Commission Regulation (EC) No 152/2009 of 27 January 2009 laying down the methods of sampling and analysis for the official control of feed (Text with EEA relevance)

	Premix	Premix feed	Mineral concentrate	Protein feed	Piglet
L	13	12	13	12	13
n	48	45	47	46	49
mean [IU/kg]	17,02  × 106	1,21  × 106	537 100	151 800	18 070
sr [IU/kg]	0,51  × 106	0,039  × 106	22 080	12 280	682
r [IU/kg]	1,43  × 106	0,109  × 106	61 824	34 384	1 910
CVr [%]	3,0	3,5	4,1	8,1	3,8
sR [IU/kg]	1,36  × 106	0,069  × 106	46 300	23 060	3 614
R [IU/kg]	3,81  × 106	0,193  × 106	129 640	64 568	10 119
CVR [%]	8,0	6,2	8,6	15	20
L: number of laboratories n: number of single values sr: standard deviation of repeatability sR:  standard deviation of reproducibility r: repeatability R;  reproducibility CVr:  coefficient of variation of repeatability CVR:  coefficient of variation of reproducibility.	L: number of laboratories n: number of single values sr: standard deviation of repeatability sR:  standard deviation of reproducibility r: repeatability R;  reproducibility CVr:  coefficient of variation of repeatability CVR:  coefficient of variation of reproducibility.	L: number of laboratories n: number of single values sr: standard deviation of repeatability sR:  standard deviation of reproducibility r: repeatability R;  reproducibility CVr:  coefficient of variation of repeatability CVR:  coefficient of variation of reproducibility.	L: number of laboratories n: number of single values sr: standard deviation of repeatability sR:  standard deviation of reproducibility r: repeatability R;  reproducibility CVr:  coefficient of variation of repeatability CVR:  coefficient of variation of reproducibility.	L: number of laboratories n: number of single values sr: standard deviation of repeatability sR:  standard deviation of reproducibility r: repeatability R;  reproducibility CVr:  coefficient of variation of repeatability CVR:  coefficient of variation of reproducibility.	L: number of laboratories n: number of single values sr: standard deviation of repeatability sR:  standard deviation of reproducibility r: repeatability R;  reproducibility CVr:  coefficient of variation of repeatability CVR:  coefficient of variation of reproducibility.

Figure 1

Extraction apparatus (point 4.8)

Image omitted; see official source

B. DETERMINATION OF VITAMIN E

The vitamin E is to be determined by:

• — the method of analysis provided for by EN 17547 Animal feeding stuffs: Methods of sampling and analysis – Determination of vitamin A, E and D ([^E0036]) content – Method using solid phase extraction (SPE) clean-up and high-performance liquid chromatography (HPLC), or

• — by reversed phase high performance liquid chromatography (RP-HPLC) using a UV or a fluorescence detector, as described in the points 1 to 9 hereafter.

1. Purpose and scope

This method makes it possible to determine the level of vitamin E in feed. The content of vitamin E is expressed as mg DL-α-tocopherol acetate per kg. 1 mg DL-α-tocopherol acetate corresponds to 0,91 mg DL-α-tocopherol (vitamin E).

The limit of quantification is 2 mg vitamin E/kg. This limit of quantification is only achievable with fluorescence detector. With an UV detector the limit of quantification is 10 mg/kg.

2. Principle

The sample is hydrolysed with ethanolic potassium hydroxide solution and the vitamin E is extracted into light petroleum. The solvent is removed by evaporation and the residue is dissolved in methanol and, if necessary, diluted to the required concentration. The content of vitamin E is determined by reversed phase high performance liquid chromatography (RP-HPLC) using a fluorescence or a UV detector.

3. Reagents

• 3.1.Ethanol, σ = 96 %.

• 3.2.Light petroleum, boiling range 40 °C–60 °C.

• 3.3.Methanol.

• 3.4.Potassium hydroxide solution, c = 50 g/100 ml.

• 3.5.Sodium ascorbate solution, c = 10 g/100 ml (see point 7.7 observations).

• 3.6.Sodium sulphide, Na2S · x H2O (x = 7 – 9).

• 3.6.1.Sodium sulphide solution, c = 0,5 mol/l in glycerol, β = 120 g/l (for x = 9) (see point 7.8 observations).

• 3.7.Phenolphthalein solution, c = 2 g/100 ml in ethanol (point 3.1).

• 3.8.Mobile phase for HPLC: mixture of methanol (point 3.3) and water, e.g. 980 + 20 (v + v). The exact ratio will be determined by the characteristics of the column employed.

• 3.9.Nitrogen, oxygen free.

• 3.10.DL-α-tocopherol acetate, extra pure, of certified activity

• 3.10.1.Stock solution of DL-α-tocopherol acetate: Weigh to the nearest 0,1 mg, 100 mg of DL-α-tocopherol acetate (point 3.10) into a 100 ml graduated flask. Dissolve in ethanol (point 3.1) and make up to the mark with the same solvent. 1 ml of this solution contains 1 mg DL-α-tocopherol acetate (UV control see point 5.6.1.3; stabilisation see point 7.4 observations).

• 3.11.DL-α-tocopherol, extra pure, of certified activity

• 3.11.1.Stock solution of DL-α-tocopherol: Weigh to the nearest 0,1 mg, 100 mg of DL-α-tocopherol (point 3.11) into a 100 ml graduated flask. Dissolve in ethanol (point 3.1) and make up to the mark with the same solvent. 1 ml of this solution contains 1 mg DL-α-tocopherol (UV control see point 5.6.2.3; stabilisation see point 7.4 observations).

• 3.12.2,6-Di-tert-butyl-4-methylphenol (BHT) (see point 7.5 observations).

4. Apparatus

• 4.1.Rotary film evaporator.

• 4.2.Amber glassware.

• 4.2.1.Flat bottom or conical flasks, 500 ml, with ground-glass socket.

• 4.2.2.Graduated flasks with ground-glass stoppers, narrow-necked, 10, 25, 100 and 500 ml.

• 4.2.3.Separating funnels, conical, 1 000  ml, with ground-glass stoppers.

• 4.2.4.Pear-shaped flasks, 250 ml, with ground-glass sockets.

• 4.3.Allihn condenser, jacket length 300 mm, with ground-glass joint, with adapter for gas feed pipe.

• 4.4.Pleated filter paper for phase separation, diameter 185 mm (e.g. Schleicher & Schuell 597 HY 1/2).

• 4.5.HPLC equipment with injection system

• 4.5.1.Liquid chromatographic column, 250 mm × 4 mm, C18, 5 or 10 μm packing, or equivalent.

• 4.5.2.Fluorescence or UV detector, with variable wavelength adjustment.

• 4.6.Spectrophotometer with 10 mm quartz cells.

• 4.7.Water-bath with magnetic stirrer.

• 4.8.Extraction apparatus (see Figure 2) consisting of:

• 4.8.1.Glass cylinder of 1 l capacity fitted with a ground glass neck and stopper.

• 4.8.2.Ground glass insert equipped with a side-arm and an adjustable tube passing through the centre. The adjustable tube shall have a U-shaped lower end and a jet at the opposite end so that the upper liquid layer in the cylinder may be transferred into a separating funnel.

5. Procedure

• Note: Vitamin E is sensitive to (UV-) light and to oxidation. All operations shall be carried out in the absence of light (using amber glassware, or glassware protected with aluminium foil) and oxygen (flush with nitrogen). During extraction air above the liquid shall be replaced by nitrogen (avoid excess pressure by loosening the stopper from time to time).

5.1.   Preparation of the sample

Grind the sample so that it passes a 1 mm mesh sieve, taking care to avoid generation of heat. Grinding must be carried out immediately before weighing and saponification otherwise there may be losses of vitamin E.

5.2.   Saponification

Depending on the vitamin E content weigh, to the nearest 0,01 g, 2 g to 25 g of the sample into a 500 ml flat bottom or conical flask (point 4.2.1). Add successively with swirling 130 ml ethanol (point 3.1), approximately 100 mg BHT (point 3.12), 2 ml sodium ascorbate solution (point 3.5) and 2 ml sodium sulphide solution (point 3.6). Fit the condenser (point 4.3) to the flask and immerse the flask in a water-bath with magnetic stirrer (point 4.7). Heat to boiling and allow to reflux for 5 minutes. Then add 25 ml potassium hydroxide solution (point 3.4) through the condenser (point 4.3) and allow to reflux for a further 25 min with stirring under a slow stream of nitrogen. Then rinse the condenser with approximately 20 ml water and cool the content of the flask to room temperature.

