kSIST FprEN 14526:2025

SLOVENSKI STANDARD
oSIST prEN 14526:2025
01-januar-2025
Živila - Določevanje toksinov iz skupine saksitoksina v školjkah - Metoda HPLC z
uporabo predkolonske derivatizacije s peroksidno ali perjodatno oksidacijo
Foodstuffs - Determination of saxitoxin-group toxins in shellfish - HPLC method using
pre-column derivatization with peroxide or periodate oxidation
Lebensmittel - Bestimmung von Toxinen der Saxitoxingruppe in Schalentieren - HPLC-
Verfahren mit Vorsäulenderivatisierung und Peroxid- oder Periodatoxidation
Produits alimentaires - Détermination de la teneur en toxines du groupe de la saxitoxine
dans les coquillages - Méthode par CLHP avec dérivation pré-colonne et par oxydation
au peroxyde ou au periodate
Ta slovenski standard je istoveten z: prEN 14526
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
67.120.30 Ribe in ribji proizvodi Fish and fishery products
oSIST prEN 14526:2025 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

oSIST prEN 14526:2025
oSIST prEN 14526:2025
DRAFT
EUROPEAN STANDARD
prEN 14526
NORME EUROPÉENNE
EUROPÄISCHE NORM
November 2024
ICS 67.120.30 Will supersede EN 14526:2017
English Version
Foodstuffs - Determination of saxitoxin-group toxins in
shellfish - HPLC method using pre-column derivatization
with peroxide or periodate oxidation
Produits alimentaires - Détermination de la teneur en Lebensmittel - Bestimmung von Toxinen der
toxines du groupe de la saxitoxine dans les coquillages Saxitoxingruppe in Schalentieren - HPLC-Verfahren mit
- Méthode par CLHP avec dérivation pré-colonne et par Vorsäulenderivatisierung und Peroxid- oder
oxydation au peroxyde ou au periodate Periodatoxidation
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 275.
If this draft becomes a European Standard, 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.

This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other
language made by translation under the responsibility 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, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 14526:2024 E
worldwide for CEN national Members.

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Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 7
3 Terms and definitions . 7
4 Principle . 7
5 Reagents . 10
6 Apparatus . 14
7 Procedure . 15
7.1 Sample preparation . 15
7.2 Extraction procedure . 15
7.3 Sample purification . 16
7.3.1 SPE-C18 clean-up . 16
7.3.2 SPE-COOH clean-up (fractionation). 16
7.3.3 Alternative weak cation exchange SPE clean-up [2], [11] . 17
7.4 Conversion of GTX6 into NEO and/or C3,4 into GTX1,4 . 18
7.4.1 General. 18
7.4.2 Hydrolysis of SPE-COOH Fraction 1 or 2 . 18
7.5 Oxidation procedure . 18
7.5.1 General. 18
7.5.2 Periodate oxidation . 18
7.5.3 Peroxide oxidation . 19
8 HPLC determination . 20
9 Calibration curve . 22
10 Identification . 22
11 Calculation . 22
11.1 General. 22
11.2 Toxin calculation . 23
11.2.1 General. 23
11.2.2 Calculation method with standard calibration curve . 23
11.3 Calculation of NEO in the presence of dcSTX [4], [5] . 23
11.3.1 General. 23
11.3.2 Method 1 . 24
11.3.3 Method 2 . 25
11.3.4 Method 3 . 26
11.4 Calculation of GTX6 . 26
11.5 Calculation of C3,4 . 26
11.6 Calculation of dcNEO in the presence of dcSTX . 27
11.6.1 General. 27
11.6.2 Method 4 . 28
11.6.3 Method 5 . 28
11.7 Calculation of GTX1,4 in the presence of GTX2,3 and dcGTX2,3 . 29
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11.7.1 General . 29
11.7.2 Method 6 . 30
11.8 Calculation of NEO in the presence of dcSTX, dcNEO and STX (optional) . 31
11.8.1 General . 31
11.8.2 Method 7 . 32
11.8.3 Method 8 . 33
11.9 Conversion to STX 2HCl equivalents . 34
12 Quality controls . 35
12.1 General . 35
12.2 Acceptance criteria series of analysis . 35
12.3 Overall recovery . 36
13 Precision . 36
14 Test report . 36
Annex A (informative) Precision data . 37
Annex B (informative) Chromatograms . 65
Bibliography . 69
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European foreword
This document (prEN 14526:2024) has been prepared by Technical Committee CEN/TC 275 “Food
analysis – Horizontal methods”, the secretariat of which is held by DIN.
