Commission Implementing Regulation (EU) 2024/771 of 29 February 2024 amending Regulation (EC) No 152/2009 laying down the methods of sampling and analysis for the official control of feed

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 ⁴² 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 ⁴³. 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-tetrahydro–3,5-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 44.
---	---	---
	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 dihydro–3,5-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.
---	---	---
	4.2.	Equipment for ternary gradient HPLC:
---	---	---
	4.2.1.	Liquid chromatographic column, Hypersil ODS, 3 μm packing, 100 mm × 4,6 mm, or equivalent.
---	---	---
	4.2.2.	UV detector with variable wavelength adjustment or diode array detector.
---	---	---
	4.3.	Rotary film evaporator.
---	---	---
	4.4.	Membrane filter (e.g. chemically resistant Nylon), 0,45 μm.
---	---	---
	4.5.	Disposable syringe, 5 ml.
---	---	---
	4.6.	Vacuum manifold.
---	---	---
	4.7.	Ultrasonic bath.

5. Procedure

5.1.   General

5.1.1.   Blank feed

A blank feed shall be analysed to check that neither diclazuril nor interfering substances are present. The blank feed shall be similar in type to that of the sample and on analysis diclazuril or interfering substances shall not be detected.

5.1.2.   Recovery test

A recovery test shall be carried out by analysing the blank feed which has been fortified by addition of a quantity of diclazuril similar to that present in the sample. To fortify at a level of 1 mg/kg add 0,1 ml of the stock standard solution (point 3.8.1) to 50 g of a blank feed, mix thoroughly and leave for 10 min, mixing again several times before proceeding (point 5.2).

Alternatively, if a blank feed similar in type to that of the sample is not available (see point 5.1.1), a recovery test can be performed by means of the standard addition method. In this case, the sample to be analysed is fortified with a quantity of diclazuril, similar to that already present in the sample. This sample is analysed, together with the unfortified sample and the recovery can be calculated by subtraction.

5.2.   Extraction

5.2.1.   Compound feed

Weigh to the nearest 0,01 g approximately 50 g of the sample. Transfer to a 500 ml conical flask, add 1,00 ml internal standard solution (point 3.9.2), 200 ml extraction solvent (point 3.12) and stopper the flask. Shake the mixture on the shaker (point 4.1) overnight. Allow to settle for 10 minutes. Transfer a 20 ml aliquot of the supernatant to a suitable glass container and dilute with 20 ml water (point 3.1). Transfer this solution on an extraction cartridge (point 3.11), and pass through by applying vacuum (point 4.6). Wash the cartridge with 25 ml of a mixture of extraction solvent (point 3.12) and water (point 3.1), 65 + 35 (V + V). Discard the collected fractions and elute the compounds with 25 ml of a mixture of extraction solvent (point 3.12) and water, 80 + 20 (V + V). Evaporate this fraction until it has just reached dryness by means of the rotary evaporator (point 4.3) at 60 °C. Dissolve the residue in 1,0 ml DMF (point 3.6), add 1,5 ml of water (point 3.1) and mix. Filter through a membrane filter (point 4.4) mounted on a disposable syringe (point 4.5). Proceed to the HPLC determination (point 5.3).

5.2.2.   Premixtures

Weigh to the nearest 0,001 g approximately 1 g of the sample. Transfer to a 500 ml conical flask, add 1,00 ml internal standard solution (point 3.9.3), 200 ml extraction solvent (point 3.12) and stopper the flask. Shake the mixture overnight on the shaker (point 4.1). Allow to settle for 10 minutes. Transfer an aliquot of 10 000/p ml (p = nominal content of diclazuril in the premix in mg/kg) of the supernatant to a round-bottomed flask of suitable size. Evaporate until it has just reached dryness, under reduced pressure at 60 °C by means of the rotary evaporator (point 4.3). Redissolve the residue in 10,0 ml DMF (point 3.6), add 15,0 ml water (point 3.1) and mix. Proceed to the HPLC determination (point 5.3).

5.3.   HPLC determination

5.3.1.   Parameters

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

• — Liquid chromatographic column (point 4.2.1): 100 mm × 4,6 mm, Hypersil ODS, 3 μm packing, or equivalent.

• — Mobile phase

• — Eluent A (point 3.13.1): Aqueous solution of ammonium acetate and tetrabutyl-ammonium hydrogen sulphate.

• — Eluent B (point 3.13.2): acetonitrile.

• — Eluent C (point 3.13.3): methanol.

• — Elution mode – linear gradient

• — initial conditions: A + B + C = 60 + 20 + 20 (V + V + V),

• — after 10 min, gradient elution during 30 min to: A + B + C = 45 + 20 + 35 (V + V + V),

• — then flush with B during 10 min.

• — Flow rate: 1,5-2 ml/min

• — Injection volume: 20 μl

• — Detector wavelength: 280 nm.

