摘要
Background Oxidative stress is a major mechanism underlying aging and health damage, and it is associated with decreased serum free thiol levels. Monitoring serum free thiol levels provides a valuable reference for assessing the body's health status. However, it is a key challenge in clinical analysis to simultaneously monitor serum total free thiols and specific low-molecular-mass free thiol compounds in a single run. Traditional methods often struggle to avoid interference from side reactions due to the high reactivity of free thiols in alkaline derivatization environments. Results In this study, we developed an integrated non-alkaline derivatization strategy using 2,2′-dithiodipyridine and high-performance liquid chromatography. 2,2′-Dithiodipyridine can react rapidly with free thiols under acidic to neutral environments, and all derivatives exhibit similar absorbance characteristics to the substrates. Taking advantage of these properties, this method combines reliable derivatization with efficient chromatographic separation, enabling the simultaneous analysis of total free thiols and five main specific low-molecular-mass thiol compounds within a single injection cycle. Eight chromatographic peaks including free thiol derivatives, the derivatization reagent, and internal standard were observed during a 14-minute analysis. The peak corresponding to 2-thiopyridone indicates the total concentration of free thiols while specific pyridyldithio derivative peaks represent the individual levels of low-molecular-mass free thiols. Using the developed method, a significant negative correlation was observed between free thiol levels and both age and health risk factors in a study involving 260 volunteers. Significance This study presents a robust and simple method for the simultaneous measurement of total free and low-molecular-mass free thiols, providing a facile technique for population analysis and for investigating the relationship between free thiols and human health.
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