Extraction and determination of free glutathione and its disulfide in various flour samples using liquid chromatography and on-line post column derivatization

In the present report we propose an HPLC method coupled to on-line post column derivatization for the simultaneous determination of glutathione and its disulfide in flour samples. The extraction of the analytes from the sample matrix was investigated in terms of efficiency, stability and compatibility with the analytical method. The selection of o-phthalaldehyde at high alkaline medium as post column reaction system enabled the determination of both analytes in a single run at reasonable separation times (<10 min). Fluorimetric detection allowed determination at the low nmol g-1 (5-75 nmol g-1) levels in the absence of matrix interferences from endogenous compounds. The LOD values were 1.2-1.3 nmol g-1 and the precision better than 7.2% in all cases. Various commercially available flour samples (wheat, corn, rice) were analyzed and were found to contain glutathione and its disulfide in the ranges of 29-243 and 8-84 nmol g-1 respectively. Only in one rice flour sample the levels of GSH were < LOQ of the method.

Automated summary

In this study, an HPLC method coupled with on-line post column derivatization was proposed for the simultaneous determination of glutathione and its disulfide in flour samples. The efficiency, stability and compatibility of analyte extraction from the sample matrix were investigated. By using o-phthalaldehyde in a high alkaline medium as the post column reaction system, both analytes could be determined in a single run with reasonable separation times. Fluorimetric detection allowed for determination at low levels without interference from endogenous compounds. The method exhibited LOD values of 1.2-1.3 nmol g-1 and precision better than 7.2% in all cases. Analysis of commercially available flour samples revealed the presence of glutathione and its disulfide in the ranges of 29-243 and 8-84 nmol g-1, respectively, with only one rice flour sample having levels of GSH below the LOQ of the method.