5.3.   Extraction

Transfer by decantation the saponification solution quantitatively by rinsing with a total volume of 250 ml water to a 1 000  ml separating funnel (point 4.2.3) or to the extraction apparatus (point 4.8). Rinse the saponification flask successively with 25 ml ethanol (point 3.1) and 100 ml light petroleum (point 3.2) and transfer the rinsings to the separating funnel or to the extraction apparatus. The proportion of water and ethanol in the combined solutions must be about 2:1. Shake vigorously for 2 min and allow to settle for 2 minutes.

5.3.1.   Extraction using a separating funnel (point 4.2.3)

When the layers have separated (see observation point 7.3) transfer the light petroleum layer to another separating funnel (point 4.2.3). Repeat this extraction twice, with 100 ml light petroleum (point 3.2) and twice, with 50 ml light petroleum (point 3.2).

Wash the combined extracts in the separating funnel twice by gently swirling (to avoid formation of emulsions) with 100 ml portions of water and then by repeated shaking with further 100 ml portions of water until the water remains colourless on addition of phenolphthalein solution (point 3.7) (washing four times is usually sufficient). Filter the washed extract through a dry pleated filter for phase separation (point 4.4) to remove any suspended water into a 500 ml graduated flask (point 4.2.2). Rinse the separating funnel and the filter with 50 ml light petroleum (point 3.2), make up to the mark with light petroleum (point 3.2) and mix well.

5.3.2.   Extraction using an extraction apparatus (point 4.8)

When the layers have separated (see observation point 7.3) replace the stopper of the glass cylinder (point 4.8.1) by the ground glass insert (point 4.8.2) and position the U-shaped lower end of the adjustable tube so that it is just above the level of the interface. By application of pressure from a nitrogen line to the side-arm, transfer the upper light petroleum-layer to a 1 000  ml separating funnel (point 4.2.3). Add 100 ml light petroleum (point 3.2) to the glass cylinder, stopper and shake well. Allow the layers to separate and transfer the upper layer to the separating funnel as before. Repeat the extraction procedure with further 100 ml of light petroleum (point 3.2), then twice with 50 ml portions of light petroleum (point 3.2) and add the light petroleum layers to the separating funnel.

Wash the combined light petroleum extracts as described in point 5.3.1 and proceed as described there.

5.4.   Preparation of the sample solution for HPLC

Pipette an aliquot portion of the light petroleum solution (from point 5.3.1 or 5.3.2) into a 250 ml pear-shaped flask (point 4.2.4). Evaporate the solvent nearly to dryness on the rotary evaporator (point 4.1) with reduced pressure at a bath temperature not exceeding 40 °C. Restore atmospheric pressure by admitting nitrogen (point 3.9) and remove the flask from the rotary evaporator. Remove the remaining solvent with a stream of nitrogen (point 3.9) and dissolve the residue immediately in a known volume (10–100 ml) of methanol (point 3.3) (the concentration of DL-α-tocopherol must be in the range 5 μg/ml to 30 μg/ml).

5.5.   Determination by HPLC

Vitamin E is separated on a C18 reversed phase column (point 4.5.1) and the concentration is measured using a fluorescence detector (excitation: 295 nm, emission: 330 nm) or a UV detector (292 nm) (point 4.5.2).

Inject an aliquot portion (e.g. 20 μl) of the methanolic solution obtained in point 5.4 and elute with the mobile phase (point 3.8). Calculate the mean peak heights (areas) of several injections of the same sample solution and the mean peak heights (areas) of several injections of the calibration solutions (point 5.6.2).

HPLC conditions

The following conditions are offered for guidance; other conditions may be used provided that they give equivalent results.

Liquid chromatographic column (point 4.5.1):	250 mm × 4 mm, C18, 5 or 10 μm packing, or equivalent
Mobile phase (point 3.8):	Mixture of methanol (point 3.3) and water, e.g. 980 + 20 (v + v).
Flow rate:	1–2 ml/min
Detector (point 4.5.2)	Fluorescence detector (excitation: 295 nm/emission: 330 nm) or UV detector (292 nm).

5.6.   Calibration (DL-α-tocopherol acetate or DL-α-tocopherol)

5.6.1.   DL-α-tocopherol acetate standard

5.6.1.1.   Preparation of the working standard solution

Transfer by pipette 25 ml of the DL-α-tocopherol acetate stock solution (point 3.10.1) into a 500 ml flat bottom or conical flask (point 4.2.1) and hydrolyse as described under point 5.2. Subsequently extract with light petroleum (point 3.2) according to point 5.3 and make up to 500 ml with light petroleum. Evaporate 25 ml of this extract on the rotary evaporator (see point 5.4) nearly to dryness, remove the remaining solvent with a stream of nitrogen (point 3.9) and redissolve the residue in 25,0 ml of methanol (point 3.3). The nominal concentration of this solution is 45,5 μg DL-α-tocopherol per ml, equivalent to 50 μg DL-α-tocopherol acetate per ml. The working standard solution shall be freshly prepared before use.

5.6.1.2.   Preparation of the calibration solutions and calibration graph

Transfer 1,0, 2,0, 4,0 and 10,0 ml of the working standard solution into a series of 20 ml graduated flasks, make up to the mark with methanol (point 3.3) and mix. The nominal concentrations of these solutions are 2,5, 5,0, 10,0 and 25,0 μg/ml DL-α-tocopherol acetate, i.e. 2,28, 4,55, 9,10 μg/ml and 22,8 μg/ml DL-α-tocopherol,

Inject 20 μl of each calibration solution several times and determine the mean peak heights (areas). Using the mean peak heights (areas) plot a calibration graph.

5.6.1.3.   UV standardisation of the DL-α-tocopherol acetate stock solution (point 3.10.1)

Dilute 5,0 ml of the DL-α-tocopherol acetate stock solution (point 3.10.1) to 25,0 ml with ethanol and measure the UV spectrum of this solution against ethanol (point 3.1) in the spectrophotometer (point 4.6) between 250 nm and 320 nm.

The absorption maximum shall be at 284 nm:

Image omitted; see official source

At this dilution an extinction value of 0,84 to 0,88 must be obtained.

5.6.2.   DL-α-tocopherol standard

5.6.2.1.   Preparation of the working standard solution

Transfer by pipette 2 ml of the DL-α-tocopherol stock solution (point 3.11.1) into a 50 ml graduated flask, dissolve in methanol (point 3.3) and make up to the mark with methanol. The nominal concentration of this solution is 40 μg DL-α-tocopherol per ml, equivalent to 44,0 μg DL-α-tocopherol acetate per ml. The working standard solution shall be freshly prepared before use.

5.6.2.2.   Preparation of the calibration solutions and calibration graph

Transfer 1,0, 2,0, 4,0 and 10,0 ml of the working standard solution into a series of 20 ml graduated flasks, make up to the mark with methanol (point 3.3) and mix. The nominal concentrations of these solutions are 2,0, 4,0, 8,0 and 20,0 μg/ml DL-α-tocopherol, i.e. 2,20, 4,40, 8,79 μg/ml and 22,0 μg/ml DL-α-tocopherol acetate.

Inject 20 μl of each calibration solution several times and determine the mean peak heights (areas). Using the mean peak heights (areas) plot a calibration graph.

5.6.2.3.   UV standardisation of the DL-α-tocopherol stock solution (point 3.11.1)

Dilute 2,0 ml of the DL-α-tocopherol stock solution (point 3.11.1) to 25,0 ml with ethanol and measure the UV spectrum of this solution against ethanol (point 3.1) in the spectrophotometer (point 4.6) between 250 nm and 320 nm. The absorption maximum shall be at 292 nm:

Image omitted; see official source

At this dilution an extinction value of 0,6 must be obtained.

6. Calculation of the results

From the mean height (area) of the vitamin E peaks of the sample solution determine the concentration of the sample solution in μg/ml (calculated as DL-α-tocopherol acetate) by reference to the calibration graph (point 5.6.1.2 or 5.6.2.2).

The vitamin E content w in mg/kg of the sample is given by the following formula:

Image omitted; see official source

in which:

c	=	vitamin E concentration (as DL-α-tocopherol acetate) of the sample solution (point 5.4) in μg/ml
---	---	---
V1	=	volume of sample solution (point 5.4), in ml
---	---	---
V2	=	volume of aliquot taken in (point 5.4), in ml
---	---	---
m	=	weight of the test portion in g.

