During this research a simple, accurate, and environmentally friendly method to determine lipoyllysine and lipoic acid in meat was developed and validated. The presented approach was based on the hydrolysis of the proteins containing lipoic acid, reduction of disulfide bonds with tris(hydroxymethyl)phosphine, and precolumn derivatization of free thiol groups with 1-benzyl-2-chloropyridinium bromide long-term followed by HPLC separation with a diode-array detector. The method has been validated in accordance with the U.S. FDA guidelines and was linear in the range of 0.1-10 μmol/L in concentration with R2 values ≥0.9997 for both analytes. For lipoyllysine and lipoic acid, intra- and interday precision values were lower than 10%. The intraday accuracy values ranged from 91.0% to 99.4% for lipoyllysine and from 99.1% to 107.3% for lipoic acid, whereas the interday accuracy values for lipoyllysine and lipoic acid were 92.0-95.6% and 93.5-98.8%, respectively. Additionally, in this research the antioxidant activity of lipoyllysine and reduced lipoyllysine compound using spectrophotometric method with 1,1-diphenyl-2-picrylhydrazyl was examined for the first time. The data showed that dihydrolipoyllysine exhibits stronger antioxidant capacity than lipoyllysine based on a lower value of concentration required to achieve a 50% antioxidant effect in 1,1-diphenyl-2-picrylhydrazyl radical scavenging test.
Keywords: 2,2-diphenyl-1-picrylhydrazyl; antioxidants; high performance liquid chromatography; hydrolysis; lipoyllysine; radical scavenging; tissue.