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)
Modified by
- Commission Regulation (EU) No 278/2012of 28 March 2012amending Regulation (EC) No 152/2009 as regards the determination of the levels of dioxins and polychlorinated biphenyls(Text with EEA relevance), 32012R0278, March 29, 2012
- Commission Regulation (EU) No 51/2013of 16 January 2013amending Regulation (EC) No 152/2009 as regards the methods of analysis for the determination of constituents of animal origin for the official control of feed(Text with EEA relevance)Corrigendum to Commission Regulation (EU) No 51/2013 of 16 January 2013 amending Regulation (EC) No 152/2009 as regards the methods of analysis for the determination of constituents of animal origin for the official control of feed(Official Journal of the European Union L 20 of 23 January 2013), 32013R005132013R0051R(01), January 23, 2013
- Commission Regulation (EU) No 691/2013of 19 July 2013amending Regulation (EC) No 152/2009 as regards methods of sampling and analysis(Text with EEA relevance), 32013R0691, July 20, 2013
- Commission Regulation (EU) No 709/2014of 20 June 2014amending Regulation (EC) No 152/2009 as regards the determination of the levels of dioxins and polychlorinated biphenyls(Text with EEA relevance), 32014R0709, June 27, 2014
- Commission Regulation (EU) 2017/645of 5 April 2017correcting the Latvian language version of Regulation (EC) No 152/2009 laying down the methods of sampling and analysis for the official control of feed(Text with EEA relevance), 32017R0645, April 6, 2017
- Commission Regulation (EU) 2017/771of 3 May 2017amending Regulation (EC) No 152/2009 as regards the methods for the determination of the levels of dioxins and polychlorinated biphenyls(Text with EEA relevance), 32017R0771, May 4, 2017
- Commission Implementing Regulation (EU) 2020/1560of 26 October 2020amending Annex VI to Regulation (EC) No 152/2009 laying down the methods of analysis for the determination of constituents of animal origin for the official control of feed(Text with EEA relevance), 32020R1560, October 27, 2020
- Commission Implementing Regulation (EU) 2022/893of 7 June 2022amending Annex VI to Regulation (EC) No 152/2009 as regards the methods of analysis for the detection of constituents of terrestrial invertebrates for the official control of feed(Text with EEA relevance), 32022R0893, June 8, 2022
- Commission Implementing Regulation (EU) 2024/771of 29 February 2024amending Regulation (EC) No 152/2009 laying down the methods of sampling and analysis for the official control of feed(Text with EEA relevance), 32024R0771, March 15, 2024
Corrected by
- Corrigendum to Commission Regulation (EU) No 51/2013 of 16 January 2013 amending Regulation (EC) No 152/2009 as regards the methods of analysis for the determination of constituents of animal origin for the official control of feed, 32013R0051R(01), March 6, 2013
Lot (or batch): an identified quantity of feed determined to have common characteristics, such as origin, variety, type of packaging, packer, consignor or labelling, and in case of a production process, a unit of production from a single plant using uniform production parameters or a number of such units, when produced in continuous order and stored together. Sampled portion: A lot or an identified part of the lot or sublot. Sealed sample: a sample sealed in such a manner as to prevent any access to the sample without breaking or removing the seal. Incremental sample: A quantity taken from one point in the sampled portion. Aggregate sample: An aggregate of incremental samples taken from the same sampled portion. Reduced sample: A part of the aggregate sample, obtained from the latter by a process of representative reduction. Final sample: A part of the aggregate sample (mixed), of the reduced sample or of the homogenised aggregate sample, depending on the type of control (see point 9.4). Laboratory sample: a sample intended for the laboratory (as received by the laboratory) and can be the final, reduced or aggregate sample. Distance selling sample: Sample of a lot or batch of feed offered for sale by means of distance communication.
The samples shall be taken by persons authorised for that purpose by the competent authority. For a distance selling sample, a quantity of the feed shall be requested from the feed business operator by the competent authority by means of distance communication. The sample has to be sealed in such a manner as to prevent any access to the sample without breaking or removing the seal.
Identification of the sample: the sample has to be indelibly marked and must be identified in such a way that there is an unambiguous link to the sampling report. From each aggregate sample or reduced sample, the following final samples are taken: one for control (enforcement) and one for the feed business operator (defence sample) must be taken. Eventually, one other final sample may be taken for reference. In case the complete aggregate sample is homogenised, the final samples are taken from the homogenised aggregate sample, unless such procedure conflicts with Member States’ rules as regards the right of the feed business operator. In accordance with Article 15, paragraphs 1 and 2, of Regulation (EU) 2017/625, when it is necessary for the performance of official sampling, feed business operators shall, where required by the competent authorities: give the staff of the competent authorities access to the equipment under their control, including, when necessary, making available the proper sampling equipment and personal protective equipment, assist and cooperate with the staff of the competent authorities in order to enable the sampling, including making feed accessible to the staff of the competent authorities.
The quantitative requirements in points 5.1 and 5.2 as regards the number of incremental samples are applicable for sampled portion sizes up to a maximum of 500 tonnes and which can be sampled in a representative way. The sampling procedure described is equally valid for quantities larger than prescribed maximum sampled portion size provided that the maximum number of incremental samples given in the following tables under points 5.1.1, 5.1.3 and 5.1.5 is ignored, the number of incremental samples being determined by the square-root formula given in the appropriate part of the procedure (see point 5.3) and the minimum aggregate sample size increased proportionally. This does not prevent a large lot being divided into smaller sublots and each sublot sampled in accordance with the procedure described in points 5.1 and 5.2. The size of the sampled portion must be such that each of its constituent parts can be sampled. For very large lots or sublots (> 500 tonnes) and for lots which are transported or stored in such a way that sampling cannot be done in accordance with the sampling procedure provided for in points 5.1 and 5.2 of this point, the sampling procedure as provided for in point 5.3 is to be applied. For distance selling samples, the size of the lot from which the quantity is requested is usually not known by the competent authority. Therefore, the procedure referred to in points 5.1 and 5.2 cannot be used. In this case, the procedure described in point 11 shall be applied. In case the feed business operator is required by legislation to comply with this Regulation within the frame of a mandatory monitoring system, the feed business operator may deviate from the quantitative requirements as provided for in this point to take into account operational characteristics on the condition that the feed business operator has demonstrated to the satisfaction of the competent authority the equivalence of the sampling procedure as regards representativeness and after authorisation from the competent authority. In exceptional cases, if it is not possible to carry out the method of sampling set out as regards the quantitative requirements because of the unacceptable commercial damage to the lot (because of packaging forms, means of transport, way of storage, etc.) an alternative method of sampling may be applied provided that it is as representative as possible and is fully described and documented.
| Size of sampled portion | Minimum number of incremental samples |
|---|---|
| ≤ | |
| > | √ ( |
| Size of sampled portion | Minimum number of incremental samples |
|---|---|
| ≤ | |
| > |
| Size of sampled portion | Minimum number of units from which (at least) one incremental sample has to be taken |
|---|---|
| > | ¼ of the √ (number of units making up the sampled portion) |
| Size of sampled portion | Minimum number of incremental samples |
|---|---|
| ≤ | |
| > | √( |
control of aflatoxins, rye ergot, other mycotoxins and harmful botanical impurities in feed materials, control of cross-contamination by a constituent, including GM material, or substance for which non-uniform distribution is expected in feed.
| Size of sampled portion | Minimum number of incremental samples |
|---|---|
| < | See quantitative requirements under 5.1. The number of incremental samples to be taken has to be multiplied by 2,5. |
| ≥ |
| Nature of feed | Minimum size of aggregate sample | |
|---|---|---|
| 6.1. | Loose feed | |
| 6.2. | Packaged feed: | |
| 6.3. | Liquid or semi-liquid feed: | |
| 6.4. | Feed blocks or mineral licks: | |
| 6.4.1. | each weighing more than 1 kg | |
| 6.4.2. | each weighing not more than 1 kg | weight of four original blocks or licks |
| 6.5. | Roughage/forage | |
| Solid feed | |
| Liquid or semi-liquid feed |
(i) completely homogenised. Afterwards from the homogenised aggregate sample the final samples (for control, defence and possibly reference) shall then be taken of approximately the same amount and conforming to the quantitative requirements of point 7; or (ii) reduced to at least 2 kg or 2 litres Except in the case of roughage/forage with low specific density. 35000 seeds/grains to enable to obtain the final samples for enforcement, defence and possibly reference of at least10000 seeds grain (see footnote (**) in point 6 and footnote (*) in point 7).