This document is currently submitted to the CEN Enquiry.
This document will supersede EN 14526:2017.
prEN 14526:2024 includes the following significant technical changes with respect to EN 14526:2017:
— new Figure 2 detailing a procedure for a large numbers of test samples added;
— mandatory Clause 3 Terms and definitions added;
— new Clause 12 specifying quality controls added;
— new Annex B with example chromatograms added.
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Introduction
Paralytic shellfish poisoning (PSP) toxins are derivatives of saxitoxin. These toxins have been detected in
filter-feeding bivalve molluscs in various parts of the world. Paralytic shellfish poisoning is characterized
by symptoms varying from slight tingling sensation or numbness around the lips to fatal respiratory
paralysis. This document describes an analytical method for the quantification of these PSP toxins by
extraction from shellfish tissue followed by several clean-up steps and a separation by high performance
liquid chromatography (HPLC) with fluorescence detection (FLD).
WARNING — The use of this document can involve hazardous materials, operations and equipment. This
document does not purport to address all the safety problems associated with its use. It is the
responsibility of the user of this document to take appropriate measures to ensure the safety and health
of personnel prior to application of this document and fulfil statutory and regulatory requirements for
this purpose.
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1 Scope
This document specifies a method [1] for the quantitative determination of saxitoxin (STX), decarbamoyl
saxitoxin (dcSTX), neosaxitoxin (NEO), decarbamoyl neosaxitoxin (dcNEO), gonyautoxin 1 and 4 (GTX1,4;
sum of isomers), gonyautoxin 2 and 3 (GTX2,3; sum of isomers), gonyautoxin 5 (GTX5 also called B1),
gonyautoxin 6 (GTX6 also called B2), decarbamoyl gonyautoxin 2 and 3 (dcGTX2,3; sum of isomers),
N-sulfocarbamoyl gonyautoxin 2 and 3 (C1,2; sum of isomers) and N-sulfocarbamoyl gonyautoxin 1 and
4 (C3,4; sum of isomers) in (raw) mussels, oysters, scallops and clams. Laboratory experience has shown
that this document can also be applied to other marine invertebrates [2], [3] and processed products of
those species, however, no complete interlaboratory validation study according to ISO 5725-2:1994 has
been carried out so far. The method described was validated in an interlaboratory study [4], [5] and was
also verified in a European Union Reference Laboratory for Marine Biotoxins (EURLMB)-performance
test aiming the total toxicity of the samples [6]. Toxins which were not available in the first
interlaboratory study [4], [5] as dcGTX2,3 and dcNEO were validated in two additional interlaboratory
studies [7], [8]. The lowest validated levels [4], [5], [8], are given in µg toxin (free base)/kg shellfish tissue
and also as µmol/kg shellfish tissue and are listed in Table 1.
Table 1 — Lowest validated levels
Toxin µg/kg µmol/kg
c c
saxitoxin (STX) [5]
22 0,07
b b
gonyautoxin 2,3 (GTX2,3) [5]
114 0,29
c c
gonyautoxin 5 (GTX5) [5]
27 0,07
c c
decarbamoyl saxitoxin (dcSTX) [5]
8 0,03
c c
neosaxitoxin (NEO) [5]
33 0,10
c c
gonyautoxin 1,4 (GTX1,4) [5]
61,4 0,15
c c
N-sulfocarbamoyl gonyautoxin 2,3 (C1,2) [5]
93 0,20
b b
N-sulfocarbamoyl gonyautoxin 1,4 (C3,4) [5]
725 1,48
gonyautoxin 6 (GTX6) direct [4] 30 0,08
b b
indirect [9]
834 2,11
a a
decarbamoyl gonyautoxin 2,3 (dcGTX2,3) [8]
271 0,77
b b
decarbamoyl neosaxitoxin (dcNEO) [8]
594 2,18
a
lowest spiked level; mean recovery: 58 % [8]
b
lowest concentration tested
c
lowest concentration tested with a HorRat < 2 [4], [5]
A quantitative determination of GTX6 was not included in the first interlaboratory study but several
laboratories detected this toxin directly after solid phase extraction with ion-exchange (SPE-COOH)
clean-up and reported a mass concentration of 30 µg/kg or higher in certain samples. For that reason,
the present method is applicable to quantify GTX6 directly, depending on the availability of the standard
substance. Whenever GTX6 standard is not commercially available, it is possible to determine GTX6 after
hydrolysis of Fraction 2 of the SPE-COOH clean-up, described in 6.4, as NEO. The indirect quantification
of GTX6 was validated in two additional interlaboratory studies [7], [8]. A study to compare direct and
indirect GTX6 quantification was conducted at the EURLMB [16].
