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)

• 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	—
Reference [^E0044]	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.

First study from 1994: Journal of AOAC International, Volume 77, No 6, 1994, p. 1359-1361; Second study from 2015: JRC Technical report ‘Re-validation of a method for the determination of diclazuril by collaborative study’ (2016).

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 ()  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  [^E0046]	16 000  [^E0046]	80 [^E0046]	105 [^E0046]	120 [^E0046]	50 [^E0047]	35 [^E0047]
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, %.

Analyst, 1995, 120, p. 2175-2180.

Content declared by manufacturer.

Feed prepared in the laboratory.

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.

• 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.

• 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

• — 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.

M. DETERMINATION OF SALINOMYCIN

The salinomycin 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.

N. DETERMINATION OF SEMDURAMYCIN

The semduramycin 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 16158 Animal feedingstuffs – Determination of semduramicin content – Liquid chromatographic method using a ‘tree’ analytical approach.

O. EN STANDARDS

For the application of Article 34(2)(a) of Regulation (EU) 2017/625 the following EN standards are relevant:

EN ISO 30024 Animal feeding stuffs – Determination of phytase activity

EN 17050 Animal feeding stuffs – methods of sampling and analysis – Determination of iodine in animal feed by ICP-MS

EN 17550 Animal feeding stuffs: Determination of carotenoids in animal compound feed and premixtures by high performance liquid chromatography – UV detection (HPLC-UV)

EN 15784 Animal feeding stuffs: Methods of sampling and analysis – Detection and enumeration of Bacillus spp.

EN 15785 Animal feeding stuffs: Methods of sampling and analysis – Isolation and enumeration of Bifidobacterium spp.

EN 15786 Animal feeding stuffs: Methods of sampling and analysis – Detection and enumeration of Pediococcus spp.

EN 15787 Animal feeding stuffs: Methods of sampling and analysis – Detection and enumeration of Lactobacillus spp. used as feed additive

EN 15788 Animal feeding stuffs: Methods of sampling and analysis – Detection and enumeration of Enterococcus (E. faecium) spp. used as feed additive

EN 15789 Animal feeding stuffs: Methods of sampling and analysis – Detection and enumeration of Saccharomyces cerevisiae used as feed additive

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 (for the analysis of the feed feed additives cobalt and molybdenum)

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 (for the analysis of the feed additive cobalt)

EN 16159 Animal feeding stuffs – Determination of selenium by hydride generation atomic absorption spectrometry (HGAAS) after microwave digestion (digestion with 65 % nitric acid and 30 % hydrogen peroxide) (for the analysis of the feed additive selenium)

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) (for the analysis of the feed additives cobalt, molybdenum, and selenium)

ANNEX V

METHODS OF ANALYSIS TO CONTROL UNDESIRABLE SUBSTANCES IN FEED

A. DETERMINATION OF THE LEVELS OF DIOXINS (PCDD/PCDF) AND PCBs

CHAPTER I

METHODS OF SAMPLING AND INTERPRETATION OF ANALYTICAL RESULTS

1. Scope and definitions

The samples intended for the official control of the levels of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), dioxin-like polychlorinated biphenyls (PCBs) ([^E0048]) and non-dioxin-like PCBs in feed shall be taken in accordance with the provisions of Annex I. The quantitative requirements in relation to the control of substances or products uniformly distributed throughout the feed as provided for in point 5.1 of Annex I shall be applied. Aggregate samples thus obtained shall be considered representative for the lots or sublots from which they are taken. Compliance with maximum levels laid down by Directive 2002/32/EC shall be established on the basis of the levels determined in the laboratory samples.

For the purposes of this Part, the definitions laid down in Annex I to Commission Implementing Regulation (EU) 2021/808 ([^E0049]) shall apply.

In addition to those definitions, the following definitions shall apply for the purpose of this Part:

‘Screening methods’ means methods used for selection of those samples with levels of PCDD/Fs and dioxin-like PCBs that exceed the maximum levels or the action thresholds. They shall allow a cost-effective high sample-throughput, thus increasing the chance to discover new incidents with high exposure and health risks to consumers. Screening methods shall be based on bioanalytical or GC-MS methods. Results from samples exceeding the cut-off value used to check compliance with the maximum level shall be verified by a full re-analysis from the original sample using a confirmatory method.

