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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)

EU Regulation No 152/2009 · Adopted 27 January 2009 · Extends to United Kingdom

Original text. legislation.gov.uk does not revise this item, so later amendments are not shown here. Changes made to it are listed under each provision when known.

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)

THE COMMISSION OF THE EUROPEAN COMMUNITIES,

Having regard to the Treaty establishing the European Community,

Having regard to Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 on official controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rulesf1, and in particular Article 11(4)(a), (b) and (c) thereof,

Whereas:

(1)

The following acts were adopted for the implementation of Directive 70/373/EEC and remain in force in accordance with Article 61(2) of Regulation (EC) No 882/2004:

  • First Commission Directive 71/250/EEC of 15 June 1971 establishing Community methods of analysis for the official control of feedingstuffsf2,

  • Second Commission Directive 71/393/EEC of 18 November 1971 establishing Community methods of analysis for the official control of feedingstuffsf3,

  • Third Commission Directive 72/199/EEC of 27 April 1972 establishing Community methods of analysis for the official control of feedingstuffsf4,

  • Fourth Commission Directive 73/46/EEC of 5 December 1972 establishing Community methods of analysis for the official control of feedingstuffsf5,

  • First Commission Directive 76/371/EEC of 1 March 1976 establishing Community methods of sampling for the official control of feedingstuffsf6,

  • Seventh Commission Directive 76/372/EEC of 1 March 1976 establishing Community methods of analysis for the official control of feedingstuffsf7,

  • Eight Commission Directive 78/633/EEC of 15 June 1978 establishing Community methods of analysis for the official control of feedingstuffsf8,

  • Ninth Commission Directive 81/715/EEC of 31 July 1981 establishing Community methods of analysis for the official control of feedingstuffsf9,

  • Tenth Commission Directive 84/425/EEC of 25 July 1984 establishing Community methods of analysis for the official control of feedingstuffsf10,

  • Commission Directive 86/174/EEC of 9 April 1986 fixing the method of calculation for the energy value of compound poultry-feedf11,

  • Eleventh Commission Directive 93/70/EEC of 28 July 1993 establishing Community methods of analysis for official control of feedingstuffsf12,

  • Twelfth Commission Directive 93/117/EC of 17 December 1993 establishing Community methods of analysis for official control of feedingstuffsf13,

  • Commission Directive 98/64/EC of 3 September 1998 establishing Community methods of analysis for the determination of amino-acids, crude oils and fats, and olaquindox in feedingstuffs and amending Directive 71/393/EECf14,

  • Commission Directive 1999/27/EC of 20 April 1999 establishing Community methods of analysis for the determination of amprolium, diclazuril and carbadox in feedingstuffs and amending Directives 71/250/EEC, 73/46/EEC and repealing Directive 74/203/EECf15,

  • Commission Directive 1999/76/EC of 23 July 1999 establishing a Community method of analysis for the determination of lasalocid sodium in feedingstuffsf16,

  • Commission Directive 2000/45/EC of 6 July 2000 establishing Community methods of analysis for the determination of vitamin A, vitamin E and tryptophan in feedingstuffsf17,

  • Commission Directive 2002/70/EC of 26 July 2002 establishing requirements for the determination of levels of dioxins and dioxin-like PCBs in feedingstuffsf18,

  • Commission Directive 2003/126/EC of 23 December 2003 on the analytical method for the determination of constituents of animal origin for the official control of feedingstuffsf19.

(2)

Since Directive 70/373/EEC was replaced by Regulation (EC) No 882/2004 it is appropriate to replace the implementing acts to that Directive by a single Regulation. At the same time the methods should be adapted in the light of developments in scientific and technological knowledge. Methods which are no longer valid for their intended purpose should be deleted. It is foreseen to update the sampling provisions in due time to take into account the recent developments in the way feed are produced, stored, transported and marketed, nevertheless it is appropriate to maintain for the time being the existing provisions on sampling.

(3)

Directives 71/250/EEC, 71/393/EEC, 72/199/EEC, 73/46/EEC, 76/371/EEC, 76/372/EEC, 78/633/EEC, 81/715/EEC, 84/425/EEC, 86/174/EEC, 93/70/EEC, 93/117/EC, 98/64/EC, 1999/27/EC, 1999/76/EC, 2000/45/EC, 2002/70/EC and 2003/126/EC should therefore be repealed.

(4)

The measures provided for in this Regulation are in accordance with the opinion of the Standing Committee on the Food Chain and Animal Health,

HAS ADOPTED THIS REGULATION:

Annotations to the whole text

Footnotes

f1

OJ L 165, 30.4.2004, p. 1, corrected by OJ L 191, 28.5.2004, p. 1.

f2

OJ L 155, 12.7.1971, p. 13.

f3

OJ L 279, 20.12.1971, p. 7.

f4

OJ L 123, 29.5.1972, p. 6.

f5

OJ L 83, 30.3.1973, p. 21.

f6

OJ L 102, 15.4.1976, p. 1.

f7

OJ L 102, 15.4.1976, p. 8.

f8

OJ L 206, 29.7.1978, p. 43.

f9

OJ L 257, 10.9.1981, p. 38.

f10

OJ L 238, 6.9.1984, p. 34.

f11

OJ L 130, 16.5.1986, p. 53.

f12

OJ L 234, 17.9.1993, p. 17.

f13

OJ L 329, 30.12.1993, p. 54.

f14

OJ L 257, 19.9.1998, p. 14.

f15

OJ L 118, 6.5.1999, p. 36.

f16

OJ L 207, 6.8.1999, p. 13.

f17

OJ L 174, 13.7.2000, p. 32.

f18

OJ L 209, 6.8.2002, p. 15.

f19

OJ L 339, 24.12.2003, p. 78.

