kSIST FprEN 17550:2021

SLOVENSKI STANDARD
oSIST prEN 17550:2020
01-november-2020
Krma: metode vzorčenja in analize - Določevanje karotenoidov v krmnih
mešanicah in premiksih s tekočinsko kromatografijo visoke ločljivosti z
ultravijolično (UV) detekcijo (HPLC-UV)

Animal feeding stuffs: Methods of sampling and analysis - Determination of carotenoids

in animal compound feed and premixtures by high performance liquid chromatography -

Aliments des animaux - Méthodes d’échantillonnage et d’analyse - Détermination de la

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

Aliments des animaux - Méthodes d'échantillonnage et Futtermittel - Probenahme- und

d'analyse - Détermination de la teneur en caroténoïdes Untersuchungsverfahren - Bestimmung von

des aliments composés et des prémélanges pour Carotinoiden in Mischfuttermitteln und

animaux par chromatographie liquide à haute Vormischungen für Tiere mittels Reverse

performance couplée à une détection UV (CLHP-UV) Hochdruckphase-Flüssigchromatographie-UV

This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee

If this draft becomes a European Standard, 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.

This draft European Standard was established by CEN 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

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, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without

© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17550:2020 E

European foreword ............................................................................................................................................ 3

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

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

3 Terms and definitions ......................................................................................................................... 4

4 Principle ................................................................................................................................................... 4

5 Reagents and materials ...................................................................................................................... 5

6 Apparatus ................................................................................................................................................ 8

7 Sampling ................................................................................................................................................. 10

8 Preparation of test sample .............................................................................................................. 10

8.1 General.................................................................................................................................................... 10

8.2 Laboratory sample ............................................................................................................................. 10

8.3 Test sample ........................................................................................................................................... 10

8.4 Test portion .......................................................................................................................................... 10

9 Procedure .............................................................................................................................................. 11

9.1 General.................................................................................................................................................... 11

9.2 Enzymatic disclosure ......................................................................................................................... 11

9.3 Extraction .............................................................................................................................................. 11

9.3.1 Extraction using PLE .......................................................................................................................... 11

9.3.2 Conventional liquid solid extraction (SLE) ................................................................................ 12

9.4 Centrifugation ...................................................................................................................................... 12

9.5 HPLC analysis ....................................................................................................................................... 13

9.5.1 Analytical conditions ......................................................................................................................... 13

9.5.2 HPLC determination .......................................................................................................................... 13

9.5.3 Criteria for acceptance of the analytical result ........................................................................ 15

10 Calculation ............................................................................................................................................. 16

11 Test report ............................................................................................................................................. 16

11.1 Interlaboratory study ........................................................................................................................ 16

11.2 Repeatability ........................................................................................................................................ 16

11.3 Reproducibility .................................................................................................................................... 17

12 Test report ............................................................................................................................................. 17

Annex A (informative) Complementary instrumental and analytical information ................. 18

A.1 Absorption coefficient values ......................................................................................................... 18

A.2 Examples of chromatographic profile patterns of isomerized carotenoids .................. 19

A.3 Results of the collaborative study ................................................................................................. 30

Bibliography ....................................................................................................................................................... 46

This document (prEN 17550:2020) has been prepared by Technical Committee CEN/TC 327 “Animal

feeding stuffs – Methods of sampling and analysis”, the secretariat of which is held by NEN.

This document has been prepared under a mandate given to CEN by the European Commission and the

This analytical procedure specifies a reverse phase high performance liquid chromatographic with UV

detection (RP-HPLC-UV) method for the simultaneous determination of four authorized carotenoids in

fish compound feed, namely astaxanthin (AXN), canthaxanthin (CXN), adonirubin (ADR) and astaxanthin

dimethyldisuccinate (AXN DMDS), and of six authorized carotenoids in poultry feed, namely

canthaxanthin (CXN); capsanthin (CSN), ethyl ester of beta-apo-8'-carotenoic acid (BACARE),

citranaxanthin (CIXN), lutein (LUT) and zeaxanthin (ZEA) at levels ranging from ca. 2 to ca. 4 500 mg/kg

(depending on the carotenoid). Beta-carotene (BCAR), authorized in compound feed for all animal

species, was also added to the scope. The analytical procedure is fit for the purpose of quantitation of

declared carotenoids and labelling confirmation. The procedure applies to natural and synthetic feed

Xanthophyll esters like those of lutein, zeaxanthin and capsanthin that might be present in feed materials

are not authorized feed additives and therefore not part of the scope of this method.

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements 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 6498, Animal feeding stuffs - Guidelines for sample preparation (ISO 6498)

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

The carotenoids are first disclosed through an enzymatic reaction at 50 °C. The samples are extracted

with acetone by means of a pressurized liquid extraction instrument or by liquid solid extraction. The

extracts are centrifuged and analysed by reverse phase HPLC with UV or Diode Array Detection. A

common isosbestic wavelength of 410 nm is selected for the determination of the target analytes, thus

ensuring that the various isomers of each of the carotenoids have similar absorbance coefficients. The

NOTE The maximum contents of the carotenoids as established by the European regulations for the

authorization of feed additives ([1], [2]), are expressed in terms of the sum of the all-trans and cis isomers.

Therefore, the areas of the corresponding isomers in the HPLC chromatogram have to be summed up prior to

WARNING 1 — Carotenoids are subject to light degradation. Protect analytical work adequately from day

light, and keep standard solutions protected from light by using amber glassware, amber vials or

WARNING 2 — Avoid inhalation of and exposure to the toxic standard materials and solutions thereof.

Work under fume hood when handling the solvents and solutions. Wear safety glasses and protective

5.1 Protease with the ability to release carotenoids from their encapsulated form

5.4 High purity diatomaceous earth suitable for PLE, e.g. Hydromatrix™, bulk support material

Milli-Q, Hydromatrix™, Alcalase® and Multifect PR 6L are examples of suitable products available commercially.

This information is given for the convenience of users of this document and does not constitute an endorsement by

The exact spectroscopic grade depends on the carotenoid for which the UV standardisation of the standard

5.13.1 Phase A: acetonitrile:methyl tert-butyl ether:water mixture 70:20:10; v:v:v, stabilized with

Using a graduated cylinder (6.17), transfer 700 ml of acetonitrile (5.7) into a 1 000 ml bottle. Measure

(6.17) and add 200 ml of methyl tert-butyl ether (5.8) and 100 ml water (5.2). Add 1,0 g of BHT (5.3).

Perform mixing and degassing for 10 min in an ultrasonic bath (6.11). This mobile phase is stable for

NOTE The retention time of the carotenoids is strongly influenced by slight differences in the composition of

mobile phase A. The use of an HPLC quality control sample (9.1) is crucial for the correct signal allocation.

Furthermore, the presence of non-targeted carotenoids might interfere with the signals of the targeted analytes

Using a graduated cylinder (6.17), transfer 700 ml of acetonitrile (5.7) into a 1 000 ml bottle. Measure

(6.17) and add 300 ml methyl tert-butyl ether (5.8). Add 1,0 g of BHT (5.3). Perform mixing and degassing

for 10 min in an ultrasonic bath (6.11). This mobile phase is stable for 28 days.

NOTE Possible carotenoids are astaxanthin (AXN), canthaxanthin (CXN), adonirubin (ADR), astaxanthin

dimethyldisuccinate (AXN DMDS), capsanthin (CSN), ethyl ester of beta-apo-8'-carotenoic acid (BACARE),

When the storage container contains more than 1,0 mg of the selected carotenoid (see 5.14.1 or 5.14.2 or

5.14.3 or 5.14.4 or 5.14.5 or 5.14.6 or 5.14.7 or 5.14.8 or 5.14.9 or 5.14.10, depending on the carotenoid

selected), make a 0,2 mg/ml standard solution by e.g. weighing 1,0 mg of this carotenoid (note down the

Dissolve and make up to the mark with THF/BHT (5.9). Mix well using a vortex mixer (6.27) and an

ultrasonic bath (6.11). The accurate mass fraction needs to be standardized using a spectrophotometer

(6.2) as described in detail in 5.15.2. Reserve a 1 ml portion for the isomerisation procedure (5.15.4), if

needed. Reserve another aliquot for the identification of the analyte as described in paragraph 9.5.3.2 and

When the storage container specifies a nominal content equal to or less than 1,0 mg of the selected

carotenoid (see 5.14.1 or 5.14.2 or 5.14.3 or 5.14.4 or 5.14.5 or 5.14.6 or 5.14.7 or 5.14.8 or 5.14.9 or

5.14.10, depending on the carotenoid selected), transfer quantitatively the whole content in a 5,0 ml

volumetric flask. Dissolve and make up to the mark with THF/BHT (5.9). Mix well using a vortex mixer

(6.27) and an ultrasonic bath (6.11). The accurate mass fraction needs to be standardized using the

spectrophotometer (6.2) as described in detail in 5.15.2. Reserve a 1 ml portion for the isomerisation

procedure (5.15.4), if needed. Reserve another aliquot for the identification of the analyte as described in

Pipette 100 μl of the selected carotenoid stock standard solution (5.15.1) into a 10 ml volumetric flask

(6.8) and make up to the mark with the appropriate solvent. The nominal value of the obtained solution

is ca. 2 μg/ml. Scan the spectrum of this solution from 300 nm to 550 nm and measure the absorption of

this solution against the pure solvent, at the maximum, using the spectrophotometer (6.2). The maximum

EXAMPLE For all-E AXN, the solvent suggested in the table in Annex A is n-hexane. For this solvent, the

Pipette with a suited pipette 100 μl of the astaxanthin stock standard solution (5.15.1) into a 10 ml

volumetric flask and make up to the mark with n-hexane. The nominal value of the obtained standard

measuring solution is ca. 2 μg/ml. Measure the UV spectrum of this solution against pure n-hexane, using

the spectrophotometer (6.2) set at the wavelength of maximum absorption (approx. 470 nm).

NOTE The solvent of the measured solution is not pure as there is a small presence of THF/BHT (5.9). However,

we consider the effect negligible and we apply the same tabulated extinction coefficient given that the contribution

A solution of the same concentration, 100 μl of the astaxanthin stock standard solution (5.15.1) pipetted

into a 10 ml volumetric flask and made up to the mark with acetone (5.5), shall be injected simultaneously

Pipette with a suited pipette 100 µl of carotenoid stock standard solution (5.15.1) into a 10 ml volumetric

It is recommended to perform this step when ambiguity occurs regarding the presence of possible

The standards prepared according to 5.15.1.1 or 5.15.1.2 could be isomerised in order to have a profile

of the all-trans and cis isomers of each carotenoid, which are present in the equilibrium phase.

Fill a LC vial with 1 ml standard. Close tightly and check that the vial is well sealed and the cap does not

turn. Heat up the vial at about 80 °C for about 2 h to achieve the equilibrium between the isomers. A vials

Weigh approximately 0,8 mg AXN (5.14.1), 0,4 mg CXN (5.14.2) and 0,5 g BHT (5.3) in a round bottom

flask (6.9). Add 200 ml of an acetone (5.5): water (5.2) 75:25 v:v solution. Fit a condenser (6.10) to the

flask and immerse the flask in a heating mantle or heated bath (6.28) with a magnetic stirrer (6.29). Heat

NOTE Alternatively, the mixture can be heated at 80°C in a pressure-resistant tube.

This reaction will cause the formation of a stable isomers ratio. Transfer to a 500 ml volumetric flask (6.8)

and make up to volume with acetone (5.5). Mix well and transfer immediately in HPLC vials. The filling

should be fast and each vial should be immediately closed to avoid evaporation. Store the vials at room

6.1.1 Pump, pulse free, capable of maintaining a volume flow rate from 0,1 ml/min to 2,0 ml/min

If an autosampler is used, a cooled one is recommended although it was not used during the validation of

6.1.3 UV/VIS detector, variable wavelength, suitable for reliable measurements at 410 nm, or UV/VIS

6.1.5 Analytical column, Supelco Suplex pKb-100 5 µm, 250 mm × 4,6 mm or equivalent

6.1.6 Guard column, 5 μm, 2 cm × 4 mm SUPELCOSIL™ Suplex™ pKb-100 Supelguard™ Cartridge or

6.5 Balances, one analytical, of 10 g capacity or greater with 0,1 mg readability, and one, of 100 g

6.7 Variable-volume positive displacement piston pipettes, suitable for pipetting volumes ranging

6.13 PLE, pressurized solvent extraction system, ASE 300 Dionex , Büchi SpeedExtractor E-914 or

Supelco® Suplex pKb-100, SUPELCOSIL™ Suplex™ pKb-100 Supelguard™ Cartridge, ASE 300 Dionex®, Büchi

SpeedExtractor E-914, Eppendorf® tubes and Techne Dri-Block Heater are examples of suitable products available

commercially. This information is given for the convenience of users of this document and does not constitute an

It is important that the laboratory receives a sample that is truly representative and has not been

Sampling is not part of the method specified in this document. A recommended sampling method is given

Grind the laboratory sample (usually 50 g) so that it passes completely through a sieve with 1 mm

The test sample consists of a representative and homogenized aliquot of the ground laboratory sample

Accurately weigh 5,0 g to the nearest 0,1 g of the thoroughly mixed test sample (8.3) into a 100 ml

polypropylene container (6.6) for extraction procedure 9.3.1 or into a 50 ml or 100 ml centrifuge tube

(6.23) for extraction procedure 9.3.2. Note down the mass expressed in g. Submit it to the analysis

The complete procedure (9.2 to 9.4) should be applied to two test portions (8.4) of the same test sample

The QCS (5.16) shall be injected before and after the complete sequence samples each day of analysis, in

NOTE Depending on the analytes to be determined a relevant carotenoid can be added to this mix.

The mixture is isomerised until a constant ratio of the isomers is reached. This solution, when protected

Add 0,2 g of BHT (5.3), 100 µl of enzyme (5.1) and 15 ml of purified water (5.2) to the test portion (8.4).

Close tightly and shake vigorously to ensure that all the feed is permeated with water. Place in an

ultrasonic bath (6.11) with the temperature set at 50 °C for 15 min to 20 min. Shake every 5 min.

Remove the lid of the test portion after step 9.2, taking care of not losing sample drops. Add

approximately 13 g of high purity diatomaceous earth suitable for PLE (5.4), close tightly and hand-shake

vigorously until the wet feed sample looks all adsorbed on the diatomaceous earth beadlets and detaches

Open the containers carefully, remove the sample particles from the lid into the container using a flat

spatula (6.12). Mix well with the spatula (6.12), making sure that there are no sample clusters left. Place

two cellulose filters (6.15) on the bottom of the PLE cell (6.14). Transfer all the material in the PLE cell

(6.14) using a funnel (6.25). Top up with diatomaceous earth (5.4) if needed. Close the cell and extract

Each feed extraction lasts approximately 30 min. The instrument can run up to 12 samples in an

Take the tube containing the raw extract. Mix well and record the total volume (V ) of extract using a

Add 50 ml of acetone (5.5) and shake for 1 min. Centrifuge (6.22) at 1 800 g to 1 900 g for 5 min. Collect

the supernatant (first extract) and transfer into a 150 ml volumetric flask (6.8).

Add 50 ml of acetone (5.5) to the solid residue of the first extraction. Use the ultra-turrax mixer (6.24) if

needed to achieve dissolution and shake for 1 min. Centrifuge (6.22) at 1 800 g to 1 900 g for 5 min.

Collect the supernatant (second extract) and pool with the first extract in the 150-ml volumetric flask.