‘Confirmatory methods’ means methods that provide full or complementary information enabling the PCDD/Fs and dioxin-like PCBs to be identified and quantified unequivocally at the maximum or in case of need at the action threshold. Such methods utilise gas chromatography/high resolution mass spectrometry (GC-HRMS) or gas chromatography/tandem mass spectrometry (GC-MS/MS).

2. Compliance of the lot or sublot with the maximum level

2.1.   As regards non-dioxin-like PCBs

The lot or sublot complies with the maximum level if the analytical result for the sum of PCB 28, PCB 52, PCB 101, PCB 138, PCB 153 and PCB 180 (hereafter referred to as non-dioxin-like PCBs) does not exceed the maximum level laid down by Directive 2002/32/EC, taking into account the expanded measurement uncertainty ([^E0050]) The lot or sublot does not comply with the maximum level as laid down by Directive 2002/32/EC, if the mean of two upper-bound ([^E0051]) analytical results obtained from duplicate analysis ([^E0052]), taking into account the expanded measurement uncertainty, exceeds the maximum level beyond reasonable doubt, i.e. the analysed concentration after deduction of the expanded measurement uncertainty is used to assess compliance.

The expanded measurement uncertainty is calculated using a coverage factor of 2 which gives a level of confidence of approximately 95 %. A lot or sublot is non-compliant if the mean of the measured values minus the expanded uncertainty of the mean is above the maximum level.

The rules, mentioned in the paragraphs above under this point, shall apply for the analytical result obtained on the sample for official control. In case of analysis for second expert opinion or reference purposes, the national rules shall apply.

2.2.   As regards PCDD/Fs and dioxin-like PCBs

The lot or sublot complies with the maximum level if the result of a single analysis:

• — performed by a screening method with a false-compliant rate below 5 %, indicates that the level does not exceed the respective maximum level of PCDD/Fs and the sum of PCDD/Fs and dioxin-like PCBs laid down by Directive 2002/32/EC,

• — performed by a confirmatory method, does not exceed the respective maximum level of PCDD/Fs and the sum of PCDD/Fs and dioxin-like PCBs laid down by Directive 2002/32/EC, taking into account the expanded measurement uncertainty.

For screening assays a cut-off value shall be established for decisions on sample compliance with the respective maximum levels set for either PCDD/Fs, or for the sum of PCDD/Fs and dioxin-like PCBs.

The lot or sublot does not comply with the maximum level as laid down by Directive 2002/32/EC if the mean of two upper-bound ([^E0053]) analytical results obtained from duplicate analysis, ([^E0054]) using a confirmatory method, taking into account the expanded measurement uncertainty, exceeds the maximum level beyond reasonable doubt, i.e. the analysed concentration after deduction of the expanded measurement uncertainty is used to assess compliance.

The expanded measurement uncertainty is calculated using a coverage factor of 2 which gives a level of confidence of approximately 95 %. A lot or sublot is non-compliant if the mean of the measured values minus the expanded uncertainty of the mean is above the maximum level.

The sum of the estimated expanded uncertainties of the separate analytical results of PCDD/Fs and dioxin-like PCBs shall be used for the sum of PCDD/Fs and dioxin-like PCBs.

The rules, mentioned in the paragraphs above under this point, shall apply for the analytical result obtained on the sample for official control. In case of analysis for defence or reference purposes, the national rules shall apply.

3. Results exceeding action thresholds as laid down in Annex II to Directive 2002/32/EC

Action thresholds serve as a tool for the selection of samples in those cases where it is necessary to identify a source of contamination and to take measures for its reduction or elimination. Screening methods shall establish the appropriate cut-off values for selection of those samples. Where significant efforts are necessary to identify a source and to reduce or eliminate the contamination, it is appropriate to confirm exceedance of the action thresholds by duplicate analysis using a confirmatory method and taking into account the expanded measurement uncertainty ([^E0055]).

