1、August 2012 Translation by DIN-Sprachendienst.English price group 16No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS
2、 67.120.30!$w4“1908417www.din.deDDIN EN 16204Foodstuffs Determination of lipophilic algal toxins (okadaic acid group toxins,yessotoxins, azaspiracids, pectenotoxins) in shellfish and shellfishproducts by LC-MS/MS;English version EN 16204:2012,English translation of DIN EN 16204:2012-08Lebensmittel B
3、estimmung der lipophilen Algentoxine (Okadasuregruppen-Toxine, Yessotoxine,Azaspirosuren, Pectenotoxine) in Schalentieren und Schalentiererzeugnissen mitLC-MS/MS;Englische Fassung EN 16204:2012,Englische bersetzung von DIN EN 16204:2012-08Produits alimentaires Dosage des toxines algales lipophiles (
4、toxines du groupe acide okadaque, yessotoxines,azaspiracides, pectnotoxines) dans les coquillages et les produits base de coquillagespar CL-SM/SM;Version anglaise EN 16204:2012,Traduction anglaise de DIN EN 16204:2012-08www.beuth.deDocument comprises 41 pagesIn case of doubt, the German-language ori
5、ginal shall be considered authoritative.07.12 DIN EN 16204:2012-08 2 A comma is used as the decimal marker. National foreword This standard has been prepared by Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany). Preliminary work was done by Working Group W
6、G 5 “Biotoxins”. The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Working Committee NA 057-01-03 AA Biotoxine. This document describes a multi-reference method for the d
7、etection and determination of a number of lipophilic toxins. These toxins are fat-soluble algal toxins produced by certain marine dinoflagellates. Such toxins include the dinophysistoxins (dinophysistoxin-1 (DTX-1) and dinophysistoxin 2 (DTX-2), okadaic acid (OA) and their esters), azaspiracids, pec
8、tenotoxins, and yessotoxins. They can accumulate in shellfish, and after consumption, may cause harm in humans. This method has been validated in an inter-laboratory study consisting of three parts via the analysis of both naturally contaminated homogenates and spiked extracts. The limit of quantifi
9、cation (LOQ) for DTX toxins, azaspiracids and pectenotoxins was determined to be 6 g/kg of shellfish meat and for yessotoxins 10 g/kg of shellfish meat. Until Commission Regulation 15/2011 “as regards recognised testing methods for detecting marine biotoxins in live bivalve molluscs” was published,
10、the mouse bioassay (MBA) was the official method for the detection of lipophilic biotoxins according to European legislation. The present standard makes it possible to detect and quantify the presence of these toxins using chemical analysis, making animal testing unnecessary and increasing consumer
11、protection. Furthermore, the lower limit of quantification for the method specified here as compared with that for the mouse bioassay now makes it possible to detect the contamination of shellfish waters at an earlier stage. EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16204 May 2012 ICS 67.
12、120.30 English Version Foodstuffs - Determination of lipophilic algal toxins (okadaic acid group toxins, yessotoxins, azaspiracids, pectenotoxins) in shellfish and shellfish products by LC-MS/MS Produits alimentaires - Dosage des toxines algales lipophiles (toxines du groupe acide okadaque, yessotox
13、ines, azaspiracides, pectnotoxines) dans les coquillages et les produits base de coquillages par CL-SM/SM Lebensmittel - Bestimmung der lipophilen Algentoxine (Okadasuregruppen-Toxine, Yessotoxine, Azaspirosuren, Pectenotoxine) in Schalentieren und Schalentiererzeugnissen mit LC-MS/MS This European
14、Standard was approved by CEN on 20 April 2012. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerni
15、ng such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into i
16、ts own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland,
17、 Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marn
18、ix 17, B-1000 Brussels 2012 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16204:2012: EEN 16204:2012 (E) 2 Contents Page Foreword 3Introduction .41 Scope 52 Normative references 53 Principle 54 Reagents .55 Apparatus .76 Procedur
