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1、DRAFT FOR DEVELOPMENT DD ENV 14194:2002 Foodstuffs Determination of saxitoxin and dc-saxitoxin in mussels HPLC method using post column derivatisation ICS 67.120.30 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW Licensed Copy: London South Bank University, London South Bank U
2、niversity, Tue Dec 12 05:31:25 GMT+00:00 2006, Uncontrolled Copy, (c) BSI DD ENV 14194:2002 This Draft for Development, having been prepared under the direction of the Consumer Products and Services Sector Policy and Strategy Committee, was published under the authority of the Standards Policy and S
3、trategy Committee on 16 May 2002 BSI 16 May 2002 ISBN 0 580 39628 2 National foreword This Draft for Development is the English language version of ENV 14194:2002. This publication is not to be regarded as a British Standard. It is being issued in the Draft for Development series of publications and
4、 is of a provisional nature because the method has not been validated. It should be applied on this provisional basis, so that information and experience of its practical application may be obtained. Comments arising from the use of this Draft for Development are requested so that UK experience can
5、be reported to the European organization responsible for its conversion into a European Standard. A review of this publication will be initiated 2 years after its publication by the European organization so that a decision can be taken on its status at the end of its three-year life. The commencemen
6、t of the review period will be notified by an announcement in Update Standards. According to the replies received by the end of the review period, the responsible BSI Committee will decide whether to support the conversion into a European Standard, to extend the life of the prestandard or to withdra
7、w it. Comments should be sent in writing to the Secretary of BSI Technical Committee AW/-/3, Horizontal analysis, at 389 Chiswick High Road, London W4 4AL, giving the document reference and clause number and proposing, where possible, an appropriate revision of the text. A list of organizations repr
8、esented on this committee can be obtained on request to its secretary. Cross-references The British Standards 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 Correspond
9、ence Index”, or by using the “Find” facility of the BSI Standards Electronic Catalogue. Summary of pages This document comprises a front cover, an inside front cover, the ENV title page, pages 2 to 11 and a back cover. The BSI copyright date displayed in this document indicates when the document was
10、 last issued. Amendments issued since publication Amd. No. DateComments Licensed Copy: London South Bank University, London South Bank University, Tue Dec 12 05:31:25 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EUROPEAN PRESTANDARD PRNORME EUROPENNE EUROPISCHE VORNORM ENV 14194 April 2002 ICS 67.120.
11、30 English version Foodstuffs - Determination of saxitoxin and dc-saxitoxin in mussels - HPLC method using post column derivatisation Produits alimentaires - Dtermination de la teneur en saxitoxine et en dc-saxitoxine dans les moules - Mthode par chromatographie liquide haute performance aprs drivat
12、ion post-colonne Lebensmittel - Bestimmung von Saxitoxin und DC-Saxitoxin in Muscheln - HPLC-Verfahren mit Nachsulenderivatisierung This European Prestandard (ENV) was approved by CEN on 14 December 2001 as a prospective standard for provisional application. The period of validity of this ENV is lim
13、ited initially to three years. After two years the members of CEN will be requested to submit their comments, particularly on the question whether the ENV can be converted into a European Standard. CEN members are required to announce the existence of this ENV in the same way as for an EN and to mak
14、e the ENV available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the ENV) until the final decision about the possible conversion of the ENV into an EN is reached. CEN members are the national standards bodies of
15、Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG
16、Management Centre: rue de Stassart, 36 B-1050 Brussels 2002 CENAll rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. ENV 14194:2002 E Licensed Copy: London South Bank University, London South Bank University, Tue Dec 12 05:31:25 GMT+00:00 2006,
17、 Uncontrolled Copy, (c) BSI ENV 14194:2002 (E) 2 Foreword This document (ENV 14194:2002) has been prepared by Technical Committee CEN /TC 275, “Food analysis - Horizontal methods“, the secretariat of which is held by DIN. According to the CEN/CENELEC Internal Regulations, the national standards orga
18、nizations of the following countries are bound to announce this European Prestandard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. 1 Scope Thi
