1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN 16204:2012Foodstuffs Determination oflipophilic algal toxins (okadaicacid group toxins, yessotoxins,azaspiracids, pectenotoxins) inshellfish and shellfish productsby LC-MS/
2、MSBS EN 16204:2012 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 16204:2012.The UK participation in its preparation was entrusted to TechnicalCommittee AW/275, Food analysis - Horizontal methods.A list of organizations represented on this committee can beobtai
3、ned on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2012. Published by BSI StandardsLimited 2012ISBN 978 0 580 72863 1ICS 67.120.30Compliance with a
4、 British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 31 May 2012.Amendments issued since publicationDate Text affectedBS EN 16204:2012EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM E
5、N 16204 May 2012 ICS 67.120.30 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
6、acide okadaque, yessotoxines, 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
7、 LC-MS/MS This European 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 bibliograph
8、ical references concerning 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 responsibilit
9、y of a CEN member into its 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, Hu
10、ngary, Iceland, Ireland, 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 Manage
11、ment Centre: Avenue Marnix 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: EBS EN 16204:2012EN 16204:2012 (E) 2 Contents Page Foreword 3Introduction .41 Scope 52 Normative references 53 Principl
12、e 54 Reagents .55 Apparatus .76 Procedure .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
13、 operating conditions (chromatography under 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 Quant
14、itative determination by means of external 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
15、B (informative) Examples for suitable MS 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
16、 . 35Annex C (informative) Typical chromatogram 37Bibliography . 38BS EN 16204:2012EN 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 shal
17、l be given the status of a national 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 do
18、cument may be the subject of patent 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
19、: Austria, Belgium, Bulgaria, Croatia, 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 Un
20、ited Kingdom. BS EN 16204:2012EN 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,
21、may cause harm in humans such 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.
22、BS EN 16204:2012EN 16204:2012 (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 chromatogra
23、phy coupled to tandem mass 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 t
24、he validation, see Annex 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
25、okadaic acid (OA), pectenotoxin-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 di
26、nophysistoxin-1 (DTX-1) and 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-
27、1 (PTX-1). The limit of quantification (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 quant
28、itatively as the corresponding 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 t
29、he referenced document (including 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 methano
30、l ( = 80 %). Separation is 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
31、If not otherwise specified, 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
32、health, appropriate precautionary 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 equiv
33、alent results can be obtained when using 100 % methanol. 4.2 Acetonitrile BS EN 16204:2012EN 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 Ammo
34、nium hydrogen carbonate 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 neces
35、sary, filter the eluent 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
36、filter, if required. 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 39
37、5 mg (to give a 5 mmol/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 est
38、imate and determine 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
39、available. If other 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 Bios
40、cience, Halifax, for 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. BS EN 16204:2012EN 16204:2012 (E) 7 4.12
41、.3 Azaspiracid-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
42、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 ng/ml. 4.12.6 Multi-toxin standard solutions in matrix Prepare a solution with a standard concentration level by dilution in a toxin-free methanolic shellfish extract (6.2) to estimate or determine matrix effects. Th
43、e recommended concentration levels are: OA, AZA-1, PTX-2: 15,0 ng/ml; YTX: 30,0 ng/ml. 5 Apparatus Usual laboratory glassware and equipment and, in particular, the following: 5.1 Mechanical high-speed blender or homogenizer (e.g. Grindomix, Ultra-Turrax)2). 5.2 Centrifuge (working at minimum 2 000 g
44、) and centrifuge tubes (volume 20 ml). 5.3 Shaker (e.g. Vortex). 5.4 Heat block. 5.5 Analytical balance, accuracy to the nearest 0,1 mg. 5.6 Volumetric flask, 20 ml or 10 ml. 5.7 Graduated cylinder and suitable pipettes. 5.8 High Performance Liquid Chromatography (HPLC) system, capable of gradient e
45、lution. 5.9 HPLC vials. 5.10 Triple-Quadrupol-LC-MS/MS system. 5.11 Analytical Reversed Phase Column, e.g. RP C18, particle size 3 m, 150 mm (length) 2 mm (diameter) or C8, 50 mm (length) x 2 mm (diameter), 3 m particle size, (only for acidic conditions). Optionally, an appropriate guard column may
46、be used. 5.12 Syringe or membrane filter (e.g. regenerated cellulose membranes with a pore diameter of 0,45 m). 2) Ultra Turrax is an example for a suitable product available commercially from various suppliers. This information is given for the convenience of users of this standard and does not con
47、stitute an endorsement by CEN of this product. BS EN 16204:2012EN 16204:2012 (E) 8 5.13 Syringe for filter system, e.g. 1 ml. 5.14 High-speed table centrifuge (up to 10 000 g). 6 Procedure 6.1 Preparation of samples 6.1.1 General Storage of shellfish samples in frozen state (at 18 C) for up to a yea
48、r has no negative influence (see 7) on the results obtained with this method. 6.1.2 Raw samples After receipt, the raw samples should be shelled and drained, and then frozen until used for analysis or shall be extracted immediately. 6.1.3 Cooked samples The sample is cooked/steamed after receipt the
49、n drained and shelled. If it is not immediately treated, it can be cooled down until 24 h. To cook the shellfish, heat a sufficient amount of water (approximately 2 l to 3 l water per 1 kg of shellfish) to the boiling point. When the water is boiling, place shellfish into the water and cook under further addition of heat for approximately 3 min. After cooking, remove the shellfish me