19、e .86.1 Preparation of samples .86.1.1 General 86.1.2 Raw samples 86.1.3 Cooked samples 86.2 Homogenization and extraction .86.3 Hydrolysis .87 HPLC-MS/MS analysis .97.1 General 97.2 HPLC operating conditions (chromatography under acidic conditions) .97.3 HPLC operating conditions (chromatography un
20、der basic conditions) . 107.4 Mass spectrometric operating conditions . 107.5 Calibration curve. 107.6 Determination of algal toxins in sample test solutions 117.7 Quality control measures for sequences . 118 Calculation . 128.1 Peak identification 128.2 Quantitative determination by means of extern
21、al calibration and matrix correction 128.3 Description of matrix correction . 138.4 Calculation of the total toxicitiy 149 Precision 1410 Test report . 14Annex A (informative) Precision data . 15A.1 Details on the inter-laboratory study 15A.2 Recovery 28Annex B (informative) Examples for suitable MS
22、 detection conditions . 29B.1 Examples suitable for SCIEX API 4000 or API 4000 Q-Trap . 29B.2 Examples suitable for Waters (Micromass) TSQ Ultima . 31B.3 Examples suitable for Thermo Fisher TSQ Quantum Ultra 33B.4 Examples suitable for Agilent 6410 or 6460 QQQ . 35Annex C (informative) Typical chrom
23、atogram 37Bibliography . 38DIN EN 16204:2012-08 EN 16204:2012 (E) 3 Foreword This document (EN 16204:2012) has been prepared by Technical Committee CEN/TC 275 “Food Analysis - Horizontal methods”, the secretariat of which is held by DIN. This European Standard shall be given the status of a national
24、 standard, either by publication of an identical text or by endorsement, at the latest by November 2012, and conflicting national standards shall be withdrawn at the latest by November 2012. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
25、 rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croat
26、ia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. DIN EN 16204:2012-08
27、EN 16204:2012 (E) 4 Introduction Lipophilic marine biotoxins are the most frequently occurring algal toxins in Europe and are produced by certain marine dinoflagellates. They can accumulate in filter-feeding bivalves reaching highly toxic levels and, after consumption, may cause harm in humans such
28、as nausea, vomiting and diarrhoea. Commission Regulation 15/2011 stipulates LC-MS/MS as the reference methodology and refers to a method validated by the EU-RL Network. The method presented in EN 16204 is proposed as an alternative method to the one validated by EU-RL. DIN EN 16204:2012-08 EN 16204:
29、2012 (E) 5 1 Scope This European Standard specifies a multi-reference method for the determination of lipophilic algal toxins (fat-soluble algal toxins produced by some dinoflagellates) in raw shellfish and shellfish products including cooked shellfish, by liquid chromatography coupled to tandem mas
30、s spectrometry LC-MS/MS 1, 2, 3. This method has been validated in an inter-laboratory study consisting of three parts via the analysis of both naturally contaminated homogenates of blue mussel and spiked extracts of blue mussel, oyster and clam. For further information on the validation, see Annex
31、A. Additional studies have investigated further matrices (see 4, 5). The detection limit for toxins of the okadaic acid group, azaspiracids and pectenotoxins was determined to be 6 g/kg shellfish meat and for yessotoxins 10 g/kg shellfish meat. Quantitative determination of okadaic acid (OA), pecten
32、otoxin-2 (PTX-2), azaspiracid-1 (AZA-1) and yessotoxin (YTX) can be carried out directly by means of standard substances available commercially. Assuming an equal response factor, okadaic acid is used for the indirect quantitative determination of the two dinophysistoxins dinophysistoxin-1 (DTX-1) a
33、nd dinophysistoxin-2 (DTX-2); likewise azaspiracid-1 (AZA-1) is used for the indirect quantitative determination of azaspiracid-2 (AZA-2) and azaspiracid-3 (AZA-3), while YTX is used for homo-yessotoxin, 45-OH-yessotoxin and 45-OH-homo-yessotoxin, and PTX-2 for pectenotoxin-1 (PTX-1). The limit of q
34、uantification (LOQ) for toxins of the okadaic acid group, azaspiracids and pectenotoxins was determined to be 20 g/kg shellfish meat and for yessotoxins 35 g/kg shellfish meat. By means of hydrolysis 6, the esters of okadaic acid, DTX-1 and DTX-2 can also be determined quantitatively as the correspo
