Abstract:

Oxidative stress, which is characterized by an imbalance between antioxidants and free radicals, plays a pivotal role in the pathogenesis of coronary heart disease, a common and serious cardiovascular condition, and contributes significantly to its development and progression. Serum free thiols are crucial components of the body’s antioxidant defense system. The accurate determination of serum free thiol levels provides a reference basis for understanding the body’s status and monitoring the risk factors associated with the occurrence and progression of coronary heart disease. In this study, a high performance liquid chromatographic (HPLC) method based on the derivatization reaction of 2,2'-dithiodipyridine was developed to simultaneously obtain the concentrations of total free thiols (Total-SH), low-molecular-mass free thiols (LMM-SH), and protein-free thiols (P-SH) in human serum. An Agilent Eclipse XDB-C18 column (150 mm×4.6 mm, 5 μm) was used for the analysis, and gradient elution was performed at a flow rate of 1 mL/min. A 0.1% formic acid aqueous solution was used as mobile phase A, and a 0.1% formic acid acetonitrile solution was used as mobile phase B. The gradient elution program was as follows: 0-0.1 min, 12%B-30%B; 0.1-2 min, 30%B; 2-2.1 min, 30%B-100%B; 2.1-6 min, 100%B; 6-6.1 min, 100%B-12%B; 6.1-7 min, 12%B. Well-separated peaks appeared after a run time of 5 min. The peak of 2-thiopyridone represented the Total-SH content of the samples, and the peak of the pyridyldithio derivative represented the LMM-SH content. The difference between these two peaks indicated the P-SH content. The derivatization reaction conditions were optimized, and the method was validated. The method demonstrated good linearity, with a correlation coefficient ≥0.9994, over the concentration range of 31.25-1000 μmol/L. The limits of detection for Total-SH and LMM-SH were 2.61 and 0.50 μmol/L, and the limits of quantification for Total-SH and LMM-SH were 8.71 and 1.67 μmol/L, respectively. The recoveries of Total-SH and LMM-SH were in the range of 91.1%-106.0%. The intra- and inter-day precisions ranged from 0.4% to 9.1%. The developed method was used to analyze serum samples from 714 volunteers. The Total-SH concentrations ranged from 376.60 to 781.12 μmol/L, with an average concentration of 555.62 μmol/L. The LMM-SH concentrations varied from 36.37 to 231.65 μmol/L,with an average of 82.34 μmol/L. The P-SH concentrations ranged from 288.36 to 687.74 μmol/L, with an average of 473.27 μmol/L. Spearman’s correlation test showed that serum thiol levels were correlated with the severity of coronary artery disease and common clinical biochemical indicators. The proposed study provides a simple and reliable HPLC method for detecting serum free thiols and exploring their relationship with coronary heart disease, offering a new reference for the study of markers related to the risk of coronary heart disease.

Key words: high performance liquid chromatography (HPLC), derivatization reaction, 2,2'-dithiodipyridine, free thiols, serum, coronary heart disease