Coudoré François, Roche Denis, Lefeuvre Sandrine, Faussot Delphine, Billaud Eliane M, Loriot Marie-Anne, Beaune Philippe
Biochemistry Department, Assistance Publique-Hôpitaux de Paris, European Hospital Georges Pompidou, France.
J Chromatogr Sci. 2012 Nov-Dec;50(10):877-84. doi: 10.1093/chromsci/bms085. Epub 2012 Jun 11.
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.
建立并验证了一种简单、快速、灵敏且特异的超高效液相色谱-串联质谱法（沃特世UPLC-MS-MS），用于定量测定人血浆中尿嘧啶（U）和5,6-二氢尿嘧啶（UH2）的水平。分析物在脱蛋白后用乙酸乙酯和异丙醇萃取，并通过高效液相色谱（HPLC）（Acquity UPLC BEH C18柱）在二元流动相系统中于梯度洗脱条件下以0.4 mL/min的流速进行分离。5-溴尿嘧啶（UBr）用作内标。检测在三重四极杆质谱仪上通过电喷雾正离子化进行。分别使用m/z 112.82 → 70.05、m/z 114.88 → 55.04和m/z 190.83 → 117.86的多反应监测模式来定量U、UH2和UBr。该方法在0.625 - 160.0 ng/mL的浓度范围内呈线性。每次进样的总运行时间为4.5分钟。采用九点校准曲线和四点质量控制。观察到具有优异的线性和精密度，相关系数（r(2)）> 0.9999。批内和批间精密度分别≤ 7.3%和≤ 8.6%，准确度≤ 17%。所建立的方法适用于临床实践中U、UH2以及5,6-二氢尿嘧啶与尿嘧啶比值的常规定量测定。
