SIST ENV 14194:2002
(Main)Foodstuffs - Determination of saxitoxin and dc-saxitoxin in mussels - HPLC method using post column derivatisation
Foodstuffs - Determination of saxitoxin and dc-saxitoxin in mussels - HPLC method using post column derivatisation
This European Prestandard specifies a method for the quantitative determination of saxitoxin, dc-saxitoxin and the qualitative determination of neo-saxitoxin, and the gonyau toxins GTX-2 and GTX-3 in mussels. The method can also be used to identify the toxins C-1, C-2, GTX-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 lowest limit of determination is for saxitoxin 0,04 mg/kg mussel meat and for dc-saxitoxin 0,03 mg/kg mussel meat (signal/noise = 10).
The upper limits of determination have not been determined. The limits of detection for C-1, C-2, GTX-2, GTX-3, GTX-5, GTX-6 and neo-saxitoxin have not been determined.
Lebensmittel - Bestimmung von Saxitoxin und DC-Saxitoxin in Muscheln - HPLC-Verfahren mit Nachsäulenderivatisierung
Diese Europäische Vornorm legt ein Verfahren zur quantitativen Bestimmung von Saxitoxin (Stx) und DC-Saxitoxin (DC-Stx) und zur qualitativen Bestimmung von Neo-Saxitoxin und den Gonyau-Toxinen GTX-2 und GTX-3 in Muscheln fest. Das Verfahren kann auch zum Nachweis der N-Sulfocarbamoyltoxine C-1, C-2, GTX-5 und GTX-6 nach Hydrolyse angewandt und bei Vorliegen dieser Toxine zum Ausschluss falsch positiver Ergebnisse für GTX-2, GTX-3, Neo-Saxitoxin und Saxitoxin verwendet werden. Bei Muscheln ist die Bestimmungsgrenze für Saxitoxin 0,04 mg/kg Muschelfleisch und für DC-Saxitoxin 0,03 mg/kg Muschelfleisch (Signal-Rausch-Verhältnis = 10).
Die Nachweisgrenzen für C-1, C-2, GTX-2, GTX-3, GTX-5, GTX-6 und Neo-Saxitoxin sind nicht ermittelt worden.
Produits alimentaires - Détermination de la teneur en saxitoxine et en dc-saxitoxine dans les moules - Méthode par chromatographie liquide haute performance apres dérivation post-colonne
Cette Prénorme européenne spécifie une méthode de détermination quantitative de la saxitoxine (STX) et de la dc-saxitoxine (dc-STX) et de détermination qualitative de la néo-saxitoxine et des gonyautoxines GTX-2 et GTX-3 présentes dans les moules. Cette méthode peut également etre utilisée pour identifier les toxines de N-sulfo-carbamoyles C-1, C-2, GTX-5 et GTX-6 apres hydrolyse et, si ces toxines sont présentes, pour identifier les résultats faux positifs pour la GTX-2, la GTX-3, la néo-saxitoxine et la saxitoxine. Pour les moules, la limite de détection est de 0,04 mg/kg de chair de moule pour la saxitoxine, de 0,03 mg/kg de chair de moule pour la dc-saxitoxine (signal/bruit = 10).
Les limites de détection pour la C-1, la C-2, la GTX-2, la GTX-3, la GTX-5, la GTX-6 et la néo-saxitoxine n'ont pas été déterminées.
