EN 14526:2017

SLOVENSKI STANDARD
01-marec-2017
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SIST EN 14526:2005
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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 - Dosage 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: EN 14526:2017
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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 14526
EUROPEAN STANDARD
NORME EUROPÉENNE
January 2017
EUROPÄISCHE NORM
ICS 67.120.30 Supersedes EN 14526:2004
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 European Standard was approved by CEN on 7 November 2016.

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 bibliographical 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 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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 14526:2017 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 6
3 Principle . 6
4 Reagents . 9
5 Apparatus . 12
6 Procedure. 14
7 HPLC determination . 18
8 Calibration curve . 20
9 Identification . 20
10 Calculation . 20
11 Precision . 35
12 Test report . 35
Annex A (informative) Precision data . 36
Bibliography . 64
European foreword
This document (EN 14526:2017) 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 July 2017, and conflicting national standards shall be
withdrawn at the latest by July 2017.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN 14526:2004.
— the applicability is greater as more samples were tested in interlaboratory studies;
— the extraction procedure in 6.2 has been revised;
— the chromatographic conditions in Clause 7 have been revised;
— guidelines for calculation in presence of several toxins were introduced;
— the method has been additionally validated in several interlaboratory studies, and the precision
data in Annex A have been revised.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.

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 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 take appropriate measures to ensure the safety and health
of personnel prior to application of the standard, and fulfil statutory and regulatory requirements for
this purpose.
1 Scope
This European standard 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 1 and 2 (C1,2; sum of isomers) and (depending on the
availability of certified reference materials (CRMs)) N-sulfocarbamoyl-gonyautoxin 3 and 4 (C3,4; sum
of isomers) in (raw) mussels, oysters, scallops and clams. Laboratory experience has shown that it is
also be applicable in other shellfish [2], [3] and cooked shellfish products. The method described was
validated in an interlaboratory study [4], [5] and was also verified in a EURL-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, B1) [5] 27 0,07
c c
dc-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 1,2 (C1,2) [5] 93 0,20
b b
N-sulfocarbamoyl-gonyautoxin 3,4 (C3,4) [5] 725 1,48
gonyautoxin 6 (GTX6, B2) Direct [4] 30 0,08
b b
Indirect [9] 834 2,11
a a
dc-gonyautoxin 2,3 (dcGTX2,3) [8] 271 0,77
b b
dc-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 (B2) 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 (B2) directly, depending on the availability of
the standard substance. Currently it is possible to determine GTX6 after a 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 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, see [10].
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 the referenced document (including any amendments) applies.
EN ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696)
3 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 (B1), dcSTX, GTX2,3 and dcGTX2,3) can be
1)
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 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 (B1) 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 toxins NEO, GTX6 (B2), GTX1,4 and C3,4 produce three peaks after periodate oxidation but
only 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). So the chromatograms
shall be carefully interpreted after a SPE C18 clean-up.

1)
This document is based on a procedure described by Lawrence et al. [4] and was also published as AOAC
Official Method 2005.06 [1].
Table 2 — Reaction products after oxidation with periodate and peroxide
Toxin Oxidation products Intensity Oxidation product at the
and HPLC-eluting same retention time as
order
peroxide periodate peroxide periodate peroxide periodate
a b
STX one one ++ + NEO {3}; GTX6 (B2) {3}
NEO {3}
dc-STX first {1} first {1} ++ -  dcNEO {1}
second second a NEO {2};
+ +
NEO {2}
{2} {2} GTX6 (B2) {2};
dcNEO {2}
NEO no first {1} — +  GTX6 (B2) {1}
second second GTX6 (B2) {2}; dcSTX {2};
- ++ dcSTX {2}
{2} {2} dcNEO {2}
third {3} third {3} - + STX STX; GTX6 (B2) {3}
C1,2 one one ++ +
C3,4 no first {1} — +  GTX1,4 {1}
no second — ++  GTX1,4 {2}; dcGTX2,3 {2}
{2}
no third {3} — +  GTX1,4 {3}; GTX2,3
GTX1,4 no first {1} — +  C3,4 {1}
no second — ++  C3,4 {2}; dcGTX2,3 {2}
{2}
third {3} third {3} - ++ GTX2,3 C3,4 {3}; GTX2,3
a
GTX2,3 one one ++ ++ C3,4 {3};
GTX1,4
GTX1,4 {3}
{3}
GTX5 one one ++ -
(B1)
GTX6 no first {1} — +  NEO {1}
(B2)
no second — ++  NEO {2}; dcSTX {2}; dcNEO
{2} {2}
no third {3} — -  NEO {3}; STX
dcGTX2,3 first {1} first {1} ++ +
second second + ++  C3,4 {2}; GTX1,4 {2}
{2} {2}
dcNEO first {1} first {1} - ++  dcSTX {1}
second second - + dcSTX {2} dcSTX {2}; NEO {2}; GTX6
{2} {2} (B2) {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.