For a distance selling sample, the feed shall be requested from the feed business operator by means of distance communication techniques. In this case, when requesting feed, the competent authority does not have to identify itself with an official identity to the feed business operator and may use a cover identity. The aggregate sample and the final samples of the distance selling sample have to be taken immediately upon receipt of the consignment by persons authorised for this purpose. For generating the aggregate sample, an appropriate number of incremental samples have to be taken randomly and evenly distributed from the total quantity obtained and carefully mixed/homogenised, in accordance, as far as possible, with the principles laid down in point 5 and points 9.2 and 9.3. If the feed is packaged in individual units, at least 4 units should be obtained from which at least one incremental sample has to be taken. Should it be shown on a case-by-case basis that the units obtained come from different lots, the number of units to be sampled has to be reduced and limited to those units originating from the same lot. In case of analysing the distance selling sample for constituents or substances which are non-uniformly distributed in feed, the number of incremental samples has to be at least 2,5 times higher than that for samples analysed for substances uniformly distributed throughout the feed. From the aggregate sample, the corresponding final samples (for control, for defence and possibly reference) are then taken in accordance, as far as feasible, with the principles laid down in point 9.4 and the sampling record indicates that the sample is a distance selling sample. The competent authority then immediately informs the feed business operator of the sampling. The feed business operator is also notified that one sample (for defence) is kept, when possible, at their disposal by the competent authority, in a specified location, for defence purposes or sent to the feed business operator or sent to the laboratory designated by the feed business operator in accordance with the national rules in place. If the sample is sent directly to the official laboratory, the final sample must be prepared and sealed in the laboratory by persons authorised for this purpose or in the presence of persons authorised for this purpose. The sampling record of the distance selling sample has to be sent immediately after the final samples have been formed to the competent authority, which informs the feed business operator of the sampling. It is considered that the quantity supplied by the feed business operator to the competent authority represents a part of a lot of feed of the same class or description. In accordance with Article 15 of Regulation (EC) No 178/2002 of the European Parliament and of the Council Regulation (EC) No 178/2002 of the European Parliament and of the Council of 28 January 2002 laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety (OJ L 31, 1.2.2002, p. 1 ).
Mix the final sample and collect it in a suitable clean, dry container fitted with an air-tight stopper. Mix again in order to ensure full homogenisation, immediately before weighing out the amount for analysis (test aliquot).
Unless otherwise specified in the methods of analysis, dry the final sample to bring its moisture content down to a level of 8 to 12 %, according to the preliminary drying procedure described under point 4.3 of the method of determination of moisture mentioned in Part A of Annex III). Then proceed as indicated in point 3.1.1.
Collect the final sample in a suitable clean, dry container, fitted with an air-tight stopper. Mix thoroughly in order to ensure full homogenisation immediately before weighing out the amount for analysis (test aliquot).
Final samples which cannot be prepared according to one of the above procedures shall be treated by any other procedure which ensures that the amounts weighed out for the analysis (test aliquot) are homogeneous and representative of the final samples.
In case of an examination by visual inspection (without making use of microscope), the whole aggregate or final sample is used for examination. In case of a microscopic examination, the laboratory may reduce the aggregate sample, or further reduce the reduced sample. The final samples for defence and possibly reference purposes are taken following a procedure equivalent to the procedure followed for the final sample for enforcement. In case the whole aggregate sample is homogenised, the final samples are taken from the homogenised aggregate sample. For the determination of rye ergot and harmful botanical impurities, the final sample has to be divided into 2 subsamples of equal weight of approximately 500 grams. One subsample is examined. In case the result of the subsamples is equal or below 50 % (analytical threshold) of the maximum level, the sample is compliant with the maximum level. If the result is above 50 % of the maximum level, another subsample needs to be examined and the average of the result of the 2 subsamples is used for checking compliance with the maximum level.
the result of the analysis is significantly (> 50 %) lower or higher than the labelling information/specification to be controlled (depending on whether the labelling information/specification is a maximum or a minimum level), the moisture content of the sampled feed is known and it can be determined that correction to the moisture content will not change the assessment, then, on the condition that the appropriate quality procedures are applied and the analysis serves only the purpose of checking compliance with legal provisions, the correction to the moisture content might be omitted (e.g. in cases there is no specification or regulatory level), unless it is required for interpretation.
(a) corrected for recovery, where appropriate and relevant, and when corrected it has to be so stated. The recovery rate is to be quoted unless intrinsic correction for bias is part of the procedure, whereby bias is the difference between the measured value and the reference concentration. The correction for recovery is not necessary in case the recovery rate is between 90-110 %; (b) as "x +/- U", whereby x is the analytical result and U is the expanded analytical measurement uncertainty, using a coverage factor of 2 The confidence interval of 95 % can be achieved by making use of another factor such as the t-factor.
exceed the maximum content taking into account expanded analytical measurement uncertainty and correction for recovery. This means, in order to assess compliance, the analysed concentration (i.e. mean of two determinations) is used after being corrected for recovery and after deduction of the expanded analytical measurement uncertainty, be lower than the minimum content taking into account the expanded analytical measurement uncertainty and correction for recovery. This means, in order to assess compliance, the analysed concentration (i.e. mean of two determinations) is used after being corrected for recovery and after the addition of the expanded analytical measurement uncertainty.
(a) corrected for recovery, where appropriate and relevant, and when corrected it has to be so stated. The recovery rate is to be quoted unless intrinsic correction for bias is part of the procedure, whereby bias is the difference between the measured value and the reference concentration. The correction for recovery is not necessary in case the recovery rate is between 90-110 %; (b) as "x +/- U", whereby x is the analytical result (mean of two determinations) and U is the expanded analytical measurement uncertainty, using a coverage factor of 2 The confidence interval of 95 % can be achieved by making use of another factor such as the t-factor.
0,4 % in absolute value, for crude protein contents of less than 20 %, 2,0 % relative to the higher value, for crude protein contents from 20 % to 40 %, 0,8 % in absolute value, for crude protein contents of more than 40 %.
1,8 % in absolute value, for crude protein contents of less than 20 %, 9,0 % relative to the higher value, for crude protein contents from 20 % to 40 %, 3,6 % in absolute value, for crude protein contents of more than 40 %.
At 420 nm: 50 % relative to the higher value, for urea contents from 3000 mg/kg to lower than5000 mg/kg,25 % relative to the higher value, for urea contents from 5000 mg/kg to lower than7000 mg/kg,20 % relative to the higher value, for urea contents of 7000 mg/kg or more.
At 435 nm: 40 % relative to the higher value, for urea contents from 3000 mg/kg to lower than5000 mg/kg,25 % relative to the higher value, for urea contents from 5000 mg/kg to lower than9000 mg/kg,5 % relative to the higher value, for urea contents of 9000 mg/kg or more.
At 420 nm: 3000 mg/kg, in absolute value, for urea contents from3000 mg/kg to lower than12000 mg/kg,4500 mg/kg, in absolute value, for urea contents of12000 mg/kg or more.