7. Observations

• 7.1.For samples with low vitamin E concentration it may be useful to combine the light petroleum-extracts of two saponification-charges (amount weighed: 25 g) to one sample solution for HPLC-determination.

• 7.2.The weight of the sample taken for the analysis shall not contain more than 2 g fat.

• 7.3.If phase separation does not occur add approximately 10 ml ethanol (point 3.1) to break the emulsion.

• 7.4.After the spectrophotometric measurement of the DL-α-tocopherol acetate or DL-α-tocopherol solution according to point 5.6.1.3 or 5.6.2.3 respectively add approximately 10 mg BHT (point 3.12) to the solution (point 3.10.1 or 3.10.2) and keep the solution in a refrigerator (storage life max. 4 weeks).

• 7.5.Hydroquinone can be used instead of BHT.

• 7.6.Using a normal phase-column the separation of α-, β-, γ- and δ-tocopherol is possible.

• 7.7.Approximately 150 mg ascorbic acid can be used instead of sodium ascorbate solution.

• 7.8.Approximately 50 mg EDTA can be used instead of sodium sulphide solution.

• 7.9.Vitamin E acetate hydrolyses very fast under alkaline conditions and is therefore very sensitive to oxidation, especially in the presence of trace elements like iron or copper. In case of the determination of vitamin E in premixtures at levels higher than 5 000  mg/kg, a degradation of vitamin E could be the consequence. Therefore, a HPLC method including an enzymatic digestion of the vitamin E formulation without an alkaline saponification step is to be recommended for confirmation.

8. Repeatability·

The difference between the results of two parallel determinations carried out on the same sample must not exceed 15 % relative to the higher result.

9. Results of collaborative study ([^E0037])

	Premix	Premix feed	Mineral concentrate	Protein feed	Piglet
L	12	12	12	12	12
n	48	48	48	48	48
mean [mg/kg]	17 380	1 187	926	315	61,3
sr [mg/kg]	384	45,3	25,2	13,0	2,3
r [mg/kg]	1 075	126,8	70,6	36,4	6,4
CVr [%]	2,2	3,8	2,7	4,1	3,8
sR mg/kg]	830	65,0	55,5	18,9	7,8
R [mg/kg]	2 324	182,0	155,4	52,9	21,8
CVR [%]	4,8	5,5	6,0	6,0	12,7
L: number of laboratories n: number of single values sr: standard deviation of repeatability sR:  standard deviation of reproducibility r: repeatability R: reproducibility CVr:  coefficient of variation of repeatability CVR:  coefficient of variation of reproducibility.	L: number of laboratories n: number of single values sr: standard deviation of repeatability sR:  standard deviation of reproducibility r: repeatability R: reproducibility CVr:  coefficient of variation of repeatability CVR:  coefficient of variation of reproducibility.	L: number of laboratories n: number of single values sr: standard deviation of repeatability sR:  standard deviation of reproducibility r: repeatability R: reproducibility CVr:  coefficient of variation of repeatability CVR:  coefficient of variation of reproducibility.	L: number of laboratories n: number of single values sr: standard deviation of repeatability sR:  standard deviation of reproducibility r: repeatability R: reproducibility CVr:  coefficient of variation of repeatability CVR:  coefficient of variation of reproducibility.	L: number of laboratories n: number of single values sr: standard deviation of repeatability sR:  standard deviation of reproducibility r: repeatability R: reproducibility CVr:  coefficient of variation of repeatability CVR:  coefficient of variation of reproducibility.	L: number of laboratories n: number of single values sr: standard deviation of repeatability sR:  standard deviation of reproducibility r: repeatability R: reproducibility CVr:  coefficient of variation of repeatability CVR:  coefficient of variation of reproducibility.

Figure 2

Extraction apparatus (point 4.8)

Image omitted; see official source

C. DETERMINATION OF THE TRACE ELEMENTS IRON, COPPER, MANGANESE AND ZINC

The iron, copper, manganese and zinc content is to be determined by:

• — the method of analysis provided for by EN 15510 Animal feeding stuffs: Methods of sampling and analysis – Determination of calcium, sodium, phosphorus, magnesium, potassium, iron, zinc, copper, manganese, cobalt, molybdenum and lead by ICP-AES, or

• — the method of analysis provided for by EN 15621 Animal feeding stuffs: Methods of sampling and analysis – Determination of calcium, sodium, phosphorus, magnesium, potassium, sulphur, iron, zinc, copper, manganese and cobalt after pressure digestion by ICP-AES, or

• — the method of analysis provided for by EN 17053 Animal feeding stuffs: Methods of sampling and analysis – Determination of trace elements, heavy metals and other elements in feed by ICP-MS (multi-method), or

• — the method of analysis provided for by EN ISO 6869 Animal feeding stuffs. Determination of the contents of calcium, copper, iron, magnesium, manganese, potassium, sodium and zinc. Method using atomic absorption spectrometry, or

• — the flame atomic absorption spectrometry (FAAS) method, as described in the points 1 to 8 hereafter.

1. Purpose and scope

The method makes it possible to determine the trace elements iron, copper, manganese and zinc in feed ([^E0038]). The limits of quantification are:

• — iron (Fe): 20 mg/kg

• — copper (Cu): 10 mg/kg

• — manganese (Mn): 20 mg/kg

• — zinc (Zn): 20 mg/kg.

2. Principle

The sample is brought into solution in hydrochloric acid after destruction of organic matter, if any. The elements iron, copper, manganese and zinc are determined, after appropriate dilution, by atomic absorption spectrometry.

3. Reagents

Introductory comments

For preparation of the reagents and analytical solutions use water free from the cations to be determined, obtained either by double distilling water in a borosilicate glass or quartz still or by double treatment on ion exchange resin.

The reagents must be of at least analytical grade. Freedom from the element to be determined must be checked in a blank experiment. If necessary, the reagents must be further purified.

In place of the standard solutions described below, commercial standard solutions may be used provided that they are guaranteed and have been checked before use.

• 3.1.Hydrochloric acid (d: 1,19 g/ml).

• 3.2.Hydrochloric acid (6 mol/litre).

• 3.3.Hydrochloric acid (0,5 mol/litre).

• 3.4.Hydrofluoric acid 38 to 40 % (v/v) having an iron (Fe) content of less than 1 mg/litre and a residue after evaporation of less than 10 mg (as sulphate)/litre.

• 3.5.Sulphuric acid. (d: 1,84 g/ml).

• 3.6.Hydrogen peroxide. (approximately 100 volumes of oxygen (30 % by weight)).

• 3.7.Standard iron solution (1 000  μg Fe/ml) prepared as follows or equivalent commercially available solution: dissolve 1 g of iron wire in 200 ml of 6 mol/litre hydrochloric acid (point 3.2), add 16 ml of hydrogen peroxide (point 3.6) and make up to one litre with water.

• 3.7.1.Working standard iron solution (100 μg Fe/ml) prepared by diluting one part of the standard solution (point 3.7) with 9 parts of water.

• 3.8.Standard copper solution (1 000  μg Cu/ml) prepared as follows or equivalent commercially available solution:

• — dissolve 1 g of copper in powder form in 25 ml of 6 mol/litre hydrochloric acid (point 3.2), add 5 ml of hydrogen peroxide (point 3.6) and make up to one litre with water.

• 3.8.1.Working standard copper solution (10 μg Cu/ml) prepared by diluting 1 part of the standard solution (point 3.8) with 9 parts of water and then diluting 1 part of the resulting solution with 9 parts of water.

• 3.9.Standard manganese solution (1 000  μg Mn/ml) prepared as follows or equivalent commercially available solution:

• — dissolve 1 g of manganese in powder form in 25 ml of 6 mol/litre hydrochloric acid (point 3.2) and make up to one litre with water.

• 3.9.1.Working standard manganese solution (10 μg Mn/ml) prepared by diluting 1 part of the standard solution (point 3.9) with 9 parts of water and then diluting 1 part of the resulting solution with 9 parts of water.

• 3.10.Standard zinc solution (1 000  μg Zn/ml) prepared as follows or equivalent commercially available solution:

• — dissolve 1 g of zinc in strip or leaf form in 25 ml of 6 mol/litre hydrochloric acid (point 3.2) and make up to one litre with water.