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A quantitative determination of C3,4 was included in the first interlaboratory study. The present method
is applicable to quantify C3,4 directly, depending on the availability of the standard substance. If no
standard substances are available, C3,4 can only be quantified as GTX1,4 if the same hydrolysis protocol
used for GTX6 (6.4) is applied to Fraction 1 of the SPE-COOH clean-up [10]. A study to compare direct and
indirect C3,4 quantification was conducted at the EURLMB [16].
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements 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)
EN ISO/IEC 17025, General requirements for the competence of testing and calibration laboratories
(ISO/IEC 17025)
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
4 Principle
WARNING — PSP toxins are neurotoxins which can be taken up by inhalation or orally. Therefore,
adequate protection measures are to be applied.
Paralytic Shellfish Poisoning (PSP) toxins are extracted from shellfish tissue homogenate by heating with
acetic acid. After centrifugation the supernatant is purified by solid phase extraction (SPE) using a C18
clean-up cartridge. It is analysed by HPLC after oxidation with periodate or peroxide with fluorescence
detection. Most toxins (STX, C1,2, GTX5, dcSTX, GTX2,3 and dcGTX2,3) can be quantified after SPE-C18
clean-up .
Oxidation of PSP toxins leads to several reaction products that are separated by reversed phase HPLC and
detected by fluorescence detection. The obtained reaction products for PSP toxins after oxidation with
peroxide and periodate are listed in Table 2. Additionally, the corresponding chromatograms are shown
in Figure 1.
The gonyautoxins GTX2 and GTX3 as well as GTX1 and GTX4 and decarbamoyl gonyautoxins dcGTX2 and
dcGTX3 and the N-sulfocarbamoyl gonyautoxins C1 and C2 as well as C3 and C4 are structural isomers
and lead with both oxidation modes to the same reaction products. The amount of structural isomers is
determined as the sum of both toxins.
STX reacts to a single specific oxidation product regardless of the kind of oxidation reaction (whether
peroxide or periodate). The same is valid for GTX2,3 as well as GTX5 and C1,2. In contrast, dcSTX and
dcGTX2,3 produce each two different oxidation products in both oxidation reactions, see also Table 2.
The toxin dcNEO is oxidized into two oxidation products only with the periodate oxidation. Each of the

This document is based on a procedure described by Lawrence et al. [4] and was also published as AOAC Official
Method 2005.06 [1].
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toxins NEO, GTX6, GTX1,4 and C3,4 produce three peaks after periodate oxidation but normally the
second eluting peak is used for quantification (peroxide oxidation cannot be used for quantification).
Co-occurrence of different PSP toxins in shellfish can influence the analytical results, because some of the
PSP toxins can (partially) lead to the same reaction products (see Table 2 and Annex B). So, the
chromatograms shall be carefully interpreted after a SPE C18 clean-up.
Table 2 — Reaction products after oxidation with periodate and peroxide
Oxidation products and Oxidation product at the same retention
Toxin Intensity
HPLC-eluting order time as
peroxide periodate peroxide periodate peroxide periodate
a b
STX one one ++ + NEO (3); GTX6 (3)
NEO (3)
dcSTX first (1) first (1) ++ -  dcNEO (1)
a
second (2) second (2) + + NEO (2); GTX6 (2);
NEO (2)
dcNEO (2)
NEO no first (1) — +  GTX6 (1)
second (2) second (2) - ++ dcSTX (2) GTX6 (2); dcSTX (2);
dcNEO (2)
third (3) third (3) - + STX STX; GTX6 (3)
C1,2 one one ++ +
C3,4 no first (1) — +  GTX1,4 (1)
no second (2) — ++  GTX1,4 (2); dcGTX2,3 (2)
no third (3) — +  GTX1,4 (3); GTX2,3
GTX1,4 no first (1) — +  C3,4 (1)
no second (2) — ++  C3,4 (2); dcGTX2,3 (2)
third (3) third (3) - ++ GTX2,3 C3,4 (3); GTX2,3
a
GTX2,3 one one ++ ++ C3,4 (3);
GTX1,4 (3)
GTX1,4 (3)
GTX5 one one ++ -
GTX6 no first (1) — +  NEO (1)
no second (2) — ++  NEO (2); dcSTX (2); dcNEO (2)
no third (3) — -  NEO (3); STX
dcGTX2,3 first (1) first (1) ++ +
second (2) second (2) + ++  C3,4 (2); GTX1,4 (2)
dcNEO first (1) first (1) - ++  dcSTX (1)
second (2) second (2) - + dcSTX (2) dcSTX (2); NEO (2); GTX6 (2)
Intensity: —  not visible
-  very low
+  low
++  high
a
High concentration of the indicated toxin may influence the quantification by simulating an increased content.
b
Numbers in curly brackets are the elution order.