At 435 nm 50 % relative to the higher value, for urea contents from 3000 mg/kg to lower than8000 mg/kg,25 % relative to the higher value, for urea contents of 8000 mg/kg or more.
| MAT 2 | MAT 5 | MAT 3 | MAT 4 | MAT 6 | |
|---|---|---|---|---|---|
| Sheep | Cattle | Sheep | Sheep | Cattle | |
| Target mass fraction (mg kg | |||||
| Average mass fraction. (mg kg | |||||
| Reproducibility standard deviation s | |||||
| Repeatability standard deviation s | |||||
| Reproducibility relative standard deviation | |||||
| Repeatability relative standard deviation | |||||
| MAT 2 | MAT 5 | MAT 3 | MAT 4 | MAT 6 | |
|---|---|---|---|---|---|
| Sheep | Cattle | Sheep | Sheep | Cattle | |
| Target mass fraction (mg kg | |||||
| Average mass fraction. (mg kg | |||||
| Reproducibility standard deviation s | |||||
| Repeatability standard deviation s | |||||
| Reproducibility relative standard deviation | |||||
| Repeatability relative standard deviation | |||||
EN ISO 13903 Animal feeding stuffs – Determination of amino acids content, EN ISO 17180 Animal feeding stuffs – Determination of lysine, methionine, and threonine in commercial amino acid products and premixtures The method of analysis provided for by EN 17180 is referred to as an alternative method to be used for official control purposes for the determination of amino acids in feed containing more than 10 % of amino acids. the method of analysis as described in points 1 to 10 hereafter.
a 100 ml round-bottomed flask (point 4.1) for open hydrolysis (point 5.3.2.3), or a 250 ml round-bottomed flask (point 4.1) if a low sodium concentration is required (point 5.3.3.1), or a 100 ml bottle fitted with a screw cap (point 4.2) (for closed hydrolysis point 5.3.2.4).
| Reference Material | Amino Acid | |||
|---|---|---|---|---|
| Threonine | Cyst(e)ine | Methionine | Lysine | |
| Mixed Pig Feed | ||||
| Broiler Compound Feed | ||||
| Protein Concentrate | ||||
| Premixture | — | |||
| Reference Material | Amino Acid | |||
|---|---|---|---|---|
| Threonine | Cyst(e)ine | Methionine | Lysine | |
| Mixed Pig Feed | ||||
| Broiler Compound Feed | ||||
| Protein Concentrate | ||||
| Premixture | — | |||
| Reference Material | Amino Acid | |||
|---|---|---|---|---|
| Threonine | Cyst(e)ine | Methionine | Lysine | |
| Mixed Pig Feed | ||||
| Broiler Compound feed | ||||
| Protein Concentrate | ||||
| Premixture | — | |||
6 % relative to the higher value, for total amino acids in case of glycine, alanine, lysine, proline, glutamic acid, isoleucine and histidine, 8 % relative to the higher value, for total amino acids in case of threonine, phenylalanine, methionine, aspartic acid and leucine, 10 % relative to the higher value, for total amino acids in case of arginine and valine, 12 % relative to the higher value, for total serine amino acid, 15 % relative to the higher value, for total cyst(e)ine amino acid.
15 % relative to the higher value, for total amino acids in case of glycine, alanine and threonine, 20 % relative to the higher value, for total amino acids in case of lysine, proline, phenylalanine, methionine and aspartic acid, 22 % relative to the higher value, for total amino acids in case of glutamic acid and leucine, 27 % relative to the higher value for total arginine amino acid, 32 % relative to the higher value, for total isoleucine amino acid, 35 % relative to the higher value, for total amino acids in case of valine and serine, 40 % relative to the higher value, for total histidine amino acid, 50 % relative to the higher value, for total cyst(e)ine amino acid.
EN ISO 13904 Animal feeding stuffs – Determination of tryptophan content, the method of analysis as described in points 1 to 9 hereafter.
| Liquid chromatographic column (point 4.2): | 125 mm × 4 mm, C |
| Column temperature: | Room temperature |
| Mobile phase (point 3.22): | 3,00 g acetic acid (point 3.18) + 900 ml water (point 3.1) + 50,0 ml solution (point 3.21) of 1,1,1-trichloro-2-methyl-2-propanol (point 3.19) in methanol (point 3.8) (1 g/100 ml). Adjust pH to 5,00 using ethanolamine (point 3.20). Make up to |
| Flow rate: | 1 ml/min |
| Total run time: | approx. 34 min |
| Detection wavelength: | excitation: 280 nm, emission: 356 nm. |
| Injection volume | 20 μl. |
| L | |||
| s | |||
| r [g/kg] | |||
| CV | |||
| S | |||
| R [g/kg] | |||
| CV |
| Mean [g/kg] | ||
| s | ||
| r [g/kg] | ||
| CV | ||
| S | ||
| R [g/kg] | ||
| CV |
| L | ||||
| n | ||||
| Mean [g/kg] | ||||
| s | ||||
| r [g/kg] | ||||
| CV | ||||
| S | ||||
| R [g/kg] | ||||
| CV |
| Liquid chromatographic column: | 125 mm × 4 mm, C | |
| Column temperature: | 32 °C | |
| Mobile phase: | A: 0,01 mol/l KH | |
| B: Methanol | ||
| Gradient programme: | 0 min 100 % A | 0 % B |
| 15 min 100 % A | 0 % B | |
| 17 min 60 % A | 40 % B | |
| 19 min 60 % A | 40 % B | |
| 21 min 100 % A | 0 % B | |
| 33 min 100 % A | 0 % B | |
| Flow rate: | 1,2 ml/min | |
| Total run time: | approx. 33 min. | |
0,2 %, in absolute value, for contents of crude oils and fats lower than 5 %, 4,0 % relative to the highest result for contents of 5 to 10 %, 0,4 % in absolute value, for contents above 10 %.
0,6 % in absolute value for crude fibre contents lower than 10 %, 6 % relative to the higher result, for crude fibre contents equal to or greater than 10 %.
1,0 % in absolute value for crude fibre contents lower than 10 %, 10 % relative to the higher result, for crude fibre contents equal to or greater than 10 %.
| ml | mg | difference | mg | difference | ml |
|---|---|---|---|---|---|
| ml | mg | difference | mg | difference | ml |
|---|---|---|---|---|---|
16,29 g for the French saccharimeters 26,00 g for the German saccharimeters 20,00 g for mixed saccharimeters.
(sugar) beet products such as (sugar)beet pulp, (sugar) beet molasses, (sugar) beet pulp – molassed, (sugar) beet vinasse, (beet) sugar, citrus pulp, linseed; linseed expeller; linseed extracted, rape seed; rape seed expeller; rape seed extracted; rape seed hulls, sunflower seed; sunflower seed extracted; sunflower seed, partially decorticated, extracted, copra expeller; copra extracted, potato pulp, dehydrated yeast, products rich in inulin (e.g. Chips and meal of Jerusalem artichokes), greaves, soybean products. In these cases the method of analysis as provided by Commission Regulation (EC) No 121/2008 Commission Regulation (EC) No 121/2008 of 11 February 2008 laying down the method of analysis for the determination of starch content in preparations of a kind used in animal feeding (CN code2309 ) (OJ L 37, 12.2.2008, p. 3 ).
the method of analysis provided for by EN 15510 Animal feeding stuffs: Methods of sampling and analysis – Determination of calcium, sodium, phosphorus, magnesium, potassium, iron, zinc, copper, manganese, cobalt, molybdenum and lead by ICP-AES, or the method of analysis provided for by EN 15621 Animal feeding stuffs: Methods of sampling and analysis – Determination of calcium, sodium, phosphorus, magnesium, potassium, sulphur, iron, zinc, copper, manganese and cobalt after pressure digestion by ICP-AES, or the photometric method, as described hereafter.
