The Feedingstuffs (Zootechnical Products) Regulations 1999

PART III DETERMINATION OF NIFURSOL [3,5-dinitro-2-(5-nitrofurfuryli dene)salicylohydrazide]

1.  SCOPE AND FIELD OF APPLICATION

The method is for the determination of the quantity of nifursol in complete feeding stuffs, protein concentrates and feed supplements. Other substances that will provide a nitro group under the conditions of the method, e.g. nitrofurazone and furazolidone, will interfere. The lower limit of the determination is 20mg/kg.

2.  PRINCIPLE

The sample is extracted with dimethylformamide and the extract is purified on a column of aluminium oxide. A portion of the purified extract containing the nifursol is treated with phenylhydrazine hydrochloride and the resulting phenylhydrazone extracted into toluene. The addition of methylbenzethonium hydroxide to the toluene solution produces a blue colour, the absorbance of which is measured as 515nm.

3.  REAGENTS

3.1 Toluene.

3.2 Aluminium oxide for column chromatography, 80 to 200 mesh, alkaline, Brockman activity 1. To 100 parts of the aluminium oxide add 6 parts of powdered magnesium hydroxide. Shake in a screw-cap bottle to mix, add 8 parts of water, and mix until free from lumps.

3.3 Sand; acid washed.

3.4 Dimethylformamide solution, 95% v/v.

3.5 Dimethylformamide solution, 50% v/v.

3.6 Phenylhydrazine, hydrochloride solution: shake 0.25±0.005g of phenylhydrazine hydrochloride in 25ml of water, add 25ml of concentrated hydrochloric acid, and shake to dissolve the solid, filtering if necessary. Prepare this reagent immediately before use.

3.7 Methylbenzethonium hydroxide solution: about 10% in methanol.

3.8 Nifursol standard solution: weigh, to the nearest 0.1mg, 25mg of pure nifursol into a 100ml graduated flask, add 5ml of 95% v/v dimethylformamide solution (3.4), and mix until all the solid has dissolved. Dilute to the mark with methanol. Prepare this solution freshly each day.

4.  APPARATUS

4.1 Chromatographic column—A glass column, internal diameter: 20 to 25mm; length: 100 to 150mm plugged at the lower end with glass wool.

4.2 Spectrophotometer, with 10mm cells.

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.

6.  CALCULATING THE RESULTS

• The nifursol content in mg/kg of sample is given by the formula:

• 

• in which:

  • A=μg of nifursol read from the calibration curve; and

  • M=mass of the test portion in g.