CHAPTER II

SAMPLE PREPARATION AND REQUIREMENTS FOR METHODS OF ANALYSIS USED IN OFFICAL CONTROL OF THE LEVELS OF DIOXINS (PCDD/FS) AND DIOXIN-LIKE PCBS IN FEED

1. Field of application

The requirements set out in this Chapter shall be applied where feed is analysed for the official control of the levels of 2,3,7,8-substituted PCDD/Fs and dioxin-like PCBs and as regards sample preparation and analytical requirements for other regulatory purposes, which includes the controls performed by the feed business operator to ensure compliance with the provisions of Regulation (EC) No 183/2005 of the European Parliament and of the Council ([^E0056]).

Monitoring for the presence of PCDD/Fs and dioxin-like PCBs in feed may be performed with two different types of analytical methods:

(a) Screening methods

The goal of screening methods is to select those samples with levels of PCDD/Fs and dioxin-like PCBs that exceed the maximum levels or the action thresholds. Screening methods shall ensure cost-effective high sample-throughput, thus increasing the chance to discover new incidents with high exposure and health risks of consumers. Their application shall aim to avoid false-compliant results. They may comprise bioanalytical and GC-MS methods.

Screening methods compare the analytical result with a cut-off value, providing a yes/no decision over the possible exceedance of the maximum level or action threshold. The concentration of PCDD/Fs and the sum of PCDD/Fs and dioxin-like PCBs in samples suspected to be non-compliant with the maximum level shall be determined or confirmed by a confirmatory method.

In addition, screening methods may give an indication of the levels of PCDD/Fs and dioxin-like PCBs present in the sample. In case of application of bioanalytical screening methods the result is expressed as Bioanalytical Equivalents (BEQ), whereas in case of application of physico-chemical GC-MS methods it is expressed as Toxic Equivalents (TEQ). The numerically indicated results of screening methods are suitable for demonstrating compliance or suspected non-compliance or exceedance of action thresholds and give an indication of the range of levels in case of follow-up by confirmatory methods. They are not suitable for purposes such as evaluation of background levels, estimation of intake, following of time trends in levels or re-evaluation of action thresholds and maximum levels.

(b) Confirmatory methods

Confirmatory methods allow the unequivocal identification and quantification of PCDD/Fs and dioxin-like PCBs present in a sample and provide full information on the level of individual congeners. Therefore, those methods allow the control of maximum levels and action thresholds, including the confirmation of results obtained by screening methods. Furthermore, results may be used for other purposes such as determination of low background levels in feed monitoring, following of time trends, exposure assessment and building of a database for possible re-evaluation of action thresholds and maximum levels. They are also important for establishing congener patterns in order to identify the source of a possible contamination. Such methods utilise GC-HRMS. For confirming compliance or non-compliance with the maximum level, also GC-MS/MS can be used.

2. Background

For calculation of TEQ concentrations, the concentrations of the individual substances in a given sample shall be multiplied by their respective Toxic Equivalency Factor (TEF) (see footnote 1 of Chapter I) and subsequently summed to give the total concentration of dioxin-like compounds expressed as TEQs.

For the purposes of this Part A, the accepted specific limit of quantification of an individual congener means the lowest content of the analyte that can be measured with reasonable statistical certainty, fulfilling the identification criteria as described in internationally recognised standards, for example, in standard EN 16215:2012 (Animal feed – Determination of dioxins and dioxin-like PCBs by GC-HRMS and of indicator PCBs by GC-HRMS) and/or in EPA methods 1613 and 1668 as revised.

The limit of quantification of an individual congener may be identified as:

• (a) the concentration of an analyte in the extract of a sample which produces an instrumental response at two different ions to be monitored with a S/N (signal/noise) ratio of 3:1 for the less intensive raw data signal; or

• (b) if for technical reasons the signal-to-noise calculation does not provide reliable results, the lowest concentration point on a calibration curve that gives an acceptable (≤ 30 %) and consistent (measured at least at the start and at the end of an analytical series of samples) deviation to the average relative response factor calculated for all points on the calibration curve in each series of samples. The limit of quantification (LOQ) is calculated from the lowest concentration point taking into account the recovery of internal standards and the sample intake.

