Ureidopropionic acid

Clinical pharmacokinetics of fluorouracil and folinic acid

This article deals with the pharmacokinetics of fluorouracil (5-FU) and folinic acid (CHO-THF). 5-FU kinetics are characterized by short serum half-life times of 4.5 to 13 minutes. The high body clearance is mainly caused by a rapid catabolism of 5-FU to dihydrofluorouracil (FUH2), alpha-fluoro-ureidopropionic acid (FUPA), and to alpha-fluoro-beta-alanine (FBAL). Due to an incomplete hepatic extraction, systemic 5-FU levels greater than 5 microM are determined even during hepatic artery infusion. 5-FU measurements during isolated liver perfusion revealed different phases of 5-FU clearance. With higher levels (greater than 430 microM) clearance behaves in accordance with zero-order kinetics, with lower concentrations (less than 150 microM) 5-FU is cleared according to first-order kinetics. The impact of 5-FU kinetics on the treatment with 5-FU is discussed. After administration of the commercially available d,l-folinic acid (d,l-CHO-THF), the biologically inactive d-form is cleared very slowly with a median half-life of 438 +/- 63 minutes. l-CHO has short elimination half-lives of 56.5 +/- 10.5 minutes. The high body clearance of 222 +/- 27 mL/min is partially caused by metabolism to l-methyltetrahydrofolic acid (l-CH3-THF). With bolus injection of 200 mg/m2 or short-term infusion of 300 mg d,l-CHO-THF, serum levels greater than 1 microM are attained for approximately 2 to 3 hours. Total reduced l-folates (l-CHO-THF plus l-CH3-THF) are above 5 microM for at least 3 hours. Continuous infusion of CHO-THF affords steady-state levels in the micromolar range for prolonged time periods. After application of the pure l-folinic acid, mean elimination half-live and area under the curve were in the same range as after administration of the d,l-CHO-THF. Higher doses of oral d,l-folinic acid are always absorbed incompletely. Since the amount of l-CHO-THF absorbed is converted rapidly to CH3-THF, serum levels of l-CHO-THF remain always below 0.1 microM. CH3-THF steady-state levels in the micromolar range, however, are attained with different oral d,l-CHO-THF protocols.

Enzymic interconversion of hydrouracil and beta-ureidopropionic acid

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 α-fluoro-β-ureidopropionic acid (FUPA) and α-fluoro-β-alanine (FβAL) 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 FβAL 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) (150×2.00mm, 3μ) column by gradient elution and analyzed by tandem mass spectrometry via electrospray ionisation in positive polarity. The analytical response was linear (r²≥0.99) in the concentration (ng/mL) ranges: 50-10 000 for FβAL 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.

The catabolism of C14-labeled uracil, dihydrouracil, and beta-ureidopropionic acid in the intact rat

Liquid chromatography-tandem mass spectrometric assay for the analysis of uracil, 5,6-dihydrouracil and beta-ureidopropionic acid in urine for the measurement of the activities of the pyrimidine catabolic enzymes

A liquid chromatography-tandem mass spectrometric assay for the determination of uracil, 5,6-dihydrouracil and beta-ureidopropionic acid in urine was developed to measure the activities of enzymes involved in pyrimidine breakdown. The assay was required to investigate the relation between the uracil-dihydrouracil ratio and toxicities observed after treatment with fluoropyrimidines drugs. After addition of stable isotopically labelled internal standards, the analytes were isolated from a 100-microl urine sample using liquid-liquid extraction with ethyl acetate-2-propanol. Compounds were separated on an Atlantis dC18 column, using ammonium acetate-formic acid in water as the eluent. The eluate was totally led into an electrospray interface with positive ionisation and the analytes were quantified using triple quadrupole mass spectrometry. The assay was validated in the range 1.6-1600 microM, using both, artificial urine and pooled urine as matrices. Intra-day precisions were < or = 8% and inter-day precisions were < or = 10%. Accuracies between 91 and 108% were found. The analytes were chemically stable under all relevant conditions and the assay was successfully applied in two clinical studies of cancer patients treated with 5-fluorouracil or capecitabine.