SIST EN 14526:2005
(Main)Foodstuffs - Determination of saxitoxin and dc-saxitoxin in mussels - HPLC method using pre-column derivatization with peroxide or periodate oxidation
Foodstuffs - Determination of saxitoxin and dc-saxitoxin in mussels - HPLC method using pre-column derivatization with peroxide or periodate oxidation
This European standard specifies a method for the quantitative determination of saxitoxin (STX) and decarbamoyl saxitoxin (dc-STX) in mussels. It may also be applicable in other shellfish, for example scallops. The limit of determination of this method (signal/noise = 10) is 0,006 mg/kg for saxitoxin and 0,02 mg/kg for dc-saxitoxin in mussel meat. The method has been tested for saxitoxin at levels at 0,4 mg/kg and 0,5 mg/kg and for dc-saxitoxin at levels at 0,4 mg/kg and 1,6 mg/kg.
Lebensmittel - Bestimmung von Saxitoxin und DC-Saxitoxin in Muscheln - HPLC-Verfahren mit Vorsäulenderivatisierung mit Peroxid- oder Periodatoxdation
Dieses Dokument legt ein Verfahren für die quantitative Bestimmung von Saxitoxin (STX) und Decarbamoyl-Saxitoxin (DCSTX) in Miesmuscheln fest. Es darf auch auf andere Muscheln angewendet werden, z. B. Jacobsmuscheln. Die Bestimmungsgrenze dieses Verfahrens (Signal/Rauschverhältnis = 10) ist 0,006 mg/kg für Saxitoxin und 0,02 mg/kg für DC-Saxitoxin in Muschelfleisch. Dieses Verfahren wurde im Ringversuch für Saxitoxingehalte von 0,4 mg/kg und 0,5 mg/kg und für DC-Saxitoxingehalte von 0,4 mg/kg und 1,6 mg/kg geprüft.
Produits alimentaires - Détermination de la teneur en saxitoxine et en dc-saxitoxine dans les moules - Méthode par CLHP avec dérivation pré-colonne et par oxydation au peroxide ou au periodate
Le présent document spécifie une méthode de détermination quantitative de la saxitoxine (STX) et de la décarbamoyle saxitoxine (dc-STX) dans les moules. Cette méthode peut également s'appliquer a d'autres mollusques, par exemple les coquilles St-Jacques. La limite de quantification de cette méthode (signal/bruit = 10) est de 0,006 mg/kg pour la saxitoxine et de 0,02 mg/kg pour la dc-saxitoxine dans la chair de moule. Cette méthode a été évaluée pour la saxitoxine aux teneurs de 0,4 mg/kg et 0,5 mg/kg et pour la dc-saxitoxine aux teneurs de 0,4 mg/kg et 1,6 mg/kg.
