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:

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.

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:

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

M=mass of the test portion in g.

(a) insofar as they relate to zootechnical products, the following European Community Directives-


     Council Directive 70/524/EEC concerning additives in feeding stuffs (OJ No. L270, 14.12.70, p. 1, OJ/SE Vol. 18, p. 4), including the amendments thereto in Council Directive 96/51/EC (OJ No. L235, 17.9.96, p. 39);

     Third Commission Directive 72/199/EC (OJ No. L123, 29.5.72, p. 6) (OJ/SE 1966-72 p. 74) establishing Community methods of analysis for the official control of feeding stuffs;

     Fifth Commission Directive 74/203/EEC (OJ No. L108, 22.4.74, p. 7) establishing Community methods of analysis for the official control of feeding stuffs;

     First Commission Directive 76/371/EEC (OJ No. L102, 15.4.76, p. 1) establishing Community methods of sampling for the official control of feeding stuffs;

     Eighth Commission Directive 78/633/EEC (OJ No. L206, 29.7.78, p. 43) establishing Community methods of analysis for the official control of feeding stuffs;

     Commission Directive 81/680/EEC (OJ No. L246, 29.8.81, p. 32) amending Directives 71/250/EEC, 71/393/EEC, 72/199/EEC, 73/46/EEC, 74/203/EEC, 75/84/EEC, 76/372/EEC and 78/633/EEC establishing Community methods of analysis for the official control of feeding stuffs;

     Commission Directive 84/4/EEC (OJ No. L15, 18.1.84, p. 28) amending Directives 71/393/EEC, 72/199/EEC and 78/633/EEC establishing Community methods of analysis for the official control of feeding stuffs;

     The requirements of Council Directive 95/53/EC fixing the principles governing the organisation of official inspections in the field of animal nutrition (OJ No. L265, 8.11.95, p. 17);

     Council Directive 95/69/EC laying down the conditions and arrangements for approving and registering certain establishments and intermediaries operating in the animal feed sector and amending Directives 70/524/EEC, 74/63/EEC, 79/373/EEC and 82/471/EEC (OJ No. L332, 30.12.95, p. 15);

     Council Directive 96/51/EC amending Directive 70/524/EEC concerning additives in feedingstuffs (OJ No. L235, 17.9.96, p. 39);

     Articles 6, 8 and 9 of Commission Directive 98/51/EC laying down certain measures for implementing Council Directive 95/69/EC laying down the conditions and arrangements for approving and registering certain establishments and intermediaries operating in the animal feed sector (OJ No. L208, 24.7.98, p. 43);

     Commission Directive 98/54/EC (OJ No. L208, 24.7.98, p. 49) amending Directives 71/250/EEC, 72/199/EEC, 73/46/EEC and repealing Directive 75/84/EEC;

     Commission Directive 98/64/EC (OJ No. L257, 19.9.98, p. 14) establishing Community methods of analysis for the determination of amino acids, crude oils and fats, and olaquindox in feedingstuffs and amending Directive 71/393/EEC;

     Council Directive 1999/20/EC (OJ No. L80, 25.3.1999, p. 20) amending Directives 70/524/EEC concerning additives in feedingstuffs, 82/471/EEC concerning certain products used in animal nutrition, 95/53/EC fixing the principles governing the organisation of official inspections in the field of animal nutrition and 95/69/EC laying down the conditions and arrangements for approving and registering certain establishments and intermediaries operating in the animal feed sector; and

     Commission Directive 1999/27/EC (OJ No. L118, 6.5.99, p. 36) establishing Community methods of analysis for the determination of amprolium, diclazuril and carbadox in feedingstuffs and amending Directives 71/250/EEC, 73/46/EEC and repealing Directive 74/203/EEC; and



(b) in full, the following Community Directives-



     Ninth Commission Directive 81/715/EEC (OJ No. L257, 10.9.81, p. 38) establishing Community methods of analysis for the official control of feedingstuffs;

     Tenth Commission Directive 84/425/EEC (OJ No. L238, 6.9.84, p. 34) establishing Community methods of analysis for the official control of feedingstuffs;

     Eleventh Commission Directive 93/70/EEC (OJ No. L234, 17.9.93, p. 17) establishing Community methods of analysis for the official control of feedingstuffs; and

     Twelfth Commission Directive 93/117/EEC (OJ No. L329, 30.12.93, p. 54) establishing Community methods of analysis for the official control of feedingstuffs.