• 3.10.1.Working standard zinc solution (10 μg Zn/ml) prepared by diluting 1 part of the standard solution (point 3.10) with 9 parts of water and then diluting 1 part of the resulting solution with 9 parts of water.

• 3.11.Lanthanum chloride solution: dissolve 12 g of lanthanum oxide in 150 ml of water, add 100 ml of 6 mol/litre hydrochloric acid (point 3.2) and make up to one litre with water.

4. Apparatus

• 4.1.Muffle furnace with temperature regulation and preferably recorder.

• 4.2.Glassware must be of resistant borosilicate type and it is recommended to use apparatus which is reserved exclusively for trace element determinations.

• 4.3.Atomic absorption spectrophotometer meeting the requirements of the method with regard to sensitivity and precision in the required range.

5. Procedure ([^E0039])

5.1.   Samples containing organic matter

5.1.1.   Ashing and preparation of the solution for analysis ([^E0040])

• 5.1.1.1.Place 5 to 10 g of sample weighed to the nearest 0,2 mg in a quartz or platinum crucible (see Note (b)), dry in an oven at 105 °C and introduce the crucible into the cold muffle furnace (point 4.1). Close the furnace (see Note (c)) and gradually raise the temperature to 450 to 475 °C over about 90 minutes. Maintain this temperature for 4 to 16 hours (e.g. overnight) to remove carbonaceous material and then open the furnace and allow to cool (see Note (d)).

• Moisten the ashes with water and transfer these in a beaker of 250 ml. Wash the crucible out with a total of about 5ml of hydrochloric acid (point 3.1) and add the latter slowly and carefully to the beaker (there may be a vigorous reaction due to CO2 formation). Add hydrochloric acid (point 3.1) dropwise with agitation until all effervescence has stopped. Evaporate to dryness, occasionally stirring with a glass rod.

• Next add 15 ml of 6 mol/litre hydrochloric acid (point 3.2) to the residue followed by about 120 ml of water. Stir with the glass rod, which shall be left in the beaker, and cover the beaker with a watch-glass. Bring gently to the boil and maintain at boiling point until no more ash can be seen to dissolve. Filter on ash-free filter paper and collect the filtrate in a 250 ml volumetric flask. Wash the beaker and filter with 5 ml of hot 6 mol/litre hydrochloric acid (point 3.2) and twice with boiling water. Fill the volumetric flask up to the mark with water (HCl concentration about 0,5 mol/litre).

• 5.1.1.2.If the residue in the filter appears black (carbon), put it back in the furnace and ash again at 450 to 475 °C. This ashing, which only requires a few hours (about three to five hours), is complete when the ash appears white or nearly white. Dissolve the residue with about 2 ml of hydrochloric acid (point 3.1), evaporate to dryness and add 5 ml of 6 mol/litre hydrochloric acid (point 3.2). Heat, filter the solution into the volumetric flask and make up to the mark with water (HCl concentration about 0,5 mol/litre).

Notes:

• (a) In determining trace elements it is important to be alert to the risks of contamination, particularly by zinc, copper and iron. For this reason, the equipment used in preparing the samples must be free of these metals. To reduce the general risk of contamination, work in a dust-free atmosphere with scrupulously clean equipment and carefully washed glassware. The determination of zinc is particularly sensitive to many types of contamination, e.g. from glassware, reagents, dust, etc.

• (b) The weight of sample to be ashed is calculated from the approximate trace element content of the feed in relation to the sensitivity of the spectrophotometer used. For certain feed low in trace elements it may be necessary to start with a 10 to 20 g sample and make up the final solution to only 100 ml.

• (c) Ashing must be carried out in a closed furnace without injection of air or oxygen.

• (d) The temperature indicated by the pyrometer must not exceed 475 °C.

5.1.2.   Spectrophotometric determination

5.1.2.1.   Preparation of calibration solutions

For each of the elements to be determined, prepare from the working standard solutions given in points 3.7.1, 3.8.1, 3.9.1 and 3.10.1 a range of calibration solutions, each calibration solution having an HCl concentration of about 0,5 mol/litre (and (in the cases of iron, manganese and zinc) a lanthanum chloride concentration equivalent to 0,1 % La (w/v).

The trace element concentrations selected must lie within the range of sensitivity of the spectrophotometer used. The tables below show, by way of example, the compositions of typical ranges of calibration solutions; depending, however, on the type and sensitivity of spectrophotometer used it may be necessary to select other concentrations.

Iron

μg Fe/ml	0	0,5	1	2	3	4	5
ml working standard solution (point 3.7.1) (1 ml = 100 μg Fe)	0	0,5	1	2	3	4	5
ml HCl (point 3.2)	7	7	7	7	7	7	7
+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.	+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.	+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.	+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.	+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.	+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.	+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.	+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.

Copper

μg Cu/ml	0	0,1	0,2	0,4	0,6	0,8	1,0
ml working standard solution (point 3.8.1) (1 ml = 10 μg Cu)	0	1	2	4	6	8	10
ml HCl (point 3.2)	8	8	8	8	8	8	8

Manganese

μg Mn/ml	0	0,1	0,2	0,4	0,6	0,8	1,0
ml working standard solution (point 3.9.1) (1 ml = 10 μg Mn)	0	1	2	4	6	8	10
ml HCl (point 3.2)	7	7	7	7	7	7	7
+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.	+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.	+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.	+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.	+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.	+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.	+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.	+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.

Zinc

μg Zn/ml	0	0,05	0,1	0,2	0,4	0,6	0,8
ml working standard solution (point 3.10.1) (1 ml = 10 μg Zn)	0	0,5	1	2	4	6	8
ml HCl (point 3.2)	7	7	7	7	7	7	7
+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.	+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.	+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.	+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.	+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.	+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.	+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.	+ 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water.

5.1.2.2.   Preparation of solution for analysis

For the determination of copper, the solution prepared from point 5.1.1 can normally be used directly. If necessary to bring its concentration within the range of the calibration solutions, an aliquot portion may be pipetted into a 100 ml volumetric flask and made up to the mark with 0,5 mol/litre hydrochloric acid (point 3.3).

For the determination of iron, manganese and zinc, pipette an aliquot portion of the solution prepared from point 5.1.1 into a 100 ml volumetric flask, add 10 ml of lanthanum chloride solution (point 3.11) and make up to the mark with 0,5 mol/litre hydrochloric acid (point 3.3) (see also point 8 ‘Observation’).

5.1.2.3.   Blank experiment

The blank experiment must include all the prescribed steps of the procedure except that the sample material is omitted. The calibration solution ‘0’ must not be used as the blank.

5.1.2.4.   Measurement of the atomic absorption

Measure the atomic absorption of the calibration solutions and of the solution to be analysed using an oxidising air-acetylene flame at the following wavelengths:

Fe: 248,3 nm

Cu: 324,8 nm

Mn: 279,5 nm

Zn: 213,8 nm.

Carry out each measurement four times.

5.2.   Mineral feed

If the sample contains no organic matter, prior ashing is unnecessary. Proceed as described in point 5.1.1.1 starting from the second paragraph. Evaporation with hydrofluoric acid may be omitted.

6. Calculation of results

Using a calibration curve, calculate the trace element concentration in the solution to be analysed and express the result in milligrams of trace element per kilogram of sample (ppm).

7. Repeatability

The difference between the results of two parallel determinations carried out on the same sample by the same analyst shall not exceed:

• — 5 mg/kg, in absolute value, for contents of the trace element concerned up to 50 mg/kg,

• — 10 % of the higher result for contents of the trace element concerned from 50 and up to 100 mg/kg,

• — 10 mg/kg, in absolute value, for contents of the trace element concerned from 100 and up to 200 mg/kg,

• — 5 % of the higher result for contents of the trace element concerned above 200 mg/kg.

8. Observation

The presence of large quantities of phosphates may interfere with the determination of iron, manganese and zinc. Such interference must be corrected by addition of lanthanum chloride solution (point 3.11). If, however, in the sample the weight ratio Ca + Mg/P is > 2, addition of lanthanum chloride solution (point 3.11) to the solution for analysis and to the calibration solutions may be omitted.