Živila - Določevanje saksitoksina in dc-saksitoksina v školjkah - Metoda HPLC z uporabo predkolonske derivatizacije s peroksidno ali perjodatno oksidacijo
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Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.NVLGDFLMRLebensmittel - Bestimmung von Saxitoxin und DC-Saxitoxin in Muscheln - HPLC-Verfahren mit Vorsäulenderivatisierung mit Peroxid- oder PeriodatoxdationProduits alimentaires - Détermination de la teneur en saxitoxine et en dc-saxitoxine dans les moules - Méthode par CLHP avec dérivation pré-colonne et par oxydation au peroxide ou au periodateFoodstuffs - Determination of saxitoxin and dc-saxitoxin in mussels - HPLC method using pre-column derivatization with peroxide or periodate oxidation67.120.30Ribe in ribji proizvodiFish and fishery productsICS:Ta slovenski standard je istoveten z:EN 14526:2004SIST EN 14526:2005en01-januar-2005SIST EN 14526:2005SLOVENSKI
STANDARD
SIST EN 14526:2005
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14526August 2004ICS 67.120.30English versionFoodstuffs - Determination of saxitoxin and dc-saxitoxin inmussels - HPLC method using pre-column derivatization withperoxide or periodate oxidationProduits alimentaires - Détermination de la teneur ensaxitoxine et en dc-saxitoxine dans les moules - Méthodepar CLHP avec dérivation pré-colonne et par oxydation auperoxide ou au periodateLebensmittel - Bestimmung von Saxitoxin und DC-Saxitoxinin Muscheln - HPLC-Verfahren mitVorsäulenderivatisierung mit Peroxid- oderPeriodatoxdationThis European Standard was approved by CEN on 21 May 2004.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards 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 any other language made by translationunder the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2004 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14526:2004: ESIST EN 14526:2005
EN 14526:2004 (E) 2 Contents Page Foreword.3 Introduction.4 1 Scope.5 2 Normative references.5 3 Principle.5 4 Reagents.6 5 Apparatus.9 6 Procedure.11 7 HPLC determination.13 8 Calibration graph.13 9 Identification.14 10 Calculation.14 11 Precision.14 12 Test report.15 Annex A (informative)
Performance data.16 Bibliography.17
SIST EN 14526:2005
EN 14526:2004 (E) 3 Foreword This document (EN 14526:2004) 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 standard, either by publication of an identical text or by endorsement, at the latest by February 2005, and conflicting national standards shall be withdrawn at the latest by February 2005.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. WARNING — The use of this standard can involve hazardous materials, operations and equipment. This standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. SIST EN 14526:2005
EN 14526:2004 (E) 4 Introduction This document is based on a procedure described by Lawrence and Ménard [1]. In this original procedure, the C18 solid phase extraction is followed by a solid phase extraction with a weak cation exchanger. In the latter step, three different groups of shellfish toxins are being separated by elution with mobile phases of different ionic strength and polarity. The different groups of toxins are then derivatized with periodate or peroxide depending on the peak responses of the different toxins to the respective derivatization reactions. The method described in this document is specifically for saxitoxin and decarbamoyl (dc)-saxitoxin, so only the C18 clean up is applied. The method described in this document offers the option to choose between an extraction with acetic acid (HAc) or an extraction with hydrochloric acid (HCl). The analyst’s choice depends on his desires and final needs. HCl extraction with boiling leads to partial hydrolysis of the PSP toxins, leading to conversion of some of the PSP toxins (C-1, C-2, GTX-5) into more toxic analogues (GTX-2, GTX-3, saxitoxin). This process also occurs more or less in the human stomach. This extraction can therefore be considered a reasonable approach to estimate potential toxicity. HAc extraction without boiling is a milder extraction procedure, which leaves the toxin profile of the sample practically intact. This extraction procedure can be applied if it is considered more important to estimate the actual toxin profile. The HAc extraction has been applied in the development of (certified) reference material for PSP toxins [2] and [3]. SIST EN 14526:2005
