Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, the Netherlands; Clinical Laboratory, Catharina Hospital Eindhoven, the Netherlands; Expert Center Clinical Chemistry Eindhoven, the Netherlands.
Clinical Laboratory, Catharina Hospital Eindhoven, the Netherlands; Central Diagnostic Laboratory, Maastricht University Medical Center, Maastricht, the Netherlands.
J Pharm Biomed Anal. 2022 Nov 30;221:115027. doi: 10.1016/j.jpba.2022.115027. Epub 2022 Sep 1.
Establishing dihydropyrimidine dehydrogenase (DPD) activity is highly important in determining the correct starting dose of fluoropyrimidines such as 5-fluorouracil and capecitabine. The concentration ratio of endogenous uracil with its metabolite dihydrouracil (DHU) is a well-known parameter that is linked to DPD activity. Concentration ratios such as thymine over its DPD-converted metabolite dihydrothymine (DHT) is less described and may serve as an alternative diagnostic biomarker for DPD activity. In this study, we describe the development and validation of an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for the quantification of uracil, DHU, thymine, and DHT in human plasma. In addition, stability experiments were performed. Uracil and thymine were quantified up to 80.0 ng/mL and DHU and DHT up to 800 ng/mL. Intra- and inter-assay precision were maximum 8.0 % and 7.6 %. respectively. Also, recovery was adequate and significant matrix-effects and carry-over were excluded. Stability experiments showed that uracil concentrations increased with 27-52 % when stored for 1 or 2 h at ambient temperatures compared to cold storage. Thymine, DHU, and DHT concentrations remained stable, thymine after 1 h in plasma excluded, showing the DHT:T ratio might be a more robust marker for DPD activity than DHU:U. In conclusion, we present here a novel assay capable of quantifying uracil, thymine, DHU and DHT in a single analytical run. We provide additional data showing increased stability for DHU, thymine and DHT compared to uracil. This assay may be used as a diagnostic test in future studies, establishing the association of these endogenous biomarker concentrations with DPD activity and safety to treatment with fluoropyrimidines. In addition, future research should also be focused on reducing pre-analytical instability. Standardization in this field is essential to set proper reference values and to allow inter-study comparison on clinical outcomes.
建立二氢嘧啶脱氢酶（DPD）活性对于确定氟嘧啶类药物（如 5-氟尿嘧啶和卡培他滨）的起始剂量非常重要。内源性尿嘧啶与其代谢物二氢尿嘧啶（DHU）的浓度比值是与 DPD 活性相关的一个众所周知的参数。胸腺嘧啶与其 DPD 转化代谢物二氢胸腺嘧啶（DHT）的浓度比值则描述较少，可能作为 DPD 活性的替代诊断生物标志物。在本研究中，我们描述了一种超高效液相色谱-串联质谱（UPLC-MS/MS）测定人血浆中尿嘧啶、DHU、胸腺嘧啶和 DHT 的方法的建立和验证。此外，还进行了稳定性实验。尿嘧啶和胸腺嘧啶的定量下限分别为 80.0ng/mL 和 800ng/mL。内、日间精密度最高分别为 8.0%和 7.6%。此外，回收率充足，且显著排除了基质效应和交叉污染。稳定性实验表明，与冷藏相比，室温下储存 1 或 2 小时，尿嘧啶浓度增加 27-52%。胸腺嘧啶、DHU 和 DHT 浓度保持稳定，排除了血浆中 1 小时的胸腺嘧啶，表明 DHT:T 比值可能比 DHU:U 更能反映 DPD 活性。总之，我们在此提出了一种新的分析方法，能够在单次分析运行中定量测定尿嘧啶、胸腺嘧啶、DHU 和 DHT。我们提供了额外的数据，表明与尿嘧啶相比，DHU、胸腺嘧啶和 DHT 更稳定。该方法可用于未来的研究中，建立这些内源性生物标志物浓度与 DPD 活性和氟嘧啶类药物治疗安全性的关联。此外，未来的研究还应集中在减少分析前的不稳定性上。该领域的标准化对于设定适当的参考值和允许对临床结果进行研究间比较至关重要。