3 %, relative to the higher result, for phosphorus contents of less than 5 %, 0,15 % in absolute value, for phosphorus contents of 5 % or more.
the method of analysis provided for by EN 17547 Animal feeding stuffs: Methods of sampling and analysis – Determination of vitamin A, E and D The method of analysis provided for by EN 17547 is referred to as an alternative method to be used for official control purposes for the determination of vitamin A and E instead of the method described for the determination of vitamin A in part A of this Annex and for vitamin E in part B of this Annex. by reversed phase high performance liquid chromatography (RP-HPLC) using a UV or a fluorescence detector, as described in the points 1 to 9 hereafter.
| Liquid chromatographic column (point 4.5.1): | 250 mm × 4 mm, C |
| Mobile phase (point 3.9): | Mixture of methanol (point 3.3) and water, e.g. 980 + 20 (v + v) |
| Flow rate: | 1–2 ml/min |
| Detector (point 4.5.2): | UV detector (325 nm) or fluorescence detector (excitation: 325 nm/emission: 475 nm). |
at saponification (point 5.2): due to the amount of fat present in the sample, increasing of potassium hydroxide solution amount (point 3.4) may be necessary, at extraction (point 5.3): due to the presence of emulsions, adaptation of the water/ethanol 2:1 ratio may be necessary.
| Premix | Premix feed | Mineral concentrate | Protein feed | Piglet | |
|---|---|---|---|---|---|
| L | |||||
| n | |||||
| mean [IU/kg] | |||||
| sr [IU/kg] | |||||
| r [IU/kg] | |||||
| CVr [%] | |||||
| sR [IU/kg] | |||||
| R [IU/kg] | |||||
| CVR [%] |
the method of analysis provided for by EN 17547 Animal feeding stuffs: Methods of sampling and analysis – Determination of vitamin A, E and D The method of analysis provided for by EN 17547 is referred to as an alternative method to be used for official control purposes for the determination of vitamin A and E instead of the method described for the determination of vitamin A in part A of this Annex and for vitamin E in part B of this Annex. by reversed phase high performance liquid chromatography (RP-HPLC) using a UV or a fluorescence detector, as described in the points 1 to 9 hereafter.
| Liquid chromatographic column (point 4.5.1): | 250 mm × 4 mm, C |
| Mobile phase (point 3.8): | Mixture of methanol (point 3.3) and water, e.g. 980 + 20 (v + v). |
| Flow rate: | 1–2 ml/min |
| Detector (point 4.5.2) | Fluorescence detector (excitation: 295 nm/emission: 330 nm) or UV detector (292 nm). |
| Premix | Premix feed | Mineral concentrate | Protein feed | Piglet | |
|---|---|---|---|---|---|
| L | |||||
| n | |||||
| mean [mg/kg] | |||||
| sr [mg/kg] | |||||
| r [mg/kg] | |||||
| CVr [%] | |||||
| sR mg/kg] | |||||
| R [mg/kg] | |||||
| CVR [%] |
the method of analysis provided for by EN 15510 Animal feeding stuffs: Methods of sampling and analysis – Determination of calcium, sodium, phosphorus, magnesium, potassium, iron, zinc, copper, manganese, cobalt, molybdenum and lead by ICP-AES, or the method of analysis provided for by EN 15621 Animal feeding stuffs: Methods of sampling and analysis – Determination of calcium, sodium, phosphorus, magnesium, potassium, sulphur, iron, zinc, copper, manganese and cobalt after pressure digestion by ICP-AES, or the method of analysis provided for by EN 17053 Animal feeding stuffs: Methods of sampling and analysis – Determination of trace elements, heavy metals and other elements in feed by ICP-MS (multi-method), or the method of analysis provided for by EN ISO 6869 Animal feeding stuffs. Determination of the contents of calcium, copper, iron, magnesium, manganese, potassium, sodium and zinc. Method using atomic absorption spectrometry, or the flame atomic absorption spectrometry (FAAS) method, as described in the points 1 to 8 hereafter.
iron (Fe): 20 mg/kg copper (Cu): 10 mg/kg manganese (Mn): 20 mg/kg zinc (Zn): 20 mg/kg.
dissolve 1 g of copper in powder form in 25 ml of 6 mol/litre hydrochloric acid (point 3.2), add 5 ml of hydrogen peroxide (point 3.6) and make up to one litre with water.
dissolve 1 g of manganese in powder form in 25 ml of 6 mol/litre hydrochloric acid (point 3.2) and make up to one litre with water.
dissolve 1 g of zinc in strip or leaf form in 25 ml of 6 mol/litre hydrochloric acid (point 3.2) and make up to one litre with water.
(a) In determining trace elements it is important to be alert to the risks of contamination, particularly by zinc, copper and iron. For this reason, the equipment used in preparing the samples must be free of these metals. To reduce the general risk of contamination, work in a dust-free atmosphere with scrupulously clean equipment and carefully washed glassware. The determination of zinc is particularly sensitive to many types of contamination, e.g. from glassware, reagents, dust, etc. (b) The weight of sample to be ashed is calculated from the approximate trace element content of the feed in relation to the sensitivity of the spectrophotometer used. For certain feed low in trace elements it may be necessary to start with a 10 to 20 g sample and make up the final solution to only 100 ml. (c) Ashing must be carried out in a closed furnace without injection of air or oxygen. (d) The temperature indicated by the pyrometer must not exceed 475 °C.
| μg Fe/ml | |||||||
| ml working standard solution (point 3.7.1) (1 ml = 100 μg Fe) | |||||||
| ml HCl (point 3.2) | |||||||
| + 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water. | |||||||
| μg Cu/ml | |||||||
| ml working standard solution (point 3.8.1) (1 ml = 10 μg Cu) | |||||||
| ml HCl (point 3.2) |
| μg Mn/ml | |||||||
| ml working standard solution (point 3.9.1) (1 ml = 10 μg Mn) | |||||||
| ml HCl (point 3.2) | |||||||
| + 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water. | |||||||
| μg Zn/ml | |||||||
| ml working standard solution (point 3.10.1) (1 ml = 10 μg Zn) | |||||||
| ml HCl (point 3.2) | |||||||
| + 10 ml of lanthanum chloride solution (point 3.11) and make up to 100 ml with water. | |||||||
Fe: 248,3 nm Cu: 324,8 nm Mn: 279,5 nm Zn: 213,8 nm. Carry out each measurement four times.
5 mg/kg, in absolute value, for contents of the trace element concerned up to 50 mg/kg, 10 % of the higher result for contents of the trace element concerned from 50 and up to 100 mg/kg, 10 mg/kg, in absolute value, for contents of the trace element concerned from 100 and up to 200 mg/kg, 5 % of the higher result for contents of the trace element concerned above 200 mg/kg.
the method of analysis provided for by EN 17299 Animal feeding stuffs: Methods of sampling and analysis – Screening and determination of authorized coccidiostats at additive and 1 % and 3 % cross-contamination level, and of non-registered coccidiostats and of one antibiotic at sub-additive levels, in compound feed with High Performance Liquid Chromatograpy – Tandem Mass Spectrometry detection (LC-MS/MS), or by reversed-phase high performance liquid chromatography (HPLC) using an UV detector, as described in points 1 to 8 hereafter.
Liquid chromatographic column (point 4.4.1) HPLC Mobile phase (point 3.21) Flow rate: 1,5 to 2 ml/min. Detection wavelength: 243 nm Injection volume: 40 to 100 μl.
(a) the wavelength of maximum absorption of the sample and of the standard spectra, recorded at the peak apex on the chromatogram, must be the same within a margin determined by the resolving power of the detection system. For diode-array detection, this is typically within ± 2 nm; (b) between 225 and 300 nm, the sample and standard spectra recorded at the peak apex on the chromatogram, must not be different for those parts of the spectrum within the range 10 to 100 % of relative absorbance. This criterion is met when the same maxima are present and at no observed point the deviation between the two spectra exceeds 15 % of the absorbance of the standard analyte; (c) between 225 and 300 nm, the spectra of the upslope, apex and downslope of the peak produced by the sample extract must not be different from each other for those parts of the spectrum within the range 10 to 100 % of relative absorbance. This criterion is met when the same maxima are present and when at all observed points the deviation between the spectra does not exceed 15 % of the absorbance of the spectrum of the apex.
| Sample B (Meal) | Sample C (Pellets) | ||||
|---|---|---|---|---|---|
| On receipt | After two months | On receipt | After two months | ||
| Mean [mg/kg] | ND | ||||
| S | — | ||||
| CV | — | ||||
| Rec. [%] | |||||
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.
