1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationDD CEN/TS 16233-2:2011Foodstuffs HPLC methodfor the determination ofxanthophylls in fish fleshPart 2: Identification of the enantiomerratio of astaxanthinDD CEN/TS 16233-2:2011 D
2、RAFT FOR DEVELOPMENTNational forewordThis Draft for Development is the UK implementation of CEN/TS16233-2:2011.This publication is not to be regarded as a British Standard.It is being issued in the Draft for Development series of publicationsand is of a provisional nature. It should be applied on th
3、isprovisional basis, so that information and experience of its practicalapplication can be obtained.Comments arising from the use of this Draft for Developmentare requested so that UK experience can be reported to theinternational organization responsible for its conversion toan international standa
4、rd. A review of this publication willbe initiated not later than 3 years after its publication by theinternational organization so that a decision can be taken on itsstatus. Notification of the start of the review period will be made inan announcement in the appropriate issue of Update Standards.Acc
5、ording to the replies received by the end of the review period,the responsible BSI Committee will decide whether to support theconversion into an international Standard, to extend the life of theTechnical Specification or to withdraw it. Comments should be sentto the Secretary of the responsible BSI
6、 Technical Committee at BritishStandards House, 389 Chiswick High Road, London W4 4AL.The UK participation in its preparation was entrusted to TechnicalCommittee AW/-/3, Food analysis - Horizontal methods.A list of organizations represented on this committee can beobtained on request to its secretar
7、y.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. BSI 2011ISBN 978 0 580 73655 1ICS 67.120.30Compliance with a British Standard cannot confer immunity fromlegal obligations.This Draft for Development was publis
8、hed under the authority ofthe Standards Policy and Strategy Committee on 31 August 2011.Amendments issued since publicationDate Text affectedDD CEN/TS 16233-2:2011TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN/TS 16233-2 July 2011 ICS 67.120.30 English Version Foodstuffs
9、 - HPLC method for the determination of xanthophylls in fish flesh - Part 2: Identification of the enantiomer ratio of astaxanthin Produits alimentaires - Mthode de dosage des xanthophylles dans la chair de poisson par CLHP - Partie 2: Identification de la distribution nantiomrique de lastaxanthine
10、Lebensmittel - HPLC-Verfahren zur Bestimmung von Xanthophyllen in Fischfleisch - Teil 2: Bestimmung des Enantiomerenverhltnisses von Astaxanthin 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 init
11、ially 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 t
12、he 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 bodi
13、es 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 Kin
14、gdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2011 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. CEN/TS 16233-2:2011:
15、EDD CEN/TS 16233-2:2011CEN/TS 16233-2:2011 (E) 2 Contents Page Foreword 3Introduction .41 Scope 42 Normative references 43 Principle 44 Reagents .55 Apparatus .66 Sample preparation and extraction77 HPLC .78 Test report . 10Bibliography . 11DD CEN/TS 16233-2:2011CEN/TS 16233-2:2011 (E) 3 Foreword Th
16、is 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 CENELE
17、C 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 Rep
18、ublic, 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. DD CEN/TS 16233-2:2011CEN/TS 16233-2:2011 (E) 4 In
19、troduction Three stereoisomers of all-E-astaxanthin (two enantiomers and a meso-form) exist, named (3R,3R)-, (3S,3S)- and (3R,3S, 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
20、 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
21、 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 farm
22、ed 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 obse
23、rved 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 describ
24、es 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
25、. 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 a
26、nd 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,3R)-, (3R,3S, meso)- and (3S,3S)-all-E-astaxanthin by chromatography on a chira
27、l HPLC column. (3R,3R) all-E-astaxanthin (3S,3S) all-E-astaxanthin DD CEN/TS 16233-2:2011CEN/TS 16233-2:2011 (E) 5 (3R,3S) 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 IS
28、O 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
29、 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 vo
30、lume 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 o
31、f astaxanthin standard solution, = 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 di
32、ssolution 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 solution to room temperature and dilute to the mark with n-heptane. This results in an astaxanthin
33、 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. DD CEN/TS 16233-2:2011CEN/TS 16233-2:2011 (E) 6 4.12 Preparation of canthaxanthin standard solution, = 1,5 mg/ml. Weigh approximately 1,5 mg to the nearest 0,1 mg of the
34、 reference substance of all-E-canthaxanthin (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 volu
35、metric flask and add approximately 85 ml of cyclohexane (4.4). 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
36、 1 part per volume of tetrahydrofuran. 4.13 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 (4.2) to the nearest 0,1 mg and dissolve in a 500 ml volumetric flask i
37、n 10 ml of tetrahydrofuran (4.3). Dilute this solution with 200 ml of a mixture of 86 parts per volume of n-heptane (4.5) 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 o
38、f 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
39、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 m to 40 m), diameter: approximately 6 cm. 5.3 Dispersing instrument. 5.3.1 Bench-top di
40、spersing 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
41、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 readabilit
42、y of 0,01 mg, precision (std dev.) of 0,015 mg, capacity of 205 g. 5.9 SPE columns, 25 ml reservoirs, plastic, equipped with a 10 micron bottom frits. DD CEN/TS 16233-2:2011CEN/TS 16233-2:2011 (E) 7 5.10 Solid phase extraction manifold, steel needles (0,90mm x 55 mm) attached to the valve outlets. 5
43、.11 HPLC chromatographic system, with column thermostat and UV/visible or diode array detector. 6 Sample preparation and extraction 6.1 Large scale extraction Cut approximately 100 g of tissue in pieces of about 1 cm in length using a knife or a pair of scissors. Weigh 10 g to 20 g of the minced tis
44、sue to the nearest 0,01 g into a 200 ml beaker. Add 5 g of magnesium sulfate (4.1), 50 mg of BHT (4.2) and 40 ml of acetone (4.7). Homogenize the mixture with a dispersing instrument (5.3.1) and filter through a sintered glass frit (5.2) using vacuum. Scrape off the residue and homogenize again with
45、 a fresh portion of approximately 40 ml of acetone. Repeat this procedure until the filtrate is colourless (usually 2 extractions). Combine the filtrates, add 20 ml ethanol (4.8) and evaporate the mixture at 50 C using a rotary evaporator (5.4). Dry the residue by addition and evaporation of approxi
46、mately 5 ml of ethanol. Dissolve the residue in 10 ml to 50 ml of mobile phase (4.9) and fill an aliquot of the turbid solution into a HPLC vial. Centrifuge the vial at approximately 2 500 g and use the clear supernatant for HPLC-analysis. 6.2 Small scale extraction Combine approximately 100 g of fi
47、sh tissue with 1 g of BHT (4.2) and homogenize the mixture with a knife mill (5.1). Weigh, to the nearest 1 mg, approximately 1 g of the homogenate and approximately 1 g of magnesium sulfate (4.1) into an empty 25 ml plastic SPE-column equipped with a 10 m frit at the bottom (5.9). Insert the column
48、 into a closed valve of a solid-phase extraction manifold (5.10). Add 8 ml of acetone (4.7) and homogenize the mixture with a dispersing instrument (5.3.2). Open the valve and suck the extract through the frit into a 35 ml test tube using vacuum. Repeat extraction and filtration with two additional
49、portions of 8 ml acetone. Evaporate the combined filtrates under a flow of nitrogen at 50 C (5.5). Dissolve the residue in 5 ml to 10 ml of the mobile phase (4.9) and fill an aliquot of the turbid solution into a HPLC vial. Centrifuge the vial at approximately 2 500 g and use the clear supernatant for HPLC-analysis. 7 HPLC 7.1 Identification Identify the racemats of the xanthophylls by injecting 20 l of the clear supernatant taken from the centrifuged HPLC vial according to 6.1 or 6.2 int
