1、BS EN 12821:2009ICS 67.050NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDFoodstuffs Determination ofvitamin D by highperformance liquidchromatography Measurement ofcholecalciferol (D3) orergocalciferol (D2)This British Standardwas published under theauthority o
2、f the StandardsPolicy and StrategyCommittee on 30 April2009 BSI 2009ISBN 978 0 580 57954 7Amendments/corrigenda issued since publicationDate CommentsBS EN 12821:2009National forewordThis British Standard is the UK implementation of EN 12821:2009. Itsupersedes BS EN 12821:2000 which is withdrawn.The
3、UK participation in its preparation was entrusted to TechnicalCommittee AW/-/3, Food analysis - Horizontal methods.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract.
4、Users are responsible for its correct application.Compliance with a British Standard cannot confer immunityfrom legal obligations.BS EN 12821:2009EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 12821April 2009ICS 67.050 Supersedes EN 12821:2000 English VersionFoodstuffs - Determination of vitamin
5、D by high performanceliquid chromatography - Measurement of cholecalciferol (D3) orergocalciferol (D2)Produits alimentaires - Dosage de la vitamine D parchromatographie liquide haute performance - Dosage ducholcalcifrol (D3) et de l ergocalcifrol (D2)Lebensmittel - Bestimmung von Vitamin D mitHochle
6、istungs-Flssigchromatographie - Bestimmung vonCholecalciferol (D3) oder Ergocalciferol (D2)This European Standard was approved by CEN on 21 February 2009.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the stat
7、us of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the CEN Management Centre or to any CEN member.This European Standard exists in three official versions (English, French, German). A v
8、ersion in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, De
9、nmark, 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 STANDARDIZATIONCOMIT EUROPEN DE NORMALISATI
10、ONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: Avenue Marnix 17, B-1000 Brussels 2009 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 12821:2009: EBS EN 12821:2009EN 12821:2009 (E) 2 Contents Page Foreword 3 1 Scope 4 2 Normative
11、 references 4 3 Principle 4 4 Reagents .4 5 Apparatus .8 6 Procedure .9 7 Calculation . 11 8 Precision 12 9 Test report . 13 Annex A (informative) Examples of suitable HPLC systems . 14 Annex B (informative) Examples of suitable extraction and saponification conditions . 15 Annex C (normative) Examp
12、les of suitable semi-preparative and analytical HPLC chromatograms . 16 Annex D (informative) Precision data . 18 Annex E (informative) Additional cleanup step for the determination of vitamin D with use of preparative TLC, column chromatography and or SPE 20 Bibliography . 24 BS EN 12821:2009EN 128
13、21:2009 (E) 3 Foreword This document (EN 12821:2009) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication of an identical text
14、or by endorsement, at the latest by October 2009, and conflicting national standards shall be withdrawn at the latest by October 2009. 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 responsibl
15、e for identifying any or all such patent rights. This document supersedes EN 12821:2000. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republi
16、c, 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. BS EN 12821:2009EN 12821:2009 (E) 4 1 Scope This Europ
17、ean Standard specifies a method for the determination of vitamin D3(cholecalciferol) or vitamin D2(ergocalciferol) in foodstuffs by high performance liquid chromatography (HPLC). Vitamin D3is primary in foodstuffs of animal origin, while vitamin D2is primary in wild mushrooms. Both vitamin D3 and vi
18、tamin D2can be present in fortified foodstuffs. This European Standard is not applicable for samples with a content of vitamin D3and vitamin D2. Apart from the vitamin D activity from the parent forms, vitamin D3and vitamin D2, the corresponding metabolites 25-hydroxy vitamin D and 1,25-dihydroxy vi
19、tamin D also contribute to the vitamin D activity. This European Standard does only include measurement of vitamin D3or vitamin D2. This European Standard provides the base for the analytical methods. It is intended to serve as a frame in which the analyst can define his own analytical work in accor