35、nding free acids. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (i
36、ncluding any amendments) applies. EN ISO 3696:1995, Water for analytical laboratory use Specification and test methods (ISO 3696:1987) 3 Principle Remove the shellfish meat from the shell and homogenize the total shellfish meat. Extraction is carried out with aqueous methanol ( = 80 %). Separation i
37、s performed on a HPLC reverse-phase column provided with a binary gradient and detection is carried out by means of tandem mass spectrometry using triple quadrupole technology. The concentration of lipophilic toxins is determined by means of external calibration. 4 Reagents If not otherwise specifie
38、d, reagents of analytical grade and solvents suitable for LC-MS/MS shall be used. Water shall be distilled in glass vessels or demineralised before use, or shall be of equivalent purity according to EN ISO 3696:1995. Since the use of this method involves reagents harmful to health, appropriate preca
39、utionary and protective measures such as avoiding skin contact and using an extractor hood shall be taken. 4.1 Aqueous methanol (= 80 %). NOTE The validation data of this method have been elaborated with 80 % aqueous methanol. However, it has been shown (see 4, 5) that equivalent results can be obta
40、ined when using 100 % methanol. 4.2 Acetonitrile DIN EN 16204:2012-08 EN 16204:2012 (E) 6 4.3 Sodium hydoxide, c(NaOH) = 2,5 mol/l. 4.4 Hydrochloric acid, c(HCl) = 2,5 mol/l. 4.5 Formic acid, 98 % to 100 % w/w. 4.6 Ammonium formate 4.7 Nitrogen, gaseous, min purity: 5,0. 4.8 Ammonium hydrogen carbon
41、ate 4.9 HPLC mobile phase 1 (chromatography under acidic conditions) 4.9.1 Eluent A1 Dissolve 126 mg (to give a 2 mmol/l solution) of ammonium formate (4.6) and 2 ml (to give a 50 mmol/l solution) of formic acid (4.5) in 50 ml of water and fill up to 1 000 ml with water. If necessary, filter the elu
42、ent using a 0,45 m membrane filter. 4.9.2 Eluent B1 Dissolve 126 mg (to give a 2 mmol/l solution) ammonium formate (4.6) and 2 ml (to give a 50 mmol/l solution) of formic acid (4.5) in 50 ml of water. Add 950 ml of acetonitrile (4.2) and filter the eluent using a 0,45 m membrane filter, if required.
43、 4.10 HPLC mobile phase 2 (chromatography under basic conditions) 4.10.1 Eluent A2 Dissolve 395 mg (to give a 5 mmol/l solution) of ammonium hydrogen carbonate (4.8) in 1 000 ml of water. If necessary, filter the eluent using a 0,45 m membrane filter. 4.10.2 Eluent B2 Dissolve 395 mg (to give a 5 mm
44、ol/l solution) of ammonium hydrogen carbonate (4.8) in 50 ml water. Add 950 ml of acetonitrile (4.2) in portions of about 100 ml. Shake vigorously after each portion added. If necessary, filter the eluent using a 0,45 m membrane filter. 4.11 Toxin-free shellfish homogenate To estimate and determine
45、matrix effects, standard solutions in matrix are prepared. The shellfish homogenate required for this purpose is prepared from shellfishes that have been proved free from toxins. 4.12 Reference substances1)NOTE During the validation study, the following reference substances were available. If other
46、certified reference substances should become available in the future, the use of these substances is recommended. 4.12.1 Okadaic acid (OA) 4.12.2 Pectenotoxin-2 (PTX-2) 1) Reference substances can be purchased from National Research Council Canada (NRC), Institute for Marine Bioscience, Halifax, for
47、 instance (http:/www.nrc-cnrc.gc.ca). This is an example for suitable products available commercially. This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of these products. DIN EN 16204:2012-08 EN 16204:2012 (E) 7 4.12.3 Azaspiracid-
48、1 (AZA-1) 4.12.4 Yessotoxin (YTX) 4.12.5 Multi-toxin standard solutions Prepare standard calibration solutions (multi-toxin standard) in aqueous methanol (4.1). The following concentrations are recommended: OA, AZA-1, PTX-2: 1,5 ng/ml; 2,5 ng/ml; 5,0 ng/ml; 10,0 ng/ml; 15,0 ng/ml and 25,0 ng/ml; YTX: 3,0 ng/ml; 5,0 ng/ml; 10,0 ng/ml; 20,0 ng/ml; 30,0 ng/ml; and 50,0 n
