{
  "url": "https://openlegislation.uk/eu-regulation-2009-152/annex-IV/division-D/division-5/division-5.2",
  "json_url": "https://openlegislation.uk/eu-regulation-2009-152/annex-IV/division-D/division-5/division-5.2.json",
  "legislation": "eur/2009/152",
  "legislation_title": "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)",
  "legislation_url": "https://openlegislation.uk/eu-regulation-2009-152",
  "path": "annex/IV/division/D/division/5/division/5.2",
  "kind": "division",
  "number": "5.2",
  "heading": "Extraction",
  "text": "Weigh to the nearest 0,1 g, 10 g of the prepared sample, into a 200 ml centrifuge tube, add 0,5 g of sodium ascorbate (3.10), 0,5 g of EDTA (3.13) and 20 ml of water and mix. Place the tube for 5 minutes in a water bath (80 oC). After cooling down to room temperature, add 20 ml of sodium carbonate solution (3.15) and mix. Add immediately 100 ml of ethyl acetate (3.4) and shake vigorously by hand for 15 seconds. Then place the tube for three minutes in the ultrasonic bath (4.1) and loosen the stopper. Centrifuge for two minutes and decant the ethyl acetate phase through a glass fibre filter (4.6), into a 500 ml separating funnel. Repeat the extraction of the sample with a second portion of 100 ml of ethyl acetate. Wash the combined extracts for one minute with 50 ml of sodium chloride saturated sodium carbonate solution (3.16) and discard the aqueous layer.\nExtract the organic layer for 1 min. with 50 ml of hydrochloric acid (3.17). Run the lower acid layer into a 250 ml separating funnel. Re-extract the organic layer for 1,5 minutes with a further 50 ml of hydrochlorid acid and combine with the first extract. Wash the combined acid extracts by swirling for approximately 10 seconds with 10 ml of ethyl acetate (3.4).\nQuantitatively transfer the aqueous layer into a 250 ml round-bottomed flask and discard the organic phase. Evaporate all the remaining ethyl acetate from the acid solution using a rotary film evaporator (4.2). The temperature of the water bath must not exceed 40 oC. Under a vacuum of approximately 25 mbar all of the residual ethyl acetate will be removed within 5 minutes at 38 oC.",
  "extent": "E+W+S+N.I.",
  "status": null,
  "version": "current",
  "annotations": [],
  "outstanding_effects": [],
  "provenance": {
    "source": "legislation.gov.uk",
    "source_url": "https://www.legislation.gov.uk/eur/2009/152/annex/IV/division/D/division/5/division/5.2",
    "retrieved_at": "2026-11-01T14:51:38.610873+00:00"
  },
  "licence": "Open Government Licence v3.0",
  "licence_url": "https://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/",
  "attribution": "Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives."
}