a) Non-hydroxylated toxins: peroxide b) Non-hydroxylated toxins:
periodate
c) N-hydroxylated toxins: peroxide d) N-hydroxylated toxins: periodate
Key
Y detection response (V)
X time (min)
Figure 1 — Reaction products after derivatization with peroxide and periodate (peaks
for quantification are marked with arrows)
For the quantitative determination of N-hydroxylated toxins, a fractionation by SPE-COOH clean-up is
necessary (shown in Table 3) because the oxidation products of some PSP toxins (NEO and GTX6 (B2),
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. The C toxins elute unretained with water, the GTX toxins (GTX1 to GTX6 as well as dcGTX2
and dcGTX3) elute with 0,05 mol/l NaCl while the saxitoxin group (STX, NEO, dcNEO and dc-STX)
requires 0,3 mol/l NaCl for elution. These fractions can be analysed by HPLC after oxidation with
periodate or peroxide.
Table 3 — Toxin elution order after SPE-COOH clean-up
Fraction Eluent Eluting toxin
1 water C1,2; C3,4
2 0,05 mol/l NaCl dcGTX2,3; GTX2,3; GTX1,4; GTX5; GTX6 (B2)
3 0,3 mol/l NaCl dcSTX; STX; NEO; dcNEO
4 Reagents
If not otherwise specified, reagents of pro analysis (p.a.) 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.
4.1 Methanol, HPLC quality.
4.2 Acetonitrile, HPLC quality.
4.3 Ammonium formate solution, substance concentration c = 0,3 mol/l.
Dissolve 1,892 g of ammonium formate (crystalline powder) in 100 ml of water.
4.4 Glacial acetic acid:
4.4.1 Acetic acid solution 1, mass fraction p ≈ 1 %.
Dilute 1 ml of glacial acetic acid (4.4) to 100 ml with water.
4.4.2 Acetic acid solution 2, c ≈ 0,1 mol/l.
Dilute 572 µl of glacial acetic acid (4.4) to 100 ml with water.
4.4.3 Acetic acid solution 3, c ≈ 0,1 mmol/l.
Dilute 100 µl of acetic acid solution 2 (4.4.2) to 100 ml with water.
4.5 Ammonium acetate:
4.5.1 Ammonium acetate solution 1, c = 0,1 mol/l.
Dissolve 0,77 g of ammonium acetate (4.5) to 100 ml with water.
4.5.2 Ammonium acetate solution 2, c = 0,01 mol/l.
Dilute 10 ml of ammonium acetate solution 1 (4.5.1) to 100 ml with water.
4.6 Sodium chloride:
4.6.1 Sodium chloride solution 1, c = 0,05 mol/l.
Dissolve 0,29 g of sodium chloride (4.6) to 100 ml with water.
4.6.2 Sodium chloride solution 2, c = 0,3 mol/l.
Dissolve 1,75 g of sodium chloride (4.6) to 100 ml with water.
4.7 Hydrochloric acid, c = 1 mol/l.
4.8 Disodium hydrogenphosphate solution c = 0,3 mol/l.
Dissolve 4,26 g of disodium hydrogenphosphate in 100 ml water or dissolve 8,04 g of disodium
hydrogenphosphate 7-hydrate in 100 ml water.
4.9 Sodium hydroxide:
4.9.1 Sodium hydroxide solution 1, c = 1 mol/l.
Dissolve 4 g of sodium hydroxide (4.9) to 100 ml with water.
4.9.2 Sodium hydroxide solution 2, c = 0,2 mol/l.
Dilute 10 ml of sodium hydroxide solution 1 (4.9.1) to 50 ml with water.
4.10 Hydrogen peroxide solution, w ≈ 10 %.
Dilute 3 ml of commercially available hydrogen peroxide solution, of mass fraction w = 30 % with 6 ml
of water. Prepare fresh every day. Store both solutions in the dark at approximately + 4 °C.
