Glutathione HPLC Kit

The Glutathione HPLC Kit is intended for the quantitative determination of glutathione in EDTA whole blood.

For research use only. Not for use in diagnostic procedures.

For Laboratory Professional Use Only.

This is the RUO version of Immundiagnostik part number KC1800.

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Test Principle of the Glutathione HPLC Kit

The determination of glutathione starts by adding a dilution solution to the sample and dividing it into two aliquots.

The addition of reaction buffer and derivatization solution measures the reduced fraction. After an incubation of 20 minutes, in which GSH is transformed into a fluorescent product, a precipitation reagent is added to separate higher molecular substances.

Adding the reduction solution, internal standard, and derivatization solution measures the total glutathione. After that, the sample is handled like the reduced fraction. The reduction solution reduces all the oxidized glutathione (GSSG) to each two molecules of GSH, resulting in the measurement of total GSH.

20 μl of the supernatant are injected into the HPLC system.

The separation via HPLC follows an isocratic method at 30 °C using a reversed-phase column in two runs. One run lasts 10 minutes. A fluorescence detector records the chromatograms. The quantification is performed with the delivered EDTAblood calibrator; the concentration is calculated via integration of the peak area/height by the external standard method for the reduced fraction and the internal standard method for the total GSH fraction. For the measurement of the reduced fraction, using the internal standard is not possible because of the production of mixed sulfides.

The amount of oxidized glutathione (GSSG) can be calculated by subtracting the reduced glutathione (GSH) from the total GSH. Please note that the obtained difference must be divided by two as oxidized glutathione (GSSG) is composed of two GSH molecules.

The formula is: (total GSH – GSH) / 2 = GSSG.