The Feedingstuffs (Zootechnical Products) Regulations 1999

PART II DETERMINATION OF METICLORPINDOL (3,5-dichloro-2,6- dimethylpyridine-4-ol)

1.  SCOPE AND FIELD OF APPLICATION

• The method is for the determination of the quantity of meticlorpindol in complete feeding stuffs, protein concentrates and feed supplements. The lower limit of the determination is 60mg/kg.

2.  PRINCIPLE

• Meticlorpindol is extracted from the feed with methanolic ammonia solution, and a portion of the extract is passed through a column of aluminium oxide onto a column of ion-exchange resin. The meticlorpindol is retained on the resin and interfering substances are removed by washing with 80% methanol. The meticlorpindol is eluted from the resin with 40% acetic acid and the absorbance is measured at 267nm.

3.  REAGENTS

• Note: The suitability of a batch of aluminium oxide and of the other reagents should be tested before use by analysing a blank feed to which a known amount of meticlorpindol has been added.

  3.1.

  Aluminium oxide for column chromatography, 100 to 250 mesh, alkaline, Brockman activity 1.

  3.2.

  Ammonia (density 0.88g per ml).

  3.3.

  Anion exchange resin, AG1-X8 or Dowex 1-X8, 100 to 200 mesh—To convert Dowex resin in the chloride form to the acetate form add 1 litre of 6M hydrochloric acid to 350g of resin in a 3 litre beaker, and heat the mixture on a steam bath for 2 to 3 hours. Pour the slurry into a glass Buchner funnel, and wash the resin with water until the washings are free from chloride (about 6 litres of water are required). Transfer the resin to a 50mm diameter glass column having a coarse sintered-glass disc at the bottom end, and wash with sodium acetate solution (5g sodium acetate, anhydrous, dissolved in water and diluted to 100ml) until the column effluent gives only a cloudy solution on addition of silver nitrate solution. Return the resin to the glass Buchner funnel, and wash with water. Transfer the resin to a 3 litre beaker, add 1 litre of 40% v/v acetic acid solution (3.4) and heat on a steam bath for 3 hours or longer. Filter, and wash the resin again with water until the washings are free from chloride. Store the resin in water.

  3.4.

  Acetic acid solution 40% v/v.

  3.5.

  Methanol.

  3.6.

  Methanol solution 80% v/v.

  3.7.

  Ammoniacal methanol solution: dilute 1 volume of ammonia (3.2) with 19 volumes of methanol (3.5).

  3.8.

  Meticlorpindol standard solution: weigh, to the nearest 0.1mg, 125mg of meticlorpindol into a beaker, add 25ml of sodium hydroxide solution (2g sodium hydroxide dissolved in water and diluted to 100ml) to dissolve the meticlorpindol, transfer the solution to a 500ml graduated flask, and dilute to the mark with water. This solution contains 250μg per ml meticlorpindol.

4.  APPARATUS

4.1 Aluminium oxide column: constructed as indicated in the diagram included in this method of analysis.

4.2 Ion exchange column: constructed as indicated in the diagram included in this method of analysis.

4.3 Spectrophotometer, recording, with 10mm silica cells.

5.  PROCEDURE

5.1 Extraction of meticlorpindol

• Weigh, to the nearest 0.001g, approximately 50g of the finely divided and mixed sample, or a suitable amount expected to contain about 12mg of meticlorpindol, transfer to a 500ml graduated flask, and add 400ml of ammoniacal methanol solution (3.7). Place a magnetic stirring bar in the flask and stir the mixture on a magnetic stirrer for 20 minutes. Remove the stirring bar from the flask, dilute to the mark with ammoniacal methanol solution (3.7), mix the contents well, and set aside for 20 to 30 minutes.