4.11 Periodic acid:
4.11.1 Periodic acid solution 1, c = 0,1 mol/l.
Dissolve 0,2279 g of periodic acid (4.11) in 10 ml of water.
4.11.2 Periodic acid solution 2, c = 0,034 mol/l.
Dilute 3,4 ml of periodic acid solution 1 (4.11.1) with 6,6 ml of water. Store in a refrigerator in the dark
at approximately + 4 °C. Prepare fresh every day.
4.12 Periodate oxidation reagent.
Mix one volume part of periodic acid solution 2 (4.11.2) with one volume part of disodium
hydrogenphosphate solution (4.8) and one volume part of ammonium formate solution (4.3). Bring the
mixture to pH 8,2 by drop wise adding sodium hydroxide solution 2 (4.9.2) and check the pH by using a
pH meter. Prepare fresh every day of analysis.
4.13 PSP toxin standard substances:
4.13.1 PSP toxin stock solutions.
For convenience, standard substances can be combined into three mixtures by appropriate dilution of
standard solutions in water. Adjust those solutions to about pH 4 with 0,1 mol/l of acetic acid solution 2
(4.4.2). For the analysis of C1,2, adjust solutions to pH 5 as otherwise degradation has been observed.
Table 4 shows suitable concentration for each PSP toxin in three 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 4 — Examples of suitable concentrations for each PSP toxin in three stock solution
mixtures
Stock solution mixtures Toxin concentration
a
nmol/ml
µg/ml
Mix 1 GTX1,4 0,192 0,467
NEO 0,189 0,600
Mix 2 GTX2,3 0,265 0,670
GTX5 (B1) 0,202 0,532
STX 0,201 0,672
dc-STX 0,054 0,211
dcGTX2,3 0,080 0,227
Mix 3 C1,2 0,203 0,427
C3,4 0,188 0,383
dcNEO 0,137 0,503
a
related to the free base of the toxins
NOTE Ampoules containing separately STX, NEO, GTX1,4, GTX2,3, C1,2, GTX5, dcGTX2,3, dcNEO, dcSTX
standard substances in hydrochloric acid or aqueous acetic acid with concentrations ranging from 100 µg/ml to
2000 µg/ml are commercially available .
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 http://aesan.msssi.gob.es/en/CRLMB/web/home.shtml and
http://aesan.msssi.gob.es/en/CRLMB/web/estandares_materiales_referencia/materiales_referencia.shtml. 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.
4.13.2 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 (4.13.1) with 0,1 mmol/l of acetic
acid solution 3 (4.4.3). 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, and dried.
For the interlaboratory study in 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 (6.5.2
and 6.5.3). 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 (4.4.3).
4.13.3 PSP-solution for recovery check.
Prepare solutions of toxins of the appropriate mass concentration (e.g. in 0,6 % acetic acid) for checking
the recovery of the toxins on the SPE-cartridges.
4.14 Matrix modifier 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 6.3.1 and adjust the extract to pH 6,5 with sodium hydroxide
solution 1 (4.9.1). The solution can be stored in a refrigerator for 2 days to 3 days to precipitate co-
extracted material. Decant the supernatant or filter it using a 0,45 µm filter (5.20) 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).
4.15 HPLC eluents:
4.15.1 Eluent A: Ammonium formate, c = 0,1 mol/l.
Dissolve 6,31 g of ammonium formate in 1 l water and adjust to pH 6,0 by adding approximately 6 ml of
acetic acid solution 2 (4.4.2). Filter through a membrane filter (5.18) using vacuum.
4.15.2 Eluent B: Ammonium formate, c = 0,1 mol/l in 5 % acetonitrile.
Dissolve 6,31 g of ammonium formate in 950 ml water and add 50 ml of acetonitrile (4.2). Adjust to
pH 6,0 by adding approximately 6 ml of acetic acid solution 2 (4.4.2). Filter through a membrane filter
(5.18) using vacuum.
5 Apparatus
Usual laboratory glassware and equipment and, in particular, the following:
5.1 Grinder.
5.2 Balance, capable of weighing to the nearest 0,01 g.
5.3 Analytical balance, capable of weighing to the nearest 0,1 mg.
5.4 Plastic centrifuge tubes, polypropylene, 50 ml, with caps.
3)
5.5 Centrifuge, capable to reach 3 600 g at the outer end of the centrifuge tubes.
5.6 Pipettes, autopipettes with disposable plastic tips.
5.7 Vortex mixer.
5.8 Water bath or hot plate.
5.9 Graduated conical test tube, (2 ml, 5 ml, 10 ml, 15 ml).
5.10 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 (4.13.3)
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. This
check is not possible for GTX6 and C3,4 as standard substances are not yet commercially available.