D. DETERMINATION OF HALOFUGINONE

DL-trans-7-bromo-6-chloro-3-[3-(3-hydroxy-2-piperidyl)acetonyl]-quinazolin-4-(3H)-one hydrobromide

The halofuginone content is to be determined by:

• — the method of analysis provided for by EN 17299 Animal feeding stuffs: Methods of sampling and analysis – Screening and determination of authorized coccidiostats at additive and 1 % and 3 % cross-contamination level, and of non-registered coccidiostats and of one antibiotic at sub-additive levels, in compound feed with High Performance Liquid Chromatograpy – Tandem Mass Spectrometry detection (LC-MS/MS), or

• — by reversed-phase high performance liquid chromatography (HPLC) using an UV detector, as described in points 1 to 8 hereafter.

1. Purpose and scope

The method makes it possible to determine the level of halofuginone in feed. The limit of quantification is 1 mg/kg.

2. Principle

After treatment with hot water, halofuginone is extracted as the free base into ethyl acetate and subsequently partitioned as the hydrochloride into an aqueous acid solution. The extract is purified by ion-exchange chromatography. The content of halofuginone is determined by reversed-phase high performance liquid chromatography (HPLC) using an UV detector.

3. Reagents

• 3.1.Acetonitrile, equivalent to HPLC grade.

• 3.2.Amberlite XAD-2 resin.

• 3.3.Ammonium acetate.

• 3.4.Ethyl acetate.

• 3.5.Acetic acid, glacial.

• 3.6.Halofuginone standard substance (DL-trans-7-bromo-6-chloro-3-[3-(3-hydroxy-2-piperidyl)acetonyl] quinazolin-4-(3H)-one hydrobromide, E 764).

• 3.6.1.Halofuginone stock standard solution, 100 μg/ml

• Weight to the nearest 0,1 mg, 50 mg of halofuginone (point 3.6) in a 500 ml graduated flask, dissolve in ammonium acetate buffer solution (point 3.18), make up to the mark with the buffer solution and mix. This solution is stable for three weeks at 5 °C if stored in the dark.

• 3.6.2.Calibration solutions

• Into a series of 100 ml graduated flasks transfer 1,0, 2,0, 3,0, 4,0 and 6,0 ml of the stock standard solution (point 3.6.1). Make up to the mark with mobile phase (point 3.21) and mix. These solutions have concentrations of 1,0, 2,0, 3,0, 4,0 and 6,0 μg/ml of halofuginone respectively. These solutions must be freshly prepared before use.

• 3.7.Hydrochloric acid (ρ20 approximately 1,16 g/ml).

• 3.8.Methanol.

• 3.9.Silver nitrate.

• 3.10.Sodium ascorbate.

• 3.11.Sodium carbonate.

• 3.12.Sodium chloride.

• 3.13.EDTA (ethylenediaminetetraacetic acid, disodium salt).

• 3.14.Water, equivalent to HPLC grade.

• 3.15.Sodium carbonate solution, c = 10 g/100 ml.

• 3.16.Sodium chloride-saturated sodium carbonate solution, c = 5 g/100 ml

• Dissolve 50 g of sodium carbonate (point 3.11) in water, dilute to 1 litre and add sodium chloride (point 3.12) until the solution is saturated.

• 3.17.Hydrochloric acid, approximately 0,1 mol/l

• Dilute 10 ml of HCI (point 3.7) with water to 1 litre.

• 3.18.Ammonium acetate buffer solution, approximately 0,25 mol/l

• Dissolve 19,3 g of ammonium acetate (point 3.3) and 30 ml of acetic acid (point 3.5) in water (point 3.14) and dilute to 1 litre.

• 3.19.Amberlite XAD-2 resin preparation

• Wash an appropriate quantity of Amberlite (point 3.2) with water until all chloride ions have been removed, as indicated by a silver nitrate (point 3.20) test performed on the discarded aqueous phase. Then wash the resin with 50 ml of methanol (point 3.8), discard the methanol and store the resin under fresh methanol.

• 3.20.Silver nitrate solution, approximately 0,1 mol/l

• Dissolve 0,17 g of silver nitrate (point 3.9) in 10 ml of water.

• 3.21.HPLC Mobile phase

• Mix 500 ml of acetonitrile (point 3.1) with 300 ml of ammonium acetate buffer solution (point 3.18) and 1 200  ml of water (point 3.14). Adjust the pH to 4,3 using acetic acid (point 3.5). Filter through a 0,22 μm filter (point 4.8) and degas the solution (e.g. by ultrasonification for 10 minutes). This solution is stable for one month, if stored in the dark in a closed container.

4. Apparatus

• 4.1.Ultrasonic bath.

• 4.2.Rotary film evaporator.

• 4.3.Centrifuge.

• 4.4.HPLC equipment with variable wavelength ultraviolet detector or diode-array detector.

• 4.4.1.Liquid chromatographic column, 300 mm × 4 mm, C18, 10 μm packaging, or an equivalent column.

• 4.5.Glass column (300 mm × 10 mm) fitted with a sintered-glass filter and a stopcock.

• 4.6.Glass-fibre filters, diameter 150 mm.

• 4.7.Membrane filters, 0,45 μm.

• 4.8.Membrane filters, 0,22 μm.

5. Procedure

• Note: Halofuginone as the free base is unstable in alkaline and ethyl acetate solutions. It shall not remain in ethyl acetate for more than 30 minutes.

5.1.   General

• 5.1.1.A blank feed shall be analysed to check that neither halofuginone nor interfering substances are present.

• 5.1.2.A recovery test shall be carried out by analysing the blank feed which has been fortified by addition of a quantity of halofuginone, similar to that present in the sample. To fortify at a level of 3 mg/kg, add 300 μl of the stock standard solution (point 3.6.1) to 10 g of the blank feed, mix and wait for 10 minutes before proceeding with the extraction step (point 5.2).

• Note: For the purpose of this method, the blank feed shall be similar in type to that of the sample and on analysis halofuginone shall not be detected.

5.2.   Extraction

Weigh to the nearest 0,1 g, 10 g of the prepared sample, into a 200 ml centrifuge tube, add 0,5 g of sodium ascorbate (point 3.10), 0,5 g of EDTA (point 3.13) and 20 ml of water and mix. Place the tube for 5 minutes in a water bath (80 °C). After cooling down to room temperature, add 20 ml of sodium carbonate solution (point 3.15) and mix. Add immediately 100 ml of ethyl acetate (point 3.4) and shake vigorously by hand for 15 seconds. Then place the tube for three minutes in the ultrasonic bath (point 4.1) and loosen the stopper. Centrifuge for two minutes and decant the ethyl acetate phase through a glass fibre filter (point 4.6), into a 500 ml separating funnel. Repeat the extraction of the sample with a second portion of 100 ml of ethyl acetate. Wash the combined extracts for one minute with 50 ml of sodium chloride saturated sodium carbonate solution (point 3.16) and discard the aqueous layer.

Extract the organic layer for 1 min with 50 ml of hydrochloric acid (point 3.17). Run the lower acid layer into a 250 ml separating funnel. Re-extract the organic layer for 1,5 minutes with a further 50 ml of hydrochlorid acid and combine with the first extract. Wash the combined acid extracts by swirling for approximately 10 seconds with 10 ml of ethyl acetate (point 3.4).

Quantitatively transfer the aqueous layer into a 250 ml round-bottomed flask and discard the organic phase. Evaporate all the remaining ethyl acetate from the acid solution using a rotary film evaporator (point 4.2). The temperature of the water bath must not exceed 40 °C. Under a vacuum of approximately 25 mbar all of the residual ethyl acetate will be removed within 5 minutes at 38 °C.

5.3.   Clean up

5.3.1.   Preparation of the Amberlite column

An XAD-2 column is prepared for each sample extract. Transfer 10 g of prepared Amberlite (point 3.19) into a glass column (point 4.5) with methanol (point 3.8). Add a small plug of glass-wool to the top of the resin bed. Drain the methanol from the column and wash the resin with 100 ml of water, stopping the flow as the liquid reaches the top of the resin bed. Allow the column to equilibrate for 10 minutes before use. Never allow the column to run dry.

