Department of Analytical Chemistry, Institute of Chemistry, University of Campinas, 13083-970, Campinas, SP, Brazil.
Department of Analytical Chemistry, Institute of Chemistry, University of Campinas, 13083-970, Campinas, SP, Brazil; Mackenzie Presbyterian University, MackMass Laboratory, Scholl of Engineering, 01302-907, São Paulo, SP, Brazil.
Talanta. 2020 Apr 1;210:120640. doi: 10.1016/j.talanta.2019.120640. Epub 2019 Dec 16.
A notable change in the body fluids nucleosides of cancer patients has been actively highlighted in searches for new biomarkers to early cancer detection. For this reason, improvements of bioanalytical methods for these compounds focused on a noninvasive sampling trend are of great importance. Therefore, this work aimed firstly to develop efficient methods for nucleoside analysis in urine and serum by liquid chromatography-tandem mass spectrometry (LC-MS/MS), applying different strategies to quantify nine nucleosides, and further identify other untargeted nucleosides. Sample preparation was based on protein precipitation and affinity-solid phase extraction (SPE), whereas quantification was performed using a triple quadrupole (QqQ) mass analyzer operating in the selected reaction monitoring (SRM) mode. Surrogates matrices were proposed as an alternative to standard addition calibration. Specifically, to quantitate creatinine, a simple LC-MS/MS method was validated and used for normalization of urinary metabolites quantitation. To identify the other nucleosides, LC methods using different MS scans modes were evaluated on a quadrupole-time of flight (Q-TOF) or a hybrid triple quadrupole linear ion trap (Q-trap). Validation was performed for nucleosides quantification using the synthetic matrices of urine and serum, and selectivity, linearity, accuracy, reproducibility, matrix effect, LOD's and LOQ's were accessed, providing trustworthy results for bioanalysis purposes. Both LC-Q-Trap/MS and LC-Q-TOF/MS methods showed proper sensitivity for structural characterization on assays with urine and serum samples from healthy volunteers and could also be used in the identification of untargeted nucleosides. The investigated approaches delivered in-depth results and seem promising for future applications on urine and serum samples analyses aiming to validate nucleosides as cancer biomarkers.
在寻找新的生物标志物以进行早期癌症检测的过程中，癌症患者体液核苷的显著变化已被积极强调。出于这个原因，改进针对这些化合物的生物分析方法，重点关注非侵入性采样趋势，具有重要意义。因此，这项工作首先旨在通过液相色谱-串联质谱法（LC-MS/MS）开发用于尿液和血清中核苷分析的有效方法，应用不同策略来定量分析 9 种核苷，并进一步鉴定其他非靶向核苷。样品制备基于蛋白质沉淀和亲和固相萃取（SPE），而定量则使用三重四极杆（QqQ）质量分析仪以选择反应监测（SRM）模式进行。替代物基质被提议作为标准添加校准的替代方法。具体来说，为了定量肌酐，验证了一种简单的 LC-MS/MS 方法，并将其用于尿液代谢物定量的归一化。为了鉴定其他核苷，评估了在四极杆飞行时间（Q-TOF）或混合三重四极杆线性离子阱（Q-trap）上使用不同 MS 扫描模式的 LC 方法。使用尿液和血清的合成基质对核苷定量进行了验证，并且评估了选择性、线性、准确性、重现性、基质效应、LOD 和 LOQ，为生物分析目的提供了可靠的结果。LC-Q-Trap/MS 和 LC-Q-TOF/MS 方法均显示出对健康志愿者尿液和血清样本分析的检测具有适当的灵敏度，并且还可用于鉴定非靶向核苷。所研究的方法提供了深入的结果，似乎有望应用于尿液和血清样本分析，以验证核苷作为癌症生物标志物。
