High-performance liquid chromatographic assay with UV detection for measurement of dihydrouracil/uracil ratio in plasma.
A rapid, robust and sensitive HPLC method for analysis of uracil (U) and dihydrouracil (UH2) in plasma was developed using solid phase extraction and ultraviolet detection. Separation was achieved with a SymmetryShield RP18 column and an Atlantis dC18 column using a 10 mM potassium phosphate buffer as mobile phase. Compounds were eluted within 15 min without interference. Recovery was 80.4 and 80.6% for U and UH2. Calibration curves were linear from 2.5 to 80 ng/mL for U and 6.75 to 200 ng/mL for UH2. The LLQ was, respectively, 2.5 ng/mL for U, and 6.75 ng/mL for UH2. Within-run and between-run precision were less than 5.94% and inaccuracy did not exceed 7.80%. The overall procedure has been applied to correlate UH2/U ratio with dihydropyrimidine dehydrogenase activity in 165 cancer patients.
Department of Pharmacokinetic, Anticancer Centre René Gauducheau, Boulevard Jacques Monod, 44805 NANTES Cedex, France. r-deporte-fety@nantes.fnclcc.fr
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Déporte
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Amiand
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Moreau
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Charbonnel
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Campion
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005472:Fluorouracil / Q000008:administration & dosage; D006801:Humans; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D014498:Uracil / Q000031:analogs & derivatives* / Q000097:blood*
C007419:dihydrouracil; D014498:Uracil; D005472:Fluorouracil