Foodstuffs - HPLC method for the determination of xanthophylls in fish flesh - Part 2: Identification of the enantiomer ratio of astaxanthin

This Technical Specification describes a method for the determination of the astaxanthin enantiomer ratio in fish flesh by high performance liquid chromatography (HPLC).

Lebensmittel - HPLC-Verfahren zur Bestimmung von Xanthophyllen in Fischfleisch - Teil 2: Bestimmung des Enantiomerenverhältnisses von Astaxanthin

Das vorliegende Dokument beschreibt ein Verfahren zur Bestimmung des Enatiomerenverhältnisses von Astaxanthin in Fischfleisch mit Hochleistungsflüssigchromatographie (HPLC).

Produits alimentaires - Méthode de dosage des xanthophylles dans la chair de poisson par CLHP - Partie 2: Identification de la distribution énantiomérique de l'astaxanthine

La présente Spécification technique décrit une méthode de détermination de la distribution énantiomérique de
l’astaxanthine dans la chair de poisson par chromatographie liquide à haute performance (CLHP).

Živila - Metoda HPCL za določevanje ksantofilov v ribjem mesu - 2. del: Določanje razmerja enantiomer astaksantina

Ta tehnična specifikacija opisuje metodo določanja razmerja enantiomer astaksantina v ribjem mesu s tekočinsko kromatografijo visoke ločljivosti (HPLC).

General Information

Status
Published
Publication Date
30-Oct-2011
Current Stage
9093 - Decision to confirm - Review Enquiry
Completion Date
16-Sep-2014

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SLOVENSKI STANDARD
SIST-TS CEN/TS 16233-2:2011
01-oktober-2011

äLYLOD0HWRGD+3&/]DGRORþHYDQMHNVDQWRILORYYULEMHPPHVXGHO'RORþDQMH

UD]PHUMDHQDQWLRPHUDVWDNVDQWLQD

Foodstuffs - HPLC method for the determination of xanthophylls in fish flesh - Part 2:

Identification of the enantiomer ratio of astaxanthin

Lebensmittel - HPLC-Verfahren zur Bestimmung von Xanthophyllen in Fischfleisch - Teil

2: Bestimmung des Enantiomerenverhältnisses von Astaxanthin

Produits alimentaires - Méthode de dosage des xanthophylles dans la chair de poisson

par CLHP - Partie 2: Identification de la distribution énantiomérique de l'astaxanthine

Ta slovenski standard je istoveten z: CEN/TS 16233-2:2011
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
SIST-TS CEN/TS 16233-2:2011 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST-TS CEN/TS 16233-2:2011
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SIST-TS CEN/TS 16233-2:2011
TECHNICAL SPECIFICATION
CEN/TS 16233-2
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
July 2011
ICS 67.120.30
English Version
Foodstuffs - HPLC method for the determination of xanthophylls
in fish flesh - Part 2: Identification of the enantiomer ratio of
astaxanthin

Produits alimentaires - Méthode de dosage des Lebensmittel - HPLC-Verfahren zur Bestimmung von

xanthophylles dans la chair de poisson par CLHP - Partie 2: Xanthophyllen in Fischfleisch - Teil 2: Bestimmung des

Identification de la distribution énantiomérique de Enantiomerenverhältnisses von Astaxanthin

l'astaxanthine

This Technical Specification (CEN/TS) was approved by CEN on 28 May 2011 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit their

comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available

promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS)

until the final decision about the possible conversion of the CEN/TS into an EN is reached.

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, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: Avenue Marnix 17, B-1000 Brussels

© 2011 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 16233-2:2011: E

worldwide for CEN national Members.
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SIST-TS CEN/TS 16233-2:2011
CEN/TS 16233-2:2011 (E)
Contents Page

Foreword ..............................................................................................................................................................3

Introduction .........................................................................................................................................................4

1 Scope ......................................................................................................................................................4

2 Normative references ............................................................................................................................4

3 Principle ..................................................................................................................................................4

4 Reagents .................................................................................................................................................5

5 Apparatus ...............................................................................................................................................6

6 Sample preparation and extraction......................................................................................................7

7 HPLC .......................................................................................................................................................7

8 Test report ........................................................................................................................................... 10

Bibliography ..................................................................................................................................................... 11

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SIST-TS CEN/TS 16233-2:2011
CEN/TS 16233-2:2011 (E)
Foreword

This document (CEN/TS 16233-2:2011) has been prepared by Technical Committee CEN/TC 275 “Food

analysis - Horizontal methods”, the secretariat of which is held by DIN.

Attention is drawn to the possibility that some of the elements of this document may be the subject of patent

rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.

According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following

countries are bound to announce this Technical Specification: 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, Romania, Slovakia, Slovenia,

Spain, Sweden, Switzerland and the United Kingdom.
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SIST-TS CEN/TS 16233-2:2011
CEN/TS 16233-2:2011 (E)
Introduction

Three stereoisomers of all-E-astaxanthin (two enantiomers and a meso-form) exist, named (3R,3’R)-,

(3S,3’S)- and (3R,3’S, meso)-all-E-astaxanthin, see Figure 1. These isomers can be determined by

chromatography on a chiral HPLC column. Salmonids receive their astaxanthin through the diet and the flesh

isomer composition reflects the dietary source of astaxanthin. Synthetically produced (racemic) astaxanthin

and alternative organic forms of astaxanthin (produced by micro-organisms like Xanthophyllomyces

dendrorhous - formerly Phaffia rhodozyma and Haematococcus pluvialis) have different stereoisomer profiles,

see Figure 2. Even though there are minor differences in bioavailability of theses forms in fish [1], [2], the

resulting flesh isomer profile can be used to distinguish wild from farmed salmon, and to determine which form

of astaxanthin that has been supplemented the feed to farmed species [3], [4], [5] . Precautions should be

taken since the method can sometimes not discriminate groups of farmed salmon that have been fed diets

with mixed astaxanthin sources or fed different sources during periods of the production cycle [6]. In addition,

racemic astaxanthin mixtures have been observed in the shrimp species Penaeus and also in some other

orders of higher crustaceans. Therefore, fish exclusively fed with shrimp meal could also contain racemic

astaxanthin [7], [8], and could erroneously be regarded as fish fed with synthetic astaxanthin.

1 Scope

This Technical Specification describes a method for the determination of the astaxanthin enantiomer ratio in

fish flesh by high performance liquid chromatography (HPLC).
2 Normative references

The following referenced documents are indispensable for the application of this document. For dated

references, only the edition cited applies. For undated references, the latest edition of the referenced

document (including any amendments) applies.

EN ISO 3696:1995, Water for analytical laboratory use – Specification and test methods (ISO 3696:1987).

3 Principle

Extract fish flesh by homogenizing the tissue in acetone. Filter the extract and evaporate at reduced pressure

using a rotary evaporator or a flow of nitrogen at 50 °C. Finally, dissolve the residue in the mobile phase, a

mixture of n-heptane, dichloromethane and ethanol.

Determine the ratio of (3R,3’R)-, (3R,3’S, meso)- and (3S,3’S)-all-E-astaxanthin by chromatography on a

chiral HPLC column.
(3R,3'R) all-E-astaxanthin
(3S,3'S) all-E-astaxanthin
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SIST-TS CEN/TS 16233-2:2011
CEN/TS 16233-2:2011 (E)
(3R,3'S) all-E-astaxanthin
Figure 1 — Enantiomers and meso-form of all-E-astaxanthin
4 Reagents

During the analysis, unless otherwise stated, use only water complying with grade 1 of EN ISO 3696:1995 and

reagents of recognized analytical grade, e.g. pro analysis (p.a.).
4.1 Magnesium sulfate, anhydrous, purity (complexometric) > 98 %
4.2 Butylated hydroxytoluene (BHT), purity (GC) > 99 %.

4.3 Tetrahydrofuran, purity (GC) > 99 %, stabilized with 0,025 % 2,6-di-tert-butyl-p-cresol (BHT).

4.4 Cyclohexane, purity (GC): > 99 %.
4.5 n-heptane, purity (GC): > 99 %.
4.6 Dichloromethane, p.a., purity (GC) : > 99 %.
4.7 Acetone, purity (GC): > 99 %.
4.8 Ethanol, absolute, purity (GC): > 99 %.
4.9 Chiral HPLC mobile phase solvent, isocratic.

Mix 24 parts per volume of n-heptane (4.5) with 58 parts per volume of dichloromethane (4.6) and 0,3 parts

per volume of ethanol (4.8).

4.10 Reference substances of all-E-astaxanthin and all-E-canthaxanthin, purity (HPLC): > 95 %.

Store reference substances under nitrogen or argon at approximately -20 °C. Traces of oxygen destroy the

substances.
4.11 Preparation of astaxanthin standard solution, ρ = 1,5 mg/ml.

Weigh approximately 1,5 mg to the nearest 0,1 mg of the reference substance of all-E-astaxanthin (4.10) and

1 g of BHT (4.2) into a 100 ml volumetric flask. Dissolve in 5 ml of tetrahydrofuran (4.3) and dilute to the mark

with tetrahydrofuran. Support dissolution by ultrasonic treatment. Transfer an aliquot of 10 ml of this solution

into a 100 ml volumetric flask and add approximately 85 ml of n-heptane (4.5). The mixture cools and

contracts. Warm the so
...

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