Check the stability of the chromatographic system, injecting several times the calibration solution (point 3.10.2), containing 2 μg/ml of diclazuril and of internal standard, until constant peak heights and retention times are achieved.

5.3.2.   Chromatographic analysis of calibration solutions

Inject 20 μl of the calibration solutions (points 3.10.1, 3.10.2, 3.10.3, 3.10.4 and 3.10.5) twice each, identify and integrate the diclazuril and internal standard peaks, and draw the calibration curve based on the ratio of the mean peak height or area of diclazuril to the mean peak height or area of internal standard versus diclazuril concentration in the calibration solutions (μg/ml).

5.3.3.   Chromatographic analysis of sample solutions

Inject 20 μl of the sample solution (point 5.2.1 or 5.2.2) twice and determine the mean peak height or area of the diclazuril and internal standard peaks.

6. Calculation of the results

6.1.   Compound feed

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

Image omitted; see official source or Image omitted; see official source

where:

• — Height(d,s) is the peak height of diclazuril in the sample solution (point 5.2.1),

• — Area(d,s) is the peak area of diclazuril in the sample solution (point 5.2.1),

• — Height(i,s) is the peak height of the internal standard in the sample solution (point 5.2.1),

• — Area(i,s) is the peak area of the internal standard in the sample solution (point 5.2.1),

• — b is the intercept of the calibration curve plotted from the calibration solutions (points 3.10.1, 3.10.2, 3.10.3, 3.10.4 and 3.10.5) according to point 5.3.2,

• — a is the slope of the calibration curve plotted from the calibration solutions (points 3.10.1, 3.10.2, 3.10.3, 3.10.4 and 3.10.5) according to point 5.3.2,

• — m is the mass of the test portion in grams,

• — V is the final volume in millilitres of the sample extract after re-dissolving according to point 5.2.1 (i.e. 2,5 ml).

6.2.   Premixtures

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

Image omitted; see official source or Image omitted; see official source

where:

• — Height(d,s) is the peak height of diclazuril in the sample solution (point 5.2.2),

• — Area(d,s) is the peak area of diclazuril in the sample solution (point 5.2.2),

• — Height(i,s) is the peak height of the internal standard in the sample solution (point 5.2.2),

• — Area(i,s) is the peak area of the internal standard in the sample solution (point 5.2.2),

• — b is the intercept of the calibration curve plotted from the calibration solutions (points 3.10.1, 3.10.2, 3.10.3, 3.10.4 and 3.10.5) according to point 5.3.2,

• — a is the slope of the calibration curve plotted from the calibration solutions (points 3.10.1, 3.10.2, 3.10.3, 3.10.4 and 3.10.5) according to point 5.3.2,

• — m is the mass of the test portion in grams,

• — V is the final volume in millilitres of the sample extract after re-dissolving according to point 5.2.2 (i.e. 25 ml),

• — p is the nominal content of diclazuril in mg/kg in the premixture.

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 (point 5.2.1 or 5.2.2) and the calibration solution (point 3.10.2) are compared.

7.1.1.   Co-chromatography

A sample extract (point 5.2.1 or 5.2.2) is fortified by addition of an appropriate amount of calibration solution (point 3.10.2). The amount of added diclazuril must be similar to the amount of diclazuril found in the sample extract.

Only the height of the diclazuril peak and the internal standard 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 height, must be within ± 10 % of the original width of the diclazuril peak or the internal standard peak of the unfortified sample extract.

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 230 and 320 nm, the sample and standard spectra recorded at the peak apex of the chromatogram, must not be different for those parts of the spectrum within the range 10 %–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 230 and 320 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 %–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 peak 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 independent measurements carried out on two sub-samples must not exceed:

• — 30 % relative, to the higher value for diclazuril contents from 0,5 mg/kg to 2,5 mg/kg,

• — 0,75 mg/kg for diclazuril contents between 2,5 mg/kg and 5 mg/kg,

• — 15 % relative to the higher value for diclazuril contents of more than 5 mg/kg.

7.3.   Recovery

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

8. Results of a collaborative study

Two collaborative studies were organised. In the first one, carried out by another group of laboratories in 1994, the samples analysed were two premixtures (O 100, A 100) and three samples of complementary feed for poultry (L1, Z1, K1). One premixture sample was mixed with an organic matrix (O 100) and the other with an inorganic matrix (A 100). The theoretical content is 100 mg diclazuril per kg. The laboratories were instructed to analyse each of the samples once or in duplicate. (More detailed information on the first collaborative study can be found in the Journal of AOAC International, Volume 77, No 6, 1994, p. 1359-1361).

In the second collaborative study, three compound feeds for poultry, containing diclazuril at concentrations of 0,9 mg/kg (MAT 1), 1,5 mg/kg (MAT 2) and blank feed (MAT 3) were analysed. Detailed information on the second study can be found in the JRC Technical report (2016) and in the Journal of AOAC International, Volume 102, No 2, 2019, p. 646-652. The results of the two collaborative studies are given in the following table.

