Development, validation and application of a novel liquid chromatography tandem mass spectrometry assay measuring uracil, 5,6-dihydrouracil, 5-fluorouracil, 5,6-dihydro-5-fluorouracil, α-fluoro-β-ureidopropionic acid and α-fluoro-β-alanine in human plasma

The plasma 5,6-dihydrouracil/uracil (UH2/U) ratio is a possible phenotypic marker of dihydropyrimidine dehydrogenase (DPD) activity, hence an index of 5-fluorouracil (5-FU) response and toxicity. Studies have re -affirmed the value of 5 -FU and 5,6-dihydro-5-fluorouracil (FUH2) for therapeutic drug monitoring (TDM). However, FUH2 has limited stability in plasma, necessitating expedited plasma separation and freezing, where routine compliance may not be easy. The metabolites alpha-fluoro-beta-ureidopropionic acid (FUPA) and alpha-fluoro-beta-alanine (F beta AL) are stable in plasma and are probable candidates for TDM. This paper describes development, validation and application of an LC-MS/MS assay quantifying U, UH2, 5-FU, FUH2, FUPA and F beta AL in human plasma. Extraction was by salt-assisted liquid-liquid extraction (LLE) in two-stages with pH adjustment. The supernatants were mixed, dried and reconstituted. Analytes were resolved on the Luna PFP (2) (150 x 2.00 mm, 3 mu) column by gradient elution and analyzed by tandem mass spectrometry via electrospray ionisation in positive polarity. The analytical response was linear (r(2) >= 0.99) in the concentration (ng/mL) ranges: 50-10 000 for F beta AL and FUH2, 50-5 000 for FUPA, 50-100 000 for 5 -FU, 5-200 for U and 10-400 for UH2. Within-and between-run accuracy and precision were <= 10.2% and <= 9.8% respectively across the QC range and inclusive of LLOQ. The internal standard (IS) normalised matrix effects were within 93-112% with CV of <= 9.7% and normalised recoveries were within 91-107% with CV of <= 9.8%. This robust assay was successfully applied to samples from rectal and colorectal cancer patients (n = 10) on 5 -FU. Deviations <= 2.0% from the mean values were observed when incurred samples were reanalysed. (C) 2017 Elsevier B.V. All rights reserved.