Živila - Določevanje saksitoksina in dc-saksitoksina v školjkah - Metoda HPLC z uporabo postkolonske derivatizacije
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST ENV 14194:2002
01-september-2002
äLYLOD'RORþHYDQMHVDNVLWRNVLQDLQGFVDNVLWRNVLQDYãNROMNDK0HWRGD+3/&]
XSRUDERSRVWNRORQVNHGHULYDWL]DFLMH
Foodstuffs - Determination of saxitoxin and dc-saxitoxin in mussels - HPLC method using
post column derivatisation
Lebensmittel - Bestimmung von Saxitoxin und DC-Saxitoxin in Muscheln - HPLC-
Verfahren mit Nachsäulenderivatisierung
Produits alimentaires - Détermination de la teneur en saxitoxine et en dc-saxitoxine dans
les moules - Méthode par chromatographie liquide haute performance apres dérivation
post-colonne
Ta slovenski standard je istoveten z: ENV 14194:2002
ICS:
67.120.30 Ribe in ribji proizvodi Fish and fishery products
SIST ENV 14194:2002 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST ENV 14194:2002
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SIST ENV 14194:2002
EUROPEAN PRESTANDARD
ENV 14194
PRÉNORME EUROPÉENNE
EUROPÄISCHE VORNORM
April 2002
ICS 67.120.30
English version
Foodstuffs - Determination of saxitoxin and dc-saxitoxin in
mussels - HPLC method using post column derivatisation
Produits alimentaires - Détermination de la teneur en Lebensmittel - Bestimmung von Saxitoxin und DC-Saxitoxin
saxitoxine et en dc-saxitoxine dans les moules - Méthode in Muscheln - HPLC-Verfahren mit
par chromatographie liquide haute performance après Nachsäulenderivatisierung
dérivation post-colonne
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 limited 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 make 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 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
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36 B-1050 Brussels
© 2002 CEN All rights of exploitation in any form and by any means reserved Ref. No. ENV 14194:2002 E
worldwide for CEN national Members.
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SIST ENV 14194:2002
ENV 14194:2002 (E)
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 organizations 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
This 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, GTX-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 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 text 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 applies (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 purified 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 derivatized 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 the 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.
2
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SIST ENV 14194:2002
ENV 14194:2002 (E)
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 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 Hydrochloric acid, volume concentration (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 water.
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.
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, 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 850 ml of water.
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SIST ENV 14194:2002
ENV 14194:2002 (E)
4.10 Helium
4.11 Saxitoxin standard solutions
4.11.1 Saxitoxin stock solution, mass concentration = 1,0 μg/ml
Prepare a calibration solution containing 1,0 μg/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 1 An ampoule containing approximately 0,2 ml of saxitoxin calibration solution, mass concentration 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 mg/ml, the content of the ampoule can be quantitatively transferred to 27,8 ml acetic
acid 3 (4.9.3) (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 2 A 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 & Testing Programme, designed
to certify the mass fractions of saxitoxin and dc-saxitoxin in mussel reference materials, see [6].
4.11.2 Saxitoxin calibration solutions, = 0,05 μg/ml to 0,30 μg/ml
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 saxitoxin stock solution (4.11.1) in acetic acid solution 3 (4.9.3). Prepare
fresh every day of analysis.
4.12 Dc-saxitoxin standard substance
Store cool and in the dark.
4.13 Dc-saxitoxin stock solution, = 1,0 μg/ml
Prepare from dc-saxitoxin standard substance (4.12) a dc-saxitoxin calibration solution 1,0 μg/ml in acetic acid
solution 3 (4.9.3) by weighing (5.9), using density of water for calculations. This solution is stable for at least
5 months (see Bibliography, [3]).
4.14 Dc-saxitoxin calibration solution, = 0,05 mg/ml to 0,30 μg/ml
Prepare for the determination of dc-saxitoxin a calibration series of increasing concentration in the mass
concentration range of 0,05 mg/ml to 0,30 μg/ml from Dc-saxitoxin stock solution in acetic acid solution 3 (4.9.3).
Prepare fresh every day of analysis.
NOTE For the quantitative determination of saxitoxin and dc-saxitoxin a combined calibration solution for these 2 toxins
can be made if the valley between the 2 toxin peaks in the HPLC chromatogram is £ 10% of the sum of the peak heights. If this
condition is fulfilled in the HPLC separation, one combined calibration solution containing saxitoxin and dc-saxitoxin can be
prepared in the mass concentration range of 0,05 μg/ml to 0,30 μg/ml (for both toxins) in acetic acid solution 3 (4.9.3).
