Second-order calibration of excitation-emission matrix fluorescence spectra for determination of glutathione in human plasma

A rapid non-separative spectroflourimetric method based on the second-order calibration of the excitation-emission data matrix was proposed for the determination of glutathione (GSH) in human plasma. In the phosphate buffer solution of pH 8.0 GSH reacts with ortho-phthaldehyde (OPA) to yield a fluorescent adduct with maximum fluorescence intensity at about 420 nm. To handle the interfering effects of the OPA adducts with aminothiols other than GSH in plasma as well as intrinsic fluorescence of human plasma, parallel factor (PARAFAC) analysis as an efficient three-way calibration method was employed. In addition, to model the indirect interfering effect of the plasma matrix, PARAFAC was coupled with standard addition method. The two-component PARAFAC modeling of the excitation-emission matrix fluorescence spectra accurately resolved the excitation and emission spectra of GSH, plasma (or plasma constituents). The concentration-related PARAFAC score of GSH represented a linear correlation with the concentration of added GSH, similar to that is obtained in simple standard addition method. Using this standard addition curve, the GSH level in plasma was found to be 6.10 ± 1.37 μmol L⁻¹. The accuracy of the method was investigated by analysis of the plasma samples spiked with 1.0 μmol L⁻¹ of GSH and a recovery of 97.5% was obtained.