Department of Analytical Chemistry, Faculty of Science, Charles University, Prague, Czech Republic.
Institute of Rheumatology, Prague, Czech Republic; Department of Rheumatology, First Faculty of Medicine, Charles University, Prague, Czech Republic.
Anal Biochem. 2020 Jan 15;589:113509. doi: 10.1016/j.ab.2019.113509. Epub 2019 Nov 17.
Allantoin is an excellent biomarker of oxidative stress in humans as the main product of uric acid oxidation by reactive oxygen species. Yet, allantoin determination is still not routinely performed in clinical laboratories. Therefore, we developed a fast, simple, selective, and sensitive UHPLC-MS/MS method for allantoin determination in human serum using an isotopically labeled internal standard. Our analytical protocol provided high sensitivity by mass spectrometry detection and high throughput by HILIC-MS/MS analysis within 4 min, with one-step serum sample preparation approximately within 7 min. Lastly, our protocol was fully validated to demonstrate its reliability in allantoin determination in human serum. The method showed an excellent linear range from 0.05 to 100 μM, with precision ranging from 1.8 to 11.3% (RSD), and with accuracy (relative error %) within ±6.0%. The method was then applied to analyze the concentration of allantoin in serum samples from 71 patients with chronic gout without treatment with xanthine oxidase inhibitors. The median serum allantoin concentration in the cohort was 2.8 μM (n = 71). Overall, our simple analytical protocol has the potential to be easily implemented in clinical routine practice for monitoring allantoin as a key oxidative stress biomarker.
尿囊素是人类氧化应激的一个极好的生物标志物，因为它是活性氧物种氧化尿酸的主要产物。然而，尿囊素的测定在临床实验室中仍然没有常规进行。因此，我们开发了一种快速、简单、选择性和灵敏的 UHPLC-MS/MS 方法，用于使用同位素标记的内标测定人血清中的尿囊素。我们的分析方案通过质谱检测提供了高灵敏度，通过亲水作用色谱-质谱/质谱分析在 4 分钟内提供了高通量，一步血清样品制备大约在 7 分钟内完成。最后，我们的方案经过全面验证，证明了其在人血清中尿囊素测定中的可靠性。该方法在 0.05 至 100 μM 的范围内表现出优异的线性关系，精密度范围为 1.8%至 11.3%（RSD），准确度（相对误差%）在±6.0%范围内。该方法随后应用于分析 71 例未经黄嘌呤氧化酶抑制剂治疗的慢性痛风患者的血清尿囊素浓度。该队列的血清尿囊素中位数浓度为 2.8 μM（n=71）。总的来说，我们的简单分析方案有可能在临床常规实践中很容易地用于监测尿囊素作为关键的氧化应激生物标志物。