4.15 Neo-Saxitoxin standard solutions
4.15.1 Neo-saxitoxin calibration solution, = 2,0 μg/ml
Prepare a calibration solution containing 2,0 μg/ml of neo-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 1 An ampoule containing 0,2 ml neo-saxitoxin calibration solution, mass concentration 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 2,0 mg/ml, the content of the ampoule can be quantitatively transferred to 13,8 ml acetic acid solution 3 (4.9.3) (total
volume 14,0 ml). The total volume can be checked and adjusted by weighing (5.9), using density of water for calculations.
4
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SIST ENV 14194:2002
ENV 14194:2002 (E)
NOTE 2 A 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 & Testing Programme, designed
to certify the mass fractions of saxitoxin and dc-saxitoxin in mussel reference materials, see [6].
4.15.2 Neo-saxitoxin calibration solution for qualitative analyses, = 1,0 μg/ml
Add 50 μl of Neo-saxitoxin calibration solution (4.15.1) to 50 μl of acetic acid solution 3 (4.9.3). Prepare fresh every
day of analysis.
4.16 GTX-2/GTX-3 standard solutions
4.16.1 GTX-2/GTX-3 calibration solution, = 2,0 μg/ml and 0,5 μg/ml respectively
Prepare a calibration solution containing 2,0 μg/ml of GTX-2 and 0,5 μg/ml of GTX-3 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 1 An ampoule containing approximately 0,2 ml GTX-2 (0,12 mg/ml) and GTX-3 (0,029 mg/ml) standard substance 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 2,0 mg/ml of GTX-2 and of 0,5 mg/ml of GTX-3, the content of the ampoule can be quantitatively
transferred to 11,8 ml acetic acid solution 3 (4.9.3) (total volume 12,0 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 2 A 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 & Testing Programme, designed
to certify the mass fractions of saxitoxin and dc-saxitoxin in mussel reference materials, see [6].
4.16.2 GTX-2/GTX-3 calibration solution for qualitative analysis, GTX-2 (0,1 μg/ml) and GTX-3 (0,024 μg/ml)
Add 50 μl of GTX-2/GTX-3 calibration solution (4.16.1) to 950 μl of acetic acid solution 3 (4.9.3). Prepare fresh
every day of analysis.
4.17 HPLC eluent A for the STX group
To 6 ml of octane sulfonic acid solution (4.5), add 20 ml of phosphoric acid solution (4.6.1). Add water to a volume
of 950 ml. Adjust to pH 7,2 with 25 % ammonia solution (4.7), dilute to 1,0 l with water and filter (5.8).
To 920 ml of this solution, add 80 ml of acetonitrile (4.3). Degas the eluent before use with helium purging (4.10) for
approximately 10 min. This solution is stable for 2 weeks.
4.18 HPLC eluent B for the GTX group
To 3 ml of octane sulfonic acid solution (4.5), add 20 ml of phosphoric acid solution (4.6.1). Add water to a volume
of 950 ml. Adjust to pH 7,0 with 25 % ammonia solution (4.7), dilute to 1,0 l with water and filter (5.8). Degas the
eluent before use with helium purging (4.10) for approximately 10 min. This solution is stable for 3 days.
4.19 Post column derivatisation reagent
Add 50 ml of periodic acid solution (4.8) to 50 ml of 1 % ammonia solution (4.7). Prepare fresh every day of
analysis.
4.20 Sodium acetate solution, c = 1,0 mol/l
Dissolve 13,6 g of sodium acetate trihydrate in 100,0 ml water.
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SIST ENV 14194:2002
ENV 14194:2002 (E)
5 Apparatus
Use usual laboratory apparatus and, in particular, the following.
5.1 Grinder
5.2 Vacuum manifold for SPE columns
5.3 C18 SPE columns, 1 ml, 100 mg sorbent
5.4 Adjustable automatic pipettes, 20 ml, 100 ml and 1000 μl
5.5 HPLC vials, 1 ml, with caps and septum
5.6 HPLC apparatus, see also figure 1 for system configuration
5.
...
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