5.2 Purification

5.2.1 Aluminium oxide column: For each column required weigh approximately 25g of aluminium oxide (3.1) into an aluminium foil dish and place in an oven at 105±5°C for 1 hour. Remove the dish from the oven and cool to room temperature in a desiccator. Make a slurry of the aluminium oxide with 25ml of ammoniacal methanol solution (3.7) and filter on a Buchner funnel. Wash the aluminium oxide with methanol (3.5) until the washings are neutral. Form a slurry of the aluminium oxide with 50ml of methanol (3.5) and pour the slurry into the column (4.1). Allow the methanol to drip through the column. Place a plug of glass wool lightly on top of the aluminium oxide and then wash with 25ml of methanol (3.5). Do not allow the liquid in the column to fall below the top of the aluminium oxide. Discard the eluate.

5.2.2 Anion exchange column: Form a slurry in acetic acid (3.4) of sufficient resin (3.3) to fill the columns required. Filter on a Buchner funnel, wash the resin with twice its own volume of acetic acid (3.4) and then with aqueous methanol (3.6) until the washings are neutral. Form a slurry of a resin with aqueous methanol (3.6) and add sufficient to a column (4.2) to give a resin bed 20 to 30 mm deep after settling. Place a small plug of glass wool on top of the resin and wash the column with two 13ml portions of aqueous methanol (3.6). Do not allow the liquid level in the column to fall below the top of the resin. Discard the eluate.

5.2.3 Chromatographic procedure: By pipette transfer 10.0ml of the extract of the feed sample (5.1) directly onto an aluminium oxide column and at the same time transfer the same volume of ammoniacal methanol solution (3.7) directly onto a second aluminium oxide column (reagent blank). Allow the solutions to drain to the top of the aluminium oxide and then wash each column with three 12ml portions of aqueous methanol (3.6), allowing the liquid to drain to the top of the aluminium oxide each time. Let all the eluate from each column drain directly into separate ion-exchange columns, and then remove the aluminium oxide columns. Allow the liquid to drain to the top of the ion-exchange resin and then wash each column with four 13ml portions of aqueous methanol (3.6). Discard the eluates.

• Elute each column with two 10ml and then one 4ml portions of acetic acid (3.4). Collect the eluates from each column in separate 25ml graduated flasks and dilute the contents of each to the mark with acetic acid (3.4).

5.3 Determination

• Record the absorption spectrum of the sample extract between 350 and 245nm in 10mm silica cells with the reagent blank solution (5.2.3) as reference. Measure the absorbance of the sample extract at 267nm above a baseline obtained by drawing a line through the absorbance at 327 and 297nm and extending it through 267nm.

• (Note: Background absorption due to the feed approaches a linear function that can be described by the points on the curve at 296 and 327nm. Occasionally this is not the case, as can be detected by absorption peaks in the region between 350 and 297nm).

• Determine the concentration of meticlorpindol in the sample by reference to the calibration curve (5.4).

5.4 Calibration curve

• By pipette transfer 1, 5, 7.5, 10, 12.5 and 15ml portions of meticlorpindol standard solution (3.8) to separate 250ml graduated flasks. Dilute the contents of each flask to the mark with acetic acid (3.4). Record the absorption spectra of these solutions in 10mm silica cells between 350 and 245nm with acetic acid (3.4) as reference. Construct a calibration curve using the absorbances at 267nm as ordinates and the corresponding concentrations of meticlorpindol in μg per ml as abscissae.

6.  CALCULATION OF RESULTS

• The meticlorpindol contents in mg/kg of sample is given by the formula:

• 

• in which:

  • C=concentration of meticlorpindol, in μg per ml, read from the calibration curve equivalent to the absorbance of the test solution;

  • 23.23=a factor that makes allowance for the volume of the feed sample in the flask;

• and

  • M=mass of test portion in g.

• Absorbance at 327 and 297nm should not differ by more than 0.05 units and both points should be below 0.2. Results should be satisfactory as long as these criteria are kept in mind along with any obvious distortion in the appearance of the curve. No maximum other than that of meticlorpindol should be present.