CEN/TS 16233-1:2011

SLOVENSKI STANDARD
01-oktober-2011
äLYLOD0HWRGD+3&/]DGRORþHYDQMHNVDQWRILORYYULEMHPPHVXGHO
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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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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.

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

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.

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.).
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.
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.
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−canthaxanthin
2 200
where
A is the absorbance value at the absorption maximum;
max
10 000 is the scaling factor;
1% 1%
2 200 is the E value of of all-E-canthaxanthin. The E value
...