Optimization of HPLC-Coulochemical Method for Determination of Homocysteine and Selected Metabolites in Plasma after Supplementation with Functional Food

Abstract

The HPLC method with coulochemical detection, used for the determination of selected aminothiol compounds (homocysteine, cysteine, reduced glutathione, methionine) in human plasma after quercetin supplementation by consuming cereal onion biscuits, was optimized. The effects of changes of organic modifier (acetonitrile and methanol in phosphate buffer) in the mobile phase and the pH value of the mobile phase on the retention and separation factors were studied. According to the results obtained, mobile phase containing 6 % acetonitrile (v/v) in 50 mmol L^–1 sodium dihydrogen phosphate solution at pH 2.6 was found to be the most suitable for the separation of selected compounds. The method was validated with respect to linearity, precision, and accuracy. Results showed that the method was sufficiently reliable, sensitive, and suitable for application to real plasma samples in clinical or epidemiological studies. Results of the pilot study pointed to the fact that the consumption of onion biscuits (with increased content of quercetine) and simultaneous drinking of green tea during two months caused decrease of homocysteine level by about 18 %. However, there were no simple correlations between quercetin and investigated parameters observed.