If the laboratory has shown over time that there is no inter-batch variation in the performance of the
SPE cartridges, the following approach may be used: Each new batch of SPE-C18 cartridges shall be
checked with sample solutions of well-known concentrations to ensure that minimum recovery of the
whole process is the minimum level of the validation data.
5.11 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 (4.13.3) to ensure
that minimum recovery obtained with the COOH-cartridge is 80 % and the correct elution patterns are
obtained according to 6.3.2. This check is necessary due to experiences gathered during method
development as it was observed that variations can occur. This check is not possible for GTX6 and
C3,4 as standard substances are not yet commercially available.
If the laboratory has shown over time that there is no inter-batch variation in the performance of the
COOH cartridges, the following approach may be used: Each new batch of COOH cartridges shall be
checked with sample solutions of well-known concentrations to ensure that minimum recovery of the
whole process is the minimum level of the validation data.
5.12 Manifold or automatic SPE station (for the SPE clean-ups).
5.13 Block heater (or similar) for the hydrolysis step.
5.14 Reaction tubes, e.g. glass tube with screw cap or vials with 1,5 ml.
5.15 pH indicator paper, able to precisely identify a pH of 6,5 ± 0,3.
5.16 pH meter.
5.17 Rotary evaporator, optionally for samples with low concentrations.
5.18 Membrane filter, for aqueous solutions, with a pore size of 0,45 μm, e.g. regenerated cellulose.

3) -2
g = 9,81 m · s .
5.19 HPLC vials, e.g. amber glass.
5.20 HPLC system, comprising the following:
5.20.1 Injector, preferably a refrigerated injector, capable of injection up to 100 µl.
5.20.2 Pump, capable of gradient elution.
5.20.3 Column oven able to heat to (40 ± 2) °C.
5.20.4 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.
5.20.5 Fluorescence detector, excitation wavelength of λ = 340 nm and emission wavelength of
λ = 395 nm and capable to detect a peroxide-processed (6.5.3) 400 pg STX standard substance as a free
base on the column with a signal-to-noise ratio of at least 10:1.
6 Procedure
6.1 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 (5.1). At least 100 g to 150 g of
pooled homogenized shellfish tissue should be taken. If not directly proceeding with the analysis the
homogenized shellfish tissue can be frozen.
6.2 Extraction procedure
Defrost the homogenized sample in the refrigerator or at room temperature or use it directly after
grinding (6.1).
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 (5.4) and mix with
3 ml of 1 % acetic acid solution 1 (4.4.1) 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.
Remove samples from the water bath, remix 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 (5.9).
Add 3 ml of 1 % acetic acid solution 1 (4.5.1) to the centrifuge tube containing the once extracted
sample (solid residue), mix well 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 (5.9) 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 extract has to be stored in a refrigerator.
6.3 Sample purification
6.3.1 SPE-C18 clean-up
Condition the cartridge according to the manufacturers' instructions, e.g. condition the 3 ml SPE-C18
cartridge (5.10) with 6 ml of methanol followed by 6 ml of water. Discard the solutions which have
passed the cartridge. Place a 5 ml graduated conical test tube (5.9) under the cartridge. Add 1 ml (0,5 g
shellfish tissue equivalent) of the crude extract (6.2) 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 with 1 mol/l of NaOH (4.9.1) using pH-indicator paper (5.15) or pH
meter (5.16) 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 usually stable for more than one year in a freezer, however, this has to be verified.
Aliquots of this extract may be used for oxidation with periodate and peroxide as described in 6.5.2 and
6.5.3.
Additionally, an aliquot of this extract will be analysed without oxidation as control-sample for the
peroxide oxidation to verify that peaks in the chromatogram of the oxidized sample are caused by PSP
toxins and not by naturally fluorescent compounds. Furthermore an aliquot of the sample extract from
SPE-C18 clean-up has to be mixed with matrix modifier and water (instead of periodate oxidant) as
control-sample for the periodate oxidation and will be analysed. The resulting chromatogram will
enable the identification of peaks arising from naturally fluorescent sample co-extractives. PSP toxins
do not produce peaks under these conditions.