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a) Non N1-hydroxylated toxins: peroxide b) Non N1-hydroxylated toxins: periodate

c) N1-hydroxylated toxins: peroxide d) N1-hydroxylated toxins: periodate
Key
X detection response (V)
Y time (min)
Figure 1 — Reaction products after derivatization with peroxide and periodate (peaks for
quantification are marked with arrows)
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For the quantitative determination of most N1-hydroxylated toxins, a fractionation by SPE-COOH clean-
up is necessary (shown in Table 4) because the oxidation products of some PSP toxins (NEO and GTX6,
GTX1,4 and C3,4) are identical. This step separates the PSP toxins into three distinct groups, namely the
C toxins, the GTX toxins and the saxitoxin group by elution with mobile phases of different ionic strength.
For example, with Bakerbond, the C toxins elute unretained with water in Fraction 1, the GTX toxins
(GTX1 to GTX6 as well as dcGTX2 and dcGTX3) elute with 0,05 mol/l NaCl in Fraction 2 while the
saxitoxin group (STX, NEO, dcNEO and dcSTX) requires 0,3 mol/l NaCl for elution in Fraction 3. These
fractions can be analysed by HPLC-FLD after oxidation with periodate or peroxide.
5 Reagents
If not otherwise specified, reagents of pro analysis and solvents suitable for HPLC-FLD shall be used.
Water shall be distilled in glass vessels or demineralized before use or shall be of equivalent purity
according to EN ISO 3696.
If not already specified, stability of solutions should be determined by the laboratory.
5.1 Methanol, HPLC quality.
5.2 Acetonitrile, HPLC quality.
5.3 Ammonium formate:
5.3.1 Ammonium formate solution, substance concentration c = 0,3 mol/l.
Dissolve 1,892 g of ammonium formate (crystalline powder) (5.3) in 100 ml of water.
5.4 Glacial acetic acid:
5.4.1 Acetic acid solution 1, mass fraction p ≈ 1 %.
Dilute 1 ml of glacial acetic acid (5.4) to 100 ml with water.
5.4.2 Acetic acid solution 2, c ≈ 0,1 mol/l.
Dilute 572 µl of glacial acetic acid (5.4) to 100 ml with water.
5.4.3 Acetic acid solution 3, c ≈ 0,1 mmol/l.
Dilute 100 µl of acetic acid solution 2 (5.4.2) to 100 ml with water.
5.4.4 Acetic acid solution 4, mass fraction p ≈ 0,6 %.
Dilute 0,6 ml of glacial acetic acid (5.4) to 100 ml with water.
5.5 Ammonium acetate:
5.5.1 Ammonium acetate solution 1, c = 0,1 mol/l.
Dissolve 0,77 g of ammonium acetate (5.5) to 100 ml with water.
5.5.2 Ammonium acetate solution 2, c = 0,01 mol/l.
Dilute 10 ml of ammonium acetate solution 1 (5.5.1) to 100 ml with water.
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5.6 Sodium chloride:
5.6.1 Sodium chloride solution 1, c = 0,05 mol/l.
Dissolve 0,29 g of sodium chloride (5.6) to 100 ml with water.
5.6.2 Sodium chloride solution 2, c = 0,3 mol/l.
Dissolve 1,75 g of sodium chloride (5.6) to 100 ml with water.
5.7 Hydrochloric acid, c = 1 mol/l.
5.8 Disodium hydrogenphosphate or disodium hydrogenphosphate 7-hydrate:
5.8.1 Disodium hydrogenphosphate solution, c = 0,3 mol/l.
Dissolve 4,26 g of disodium hydrogenphosphate (5.8) in 100 ml water or dissolve 8,04 g of disodium
hydrogenphosphate 7-hydrate (5.8) in 100 ml water.
5.9 Sodium hydroxide:
5.9.1 Sodium hydroxide solution 1, c = 1 mol/l.
Dissolve 4 g of sodium hydroxide (5.9) to 100 ml with water.