	Sample 1A 100	Sample 2O 100	Sample 3 L1	Sample 4 Z1	Sample 5 K1	Sample 6 MAT 1	Sample 7 MAT 2	Sample 8 MAT 3
L	11	11	11	11	6	10	9	10
n	19	18	19	19	12	20	18	10
Mean (mg/kg)	100,8	103,5	0,89	1,15	0,89	1,0	1,5	< LOQ
Sr (mg/kg)	5,88	7,64	0,15	0,02	0,03	0,11	0,07	—
CVr (%)	5,83	7,38	17,32	1,92	3,34	11,2	4,5	—
SR (mg/kg)	7,59	7,64	0,17	0,11	0,12	0,18	0,21	—
CVR (%)	7,53	7,38	18,61	9,67	13,65	18,1	14,3	—
Nominal content (mg/kg)	100	100	1,0	1,0	1,0	0,9	1,5	—
Reference45	First study from 1 994	First study from 1 994	First study from 1 994	First study from 1 994	First study from 1 994	Second study from 2 015	Second study from 2 015	Second study from 2 015
L = number of laboratories n = number of single values Sr = standard deviation of repeatability CVr = coefficient of variation of repeatability SR = standard deviation of reproducibility CVR = coefficient of variation of reproducibility LOQ = Limit of quantification.	L = number of laboratories n = number of single values Sr = standard deviation of repeatability CVr = coefficient of variation of repeatability SR = standard deviation of reproducibility CVR = coefficient of variation of reproducibility LOQ = Limit of quantification.	L = number of laboratories n = number of single values Sr = standard deviation of repeatability CVr = coefficient of variation of repeatability SR = standard deviation of reproducibility CVR = coefficient of variation of reproducibility LOQ = Limit of quantification.	L = number of laboratories n = number of single values Sr = standard deviation of repeatability CVr = coefficient of variation of repeatability SR = standard deviation of reproducibility CVR = coefficient of variation of reproducibility LOQ = Limit of quantification.	L = number of laboratories n = number of single values Sr = standard deviation of repeatability CVr = coefficient of variation of repeatability SR = standard deviation of reproducibility CVR = coefficient of variation of reproducibility LOQ = Limit of quantification.	L = number of laboratories n = number of single values Sr = standard deviation of repeatability CVr = coefficient of variation of repeatability SR = standard deviation of reproducibility CVR = coefficient of variation of reproducibility LOQ = Limit of quantification.	L = number of laboratories n = number of single values Sr = standard deviation of repeatability CVr = coefficient of variation of repeatability SR = standard deviation of reproducibility CVR = coefficient of variation of reproducibility LOQ = Limit of quantification.	L = number of laboratories n = number of single values Sr = standard deviation of repeatability CVr = coefficient of variation of repeatability SR = standard deviation of reproducibility CVR = coefficient of variation of reproducibility LOQ = Limit of quantification.	L = number of laboratories n = number of single values Sr = standard deviation of repeatability CVr = coefficient of variation of repeatability SR = standard deviation of reproducibility CVR = coefficient of variation of reproducibility LOQ = Limit of quantification.

9. General comments

The diclazuril response must have been previously demonstrated to be linear over the range of concentrations being measured.

At least for the analysis of diclazuril in compound feed with a high fat content (for this purpose exceeding 12 % fat) the analytical method may be substituted by other HPLC based methods, e.g. a high performance liquid chromatography coupled to mass spectrometry (HPLC-MS) based method, provided that the alternative method has equivalent performance characteristics (recovery rate, precision at repeatability and reproducibility conditions).

G. DETERMINATION OF LASALOCID SODIUM

Sodium salt of a polyether monocarboxylic acid produced by Streptomyces lasaliensis.

The lasolocid sodium 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 a spectrofluorometric (fluorescence) detector, as described in points 1 to 8 hereafter.

1. Purpose and scope

The method makes it possible to determine the level of lasalocid sodium in feed. The limit of detection is 5 mg/kg, the limit of quantification is 10 mg/kg.

2. Principle

Lasalocid sodium is extracted from the sample into acidified methanol and determined by reversed-phase high performance liquid chromatography (HPLC) using a spectrofluorometric (fluorescence) detector.

