Svobaite Rūta, Solassol Isabelle, Pinguet Frederic, Ivanauskas Liudas, Brès Janine, Bressolle Françoise M M
Pharmacokinetic Laboratory, Faculty of Pharmacy, University Montpellier I, Montpellier, France.
Clin Chem. 2008 Sep;54(9):1463-72. doi: 10.1373/clinchem.2007.102251. Epub 2008 Jul 17.
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.
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.
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.
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.
我们开发并比较了两种不同的方法来定量牛血清白蛋白（BSA）和人血浆中的尿嘧啶（U）和二氢尿嘧啶（UH₂）。特别关注了选择性/特异性以及基质效应的不存在。UH₂/U 比值旨在作为一种生物标志物，用于识别 5-氟尿嘧啶代谢缺陷的患者。
我们在固相萃取后，用两种液相色谱方法对 U 和 UH₂进行定量，一种采用紫外检测（LC-UV），另一种采用质谱检测（LC-MS）。我们选择了两种内标以防止干扰风险。使用 Waters Atlantis dC18 柱（LC-MS）或与 Atlantis dC18 相连的 Waters SymmetryShield RP18 柱（LC-UV）实现分离。质谱数据在单离子监测模式下采集。
在 BSA 溶液中，测定的不精密度小于 15%（LC-UV）和小于 12%（LC-MS）；在血浆中，测定的不精密度分别小于 9.5%和小于 9.0%。回收率为 88.2% - 110%（LC-UV）和 94.8% - 107%（LC-MS）。提取效率大于或等于 89.0%。在 BSA 中，对于 LC-UV 方法，U 和 UH₂的定量下限分别为 2.5 μg/L 和 6.25 μg/L，对于 LC-MS 为 2.5 μg/L 和 3.1 μg/L。血浆中的相应值分别为 11.6 μg/L 和 21.5 μg/L，以及 4.1 μg/L 和 12.1 μg/L。
为了估计内源性 U 和 UH₂的浓度及其比值，我们建议使用一个无药物的人血浆池，其中基线 U 和 UH₂浓度先前已用标准加入法测量。当有该设备时，我们的 LC-MS 方法具有更好的测试性能，可用于测量癌症患者的 UH₂/U 比值，因此更受青睐。