Bioanalytical screening methods will not give results at the congener level but merely an indication ([^E0057]) of the TEQ level, expressed in BEQ to acknowledge the fact that not all compounds present in a sample extract that produce a response in the test may fulfill or meet all requirements of the TEQ-principle.

Screening and confirmatory methods may only be applied for control of a certain matrix if the methods are sensitive enough to detect levels reliably at the action threshold or maximum level.

3. Quality assurance requirements

• 3.1.Measures shall be taken to avoid cross-contamination at each stage of the sampling and analysis procedure.

• 3.2.The samples shall be stored and transported in glass, aluminum, polypropylene or polyethylene containers suitable for storage without any influence on the levels of PCDD/Fs and dioxin-like PCBs in the samples. Traces of paper dust shall be removed from the sample container.

• 3.3.The sample storage and transportation shall be performed in a way that maintains the integrity of the feed sample.

• 3.4.Insofar as relevant, each laboratory sample shall be finely grinded and mixed thoroughly using a process that has been demonstrated to achieve complete homogenisation (for example, ground to pass a 1 mm sieve). Samples shall be dried before grinding if the moisture content is too high.

• 3.5.Control of reagents, glassware and equipment for possible influence of TEQ- or BEQ-based results shall be carried out.

• 3.6.A blank analysis shall be performed by carrying out the entire analytical procedure omitting only the sample.

• 3.7.For bioanalytical methods, all glassware and solvents used in analysis shall be tested to be free of compounds that interfere with the detection of target compounds in the working range. Glassware shall be rinsed with solvents or heated at temperatures suitable to remove traces of PCDD/Fs, dioxin-like compounds and interfering compounds from its surface.

• 3.8.Sample quantity used for the extraction shall be sufficient to fulfill the requirements with respect to a sufficiently low working range including the concentrations of maximum levels or action threshold.

• 3.9.The specific sample preparation procedures used for the products under consideration shall follow internationally accepted guidelines, i.e. EN ISO 6498.

4. Requirements for laboratories

• 4.1.In accordance with the provisions of Regulation (EU) 2017/625, laboratories shall be accredited by a recognised body operating in accordance with ISO/IEC Guide 58 to ensure that they are applying analytical quality assurance. Laboratories shall be accredited following the EN ISO/IEC 17025 standard. The principles as described in the Technical Guidelines for the estimation of measurement uncertainty and limits of quantification for PCDD/F and PCB analysis shall be followed ([^E0058]).

• 4.2.Laboratory proficiency shall be proven by the continuous successful participation in inter-laboratory studies for the determination of PCDD/Fs and dioxin-like PCBs in relevant feed matrices and concentration ranges.

• 4.3.Laboratories applying screening methods for the routine control of samples shall establish a close cooperation with laboratories applying the confirmatory method, both for quality control and confirmation of the analytical result of suspected samples.

5. Basic requirements to be met by analytical procedure for dioxins (PCDD/Fs) and dioxin-like PCBs

5.1.   Low working range and limits of quantification

For PCDD/Fs, detectable quantities shall be in the upper femtogram (10^-15 g) range because of extreme toxicity of some of these compounds. For most PCB congeners a limit of quantification in the nanogram (10^-9 g) range is already sufficient. For the measurement of the more toxic dioxin-like PCB congeners (in particular non-ortho-substituted congeners), the lower end of the working range shall reach the low picogram (10^-12 g) levels. For all other PCB congeners a limit of quantification in the nanogram (10^-9 g) range is sufficient.

5.2.   High selectivity (specificity)

• 5.2.1.A distinction is required between PCDD/Fs and dioxin-like PCBs and a multitude of other, coextracted and possibly interfering compounds present at concentrations up to several orders of magnitude higher than those of the analytes of interest. For GC-MS methods, a differentiation among various congeners is required, such as between toxic (for example, the seventeen 2,3,7,8-substituted PCDD/Fs, and twelve dioxin-like PCBs) and other congeners.

