Step 1: Add 1 g of sample (or homogenised sample) to 4 mL of water, mix then add 1 mL of 10 mg/mL sodium borohydride (dissolved in 50 mM NaOH and less than 5 hours old). Incubate this solution in a sealed plastic container at 40°C for 30 min then neutralise by the addition of 2.5 mL of 0.2 M acetic acid. Transfer all of the borohydride reduced sample (~ 8.5 mL) to a 10 mL volumetric flask and make the final volume to 10 mL with distilled water. Filter through Whatman No. 1 filter paper or centrifuge in a microfuge at 13000 x g and use the filtrate or supernatant directly in the assay or with an appropriate dilution in distilled water (if required). The filtrate may be hazy but this is stable in the assay and contributes very little to the absorbance. Typically use a sample volume of 0.2 mL in Step 2 of PROCEDURE B.
For analysis of results the dilution is 1 and the concentration of the prepared sample is 100 g/L (i.e. 1 g prepared in 10 mL).


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