Contents

  1. 1Article 1
  2. 2Article 2
  3. 3Article 3
  4. 4Article 4
  5. 5Article 5
  6. 6Article 6
  7. 7Article 7
  8. Signature
  9. Annex I: METHODS OF SAMPLING
  10. 1PURPOSE AND SCOPE
  11. 2DEFINITIONS
  12. 3GENERAL PROVISIONS
  13. APPARATUS
  14. 4.1Division 4.1
  15. Apparatus recommended for the sampling of solid feed
  16. Manual sampling
  17. Flat-bottomed shovel with vertical sides
  18. 4.2.1.2Division 4.2.1.2
  19. 4.2.2Mechanical sampling
  20. 4.2.3Divider
  21. 5QUANTITATIVE REQUIREMENTS AS REGARDS NUMBER OF INCREMENTAL SAMPLES
  22. Quantitative requirements as regards incremental samples in relation to the control of substances or products uniformly distributed throughout the feed
  23. 5.1.1Loose solid feed
  24. 5.1.2Loose liquid feed
  25. 5.1.3Packaged feed
  26. 5.1.4Feed blocks and mineral licks
  27. 5.1.5Roughages/forage
  28. 5.2Quantitative requirements as regards incremental samples in relation to the control of constituents or substances likely to be distributed non-uniformly in feed
  29. 5.3Quantitative requirements as regards the incremental samples in the case of very large lots
  30. 6QUANTITATIVE REQUIREMENTS AS REGARDS AGGREGATE SAMPLE
  31. QUANTITATIVE REQUIREMENTS AS REGARDS FINAL SAMPLES
  32. Final samples
  33. METHOD OF SAMPLING FOR VERY LARGE LOTS OR LOTS STORED OR TRANSPORTED IN A WAY WHEREBY SAMPLING THROUGHOUT THE LOT IS NOT FEASIBLE
  34. 8.1General principles
  35. Large lots transported by ship
  36. 8.2.1Dynamic sampling of large lots transported by ship
  37. 8.2.2Sampling of lots transported by ship by static sampling
  38. 8.3Sampling of large lots stored in warehouses
  39. Sampling of storage facilities (silos)
  40. 8.4.1Sampling of silos (easily) accessible from above
  41. Sampling of silos not accessible from above (closed silos)
  42. 8.4.2.1Silos not accessible from above (closed silos) with size > 100 tonnes
  43. 8.4.2.2Silos not accessible from above (closed silos) with size < 100 tonnes
  44. 8.5Sampling of loose feed in large closed containers
  45. INSTRUCTIONS FOR TAKING, PREPARING AND PACKAGING THE SAMPLES
  46. 9.1General
  47. 9.2Incremental samples
  48. 9.2.1Loose feed
  49. 9.2.2Packaged feed
  50. 9.2.3Homogeneous or homogenisable liquid or semi-liquid feed
  51. 9.2.4Non-homogenisable, liquid or semi-liquid feed
  52. 9.2.5Feed blocks and mineral licks
  53. 9.3Preparation of aggregate samples
  54. 9.4Preparation of final samples
  55. 9.5Packaging of samples
  56. 9.6Sending of samples to the laboratory
  57. 10SAMPLING RECORD
  58. Annex II: GENERAL PROVISIONS ON METHODS OF ANALYSIS FOR FEED
  59. PREPARATION OF SAMPLES FOR ANALYSIS
  60. 1Purpose
  61. 2Precautions to be taken
  62. Procedure
  63. 3.1General procedure
  64. 3.1.1Feed which can be ground as such
  65. 3.1.2Feed which can be ground after drying
  66. 3.1.3Liquid or semi-liquid feed
  67. 3.1.4Other feed
  68. 3.2Specific procedure in case of examination by visual inspection or by microscopy or in cases where the whole aggregate sample is homogenised
  69. 4Storage of samples
  70. PROVISIONS RELATING TO REAGENTS AND APPARATUS USED IN METHODS OF ANALYSIS
  71. 1Division 1
  72. 2Division 2
  73. 3Division 3
  74. APPLICATION OF METHODS OF ANALYSIS AND EXPRESSION OF THE RESULTS
  75. 1Extraction procedure
  76. 2Clean-up procedure
  77. 3Number of determinations
  78. 4Reporting of the method of analysis used
  79. 5Reporting of the analytical result
  80. 6Measurement uncertainty and recovery rate in case of analysis of undesirable substances
  81. Annex III: METHODS OF ANALYSIS TO CONTROL THE COMPOSITION OF FEED MATERIALS AND COMPOUND FEED
  82. DETERMINATION OF MOISTURE
  83. 1Purpose and Scope
  84. 2Principle
  85. Apparatus
  86. 3.1Division 3.1
  87. 3.2Division 3.2
  88. 3.3Division 3.3
  89. 3.4Division 3.4
  90. 3.5Division 3.5
  91. 3.6Division 3.6
  92. 4Procedure
  93. Preparation
  94. 4.1.1Feed other than those coming under 4.1.2 and 4.1.3
  95. 4.1.2Cereals and groats
  96. 4.1.3Feed in liquid or paste form, feed predominantly composed of oils and fats
  97. Drying
  98. 4.2.1Feed other than those coming under 4.2.2 and 4.2.3
  99. 4.2.2Cereals, flour, groats and meal
  101. 4.2.3Compound feed containing more than 4 % of sucrose or lactose: feed materials such as locust beans, hydrolysed cereal products, malt seeds, dried beet chips, fish and sugar solubles; compound feed containing more than 25 % of mineral salts including water of crystallisation.
  102. Preliminary drying
  103. 4.3.1Feed other than those coming under 4.3.2
  104. 4.3.2Cereals
  105. 5Calculation of results
  106. 5.1Drying without preliminary drying
  107. 5.2Drying with preliminary drying
  108. 5.3Repeatability
  109. 6Observation
  110. DETERMINATION OF MOISTURE IN ANIMAL AND VEGETABLE FATS AND OILS
  111. 1Purpose and scope
  112. 2Principle
  113. Apparatus
  114. 3.1Division 3.1
  115. 3.2Division 3.2
  116. 3.3Division 3.3
  117. 3.4Division 3.4
  118. 3.5Division 3.5
  119. 4Procedure
  120. Division
  121. 5Calculation of results
  122. Repeatability
  123. DETERMINATION OF THE CONTENT OF CRUDE PROTEIN
  124. 1Purpose and scope
  125. 2Principle
  126. Reagents
  127. 3.1Division 3.1
  128. 3.2Division 3.2
  129. 3.3Division 3.3
  130. 3.4Division 3.4
  131. 3.5Division 3.5
  132. 3.6Division 3.6
  133. 3.7Division 3.7
  134. 3.8Division 3.8
  135. 3.9Division 3.9
  136. 3.10Division 3.10
  137. 3.11Division 3.11
  138. 3.12Division 3.12
  139. 3.13Division 3.13
  140. 3.14Division 3.14
  141. 3.15Division 3.15
  142. 3.16Division 3.16
  143. 3.17Division 3.17
  144. 3.18Boric acid solution (10 g/l to 40 g/l depending on the apparatus used).
  145. Division
  146. Hydrochloric acid standard volumetric solution c(HCl) = 0,1 mol/l.
  147. Division
  148. 4Apparatus
  149. Procedure
  150. 5.1Digestion
  151. 5.2Distillation
  152. 5.2.1Distillation into sulphuric acid
  153. 5.2.2Distillation into boric acid
  154. Division
  155. 5.3Titration
  156. 5.3.1Sulphuric acid
  157. 5.3.2Boric acid
  158. Division
  159. 5.4Blank test
  160. 6Calculation of results
  161. 6.1Calculation for titration according to 5.3.1
  162. Calculation for titration according to 5.3.2
  163. 6.2.1Titration with hydrochloric acid
  164. 6.2.2Titration with sulphuric acid
  165. Verification of the method
  166. 7.1Repeatability
  167. 7.2Accuracy
  168. Observations
  169. 8.1Division 8.1
  170. 8.2Division 8.2
  171. 8.3Division 8.3
  172. 8.4Division 8.4
  173. DETERMINATION OF UREA
  174. 1Purpose and scope
  175. 2Principle
  176. Reagents
  177. 3.1Division 3.1
  178. 3.2Division 3.2
  179. 3.3Division 3.3
  180. 3.4Division 3.4
  181. 3.5Division 3.5
  182. Apparatus
  183. 4.1Division 4.1
  184. 4.2Division 4.2
  185. 4.3Division 4.3
  186. Procedure
  187. 5.1Analysis of sample
  188. 5.2Calibration curve
  189. 6Calculation of results
  190. Observations
  191. 7.1Division 7.1
  192. 7.2Division 7.2
  193. 7.3Division 7.3
  194. DETERMINATION OF VOLATILE NITROGENOUS BASES
  195. BY MICRODIFFUSION
  196. 1Purpose and scope
  197. 2Principle
  198. Reagents
  199. 3.1Division 3.1
  200. 3.2Division 3.2
  201. 3.3Division 3.3
  202. 3.4Division 3.4
  203. 3.5Division 3.5
  204. Apparatus
  205. 4.1Division 4.1
  206. 4.2Division 4.2
  207. 4.3Division 4.3
  208. 5Procedure
  209. 6Calculation of results
  210. Repeatability
  211. 7Observation