     2. The Regulations also provide for the enforcement of the following European Community Regulations-

Commission Regulation (EC) No. 2788/98 (OJ No. L347, 23.12.98, p. 31) amending Council Directive 70/524/EEC concerning additives in feedingstuffs as regards the withdrawal of authorisation for certain growth promoters;

Council Regulation (EC) No. 2821/98 (OJ No. L351, 29.12.98, p. 4) amending, as regards withdrawal of the authorisation of certain antibiotics, Directive 70/524/EEC concerning additives in feedingstuffs; and

Commission Regulation (EC) No. 45/1999 (OJ No. L6, 12.1.1999, p. 3) amending Council Directive 70/524/EEC concerning additives in feedingstuffs as regards withdrawal of the authorisation for certain additives belonging to the group of coccidiostats and other medicinal substances.

     3. In implementation of Directive 70/524/EEC as amended (other than Article 13), the Regulations prescribe the requirements for Community authorisation of zootechnical additives (regulations 5 to 9), control the marketing, as regards certain compositional and related matters and labelling, of zootechnical additives, zootechnical premixtures and zootechnical compound feeding stuffs and make provision relating to tests and use of information (regulations 35, 36, 41(1) and (3), 42, 43(1) to (4), 44, 46 to 8, 51, 52, 57(2), 58(2), 59(2), 65 to 70(1) and 72 to 4). In both cases the Regulations re-enact (or re-enact with modifications) provisions previously contained in Regulations revoked by these Regulations - the Feedingstuffs (Zootechnical Products) Regulations 1998 ("the 1998 Regulations" - S.I. 1998/1047).

     4. There exists a category of additive producers who are not "eligible persons" within the meaning of regulation 5 but who are putting an additive into circulation and require Community authorisation under Directive 70/524/EEC as amended to continue marketing the additive on or after 1st October 1999. To enable such producers to apply before 1st October 1999 for Community authorisation of the additive they are currently putting into circulation, regulation 6 has been introduced. A fee is payable by an applicant when the Minister forwards the dossier, in support of the application for Community authorisation, to the Commission and other member States (regulation 6(4) and Schedule 3).

     5. In implementation of Article 13 of Directive 70/524/EEC as amended, the Regulations also re-enact provisions in the 1998 Regulations regulating, in relation to establishments and intermediaries requiring approval-

(a) the putting into circulation of zootechnical additives, zootechnical premixtures and zootechnical compound feeding stuffs (regulations 39(1), 55(1) and 71);

(b) the supply of zootechnical additives, or of zootechnical premixtures (regulations 41(2) and 57(1)); and

(c) the incorporation of zootechnical additives, and of zootechnical premixtures, in compound feeding stuffs (regulations 43(5), 58(1) and 59(1)).

     6. In implementation of Directive 95/69/EC, the Regulations re-enact requirements previously contained in the 1998 Regulations under which-

(a) "establishments" (as defined in Article 1.3 of Directive 95/69/EC) in the United Kingdom must be approved by the relevant competent authority (the Royal Pharmaceutical Society of Great Britain in Great Britain and the Department of Agriculture for Northern Ireland in Northern Ireland) for the manufacture with a view to putting them into circulation of zootechnical additives, zootechnical premixtures and zootechnical compound feeding stuffs,

(b) "intermediaries" based in the United Kingdom (also defined in Article 1.3 of Directive 95/69/EC) must be approved by the same competent authorities for the wrapping, packaging, storing and "putting into circulation" (see definition in Article 1.3 of Directive 95/69/EC) of zootechnical additives and zootechnical premixtures, and

(c) intermediaries based in a member State other than the United Kingdom, and putting into circulation in the United Kingdom products of the kinds referred to in sub-paragraph (b), must be approved by the competent authorities in the member State concerned,

and each category must comply with approval conditions in relation to the activities in question (regulations 33, 34, 37, 38, 39(2), 40, 49, 50, 53, 54, 55(2), 56 and 61 to 64).

     7. Both establishments and intermediaries must comply with detailed "quality control"requirements specified in the Annex to Directive 95/69/EC, and that is a precondition before approval (which can only be given following an inspection by the competent authority) can be given.

     8. Application for approval (which may now be made in the Welsh language in certain circumstances) must be made to the competent authority, and the application must contain specified information. The competent authority must keep registers of approved establishments and intermediaries and must update them as necessary. Applicants may apply for approval in respect of activities additional to, or replacing, any for which they are already approved, and the competent authority can cancel approval in cases where an activity is no longer being exercised, or where the quality control requirements are not being met (regulations 10 to 25).

     9. Transitional arrangements apply in the case of establishments and intermediaries which were already exercising, on 1st April 1998, activities of a kind for which approval is necessary. In particular, they may continue to exercise the activities concerned until their application is processed, provided they applied before 1st September 1998 - (see the definitions in regulations 3 and 4 commencing with "EC permitted Chapter" or "UK permitted Chapter").