19、s European Prestandard specifies a method for the quantitative determination of saxitoxin (STX), dc-saxitoxin (dc-STX) and the qualitative determination of neo-saxitoxin, and the gonyautoxins GTX-2 and GTX-3 in mussels. The method can also be used to identify the N-sulfocarbamoyl toxins C-1, C-2, GT
20、X-5 and GTX-6 after hydrolysis and, if these toxins are present, to exclude false positive results for GTX-2, GTX-3, neo-saxitoxin and saxitoxin. For mussel the limit of quantification is for saxitoxin 0,04 mg/kg mussel meat and for dc-saxitoxin 0,03 mg/kg mussel meat (signal/noise = 10). The limits
21、 of detection for C-1, C-2, GTX-2, GTX-3, GTX-5, GTX-6 and neo-saxitoxin have not been determined. 2 Normative references This European Prestandard incorporates by dated or undated reference, provisions from other publications. These normative references are cited at the appropriate places in the te
22、xt and the publications are listed hereafter. For dated references, subsequent amendments to or revisions of any of these publications apply to this European Prestandard only when incorporated in it by amendment or revision. For undated references the latest edition of the publication referred to ap
23、plies (including amendments). EN ISO 3696:1995, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987) 3 Principle PSP (Paralytic Shellfish Poisoning) toxins are extracted from mussels homogenate with an acidic aqueous solution. After centrifugation the supernatant is p
24、urified by solid phase extraction (SPE) over a C18 clean-up cartridge. Part of the extract is directly injected on the HPLC for toxin determination. After the separation on the analytical column post column derivatisation is used, with oxidation with periodic acid and after acidification the derivat
25、ized toxins are detected using fluorimetric detection. HPLC analysis is separated in two parts: a system to separate the saxitoxin group toxins (saxitoxin, dc-saxitoxin and neo-saxitoxin) and a system to separate the GTX group toxins (GTX-2 and GTX-3 ). Identification after hydrolysis is based on th
26、e transformation of the N-sulfo-carbamoyl toxins to their corresponding carbamoyl toxins. WARNING - PSP toxins are strong neurotoxins. Gloves and safety glasses should be worn at all times, and all standard and sample preparation stages should be carried out in a fume cupboard. Licensed Copy: London
27、 South Bank University, London South Bank University, Tue Dec 12 05:31:25 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ENV 14194:2002 (E) 3 4 Reagents 4.1 General During the analysis, unless otherwise stated, use only water according to grade 1 of EN ISO 3696:1995. All chemicals shall be of pro analys
28、is (p.a) quality, unless otherwise indicated. Reference materials (calibrants of the toxins) originating from other sources than indicated may also be used if well-characterised and with a well-defined mass concentration. 4.2 Methanol 4.3 Acetonitrile, HPLC quality 4.4 Hydrochloric acid, volume conc
29、entration ?(HCl) 25 %, (acidimetric) 4.4.1 Hydrochloric acid solution, substance concentration c = 1,0 mol/l Dilute 130 ml of hydrochloric acid solution (4.4) with 870 ml of water. 4.4.2 Hydrochloric acid solution, c = 0,1 mol/l Dilute 100 ml of hydrochloric acid solution (4.4.1) with 900 ml of wate
30、r. 4.5 Octane sulfonic acid solution, c = 0,5 mol/l Dissolve 2,9 g of octane sulfonic acid sodium salt monohydrate in 25,0 ml of water. 4.6 Phosphoric acid, the mass fraction is 85 % 4.6.1 Phosphoric acid solution, c = 0,5 mol/l Take 6,7 ml of phosphoric acid (4.6) and dilute to 200,0 ml with water.
31、 4.7 Ammonia solution, ? = 25 % and ? = 1 % For the 1 % solution, dilute 40 ml ammonia solution ?= 25 % with 960 ml of water. 4.8 Periodic acid solution, c = 0,01 mol/l Dissolve 1,14 g of periodic acid in water and dilute up to 500 ml with water. 4.9 Acetic acid, 100 % 4.9.1 Acetic acid solution 1,
32、c = 1 mol/l Take 57,2 ml of acetic acid solution (4.9) and dilute to 1,0 l with water. 4.9.2 Acetic acid solution 2, c = 0,2 mol/l Dilute 200 ml of acetic acid solution 1 (4.9.1) with 800 ml of water. 4.9.3 Acetic acid solution 3, c = 0,03 mol/l Dilute 150 ml of acetic acid solution 2 (4.9.2) with 8
33、50 ml of water. Licensed Copy: London South Bank University, London South Bank University, Tue Dec 12 05:31:25 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ENV 14194:2002 (E) 4 4.10 Helium 4.11 Saxitoxin standard solutions 4.11.1 Saxitoxin stock solution, mass concentration ? = 1,0 g/ml Prepare a cali
34、bration solution containing 1,0 g/ml of saxitoxin in acetic acid solution 3 (4.9.3). Store the calibration solution cool (at approximately + 4 C) and in the dark. This solution is stable for at least 6 months. NOTE 1An ampoule containing approximately 0,2 ml of saxitoxin calibration solution, mass c
