Department of Chemistry, Missouri University of Science and Technology, Rolla, MO, 65409, United States.
Department of Computer Science, Missouri University of Science and Technology, Rolla, MO, 65409, United States.
Anal Biochem. 2021 Sep 15;629:114295. doi: 10.1016/j.ab.2021.114295. Epub 2021 Jun 26.
Oxidative stress is reported to be part of the pathology of many ocular diseases. For the diagnosis of ocular diseases, tear fluid has unique advantages. Although numerous analytical methods exist for the measurement of different types of biomolecules in tear fluid, few have been reported for comprehensive understanding of oxidative stress-related thiol redox signaling. In this study, a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed to determine a panel of twelve metabolites that systematically covered several thiol metabolic pathways. With optimization of MS/MS parameters and HPLC mobile phases, this method was sensitive (LOQ as low as 0.01 ng/ml), accurate (80-125% spike recovery) and precise (<10% RSD). This LC-MS/MS method combined with a simple tear fluid collection with Schirmer test strip followed by ultrafiltration allowed the high-throughput analysis for efficient determination of metabolites associated with thiol redox signaling in human tear fluids. The method was then applied to a small cohort of tear fluids obtained from healthy individuals. The method presented here provides a new technique to facilitate future work aiming to determine the complex thiol redox signaling in tear fluids for accurate assessment and diagnosis of ocular diseases.
氧化应激被认为是许多眼部疾病病理的一部分。对于眼部疾病的诊断，泪液具有独特的优势。尽管有许多分析方法可用于测量泪液中的不同类型生物分子，但很少有方法可用于全面了解与氧化应激相关的硫醇氧化还原信号。在这项研究中，开发了一种高效液相色谱-串联质谱（HPLC-MS/MS）方法来测定十二种代谢物，这些代谢物系统地涵盖了几种硫醇代谢途径。通过优化 MS/MS 参数和 HPLC 流动相，该方法具有较高的灵敏度（最低定量限低至 0.01ng/ml）、准确性（80-125%的加标回收率）和精密度（<10%RSD）。该 LC-MS/MS 方法结合了简单的泪液采集和 Schirmer 测试条，然后进行超滤，可实现高通量分析，从而有效地确定与人类泪液中硫醇氧化还原信号相关的代谢物。然后将该方法应用于从小部分健康个体中获得的泪液。本文介绍的方法为确定泪液中复杂的硫醇氧化还原信号提供了一种新技术，有助于对眼部疾病进行准确评估和诊断。