5.9.2 Sodium hydroxide solution 2, c = 0,2 mol/l.
Dilute 10 ml of sodium hydroxide solution 1 (5.9.1) to 50 ml with water.
5.10 Hydrogen peroxide, w = 30 %:
5.10.1 Hydrogen peroxide solution, w ≈ 10 %.
Dilute 3 ml of hydrogen peroxide solution (5.10), of mass fraction w = 30 % with 6 ml of water. Prepare
fresh every day. Store both solutions in the dark at approximately +4 °C.
5.11 Periodic acid:
5.11.1 Periodic acid solution 1, c = 0,1 mol/l.
Dissolve 0,2279 g of periodic acid (5.11) in 10 ml of water.
5.11.2 Periodic acid solution 2, c = 0,034 mol/l.
Dilute 3,4 ml of periodic acid solution 1 (5.11.1) with 6,6 ml of water. Store in a refrigerator in the dark
at approximately +4 °C. Prepare fresh every day.
5.12 Periodate oxidation reagent.
Mix one volume part of periodic acid solution 2 (5.11.2) with one volume part of disodium
hydrogenphosphate solution (5.8.1) and one volume part of ammonium formate solution (5.3.1). Bring
the mixture to pH 8,2 ± 0,3 by drop wise adding sodium hydroxide solution 2 (5.9.2) and check the pH by
using a pH meter. Prepare fresh every day of analysis.
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5.13 PSP toxin standard substances:
5.13.1 PSP toxin stock solutions.
For convenience, standard substances can be combined into four or more mixtures by appropriate
dilution of standard solutions in water. Table 3 shows suitable concentration for each PSP toxin in four
stock solution mixtures. Store the solutions in the dark at approximately +4 °C and check the mass
concentrations regularly after 2 weeks or store in the dark at approximately –18 °C or below and check
the mass concentrations regularly after 6 months.
Table 3 — Example of suitable compositions and concentrations for each PSP toxin in four stock
solution mixtures
Toxin concentration
Stock solution mixtures
a
nmol/ml
µg/ml
GTX1,4 0,41 1,0
Mix 1
NEO 0,32 1,0
GTX2,3 0,30 1,0
GTX5 0,40 1,0
STX 0,38 1,0
Mix 2
dcSTX 0,26 1,0
dcGTX2,3 0,35 1,0
C1,2 0,48 1,0
Mix 3 dcNEO 0,27 1,0
GTX6 0,40 1,0
Mix 4
C3,4 0,49 1,0
a
related to the free base of the toxins
NOTE 1 Ampoules containing separately GTX1,4, NEO, GTX2,3, GTX5, STX, dcSTX, dcGTX2,3, C1,2, dcNEO, GTX6,
C3,4 standard substances in aqueous hydrochloric acid or aqueous acetic acid with concentrations ranging from
10 µmol/l to 200 µmol/l are currently commercially available .
NOTE 2 See chromatograms for the mixtures in Table 3 in Annex B.
Some of the standard substances can be contaminated with other PSP toxins; therefore, the impurities
shall be taken into account for calibration purposes (by quantifying impurities, running different
calibration curves or including it in uncertainty measurements).

Suitable calibration solutions can be obtained from the National Research Council Canada, Halifax, Canada.
Further information on suitable calibration solutions is e. g. available on the homepage of the European
Reference Laboratory on Marine Biotoxins
https://www.aesan.gob.es/en/CRLMB/web/public_documents/seccion/materiales_referencia.htm. This
information is given for the convenience of the users of this European Standard and does not constitute an
endorsement by CEN of this source of supply. Equivalent products may be used if they can be shown to lead
to the same results.
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5.13.2 PSP toxin calibration solutions.
Prepare a calibration with at least five points for the determination of PSP toxins for example undiluted,
2,5 fold, 5 fold, 7,5 fold and 10 fold dilution of the PSP stock solution (5.13.1) with 0,1 mmol/l of acetic
acid solution 3 (5.4.3). PSP toxin calibration solutions may be also prepared by diluting stock solution
mixtures with water (as long as the pH is acidic). Store in the dark at –18 °C and check the mass
concentration regularly after 6 months.
NOTE 1 It is important to store diluted standard solutions in plastic vials or in deactivated glass containers which
can e.g. be achieved by soaking the vials overnight in sodium hydroxide, rinsed with water followed by methanol
(5.1), and dried.
For the interlaboratory study described in Clause A.1 [4], [5], three calibration points were used.
However, in order to increase the robustness of the method, it is advised to use at least five calibration
points.