3. Reagents

	3.1.	Potassium dihydrogen phosphate (KH2PO4).
---	---	---
	3.2.	Orthophosphoric acid, w (w/w) = 85 %.
---	---	---
	3.3.	Orthophosphoric acid solution, c = 20 % Dilute 23,5 ml of orthophosphoric acid (point 3.2) to 100 ml with water.
---	---	---
	3.4.	6-Methyl-2-heptylamine (1,5-dimethylhexylamine), w (w/w) = 99 %.
---	---	---
	3.5.	Methanol, equivalent to HPLC grade.
---	---	---
	3.6.	Hydrochloric acid, density = 1,19 g/ml.
---	---	---
	3.7.	Phosphate buffer solution, c = 0,01 mol/l Dissolve 1,36 g of KH2PO4 (point 3.1) in 500 ml of water (point 3.11), add 3,5 ml of orthophosphoric acid (point 3.2) and 10,0 ml of 6-methyl-2-heptylamine (point 3.4). Adjust the pH to 4,0 with orthophosphoric acid solution (point 3.3) and dilute to 1 000 ml with water (point 3.11).
---	---	---
	3.8.	Acidified methanol Transfer 5,0 ml of hydrochloric acid (point 3.6) into a 1 000 ml graduated flask, make up to the mark with methanol (point 3.5) and mix. This solution must be prepared freshly before use.
---	---	---
	3.9.	HPLC mobile phase, phosphate buffer-methanol solution 5 + 95 (V + V) Mix 5 ml of phosphate buffer solution (point 3.7) with 95 ml of methanol (point 3.5).
---	---	---
	3.10.	Lasalocid sodium standard substance with guaranteed purity, C34H53O8Na (sodium salt of a polyether monocarboxylic acid produced by Streptomyces lasaliensis), E763
---	---	---
	3.10.1.	Lasalocid sodium stock standard solution, 500 μg/ml Weigh to the nearest 0,1 mg, 50 mg of lasalocid sodium (point 3.10) into a 100 ml graduated flask, dissolve in acidified methanol (point 3.8), make up to the mark with the same solvent and mix. This solution must be freshly prepared before use.
---	---	---
	3.10.2.	Lasalocid sodium intermediate standard solution, 50 μg/ml Pipette 10,0 ml of stock standard solution (point 3.10.1) into a 100 ml graduated flask, make up to the mark with acidified methanol (point 3.8) and mix. This solution must be prepared freshly before use.
---	---	---
	3.10.3.	Calibration solutions Into a series of 50 ml graduated flasks transfer 1,0, 2,0, 4,0, 5,0 and 10,0 ml of the intermediate standard solution (point 3.10.2). Make up to the mark with acidified methanol (point 3.8) and mix. These solutions correspond to 1,0, 2,0, 4,0, 5,0 and 10,0 μg of lasalocid sodium per ml respectively. These solutions must be prepared freshly before use.
---	---	---
	3.11.	Water, equivalent to HPLC grade.

4. Apparatus

	4.1.	Ultrasonic bath (or shaking water-bath) with temperature control.
---	---	---
	4.2.	Membrane filters, 0,45 μm.
---	---	---
	4.3.	HPLC equipment with injection system, suitable for injecting volumes of 20 μl
---	---	---
	4.3.1.	Liquid chromatographic column 125 mm × 4 mm, reversed-phase C18, 5 μm packing or equivalent.
---	---	---
	4.3.2.	Spectrofluorometer with variable wavelength adjustment of excitation and emission wavelengths.

5. Procedure

5.1.   General

5.1.1.   Blank feed

For the performance of the recovery test (point 5.1.2) a blank feed shall be analysed to check that neither lasalocid sodium nor interfering substances are present. The blank feed shall be similar in type to that of the sample and lasalocid sodium or interfering substances shall not be detected.

5.1.2.   Recovery test

A recovery test shall be carried out by analysing the blank feed which has been fortified by addition of a quantity of lasalocid sodium, similar to that present in the sample. To fortify at a level of 100 mg/kg, transfer 10,0 ml of the stock standard (point 3.10.1) to a 250 ml conical flask and evaporate the solution to approximately 0,5 ml. Add 50 g of the blank feed, mix thoroughly and leave for 10 minutes mixing again several times before proceeding with the extraction step (point 5.2).

Alternatively, if a blank feed similar in type to that of the sample is not available (see point 5.1.1), a recovery test can be performed by means of the standard addition method. In this case the sample to be analysed is fortified with a quantity of lasalocid sodium similar to that already present in the sample. This sample is analysed together with the unfortified sample and the recovery calculated by subtraction.

5.2.   Extraction

5.2.1.   Feed

Weigh to the nearest 0,01 g, from 5 g to 10 g of the sample into a 250 ml conical flask with stopper. Add 100,0 ml of acidified methanol (point 3.8) by pipette. Stopper loosely and swirl to disperse. Place the flask in an ultrasonic bath (point 4.1) at approximately 40 °C for 20 minutes, then remove and cool to room temperature. Allow to stand for about 1 hour until the suspended matter has settled, then filter an aliquot portion through a 0,45 μm membrane filter (point 4.2) into a suitable vessel. Proceed to the HPLC determination (point 5.3).