  212. CONWAY CELLScale 1/1
  213. BY DISTILLATION
  214. 1Purpose and Scope
  215. 2Principle
  216. Reagents
  217. 3.1Division 3.1
  218. 3.2Division 3.2
  219. 3.3Division 3.3
  220. 3.4Division 3.4
  221. 3.5Division 3.5
  222. 3.6Division 3.6
  223. Apparatus
  224. 4.1Division 4.1
  225. 4.2Division 4.2
  226. 5Procedure
  227. 6Calculation of results
  228. Repeatability
  229. DETERMINATION OF AMINO ACIDS (EXCEPT TRYPTOPHANE)
  230. 1Purpose and scope
  231. Principle
  232. 2.1Free amino acids
  233. 2.2Total amino acids
  234. 3Reagents
  235. 3.1Division 3.1
  236. 3.2Division 3.2
  237. 3.3Division 3.3
  238. 3.4Division 3.4
  239. 3.5Division 3.5
  240. 3.6Division 3.6
  241. 3.7Division 3.7
  242. 3.8Division 3.8
  243. 3.9Division 3.9
  244. 3.10Division 3.10
  245. 3.11Division 3.11
  246. 3.12Division 3.12
  247. 3.13Division 3.13
  248. 3.14Division 3.14
  249. 3.15Division 3.15
  250. 3.16Division 3.16
  251. 3.16.1Division 3.16.1
  252. 3.16.2Division 3.16.2
  253. 3.16.3Division 3.16.3
  254. 3.17Sodium hydroxide solution, c = 7,5 mol/l:
  255. 3.18Sodium hydroxide solution, c = 1 mol/l:
  256. 3.19Formic acid — phenol solution:
  257. 3.20Hydrolysis mixture, c = 6 mol HCl/l containing 1 g phenol/l:
  258. 3.21Division 3.21
  259. 3.225-Sulfosalicylic acid, ß = 6 %:
  260. 3.23Oxidation mixture (Performic acid — phenol):
  261. 3.24Citrate buffer, c = 0,2 mol Na+/l, pH 2,2:
  262. 3.25Division 3.25
  263. 3.26Division 3.26
  264. 3.27Division 3.27
  265. 3.27.1Stock standard solution of amino acids (3.16.1).
  266. 3.27.2Stock standard solution of cysteic acid and methionine sulphone, c = 1,25 μmol/ml.
  267. 3.27.3Stock standard solution of the internal standard e.g. norleucine, c = 20 μmol/ml.
  268. 3.27.4Calibration solution of standard amino acids for use with hydrolysates, c = 5 nmol/50 μl of cysteic acid and methionine sulphone and c = 10 nmol/50 μl of the other amino acids. Dissolve 2,2 g sodium chloride (3.10) in 100 ml beaker with 30 ml citrate buffer (3.24). Add 4,0 ml stock standard solution of amino acids (3.27.1), 4,0 ml stock standard solution of cysteic acid and methionine sulphone (3.27.2) and 0,5 ml stock standard solution of internal standard (3.27.3) if used. Adjust pH to 2,2 with sodium hydroxide (3.18).
  269. 3.27.5Calibration solution of standard amino acids for use with hydrolysates prepared according to paragraph 5.3.3.1 and for use with extracts (5.2). The calibration solution is prepared according to 3.27.4 but omitting sodium chloride.
  270. Apparatus
  271. 4.1Division 4.1
  272. 4.2Division 4.2
  273. 4.3Division 4.3
  274. 4.4Division 4.4
  275. 4.5Division 4.5
  276. 4.6Division 4.6
  277. 4.7Division 4.7
  278. 4.8Division 4.8
  279. 4.9Amino acid analyser or HPLC equipment with ion exchange column, device for ninhydrin, post column derivatisation and photometric detector.
  280. Procedure
  281. 5.1Preparation of the sample
  282. 5.2Determination of free amino acids in feed and premixtures
  283. Determination of total amino acids
  284. 5.3.1Oxidation
  285. Hydrolysis
  286. 5.3.2.1Hydrolysis of oxidised samples
  287. 5.3.2.2Hydrolysis of unoxidised samples
  288. 5.3.2.3Open hydrolysis
  289. 5.3.2.4Closed Hydrolysis
  290. 5.3.3Adjustment of pH
  291. 5.3.3.1For Chromatographic Systems (4.9) requiring a low sodium concentration
  292. 5.3.3.2For all other Amino Acid Analysers (4.9)
  293. 5.3.4Sample solution for chromatography
  294. 5.4Chromatography
  295. 6Calculation of results
  296. 6.1The total dilution volume of extracts (F) for determination of free amino acids (5.2) is calculated as follows:
  297. 7Evaluation of the method
  298. 7.1Repeatability
  299. 7.2Reproducibility
  300. 8Use of reference materials
  301. Observations
  302. 9.1Because of differences between amino acid analysers the final concentrations of the calibration solutions of standard amino acids (see 3.27.4 and 3.27.5) and of the hydrolysate (see 5.3.4) shall be taken as a guideline.
  303. 9.2Division 9.2
  304. 9.3Division 9.3
  305. DETERMINATION OF TRYPTOPHAN
  306. 1Purpose and scope
  307. 2Principle
  308. Reagents
  309. 3.1Division 3.1
  310. 3.2Division 3.2
  311. 3.3Division 3.3
  312. 3.4Division 3.4
  313. 3.5Division 3.5
  314. 3.6Division 3.6
  315. 3.7Division 3.7
  316. 3.8Division 3.8
  317. 3.9Division 3.9
  318. 3.10Sodium hydroxide solution, c = 1 mol/l:
  319. 3.11Hydrochloric acid, c = 6 mol/l:
  320. 3.12Hydrochloric acid, c = 1 mol/l:
  321. 3.13Hydrochloric acid, c = 0,1 mol/l:
  322. 3.14Ortho-phosphoric acid, c = 0,5 mol/l:
  323. 3.15Concentrated solution of tryptophan (3.2), c = 2,5 μmol/ml:
  324. 3.16Concentrated internal standard solution, c = 2,5 μmol/ml:
  325. 3.17Calibration standard solution of tryptophan and internal standard:
  326. 3.18Division 3.18
  327. 3.19Division 3.19
  328. 3.20Division 3.20
  329. 3.21Division 3.21
  330. 3.22Division 3.22
  331. Apparatus
  332. 4.1Division 4.1
  333. 4.2Division 4.2
  334. 4.3Division 4.3
  335. 4.4Division 4.4
  336. 4.5Division 4.5
  337. 4.6Division 4.6
  338. 4.7Division 4.7
  339. 4.8Division 4.8
  340. Procedure
  341. 5.1Preparation of samples
  342. 5.2Determination of free tryptophan (extract)
  343. 5.3Determination of total tryptophan (hydrolysate)
  344. 5.4HPLC determination
  345. 6Calculation of results
  346. 7Repeatability
  347. 8Results of a collaborative study
  348. Observations
  349. 9.1Following special chromatographic conditions may give better separation between tryptophan and α-methyl-tryptophan.
  350. 9.2The chromatography will vary according to the type of HPLC and column packing material used. The chosen system must be capable of giving baseline separation between the tryptophan and the internal standard. Moreover it is important that degradation products are well separated from the tryptophan and the internal standard. Hydrolysates without internal standard shall be run in order to check the base line under the internal standard for impurities. It is important that the run time is sufficiently long for the elution of all the degradation products, otherwise late eluting peaks may interfere with subsequent chromatographic runs.
  351. 9.3Barium hydroxide
  352. DETERMINATION OF CRUDE OILS AND FATS
  353. 1Purpose and scope
  354. 1.1Procedure A — Directly extractable crude oils and fats
  355. 1.2Procedure B — Total crude oils and fats
  356. 1.3Interpretation of results
  357. Principle
  358. 2.1Procedure A
  359. 2.2Procedure B
  360. Reagents
  361. 3.1Division 3.1
  362. 3.2Division 3.2
  363. 3.3Division 3.3
  364. 3.4Division 3.4
  365. Apparatus
  366. 4.1Division 4.1
  367. 4.2Division 4.2
  368. 4.3Division 4.3
  369. Procedure
  370. 5.1Procedure A (see point 8.1)
  371. 5.2Procedure B
  372. 6Expression of result
  373. 7Repeatability
  374. Observations
  375. 8.1For products with a high content of oils and fats, which are difficult to crush or unsuitable for drawing a homogeneous reduced test sample, proceed as follows.
  376. 8.2Division 8.2
  377. 8.3Division 8.3
  378. 8.4Division 8.4
  379. 8.5Division 8.5
  380. 8.6Division 8.6
  381. DETERMINATION OF CRUDE FIBRE
  382. 1Purpose and scope
  383. 2Principle
  384. Reagents
  385. 3.1Division 3.1
  386. 3.2Division 3.2
  387. 3.3Division 3.3
  388. 3.4Division 3.4
  389. 3.5Division 3.5
  390. 3.6Division 3.6
  391. 3.7Division 3.7
  392. Apparatus
  393. 4.1Division 4.1
  394. 4.2Division 4.2
  395. 4.3Division 4.3
  396. 4.4Division 4.4
  397. 4.5Division 4.5
  398. 4.6Division 4.6
  399. 4.7Division 4.7
  400. 5Procedure
  401. 6Calculation of results
  402. 7Repeatability
  403. Observations
  404. 8.1Division 8.1
  405. 8.2Division 8.2
  406. 8.3Division 8.3
  407. 8.4Division 8.4
  408. 8.5Division 8.5
  409. 8.6Division 8.6
  410. DETERMINATION OF SUGAR
  411. 1Purpose and scope
  412. 2Principle
  413. Reagents
  414. 3.1Division 3.1
  415. 3.2Division 3.2
  416. 3.3Division 3.3
  417. 3.4Division 3.4
  418. 3.5Division 3.5
  419. 3.6Division 3.6
  420. 3.7Division 3.7
  421. 3.8Luff-Schoorl reagent:
  422. 3.8.1Division 3.8.1
  423. 3.8.2Division 3.8.2
  424. 3.8.3Division 3.8.3
  425. 3.9Division 3.9
  426. 3.10Division 3.10
  427. 3.11Division 3.11
  428. 3.12Division 3.12
  429. 3.13Division 3.13
  430. 3.14Division 3.14
  431. 4Apparatus
  432. Procedure
  433. 5.1Extraction of sample
  434. 5.2Determination of reducing sugars
  435. 5.3Determination of total sugars after inversion
  436. 5.4Titration by the Luff-Schoorl method
  437. 6Calculation of results
  438. Special procedures
  439. 7.1In the case of feed which are rich in molasses and other feed which are not particularly homogeneous, weigh out 20 g and place with 500 ml of water in a 1 litre volumetric flask. Mix for one hour in the tumbler. Clarify using Carrez 1 (3.2) and II (3.3) reagents as described under 5.1, this time however using four times the quantities of each reagent. Bring up to volume with 80 % ethanol (v/v).
  440. 7.2Division 7.2
  441. 8Observations
  442. 8.1Division 8.1
  443. 8.2Division 8.2
  444. 8.3Division 8.3
  445. 8.3.1Division 8.3.1
  446. 8.3.2For an accurate calculation of reducing sugars, excluding lactose, the same sample must be used for the two final determinations. One of the analyses is carried out on part of the solution obtained under 5.1, the other on part of the solution obtained during the determination of lactose by the method laid down for that purpose (after fermenting the other types of sugar and clarifying).
  447. Example:
  448. DETERMINATION OF LACTOSE
  449. 1Purpose and scope
  450. 2Principle
  451. Reagents
  452. 3.1Division 3.1
  453. 3.2Division 3.2
  454. 3.3Division 3.3
  455. 3.4Luff-Schoorl reagent:
  456. 3.4.1Division 3.4.1
  457. 3.4.2Division 3.4.2
  458. 3.4.3Division 3.4.3
  459. 3.5Division 3.5
  460. 3.6Division 3.6
  461. 3.7Division 3.7
  462. 3.8Division 3.8
  463. 3.9Division 3.9
  464. 4Apparatus
  465. 5Procedure
  466. 6Calculation of results
  467. 7Observation
  468. DETERMINATION OF STARCH
  469. POLARIMETRIC METHOD
  470. 1Purpose and scope
  471. 2Principle
  472. Reagents
  473. 3.1Division 3.1
  474. 3.2Hydrochloric acid. solution 1,13 % (w/v)
  475. 3.3Division 3.3
  476. 3.4Division 3.4
  477. 3.5Division 3.5
  478. Apparatus
  479. 4.1Division 4.1
  480. 4.2Division 4.2
  481. Procedure
  482. 5.1Preparation of the sample
  483. 5.2Determination of the total optical rotation (P or S) (see observation 7.1)
  484. 5.3Determination of the optical rotation (P' or S') of substances soluble in 40 % ethanol
  485. 6Calculation of results
  486. 6.1Measurement by polarimeter
  487. 6.2Measurement by saccharimeter
  488. 6.3Repeatability
  489. Observations
  490. 7.1Division 7.1
  491. 7.2Division 7.2
  492. 7.3The following feed materials, where they are present in significant amounts in feed, are known to give rise to interferences when determining the starch content by the polarimetric method and thereby incorrect results could be yielded:
  493. DETERMINATION OF CRUDE ASH
  494. 1Purpose and Scope
  495. 2Principle
  496. 3Reagents
  497. Apparatus
  498. 4.1Division 4.1
  499. 4.2Division 4.2
  500. 4.3Division 4.3
  501. 5Procedure
  502. 5.1Division 5.1
  503. 5.2Division 5.2
  504. 6Calculation of results
  505. Observations
  506. 7.1Division 7.1
  507. 7.2Division 7.2
  508. 7.3Division 7.3
  509. DETERMINATION OF ASH WHICH IS INSOLUBLE IN HYDROCHLORIC ACID
  510. 1Purpose and Scope
  511. 1.1Division 1.1
  512. 1.2Division 1.2
  513. Principle
  514. 2.1Division 2.1
  515. 2.2Division 2.2
  516. Reagents
  517. 3.1Division 3.1
  518. 3.2Division 3.2
  519. 3.3Division 3.3
  520. Apparatus
  521. 4.1Division 4.1
  522. 4.2Division 4.2
  523. 4.3Division 4.3
  524. Procedure
  525. 5.1Method A
  526. 5.2Method B
  527. 6Calculation of results
  528. 7Observation
  529. DETERMINATION OF CARBONATES
  530. 1Purpose and Scope
  531. 2Principle
  532. Reagents
  533. 3.1Division 3.1
  534. 3.2Division 3.2
  535. 3.3Division 3.3
  536. 4Apparatus
  537. 5Procedure
  538. 6Calculation of results
  539. Observations
  540. 7.1Division 7.1
  541. 7.2If the apparatus used has a different volume from that of the Scheibler-Dietrich apparatus, the portions taken from the sample and from the control substance and the calculation of the results must be adapted accordingly.
  542. DETERMINATION OF TOTAL PHOSPHORUS
  543. PHOTOMETRIC METHOD
  544. 1Purpose and Scope
  545. 2Principle
  546. Reagents
  547. 3.1Division 3.1
  548. 3.2Division 3.2
  549. 3.3Division 3.3
  550. 3.4Division 3.4
  551. 3.5Division 3.5
  552. 3.6Division 3.6
  553. 3.6.1Division 3.6.1
  554. 3.6.2Division 3.6.2
  555. 3.7Division 3.7
  556. Apparatus
  557. 4.1Division 4.1
  558. 4.2Division 4.2
  559. 4.3Division 4.3
  560. 4.4Division 4.4
  561. 4.5Division 4.5
  562. 4.6Division 4.6
  563. Procedure
  564. 5.1Preparation of the solution
  565. 5.1.1Usual procedure
  566. 5.1.2Samples containing organic substances and free from calcium and magnesium dihydrogen phosphates
  567. 5.2Development of coloration and measurement of optical density
  568. 5.3Calibration curve
  569. 6Calculation of results
  570. Repeatability
  571. DETERMINATION OF CHLORINE FROM CHLORIDES
  572. 1Purpose and Scope
  573. 2Principle
  574. Reagents
  575. 3.1Division 3.1
  576. 3.2Division 3.2
  577. 3.3Division 3.3
  578. 3.4Division 3.4
  579. 3.5Division 3.5
  580. 3.6Division 3.6
  581. 3.7Division 3.7
  582. 3.8Division 3.8
  583. 3.9Division 3.9
  584. 4Apparatus
  585. Procedure
  586. 5.1Preparation of the solution
  587. 5.1.1Samples free from organic matter
  588. 5.1.2Samples containing organic matter, excluding the products listed under 5.1.3.
  589. 5.1.3Cooked feed, flax cakes and flour, products rich in flax flour and other products rich in mucilage or in colloidal substances (for example, dextrinated starch)
  590. 5.2Titration
  591. 6Calculation of results
  592. Observations
  593. 7.1Division 7.1
  594. 7.2Division 7.2
  595. 7.3Division 7.3
  596. Annex IV: METHODS OF ANALYSIS TO CONTROL THE LEVEL OF AUTHORISED ADDITIVES IN FEED
  597. DETERMINATION OF VITAMIN A
  598. 1Purpose and Scope
  599. 2Principle
  600. Reagents
  601. 3.1Division 3.1
  602. 3.2Division 3.2
  603. 3.3Division 3.3
  604. 3.4Division 3.4
  605. 3.5Division 3.5
  606. 3.6Division 3.6
  607. 3.6.1Division 3.6.1
  608. 3.7Division 3.7
  609. 3.8Division 3.8
  610. 3.9Division 3.9
  611. 3.10Division 3.10
  612. 3.11Division 3.11
  613. 3.11.1Division 3.11.1
  614. 3.12Division 3.12
  615. 3.12.1Division 3.12.1
  616. 3.13Division 3.13
  617. Apparatus
  618. 4.1Division 4.1
  619. 4.2Division 4.2
  620. 4.2.1Division 4.2.1
  621. 4.2.2Division 4.2.2
  622. 4.2.3Division 4.2.3
  623. 4.2.4Division 4.2.4
  624. 4.3Division 4.3
  625. 4.4Division 4.4
  626. 4.5Division 4.5
  627. 4.5.1Division 4.5.1
  628. 4.5.2Division 4.5.2
  629. 4.6Division 4.6
  630. 4.7Division 4.7
  631. 4.8Division 4.8
  632. 4.8.1Division 4.8.1
  633. 4.8.2Division 4.8.2
  634. Procedure
  635. Division
  636. 5.1Preparation of the sample
  637. 5.2Saponification
  638. 5.3Extraction
  639. 5.3.1Extraction using a separating funnel (4.2.3)
  640. 5.3.2Extraction using an extraction apparatus (4.8)
  641. 5.4Preparation of the sample solution for HPLC
  642. 5.5Determination by HPLC
  643. HPLC conditions
  644. Calibration
  645. 5.6.1Preparation of the working standard solutions
  646. 5.6.2Preparation of the calibration solutions and calibration graph
  647. UV standardisation of the standard solutions
  648. 5.6.3.1Vitamin A acetate stock solution
  649. 5.6.3.2Vitamin A palmitate stock solution
  650. 5.6.3.3Vitamin A working standard solution
  651. 6Calculation of the results
  652. Observations
  653. 7.1Division 7.1
  654. 7.2Division 7.2
  655. 7.3Division 7.3
  656. 7.4Division 7.4
  657. 7.5Division 7.5
  658. 7.6Division 7.6
  659. 7.7Division 7.7
  660. 7.8Division 7.8
  661. 7.9In cases of analysis of vitamin A in milk replacers, specific attention has to be paid
  662. 8Repeatability
  663. 9Results of a collaborative study
  664. DETERMINATION OF VITAMIN E
  665. 1Purpose and Scope
  666. 2Principle
  667. Reagents
  668. 3.1Division 3.1
  669. 3.2Division 3.2
  670. 3.3Division 3.3
  671. 3.4Division 3.4
  672. 3.5Division 3.5
  673. 3.6Division 3.6
  674. 3.6.1Division 3.6.1
  675. 3.7Division 3.7
  676. 3.8Division 3.8
  677. 3.9Division 3.9
  678. 3.10Division 3.10
  679. 3.10.1Division 3.10.1
  680. 3.11Division 3.11
  681. 3.11.1Division 3.11.1
  682. 3.12Division 3.12
  683. Apparatus
  684. 4.1Division 4.1
  685. 4.2Division 4.2
  686. 4.2.1Division 4.2.1
  687. 4.2.2Division 4.2.2
  688. 4.2.3Division 4.2.3
  689. 4.2.4Division 4.2.4
  690. 4.3Division 4.3
  691. 4.4Division 4.4
  692. 4.5Division 4.5
  693. 4.5.1Division 4.5.1
  694. 4.5.2Division 4.5.2
  695. 4.6Division 4.6
  696. 4.7Division 4.7
  697. 4.8Division 4.8
  698. 4.8.1Division 4.8.1
  699. 4.8.2Division 4.8.2
  700. 5Procedure
  701. 5.1Preparation of the sample
  702. 5.2Saponification
  703. 5.3Extraction
  704. 5.3.1Extraction using a separating funnel (4.2.3)
  705. 5.3.2Extraction using an extraction apparatus (4.8)
  706. 5.4Preparation of the sample solution for HPLC
  707. 5.5Determination by HPLC
  708. HPLC conditions
  709. Calibration (DL-α-tocopherol acetate or DL-α-tocopherol)
  710. DL-α-tocopherol acetate standard
  711. 5.6.1.1Preparation of the working standard solution
  712. 5.6.1.2Preparation of the calibration solutions and calibration graph
  713. 5.6.1.3UV standardisation of the DL-α-tocopherol acetate stock solution (3.10.1)
  714. DL-α-tocopherol standard
  715. 5.6.2.1Preparation of the working standard solution
  716. 5.6.2.2Preparation of the calibration solutions and calibration graph
  717. 5.6.2.3UV standardisation of the DL-α-tocopherol stock solution (3.11.1)
  718. 6Calculation of the results
  719. Observations
  720. 7.1Division 7.1
  721. 7.2Division 7.2
  722. 7.3Division 7.3
  723. 7.4Division 7.4
  724. 7.5Division 7.5
  725. 7.6Division 7.6
  726. 7.7Division 7.7
  727. 7.8Division 7.8
  728. 7.9Division 7.9
  729. 8Repeatability·
  730. 9Results of a collaborative study
  731. DETERMINATION OF THE TRACE ELEMENTS IRON, COPPER, MANGANESE AND ZINC
  732. 1Purpose and scope
  733. 2Principle
  734. Reagents
  735. Introductory comments
  736. 3.1Division 3.1
  737. 3.2Division 3.2
  738. 3.3Division 3.3
  739. 3.4Division 3.4
  740. 3.5Division 3.5
  741. 3.6Division 3.6
  742. 3.7Division 3.7
  743. 3.7.1Division 3.7.1
  744. 3.8Standard copper solution (1 000 μg Cu/ml) prepared as follows or equivalent commercially available solution:
  745. 3.8.1Division 3.8.1
  746. 3.9Standard manganese solution (1 000 μg Mn/ml) prepared as follows or equivalent commercially available solution:
  747. 3.9.1Division 3.9.1
  748. 3.10Standard zinc solution (1 000 μg Zn/ml) prepared as follows or equivalent commercially available solution:
  749. 3.10.1Division 3.10.1
  750. 3.11Division 3.11
  751. Apparatus
  752. 4.1Division 4.1
  753. 4.2Division 4.2
  754. 4.3Division 4.3
  755. Procedure
  756. Samples containing organic matter
  757. Ashing and preparation of the solution for analysis
  758. 5.1.1.1Place 5 to 10 g of sample weighed to the nearest 0,2 mg in a quartz or platinum crucible (see Note (b)), dry in an oven at 105 oC and introduce the crucible into the cold muffle furnace (4.1). Close the furnace (see Note (c)) and gradually raise the temperature to 450 to 475 oC over about 90 minutes. Maintain this temperature for 4 to 16 hours (e.g. overnight) to remove carbonaceous material and then open the furnace and allow to cool (see Note (d)).
  759. If the residue in the filter appears black (carbon), put it back in the furnace and ash again at 450 to 475 oC. This ashing, which only requires a few hours (about three to five hours), is complete when the ash appears white or nearly white. Dissolve the residue with about 2 ml of hydrochloric acid (3.1), evaporate to dryness and add 5 ml of 6 mol/litre hydrochloric acid (3.2). Heat, filter the solution into the volumetric flask and make up to the mark with water (HCl concentration about 0,5 mol/litre.
  760. Notes:
  761. Spectrophotometric determination
  762. 5.1.2.1Preparation of calibration solutions
  763. Iron
  764. Copper
  765. Manganese
  766. Zinc
  767. 5.1.2.2Preparation of solution for analysis
  768. 5.1.2.3Blank experiment
  769. 5.1.2.4Measurement of the atomic absorption
  770. 5.2Mineral feed
  771. 6Calculation of results
  772. 7Repeatability
  773. 8Observation
  774. DDETERMINATION OF HALOFUGINONE
  775. 1Purpose and scope
  776. 2Principle
  777. Reagents
  778. 3.1Division 3.1
  779. 3.2Division 3.2
  780. 3.3Division 3.3
  781. 3.4Division 3.4
  782. 3.5Division 3.5
  783. 3.6Division 3.6
  784. 3.6.1Halofuginone stock standard solution, 100 μg/ml
  785. 3.6.2Calibration solutions
  786. 3.7Division 3.7
  787. 3.8Division 3.8
  788. 3.9Division 3.9
  789. 3.10Division 3.10
  790. 3.11Division 3.11
  791. 3.12Division 3.12
  792. 3.13Division 3.13
  793. 3.14Division 3.14
  794. 3.15Division 3.15
  795. 3.16Sodium chloride-saturated sodium carbonate solution, c = 5 g/100 ml.
  796. 3.17Hydrochloric acid, approximately 0,1 mol/l.
  797. 3.18Ammonium acetate buffer solution, approximately 0,25 mol/l.
  798. 3.19Amberlite XAD-2 resin preparation.
  799. 3.20Silver nitrate solution, approximately 0,1 mol/l.
  800. 3.21HPLC Mobile phase.
  801. Apparatus
  802. 4.1Division 4.1
  803. 4.2Division 4.2
  804. 4.3Division 4.3
  805. 4.4Division 4.4
  806. 4.4.1Division 4.4.1
  807. 4.5Division 4.5
  808. 4.6Division 4.6
  809. 4.7Division 4.7
  810. 4.8Division 4.8
  811. Procedure
  812. Division
  813. General
  814. 5.1.1Division 5.1.1
  815. 5.1.2A recovery test shall be carried out by analysing the blank feed which has been fortified by addition of a quantity of halofuginone, similar to that present in the sample. To fortify at a level of 3 mg/kg, add 300 μl of the stock standard solution (3.6.1) to 10 g of the blank feed, mix and wait for 10 minutes before proceeding with the extraction step (5.2).
  816. 5.2Extraction
  817. Clean up
  818. 5.3.1Preparation of the Amberlite column
  819. 5.3.2Sample clean up
  820. HPLC determination
  821. 5.4.1Parameters
  822. 5.4.2Calibration graph
  823. 5.4.3Sample solution
  824. 6Calculation of results
  825. Validation of the results
  826. 7.1Identity
  827. 7.1.1Co-chromatography
  828. 7.1.2Diode-array detection
  829. 7.2Repeatability
  830. 7.3Recovery
  831. 8Results of a collaborative study
  832. EDETERMINATION OF ROBENIDINE
  833. 1Purpose and scope
  834. 2Principle
  835. Reagents
  836. 3.1Division 3.1
  837. 3.2Acidified methanol.
  838. 3.3Division 3.3
  839. 3.4Molecular sieve.
  840. 3.5Aluminium oxide acidic activity grade I for column chromatography.
  841. 3.6Potassium dihydrogen phosphate solution, c = 0,025 mol/l.
  842. 3.7Di-sodium hydrogen phosphate solution, c = 0,025 mol/l.
  843. 3.8HPLC mobile phase.
  844. 3.9Standard substance.
  845. 3.9.1Robenidine stock standard solution: 300 μg/ml
  846. 3.9.2Robenidine intermediate standard solution: 12 μg/ml
  847. 3.9.3Calibration solutions
  848. 3.10Division 3.10
  849. Apparatus
  850. 4.1Glass column.
  851. 4.2Division 4.2
  852. 4.3Division 4.3
  853. 4.4Division 4.4
  854. 4.4.1Division 4.4.1
  855. 4.5Division 4.5
  856. 4.6Division 4.6
  857. 4.7Division 4.7
  858. Procedure
  859. Division
  860. General
  861. 5.1.1Division 5.1.1
  862. 5.1.2A recovery test shall be carried out by analysing the blank feed (5.1.1) which has been fortified by addition of a quantity of robenidine, similar to that present in the sample. To fortify at a level of 60 mg/kg, transfer 3,0 ml of the stock standard solution (3.9.1) to a 250 ml conical flask. Evaporate the solution to ca. 0,5 ml in a stream of nitrogen. Add 15 g of the blank feed, mix and wait for 10 minutes before proceeding with the extraction step (5.2).
  863. 5.2Extraction
  864. Purification
  865. 5.3.1Preparation of the aluminium-oxide column
  866. 5.3.2Sample purification
  867. HPLC determination
  868. 5.4.1Parameters
  869. 5.4.2Calibration graph
  870. 5.4.3Sample solution
  871. 6Calculation of results
  872. Validation of the results
  873. 7.1Identity
  874. 7.1.1Co-chromatography
  875. 7.1.2Diode-array detection
  876. 7.2Repeatability
  877. 7.3Recovery
  878. 8Results of a collaborative study
  879. FDETERMINATION OF DICLAZURIL
  880. 1Purpose and scope
  881. 2Principle
  882. Reagents
  883. 3.1Division 3.1
  884. 3.2Division 3.2
  885. 3.3Division 3.3
  886. 3.4Division 3.4
  887. 3.5Division 3.5
  888. 3.6Division 3.6
  889. 3.7Division 3.7
  890. 3.8Division 3.8
  891. 3.8.1Diclazuril stock standard solution, 500 μg/ml
  892. 3.8.2Diclazuril standard solution, 50 μg/ml
  893. 3.9Division 3.9
  894. 3.9.1Internal stock standard solution, 500 μg/ml
  895. 3.9.2Internal standard solution, 50 μg/ml
  896. 3.9.3Internal standard solution for premixtures, p/1 000 mg/ml
  897. 3.10Calibration solution, 2 μg/ml.
  898. 3.11Division 3.11
  899. 3.12Extraction solvent: acidified methanol.
  900. 3.13Division 3.13
  901. 3.13.1Eluent A: ammonium acetate — tetrabutylammonium hydrogen sulphate solution.
  902. 3.13.2Division 3.13.2
  903. 3.13.3Division 3.13.3
  904. Apparatus
  905. 4.1Division 4.1
  906. 4.2Division 4.2
  907. 4.2.1Division 4.2.1
  908. 4.2.2Division 4.2.2
  909. 4.3Division 4.3
  910. 4.4Division 4.4
  911. 4.5Division 4.5
  912. 4.6Division 4.6
  913. Procedure
  914. General
  915. 5.1.1Blank feed
  916. 5.1.2Recovery test
  917. Extraction
  918. 5.2.1Feed
  919. 5.2.2Premixtures
  920. HPLC determination
  921. 5.3.1Parameters
  922. 5.3.2Calibration solution
  923. 5.3.3Sample solution
  924. Calculation of the results
  925. 6.1Feeds
  926. 6.2Premixtures
  927. Validation of the results
  928. 7.1Identity
  929. 7.1.1Co-chromatography
  930. 7.1.2Diode-array detection
  931. 7.2Repeatability
  932. 7.3Recovery
  933. 8Results of a collaborative study
  934. 9Observations
  935. GDETERMINATION OF LASALOCID SODIUM
  936. 1Purpose and scope
  937. 2Principle
  938. Reagents
  939. 3.1Division 3.1
  940. 3.2Division 3.2
  941. 3.3Orthophosphoric acid solution, c = 20 %.
  942. 3.4Division 3.4
  943. 3.5Division 3.5
  944. 3.6Division 3.6
  945. 3.7Phosphate buffer solution, c = 0,01 mol/l.
  946. 3.8Acidified methanol.
  947. 3.9HPLC mobile phase, phosphate buffer-methanol solution 5 + 95 (V + V).
  948. 3.10Division 3.10
  949. 3.10.1Lasalocid sodium stock standard solution, 500 μg/ml
  950. 3.10.2Lasalocid sodium intermediate standard solution, 50 μg/ml
  951. 3.10.3Calibration solutions
  952. 3.11Division 3.11
  953. Apparatus
  954. 4.1Division 4.1
  955. 4.2Division 4.2
  956. 4.3Division 4.3
  957. 4.3.1Division 4.3.1
  958. 4.3.2Division 4.3.2
  959. Procedure
  960. General
  961. 5.1.1Blank feed
  962. 5.1.2Recovery test
  963. Extraction
  964. 5.2.1Feed
  965. 5.2.2Premixtures
  966. HPLC determination
  967. 5.3.1Parameters
  968. 5.3.2Calibration graph
  969. 5.3.3Sample solution
  970. 6Calculation of results
  971. 6.1Feed
  972. 6.2Premixtures
  973. Validation of the results
  974. 7.1Identity
  975. 7.1.1Co-chromatography
  976. 7.2Repeatability
  977. 7.3Recovery
  978. 8Results of a collaborative study
  979. Annex V: METHODS OF ANALYSIS TO CONTROL UNDESIRABLE SUBSTANCES IN FEED
  980. DETERMINATION OF FREE AND TOTAL GOSSYPOL
  981. 1Purpose and scope
  982. 2Principle
  983. Reagents
  984. 3.1Division 3.1
  985. 3.2Division 3.2
  986. 3.3Division 3.3
  987. 3.4Division 3.4
  988. 3.5Division 3.5
  989. 3.6Standard gossypol solution B: Place 27,9 mg of gossypol acetate in a 50 ml graduated flask, Dissolve and make up to volume with solvent B (3.3). The gossypol concentration of this solution is 0,5 mg/ml.
  990. Apparatus
  991. 4.1Division 4.1
  992. 4.2Division 4.2
  993. Procedure
  994. 5.1Test sample
  995. 5.2Determination of free gossypol
  996. 5.3Determination of total gossypol
  997. 6Calculation of results
  998. 6.1From the specific optical density
  999. From a calibration curve
  1000. 6.2.1Free gossypol
  1001. 6.2.2Total gossypol
  1002. 6.3Repeatability
  1003. DETERMINATION OF THE LEVELS OF DIOXINS (PCDD/PCDF) AND PCBs
  1004. Methods of sampling and interpretation of analytical results
  1005. 1Scope and definitions
  1006. Compliance of the lot or sublot with the maximum level
  1007. 2.1As regards non-dioxin-like PCBs
  1008. 2.2As regards PCDD/Fs and dioxin-like PCBs
  1009. 3Results exceeding action thresholds as laid down in the CMU Regulations
  1010. 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
  1011. 1Field of application
  1012. 2Background
  1013. Quality assurance requirements
  1014. 3.1Division 3.1
  1015. 3.2Division 3.2
  1016. 3.3Division 3.3
  1017. 3.4Division 3.4
  1018. 3.5Division 3.5
  1019. 3.6Division 3.6
  1020. 3.7Division 3.7
  1021. 3.8Division 3.8
  1022. 3.9Division 3.9
  1023. Requirements for laboratories
  1024. 4.1Division 4.1
  1025. 4.2Division 4.2
  1026. 4.3Division 4.3
  1027. Basic requirements to be met by analytical procedure for dioxins (PCDD/Fs) and dioxin-like PCBs
  1028. 5.1Low working range and limits of quantification
  1029. High selectivity (specificity)
  1030. 5.2.1Division 5.2.1
  1031. 5.2.2Division 5.2.2
  1032. High accuracy (trueness and precision, bioassay apparent recovery)
  1033. 5.3.1Division 5.3.1
  1034. 5.3.2Division 5.3.2
  1035. Validation in the range of maximum level and general quality control measures
  1036. 5.4.1Division 5.4.1
  1037. 5.4.2Division 5.4.2
  1038. Limit of quantification
  1039. 5.5.1Division 5.5.1
  1040. 5.5.2Division 5.5.2
  1041. 5.6Analytical criteria
  1042. Specific requirements for screening methods
  1043. 5.7.1Division 5.7.1
  1044. 5.7.2Division 5.7.2
  1045. 5.7.3Division 5.7.3
  1046. 5.7.4Division 5.7.4
  1047. 5.7.5Division 5.7.5
  1048. 5.7.6Division 5.7.6
  1049. 5.7.7Division 5.7.7
  1050. 5.7.8Division 5.7.8
  1051. SPECIFIC requirements for GC-MS methods to be complied with for screening or confirmatory purposes
  1052. 6.1Acceptable differences between upper-bound and lower-bound WHO-TEQ results
  1053. Control of recoveries
  1054. 6.2.1Division 6.2.1
  1055. 6.2.2Division 6.2.2
  1056. 6.2.3Division 6.2.3
  1057. 6.2.4Division 6.2.4
  1058. 6.2.5Division 6.2.5
  1059. 6.3Removal of interfering substances
  1060. 6.4Calibration with standard curve
  1061. 6.5Specific criteria for confirmatory methods
  1062. 7Specific requirements for bioanalytical methods
  1063. Evaluation of the test response
  1064. 7.1.1General requirements
  1065. Calibration
  1066. 7.1.2.1Calibration with standard curve
  1067. 7.1.2.2Calibration with reference samples
  1068. 7.1.3Separate determination of PCDD/Fs and dioxin-like PCBs
  1069. 7.1.4Bioassay apparent recoveries
  1070. 7.1.5Control of recoveries for clean-up
  1071. 7.1.6Reporting limit
  1072. Use of reference samples
  1073. 7.2.1Division 7.2.1
  1074. 7.2.2Division 7.2.2
  1075. 7.2.3Division 7.2.3
  1076. 7.2.4Division 7.2.4
  1077. 7.3Determination of cut-off values
  1078. 7.3.1Division 7.3.1
  1079. 7.3.2Division 7.3.2
  1080. 7.3.3Division 7.3.3
  1081. Figure 1
  1082. 7.3.4Restrictions to cut-off values
  1083. Performance characteristics
  1084. 7.4.1Division 7.4.1
  1085. 7.4.2Division 7.4.2
  1086. 7.4.3Division 7.4.3
  1087. 7.4.4Division 7.4.4
  1088. 7.4.5Division 7.4.5
  1089. 7.4.6Division 7.4.6
  1090. 7.4.7Division 7.4.7
  1091. 7.4.8Division 7.4.8
  1092. 7.4.9Division 7.4.9
  1093. Reporting of the results
  1094. Confirmatory methods
  1095. 8.1.1Division 8.1.1
  1096. 8.1.2Division 8.1.2
  1097. 8.1.3Division 8.1.3
  1098. 8.1.4Division 8.1.4
  1099. 8.1.5Division 8.1.5
  1100. Bioanalytical screening methods
  1101. 8.2.1Division 8.2.1
  1102. 8.2.2Division 8.2.2
  1103. 8.2.3Division 8.2.3
  1104. 8.2.4Division 8.2.4
  1105. 8.2.5Division 8.2.5
  1106. 8.2.6Division 8.2.6
  1107. 8.2.7Division 8.2.7
  1108. 8.2.8Division 8.2.8
  1109. Physico-chemical screening methods
  1110. 8.3.1Division 8.3.1
  1111. 8.3.2Division 8.3.2
  1112. 8.3.3Division 8.3.3
  1113. 8.3.4Division 8.3.4
  1114. 8.3.5Division 8.3.5
  1115. 8.3.6Division 8.3.6
  1116. 8.3.7Division 8.3.7
  1117. 8.3.8Division 8.3.8
  1118. Sample preparation and requirements for methods of analysis used in offical control of the levels of non dioxin-like PCBs in feed
  1119. 1Field of application
  1120. 2Applicable detection methods
  1121. Identification and confirmation of analytes of interest
  1122. 3.1Division 3.1
  1123. 3.2Division 3.2
  1124. 3.3Division 3.3
  1125. 3.4Division 3.4
  1126. 4Demonstration of performance of method
  1127. 5Limit of quantification
  1128. 6Quality control
  1129. Control of recoveries
  1130. 7.1Division 7.1
  1131. 7.2Division 7.2
  1132. 7.3Division 7.3
  1133. 7.4Division 7.4
  1134. 7.5Division 7.5
  1135. 8Requirements for laboratories
  1136. 9Performance characteristics: criteria for the sum of non-dioxin-like PCBs at the maximum level
  1137. Reporting of the results
  1138. 10.1Division 10.1
  1139. 10.2Division 10.2
  1140. 10.3Division 10.3
  1141. 10.4Division 10.4
  1142. 10.5Division 10.5
  1143. Annex VI: METHODS OF ANALYSIS FOR THE DETERMINATION OF CONSTITUENTS OF ANIMAL ORIGIN FOR THE OFFICIAL CONTROL OF FEED
  1144. 1PURPOSE AND SCOPE
  1145. METHODS
  1146. Light microscopy
  1147. 2.1.1Principle
  1148. Reagents and equipment
  1149. Reagents
  1150. Concentrating agent
  1151. Tetrachloroethylene (specific gravity 1,62)
  1152. Staining reagent
  1153. 2.1.2.1.2.1Division 2.1.2.1.2.1
  1154. Mounting media
  1155. Lye (NaOH 2,5 % w/v or KOH 2,5 % w/v)
  1156. Glycerol (undiluted, viscosity: 1 490 cP) or a mounting medium with equivalent properties for non-permanent slide preparation
  1157. Norland ® Optical Adhesive 65 (viscosity: 1 200 cP) or a resin with equivalent properties for permanent slide preparation
  1158. Mounting media with staining properties
  1159. 2.1.2.1.4.1Division 2.1.2.1.4.1
  1160. Cystine reagent (2 g lead acetate, 10 g NaOH/100 ml water)
  1161. 2.1.2.1.4.3Division 2.1.2.1.4.3
  1162. 2.1.2.1.4.4Division 2.1.2.1.4.4
  1163. Rinsing agents
  1164. Ethanol ≥ 96 % (technical grade)
  1165. Acetone (technical grade)
  1166. Bleaching reagent
  1167. Commercial sodium hypochlorite solution (9 - 14 % active chlorine)
  1168. Equipment
  1169. Analytical balance with an accuracy of 0,001 g
  1170. Grinding equipment: knife or rotor mill. If a rotor mill is used, mill sieves ≤ 0,5 mm shall be prohibited
  1171. Sieves with square meshes of 0,25 mm and 1 mm width. With the exception of sample pre-sieving, the diameter of the sieves should not exceed 10 cm to avoid loss of materials. Calibration of sieves is not required
  1172. 2.1.2.2.4Division 2.1.2.2.4
  1173. Separation funnel
  1174. 2.1.2.2.5Division 2.1.2.2.5
  1175. 2.1.2.2.6Division 2.1.2.2.6
  1176. Standard laboratory glassware
  1177. Equipment for slide preparation: classical microscope slides, hollow slides, coverslips (20 × 20 mm), tweezers, fine spatula
  1178. Laboratory oven
  1179. Centrifuge
  1180. Filter paper: qualitative cellulose filter (pore size 4-11 μm)
  1181. Sampling and sample preparation
  1182. 2.1.3.1Sampling
  1183. 2.1.3.2Precautions to be taken
  1184. Preparation of samples other than fat or oil
  1185. 2.1.3.3.1Division 2.1.3.3.1
  1186. 2.1.3.3.2Division 2.1.3.3.2
  1187. 2.1.3.3.3Division 2.1.3.3.3
  1188. 2.1.3.3.4Division 2.1.3.3.4
  1189. 2.1.3.3.5Division 2.1.3.3.5
  1190. 2.1.3.3.6Division 2.1.3.3.6
  1191. 2.1.3.4Preparation of samples consisting of fat or oil
  1192. 2.1.3.5Use of staining reagents
  1193. Microscopic examination
  1194. 2.1.4.1Slide preparation
  1195. 2.1.4.2Observation flowchart for the detection of animal particles in compound feed and feed material
  1196. 2.1.4.3Number of determinations
  1197. 2.1.5Expression of the results
  1198. PCR
  1199. 2.2.1Principle
  1200. Reagents and equipment
  1201. Reagents
  1202. 2.2.2.1.1Reagents for DNA extraction step
  1203. Reagents for genetic amplification step
  1204. 2.2.2.1.2.1Primers and probes
  1205. 2.2.2.1.2.2Master Mix
  1206. Decontamination reagents
  1207. Hydrochloric acid solution (0,1 N)
  1208. Bleach (solution of sodium hypochlorite at 0,15 % of active chlorine)
  1209. Non-corrosive reagents for decontaminating costly devices like analytical balances (e.g. DNA Erase TM of MP Biomedicals)
  1210. Equipment
  1211. Analytical balance with an accuracy of 0,001 g
  1212. Grinding equipment
  1213. Thermocycler enabling real-time PCR
  1214. Microcentrifuge for microfuge tubes
  1215. Set of micropipettes allowing to pipet from 1 μl up to 1 000 μl
  1216. 2.2.2.2.6Division 2.2.2.2.6
  1217. Freezers to store samples and reagents
  1218. Sampling and sample preparation
  1219. 2.2.3.1Sampling
  1220. 2.2.3.2Sample preparation
  1221. 2.2.4DNA extraction
  1222. 2.2.5Genetic amplification
  1223. 2.2.6Interpretation and expression of results
  1224. 2.2.6.1Negative result
  1225. 2.2.6.2Positive result
  1226. Annex VII: METHOD OF CALCULATING THE ENERGY VALUE OF POULTRYFEED
  1227. 1Method of calculation and expression of energy value
  1228. 2Tolerances applicable to declared values
  1229. 3Expression of result
  1230. 4Sampling and analysis methods
  1231. Annex VIII: METHODS OF ANALYSIS TO CONTROL ILLEGAL PRESENCE OF NO LONGER AUTHORISED ADDITIVES IN FEED
  1232. Important notes:
  1233. ADETERMINATION OF METHYL BENZOQUATE
  1234. 1Purpose and scope
  1235. 2Principle
  1236. Reagents
  1237. 3.1Division 3.1
  1238. 3.2Division 3.2
  1239. 3.3HPLC mobile phase
  1240. 3.4Methanesulfonic acid solution, c = 2 %
  1241. 3.5Hydrochloric acid solution, c = 10 %
  1242. 3.6Cation-exchange resin Amberlite CG-120 (Na), 100 to 200 mesh
  1243. 3.7Division 3.7
  1244. 3.7.1Methyl benzoquate stock standard solution, 500 μg/ml
  1245. 3.7.2Methyl benzoquate intermediate standard solution, 50 μg/ml
  1246. 3.7.3Calibration solutions
  1247. Apparatus
  1248. 4.1Division 4.1
  1249. 4.2Division 4.2
  1250. 4.3Division 4.3
  1251. 4.4Division 4.4
  1252. 4.4.1Division 4.4.1
  1253. 4.5Division 4.5
  1254. 4.6Division 4.6
  1255. Procedure
  1256. General
  1257. 5.1.1Division 5.1.1
  1258. 5.1.2A recovery test shall be carried out by analysing the blank feed which has been fortified by addition of a quantity of methyl benzoquate, similar to that present in the sample. To fortify at a level of 15 mg/kg, add 600 μl of the stock standard solution (3.7.1) to 20 g of the blank feed, mix and wait for 10 minutes before proceeding with the extraction step (5.2).
  1259. 5.2Extraction
  1260. 5.3Liquid-liquid partition
  1261. Ion-exchange chromatography
  1262. 5.4.1Preparation of the cation-exchange column
  1263. 5.4.2Column chromatography
  1264. 5.5Liquid-liquid partition
  1265. HPLC determination
  1266. 5.6.1Parameters
  1267. 5.6.2Calibration graph
  1268. 5.6.3Sample solution
  1269. 6Calculation of results
  1270. Validation of the results
  1271. 7.1Identity
  1272. 7.1.1Co-chromatography
  1273. 7.1.2Diode-array detection
  1274. 7.2Repeatability
  1275. 7.3Recovery
  1276. 8Results of a collaborative study
  1277. BDETERMINATION OF OLAQUINDOX
  1278. 1Purpose and scope
  1279. 2Principle
  1280. Reagents
  1281. 3.1Division 3.1
  1282. 3.2Division 3.2
  1283. 3.3Division 3.3
  1284. 3.4Mobile phase for HPLC.
  1285. 3.5Division 3.5
  1286. 3.5.1Olaquindox stock standard solution, 250 μg/ml
  1287. 3.5.2Olaquindox intermediate standard solution, 25 μg/ml
  1288. 3.5.3Calibration solutions
  1289. Apparatus
  1290. 4.1Division 4.1
  1291. 4.2Division 4.2
  1292. 4.3Division 4.3
  1293. 4.3.1Division 4.3.1
  1294. 4.4Division 4.4
  1295. Procedure
  1296. Division
  1297. General
  1298. 5.1.1Division 5.1.1
  1299. 5.1.2A recovery test shall be carried out by analysing the blank feed which has been fortified by addition of a quantity of olaquindox, similar to that present in the sample. To fortify at a level of 50 mg/kg, transfer 10,0 ml of the stock standard solution (3.5.1) to a 250 ml conical flask and evaporate the solution to ca. 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 (5.2).
  1300. 5.2Extraction
  1301. HPLC determination
  1302. 5.3.1Parameters:
  1303. 5.3.2Calibration graph
  1304. 5.3.3Sample solution
  1305. 6Calculation of the results
  1306. Validation of the results
  1307. 7.1Identity
  1308. 7.1.1Co-chromatography
  1309. 7.1.2Diode array detection
  1310. 7.2Repeatability
  1311. 7.3Recovery
  1312. 8Results of a collaborative study
  1313. 9Observation
  1314. CDETERMINATION OF AMPROLIUM
  1315. 1Purpose and Scope
  1316. 2Principle
  1317. Reagents
  1318. 3.1Division 3.1
  1319. 3.2Division 3.2
  1320. 3.3Division 3.3
  1321. 3.4Sodium dihydrogen phosphate solution, c = 0,1 mol/l.
  1322. 3.5Sodium perchlorate solution, c = 1,6 mol/l.
  1323. 3.6Mobile phase for HPLC (see observation 9.1).
  1324. 3.7Division 3.7
  1325. 3.7.1Amprolium stock standard solution, 500 μg/ml
  1326. 3.7.2Amprolium intermediate standard solution, 50 μg/ml
  1327. 3.7.3Calibration solutions
  1328. 3.8Extraction solvent.
  1329. Apparatus
  1330. 4.1Division 4.1
  1331. 4.1.1Division 4.1.1
  1332. 4.1.2Division 4.1.2
  1333. 4.2Division 4.2
  1334. 4.3Division 4.3
  1335. 4.4Division 4.4
  1336. 4.5Division 4.5
  1337. Procedure
  1338. General
  1339. 5.1.1Blank feed
  1340. 5.1.2Recovery test
  1341. Extraction
  1342. 5.2.1Premixtures (content < 1 % amprolium) and feed
  1343. 5.2.2Premixtures (content ≥ 1 % amprolium)
  1344. HPLC determination
  1345. 5.3.1Parameters:
  1346. 5.3.2Calibration graph
  1347. 5.3.3Sample solution
  1348. 6Calculation of the results
  1349. Validation of the results
  1350. 7.1Identity
  1351. 7.1.1Co-chromatography
  1352. 7.1.2Diode array detection
  1353. 7.2Repeatability
  1354. 7.3Recovery
  1355. 8Results of a collaborative study
  1356. Observations
  1357. 9.1Division 9.1
  1358. 9.2Division 9.2
  1359. 9.3Division 9.3
  1360. DDETERMINATION OF CARBADOX
  1361. 1Purpose and scope
  1362. 2Principle
  1363. Reagents
  1364. 3.1Division 3.1
  1365. 3.2Division 3.2
  1366. 3.3Division 3.3
  1367. 3.4Division 3.4
  1368. 3.5Methanol-acetonitrile 1 + 1 (v + v).
  1369. 3.6Acetic acid, σ = 10 %.
  1370. 3.7Division 3.7
  1371. 3.8Division 3.8
  1372. 3.9Acetate buffer solution, c = 0,01 mol/l, pH = 6,0.
  1373. 3.10Mobile phase for HPLC.
  1374. 3.11Standard substance.
  1375. 3.11.1Carbadox stock standard solution, 100 μg/ml (see Note 5. Procedure):
  1376. 3.11.2Calibration solutions
  1377. 3.12Water-[methanol-acetonitrile] (3.5) mixture, 300 + 700 (v + v).
  1378. Apparatus
  1379. 4.1Division 4.1
  1380. 4.2Division 4.2
  1381. 4.3Glass column (length 300 to 400 mm, internal diameter approximately 10 mm) with sintered glass frit and draw-off valve.
  1382. 4.4Division 4.4
  1383. 4.4.1Division 4.4.1
  1384. 4.4.2Division 4.4.2
  1385. 4.5Division 4.5
  1386. 4.6Division 4.6
  1387. 4.7Division 4.7
  1388. Procedure
  1389. Division
  1390. General
  1391. 5.1.1Blank feed
  1392. 5.1.2Recovery test
  1393. Extraction
  1394. 5.2.1Feed
  1395. 5.2.2Premixtures (0,1 %-2,0 %)
  1396. 5.2.3Preparations (> 2 %)
  1397. Purification
  1398. 5.3.1Preparation of the aluminium oxide column
  1399. 5.3.2Sample purification
  1400. HPLC determination
  1401. 5.4.1Parameters
  1402. 5.4.2Calibration graph
  1403. 5.4.3Sample solution
  1404. 6Calculation of the results
  1405. 6.1Feed
  1406. 6.2Premixtures and preparations
  1407. Validation of the results
  1408. 7.1Identity
  1409. 7.1.1Co-chromatography
  1410. 7.1.2Diode-array detection
  1411. 7.2Repeatability
  1412. 7.3Recovery
  1413. 8Results of a collaborative study
  1414. Annex IX: CORRELATION TABLES REFERRED TO IN ARTICLE 6
  1415. 1Directive 71/250/EEC
  1416. 2Directive 71/393/EEC
  1417. 3Directive 72/199/EEC
  1418. 4Directive 73/46/EEC
  1419. 5Directive 76/371/EEC
  1420. 6Directive 76/372/EEC
  1421. 7Directive 78/633/EEC
  1422. 8Directive 81/715/EEC
  1423. 9Directive 84/425/EEC
  1424. 10Directive 86/174/EEC
  1425. 11Directive 93/70/EEC
  1426. 12Directive 93/117/EC
  1427. 13Directive 98/64/EC
  1428. 14Directive 1999/27/EC
  1429. 15Directive 1999/76/EC
  1430. 16Directive 2000/45/EC
  1431. 17Directive 2002/70/EC
  1432. 18Directive 2003/126/EC