Department of Chemistry and Center for Biomedical Science & Engineering, Missouri University of Science and Technology, 400 West 11th Street, Rolla, MO 65409, United States.
Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
Anal Chim Acta. 2015 Jan 1;853:442-450. doi: 10.1016/j.aca.2014.10.044. Epub 2014 Oct 31.
Pteridines are a diverse family of endogenous metabolites that may serve as useful diagnostic biomarkers for disease. While many preparative and analytical techniques have been described for analysis of selected pteridines in biological fluids, broad intracellular pteridine detection remains a significant analytical challenge. In this study, a novel, specific and sensitive extraction and high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF MS) method was developed to simultaneously quantify seven intracellular pteridines and monitor 18 additional, naturally-occurring intracellular pteridines. The newly developed method was validated through evaluation of spiked recoveries (84.5-109.4%), reproducibility (2.1-5.4% RSD), method detection limits (0.1-3.0 μg L(-1)) and limits of quantitation (0.1-1 μg L(-1)), and finally application to non-small cell lung cancer A549 cells. Twenty-three pteridine derivatives were successfully detected from cell lysates with an average RSD of 12% among culture replicates. Quantified intracellular pteridine levels ranged from 1 to 1000 nM in good agreement with previous studies. Finally, this technique may be applied to cellular studies to generate new biological hypotheses concerning pteridine physiological and pathological functions as well as to discovery new pteridine-based biomarkers.
蝶啶类化合物是一类内源性代谢物，它们可能成为疾病诊断的有用生物标志物。虽然已经有许多用于分析生物体液中特定蝶啶类化合物的制备和分析技术，但广泛的细胞内蝶啶检测仍然是一个重大的分析挑战。在这项研究中，开发了一种新颖、特异和灵敏的提取和高效液相色谱-四极杆飞行时间质谱（HPLC-QTOF MS）方法，用于同时定量七种细胞内蝶啶类化合物，并监测另外 18 种天然存在的细胞内蝶啶类化合物。通过评估加标回收率（84.5-109.4%）、重现性（2.1-5.4%RSD）、方法检测限（0.1-3.0μg L(-1))和定量限（0.1-1μg L(-1))，对新开发的方法进行了验证，最后应用于非小细胞肺癌 A549 细胞。从细胞裂解物中成功检测到 23 种蝶啶衍生物，培养重复之间的平均 RSD 为 12%。定量的细胞内蝶啶水平在 1 至 1000nM 之间，与先前的研究结果一致。最后，该技术可应用于细胞研究，以产生关于蝶啶生理和病理功能的新生物学假设，并发现新的蝶啶基生物标志物。