5.2.2.   Premixtures

Weigh to the nearest 0,001 g about 2 g of the unground premix into a 250 ml graduated flask. Add 100,0 ml of acidified methanol (point 3.8) and swirl to disperse. Place the flask and contents in an ultrasonic bath (point 4.1) at approximately 40 °C for 20 minutes, then remove and cool to room temperature. Dilute to the mark with acidified methanol (point 3.8) and mix thoroughly. Allow to stand for 1 hour until the suspended matter has settled, then filter an aliquot portion through a 0,45 μm membrane filter (point 4.2). Dilute an appropriate volume of the clear filtrate with acidified methanol (point 3.8) to produce a final test solution containing about 4 μg/ml of lasalocid sodium. Proceed to the HPLC determination (point 5.3).

5.3.   HPLC determination

5.3.1.   Parameters

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

Liquid chromatographic column (point 4.3.1):	125 mm × 4 mm, reversed-phase C18, 5 μm packing or equivalent
Mobile phase (point 3.9):	Mixture of phosphate buffer solution (point 3.7) and methanol (point 3.5), 5 + 95 (V + V)
Flow rate:	1,2  ml/min
Detection wavelengths:	Excitation: 310 nm
	Emission: 419 nm
Injection volume:	20 μl

Check the stability of the chromatographic system, injecting the calibration solution (point 3.10.3) containing 4,0 μg/ml several times, until constant peak heights (or areas) and retention times are achieved.

5.3.2.   Calibration graph

Inject each calibration solution (point 3.10.3) several times and determine the mean peak heights (areas) for each concentration. Plot a calibration graph using the mean peak heights (areas) as the ordinates and the corresponding concentrations in μg/ml as the abscissae.

5.3.3.   Sample solution

Inject the sample extracts obtained in point 5.2.1 or 5.2.2 several times, using the same volume as taken for the calibration solution and determine the mean peak heights (areas) of the lasalocid sodium peaks.

6. Calculation of results

From the mean peak height (area) produced by injection of the sample solution (point 5.3.3) determine the concentration of lasalocid sodium (μg/ml) by reference to the calibration graph.

6.1.   Feed

The lasalocid sodium content, w (mg/kg) in the sample is given by the following formula:

Image omitted; see official source

where:

c	=	lasalocid sodium concentration of the sample solution (point 5.2.1) in μg/ml
V1	=	volume of the sample extract according to point 5.2.1 in ml (i.e. 100)
m	=	weight of the test portion in g.

6.2.   Premixtures

The lasalocid sodium content, w (mg/kg) in the sample is given by the following formula:

Image omitted; see official source

where:

c	=	lasalocid sodium concentration of the sample solution (point 5.2.2) in μg/ml
V2	=	volume of the sample extract according to point 5.2.2 in ml (i.e. 250)
f	=	dilution factor according to point 5.2.2
m	=	weight of the test portion in g.

7. Validation of the results

7.1.   Identity

Methods based on spectrofluorometry are less subject to interference than those in which UV detection is used. The identity of the analyte can be confirmed by co-chromatography.

7.1.1.   Co-chromatography

A sample extract (point 5.2.1 or 5.2.2) is fortified by the addition of an appropriate amount of a calibration solution (point 3.10.3). The amount of added lasalocid sodium must be similar to the amount of lasalocid sodium found in the sample extract. Only the height of the lasalocid sodium peak shall be enhanced after taking into account the amount of lasalocid sodium added and the dilution of the extract. The peak width, at half height, must be within ± 10 % of the original peak width produced by the unfortified sample extract.

7.2.   Repeatability

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

• — 15 % relative to the higher value for lasalocid sodium contents from 30 mg/kg to 100 mg/kg,

• — 15 mg/kg for lasalocid sodium contents from 100 mg/kg to 200 mg/kg,

• — 7,5 % relative to the higher value for lasalocid sodium contents of more than 200 mg/kg.

7.3.   Recovery

For the fortified (blank) feed sample, the recovery shall be at least 80 %. For the fortified premixture samples, the recovery shall be at least 90 %.

8. Results of a collaborative study

A collaborative study ⁴⁶ was arranged in which 2 premixtures (samples 1 and 2) and 5 feeds (samples 3-7) were analysed by 12 laboratories. Duplicate analyses were performed on each sample. The results are given in the following table:

	Sample 1 Chicken premix	Sample 2 Turkey premix	Sample 3 Turkey pellets	Sample 4 Chicken crumbs	Sample 5 Turkey Feed	Sample 6 Poultry Feed A	Sample 7 Poultry Feed B
L	12	12	12	12	12	12	12
n	23	23	23	23	23	23	23
Mean [mg/kg]	5 050	16 200	76,5	78,4	92,9	48,3	32,6
sr [mg/kg]	107	408	1,71	2,23	2,27	1,93	1,75
CVr [%]	2,12	2,52	2,24	2,84	2,44	4,00	5,37
sR [mg/kg]	286	883	3,85	7,32	5,29	3,47	3,49
CVR [%]	5,66	5,45	5,03	9,34	5,69	7,18	10,70
Nominal content [mg/kg]	5 000 47	16 000 47	80 47	105 47	120 47	50 48	35 48
L = number of laboratories n = number of single results sr = standard deviation of repeatability sR = standard deviation of reproducibility CVr = coefficient of variation of repeatability, % CVR = coefficient of variation of reproducibility, %.	L = number of laboratories n = number of single results sr = standard deviation of repeatability sR = standard deviation of reproducibility CVr = coefficient of variation of repeatability, % CVR = coefficient of variation of reproducibility, %.	L = number of laboratories n = number of single results sr = standard deviation of repeatability sR = standard deviation of reproducibility CVr = coefficient of variation of repeatability, % CVR = coefficient of variation of reproducibility, %.	L = number of laboratories n = number of single results sr = standard deviation of repeatability sR = standard deviation of reproducibility CVr = coefficient of variation of repeatability, % CVR = coefficient of variation of reproducibility, %.	L = number of laboratories n = number of single results sr = standard deviation of repeatability sR = standard deviation of reproducibility CVr = coefficient of variation of repeatability, % CVR = coefficient of variation of reproducibility, %.	L = number of laboratories n = number of single results sr = standard deviation of repeatability sR = standard deviation of reproducibility CVr = coefficient of variation of repeatability, % CVR = coefficient of variation of reproducibility, %.	L = number of laboratories n = number of single results sr = standard deviation of repeatability sR = standard deviation of reproducibility CVr = coefficient of variation of repeatability, % CVR = coefficient of variation of reproducibility, %.	L = number of laboratories n = number of single results sr = standard deviation of repeatability sR = standard deviation of reproducibility CVr = coefficient of variation of repeatability, % CVR = coefficient of variation of reproducibility, %.

H. DETERMINATION OF AMPROLIUM HYDROCHLORIDE

1-[(4-amino-2-propyl-5-pyrimidinyl)methyl]-2-methylpyridinium chloride monohydrochloride

1. Purpose and scope

This method makes it possible to determine the level of amprolium in feed. The detection limit is 1 mg/kg, the limit of quantification is 5 mg/kg.

2. Principle

The sample is extracted with a methanol-water mixture. After dilution with the mobile phase and membrane filtration the content of amprolium is determined by cation exchange high performance liquid chromatography (HPLC) using a UV detector.

3. Reagents

	3.1.	Methanol.
---	---	---
	3.2.	Acetonitrile, equivalent to HPLC grade.
---	---	---
	3.3.	Water, equivalent to HPLC grade.
---	---	---
	3.4.	Sodium dihydrogen phosphate solution, c = 0,1 mol/l Dissolve 13,80 g of sodium dihydrogen phosphate monohydrate in water (point 3.3) in a 1 000 ml graduated flask, make up to the mark with water (point 3.3) and mix.
---	---	---
	3.5.	Sodium perchlorate solution, c = 1,6 mol/l Dissolve 224,74 g of sodium perchlorate monohydrate in water (point 3.3) in a 1 000 ml graduated flask, make up to the mark with water (point 3.3) and mix.
---	---	---
	3.6.	Mobile phase for HPLC (see observation point 9.1). Mixture of acetonitrile (point 3.2), sodium dihydrogen phosphate solution (point 3.4) and sodium perchlorate solution (point 3.5), 450 + 450 + 100 (v+v+v). Prior to use filter through a 0,22 μm membrane filter (point 4.3) and degas the solution (e.g. in the ultrasonic bath (point 4.4) for at least 15 minutes).
---	---	---
	3.7.	Standard substance: pure amprolium, 1-[(4-amino-2-propyl-5-pyrimidinyl)methyl]-2-methylpyridinium chloride, E 750 (see point 9.2)
---	---	---
	3.7.1.	Amprolium stock standard solution, 500 μg/ml Weigh to the nearest 0,1 mg, 50 mg of amprolium (point 3.7) in a 100 ml graduated flask, dissolve in 80 ml methanol (point 3.1) and place the flask for 10 min in an ultrasonic bath (point 4.4). After ultrasonic treatment bring the solution to room temperature, make up to the mark with water and mix. At a temperature of ≤ 4 °C the solution is stable for 1 month.
---	---	---
	3.7.2.	Amprolium intermediate standard solution, 50 μg/ml Pipette 5,0 ml of the stock standard solution (point 3.7.1) into a 50 ml graduated flask, make up to the mark with the extraction solvent (point 3.8) and mix. At a temperature of ≤ 4 °C the solution is stable for 1 month.
---	---	---
	3.7.3.	Calibration solutions Transfer 0,5, 1,0 and 2,0 ml of the intermediate standard solution (point 3.7.2) into a series of 50 ml graduated flasks. Make up to the mark with the mobile phase (point 3.6) and mix. These solutions correspond to 0,5, 1,0 and 2,0 μg of amprolium per ml respectively. These solutions must be prepared freshly before use.
---	---	---
	3.8.	Extraction solvent Methanol (point 3.1)-water mixture 2 + 1 (v+v).

