Validation of an ultra-high performance liquid chromatography tandem mass spectrometric method for quantifying uracil and 5,6-dihydrouracil in human plasma.
A simple, rapid, sensitive and specific ultra-high-performance liquid chromatography-tandem mass spectrometry method (Waters UPLC-MS-MS) is developed and validated for the quantification of uracil (U) and 5,6-dihydrouracil (UH2) levels in human plasma. Analytes are extracted using ethyl acetate and isopropanol after deproteination, and separated by high-performance liquid chromatography (HPLC) (Acquity UPLC BEH C18 column) in a binary mobile phase system under gradient elution conditions at a flow rate of 0.4 mL/min. 5-Bromo-uracil (UBr) is used as the internal standard. The detection is performed on a triple-quadrupole mass spectrometer via electrospray positive ionization. Multiple reaction monitoring mode using the transitions m/z 112.82 → 70.05, m/z 114.88 → 55.04 and m/z 190.83 → 117.86 is used to quantify U, UH2 and UBr, respectively. The method is linear in the concentration range of 0.625-160.0 ng/mL. The total run time is 4.5 min per injection. Nine-point calibration curve and four-points quality controls are used. Excellent linearity and precision are observed with correlation coefficient (r(2)) > 0.9999. The intra-batch and inter-batch precisions are ≤ 7.3% and ≤ 8.6%, and accuracy is ≤ 17%. The developed method is shown to be suitable for routine quantitative determination of U, UH2 and 5,6-dihydrouracil-to-uracil ratio in clinical practice.
Biochemistry Department, Assistance Publique-Hôpitaux de Paris, European Hospital Georges Pompidou, France. francois.coudore@egp.aphp.fr
François
F
Coudoré
Denis
D
Roche
Sandrine
S
Lefeuvre
Delphine
D
Faussot
Eliane M
EM
Billaud
Marie-Anne
MA
Loriot
Philippe
P
Beaune
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D016014:Linear Models; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014498:Uracil / Q000031:analogs & derivatives* / Q000097:blood* / Q000737:chemistry
C007419:dihydrouracil; D014498:Uracil