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).
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.
(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.
| Poultry | Rabbit | |||
|---|---|---|---|---|
| Meal | Pellet | Meal | Pellet | |
| Mean [mg/kg] | ||||
| s | ||||
| CV | ||||
| S | ||||
| CV | ||||
| Recovery [%] | ||||
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.
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.
or 
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).
or 
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.
(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.
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.
| Sample 1A 100 | Sample 2O 100 | Sample 3 L1 | Sample 4 Z1 | Sample 5 K1 | ||||
|---|---|---|---|---|---|---|---|---|
| L | ||||||||
| n | ||||||||
| Mean (mg/kg) | < LOQ | |||||||
| S | — | |||||||
| CV | — | |||||||
| S | — | |||||||
| CV | — | |||||||
| Nominal content (mg/kg) | — | |||||||
| Reference | First study from | First study from | First study from | First study from | First study from | Second study from | Second study from | Second study from |
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.
| Liquid chromatographic column (point 4.3.1): | 125 mm × 4 mm, reversed-phase C |
| Mobile phase (point 3.9): | Mixture of phosphate buffer solution (point 3.7) and methanol (point 3.5), 5 + 95 (V + V) |
| Flow rate: | |
| Detection wavelengths: | Excitation: 310 nm |
| Emission: 419 nm | |
| Injection volume: |
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.
| L | |||||||
| n | |||||||
| Mean [mg/kg] | |||||||
| s | |||||||
| CV | |||||||
| s | |||||||
| CV | |||||||
| Nominal content [mg/kg] |
| 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: | |
| Detection wavelength: | |
| Injection volume: |
(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.
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 1000 mg/kg,7,5 % relative to the higher value for amprolium contents of more than 1000 mg/kg.
| sample 1 (blank feed) | sample 2 | sample 3 | sample 4 | sample 5 | |
|---|---|---|---|---|---|
| L | |||||
| n | |||||
| mean [mg/kg] | — | ||||
| s | — | ||||
| CVr [%] | — | ||||
| s | — | ||||
| CV | — | ||||
| nominal content [mg/kg] | — |
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.
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.
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.
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.
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.
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.
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 additiveEN 15788 Animal feeding stuffs: Methods of sampling and analysis – Detection and enumeration of Enterococcus (E. faecium ) spp. used as feed additiveEN 15789 Animal feeding stuffs: Methods of sampling and analysis – Detection and enumeration of Saccharomyces cerevisiae used as feed additiveEN 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)
| Congener | TEF value | Congener | TEF value |
|---|---|---|---|
| Dibenzo-p-dioxins ("PCDDs") and Dibenzo-p-furans ("PCDFs") | |||
| 2,3,7,8-TCDD | |||
| 1,2,3,7,8-PeCDD | Non-ortho PCBs | ||
| 1,2,3,4,7,8-HxCDD | PCB 77 | ||
| 1,2,3,6,7,8-HxCDD | PCB 81 | ||
| 1,2,3,7,8,9-HxCDD | PCB 126 | ||
| 1,2,3,4,6,7,8-HpCDD | PCB 169 | ||
| OCDD | Mono-ortho PCBs | ||
| 2,3,7,8-TCDF | PCB 105 | ||
| 1,2,3,7,8-PeCDF | PCB 114 | ||
| 2,3,4,7,8-PeCDF | PCB 118 | ||
| 1,2,3,4,7,8-HxCDF | PCB 123 | ||
| 1,2,3,6,7,8-HxCDF | PCB 156 | ||
| 1,2,3,7,8,9-HxCDF | PCB 157 | ||
| 2,3,4,6,7,8-HxCDF | PCB 167 | ||
| 1,2,3,4,6,7,8-HpCDF | PCB 189 | ||
| 1,2,3,4,7,8,9-HpCDF | |||
| OCDF | |||
"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).
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.
(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.
| Screening with bioanalytical or physico-chemical methods | Confirmatory methods | |
|---|---|---|
| False-compliant rate | < | |
| Trueness | ||
| Repeatability (RSD | < | |
| Intermediate precision (RSD | < | < |
Separation of PCDD/Fs from interfering chlorinated compounds such as non-dioxin-like PCBs and chlorinated diphenyl ethers shall be carried out by suitable chromatographic techniques (preferably with a florisil, alumina and/or carbon column). Gas-chromatographic separation of isomers shall be < 25 % peak to peak between 1,2,3,4,7,8-HxCDF and 1,2,3,6,7,8-HxCDF.
For GC-HRMS: In HRMS, the resolution shall typically be greater than or equal to 10000 for the entire mass range at 10 % valley.Fulfilment of further identification and confirmation 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.
For GC-MS/MS: Monitoring of at least 2 specific precursor ions, each with one specific corresponding transition product ion for all labelled and unlabelled analytes in the scope of analysis. Maximum permitted tolerance of relative ion intensities of ± 15 % for selected transition product ions in comparison to calculated or measured values (average from calibration standards), applying identical MS/MS conditions, in particular collision energy and collision gas pressure, for each transition of an analyte. Resolution for each quadrupole to be set equal to or better than unit mass resolution (unit mass resolution: sufficient resolution to separate two peaks one mass unit apart) in order to minimise possible interferences on the analytes of interest. Fulfilment of the further 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, except the obligation to use GC-HRMS.
When calculating the concentrations from a TCDD calibration curve, values at the higher end of the curve will show a high variation (high coefficient of variation (CV)). The working range is the area where this CV is smaller than 15 %. The lower end of the working range (reporting limit) shall be set at least by a factor of three above the procedure blanks. The upper end of the working range is usually represented by the EC 70 value (70 % of maximal effective concentration), but lower if the CV is higher than 15 % in this range. The working range shall be established during validation. Cut-off values (see point 7.3) shall be well within the working range.Standard solutions and sample extracts shall be tested in triplicate or at least in duplicate. When using duplicates, a standard solution or a control extract tested in four to six wells divided over the plate shall produce a response or concentration (only possible in the working range) based on a CV < 15 %.
Levels in samples shall be estimated by comparison of the test response with a calibration curve of TCDD (or PCB 126 or a PCDD/PCDF/dioxin-like PCB standard mixture) to calculate the BEQ level in the extract and subsequently in the sample. Calibration curves shall contain 8 to 12 concentrations (at least in duplicates), with enough concentrations in the lower part of the curve (working range). Special attention shall be paid to the quality of the curve-fit in the working range. As such, the R2 value is of little or no value in estimating the goodness of fit in non-linear regression. A better fit shall be achieved by minimising the difference between calculated and observed levels in the working range of the curve, for example by minimising the sum of squared residuals. The estimated level in the sample extract shall be subsequently corrected for the BEQ level calculated for a matrix or solvent blank sample (to account for impurities from solvents and chemicals used), and the apparent recovery (calculated from the BEQ level of suitable reference samples with representative congener patterns around the maximum level or action threshold). To perform a recovery correction, the apparent recovery shall be within the required range (see point 7.1.4). Reference samples used for recovery correction shall comply with the requirements laid down in point 7.2.
(1) from the lower band of the 95 % prediction interval at the decision limit of the confirmatory method;(2) from multiple analysis of samples (n ≥ 6) contaminated at the decision limit of the confirmatory method as the lower end-point of the data distribution (represented in Figure 1 by a bell-shaped curve) at the corresponding mean BEQ value.
(a) two specific ions for HRMS; (b) three specific ions for LRMS; (c) two specific precursor ions, each with one specific corresponding transition product ion for for MS-MS.
