1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationDD CEN/TS 16233-1:2011Foodstuffs HPLC methodfor the determination ofxanthophylls in fish fleshPart 1: Determination of astaxanthin andcanthaxanthinDD CEN/TS 16233-1:2011 DRAFT FO
2、R DEVELOPMENTNational forewordThis Draft for Development is the UK implementation of CEN/TS16233-1:2011.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 thisprovi
3、sional 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 standard. A r
4、eview 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.According
5、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 Techni
6、cal 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 secretary.This
7、publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. BSI 2011ISBN 978 0 580 73654 4ICS 67.120.30Compliance with a British Standard cannot confer immunity fromlegal obligations.This Draft for Development was published und
8、er the authority ofthe Standards Policy and Strategy Committee on 31 August 2011.Amendments issued since publicationDate Text affectedDD CEN/TS 16233-1:2011TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN/TS 16233-1 July 2011 ICS 67.120.30 English Version Foodstuffs - HPLC
9、 method for the determination of xanthophylls in fish flesh - Part 1: Determination of astaxanthin and canthaxanthin Produits alimentaires - Mthode de dosage des xanthophylles dans la chair de poisson par CLHP - Partie 1: Dosage de lastaxanthine et de la canthaxantine Lebensmittel - HPLC-Verfahren f
10、r die Bestimmung von Xanthophyllen in Fischfleisch - Teil 1: Bestimmung von 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 t
11、he 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 nationa
12、l 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, Croat
13、ia, 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 STANDARDIZ
14、ATION 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 16233-1:2011: EDD CEN/TS 16233-1:2011CEN/TS 16233-1:2
15、011 (E) 2 Contents Page Foreword 31 Scope 42 Normative references 43 Principle 44 Reagents .45 Apparatus .66 Calibration 77 Procedure .88 HPLC .99 Calculation . 1010 Test report . 11Annex A (informative) Validation for astaxanthin . 12Annex B (informative) Typical chromatograms . 14Bibliography . 16
16、DD CEN/TS 16233-1:2011CEN/TS 16233-1:2011 (E) 3 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 docu
17、ment 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 Specificat
18、ion: 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
19、Kingdom. DD CEN/TS 16233-1:2011CEN/TS 16233-1:2011 (E) 4 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 sh
20、ould 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 indi
21、spensable 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 36
22、96:1987). 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
23、HPLC system described effectively separates astaxanthin and canthaxanthin and these both xanthophylls from other carotenoids found in fish flesh as e.g. -carotene, lutein and zeaxanthin. The main geometrical isomers of astaxanthin are separated from each other and from oxidation products of astaxant
24、hin, 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 analytic
25、al grade, e.g. pro analysis (p.a.). DD CEN/TS 16233-1:2011CEN/TS 16233-1:2011 (E) 5 4.1 Magnesium sulfate, anhydrous, purity (complexometric) 98 %. 4.2 Phosphoric acid, volume fraction is 85 %, purity (acidimetric) 85 %. 4.3 Butylated hydroxytoluene (BHT), purity (GC) 99 %. 4.4 Tetrahydrofuran, puri
26、ty (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
27、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 Preparat
28、ion of 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 (4.3) into a 100 ml volumetric flask. Dissolve in 5 ml of tetrahydrofuran (4.4) and dilute to the mark with tetrahydrofuran. Suppo
29、rt 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 astaxa
30、nthin 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, = 1,5 mg/ml. Weigh approximately 1,5 mg to the nearest 0,1 mg of the reference substance of all-E-canthaxanthin
31、 (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
32、 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. DD C
33、EN/TS 16233-1:2011CEN/TS 16233-1:2011 (E) 6 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 fla
34、sk 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 mixtu
35、re 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 2
36、3 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 m to 40 m), diameter: approximately 6 cm. 5.3 Dispersing instrument. 5.3.1 Bench-to
37、p 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 hol
38、der 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 readabi
39、lity 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 m bottom fritts. 5.11 HPLC chromatographic system, with column therm
40、ostat and UV/visible or diode array detector. DD CEN/TS 16233-1:2011CEN/TS 16233-1:2011 (E) 7 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
41、.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 sys
42、tem. 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 sol
43、ution 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 () in milligram per millilitre of the standard solution
44、using Equations (1) and (2): 100200010maxnastaxanthiEall=A (1) where Amaxis the absorbance value at the absorption maximum; 10 000 is the scaling factor; 2 100 is the 1%1cmE value of all-E-astaxanthin. The 1%1cmE value is the theoretical absorption of a 1 % solution of all-E-astaxanthin in a 1 cm ce
45、ll at approximately 470 nm (max) in n-heptane, see 1. 200200010maxhincanthaxantEall=A (2) where Amaxis the absorbance value at the absorption maximum; 10 000 is the scaling factor; 2 200 is the 1%1cmE value of of all-E-canthaxanthin. The 1%1cmE value is the theoretical absorption of a 1 % solution o
46、f all-E-canthaxanthin in a 1 cm cell at approximately 466 nm (max) in cyclohexane, see 2. 6.3 Determination response factor of standard solution with HPLC Within 3 h after preparation, inject at least six aliquots of 20 l of each standard solution into the HPLC system. Determine the total peak areas
47、 of the chromatograms (including the peaks of the all-E-isomer, of possibly present Z-isomers and impurities, but excluding the solvent peak). The peak area of all-E-astaxanthin or all-E-canthaxanthin should exceed 95 % of the respective total peak area of the chromatogram. DD CEN/TS 16233-1:2011CEN
48、/TS 16233-1:2011 (E) 8 Calculate the response factors (RF), in area units l/mg, for all-E-astaxanthin and all-E-canthaxanthin from the mean total peak areas obtained from all chromatograms and from the astaxanthin or canthaxanthin concentrations measured by spectrometry using Equation (3): totalARF
49、= (3) where Atotalis the mean total peak area from all chromatograms of the astaxanthin or canthaxanthin standard solutions, in area un is the spectrometrically measured astaxanthin or canthaxanthin concentration in the standard solution, in mg/l. The standard solutions should only be used at the day of preparation. 6.4 Control of the response of the HPLC system It is not necessary to re-calibrate the HPLC every day if the system is not changed. However, the constancy of response of
