Step 2. Add 40 ml of freshly prepared solution B to the residue from step 1 in the centrifuge tube. Resuspend by manual shaking, stopper and then extract by mechanical shaking for 16 h at 22±5 °C (overnight). No delay should
occur between the addition of the extractant solution and the
beginning of the shaking. Separate the extract from the solid residue by centrifugation and decantation as in step 1. Retain the extract in a stoppered polyethylene container, as before, for analysis. Wash the residue by adding 20 ml of distilled water, shaking for 15 min on the end-over-end shaker and centrifuging for 20 min at 3000 g. Decant the supernatant and discard, taking care not to discard any of the solid residue.