Received on December 17, 2007
Accepted on June 30, 2008

Cancer Diagnostics

HPLC with UV or Mass Spectrometric Detection for Quantifying Endogenous Uracil and Dihydrouracil in Human Plasma

Rta vobait ¹, Isabelle Solassol ², Frederic Pinguet ², Liudas Ivanauskas ³, Janine Bres ⁴, Françoise M. M. Bressolle ^5*

¹ Pharmacokinetic Laboratory, Faculty of Pharmacy, University Montpellier I, Montpellier, France, and Department of Analytical and Toxicological Chemistry, Kaunas University of Medicine, Kaunas, Lithuania
² Oncopharmacology Department, Pharmacy Service, Val d'Aurelle Anticancer Centre, Montpellier, France
³ Department of Analytical and Toxicological Chemistry, Kaunas University of Medicine, Kaunas, Lithuania
⁴ Pharmacokinetic Laboratory, Faculty of Pharmacy, University Montpellier I, Montpellier, France
⁵ Pharmacokinetic Laboratory, Faculty of Pharmacy, University Montpellier I, Montpellier, France, and Oncopharmacology Department, Pharmacy Service, Val d'Aurelle Anticancer Centre, Montpellier, France

^* To whom correspondence should be addressed. E-mail: Fbressolle{at}aol.com.

BACKGROUND: We developed and compared 2 different methods for quantifying uracil (U) and dihydrouracil (UH₂) in BSA and human plasma. Special attention was paid to the selectivity/specificity and the absence of a matrix effect. The UH₂/U ratio is intended as a biomarker to identify patients with deficiency in 5-fluorouracil metabolism.

METHODS: We quantified U and UH₂ with 2 liquid chromatography methods after solid-phase extraction, one with UV detection (LC-UV) and the other with mass spectrometric detection (LC-MS). We selected 2 internal standards to prevent the risk of interferences. Separation was achieved with a Waters Atlantis dC18 column (LC-MS) or a Waters SymmetryShield RP18 column connected with an Atlantis dC18 (LC-UV). Mass spectrometric data were acquired in single-ion monitoring mode.

RESULTS: Assay imprecision in BSA solution was <15% (LC-UV) and <12% (LC-MS); in plasma, assay imprecision was <9.5% and <9.0%, respectively. Recoveries were 88.2%–110% (LC-UV) and 94.8%–107% (LC-MS). Extraction efficiencies were 89.0%. In BSA, the lower limits of quantification for U and UH₂ were 2.5 µg/L and 6.25 µg/L, respectively, for the LC-UV method and 2.5 µg/L and 3.1 µg/L for LC-MS. The corresponding values in plasma were 11.6 µg/L and 21.5 µg/L, and 4.1 µg/L and 12.1 µg/L.

CONCLUSIONS: To estimate endogenous U and UH₂ concentrations and their ratio, we recommend the use of a drug-free human plasma pool in which baseline U and UH₂ concentrations have previously been measured with the standard-addition method. Our LC-MS method, which has the better test performance and is useful for measuring UH₂/U ratios in cancer patients, is preferred when this equipment is available.