NOTE 2 Another method to prepare the calibration solution is to implement this in the oxidation step (7.5.2 and
7.5.3). Different aliquots from the PSP toxin stock solution are used and made up to 100 µl final volume with
0,1 mmol/l acetic acid solution 3 (5.4.3).
5.13.3 PSP-solution for checking the efficiency of the cartridges
Prepare solutions of toxins of the appropriate mass concentration for checking the recovery of the toxins
on the SPE-cartridges.
One option is to prepare the mix in 0,6 % acetic acid solution 4 (5.4.4) to check recovery for SPE-C18 and
prepare the mix in water adjusted to pH 6,5 ± 0,3 to check SPE-COOH.
A second option is to prepare the mix in 0,6 % acetic acid solution 4 (5.4.4); Toxins quantified after
peroxide oxidation: dcGTX2,3; C1,2; dcSTX; GTX2,3; GTX5 and STX will be only checked after SPE-C18.
For all other toxins checks will be done after both SPE-C18 and SPE-COOH clean-ups. For this, standard
solutions will be passed through both clean-ups sequentially after the SPE-C18 the pH is adjusted to
6,5 ± 0,3 and recovery will be evaluated after each clean-up. For example, the standards used for these
checks can have a 0,8 µM concentration.
5.14 Matrix modifier (MM) for periodate oxidation.
Use a blank extract (PSP free) from oysters as described in 6.1 and 6.2. If stored frozen at −20 °C, this
initial PSP-free crude oyster extract is stable and can be used within at least two months. For use as matrix
modifier, clean-up according to 7.3.1 and adjust the extract to pH 6,5 ± 0,3 with sodium hydroxide
solution 1 (5.9.1). Filter the supernatant using a 0,45 µm filter (6.18) and store the obtained matrix
modifier in a refrigerator. Analyse the matrix modifier for PSP toxins by periodate and peroxide oxidation
to ensure absence of toxins before use. It shall be prepared every two weeks (i.e. again cleaned up from
the crude extract).
NOTE Different batches of matrix modifier could affect enormously the recovery of N-1 hydroxylated toxins, in
particular NEO, due to matrix effects. Therefore, when selecting matrix modifier for periodate oxidation it is
important to check if the matrix modifier is affecting the recovery of these toxins.
5.15 HPLC eluents:
Prepare eluents A and B fresh before each analytical series [12].
5.15.1 Eluent A: Ammonium formate, c = 0,1 mol/l.
Dissolve 6,31 g of ammonium formate (5.3) in 1 l water and adjust to pH 6,0 ± 0,3 by adding
approximately 6 ml of acetic acid solution 2 (5.4.2).
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5.15.2 Eluent B: Ammonium formate, c = 0,1 mol/l in 5 % acetonitrile.
Dissolve 6,31 g of ammonium formate (5.3) in 950 ml water and add 50 ml of acetonitrile (5.2). Adjust to
pH 6,0 ± 0,3 by adding approximately 6 ml of acetic acid solution 2 (5.4.2). The composition can be
adjusted.
6 Apparatus
Usual laboratory glassware and equipment and, in particular, the following:
6.1 Grinder.
6.2 Balance, capable of weighing to the nearest 0,01 g.
6.3 Analytical balance, capable of weighing to the nearest 0,1 mg.
6.4 Plastic centrifuge tubes, polypropylene, 50 ml, with caps.
6.5 Centrifuge, capable to reach 3 600 g at the outer end of the centrifuge tubes.
6.6 Pipettes, autopipettes with disposable plastic tips.
6.7 Vortex mixer.
6.8 Water bath or hot plate.
6.9 Graduated conical test tube, 15 ml.
6.10 SPE-C18 cartridges, e.g. 500 mg per 3 ml volume.
Check each new batch of SPE-C18 cartridges (e.g. Supelcoclean LC18) with standard solutions (5.13.3) to
ensure that minimum recovery obtained with the C18-cartridge is 80 %. This check is necessary due to
experiences gathered during method development as it was observed that variations can occur.
6.11 SPE-COOH ion exchange cartridges, e.g. 500 mg per 3 ml volume.
Check each new batch of SPE-COOH cartridges (e.g. Bakerbond Carboxylic Acidsilane or, optional, a weak ®
cation exchanger, e.g. Strata-X from Phenomenex) with standard solutions (5.13.3) to ensure that
minimum recovery obtained with the COOH-cartridge is 80 % and the correct elution patterns are
obtained according to 7.3.2. This check is necessary due to experiences gathered during method
development as it was observed that variations can occur.