If N-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 recovery of GTX1,4 and NEO [3].
6.3.2 SPE-COOH clean-up (fractionation)
Fractionate only extracts from SPE-C18 clean-up that contain N-hydroxylated PSP toxins (e.g. NEO,
dcNEO, C3,4; GTX6 (B2) 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 (5.11) by passing 10 ml of 0,01 mol/l ammonium acetate solution 2 (4.6.2) 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 (6.3.1) through the cartridge and collect the eluate in a 10 ml graduated conical test tube
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 6.5.2 and/or 6.5.3 for
the oxidation steps. If C3,4 is present, proceed to 6.4.
Fraction 2: Pass 4 ml of 0,05 mol/l NaCl solution 1 (4.6.1) through the same cartridge and collect the
eluate (labelled as Fraction 2) in a 5 ml graduated conical test tube. Adjust final volume to 4 ml. This
fraction contains the toxins GTX1,4, GTX2,3, GTX5 (B1), GTX6 (B2) and dcGTX2,3. Proceed to 6.5.2
and/or 6.5.3 for the oxidation steps. If GTX6 (B2) is present, proceed to 6.4.
Fraction 3: Pass 5 ml of 0,3 mol/l NaCl solution 2 (4.6.2) through the cartridge and collect in 5 ml
graduated conical test tube marked as Fraction 3. Adjust final volume to 5 ml. This fraction contains
STX, NEO, dcNEO and dcSTX. Proceed to 6.5.2 and/or 6.5.3 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 50 ml round bottom flasks
instead of graduated conical test tubes 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 5 ml
graduated conical test tube using a Pasteur pipette. Rinse the 50 ml round bottom flask 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 6.5.2 and/or 6.5.3.
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 6.3.3).
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 refrigerator for not more than one week or
not more than one year in a freezer.
6.3.3 Alternative weak cation exchange SPE clean-up [2], [11]
To improve the sensitivity and to reduce the carry over in the adjoining fractions of low PSP-toxin
contents, the following modified clean-up after the SPE C18 clean-up can be used, however, this
procedure has only been in-house-validated.
The fractionation procedure is the same as described in 6.3.2. In contrast to [1], only the concentration
of the sodium chloride solutions and the volume for the elution is different (see Table 5).
Table 5 — Comparison of fractionation conditions [2], [11]
COOH-SPE (e.g. Bakerbond Weak cation exchanger
Carboxylic Acidsilane) (e.g. Strata-X-CW, Phenomenex)
Fraction Toxins Elution solvent Volume Elution solvent Volume
F1 C1,2; C3,4 Water 6,0 ml Water 5,0 ml
F2 GTX1,4; 0,05 mol/l NaCl 4,0 ml 0,3 mol/l NaCl 3,0 ml
GTX2,3;
dcGTX2,3;
GTX5
GTX6
F3 STX; NEO; 0,3 mol/l NaCl 5,0 ml 2,0 mol/l NaCl 3,0 ml
dcNEO; dcSTX
6.4 Conversion of GTX6 (B2) into NEO and/or C3,4 into GTX1,4
6.4.1 General
Currently a GTX6 (B2) standard substance is not available. Therefore, in order to analyse GTX6 (B2), the
toxin shall be hydrolysed into NEO which can be quantified [7], [8]. GTX6 (B2) content can be indirectly
determined by hydrolysis of Fraction 2 of the SPE-COOH clean-up where NEO does not occur.
Currently a C3,4 standard substance is not commercially available. The toxin, present in Fraction 1 of
the SPE-COOH clean-up, can be indirectly determined by hydrolysis to GTX1,4 [10].
6.4.2 Hydrolysis of SPE-COOH Fraction 1 or 2
Add 75 μl of 1 mol/l HCl to 300 μl of SPE-COOH Fraction 2 (6.3.2) in an HPLC vial or in a glass tube with
screw cap. Close the vial/tube well and weigh it. Heat the mixture for 20 min at 90 °C in a water bath or
block heater. Cool down the mixture to room temperature and weigh the vial again to check for possible
evaporation (if evaporation has occurred add water to the original weight). Add e.g. five times small
volumes of 15 µl of 1 mol/l NaOH (4.9.1) and mix each time until the added volume is in total 75 μl.
These steps are necessary to neutralize the acid while avoiding an over-alkalinisation of the hydrolysed
Fraction 2. Adjust to neutral pH.
