Paragraph 5 Procedure
Extraction of Uric Acid
From dried poultry waste:
Weigh to the nearest 0.001 g, about 0.4 g dried poultry waste and place in a 150 ml round-bottomed flask. Add 60 ml ethanolic formaldehyde solution (3.3), fit a reflux condenser onto the flask and heat on a steam bath for 1 hour. Cool and filter by suction through a sintered glass crucible (porosity 4) into a 100 ml graduated flask. Wash out the round-bottomed flask with 3 × 10 ml portions of ethanolic formaldehyde solution (3.3) passing each portion through the crucible into the graduated flask. Dilute to 100 ml with ethanolic formaldehyde solution and mix.
From feeding stuffs:
Weigh to the nearest 0.001 g, between 4 g and 5 g of prepared sample. Transfer to a glass percolation tube (4.2) fitted with a small paper cup to retain the feed. Remove the fat from the feed by extraction with light petroleum (3.10). Transfer quantitatively the defatted sample to a 150 ml round-bottomed flask and remove the residual solvent with a slow current of air. Continue as in 5.1.1, second sentence “… Add 60 ml ethanolic formaldehyde solution (3.3) …”.
Determination
Transfer by pipette 20 ml of the sample extract prepared as in 5.1.1 or 5.1.2 to a 50 ml centrifuge tube. Add 10 ml of Benedict and Hitchcock reagent 93.8), mix well and allow to stand in the dark for 1 hour. Centrifuge at 2,000 rpm for 15 minutes, pour off the supernatant liquid and allow to drain for 10 minutes. Carefully wipe off any remaining liquid without disturbing the precipitate, and add 20 ml sodium thiosulphate solution (3.5) to each tube. Dissolve the precipitate by stirring with a thin glass rod. Transfer by pipette 5 ml of this solution into a 200 mg graduated flask containing 40 ml succinate buffer solution (3.4). Dilute to 200 ml with water and mix well. Measure the absorbance of the solution at 294 mm in 10 mm silica cells against a solution prepared by mixing 5 ml sodium thiosulphate solution (3.5) with 40 ml succinate buffer solution (3.4) and diluting to 200 ml with water. Determine the quantity of uric acid present by reference to the calibration curve (5.3).
Calibration Curve
Into a series of 50 ml centrifuge tubes, transfer by pipette 2, 4, 6, 8, 10 and 12 ml standard uric acid solution (3.9) (corresponding to 2, 4, 6, 8, 10 and 12 mg of uric acid) and make up to 20 ml with ethanolic formaldehyde solution (3.3). Add to each tube 10 ml Benedict and Hitchcock reagent (3.8), mix well and stand in the dark for 1 hour. Continue as in 5.2 from “… Centrifuge at 2,000 rpm. …”, Measure the absorbances of the solutions and plot the calibration curve using absorbances as the ordinates and the corresponding quantities of uric acid, in mg (as shown above) as the abseissae.