(a) results shall be corrected for recoveries of internal standards; (b) recoveries of isotope-labelled internal standards shall be between 60 and 120 %; (c) lower or higher recoveries for individual congeners with a contribution to the sum of non-dioxin-like PCBs below 10 % are acceptable.
(a) recovery of internal standard(s) shall be controlled for every sample; (b) recoveries of internal standard(s) shall be between 60 and 120 %; (c) results shall be corrected for recoveries of internal standards.
| Isotope dilution mass spectrometry | Other techniques | |
|---|---|---|
| Trueness | ||
| Intermediate precision (RSD %) | ≤ | ≤ |
| Difference between upper and lower-bound calculation | ≤ | ≤ |
EN 17194: Animal feeding stuffs: Methods of sampling and analysis – Determination of Deoxynivalenol, Aflatoxin B1, Fumonisin B1 & B2, T-2 & HT-2 toxins, Zearalenone and Ochratoxin A in feed materials and compound feed by LC-MS/MS EN 17270: Animal feeding stuffs: Methods of sampling and analysis – Determination of theobromine in feed materials and compound feed, including cocoa derived ingredients, by liquid chromatography EN 17504 Animal feeding stuffs: Methods of sampling and analysis – Determination of gossypol in cotton seed and feeding stuff by LC-MS/MS EN 17362 Animal feeding stuffs: Methods of sampling and analysis – Determination of pentachlorophenol (PCP) in feed materials and compound feed by LCMS/MS EN 16279: Animal feeding stuffs – Determination of fluoride content after hydrochloric acid treatment by ion-sensitive electrode method (ISE) 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) EN 15550 Animal feeding stuffs – Methods of sampling and analysis – Determination of cadmium and lead by graphite furnace atomic absorption spectrometry (GF-AAS) after pressure digestion EN 16206 Animal feeding stuffs – Determination of arsenic by hydride generation atomic absorption spectrometry (HGAAS) after microwave pressure digestion (digestion with 65 % nitric acid and 30 % hydrogen peroxide EN 16277 Animal feeding stuffs – Determination of mercury by cold-vapour atomic absorption spectrometry (CVAAS) after microwave pressure digestion (extraction with 65 % nitric acid and 30 % hydrogen peroxide) EN 16278 Animal feeding stuffs – Determination of inorganic arsenic by hydride generation atomic absorption spectrometry (HG-AAS) after microwave extraction and separation by solid phase extraction (SPE) EN 17374 Animal feeding stuffs: Methods of sampling and analysis – Determination of inorganic arsenic in animal feed by anion-exchange HPLC-ICP-MS
(a) for the detection of constituents of animal origin other than terrestrial invertebrates, a single Tetrachloroethylene (TCE) sedimentation step as detailed in point 2.1.3.4.3; (b) for the detection of constituents of terrestrial invertebrates, a double Petroleum ether/Tetrachloroethylene (PE/TCE) sedimentation step as detailed in point 2.1.3.4.4.
Tetrachloroethylene (specific gravity 1,62). Petroleum ether (PE) boiling point 40– 60 °C (specific gravity 0,65).
Alizarin Red solution (dilute 2,5 ml 1M hydrochloric acid in 100 ml water and add 200 mg Alizarin Red to this solution).
Lye (NaOH 2,5 % w/v or KOH 2,5 % w/v). Glycerol (undiluted, viscosity: 1490 cP) or a mounting medium with equivalent properties for non-permanent slide preparation.Norland ® Optical Adhesive 65 (viscosity: 1200 cP) or a resin with equivalent properties for permanent slide preparation.
Lugol solution (dissolve 2 g potassium iodide in 100 ml water and add 1 g iodine while frequently shaking). Cystine reagent (2 g lead acetate, 10 g NaOH/100 ml water). Fehling’s reagent (prepared before use from equal parts (1/1) of two-stock solutions A and B: solution A (dissolve 6,9 g copper (II) sulphate pentahydrate in 100 ml water); solution B (dissolve 34,6 g potassium sodium tartrate tetrahydrate and 12 g NaOH in 100 ml water). Tetramethylbenzidine/Hydrogen peroxide (dissolve 1 g 3,3',5,5’ tetramethylbenzidine (TMB) in 100 ml glacial acetic acid and 150 ml water. Before use, mix 4 parts of this TMB solution with 1 part 3 % hydrogen peroxide).
Ethanol ≥ 96 % (technical grade). Acetone (technical grade).
Commercial sodium hypochlorite solution (9 – 14 % active chlorine).
Analytical balance with an accuracy of 0,001 g. Grinding equipment: knife or rotor mill. If a rotor mill is used, mill sieves ≤ 0,5 mm shall be prohibited. Sieves with square meshes of 0,25 mm and 1 mm width. With the exception of sample pre-sieving, the diameter of the sieves shall not exceed 10 cm to avoid loss of materials. Calibration of sieves is not required. Conical glass separation funnel with a content of 250 ml with Teflon or ground glass stopcock at the base of the cone. Stopcock opening diameter shall be ≥ 4mm. Alternatively, for single TCE sedimentation only, a conical bottomed settling beaker may be used provided the laboratory has demonstrated that detection levels are equivalent to that obtained using the conical glass separation funnel. 
Separation funnel Stereomicroscope covering at least a 6,5x to 40x final magnification range. Compound microscope covering at least a 100x to 400x final magnification range with transmitted light bright field. Polarised light, differential interferential contrast can additionally be used. Standard laboratory glassware. Equipment for slide preparation: classical microscope slides, hollow slides, coverslips (20x20 mm), tweezers, fine spatula. Laboratory oven. Centrifuge. Filter paper: qualitative cellulose filter (pore size 4-11 μm).
If the fat is solid, it shall be warmed in an oven until it is liquid. By using a pipette, 40 ml of fat shall be transferred from the bottom of the sample to a centrifugation tube. The sample shall be centrifuged during 10 min at 4000 r.p.m.If the fat is solid after centrifugation, it shall be warmed in an oven until it is liquid. The centrifugation shall be repeated during 5 min at 4000 r.p.m.By using a small spoon or a spatula, one half of the decanted impurities shall be transferred to microscopic slides for examination. Glycerol is recommended as mounting medium. The remaining impurities shall be used for preparing the sediment as described in point 2.1.3.4.3, first indent.