During periodate oxidation (combination of hydrolyzed Fraction 2, matrix modifier and periodate
derivatising agent) the mixture should change to pH 8,2. However if the oxidized hydrolized fraction
does not reach pH 8,2 then the hydrolyzed Fraction 2 needs to be adjusted by adding a little more NaOH
and the extra amount noted for calculations [12].
Analyse the sample by HPLC after periodate oxidation as described in 6.5.2.
The procedure can also be applied to Fraction 1 of the SPE-COOH clean-up.
NOTE Depending on the minimum measured volume of the used pH-meter it is possible to scale up all the
reagents. (i.e. 900 μl of SPE-COOH Fraction 2 (filtered) +225 μl of 1 mol/l HCl and after reaction 225 μl of 1 mol/l
NaOH solution 1).
6.5 Oxidation procedure
6.5.1 General
During the oxidation reactions, it is important that the time of the reaction is kept the same for all
sample and standard mixtures, since variations can influence the efficiency of the oxidation and the
yield of oxidation products thus affecting the repeatability of the results. A control sample to prove the
absence of naturally fluorescent compounds is necessary as indicated in paragraph 6 of 6.3.1.
For investigation of whole king scallops (Pecten maximus) and whole queen scallops (Aequipecten
opercularis) contaminated with NEO and GTX1,4, this approach can decrease the sensitivity. To improve
the sensitivity the modification in the periodate oxidation step described in Note 3 of 6.5.2 can be used.
6.5.2 Periodate oxidation
All reagents and solutions used in the oxidation reactions are dispensed using auto pipettes with
disposable plastic tips.
It is very important to adjust the pH to 6.5 prior to the periodate oxidation [12].
Add 100 µl of standard solution or sample extract after SPE C18 clean-up (6.3.1) or fraction of SPE-
COOH clean-up (6.3.2) to 100 µl of matrix modifier solution in a 1,5 ml vial. Then add 500 µl of
periodate oxidant (4.13) and mix well on a vortex mixer. Allow the solution to react at room
temperature for 1 min and then add 5 µl of glacial acetic acid (4.4) and mix. Allow the mixture to stand
for at least 10 min at room temperature before injecting 50 µl to 100 µl into the HPLC system.
The solution is stable for at least one day for the non-hydroxylated toxins. However, for NEO, GTX6
(B2), GTX1,4, a slow degradation of oxidation products occurs. Despite this degradation, the toxins may
be quantified if the standard solutions and test solutions are stored and analysed under the same
conditions, see [5].
NOTE 3 For investigation of whole king scallops (Pecten maximus) and whole queen scallops (Aequipecten
opercularis) contaminated with NEO and GTX1,4, the use of a king scallop matrix modifier instead of oyster
modifier (4.14) for the oxidation of the calibration standards was shown to give good recoveries. A modified
periodate reagent containing 5 times the normal relative amounts of periodic acid showed good sensitivity. The
periodate used for oxidation of all standard solutions and samples was prepared using 0,03 mol/l periodic acid
(4.11.2), 0,3 mol/l ammonium formate (4.3) and 0,3 mol/l sodium hydrogen phosphate (4.8) with proportions of
5:1:1 respectively, adjusting the pH to 8,2 prior to use. Also the sensitivity was raised with the use of high injection
volumes (100 μl) [3].
6.5.3 Peroxide oxidation
Add 25 µl of 10 % (w/v) aqueous H O (4.10) to 250 µl of 1 mol/l NaOH (4.9.1) in a 1,5 ml vial and mix.
2 2
Then add 100 µl of standard solution or sample extract after SPE C18 (6.3.1) or SPE-COOH clean-up
(6.3.2). Mix and allow reacting for 2 min at room temperature. Add 20 µl of glacial acetic acid (4.4) and
mix the solution. Inject 25 µl to 50 µl of this solution into the HPLC system. The solution is stable for at
least 8 h at room temperature, see [5].
NOTE Injecting more than 50 µl can cause peak broadening.
7 HPLC determination
Check your system stability (% of peak area variation and % of retention time variation) against your
own HPLC specifications. Inject PSP oxidation products and chromatographically separate to allow
individual identification and quantification of the toxins.
Note that dcGTX2,3 and dcSTX produce each two peaks as the peroxide oxidation leads to two different
oxidation products, which shall be chromatographically separated with a resolution of greater than 1,5.
The FLD sho
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