5.3.2.   Sample clean up

Transfer the extract (point 5.2) quantitatively to the top of the prepared Amberlite column (point 5.3.1) and elute, discarding the eluate. The rate of elution must not exceed 20 ml/min. Rinse the round-bottomed flask with 20 ml of hydrochlorid acid (point 3.17) and use this to wash the resin column. Blow through any remaining acid solution with a stream of air. Discard the washings. Add 100 ml of methanol (point 3.8) to the column and allow 5 to 10 ml to elute, collecting the eluate in a 250 ml round-bottomed flask. Leave the remaining methanol for 10 minutes to equilibrate with the resin and continue the elution at a rate not exceeding 20 ml/min, collecting the eluate in the same round-bottomed flask. Evaporate the methanol on the rotary film evaporator (point 4.2), the temperature of the water bath must not exceed 40 °C. Transfer the residue quantitatively into a 10 ml calibrated flask using the mobile phase (point 3.21). Make up to the mark with mobile phase and mix. An aliquot is filtered through a membrane filter (point 4.7). Reserve this solution for the HPLC determination (point 5.4)

5.4.   HPLC determination

5.4.1.   Parameters

The following conditions are offered for guidance, other conditions may be used provided they yield equivalent results.

Liquid chromatographic column (point 4.4.1)

HPLC Mobile phase (point 3.21)

Flow rate: 1,5 to 2 ml/min.

Detection wavelength: 243 nm

Injection volume: 40 to 100 μl.

Check the stability of the chromatographic system, injecting the calibration solution (point 3.6.2) containing 3,0 μg/ml several times, until constant peak heights (or areas) and retention times are achieved.

5.4.2.   Calibration graph

Inject each calibration solution (point 3.6.2) several times and measure the peak heights (areas) for each concentration. Plot a calibration graph using the mean peak heights or areas of the calibration solutions as the ordinates and the corresponding concentrations in μg/ml as the abscissae.

5.4.3.   Sample solution

Inject the sample extract (point 5.3.2) several times, using the same volume as taken for the calibration solutions and determine the mean peak height (area) of the halofuginone peaks.

6. Calculation of results

Determine the concentration of the sample solution in μg/ml, from the mean height (area) of the halofuginone peaks of the sample solution by reference to the calibration graph (point 5.4.2).

The content of halofuginone w (mg/kg) of the sample is given by the following formula:

Image omitted; see official source

in which:

c	:	halofuginone concentration of the sample solution in μg/ml,
---	---	---
m	:	weight of the test portion in grams.

7. Validation of the results

7.1.   Identity

The identity of the analyte can be confirmed by co-chromatography, or by using a diode-array detector by which the spectra of the sample extract and the calibration solution (point 3.6.2) containing 6,0 μg/ml are compared.

7.1.1.   Co-chromatography

A sample extract is fortified by addition of an appropriate amount of a calibration solution (point 3.6.2). The amount of added halofuginone must be similar to the estimated amount of halofuginone found in the sample extract.

Only the height of the halofuginone peak shall be enhanced after taking into account both the amount added and the dilution of the extract. The peak width, at half of its maximum height, must be within ± 10 % of the original width.

7.1.2.   Diode-array detection

The results are evaluated according to the following criteria:

• (a) the wavelength of maximum absorption of the sample and of the standard spectra, recorded at the peak apex on the chromatogram, must be the same within a margin determined by the resolving power of the detection system. For diode-array detection, this is typically within ± 2 nm;

• (b) between 225 and 300 nm, the sample and standard spectra recorded at the peak apex on the chromatogram, must not be different for those parts of the spectrum within the range 10 to 100 % of relative absorbance. This criterion is met when the same maxima are present and at no observed point the deviation between the two spectra exceeds 15 % of the absorbance of the standard analyte;

• (c) between 225 and 300 nm, the spectra of the upslope, apex and downslope of the peak produced by the sample extract must not be different from each other for those parts of the spectrum within the range 10 to 100 % of relative absorbance. This criterion is met when the same maxima are present and when at all observed points the deviation between the spectra does not exceed 15 % of the absorbance of the spectrum of the apex.

If one of these criteria is not met the presence of the analyte has not been confirmed.

7.2.   Repeatability

The difference between results of two parallel determinations carried out on the same sample must not exceed 0,5 mg/kg for halofuginone contents up to 3mg/kg.

7.3.   Recovery

For the fortified blank sample the recovery shall be at least 80 %.

8. Results of a collaborative study

A collaborative study ([^E0041]) was arranged in which three samples were analysed by eight laboratories.

Results

	Sample A (blank) On receipt	Sample B (Meal)	Sample B (Meal)	Sample C (Pellets)	Sample C (Pellets)
		On receipt	After two months	On receipt	After two months
Mean [mg/kg]	ND	2,80	2,42	2,89	2,45
SR [mg/kg]	—	0,45	0,43	0,40	0,42
CVR [%]	—	16	18	14	17
Rec. [%]		86	74	88	75
ND =  not detected SR =  standard deviation of reproducibility CVR =  coefficient of variation of reproducibility (%) Rec. =  recovery (%).	ND =  not detected SR =  standard deviation of reproducibility CVR =  coefficient of variation of reproducibility (%) Rec. =  recovery (%).	ND =  not detected SR =  standard deviation of reproducibility CVR =  coefficient of variation of reproducibility (%) Rec. =  recovery (%).	ND =  not detected SR =  standard deviation of reproducibility CVR =  coefficient of variation of reproducibility (%) Rec. =  recovery (%).	ND =  not detected SR =  standard deviation of reproducibility CVR =  coefficient of variation of reproducibility (%) Rec. =  recovery (%).	ND =  not detected SR =  standard deviation of reproducibility CVR =  coefficient of variation of reproducibility (%) Rec. =  recovery (%).

E. DETERMINATION OF ROBENIDINE

1,3-bis [(4-chlorobenzylidene)amino]guanidine – hydrochloride

The robenidine content is to be determined by

• — the method of analysis provided for by EN 17299 Animal feeding stuffs: Methods of sampling and analysis – Screening and determination of authorized coccidiostats at additive and 1 % and 3 % cross-contamination level, and of non-registered coccidiostats and of one antibiotic at sub-additive levels, in compound feed with High Performance Liquid Chromatography – Tandem Mass Spectrometry detection (LC-MS/MS), or

• — by reversed-phase high performance liquid chromatography (HPLC) using an UV detector, as described in points 1 to 8 hereafter.

1. Purpose and scope

This method makes it possible to determine the levels of robenidine in feed. The limit of quantification is 5 mg/kg.

2. Principle

The sample is extracted with acidified methanol. The extract is dried and an aliquot portion subjected to a clean-up on an aluminium oxide column. Robenidine is eluted from the column with methanol, concentrated, and made up to a suitable volume with mobile phase. The content of robenidine is determined by reversed-phase high-performance liquid chromatography (HPLC) using an UV detector.

3. Reagents

• 3.1.Methanol.

• 3.2.Acidified methanol

• Transfer 4,0 ml hydrochloric acid (ρ20 = 1,18 g/ml) into a 500 ml graduated flask, make up to the mark with methanol (point 3.1) and mix. This solution shall be freshly prepared before use.

• 3.3.Acetonitrile, equivalent to HPLC grade.

• 3.4.Molecular sieve

• Type 3A, 8 to 12 mesh beads (1,6–2,5 mm beads, crystalline alumino-silicate, diameter of pores 0,3 mm).

• 3.5.Aluminium oxide acidic activity grade I for column chromatography

• Transfer 100 g aluminium oxide into a suitable container and add 2,0 ml of water. Stopper and shake for approximately 20 minutes. Store in a well -stoppered container.

• 3.6.Potassium dihydrogen phosphate solution, c = 0,025 mol/l

• Dissolve 3,40 g of potassium dihydrogen phosphate in water (HPLC grade) in a 1 000  ml graduated flask, make up to the mark and mix.

• 3.7.Di-sodium hydrogen phosphate solution, c = 0,025 mol/l

• Dissolve 3,55 g of anhydrous (or 4,45 g of dihydrate or 8,95 g of dodecahydrate) di-sodium hydrogen phosphate in water (equivalent to HPLC grade) in a 1 litre graduated flask, make up to the mark and mix.

• 3.8.HPLC mobile phase

• Mix together the following reagents:

650 ml acetonitrile (point 3.3),

250 ml water (equivalent to HPLC-grade),

50 ml potassium di-hydrogen phosphate solution (point 3.6),

50 ml di-sodium hydrogen phosphate solution (point 3.7).

• Filter through a 0,22 μm filter (point 4.6) and degas the solution (e.g. by ultrasonification for 10 minutes).

• 3.9.Standard substance

• Pure robenidine: 1,3-bis [(4-chlorobenzylidene)amino]guanidine – hydrochloride.

• 3.9.1.Robenidine stock standard solution: 300 μg/ml

• Weigh to the nearest 0,1 mg, 30 mg of robenidine standard substance (point 3.9). Dissolve in acidified methanol (point 3.2) in a 100 ml graduated flask, make up to the mark with the same solvent and mix. Wrap the flask with aluminium foil and store in a dark place.