Extraction and preparation of the sediment: A portion of 10 g (accurate to 0,01 g) of the ground sub-sample shall be transferred into the separation funnel or conical bottomed settling beaker and 50 ml of TCE shall be added. The portion transferred into the funnel shall be limited to 3 g in case of fishmeal or other pure animal products, mineral ingredients or premixtures which generate more than 10 % of sediment. The mixture shall be vigorously shaken for at least 30 s and 50 ml more of TCE shall be added cautiously while washing down the inside surface of the funnel to remove any adhering particles. The resulting mixture shall be left to stand for at least 5 min before the sediment is separated off by opening the stopcock. If a conical bottomed settling beaker is used then the mixture shall be vigorously stirred for at least 15 s and any particles adhering to the side of the beaker shall be carefully washed down the inside surface with at least 10 ml of clean TCE. The mixture shall be left to stand for 3 min and then stirred again for 15 s and any particles adhering to the side of the beaker shall be carefully washed down the inside surface with at least 10 ml of clean TCE. The resulting mixture shall be left to stand for at least 5 min and then the liquid fraction is removed and discarded by careful decanting, taking care not to lose any of the sediment. The sediment shall be collected on a filter paper placed into a funnel to allow the separation of the remaining TCE while avoiding fat deposition into the sediment. The sediment shall be dried. It is recommended to subsequently weigh the sediment (accurate to 0,001 g) to control the sedimentation step. Lastly, the sediment shall be sieved at 0,25 mm and the two resulting fractions shall be examined, unless sieving is not deemed necessary. Extraction and preparation of the flotate: After recovery of the sediment with the method described above, two phases shall remain in the separation funnel: a liquid one consisting of TCE and a solid one made of floating material. This solid phase is the flotate and shall be recovered by pouring off completely TCE from the funnel by opening the stopcock. By inverting the separation funnel, the flotate shall be transferred into a large petri dish and air dried in a fume hood. It shall be sieved at 0,25 mm and the two resulting fractions shall be examined. Use of staining reagents: In order to facilitate the correct identification of the constituents of animal origin, the operator may use staining reagents during the sample preparation in accordance with guidelines issued by the EURL-AP and published on its website. In case Alizarin Red solution is used to colour the sediment, the following protocol shall apply: The dried sediment shall be transferred into a glass test tube and rinsed twice with approximately 5 ml of ethanol (each time a vortex of 30 s shall be used, the solvent shall be let settle about 1 min 30 s and poured off). The sediment shall be bleached by adding at least 1 ml sodium hypochlorite solution. The reaction shall be allowed to continue for 10 min. The tube shall be filled with water, the sediment shall be let settle 2-3 min, and the water and the suspended particles shall be poured off gently. The sediment shall be rinsed twice more with about 10 ml of water (a vortex shall be used for 30 s, let settle, and pour off the water each time). 2 to 10 drops of the Alizarin Red solution shall be added and the mixture shall be vortexed. The reaction shall be let occur for 30 s and the coloured sediment shall be rinsed twice with approximately 5 ml ethanol followed by one rinse with acetone (each time a vortex of 30 s shall be used, the solvent shall be let settle about 1 min and poured off). The coloured sediment shall be dried.
A portion of 10 g (accurate to 0,01 g) of the ground sub-sample shall be transferred into the separation funnel and submitted first to a single TCE sedimentation as described in point 2.1.3.4.3 including the recovery of the sediment on a filter paper placed on a funnel. This sediment may be used as the one obtained from point 2.1.3.4.3. The small volume of TCE drained together with the sediment shall be transferred into a graduated cylinder. By opening the stopcock of the separation funnel the graduated cylinder has to be filled further until obtaining 30 ml of TCE. Once this volume is achieved, the stopcock shall be closed. This collected volume of TCE shall be substituted by adding a volume of 30 ml of petroleum ether boiling point 40– 60 °C into the separation funnel. The content of the separation funnel shall be mixed thoroughly to obtain a 30 % PE/70 % TCE mixture (with a density of approximately 1,26 g.cm -3 ). Allow the material to settle down for 10 min. Two new fractions will segregate: a second sediment and a final flotate (< 1,26 g.cm-3 ). The second sediment is to recover in a petri dish (or a filter paper placed on a funnel) by opening the stopcock until only a few solvent mixture and the final flotate remain in the separation funnel. The remaining liquid and the final flotate shall be collected separately on a filter paper placed on a funnel. The wall of the separation funnel shall be rinsed with a flush of PE to collect all material from the final flotate. The final flotate shall be allowed to dry. The final flotate shall be sieved at 0,25 mm and the two resulting fractions shall be examined for the detection of terrestrial invertebrate constituents, unless sieving is not deemed necessary.
"As far as was discernible using a light microscope, no particle derived from terrestrial vertebrates was detected in the submitted sample." "As far as was discernible using a light microscope, no particle derived from fish was detected in the submitted sample." "As far as was discernible using a light microscope, no particle derived from terrestrial invertebrates was detected in the submitted sample."
"As far as was discernible using a light microscope, no more than 5 particles derived from terrestrial vertebrates were detected in the submitted sample. The particles were identified as … [bone, cartilage, muscle, hair, horn, other (please specify as appropriate)]. This low level presence is below the decision limit established for this microscopic method." "As far as was discernible using a light microscope, no more than 5 particles derived from fish were detected in the submitted sample. The particles were identified as … [fishbone, fish scale, cartilage, muscle, otolith, gill, other (please specify as appropriate)]. This low level presence is below the decision limit established for this microscopic method."
"As far as was discernible using a light microscope, no more than 10 particles derived from terrestrial vertebrates were detected over the two determinations in the submitted sample. The particles were identified as … [bone, cartilage, muscle, hair, horn, other (please specify as appropriate)]. This low level presence is below the decision limit established for this microscopic method." "As far as was discernible using a light microscope, no more than 10 particles derived from fish were detected over the two determinations in the submitted sample. The particles were identified as … [fishbone, fish scale, cartilage, muscle, otolith, gill, other (please specify as appropriate)]. This low level presence is below the decision limit established for this microscopic method." "As far as was discernible using a light microscope, no more than 10 particles derived from terrestrial invertebrates were detected over the two determinations in the submitted sample. The particles were identified as … [cuticle fragments, mouthparts, muscles, tracheal structures, other (please specify as appropriate)]. This low level presence is below the decision limit established for this microscopic method."
In case of sample pre-sieving, the laboratory report shall mention in which fraction (sieved fraction, pelleted fraction or kernels) the animal particles have been detected insofar as the detection of animal particles only in the sieved fraction may be the sign of an environmental contamination. When only animal particles which cannot be categorised as either terrestrial vertebrates or fish are detected (e.g. muscle fibres), the report shall mention that only such animal particles were detected and that it cannot be excluded that they originate from terrestrial vertebrates.
"As far as was discernible using a light microscope, more than 5 particles derived from terrestrial vertebrates were detected in the submitted sample. The particles were identified as … [bone, cartilage, muscle, hair, horn, other (please specify as appropriate)]." "As far as was discernible using a light microscope, more than 5 particles derived from fish were detected in the submitted sample. The particles were identified as … [fishbone, fish scale, cartilage, muscle, otolith, gill, other (please specify as appropriate)]." "As far as was discernible using a light microscope, more than 5 particles derived from terrestrial invertebrates were detected in the submitted sample. The particles were identified as … [cuticle fragments, mouthparts, muscles, tracheal structures, other (please specify as appropriate)]."
"As far as was discernible using a light microscope, more than 10 particles derived from terrestrial vertebrates were detected over the two determinations in the submitted sample. The particles were identified as … [bone, cartilage, muscle, hair, horn, other (please specify as appropriate)]." "As far as was discernible using a light microscope, more than 10 particles derived from fish were detected over the two determinations in the submitted sample. The particles were identified as … [fishbone, fish scale, cartilage, muscle, otolith, gill, other (please specify as appropriate)]." "As far as was discernible using a light microscope, more than 10 particles derived from terrestrial invertebrates were detected over the two determinations in the submitted sample. The particles were identified as … [cuticle fragments, mouthparts, muscles, tracheal structures, other (please specify as appropriate)]."
In case of sample pre-sieving, the laboratory report shall mention in which fraction (sieved fraction, pelleted fraction or kernels) the animal particles have been detected insofar as the detection of animal particles only in the sieved fraction may be the sign of an environmental contamination. When only animal particles which cannot be categorised as either terrestrial vertebrates or fish are detected (e.g. muscle fibres), the report shall mention that only such animal particles were detected and that it cannot be excluded that they originate from terrestrial vertebrates.
an extraction blank control, a positive DNA extraction control.
a positive DNA target control shall be used for each plate or series of PCR assays, an amplification reagent control (also called no template control) shall be used for each plate or series of PCR assays.
for determining the crude fat content: procedure B of the method for the determination of crude oils and fats, laid down in Part G of Annex III, for determining the starch content: the polarimetric method, laid down in Part K of Annex III
liquid chromatographic column (4.4.1), HPLC mobile phase: methanol-water mixture (3.3), flow rate: 1 to 1,5 ml/minute,detection wavelength: 265 nm, Injection volume: 20 to 50 μl.
