Fluoropyrimidines-based chemotherapies are the backbone in the treatment of many cancers. However, the use of 5-fluorouracil and its oral pre-prodrug, capecitabine, is associated with an important risk of toxicity. This toxicity is mainly due to a deficiency of dihydropyrimidine dehydrogenase (DPD). This deficiency may be detected by using a phenotypic approach that consists in the measurement of uracilemia or the calculation of dihydrouracil (UH2)/uracil (U) ratio. For uracilemia, a threshold value of 16 ng/ml has been proposed for partial deficiency, while a value of 150 ng/ml has been proposed for complete deficiency. We have developed a rapid, accurate and fully-automated procedure for the quantification of U and UH2 in plasma. Sample extraction was carried out by a programmable liquid handler directly coupled to a liquid chromatography - tandem mass spectrometry (LC-MS/MS) system. The method was validated according to the EMA guidelines and ISO 15189 requirements and was applied to real patient samples (n = 64). The limit of quantification was 5 and 10 ng/ml for U and UH2 respectively. Imprecision and inaccuracy were less than 15% for inter and intra-assay tests. Comparison with dedicated routine method showed excellent correlation. An automated procedure perfectly fulfills the need of low inaccuracy and CVs at the threshold values (less than 5% at 16 ng/ml) and is highly suitable for the characterization of DPD deficiency. Automatization should guaranty reliable and robust performances by minimizing the sources of variation such as volume inaccuracies, filtration or manual extraction related errors.

中文翻译：

---

自动定量血浆中尿嘧啶和二氢尿嘧啶。

基于氟嘧啶的化学疗法是许多癌症的治疗基础。但是，使用5-氟尿嘧啶及其口服前药卡培他滨会带来重大的毒性风险。该毒性主要是由于缺乏二氢嘧啶脱氢酶（DPD）。可以通过使用表型方法检测这种缺陷，该方法包括尿酸血症的测量或二氢尿嘧啶（UH2）/尿嘧啶（U）比的计算。对于尿毒症，已提出部分缺乏的阈值为16 ng / ml，而完全缺乏的阈值为150 ng / ml。我们已经开发了一种快速，准确和全自动的程序，用于定量测定血浆中的U和UH2。样品提取是通过直接与液相色谱-串联质谱（LC-MS / MS）系统耦合的可编程液体处理器进行的。该方法已根据EMA指南和ISO 15189要求进行了验证，并已应用于实际患者样本（n = 64）。U和UH2的定量限分别为5和10 ng / ml。批间和批内测试的不准确性和不准确性均低于15％。与专用常规方法的比较显示出极好的相关性。自动化程序完全可以满足阈值（在16 ng / ml时小于5％）的低不准确性和CV的需求，非常适合于DPD缺乏症的表征。自动化应该通过最大程度地减少变化源（例如音量不准确，