Since it is photosensitive, and its absorption decreases over time, the anthrone-sulfuric acid reagent is prepared freshly on the day of analysis. One hundred micro liters of a glucose standard, or the sample, is added to separate wells of the same 96-well microplate (Corning), and then 200 μL of 0.1% or 0.2% anthrone in concentrated H2SO4 was added to each well. The solution was well mixed using a horizontal shaker for 1 min at room temperature, and the reaction incubated for 30 min at 80 °C in an oven; the microplate was covered and protected from light during the incubation. The plate was then cooled to room temperature before the absorbance was read at 625 nm using a microplate reader (TECAN, Infinite 200 PRO).