6.12 Manifold or automatic SPE station (for the SPE clean-ups).
6.13 Block heater (or similar) for the hydrolysis step.
6.14 Reaction tubes, e.g. polypropylene vials with 1,5 ml.
6.15 pH indicator paper, able to precisely identify a pH of 6,5 ± 0,3.
6.16 pH meter, with micro-electrode.

−2
g = 9,81 m · s .
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6.17 Rotary evaporator or similar equipment with suitable glass containers, optionally for samples
with low concentrations.
6.18 Membrane filter, for aqueous solutions, with a pore size of 0,45 μm, e.g. regenerated cellulose.
6.19 HPLC vials, e.g. amber glass or polypropylene vials.
6.20 HPLC system, comprising the following:
6.20.1 Injector, preferably a refrigerated injector, capable of refrigerating at (6 ± 2) °C and injection up
to 100 µl.
NOTE Some toxin oxidation products are unstable when using an auto sampler at room temperature, therefore
the responses of these toxins (e.g. dcNEO and NEO) can decrease during a sequence. This could be improved by
using a cooled auto sampler [12].
6.20.2 Pump, capable of gradient elution.
6.20.3 Column oven able to heat between (30 ± 2) °C and (45 ± 2) °C.
6.20.4 Analytical column, e.g. RP C18, particle size 5 µm, 150 mm (length) × 4,6 mm (diameter).
The measurement may be carried out with different separation columns (dimension, manufacturer).
However, the PSP oxidation products shall be chromatographically baseline separated.
6.20.5 Fluorescence detector, excitation wavelength of λ = 340 nm and emission wavelength of
λ = 395 nm.
7 Procedure
7.1 Sample preparation
Thoroughly clean outside of the shellfish with tap water. Open by cutting adductor muscle. Rinse shells
with tap water once to remove sand and foreign material. Remove the shellfish tissue from shells by
separating adductor muscles and tissue connecting at the hinge. After removal from shellfish, drain
tissues 5 min in a sieve. Homogenize the shellfish tissue in a grinder (6.1). At least 100 g of pooled
homogenized shellfish tissue should be taken. If not directly proceeding with the analysis it is advised to
store the homogenized shellfish in the freezer.
7.2 Extraction procedure
Defrost the homogenized sample in the refrigerator or at room temperature or use it directly after
grinding (7.1).
Do not heat the sample.
Keep all extracts and solutions refrigerated when not in use.
Weigh a test portion of 5 g ± 0,1 g of homogenized shellfish in a 50 ml centrifuge tube (6.4) and mix for at
least 30 s with 3 ml of 1 % acetic acid solution 1 (5.4.1) on a vortex mixer. Cap it loosely to avoid pressure
build up during heating and place in a boiling water bath (100 °C) so that the contents of the tube are
below the water line. Heat the samples for 5 min.
Make sure that the water bath has reached the boiling point before inserting samples into it and starting
to count the heating time. Do not place too many tubes in the bath at once in order to avoid that the water
bath stops boiling for more than 30 s. Start measuring the 5 min as soon as water resumed boiling.
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Remove samples from the water bath, remix for at least 30 s on a Vortex mixer and cool it by placing in a
refrigerator or a beaker of cold water for 5 min and centrifuge for 10 min at 3 600 g. Decant the
supernatant into a 15 ml graduated conical test tube (6.9).
Add 3 ml of 1 % acetic acid solution 1 (5.4.1) to the centrifuge tube containing the once extracted sample
(solid residue), mix for at least 30 s on a Vortex mixer and centrifuge again for 10 min at 3 600 g. Decant
and collect the supernatant into the same graduated conical test tube (6.9) that contains the first portion
of the crude extract and adjust accurately to 10 ml with water.
The procedure can be stopped at this point and the crude extract can be stored for at least of 5 days in a
refrigerator 2 °C to 8 °C or frozen a longer period.
NOTE Crude extracts can be kept frozen (−20 °C) for at least 3 years except for bivalve mollusc species with
high PSP biotransformation ability like Spisula solida [17], [18] or razor clams (Ensis siliqua, Ensis ensis and Solen
marginatus).