EN 14526:2004 (E) 5 1 Scope This document specifies a method for the quantitative determination of saxitoxin (STX) and decarbamoyl saxitoxin (dc-STX) in mussels. It may also be applicable in other shellfish, for example scallops. The limit of determination of this method (signal/noise = 10) is 0,006 mg/kg for saxitoxin and 0,02 mg/kg for dc-saxitoxin in mussel meat. The method has been tested for saxitoxin at levels at 0,4 mg/kg and 0,5 mg/kg and for dc-saxitoxin at levels at 0,4 mg/kg and 1,6 mg/kg. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987) 3 Principle Paralytic Shellfish Poisoning (PSP) toxins are extracted from mussel meat homogenate with acetic acid (HAc) or with hydrochloric acid (HCl). After centrifugation the supernatant is purified by solid phase extraction (SPE) over a C18 clean-up cartridge. Part of the extract is derivatized with hydrogen peroxide. WARNING — PSP toxins are strong neurotoxins. Gloves and safety glasses shall be worn at all times, and all standard and sample preparation stages shall be carried out in a fume cupboard. Derivatization of STX and dc-STX leads to several oxidation products that are separated by reverse phase HPLC with fluorescence detection. Table 1 gives the reaction products for STX and dc-STX after derivatization with hydrogen peroxide. Table 1 — Reaction products after derivatization with hydrogen perioxide Toxin Eluting order Intensity Peak name of reaction product STX first + dc-STX oxid. (1)
second + dc-STX oxid. (2)
third ++ STX oxid. dc-STX first ++ dc-STX oxid. (1)
second + dc-STX oxid. (2)
The method described in this document is suitable for the quantitative determination of the PSP toxins STX and dc-STX. The following should however be considered. Co-occurrence of different PSP toxins in mussels could influence the analytical results, because some of the PSP toxins could (partially) lead to the same reaction products. So the chromatograms shall be carefully interpreted. The analytical result obtained for STX with hydrogen peroxide oxidation is not influenced by the presence of other toxins. SIST EN 14526:2005
EN 14526:2004 (E) 6 The analytical result obtained for dc-STX with hydrogen peroxide oxidation is slightly influenced by the presence of STX, because the ''dc-STX oxid. (1)'' reaction product of STX is the same as the main reaction product of dc-STX (see Table 1). The contribution of STX to the dc-STX peak is approximately 4 % of the peak area of the STX peak [4] and [5]. If oxidised with peroxide, the gonyautoxins GTX-2 and GTX-3 lead to the same reaction product, whereas GTX-1 and GTX-4 can not be detected. If oxidised with periodate, GTX-1, GTX-2, GTX-3 and GTX-4 lead to the same reaction products. GTX-5 can be detected with peroxide oxidation, neo-STX only with periodate oxidation. 4 Reagents 4.1 General During the analysis, unless otherwise stated, use only water according to grade 1 of EN ISO 3696. All chemicals shall be of pro analysis (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 Ammonium formate 4.4.1 Ammonium formate solution 1, substance concentration c = 0,3 mol/l Dissolve 18,9 g ammonium formate (4.4) in water and make up to 1 l with water. 4.4.2 Ammonium formate solution 2, c = 0,1 mol/l Dilute 350 ml of ammonium formate solution 1 (4.4.1) with 700 ml of water. Adjust to pH 6 with acetic acid (pH paper) and filter over a membrane filter (5.15). Prepare fresh every 2 days of analysis. 4.5 Hydrochloric acid solution 4.5.1 General volume fraction 3 ≈ 25 % (acidimetric) 4.5.2 Hydrochloric acid solution 1, c = 5 mol/l Dilute 100 ml of hydrochloric acid solution (4.5) with 54 ml of water. 4.5.3 Hydrochloric acid solution 2, c = 0,1 mol/l Dilute 5 ml of hydrochloric acid solution 1 (4.5.2) with 245 ml of water. 4.6 Sodium hydroxide 4.6.1 Sodium hydroxide solution 1, c = 5 mol/l Dissolve 20 g of sodium hydroxide (4.6) in 100 ml of water. SIST EN 14526:2005
