# The Feedingstuffs (Zootechnical Products) Regulations 1999, Paragraph 5


PROCEDURE
5.1 Extraction
Weigh to the nearest 0.001g, approximately 5g of the finely divided and mixed sample, or a suitable amount expected to contain about 350μg of nifursol and transfer to a 125ml conical flask. Add 50.0ml of 95% v/v dimethylformamide solution (3.4), insert a stopper loosely, and place the flask in a water-bath at 60°C±5°C for 30 minutes. Swirl the contents of the flask occasionally during this period. Shake the flask on a mechanical shaker for 30 minutes and then filter the contents through a rapid filter-paper, preferably under reduced pressure on a Buchner funnel. Transfer 40.0ml of water, and stir. Set the beaker aside, protected from light, for 30 minutes.
5.2 Purification
Pack the chromatographic column (4.1) to a depth of 70mm with the prepared aluminium oxide (3.2) and on top of the aluminium oxide add a layer of sand (3.3) 15mm deep. Wash the column with 50ml of 50% v/v dimethylformamide solution (3.5) and then pass the dimethylformamide extract of the test sample through the column; reject the first 45ml of eluate and collect the next 17ml.
5.3 Determination
Pipette 5.0ml of the eluate to a 20ml centrifuge tube, add 5ml of phenylhydrazine hydrochloride solution (3.6), mix, and place the tube in a water-bath at 40°C±2°C for 20 minutes. Remove the tube from the water-bath and cool it in running water for 5 minutes. Add 5.0ml of toluene (3.1) to the contents of the tube, insert a glass or plastic stopper (a rubber stopper must not be used), and shake vigorously 40 times. Centrifuge for 5 minutes to clear the toluene layer, and transfer 3.0ml of the toluene layer to a 10mm spectrophotometer cell. Add 0.2ml of methylbenzethonium hydroxide solution (3.7), mix immediately, and measure the absorbance of the solution within one minute at 515nm with toluene as reference. Determine the quantity of nifursol by reference to the calibration curve (5.4).
5.4 Calibration curve
Pipette 5.0ml of nifursol standard solution (3.8) to a 200ml graduated flask, add 100ml of 95% v/v dimethylformamide solution (3.4), dilute to the mark with water and mix. Into separate 20ml centrifuge tubes transfer by pipette 1, 2, 3, 4 and 5ml portions of this solution and dilute the contents of each tube to 5ml with 50% v/v dimethylformamide solution (3.5).
Treat the contents of each tube as described under “Determination” (5.3) beginning at “… add 5ml of phenylhydrazine hydrochloride solution (3.6) …”. Plot the calibration curve using the absorbance as the ordinates and the corresponding quantities of nifursol in μg as abscissae.

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Source: https://www.legislation.gov.uk/uksi/1999/1871/schedule/5/part/III/paragraph/5 (the official version)
Page: https://openlegislation.uk/feedingstuffs-zootechnical-products-regulations-1999/schedule-5/part-III/paragraph-5
Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives.