EN 14526:2017

Foodstuffs - Determination of saxitoxin-group toxins in shellfish - HPLC method using pre-column derivatization with peroxide or periodate oxidation

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) per kg shellfish tissue and also as µmol/kg shellfish tissue and are listed in Table 1.
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].

Lebensmittel - Bestimmung von Toxinen der Saxitoxingruppe in Schalentieren - HPLC-Verfahren mit Vorsäulenderivatisierung und Peroxid- oder Periodatoxidation

Diese Europäische Norm legt ein Verfahren [1] für die quantitative Bestimmung von Saxitoxin (STX), Decarbamoyl-Saxitoxin (dcSTX), Neosaxitoxin (NEO), Decarbamoyl-Neosaxitoxin (dcNEO), Gonyautoxin 1 und 4 (GTX1,4; Summe der Isomere), Gonyautoxin 2 und 3 (GTX2,3; Summe der Isomere), Gonyautoxin 5 (GTX5, auch als B1 bezeichnet), Gonyautoxin 6 (GTX6, auch als B2 bezeichnet), Decarbamoyl-Gonyautoxin 2 und 3 (dcGTX2,3; Summe der Isomere), N-Sulfocarbamoyl-Gonyautoxin 1 und 2 (C1,2; Summe der Isomere) und (je nach Verfügbarkeit eines zertifizierten Referenzmaterials (CRMs)) N-Sulfocarbamoyl-Gonyautoxin 3 und 4 (C3,4; Summe der Isomere) in (rohen) Miesmuscheln, Austern, Jakobsmuscheln und Venusmuscheln fest. Laborerfahrungen haben gezeigt, dass das Verfahren auch auf andere Schalentiere [2], [3] und gekochte Schalentiererzeugnisse anwendbar ist. Das beschriebene Verfahren wurde in einem Ringversuch [4], [5] validiert und auch in einer EURL-Leistungsprüfung verifiziert, die auf die Gesamttoxizität der Proben ausgerichtet war [6]. Toxine, die nicht im ersten Ringversuch [4], [5] verfügbar waren, wie z. B. dcGTX2,3 und dcNEO, wurden in zwei weiteren Ringversuchen [7], [8] validiert. Die niedrigsten validierten Werte [4], [5], [8] sind in µg Toxin (freie Base) je kg Schalentierfleisch und auch in µmol/kg Schalentierfleisch angegeben und in Tabelle 1 aufgeführt.
GTX6 (B2) wurde im ersten Ringversuch nicht quantitativ bestimmt, jedoch wiesen mehrere Laboratorien dieses Toxin direkt nach der Reinigung mit Festphasenextraktion an einem Ionenaustauscher (SPE-COOH) nach. Diese Laboratorien gaben eine Massenkonzentration von 30 µg/kg oder höher in einigen Proben an. Aus diesem Grund ist das vorliegende Verfahren, abhängig von der Verfügbarkeit einer Vergleichssubstanz, für die direkte Quantifizierung von GTX6 (B2) anwendbar. Derzeit ist es möglich, GTX6 nach einer Hydrolyse der Fraktion 2 der SPE-COOH-Reinigung als NEO wie in 6.4 beschrieben zu bestimmen. Die indirekte Quantifizierung von GTX6 wurde in zwei zusätzlichen Ringversuchen [7], [8] validiert.
C3,4 wurde im ersten Ringversuch quantitativ bestimmt. Das vorliegende Verfahren ist, abhängig von der Verfügbarkeit einer Vergleichssubstanz, für die direkte Quantifizierung von C3,4 anwendbar. Wenn keine Vergleichssubstanz verfügbar ist, kann C3,4 nur als GTX1,4 quantifiziert werden, wenn die gleiche
Hydrolysevorschrift wie für GTX6 (6.4) auf die Fraktion 1 der SPE COOH-Reinigung angewendet wird, siehe [10].

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

La présente Norme européenne spécifie une méthode [1] de détermination de la teneur en saxitoxine (STX), décarbamoyle saxitoxine (dcSTX), néosaxitoxine (NEO), décarbamoyle néosaxitoxine (dcNEO), gonyautoxines 1 et 4 (GTX1,4 ; somme des isomères), gonyautoxines 2 et 3 (GTX2,3 ; somme des isomères), gonyautoxine 5 (GTX5, également appelée B1), gonyautoxine 6 (GTX6, également appelée B2), décarbamoyle gonyautoxines 2 et 3 (dcGTX2,3 ; somme des isomères), N-sulfocarbamoyle gonyautoxines 1 et 2 (C1,2 ; somme des isomères) et, sous réserve de la disponibilité de matériaux de référence certifiés, N-sulfocarbamoyle gonyautoxines 3 et 4 (C3,4 ; somme des isomères) dans les moules, les huîtres, les coquilles Saint-Jacques et les palourdes (crues). L’expérience en laboratoire a montré que cette méthode peut également être appliquée à d’autres coquillages [2], [3] et produits cuits à base de coquillages. La méthode décrite a été validée dans le cadre d’une étude interlaboratoires [4], [5] et a également été vérifiée lors d’un essai de performance EURL portant sur la toxicité globale des échantillons [6]. Les toxines non disponibles à l’occasion de la première étude interlaboratoires [4], [5], par exemple dcGTX2,3 et dcNEO, ont été validées lors de deux nouvelles études interlaboratoires [7], [8]. Les plus bas taux validés [4], [5], [8], sont exprimés en µg de toxine (en tant que base libre) par kg de chair de coquillage, et en µmol/kg de chair du coquillage. Ils sont indiqués dans le Tableau 1.
La détermination de la teneur en GTX6 (B2) n’était pas incluse dans la première étude interlaboratoires, mais plusieurs laboratoires ont détecté directement cette toxine après l’extraction en phase solide avec purification par échange d’ions (SPE-COOH). Ils ont indiqué une concentration massique de 30 µg/kg ou plus dans certains échantillons. En conséquence, la présente méthode peut être employée pour doser directement GTX6 (B2), à condition de disposer de l’étalon. Il est actuellement possible de déterminer la teneur en GTX6 après hydrolyse de la fraction 2 de la purification SPE-COOH, décrite au 6.4 en tant que NEO. La quantification indirecte de GTX6 a été validée dans le cadre de deux études interlaboratoires supplémentaires [7], [8].
La première étude interlaboratoires incluait une détermination de la teneur en C3,4. La présente méthode peut être employée pour quantifier directement C3,4, à condition de disposer de l’étalon. Si aucun étalon n’est disponible, les N-sulfocarbamoyle gonyautoxines C3,4 peuvent être quantifiées seulement en tant que GTX1,4 si le même protocole d’hydrolyse que celui utilisé pour GTX6 (6.4) est appliqué à la fraction 1 de la purification SPE-COOH (voir [10]).

Živila - Določevanje toksinov iz skupine saksitoksina v školjkah - Metoda HPLC z uporabo predkolonske derivatizacije s peroksidno ali perjodatno oksidacijo

Ta dokument določa metodo za kvantitativno določevanje saksitoksina (STX), dekarbamoil saksitoksina (dcSTX), neosaksitoksina (NEO), dekarbamoil neosaksitoksina (dcNEO), goniautoksina 1 in 4 (GTX1,4; vsota izomerov), goniautoksina 2 in 3 (GTX2,3; vsota izomerov), goniautoksina 5 (GTX5, imenovan tudi B1), goniautoksina 6 (GTX6, imenovan tudi B2), dekarbamoil goniautoksina 2 in 3 (dcGTX2,3; vsota izomerov), N-sulfokarbamoil-goniautoksina 1 in 2 (C1,2; vsota izomerov) in (odvisno od razpoložljivosti overjenih referenčnih materialov (CRM-ji)) N-sulfokarbamoil-goniautoksina 3 in 4 (C3,4; vsota izomerov) v (surovih) klapavicah, ostrigah, pokrivačah in školjkah. Izkušnje v laboratoriju kažejo, da se lahko ta standard uporablja tudi za druge proizvode iz školjk [10], [13] in kuhane proizvode iz školjk. Opisana metoda je bila potrjena v medlaboratorijski študiji [1], [2] in objavljena kot uradna metoda AOAC [3]. Ta metoda je bila preverjena tudi s preskusom učinkovitosti EURL za skupno toksičnost vzorcev [4]. Toksini, ki niso bili na voljo pri prvi medlaboratorijski študiji [1], [2], kot so dcGTX2,3 in dcNEO, so bili potrjeni v dveh dodatnih študijah [5], [6]. Najnižje potrjene vrednosti [1], [2], [6] so podane v μg toksina (prosta baza) na kg mesa školjk in tudi kot μmol/kg mesa školjk ter so navedene v preglednici 1.
Kvantitativno določevanje GTX6 (B2) ni bilo vključeno v prvi študiji, vendar pa je več laboratorijev odkrilo ta toksin neposredno po čiščenju z ionsko izmenjavo, pri čemer je masna koncentracija v nekaterih vzorcih znašala 30 μg/kg ali več. Zaradi tega se ta metoda uporablja za neposredno kvantifikacijo GTX6 (B2), odvisno od razpoložljivosti standardnega materiala. Trenutno je mogoče določiti GTX6 po hidrolizi kot NEO. Posredna kvantifikacija GTX6 je bila potrjena v dveh dodatnih študijah [5], [6].
Kvantitativno določevanje C3,4 je bilo vključeno v prvi študiji. Ta metoda se uporablja za neposredno kvantifikacijo C3,4, odvisno od razpoložljivosti standardnega materiala. Enak protokol za hidrolizo, ki se uporablja za GTX6, se lahko uporabi za kvantifikacijo tega toksina kot GTX1,4 [8] pri deležu 1 SPE-COOH, če je prisoten C3,4.

General Information

• Status: Published
• Publication Date: 17-Jan-2017
• Withdrawal Date: 30-Jul-2017
• ICS: 67.120.30 - Fish and fishery products
• Technical Committee: CEN/TC 275 - Food analysis - Horizontal methods
• Drafting Committee: CEN/TC 275/WG 14 - Marine Biotoxins
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Completion Date: 09-Jan-2023
• Directive: 882/2004 - Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 onofficial controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules
• Ref Project: SIST EN 14526:2017 - Foodstuffs - Determination of saxitoxin-group toxins in shellfish - HPLC method using pre-column derivatization with peroxide or periodate oxidation

Relations

• Replaces: EN 14526:2004 - Foodstuffs - Determination of saxitoxin and dc-saxitoxin in mussels - HPLC method using pre-column derivatization with peroxide or periodate oxidation
• Effective Date: 06-Jun-2012

Standards Content (Sample)

SLOVENSKI STANDARD
01-marec-2017
1DGRPHãþD
SIST EN 14526:2005
äLYLOD'RORþHYDQMHWRNVLQRYL]VNXSLQHVDNVLWRNVLQDYãNROMNDK0HWRGD+3/&]
XSRUDERSUHGNRORQVNHGHULYDWL]DFLMHVSHURNVLGQRDOLSHUMRGDWQRRNVLGDFLMR
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
...