7.3 Sample purification
7.3.1 SPE-C18 clean-up
Condition the cartridge according to the manufacturers' instructions, e.g. condition the 3 ml SPE-C18
cartridge (6.10) with 6 ml of methanol (5.1) followed by 6 ml of water. Discard the solutions which have
passed the cartridge. Place a graduated conical test tube (6.9) under the cartridge. Add 1 ml (0,5 g
shellfish tissue equivalent) of the crude extract (7.2) to the cartridge. Keep the flow rate between
2 ml/min to 3 ml/min for all elutions. Collect the eluate in the graduated conical test tube. Wash the
cartridge with 2 ml of water and combine the washings with the eluate to get the purified extract.
Avoid running dry the cartridges during the complete process.
Adjust this purified extract to pH 6,5 ± 0,3 with 1 mol/l of NaOH (5.9.1) using pH meter (6.16) and then
adjust the volume exactly to 4 ml with water.
For screening purposes the sample purification can be stopped at this point. The extract from SPE-C18
clean-up is stable for at least one year at ≤ −18 °C.
Aliquots of this extract may be used for oxidation with periodate and peroxide as described in 7.5.2 and
7.5.3.
If N1-hydroxylated PSP toxins are detected in this extract, continue with the SPE-COOH ion exchange
clean-up as described below.
NOTE For investigation of whole king scallops (Pecten maximus) and whole queen scallops (Aequipecten
opercularis) contaminated with NEO and GTX1,4, the use of 1,5 ml of the crude extract for the SPE-C18 clean-up
increases the sensitivity of GTX1,4 and NEO [3].
7.3.2 SPE-COOH clean-up (fractionation)
Fractionate only extracts from SPE-C18 clean-up that contain N1-hydroxylated PSP toxins (e.g. NEO,
dcNEO, C3,4; GTX6 and GTX1,4) after SPE-C18 clean-up.
Condition the cartridge according to the manufacturers’ instructions, e.g. condition the 3 ml SPE-COOH
cartridge (6.11) by passing 10 ml of 0,01 mol/l ammonium acetate solution 2 (5.5.2) through it. Keep the
flow rate between 2 ml/min to 3 ml/min for all elutions. Discard the eluate.
Fraction 1: Pass a 2 ml aliquot (0,25 g shellfish tissue equivalent) of shellfish extract from SPE-C18 clean-
up (7.3.1) through the cartridge and collect the eluate in a graduated conical test tube (6.9) labelled as
Fraction 1. Then pass 4 ml of water through the cartridge and collect into the same tube. Adjust final
volume to 6 ml in total. This fraction contains the C toxins. Proceed to 7.5.2 and/or 7.5.3 for the oxidation
steps. If C3,4 is present and C3,4 standards are not commercially available, proceed to 7.4.
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Fraction 2: Pass 4 ml of 0,05 mol/l NaCl solution 1 (5.6.1) through the same cartridge and collect the
eluate (labelled as Fraction 2) in a graduated conical test tube (6.9). Adjust final volume to 4 ml. This
fraction contains the toxins GTX1,4, GTX2,3, GTX5, GTX6 and dcGTX2,3. Proceed to 7.5.2 and/or 7.5.3 for
the oxidation steps. If GTX6 is present and GTX6 standards are not commercially available, proceed to
7.4.
Fraction 3: Pass 5 ml of 0,3 mol/l NaCl solution 2 (5.6.2) through the cartridge and collect in a graduated
conical test tube (6.9) marked as Fraction 3. Adjust final volume to 5 ml. This fraction contains STX, NEO,
dcNEO and dcSTX. Proceed to 7.5.2 and/or 7.5.3 for the oxidation steps.
Avoid running dry the cartridges during the complete process.
If problems with detector sensitivity are encountered each fraction can be concentrated. A suggested
concentration step is to collect each fraction from SPE-COOH-clean-up into a glass container suitable for
the evaporation equipment (6.17) and evaporate to approximately 1 ml on e.g. a rotary evaporator or
other adequate evaporators with a water bath set at 45 °C. Transfer the solution into a graduated conical
test tube (6.9) using a Pasteur pipette. Rinse the glass container 3 times with about 0,2 ml to 0,3 ml of
water each time, transferring the rinse into the graduated tube just so the final volume of SPE-COOH
cleaned-up fraction is 2 ml. Analyse Fractions 1, 2 and 3 by HPLC after periodate and peroxide oxidations
as described in 7.5.2 and/or 7.5.3.
NOTE To improve the sensitivity of the method, an alternative ion-exchange SPE-clean-up procedure was
developed during a single-laboratory validation [2], [11], (procedure see 7.3.3).
The sample purification can be stopped at this point. Continue immediately with oxidation and HPLC-FLD
analysis or store the SPE-COOH cleaned-up fraction in a refrige
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