Clinical Chemistry
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Clinical Chemistry 54: 1463-1472, 2008. First published July 17, 2008; 10.1373/clinchem.2007.102251
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(Clinical Chemistry. 2008;54:1463-1472.)
© 2008 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

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

Ruta Svobaite1,2,1, Isabelle Solassol3,1, Frederic Pinguet3, Liudas Ivanauskas2, Janine Brès1 and Françoise M. M. Bressolle1,3,a

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

aAddress correspondence to this author at: Laboratoire de Pharmacocinétique Clinique, Faculté de Pharmacie, B.P. 14491, 34093 Montpellier Cedex 5, France. Fax (33) 4 67 54 80 75; 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.






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