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


METHODS
We quantified U and UH(2) 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 >or=89.0%. In BSA, the lower limits of quantification for U and UH(2) were 2.5 microg/L and 6.25 microg/L, respectively, for the LC-UV method and 2.5 microg/L and 3.1 microg/L for LC-MS. The corresponding values in plasma were 11.6 microg/L and 21.5 microg/L, and 4.1 microg/L and 12.1 microg/L.


CONCLUSIONS
To estimate endogenous U and UH(2) concentrations and their ratio, we recommend the use of a drug-free human plasma pool in which baseline U and UH(2) 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 UH(2)/U ratios in cancer patients, is preferred when this equipment is available.
Svobaite
Rūta
R
Pharmacokinetic Laboratory, Faculty of Pharmacy, University Montpellier I, Montpellier, France.
Solassol
Isabelle
I
Pinguet
Frederic
F
Ivanauskas
Liudas
L
Brès
Janine
J
Bressolle
Françoise M M
FM
D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D006900:Hydroxylation; D013058:Mass Spectrometry / Q000379:methods*; D015394:Molecular Structure; D009369:Neoplasms / Q000097:blood; D012997:Solvents; D013053:Spectrophotometry / Q000379:methods*; D014498:Uracil / Q000097:blood* / Q000737:chemistry*
D012997:Solvents; D014498:Uracil