35、oncentration of saxitoxin of 0,14 mg/ml is for example available from the National Research Council Canada, Halifax, Canada (set of calibrants PSP-1). In order to obtain a calibration solution of 1,0 g/ml, the content of the ampoule can be quantitatively transferred to 27,8 ml acetic acid 3 (4.9.3)
36、(total volume 28,0 ml). The total volume can be checked and adjusted by weighing (5.9), using density of water for calculations. For this purpose weigh the ampoule before opening, take care that all glass is preserved after opening and weigh again after the calibrant solution is transferred. NOTE 2A
37、 calibration solution containing GTX-1 and GTX-4 is also available and may be used for qualitatively determination of these toxins. This has not been tested in the certification study of the Standard Measurements anodised aluminium spring clamp in combination with a filtering flask of 1 l. 5.9 Analy
38、tical balance 6 Procedure 6.1 Sample preparation Let mussels thaw if necessary. Rinse shell with tap-water once to remove sand and let the mussels drain to remove excess of water. Remove the meat from the shells. Homogenize approximately 100 g of the mussel meat in a grinder (5.1). Freeze the homoge
39、nised mussels if not directly proceeding with the analysis. 6.2 Extraction procedure Weigh a test portion of 25 g to the nearest 100 mg of the thawed homogeneous mussel mixture in a beaker. Add 25 ml of acetic acid solution 2 (4.9.2) to the beaker. Mix 10 min under constant stirring with a magnetic
40、stirrer. Filter the total mixture of a fluted filter paper. Check the pH with pH paper. If the pH of the mixture is not between pH = 3 and pH = 5 dropwise add hydrochloric acid solution (4.4.1) until it is. If not proceeding directly with the analysis, the extract may be stored cool (approximately +
41、 4 C) and in the dark for a maximum of 5 days (risk of fungal growth in extract). Licensed Copy: London South Bank University, London South Bank University, Tue Dec 12 05:31:25 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ENV 14194:2002 (E) 7 NOTEIf stored longer than 5 days, there is a risk of fungal
42、 growth in the extract. 6.3 Sample purification Place an SPE-column (5.3) on a vacuum manifold system (5.2) and apply a mild vacuum. Condition the column (5.3) successively with: 3 ml of methanol (4.2), 3 ml of water and 1,5 ml of hydrochloric acid solution (4.4.2). Ensure that the column does not r
43、un dry during these operations. Remove vacuum and place a collection tube under the column and apply vacuum. Apply 0,5 ml (Vc) of the extract on the column. Let the extract be absorbed on the column and collect the effluent in a collection tube and take care that the column does not run dry during t
44、hese steps. Rinse the column with 2,0 ml of water and combine the eluate with the effluent in the collection tube. After this step let the column run dry to remove all the liquid from the column. Remove vacuum and remove the collection tube containing the purified extract from the vacuum manifold sy
45、stem. Take the combined fractions and dilute to 3,0 ml with water and mix well (Ve). Transfer approximately 0,5 ml of the extract to an HPLC vial for analysis. This is the sample test solution. 7 HPLC 7.1 HPLC conditions The HPLC conditions are different for the two groups of toxins. For a schematic
46、 overview of the system, see figure 1, 4. Key 1HPLC pump 2Column 3Block heater 4Data system 5Detector 6Reaction coil 7Pump A 8Helium 9Oxidant 10Pump B 11Acid Figure 1 Configuration of HPLC system 1 1 54 3 1 0 6 1 2 7 8 9 Licensed Copy: London South Bank University, London South Bank University, Tue
47、Dec 12 05:31:25 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ENV 14194:2002 (E) 8 Allow the instruments to stabilise before starting the measurements. Check if the system is stable by injecting the same calibration solution 2 to 3 times. The system is considered stable if the peak areas of 2 subsequen
48、t injections do not differ more than 6% from each other. For the saxitoxin group, use eluent A (4.17) at a flow of 0,8 ml/min. Use the 10 cm C18 column (5.6.5). For the GTX group, use eluent B (4.18) at a flow of 0,6 ml/min. Use the 20 cm C18 column (5.6.6). For post column derivatisation, adjust th
49、e temperature of the postcolumn reaction system (5.6.7.2) to 30 C. Take the post column derivatisation reagent (4.19) and add this reagent to the eluent just after the column at a flow of 0,3 ml/min (5.6.7). Just after the reaction coil (just before the detector) add acetic acid 1 (4.9.1). Thus the pH of the solution just before the detector is 3, than the concerned sample is possibly positive for that toxin. For confirmation inject a combination of the calibration solution and the extract. Confirm the presence of neo-saxitoxin by re-injecting the parti