     10. The Regulations provide for examination of dossiers (regulations 5 to 8) and set fees for the examination (regulation 9), for the approval of establishments (regulation 15) and for the approval of intermediaries (regulation 23), all as read with Schedule 3. The fees replicate those in the 1998 Regulations, with the exception that the fee for applications under regulation 6 (a new type of application) and the fee for acting under regulation 7 in relation to new Community authorisation of an additive (£10,000 under the 1998 Regulations) are both aligned with that for acting under regulation 5 (£25,000).

     11. The Regulations exclude the application of the Medicines Act 1968 to zootechnical additives, except in relation to-

(a) any advisory function of a committee relating to veterinary medicinal products, and

(b) animal test certificates for unauthorised zootechnical additives (regulation 92).

     12. The Regulations provide for their enforcement by the competent authority and contain detailed provisions for that purpose, including provision for the taking of samples of products controlled by the Regulations, analysis of such samples, offences and penalties (regulations 75 to 91 and Schedules 4 and 5).

     13. The principal changes effected by the Regulations are as follows-

(a) among the provisions referred to in paragraph 12 there are included a number which, for the purposes of control of zootechnical products, and in relation to substances covered by the instruments referred to in paragraph 1, give effect to certain of the requirements of Directive 95/53/EC, including those relating to sampling and analysis and the manner of carrying out, and the frequency of, enforcement checks to be carried out by competent authorities;

(b) in accordance with Article 6 of Directive 98/51/EC, the Regulations introduce, in relation to establishments located in third countries and their UK based representatives, as regards the products referred to in sub-paragraph (a), requirements similar to those described in paragraphs 5(a) and 6 to 8, the main differences being that-


(i) approval is granted by the Minister of Agriculture, Fisheries and Food and is for the importation into the United Kingdom of the products concerned. (It is made an offence to import those products from a third country into the United Kingdom, unless such approval has been obtained or the transitional arrangements described in sub-paragraph (iv) apply, or importation is permitted by virtue of parallel arrangements operating in another member State - regulations 45, 60 and 70(2));

(ii) application for approval, including amendment applications, is made by the UK based representative to the Minister, who will maintain the register of approved third country establishments, and have the power to withdraw approvals where there is non-compliance by an establishment or its representative with quality control requirements (regulations 26 to 32);

(iii) no prior inspection by the competent authority (the Royal Pharmaceutical Society for Great Britain in Great Britain and the Department of Agriculture for Northern Ireland in Northern Ireland) is necessary before approval is granted;

(iv) the applicable transitional provisions operate in the case of third country establishments manufacturing the product concerned on 1st December 1998, and which at all times since have had a representative established in the European Community. Importation from such establishments may lawfully continue after the Regulations come into force, (provided the necessary application is made by 30th September 1999), until the application is processed. (See the definitions in regulation 3 commencing with "EC permitted third country" and "UK permitted third country");



(c) certain provisions in the 1998 Regulations applied to establishments "located in a third country". In the corresponding provisions in these Regulations there is substituted for that expression reference to the relevant type of approved or permitted third country establishment provided for in these Regulations (regulations 39(1), 43(5), 55(1) and 71);

(d) in implementation of Articles 8 and 9 of Directive 98/51/EC, the Regulations prescribe the formats of the register and approval numbers provided for in Article 5 of Directive 95/69/EC (regulations 11(2), 19(3)(b) and 27(1)(b));

(e) for enforcement purposes, the Regulations include reference to European Community Regulations withdrawing Community authorisations previously accorded to the (growth promoter) additives carbadox and olaquindox, to the (antibiotic) additives bacitracin zinc, spiramycin, virginiamycin and tylosin phosphate and, with effect from 30th September 1999, to the (coccidiostats and other medicinal substances) additives arprinocide, dinitolmide and ipronidazole (regulation 2(1) and Schedule 1);

(f) the Regulations create a new offence of making a false statement in connection with the Regulations (regulation 85(c));

(g) the Regulations omit from those definitions in regulations 3 and 4 which also appear in the 1998 Regulations all wording which is now spent; and

(h) the Regulations amend regulation 3 of the Feeding Stuffs (Sampling and Analysis) Regulations 1999 (S.I. 1999/1663) in order to rectify drafting errors (regulation 93).

     14. The provisions referred to in paragraph 1 implemented by these Regulations are implemented, so far as relevant to certain feeding stuffs and products not containing zootechnical additives, by the Feeding Stuffs (Establishments and Intermediaries) Regulations 1999.

     15. A Regulatory Appraisal has been prepared and a copy has been placed in the library of each House of Parliament.