims to elucidate the degradation pattern over time. This data on stability is crucial for understanding the drug's performance in Therapeutic Drug Monitoring practices d