Rapid 5-fluorouracil plasma quantification by immunoassay: Validation with FOLFOX6 clinical samples.

Abstract

563 Background: 5-fluorouracil (5-FU) is one of the most widely used chemotherapy agents in the treatment of colorectal cancer. Studies over the last 30 years have demonstrated wide pharmacokinetic variability of 5-FU which can lead to undue toxicity and suboptimal treatment. Recent clinical studies have demonstrated that managing plasma 5-FU levels and adjusting doses to target steady state concentrations effectively minimizes toxicity and improves outcome. Methods: An existing immunoassay using a novel antibody to 5-FU was modified to directly quantify 5-FU in patient plasma samples without any sample pretreatment. The assay was adapted to Beckman Coulter AU analyzers and to Roche COBAS c 111 analyzer. Method comparison to LC-MS/MS, limit of detection (LoD), lower limit of quantitation (LLoQ), precision, and linearity of the assay were evaluated. The assay was also validated by testing samples of patients being treated by modified FOLFOX6 from an on-going clinical trial ( NCT00943137 ). Results: The modified immunoassay was confirmed to have a linear reportable range from 85 to 18,000ng/mL and a LoD of 52ng/mL. The immunoassay was established to have good precision (CV<6%) around the medical decision points for FOLFOX and FOLFIRI regimens. Method comparison of the immunoassay obtained by testing 58 clinical samples from patients under 5-FU treatment provides excellent correlation to LC- MS/MS: Deming slope 1 ± 0.05, R > 0.98. Another 82 samples of patients on modified FOLFOX6 regimen from clinical trial (( NCT00943137 ) were tested using the immunoassay and range of these sample were reported to be from 95 to 2,970 ng/mL. Conclusions: The assay provided the performance required to rapidly quantify 5-FU plasma concentrations. It was fast (time to 1st result < 10 minutes), precise, correlated well to a physical method, required only 100uL sample on the analyzer and 7ul for actual testing per assay, and was easily adapted to a variety of clinical analyzers. Clinical and pharmacokinetic laboratories could use this assay to introduce an evidence-based approach to optimize 5-FU dosing that would have higher throughput, simpler methodology, and require less labor, space or expense than the physical methods. [Table: see text]