• 5.2.2.Bioanalytical methods shall be able to detect the target compounds as the sum of PCDD/Fs, and/or dioxin-like PCBs. Sample clean-up shall aim at removing compounds causing false non-compliant results or compounds that may decrease the response, causing false compliant results.

5.3.   High accuracy (trueness and precision, bioassay apparent recovery)

• 5.3.1.For GC-MS methods, the determination shall provide a valid estimate of the true concentration in a sample. High accuracy is required to avoid the rejection of a sample analysis result on the basis of poor reliability of the determined TEQ level. Accuracy is expressed as trueness (difference between the mean value measured for an analyte in a certified material and its certified value, expressed as a percentage of this value) and precision (RSDR relative standard deviation calculated from results generated under reproducibility conditions).

• 5.3.2.For bioanalytical methods, the bioassay apparent recovery shall be determined. Bioassay apparent recovery means the BEQ level calculated from the TCDD or PCB 126 calibration curve corrected for the blank and then divided by the TEQ level determined by the confirmatory method. It aims at correcting factors like the loss of PCDD/Fs and dioxin-like compounds during the extraction and clean-up steps, co-extracted compounds increasing or decreasing the response (agonistic and antagonistic effects), the quality of the curve fit, or differences between the TEF values and the Relative Potency (REP) values. The bioassay apparent recovery is calculated from suitable reference samples with representative congener patterns around the level of interest.

5.4.   Validation in the range of maximum level and general quality control measures

• 5.4.1.Laboratories shall demonstrate the performance of a method in the range of the maximum level, for example, 0,5x, 1x and 2x the maximum level with an acceptable coefficient of variation for repeated analysis, during the validation procedure and during routine analysis.

• 5.4.2.Regular blank controls and spiking experiments or analysis of control samples (preferably, if available, certified reference material) shall be performed as internal quality control measures. Quality control charts for blank controls, spiking experiments or analysis of control samples shall be recorded and checked to make sure the analytical performance is in accordance with the requirements.

5.5.   Limit of quantification

• 5.5.1.For a bioanalytical screening method, the establishment of the limit of quantification (LOQ) is not an indispensable requirement but the method shall prove that it can differentiate between the blank and the cut-off value. When providing a BEQ level, a reporting level shall be established to deal with samples showing a response below this level. The reporting level shall be demonstrated to be different from procedure blank samples at least by a factor of three, with a response below the working range. It shall therefore be calculated from samples containing the target compounds around the required minimum level, and not from an S/N ratio or an assay blank.

• 5.5.2.The LOQ for a confirmatory method shall be about one fifth of the maximum level.

5.6.   Analytical criteria

For reliable results from confirmatory or screening methods, the following criteria shall be met in the range of the maximum level for the TEQ or BEQ value, respectively, whether determined as total TEQ or total BEQ (as the sum of PCDD/Fs and dioxin-like PCBs) or separately for PCDD/Fs and dioxin-like PCBs:

	Screening with bioanalytical or physico-chemical methods	Confirmatory methods
False-compliant rate [^E0059]	< 5  %
Trueness		-20  % to 20  %
Repeatability (RSDr)	< 20  %
Intermediate precision (RSDR)	< 25  %	< 15  %

With respect to the maximum levels.

5.7.   Specific requirements for screening methods

• 5.7.1.Both GC-MS and bioanalytical methods may be used for screening. For GC-MS methods the requirements laid down in point 6 shall be met. For cell based bioanalytical methods specific requirements are laid down in point 7.

• 5.7.2.Laboratories applying screening methods for the routine control of samples shall establish a close cooperation with laboratories applying the confirmatory method.

• 5.7.3.Performance verification of the screening method is required during routine analysis, by analytical quality control and ongoing method validation. There shall be a continuous programme for the control of compliant results.

• 5.7.4.Check on possible suppression of the cell response and cytotoxicity: