1、STD-BSI BS EN IS0 LSL41-1-ENGL 1998 Lb24669 0734763 298 m BRITISH STANDARD Foodstuffs - Determination of ochratoxin A in cereals and cereal products - Part 1: High performance liquid chromatographic method with silica gel clean up The European Standard EN IS0 15141-1:1998 has the status of a British
2、 Standard ICs 67.060 BS EN IS0 15141-1:1998 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW - - - - - - - _ _- - - - - STDOBSI BS EN IS0 15141-L-ENGL 1778 = Lb24667 0734764 124 W BS EN IS0 15141-11998 National foreword This British Standard is the English language version of E
3、N IS0 15141-1: 1998. The UK participation in its preparation was entrusted to Technical Panel AW/-/3, Food analysis - Horizontal methods, which has the responsibility to: - aid enquirem to understand the text; - present to the responsible internationallEuropean committee any enquiries on the interpr
4、etation, or proposals for change, and keep the UK interests informed - monitor related international and European developments and promulgate them in the UK A list of organizations represented on this panel can be obtained on request to its secretary. Technical Committee AW/4, Cereals and pulses, ha
5、s also actively participated in the development of this standard. Cross-references Attention is drawn to the fact that CEN and CENELEC standards normally include an annex which lists normative references to international publications with their corresponding European publications. The British Standa
6、rds which implement international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by wing the “Find facility of the BSI Standards Electronic Catalogue. A British Standard d
7、oes not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an in
8、side front cover, the EN IS0 title page, pages 2 to 11 and a back cover. This British Standard, having Amendments issued since publication been prepared under the direction of the Consumer Products and Services Sector Committee, was published under the authority of the Standards Committee and comes
9、into effect on 15 December 1998 Amd. No. O BSI 1998 ISBN O 680 30234 2 Date Text affected EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN IS0 15141-1 October 1998 ICs 67.060 Descriptors: food products, cereals, chemical analysis, determination of content, ochratoxin, chromatographic analysis, h
10、igh performance liquid chromatography, extraction, silicon dioxid English version Foodstuffs - Determination of ochratoxin A in cereals and cereal products - Part 1 : High performance liquid chromatographic method with silica gel clean up (IS0 151 41 -1 :I 998) Produits alimentaires - Dosage de Ioch
11、ratoxine A dans les crales et produits drivs - Partie 1 : MBthode par chromatographie liquide haute performance comprenant une tape dextraction par chromatographie sur gel de silice (IS0 15141-1:1998) Lebensmittel - Bestimmung von Ochratoxin A in Getreide und Getreideerzeugnicsen - Teil 1 : Hochleis
12、tungs- flssigchromatographisches Verfahren mit Kieselgelreinigung (IS0 151 41 -1 :1998) This European Standard was approved by CEN on 1 July 1998. CEN members are bound to comply with the CENKENELEC Intemal Regulations which stipulate the conditions for giving this European Standard the status of a
13、national standard without any alteration. Up-todate lists and bibliographical references concerning such national standards may be obtained on application to the Central Secretariat or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in
14、 any other language made by translation under the responsibility of a GEN member into its own language and notified to the Central Secretariat has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Ge
15、rmany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITEE FOR STANDARDIZATION COMITE EUROPEEN DE NORMALISATION EUROPAISCHES KOMITEE FUR NORMUNG Central Secretariat: rue de Stassart. 36 8-1050 Brussels 8 1998 CE
16、N All rights of exploitation in any form and by any means resewed worldwide for CEN national Members. Ref. No. EN IC0 15141-1:1998 E STD*BSI BS EN IS0 L514L-L-ENGL 1998 162L)bbq 0734766 TT7 Page 2 EN IS0 15141-1:1998 Contents Foreword . 2 1 Scope 3 2 Normative references . 3 3 Principle . 3 4 Reagen
17、ts . 3 6 Procedure 6 7 Calculation . 8 8 Precision 9 9 Test report . 9 Annex A (informative) Precision data 10 5 Apparatus and equipment . 5 Annex B (informative) Bibliography 1 1 Foreword The text of EN IS0 151 41 -1 :1998 has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizon
18、tal methods“, the secretariat of which is held by DIN, in collaboration with Technical Committee ISO/TC 34 “Agricultural food products“. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by April 1999,
19、 and conflicting national standards shall be withdrawn at the latest by April 1999. This European Standard ,Foodstuffs - Determination of ochratoxin A in cereal and cereal products“ consists of two parts: Part 1 : High performance liquid chromatographic method with silica gel clean up Part 2: High p
20、erformance liquid chromatographic method with bicarbonate clean up According to the CENKENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Gre
21、ece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. STD.BSI BS EN IS0 15141-1-ENGL 1998 H Lb24669 O734767 933 Page 3 EN IS0 15141-1:1998 1 Scope This European Standard specifies a method for the determination of ochratoxin A at
22、levels greater than 0,4 pg/kg. The method has been successfully validated in 2 interlaboratory studies according to IS0 5725:1996 I on wheat whole meal containing 0,4 pg/kg and 1,2 pg/kg of ochratoxin A. NOTE: Numerous laboratory experiences have shown that this method is also applicable to cereais,
23、 dried fruits, oilseeds, pulses, wine, beer, fruit juices and raw coffee, see 21, 31, 141. 2 Normative references This draft European Standard incorporates by dated or undated reference, provisions from other publications. These normative references are cited at the appropriate places in the text an
24、d the publications are listed hereafter. For dated references, subsequent amendments to or revisions of any of these publications apply to this draft European Standard only when incorporated in it by amendment or revision. For undated references the latest edition of the publication referred to appl
25、ies. EN IS0 3696:1995 Water for analytical laboratory use - Specification and test methods (IS0 3696:1987). 3 Principle Ochratoxin A (OTA) is extracted with toluene after acidification with hydrochloric acid and after the ionic strength has been increased by adding magnesium chloride. The extract is
26、 purified using a mini silica gel column and ochratoxin A is determined by high performance liquid chromatography (HPLC) on a reversed phase column and identified and modified by fluorescence. The result is verified, if required, by derivatization with boron trifluoride in methanolic solution 51, 16
27、1. WARNING: Ochratoxin A causes kidney and liver damage and is a probable carcinogen. Observe appropriate safety precautions 171 for handling such compounds and in particular avoid handling in dry form as the electrostatic nature can result in dispersion and inhalation. Glassware can be decontaminat
28、ed with 4 % sodium hypochlorite solution. Attention is drawn to the statement made by the International Agency for Research on Cancer (WHO) 181, 91. 4 Reagents During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and only distilled water or water of grade 1
29、according to EN IS0 3696. Solvent shall be of quality for HPLC analysis. 4.1 Sodium sulfate, anhydrous 4.2 Glacial acetic acid p(CH,COOH) z 98 % 4.3 Solution of hydrochloric acid c(HCI) = 2 mol/l 4.4 Magnesium chloride solution c(MgCI,) = 0,4 mol/l 4.5 Acetonitrile 4.6 Toluene 4.7 n-Hexane STD-BSI B
30、S EN IS0 L5LYL-L-ENGL 1998 Lb24bb9 073Y7b8 87T - Page 4 EN IS0 15141-1:1998 4.8 Dichloromethane 4.9 Acetone 4.1 O Methanol 4.1 1 Solvent mixture I: toluene (4.6) and glacial acetic acid (4.2) 99 + 1 parts per volume (V+ V) 4.12 Solvent mixture II: acetone (4.9) and toluene (4.6) 5 +95 (V+ V) 4.13 So
31、lvent mixture 111: toluene (4.6) and glacial acetic acid (4.2) 90+ 10 (V+ V) 4.14 Mobile phase Mix 99 volume parts of acetonitrile (4.5) with 99 volume parts of water and 2 volume parts of glacial acetic acid (4.2) and degas this solution before use. 4.15 Boron trifluoride 4.1 6 Boron trifluoride in
32、 methanol solution, p(BF,) = 14 g/l O0 ml WARNING: Use a well maintained fume hood. Avoid contact with skin, eyes, and respiratory tract. 4.17 Ochratoxin A, in crystal form or as a film in ampoules 4.18 Ochratoxin A stock solution Dissolve 1 mg of the ochratoxin A (crystals) (4.17) or the contents o
33、f 1 ampoule (if ochratoxin A has been obtained as a film) in solvent mixture i (4.1 1) to give a solution containing approximately 20 pg/mi to 30 pg/ml of ochratoxin A. To determine the exact concentration, record the absorption curve between a wavelength of 300 nm and 370 nm in 5 nm steps in a 1 cm
34、 quartz cell (5.5) with solvent mixture I (4.1 1) as reference. Identify the wavelength for maximum absorption by recording in 1 nm steps around the maximum as reference. Calculate the mass concentration of ochratoxin A, poTA, in micrograms per millilitre of solution using equation 1 : where A, is t
35、he absorption determied at 1,.e maximum of the absorption curve (here: at 333 nm); M is the relative molecular mass of ochratoxin A (M = 403 g/mol); K is the molar absorption coefficient of ochratoxin A, in solvent mixture I (here: 544 m2/mol); 6 is the path length of the cell in centimetres. 4.19 O
36、chratoxin A standard solution p(0TA) = 1 pg/ml Evaporate under a nitrogen flow 1 ml of the stock solution (4.18) or the aliquot portion which is equivalent to an absolute amount of 1 O0 pg of Ochratoxin A to dryness and dilute to 1 O0 ml with the mobile phase (4.14). STD.BS1 BS EN IS0 LSL4L-L-ENGL 1
37、778 = Lb24bb9 0734769 706 Page 5 EN IS0 15141-11998 This solution can be stored in a refrigerator at 4 T. Stability shall be checked. 4.20 Ochratoxin A calibration solutions Pipette suitable volumes of ochratoxin A standard solution (4.191, e.g. 1 mi, 2,5 mi, 4 ml and 5 ml into e.g. a 100 rnl volume
38、tric flask (5.12) and dilute to the mark with the mobile phase (4.14). The amount of ochratoxin A in the calibration solutions should cover the range of 0,2 ng to 1 ,O ng per 20-pl-injection volume. 4.21 Sodium hypochlorite solution, p(Na0CI) = 4 g/lOO ml 5 Apparatus and equipment Usual laboratory e
39、quipment and, in particular, the following: 5.1 Laboratory mill, suitable to grind to 1 mm 5.2 Rotary evaporator, with a water bath capable of being controlled between 20 OC and 50 OC 5.3 Mechanical shaker 5.4 Spectrometer, suitable for measurement at Wavelengths of 300 nm up to 370 nm, having a spe
40、ctral band width of not more than f 2 nm 5.5 Quartz cells, with 1 cm optical path length and no significant absorption between wavelengths of 300 nm and 370 nm 5.6 Centrifuge tubes, e.g. of capacity 250 ml, plastic made of high density polyethylene HDPE), with screw cap 5.7 Cooling centrifuge, prefe
41、rably a refrigerated centrifuge, capable of producing a gravitational force of at least 3500 g at the base of the centrifuge tubes (5.6) 5.8 Solid phase extraction columns, e.g. SEP-PAK) disposable silica gel After the pack has been opened, condition at 105 OC for 2 h and store over activated silica
42、 gel with moisture indicator. Before use, wash with 10 ml of toluene (4.6). Check the recovery with each new batch. In the case of use of SEP-PAK columns, the cartridges have the following specification: mean mass of the packing material: 690 mg pore size: 12,5 nm particle size: 55 pm to 105 pm in a
43、 3 mi polypropylene tube. 5.9 Solvent containers, such as syringes, e.g. of 50 mi capacity with central opening and Stop-cock 5.10 Pear-shaped flasks, 50 ml, with ground glass joint ) SEP PAK0 is an example of a suitable product available commercially. This information is given for the convenience o
44、f users of this Standard and does not constitute an endorsement by CEN of these products. STDmBSI BS EN IS0 LSL4L-L-ENGL 1998 m Lb24669 0734770 428 = Page 6 EN IS0 15141-1:1998 5.1 1 Separating funnel, 50 ml 5.1 2 Volumetric flask, 1 O0 ml 5.1 3 Membrane filter for aqueous solutions, made of polytet
45、rafluoroethylene (PTFE), with a diameter of 4 mm and a pore size of 0,45 pm 5.14 Sieve, with an aperture size of not more than 1 mm 5.15 Vials with crimped caps or screw cap vials 5.16 Microsyringe, of capacity 500 pl 5.17 HPLC apparatus, comprising the following 5.1 7.1 High performance liquid chro
46、matograph, eluent reservoir, a pump, an injection system, a fluorescence detector with variable wavelength setting and a data processing, e.g. an integrator with plotter. 5.17.2 Analytical reversed phase HPLC separating column, C which ensures a baseline resolved resolution of the ochratoxin A peak
47、from all other peaks. e.g. Lichrospher“ 100 RP 18 1 length: 250 mm internal diameter: 4 mm spherical particles of size: 5 pm NOTE: Shorter columns can also be used (e.g. a column with a length of 120 mm to 150 mm) 5.17.3 Precolumn, C length: 40 mm internal diameter: 4 mm spherical particles of size:
48、 5 pm 6 Procedure 6.1 General The whole analytical procedure should be performed in one working day. If several samples are processed at the same time all samples should be analysed during the following night using an automatic sample injector. 6.2 Preparation of the test samples Grind the laborator
49、y sample using a laboratory mill (5.1) until it passes through the sieve (5.14) and mix it thoroughly. NOTE: Grinding is not necessary for wheat flour with a maximum size of 250 pm. * ) information is given for the convenience of users of this Standard and does not constitute an endorsement by CEN of these products. LichrospherO 100 RP 18 is an example of a suitable product available commercially. This STD-BSI BS EN IS0 LSL4L-L-ENGL 1998 1624669 0734773 364 Page 7 EN IS0 15141-1:1998 6.3 Extraction of ochratoxin A from the sample Place 20 g (mo), weighed to the n