EN 14526:2004 (E) 7 4.6.2 Sodium hydroxide solution 2, c = 1 mol/l Dilute 1 ml of sodium hydroxide solution 1 (4.6.1) with 4 ml of water. 4.6.3 Sodium hydroxide solution 3, c = 0,1 mol/l Dilute 1 ml of sodium hydroxide solution 2 (4.6.2) with 9 ml of water. 4.7 Hydrogen peroxide solution, volume fraction 3 = 10 % Dilute 1 ml of commercially available hydrogen peroxide solution, of mass fraction
= 30 % with 2 ml of water. Store cool and in the dark. Prepare fresh every week. 4.8 Periodic acid solution, c = 0,03 mol/l Dissolve 0,68 g of periodic acid in 100 ml of water. 4.9 Disodium hydrogenphosphate solution, c = 0,3 mol/l Dissolve 0,53 g of disodium hydrogenphosphate dihydrate in 10 ml of water. 4.10 Acetic acid, 100 % 4.10.1 Acetic acid solution 1, c = 1 mol/l Dilute 57,2 ml of acetic acid solution (4.10) to 1,0 l with water. 4.10.2 Acetic acid solution 2, c = 0,1 mol/l Dilute 10 ml of acetic acid solution 1 (4.10.1) with 90 ml of water. 4.10.3 Acetic acid solution 3, c = 0,2 mol/l Dilute 200 ml of acetic acid solution 1 (4.10.1) with 800 ml of water. 4.10.4 Acetic acid solution 4, c = 0,03 mol/l Dilute 300 ml of acetic acid solution 2 (4.10.2) with 700 ml of water. 4.11 Helium 4.12 Peroxide derivatization reagent Dilute 400 µl of hydrogen peroxide solution (4.7) with 4 ml of sodium hydroxide solution 2 (4.6.2) and mix well. Prepare fresh every day of analysis. 4.13 Periodate derivatization reagent Mix 1 volume part of periodic acid solution (4.8) with 1 volume part of disodium hydrogenphosphate solution (4.9) and 1 volume part of ammonium formate solution 1 (4.4.1). Bring the mixture to about pH 8 by dropwise adding sodium hydroxide solution 2 (4.6.2) and check the pH by using pH paper. Prepare fresh every day of analysis just before use. SIST EN 14526:2005
EN 14526:2004 (E) 8 4.14 Saxitoxin standard substance 4.14.1 Saxitoxin stock solution, mass concentration
= 1,0 µg/ml Prepare a stock solution containing 1,0 µg/ml of saxitoxin in acetic acid solution 4 (4.10.4) by weighing with the analytical balance (5.3). Store the solution cool (approximately + 4 °C) and in the dark. This solution is stable for at least 5 months (see [6]). NOTE Ampoules containing saxitoxin (STX·2HCl) standard are for example available from the National Research Council Canada, Halifax, Canada (set of calibrants PSP-1) 1) . 4.14.2 Saxitoxin calibration solutions Prepare for the determination of saxitoxin a calibration series of increasing concentration in the mass concentration range of 0,05 µg/ml to 0,30 µg/ml from the saxitoxin stock solution (4.14.1) in acetic acid solution 4 (4.10.4). Prepare fresh for every series of analysis. 4.15 Dc-saxitoxin standard substance 4.15.1 Dc-saxitoxin stock solution,
= 1,0 µg/ml Prepare a stock solution containing 1,0 µg/ml of dc saxitoxin in acetic acid solution 4 (4.10.4) by weighing with the analytical balance (5.3). Store the solution cool (approximately +4 °C) and in the dark. This solution is stable for at least 5 months (see [6]). NOTE Ampoules containing saxitoxin (dc-STX) standard are for example available from the National Research Council Canada, Halifax, Canada (set of calibrants PSP-1) 1). 4.15.2 Dc-saxitoxin calibration solutions Prepare for the determination of dc-saxitoxin a calibration series of increasing concentration in the mass concentration range of 0,05 µg/ml to 0,30 µg/ml from the dc-saxitoxin stock solution (4.15.1) in acetic acid solution 4 (4.10.4). Prepare fresh for every series of analysis. 4.16 HPLC mobile phase 4.16.1 Ammonium formate solution Filter the ammonium formate solution 2 (4.4.2) before use through a membrane filter (5.15) using vacuum and degas (e.g. using helium sparging). Store in the dark at approximately 4 °C and make fresh every 2 days. 4.16.2 Mixture of acetonitrile and ammonium formate solution Mix 50 ml of acetonitrile (4.3) with 950 ml of filtered ammonium formate solution (4.16.1). Degas (e.g. using
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