(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 220 and 350 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 220 and 350 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 no observed points the deviation between the spectra does not exceed 15 % of the absorbance of the spectrum of the apex.
| Blank | Meal 1 | Pellet 1 | Meal 2 | Pellet2 | |
|---|---|---|---|---|---|
| Mean [mg/kg] | ND | ||||
| s | — | ||||
| CV | — | ||||
| s | — | ||||
| CV | — | ||||
| Recovery [%] | — |
| Analytical column (4.3.1) | |
| Mobile Phase (3.4): | water (3.3)-methanol (3.2) mixture, 900 + 100 (V + V) |
| Flow rate: | |
| Detection wavelength: | 380 nm |
| Injection volume: | 20 μl –100 μl |
(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 220 and 400 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 220 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 %-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.
| Sample 1 | Sample 2 | Sample 3 | Sample 4 | |
|---|---|---|---|---|
| L | 13 | 10 | 11 | 11 |
| n | 40 | 40 | 44 | 44 |
| mean [mg/kg] | — | |||
| S | — | |||
| S | — | |||
| CV | — | |||
| CV | — | |||
| Nominal content | ||||
| [mg/kg] | — | 15 | 50 | 100 |
| recovery % | — |
| Liquid chromatographic | |
| column (4.1.1): | 125 mm × 4 mm, cation exchange Nucleosil 10 SA, 5 or 10 μm packing, or equivalent |
| Mobile phase (3.6): | Mixture of acetonitrile (3.2), sodium dihydrogen phosphate solution (3.4) and sodium perchlorate solution (3.5), 450+450+100 (v+v+v). |
| Flow rate: | |
| Detection wavelength: | 264 nm |
| Injection volume: | 100 μl |
(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.
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 1000 mg/kg,7,5 % relative to the higher value for amprolium contents of more than1000 mg/kg.
| 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] | — | 188 | |||
| s | — | 178 | 550 | ||
| CVr [%] | — | ||||
| s | — | 266 | 760 | ||
| CV | — | ||||
| nominal content [mg/kg] | — | 50 | 200 |
| Liquid chromatographic | |
| column (4.4.1): | 300 mm × 4 mm, C |
| Mobile phase (3.10): | Mixture of acetate buffer solution (3.9) and acetonitrile (3.2), 825 + 175 (v+v) |
| Flow rate: | |
| Detection wavelength: | 365 nm |
| Injection volume: | 20 μl |
(a) the wavelength of maximum absorption of the sample and of the standard spectra, recorded at the peak apex on the chromatogram, must be the same within a margin determined by the resolving power of the detection system. For diode-array detection, this is typically within + 2 nm; (b) between 225 and 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 225 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.
| Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | |
|---|---|---|---|---|---|---|
| L | 8 | 8 | 8 | 8 | 8 | 8 |
| n | 15 | 14 | 15 | 15 | 15 | 15 |
| Mean (mg/kg) | ||||||
| Sr (mg/kg) | ||||||
| CVr (%) | ||||||
| SR (mg/kg) | ||||||
| CVR (%) | ||||||
| Nominal content (mg/kg) |
| Premixtures | Preparations | ||||||
|---|---|---|---|---|---|---|---|
| A | B | C | D | A | B | C | |
| L | 7 | 7 | 7 | 7 | 8 | 8 | 8 |
| n | 14 | 14 | 14 | 14 | 16 | 16 | 16 |
| Mean (g/kg) | 104 | ||||||
| Sr (g/kg) | |||||||
| CVr (%) | |||||||
| SR (g/kg) | |||||||
| CVR (%) | |||||||
| Nominal content (g/kg) | 100 | 100 | 100 | ||||
| Directive 71/250/EEC | This Regulation |
|---|---|
| Article 1 first subparagraph | Article 3 |
| Article 1 second subparagraph | Article 2 |
| Article 2 | — |
| Article 3 | — |
| Annex, part 1 | Annex II |
| Annex, part 2 | — |
| Annex, part 3 | — |
| Annex, part 4 | Annex III, part O |
| Annex, part 5 | Annex III, part M |
| Annex, part 6 | Annex III, part N |
| Annex, part 7 | Annex III, part Q |
| Annex, part 9 | Annex III, part K |
| Annex, part 10 | — |
| Annex, part 11 | — |
| Annex, part 12 | Annex III, part J |
| Annex, part 14 | Annex III, part D |
| Annex, part 16 | — |
| Directive 71/393/EEC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 2 | — |
| Article 3 | — |
| Annex, part I | Annex III, part A |
| Annex, part II | Annex III, part E |
| Annex, part III | Annex III, part P |
| Annex, part IV | Annex III, part H |
| Directive 72/199/EEC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 2 | — |
| Article 3 | — |
| Article 4 | — |
| Annex I, part 1 | Annex III, part L |
| Annex I, part 2 | Annex III, part C |
| Annex I, part 3 | — |
| Annex I, part 4 | — |
| Annex I, part 5 | Annex V, part A |
| Annex II | — |
| Directive 73/46/EEC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 3 | — |
| Article 4 | — |
| Annex I, part 1 | Annex III, part B |
| Annex I, part 2 | — |
| Annex I, part 3 | Annex III, part I |
| Directive 76/371/EEC | This Regulation |
|---|---|
| Article 1 | Article 1 |
| Article 2 | — |
| Article 3 | — |
| Annex | Annex I |
| Directive 76/372/EEC | This Regulation |
|---|---|
| Article 1 | — |
| Article 2 | — |
| Article 3 | — |
| Annex | — |
| Directive 78/633/EEC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 2 | — |
| Article 3 | — |
| Annex, part 1 | — |
| Annex, part 2 | — |
| Annex, part 3 | Annex IV, part C |
| Directive 81/715/EEC | This Regulation |
|---|---|
| Article 1 | — |
| Article 2 | — |
| Article 3 | — |
| Annex | — |
| Directive 84/425/EEC | This Regulation |
|---|---|
| Article 1 | — |
| Article 2 | — |
| Article 3 | — |
| Annex | — |
| Directive 86/174/EEC | This Regulation |
|---|---|
| Article 1 | Article 4 |
| Article 2 | — |
| Article 3 | — |
| Annex | Annex VII |
| Directive 93/70/EEC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 2 | — |
| Article 3 | — |
| Annex | Annex IV, part D |
| Directive 93/117/EC | This Regulation |
|---|---|
| Article 1 | Articles 3 and 5 |
| Article 2 | — |
| Article 3 | — |
| Annex, part 1 | Annex IV, part E |
| Annex, part 2 | Annex VIII, part A |
| Directive 98/64/EC | This Regulation |
|---|---|
| Article 1 | Articles 3 and 5 |
| Article 2 | — |
| Article 3 | — |
| Article 4 | — |
| Annex, part A | Annex III, part F |
| Annex, part C | Annex VIII, part B |
| Directive 1999/27/EC | This Regulation |
|---|---|
| Article 1 | Articles 3 and 5 |
| Article 2 | — |
| Article 3 | — |
| Article 4 | — |
| Article 5 | — |
| Article 6 | — |
| Article 7 | — |
| Annex, part A | Annex VIII, part C |
| Annex, part B | Annex IV, part F |
| Annex, part C | Annex VIII, part D |
| Directive 1999/76/EC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 2 | — |
| Article 3 | — |
| Article 4 | — |
| Annex | Annex IV, part G |
| Directive 2000/45/EC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 2 | — |
| Article 3 | — |
| Article 4 | — |
| Annex, part A | Annex IV, part A |
| Annex, part B | Annex IV, part B |
| Annex, part C | Annex III, part G |
| Directive 2002/70/EC | This Regulation |
|---|---|
| Article 1 | Article 1 |
| Article 2 | Articles 2 and 3 |
| Article 3 | — |
| Article 4 | — |
| Article 5 | — |
| Annex I | Annex I and Annex V part B(I) |
| Annex II | Annex II and Annex V part B(II) |
| Directive 2003/126/EC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 2 | — |
| Article 3 | — |
| Article 4 | — |
| Article 5 | — |
| Article 6 | — |
| Annex | Annex VI |
Loading ...