• 3.9.2.Robenidine intermediate standard solution: 12 μg/ml

• Transfer 10,0 ml of the stock standard solution (point 3.9.1) into a 250 ml graduated flask, make up to the mark with the mobile phase (point 3.8) and mix. Wrap the flask with aluminium foil and store in a dark place.

• 3.9.3.Calibration solutions

• Into a series of 50 ml calibrated flasks, transfer 5,0, 10,0, 15,0, 20,0 and 25,0 ml of the intermediate standard solution (point 3.9.2). Make up to the mark with mobile phase (point 3.8) and mix. These solutions correspond to 1,2, 2,4, 3,6, 4,8 and 6,0 μg/ml of robenidine respectively. These solutions must be freshly prepared before use.

• 3.10.Water equivalent to HPLC grade.

4. Apparatus

• 4.1.Glass column

• Constructed of amber glass fitted with a stopcock and a reservoir of approximately 150 ml capacity, internal diameter 10 to 15 mm, length 250 mm.

• 4.2.Mechanical shaker or magnetic stirrer.

• 4.3.Rotary film evaporator.

• 4.4.HPLC equipment with variable wavelength ultraviolet detector or diode array detector operating in the range of 250 to 400 nm

• 4.4.1.Liquid chromatographic column: 300 mm × 4 mm, C18 10 μm packing or equivalent.

• 4.5.Glass fibre filter paper (Whatman GF/A or equivalent).

• 4.6.Membrane filters, 0,22 μm.

• 4.7.Membrane filters, 0,45 μm.

5. Procedure

• Note: Robenidine is light-sensitive. Amber glassware shall be used in all operations.

5.1.   General

• 5.1.1.A blank feed shall be analysed to check that neither robenidine nor interfering substances are present.

• 5.1.2.A recovery test shall be carried out by analysing the blank feed (point 5.1.1) which has been fortified by addition of a quantity of robenidine, similar to that present in the sample. To fortify at a level of 60 mg/kg, transfer 3,0 ml of the stock standard solution (point 3.9.1) to a 250 ml conical flask. Evaporate the solution to ca. 0,5 ml in a stream of nitrogen. Add 15 g of the blank feed, mix and wait for 10 minutes before proceeding with the extraction step (point 5.2).

• Note:For the purpose of this method, the blank feed shall be similar in type to that of the sample and on analysis robenidine shall not be detected.

5.2.   Extraction

Weigh to the nearest 0,01 g, approximately 15 g of the prepared sample. Transfer to a 250 ml conical flask and add 100,0 ml of acidified methanol (point 3.2), stopper and shake for one hour on the shaker (point 4.2). Filter the solution through a glass fibre filter paper (point 4.5) and collect the whole filtrate in a 150 ml conical flask. Add 7,5 g molecular sieve (point 3.4), stopper and shake for five minutes. Filter immediately through a glass-fibre filter paper. Retain this solution for the purification step (point 5.3).

5.3.   Purification

5.3.1.   Preparation of the aluminium-oxide column

Insert a small glass-wool plug into the lower end of a glass column (point 4.1) and tamp it down using a glass rod. Weigh out 11,0 g of the prepared aluminium oxide (point 3.5) and transfer to the column. Care shall be taken to minimise the exposure to the atmosphere during this stage. Gently tap the loaded column at its lower end to settle the aluminium oxide.

5.3.2.   Sample purification

Transfer onto the column by pipette 5,0 ml of the sample extract prepared in (point 5.2). Rest the pipette tip close to the column wall and allow the solution to be absorbed onto the aluminium oxide. Elute the robenidine from the column using 100 ml methanol (point 3.1), at a flow rate of 2 to 3 ml/minute and collect the eluate in a 250 ml round-bottomed flask. Evaporate the methanol solution to dryness under reduced pressure at 40 °C by means of a rotary film evaporator (point 4.3). Re-dissolve the residue in 3 to 4 ml of mobile phase (point 3.8) and transfer quantitatively to a 10 ml graduated flask. Rinse the flask with several 1 to 2 ml portions of mobile phase and transfer these rinsings to the graduated flask. Make up to the mark with the same solvent and mix. An aliquot is filtered through a 0,45 μm membrane filter (point 4.7). Reserve this solution for HPLC determination (point 5.4).

5.4.   HPLC determination

5.4.1.   Parameters

The following conditions are offered for guidance, other conditions may be used provided they yield equivalent results:

Liquid chromatographic column (point 4.4.1),

HPLC mobile phase (point 3.8),

Flow rate: 1,5 to 2 ml/minute,

Detector wavelength: 317 nm,

Injection volume: 20 to 50 μl.

Check the stability of the chromatographic system, injecting the calibration solution (point 3.9.3) containing 3,6 μg/ml several times, until constant peak heights and retention times are achieved.

5.4.2.   Calibration graph

Inject each calibration solution (point 3.9.3) several times and measure the peak heights (areas) for each concentration. Plot a calibration curve using the mean peak heights or areas of the calibration solutions as the ordinates and corresponding concentrations in μg per ml as abscissae.

5.4.3.   Sample solution

Inject the sample extract (point 5.3.2) several times, using the same volume as taken for the calibration solutions and determine the mean peak height (area) of the robenidine peaks.

6. Calculation of results

From the mean height (area) of the robenidine peaks of the sample solution determine the concentration of the sample solution in μg/ml by reference to the calibration graph (point 5.4.2).

The content of robenidine w (mg/kg) in the sample is given by the following formula:

Image omitted; see official source

in which:

c	=	robenidine concentration of the sample solution in μg/ml,
---	---	---
m	=	weight of the test portion in grams.

7. Validation of the results

7.1.   Identity

The identity of the analyte can be confirmed by co-chromatography, or by using a diode-array detector by which the spectra of the sample extract and the calibration solution (point 3.9.3) containing 6 μg/ml are compared.

7.1.1.   Co-chromatography

A sample extract is fortified by addition of an appropriate amount of calibration solution (point 3.9.3). The amount of added robenidine must be similar to the estimated amount of robenidine found in the sample extract.

Only the height of the robenidine peak shall be enhanced after taking into account both the amount added and the dilution of the extract. The peak width, at half of its maximum height, must be within approximately 10 % of the original width.

7.1.2.   Diode-array detection

The results are evaluated according to the following criteria:

• (a) the wavelength of maximum absorption of the sample and of the standard spectra, recorded at the peak apex on the chromatogram, must be the same within a margin determined by the resolving power of the detection system. For diode-array detection, this is typically within approximately 2 nm;

• (b) between 250 and 400 nm, the sample and standard spectra recorded at the peak apex on the chromatogram, must not be different for those parts of the spectrum within the range 10 to 100 % of relative absorbance. This criterion is met when the same maxima are present and at no observed point the deviation between the two spectra exceeds 15 % of the absorbance of the standard analyte;

• (c) between 250 and 400 nm, the spectra of the upslope, apex and downslope of the peak produced by the sample extract must not be different from each other for those parts of the spectrum within the range 10 to 100 % of relative absorbance. This criterion is met when the same maxima are present and when at all observed points the deviation between the spectra does not exceed 15 % of the absorbance of the spectrum of the apex.

If one of these criteria is not met the presence of the analyte has not been confirmed.

7.2.   Repeatability

The difference between the results of two parallel determinations carried out on the same sample must not exceed 10 % of the higher result for robenidine content higher than 15 mg/kg.

7.3.   Recovery

For a fortified blank sample the recovery shall be at least 85 %.

8. Results of a collaborative study

An EU collaborative study was arranged in which four samples of poultry and rabbit feed, in meal or pelleted form were analysed by 12 laboratories. Duplicate analyses were performed on each sample. The results are given in the following table:

	Poultry	Poultry	Rabbit	Rabbit
	Meal	Pellet	Meal	Pellet
Mean [mg/kg]	27,00	27,99	43,6	40,1
sr [mg/kg]	1,46	1,26	1,44	1,66
CVr [%]	5,4	4,5	3,3	4,1
SR [mg/kg]	4,36	3,36	4,61	3,91
CVR [%]	16,1	12,0	10,6	9,7
Recovery [%]	90,0	93,3	87,2	80,2
sr =  standard deviation of repeatability CVr =  coefficient of variation of repeatability, % SR =  standard deviation of reproducibility CVR =  coefficient of variation of reproducibility, %.	sr =  standard deviation of repeatability CVr =  coefficient of variation of repeatability, % SR =  standard deviation of reproducibility CVR =  coefficient of variation of reproducibility, %.	sr =  standard deviation of repeatability CVr =  coefficient of variation of repeatability, % SR =  standard deviation of reproducibility CVR =  coefficient of variation of reproducibility, %.	sr =  standard deviation of repeatability CVr =  coefficient of variation of repeatability, % SR =  standard deviation of reproducibility CVR =  coefficient of variation of reproducibility, %.	sr =  standard deviation of repeatability CVr =  coefficient of variation of repeatability, % SR =  standard deviation of reproducibility CVR =  coefficient of variation of reproducibility, %.

