CEN/TS 16233-1:2011

Foodstuffs - HPLC method for the determination of xanthophylls in fish flesh - Part 1: Determination of astaxanthin and canthaxanthin

This Technical Specification specifies a method for the determination of canthaxanthin and astaxanthin in fish flesh by high performance liquid chromatography (HPLC). The method can be applied at a range above 0,02 mg/kg. The method should not be applied to the determination of canthaxanthin in poultry tissues, egg yolks and shrimp tissues due to a possible interference of canthaxanthin with cryptoxanthin and xanthophyll esters sometimes present in these matrices.

Lebensmittel - HPLC-Verfahren für die Bestimmung von Xanthophyllen in Fischfleisch - Teil 1: Bestimmung von Astaxanthin und Canthaxanthin

Diese Technische Spezifikation legt ein Verfahren zur Bestimmung von Canthaxanthin und Astaxanthin in Fischfleisch mit Hochleistungsflüssigchromatographie (HPLC) fest. Das Verfahren kann bei Gehalten von mehr als 0,02 mg/kg angewendet werden. Das Verfahren sollte nicht zur Bestimmung von Canthaxanthin in Geflügelfleisch, Eigelb und Garnelenfleisch angewendet werden, weil Canthaxanthin mit Cryptoxanthin und Xanthophyllestern, die in diesen Matrices zuweilen vorliegen, interferieren kann.

Produits alimentaires - Méthode de dosage des xanthophylles dans la chair de poisson par CLHP - Partie 1: Dosage de l'astaxanthine et de la canthaxantine

La présente Spécification technique spécifie une méthode de dosage de la canthaxanthine et de
l’astaxanthine dans la chair de poisson par chromatographie liquide à haute performance (CLHP). La
méthode peut être appliquée à une gamme supérieure à 0,02 mg/kg. Il convient de ne pas appliquer la
méthode au dosage de la canthaxanthine dans les tissus de volailles, les jaunes d'oeufs et les tissus de
crevettes en raison d’une éventuelle interférence de la canthaxanthine avec les esters de cryptoxanthine et de
xanthophylle parfois présents dans ces matrices.

Živila - Metoda HPCL za določevanje ksantofilov v ribjem mesu - 1. del: Določevanje astaksantina in kantaksantina

Ta tehnična specifikacija opredeljuje metodo določevanja kantaksantina in astaksantina v ribjem mesu s tekočinsko kromatografijo visoke ločljivosti (HPLC). Metoda se lahko uporablja v razponu nad 0,02 mg/kg. Metoda se ne sme uporabljati za določevanje kantaksantina v perutninskih tkivih, jajčnih rumenjakih in tkivih kozic (morskih rakcev) zaradi morebitne interference kantaksantina s kriptoksantinovimi in ksantofilnimi estri, ki so včasih v teh matrikah.

General Information

• Status: Published
• Publication Date: 30-Oct-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
• Completion Date: 16-Sep-2014
• Ref Project: SIST-TS CEN/TS 16233-1:2011 - Foodstuffs - HPLC method for the determination of xanthophylls in fish flesh - Part 1: Determination of astaxanthin and canthaxanthin

Standards Content (Sample)

SLOVENSKI STANDARD
SIST-TS CEN/TS 16233-1:2011
01-oktober-2011
äLYLOD0HWRGD+3&/]DGRORþHYDQMHNVDQWRILORYYULEMHPPHVXGHO
'RORþHYDQMHDVWDNVDQWLQDLQNDQWDNVDQWLQD

Foodstuffs - HPLC method for the determination of xanthophylls in fish flesh - Part 1:

Determination of astaxanthin and canthaxanthin

Lebensmittel - HPLC-Verfahren für die Bestimmung von Xanthophyllen in Fischfleisch -

Teil 1: Bestimmung von Astaxanthin und Canthaxanthin

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

par CLHP - Partie 1: Dosage de l'astaxanthine et de la canthaxantine
Ta slovenski standard je istoveten z: CEN/TS 16233-1:2011
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 16233-1:2011 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 16233-1:2011
TECHNICAL SPECIFICATION
CEN/TS 16233-1
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
July 2011
ICS 67.120.30
English Version
Foodstuffs - HPLC method for the determination of xanthophylls
in fish flesh - Part 1: Determination of astaxanthin and
canthaxanthin

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

xanthophylles dans la chair de poisson par CLHP - Partie 1: Xanthophyllen in Fischfleisch - Teil 1: Bestimmung von

Dosage de l'astaxanthine et de la canthaxantine Astaxanthin und Canthaxanthin

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 16233-1:2011: E

worldwide for CEN national Members.
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Contents Page

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

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

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

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

4 Reagents .................................................................................................................................................4

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

6 Calibration ..............................................................................................................................................7

7 Procedure ...............................................................................................................................................8

8 HPLC .......................................................................................................................................................9

9 Calculation ........................................................................................................................................... 10

10 Test report ........................................................................................................................................... 11

Annex A (informative) Validation for astaxanthin ....................................................................................... 12

Annex B (informative) Typical chromatograms ........................................................................................... 14

Bibliography ..................................................................................................................................................... 16

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CEN/TS 16233-1:2011 (E)
Foreword

This document (CEN/TS 16233-1:2011) 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 16233-1:2011
CEN/TS 16233-1:2011 (E)
1 Scope

This Technical Specification specifies a method for the determination of canthaxanthin and astaxanthin in fish

flesh by high performance liquid chromatography (HPLC). The method can be applied at a range above

0,02 mg/kg. The method should not be applied to the determination of canthaxanthin in poultry tissues, egg

yolks and shrimp tissues due to a possible interference of canthaxanthin with cryptoxanthin and xanthophyll

esters sometimes present in these matrices.
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 homogenising the tissue in acetone. Filter the extract and evaporate at reduced pressure

using a rotary evaporator or a flow of nitrogen at 50 °C. Dissolve the residue in a mixture of n-heptane and

acetone and analyse by an isocratic normal-phase HPLC.

The HPLC system described effectively separates astaxanthin and canthaxanthin and these both xanthophylls

from other carotenoids found in fish flesh as e.g. β-carotene, lutein and zeaxanthin. The main geometrical

isomers of astaxanthin are separated from each other and from oxidation products of astaxanthin, astacene

and semi-astacene. In contrast, the isomers of canthaxanthin are not separated.
Figure 1 — all-E-Astaxanthin
Figure 2 — all-E-Canthaxanthin
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.).
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4.1 Magnesium sulfate, anhydrous, purity (complexometric) > 98 %.
4.2 Phosphoric acid, volume fraction ϕ is 85 %, purity (acidimetric) ≥ 85 %.
4.3 Butylated hydroxytoluene (BHT), purity (GC) > 99 %.

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

4.5 Cyclohexane, purity (GC): > 99 %.
4.6 n-heptane, purity (GC): > 99 %.
4.7 Acetone, purity (GC): > 99 %.
4.8 Ethanol, absolute, purity (GC): > 99 %.
4.9 Methanol, purity (GC): > 99 %.

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 HPLC mobile phase solvent, isocratic.

Mix 86 parts per volume of n-heptane (4.6) with 14 parts per volume of acetone (4.7).

4.12 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 (4.3) into a 100 ml volumetric flask. Dissolve in 5 ml of tetrahydrofuran (4.4) 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 (4.6). The mixture cools and

contracts. Warm the solution to room temperature and dilute to the mark with n-heptane. This results in an

astaxanthin concentration of approximately 1,5 mg/l in a mixture of 9 parts per volume of n-heptane and 1 part

per volume of tetrahydrofuran.
4.13 Preparation of canthaxanthin 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-canthaxanthin (4.10)

and 1 g of BHT (4.3) into a 100 ml volumetric flask. Dissolve in 5 ml of tetrahydrofuran (4.4) 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 cyclohexane (4.5). The mixture cools

and contracts. Warm the solution to room temperature and dilute to the mark with cyclohexane. This results in

a canthaxanthin concentration of approximately 1,5 mg/l in a mixture of 9 parts per volume of cyclohexane

and 1 part per volume of tetrahydrofuran.
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4.14 Preparation of solution of heat-isomerized carotenoids (control solution)

Weigh approximately 1,5 mg of all-E-astaxanthin (4.10), 1,5 mg of all-E-canthaxanthin (4.10) and 0,5 g of BHT

(4.3) to the nearest 0,1 mg and dissolve in a 500 ml volumetric flask in 10 ml of tetrahydrofuran (4.4). Dilute

this solution with 200 ml of a mixture of 86 parts per volume of n-heptane (4.6) and 14 parts per volume of

acetone (4.7). Reflux for 1 h in a water bath at a temperature of 80 °C. Cool to room temperature and dilute

the solution to the mark with the mixture of n-heptane and acetone. Pour the mixture into a dispenser bottle,

mix well, leave at room temperature overnight and dispense into a large number of HPLC vials. Immediately

seal the vials carefully with septa made from polytetrafluoroethylene (PTFE) and silicone and store them at

approximately 23 °C in the dark.
5 Apparatus
Usual laboratory apparatus, glassware, and the following:

5.1 Knife mill, suitable for food with grinding chamber volume of approximately 1 000 ml.

5.2 Sintered glass frit, porosity 3 (16 µm to 40 µm), diameter: approximately 6 cm.

5.3 Dispersing instrument.

5.3.1 Bench-top dispersing instrument for approximately 1 ml to 2 000 ml e.g. with 20 mm diameter

aggregate.

5.3.2 Hand-held dispersing instrument for approximately 1 ml to 250 ml e.g. with 12 mm diameter

aggregate.
5.4 Rotary evaporator e.g. 20 °C to 100 °C.
5.5 Nitrogen flow evaporator, with heating block and holder for pipettes.

5.6 Spectrometer, wavelength range 190 nm to 900 nm, wavelength accuracy: ≤ 1 nm.

5.7 Centrifuge, bench laboratory centrifuge for at least 2 500 g.
5.8 Balances.

5.8.1 Balance with readability of 0,01 g, precision (std dev.) of ± 0,005 g, capacity of 2 100 g.

5.8.2 Balance with readability of 0,01 mg, precision (std dev.) of ± 0,015 mg, capacity of 205 g.

5.9 Solid phase extraction manifold, steel needles (0,90 mm x 55 mm) attached to the valve outlets.

5.10 SPE columns, 25 ml reservoirs, plastic, equipped with 10 µm bottom fritts.

5.11 HPLC chromatographic system, with column thermostat and UV/visible or diode array detector.

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6 Calibration
6.1 General

Prepare standard solutions at single concentrations (4.12 and 4.13), measure by spectrometry immediately

after preparation (6.2), and inject the standard solutions into the HPLC (6.3). Determine the response factors

of the carotenoids from the total peak areas of the chromatograms and the concentrations measured by

spectrometry.

Since the method involves one-level calibrations it is recommended to control the linearity of the HPLC after

implementation or any change of the system. For this purpose, the standard solutions can be diluted with a

mixture of 86 parts per volume of n-heptane (4.6) and 14 parts per volume of acetone (4.7). The correlation

coefficient of the regression line (forced through zero) should exceed 0,98.
6.2 Determination concentration of standard solution with spectrometry

Within 1 h after preparation, measure the concentration of all-E-astaxanthin or all-E-canthaxanthin by

spectrometry at the respective absorption maximum using n-heptane (4.6) as a blank. Calculate the mass

concentration (ρ) in milligram per millilitre of the standard solution using Equations (1) and (2):

A ×10 000
max
ρ = (1)
all−E−astaxanthin
2 100
where
A is the absorbance value at the absorption maximum;
max
10 000 is the scaling factor;
1% 1%

2 100 is the E value of all-E-astaxanthin. The E value is the theoretical absorption of a 1 %

1cm 1cm

solution of all-E-astaxanthin in a 1 cm cell at approximately 470 nm (λ ) in n-heptane, see [1].

max
A ×10 000
max
ρ = (2)
all−E−canthaxant
...