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The sample (20 L of 1: 20 hemolysate + 20 L of KH 2 PO 4 buffer), 220 L of the main reagent and 5 L of FAD 0.315 mM + 10 L of KH 2 PO 4 buffer were added to the cuvette and the absorbance was monitored at 340 nm for 200 s (step A)