CEN/TS 16233-2:2011

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: 12-Jul-2011
• ICS: 67.120.30 - Fish and fishery products
• Technical Committee: CEN/TC 275 - Food analysis - Horizontal methods
• Drafting Committee: CEN/TC 275/WG 9 - Vitamins and Carotenoids
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Start Date: 16-Sep-2014
• Completion Date: 14-Apr-2025
• Ref Project: SIST-TS CEN/TS 16233-2:2011 - Foodstuffs - HPLC method for the determination of xanthophylls in fish flesh - Part 2: Identification of the enantiomer ratio of astaxanthin

Overview

CEN/TS 16233-2:2011 specifies a chiral HPLC method for determining the enantiomer ratio of astaxanthin in fish flesh. The Technical Specification targets quantifying the three stereoisomers of all-E-astaxanthin - (3R,3’R), (3R,3’S, meso) and (3S,3’S) - using a chiral column and defined sample-extraction and chromatographic conditions. Results help distinguish dietary sources of astaxanthin (wild, synthetic/racemic, or microbial) and support food analysis, aquaculture quality control and research.

Key topics and technical requirements

• Scope: Determination of astaxanthin enantiomer ratios in fish flesh by HPLC (chiral separation).
• Sample preparation:
  • Large- and small-scale extraction procedures using acetone, magnesium sulfate and BHT antioxidant.
  • Evaporation (rotary or nitrogen flow) and reconstitution in the HPLC mobile phase; centrifugation prior to injection.
• Reagents and standards:
  • Use analytical-grade solvents (n-heptane, dichloromethane, ethanol, tetrahydrofuran, etc.) and reference substances of all-E-astaxanthin and all-E-canthaxanthin (HPLC purity >95%).
  • Mobile phase composition example: mix 24 volumes n-heptane : 58 volumes dichloromethane : 0.3 volumes ethanol.
• HPLC conditions:
  • Example column: chiral Sumichiral OA-2000 (250 × 4.6 mm, 5 µm) or equivalent.
  • Flow rate 1.0 ml/min, column temperature 20 °C, detection VIS at 474 nm.
  • Typical retention window for the meso-form: 11–13 min; relative retention factors provided for the three isomers.
• Calculation and reporting:
  • Enantiomer ratio calculated from peak areas: A(3R,3’R) : A(meso) : A(3S,3’S).
  • Test report must include sample ID, reference to the CEN/TS method, dates, results, and any deviations or observations.
• Limitations noted:
  • Method can be limited when fish were fed mixed astaxanthin sources or shrimp-containing diets (shrimp can introduce racemic astaxanthin).

Applications and users

• Food analysis laboratories performing carotenoid profiling and authenticity testing.
• Aquaculture/feed manufacturers monitoring pigmentation sources in farmed salmonids.
• Regulatory bodies verifying product labeling and investigating origin of pigmentation.
• Academic and applied researchers studying carotenoid metabolism, bioavailability and food authenticity.

Related standards

• EN ISO 3696:1995 - Water for analytical laboratory use (referenced for water quality in reagents).

Keywords: astaxanthin, enantiomer ratio, HPLC, chiral column, xanthophylls, fish flesh, all‑E‑astaxanthin, salmon, food analysis, aquaculture quality control.

Standards Content (Sample)

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

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.

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

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.
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
(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
...