F. DETERMINATION OF DICLAZURIL

(+)-4-chlorphenyl [2,6-dichloro-4-(2,3,4,5-tetrahydro-3,5-dioxo-1,2,4-triazin-2-yl)phenyl] acetonitrile.

The diclazuril content is to be determined by:

• — the method of analysis provided for by EN 17299 Animal feeding stuffs: Methods of sampling and analysis – Screening and determination of authorized coccidiostats at additive and 1 % and 3 % cross-contamination level, and of non-registered coccidiostats and of one antibiotic at sub-additive levels, in compound feed with High Performance Liquid Chromatography – Tandem Mass Spectrometry detection (LC-MS/MS), or

• — by ternary gradient reversed-phase high performance liquid chromatography (HPLC) using an UV detector, as described in points 1 to 9 hereafter.

1. Purpose and scope

The method makes it possible to determine the level of diclazuril in compound feed and premixtures ([^E0042]). The limit of detection is 0,1 mg/kg, the limit of quantification is 0,5 mg/kg. Lower limits of quantification are achievable but this is to be validated by the user.

2. Principle

After addition of an internal standard, the sample is extracted with acidified methanol. For feed, an aliquot of the extract is purified on a C18 solid phase extraction cartridge. Diclazuril is eluted from the cartridge with a mixture of acidified methanol and water. After evaporation, the residue is dissolved in DMF/water. For premixtures, the extract is evaporated and the residue is dissolved in DMF/water. The content of diclazuril is determined by ternary gradient reversed-phase high-performance liquid chromatography (HPLC) using a UV detector.

3. Reagents

• 3.1.Water, equivalent to HPLC-grade.

• 3.2.Ammonium acetate.

• 3.3.Tetrabutylammonium hydrogen sulphate (TBHS).

• 3.4.Acetonitrile, equivalent to HPLC grade.

• 3.5.Methanol, equivalent to HPLC grade.

• 3.6.N, N-dimethylformamide (DMF).

• 3.7.Hydrochloric acid, ρ20 = 1,19 g/ml.

• 3.8.Standard substance: diclazuril: (+)-4-chlorphenyl [2,6-dichloro-4-(2,3,4,5-tetrahydro3,5 -dioxo-1,2,4-triazin-2-yl) phenyl] acetonitrile with guaranteed purity

• 3.8.1.Diclazuril stock standard solution, 500 μg/ml

• Weigh to the nearest 0,1 mg, 25 mg of diclazuril standard substance (point 3.8) in a 50 ml graduated flask. Dissolve in DMF (point 3.6), make up to the mark with DMF (point 3.6) and mix. Wrap the flask with aluminium foil or use amber flask and store in the refrigerator. At a temperature of less than or equal to 4 °C the solution is stable for 1 month ([^E0043]).

• 3.8.2.Diclazuril standard solution, 50 μg/ml

• Transfer 5,00 ml of the stock standard solution (point 3.8.1) into a 50 ml graduated flask, make up to the mark with DMF (point 3.6) and mix. Wrap the flask with aluminium foil or use amber flask and store in the refrigerator. At a temperature of less than or equal to 4 °C the solution is stable for 1 month.

• 3.9.Internal standard substance: 2,6 dichloro-α-(4-chlorophenyl)-4-(4,5 dihydro3,5 -dioxo-1,2,4-triazine-2 (3H) —yl) α-methylbenzene-acetonitrile (methyl diclazuril)

• 3.9.1.Internal stock standard solution, 500 μg/ml

• Weigh to the nearest 0,1 mg 25 mg of internal standard substance (point 3.9) in a 50 ml graduated flask. Dissolve in DMF (point 3.6), make up to the mark with DMF (point 3.6) and mix. Wrap the flask with aluminium foil or use amber flask and store in the refrigerator. At a temperature of less than or equal to 4 °C the solution is stable for 1 month.

• 3.9.2.Internal standard solution, 50 μg/ml

• Transfer 5,00 ml of the internal stock standard solution (point 3.9.1) into a 50 ml graduated flask, make up to the mark with DMF (point 3.6) and mix. Wrap the flask with aluminium foil or use amber flask and store in the refrigerator. At a temperature of less than or equal to 4 °C the solution is stable for 1 month.

• 3.9.3.Internal standard solution for premixtures, p/1 000  mg/ml (p = nominal content of diclazuril in the premixture in mg/kg)

• Weigh to the nearest 0,1 mg p/10 mg of the internal standard substance in a 100 ml graduated flask, dissolve in DMF (point 3.6) in a ultrasonic bath (point 4.7), make up to the mark with DMF and mix. Wrap the flask with aluminium foil or use amber flask and store in a refrigerator. At a temperature of less than or equal to 4 °C the solution is stable for 1 month.

• 3.10.Calibration solutions

• 3.10.1.Calibration solution, 1 μg/ml (diclazuril)

• Pipette 1,00 ml diclazuril standard solution (point 3.8.2) and 2,00 ml internal standard solution (point 3.9.2) into a 50 ml graduated flask. Add 17 ml DMF (point 3.6), make up to the mark with water (point 3.1) and mix. This solution must be prepared freshly before use.

• 3.10.2.Calibration solution, 2 μg/ml (diclazuril)

• Pipette 2,00 ml diclazuril standard solution (point 3.8.2) and 2,00 ml internal standard solution (point 3.9.2) into a 50 ml graduated flask. Add 16 ml DMF (point 3.6), make up to the mark with water (point 3.1) and mix. This solution must be prepared freshly before use.

• 3.10.3.Calibration solution, 3 μg/ml (diclazuril)

• Pipette 3,00 ml diclazuril standard solution (point 3.8.2) and 2,00 ml internal standard solution (point 3.9.2) into a 50 ml graduated flask. Add 15 ml DMF (point 3.6), make up to the mark with water (point 3.1) and mix. This solution must be prepared freshly before use.

• 3.10.4.Calibration solution, 4 μg/ml (diclazuril)

• Pipette 4,00 ml diclazuril standard solution (point 3.8.2) and 2,00 ml internal standard solution (point 3.9.2) into a 50 ml graduated flask. Add 14 ml DMF (point 3.6), make up to the mark with water (point 3.1) and mix. This solution must be prepared freshly before use.

• 3.10.5.Calibration solution, 5 μg/ml (diclazuril)

• Pipette 5,00 ml diclazuril standard solution (point 3.8.2) and 2,00 ml internal standard solution (point 3.9.2) into a 50 ml graduated flask. Add 13 ml DMF (point 3.6), make up to the mark with water (point 3.1) and mix. This solution must be prepared freshly before use.

• Note:The calibration solutions (points 3.10.1, 3.10.2, 3.10.3, 3.10.4 and 3.10.5) are covering diclazuril concentration in feed ranging from 0,5 to 2,5 mg/kg when using the current protocol.

• 3.11.C18 solid phase extraction cartridge, e.g. Mega Bond Elut, size: 20 cc, sorbent weight: 5 000  mg (pre-conditioning following the supplier guidelines).

• 3.12.Extraction solvent: acidified methanol

• Pipette 5,0 ml hydrochloric acid (point 3.7) into 1 000  ml of methanol (point 3.5), and mix.

• 3.13.Mobile phase for HPLC

• 3.13.1.Eluent A: ammonium acetate – tetrabutylammonium hydrogen sulphate solution

• Dissolve 5 g ammonium acetate (point 3.2) and 3,4 g TBHS (point 3.3) in 1 000  ml water (point 3.1) and mix.

• 3.13.2.Eluent B: acetonitrile (point 3.4).

• 3.13.3.Eluent C: methanol (point 3.5).

4. Apparatus

• 4.1.Mechanical shaker.