In theory, the calibration curve of GSH and Total GSH should match fairly closely. An important thing to keep in mind is that [GSH] is 5, 2.5, 1.25, 0.625, 0.3125, 0.1563, 0.0781, and 0 µM after serial dilution whereas [TGSH] is 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.1563, and 0 µM. after the same serial dilution. This is because of the GSSG/GSH molar ratio: 1 GSSG ⟶ 2 GSH. Thus when plotting the standard curves, the x-axis values for TGSH needs to be twice that of GSH.