4. Apparatus

	4.1.	HPLC equipment with injection system, suitable for injection volumes of 100 μl
---	---	---
	4.1.1.	Liquid chromatographic column 125 mm × 4 mm, cation exchange Nucleosil 10 SA, 5 or 10 μm packing, or equivalent.
---	---	---
	4.1.2.	UV detector with variable wavelength adjustment or diode array detector.
---	---	---
	4.2.	Membrane filter, PTFE material, 0,45 μm.
---	---	---
	4.3.	Membrane filter, 0,22 μm.
---	---	---
	4.4.	Ultrasonic bath.
---	---	---
	4.5.	Mechanical shaker or magnetic stirrer.

5. Procedure

5.1.   General

5.1.1.   Blank feed

For the performance of the recovery test (point 5.1.2) a blank feed shall be analysed to check that neither amprolium nor interfering substances are present. The blank feed shall be similar in type to that of the sample and amprolium or interfering substances must not be detected.

5.1.2.   Recovery test

A recovery test shall be carried out by analysing the blank feed which has been fortified by addition of a quantity of amprolium, similar to that present in the sample. To fortify at a level of 100 mg/kg, transfer 10,0 ml of the stock standard solution (point 3.7.1) to a 250 ml conical flask and evaporate the solution to approximately 0,5 ml. Add 50 g of the blank feed, mix thoroughly and leave for 10 min mixing again several times before proceeding with the extraction step (point 5.2).

Alternatively, if a blank feed similar in type to that of the sample is not available (see point 5.1.1), a recovery test can be performed by means of the standard addition method. In this case, the sample to be analysed is fortified with a quantity of amprolium similar to that already present in the sample. This sample is analysed together with the unfortified sample and the recovery can be calculated by subtraction.

5.2.   Extraction

5.2.1.   Premixtures (content < 1 % amprolium) and feed

Weigh to the nearest 0,01 g, 5–40 g of the sample depending on the amprolium content into a 500 ml conical flask and add 200 ml extraction solvent (point 3.8). Place the flask in the ultrasonic bath (point 4.4) and leave for 15 minutes. Remove the flask from the ultrasonic bath and shake it for 1 h on the shaker or stir on the magnetic stirrer (point 4.5). Dilute an aliquot of the extract with the mobile phase (point 3.6) to an amprolium content of 0,5–2 μg/ml and mix (see observation point 9.3). Filter 5–10 ml of this diluted solution on a membrane filter (point 4.2). Proceed to the HPLC determination (point 5.3).

5.2.2.   Premixtures (content ≥ 1 % amprolium)

Weigh to the nearest 0,001 g, 1–4 g of the premixture depending on the amprolium content into a 500 ml conical flask and add 200 ml extraction solvent (point 3.8). Place the flask in the ultrasonic bath (point 4.4) and leave for 15 minutes. Remove the flask from the ultrasonic bath and shake it for 1 h on the shaker or stir on the magnetic stirrer (point 4.5). Dilute an aliquot of the extract with the mobile phase (point 3.6) to an amprolium content of 0,5–2 μg/ml and mix. Filter 5–10 ml of this diluted solution on a membrane filter (point 4.2). Proceed to the HPLC determination (point 5.3).

5.3.   HPLC determination

5.3.1.   Parameters

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

Liquid chromatographic column (point 4.1.1):	125 mm × 4 mm, cation exchange Nucleosil 10 SA, 5 or 10 μm packing, or equivalent
Mobile phase (point 3.6):	Mixture of acetonitrile (point 3.2), sodium dihydrogen phosphate solution (point 3.4) and sodium perchlorate solution (point 3.5), 450 + 450 + 100 (v + v + v).
Flow rate:	0,7 –1 ml/min
Detection wavelength:	264 nm
Injection volume:	100 μl

Check the stability of the chromatographic system, injecting several times the calibration solution (point 3.7.3) containing 1,0 μg/ml, until constant peak heights and retention times are achieved.

5.3.2.   Calibration graph

Inject each calibration solution (point 3.7.3) several times and determine the mean peak heights (areas) for each concentration. Plot a calibration graph using the mean peak heights (areas) of the calibration solutions as the ordinates and the corresponding concentrations in μg/ml as the abscissae.

5.3.3.   Sample solution

Inject the sample extract (point 5.2) several times using the same volume as taken for the calibration solutions and determine the mean peak height (area) of the amprolium peaks.

6. Calculation of the results

From the mean height (area) of the amprolium peaks of the sample solution determine the concentration of the sample solution in μg/ml by reference to the calibration graph (point 5.3.2).

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

Image omitted; see official source

in which:

V	=	volume of the extraction solvent (point 3.8) in ml according to point 5.2 (i.e. 200 ml)
c	=	amprolium concentration of the sample extract (point 5.2) in μg/ml
f	=	dilution factor according to point 5.2
m	=	weight of the test portion in g.

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 (point 5.2) and the calibration solution (point 3.7.3) containing 2,0 μg/ml are compared.

7.1.1.   Co-chromatography

A sample extract (point 5.2) is fortified by addition of an appropriate amount of calibration solution (point 3.7.3). The amount of added amprolium must be similar to the amount of amprolium found in the sample extract.

Only the height of the amprolium 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 height, must be within ± 10 % of the original width of the amprolium peak of the unfortified sample extract.

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 210 and 320 nm, the sample and standard spectra recorded at the peak apex of the chromatogram, must not be different for those parts of the spectrum within the range 10-100 % 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 210 and 320 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 10-100 % 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 peak 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:

• — 15 % relative to the higher value for amprolium contents from 25 mg/kg to 500 mg/kg,

• — 75 mg/kg for amprolium contents between 500 mg/kg and 1 000 mg/kg,

• — 7,5 % relative to the higher value for amprolium contents of more than 1 000 mg/kg.

7.3.   Recovery

For a fortified (blank) sample the recovery shall be at least 90 %.

8. Results of a collaborative study

A collaborative study was arranged in which three poultry feeds (sample 1-3), one mineral feed (sample 4) and one premix (sample 5) were analysed. The results are given in the following table:

	sample 1 (blank feed)	sample 2	sample 3	sample 4	sample 5
L	14	14	14	14	15
n	56	56	56	56	60
mean [mg/kg]	—	45,5	188	5 129	25 140
sr [mg/kg]	—	2,26	3,57	178	550
CVr [%]	—	4,95	1,90	3,46	2,20
sR [mg/kg]	—	2,95	11,8	266	760
CVR [%]	—	6,47	6,27	5,19	3,00
nominal content [mg/kg]	—	50	200	5 000	25 000
L: number of laboratories n: number of single values sr: standard deviation of repeatability CVr: coefficient of variation of repeatability sR: standard deviation of reproducibility CVR: coefficient of variation of reproducibility.	L: number of laboratories n: number of single values sr: standard deviation of repeatability CVr: coefficient of variation of repeatability sR: standard deviation of reproducibility CVR: coefficient of variation of reproducibility.	L: number of laboratories n: number of single values sr: standard deviation of repeatability CVr: coefficient of variation of repeatability sR: standard deviation of reproducibility CVR: coefficient of variation of reproducibility.	L: number of laboratories n: number of single values sr: standard deviation of repeatability CVr: coefficient of variation of repeatability sR: standard deviation of reproducibility CVR: coefficient of variation of reproducibility.	L: number of laboratories n: number of single values sr: standard deviation of repeatability CVr: coefficient of variation of repeatability sR: standard deviation of reproducibility CVR: coefficient of variation of reproducibility.	L: number of laboratories n: number of single values sr: standard deviation of repeatability CVr: coefficient of variation of repeatability sR: standard deviation of reproducibility CVR: coefficient of variation of reproducibility.

9. Observations

	9.1.	If the sample contains thiamine, the thiamine peak in the chromatogram appears shortly before the amprolium peak. Following this method amprolium and thiamine must be separated. If the amprolium and thiamine are not separated by the column (point 4.1.1) used in this method, replace up to 50 % of the acetonitrile portion of the mobile phase (point 3.6) by methanol.
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	9.2.	According to the British Pharmacopoeia, the spectrum of an amprolium solution (c = 0,02 mol/l) in hydrochloric acid (c = 0,1 mol/l) shows maxima at 246 nm and 262 nm. The absorbance shall amount to 0,84 at 246 nm and 0,80 at 262 nm.
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	9.3.	The extract must always be diluted with the mobile phase, because otherwise the retention time of the amprolium peak may shift significantly, due to changes in the ionic strength.

I. DETERMINATION OF NARASIN

The narasin 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

• — the method provided by EN ISO 14183 Animal feedingstuffs – Determination of monensin, narasin and salinomycin contents – Liquid chromatographic method using post-column derivatisation.

J. DETERMINATION OF NICARBAZIN

The nicarbazin 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

• — the method provided by EN 15782 Animal feedingstuffs – Determination of nicarbazin – High-performance liquid chromatographic method.

K. DETERMINATION OF DECOQUINATE

The decoquinate 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

• — the method provided by EN 16162 Animal feedingstuffs – Determination of decoquinate by HPLC with fluorescence detection.

L. DETERMINATION OF MONENSIN

The monensin content is to be determined by