20、dance to the standard procedure. This method has been validated in inter-laboratory tests on fortified and non-fortified samples such as margarine, milk, milk powder, liquid infant formula, infant formula, cooking oil, and fish oil at levels from 0,4 g/100 g to 14 g/100 g. Further information on the
21、 validation data is given in Annex D. 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 amendment
22、s) applies. EN ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987). 3 Principle Vitamin D3and vitamin D2are saponified in the foodstuffs using alcoholic potassium hydroxide solution and extracted by an appropriate solvent. The determination of vitamin D3or
23、vitamin D2in an appropriate sample extract solution is carried out by semi-preparative normal phase HPLC followed by reverse-phase analytical HPLC. If vitamin D3is to be determined, then vitamin D2is used as an internal standard. If vitamin D2is to be determined, then vitamin D3is used as an interna
24、l standard. Vitamin D is detected by ultraviolet (UV) spectrometry and peaks are identified on the basis of retention times and additionally by UV spectral profile if diode-array detection is used. The determination is carried out by the internal standard procedure using peak areas or peak heights,
25、see 1 to 8. 4 Reagents 4.1 General During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and water of at least grade 1 according to EN ISO 3696. BS EN 12821:2009EN 12821:2009 (E) 5 4.2 Methanol 4.3 Ethanol, volume fraction (C2H5OH) = 100 %. 4.4 Ethanol, (C2H5
26、OH) = 96 %. 4.5 Sodium sulfate, anhydrous. 4.6 KOH solutions for saponification, in suitable concentrations, e.g. mass concentration (KOH) = 50 g/100 ml or (KOH) = 60 g/100 ml, or alcoholic solutions, e.g. 28 g of KOH in 100 ml of an ethanol and water mixture with a volume fraction of ethanol of 90
27、%. 4.7 Antioxidants, such as ascorbic acid (AA), sodium ascorbate, pyrogallol, sodium sulfide (Na2S) or butylated hydroxytoluene (BHT). 4.8 Solvents and extraction solvents, such as diethyl ether (peroxide-free), dichloromethane, light petroleum, n-hexane, ethylacetate or appropriate mixtures thereo
28、f. 4.9 HPLC Mobile phases 4.9.1 Examples of solvent mixtures for normal phase semi-preparative HPLC Examples of appropriate solvent mixtures (given as volume fractions) for normal phase semi-preparative HPLC include: n-hexane and 2-propanol (98 + 2), (99 + 1) or (95 + 5); n-hexane and isoamyl alcoho
29、l (99 + 1); n-hexane, 2-propanol and tetrahydrofuran (98 + 1 + 1); iso-octane and iso-butanol (99 + 1); n-heptane and 2-propanol (97 + 3). 4.9.2 Examples of solvent and solvent mixtures for reverse-phase analytical HPLC Examples of appropriate solvent and solvent mixtures (given as volume fractions)
30、 for reverse-phase analytical HPLC include: methanol; methanol and water (95 + 5) or (93 + 7); acetonitrile and methanol (80 + 20), (90 + 10) or (70 + 30); acetonitrile, chloroform and methanol (93 + 4 + 3). BS EN 12821:2009EN 12821:2009 (E) 6 4.10 Standard substances 4.10.1 Ergocalciferol standard
31、substance (vitamin D2), M(C28H44O) = 396,7 g/mol Vitamin D2standard substance shall be of the highest purity obtainable (having a mass fraction of greater than 98 %) and shall be stored according to the suppliers instructions (in the absence of light, typically less than 4 C). 4.10.2 Cholecalciferol
32、 standard substance (vitamin D3), M(C27H44O) = 384,6 g/mol Vitamin D3standard substance shall be of the highest purity obtainable (having a mass fraction of greater than 98 %) and shall be stored according to the suppliers instructions (in the absence of light, typically less than 4 C). 4.11 Stock s
33、olutions 4.11.1 Vitamin D2stock solution Weigh about 100 mg of vitamin D2(4.10.1) to the nearest milligram into a one mark 100 ml volumetric flask, dissolve in ethanol (4.4) and dilute to the mark with ethanol. This solution contains approximately 1 mg/ml of vitamin D2. Store below 4 C and protect f
34、rom light. Calculate the mass concentration of the stock solution and the mass fraction of the vitamin D2standard by the procedure described in 4.12.1. This solution is stable for 6 months at - 18C. 4.11.2 Vitamin D3stock solution Weigh about 100 mg of vitamin D3(4.10.2) to the nearest milligram int
35、o a one mark 100 ml volumetric flask, dissolve in ethanol (4.4) and dilute to the mark with ethanol. This solution contains approximately 1 mg/ml of vitamin D3. Store below 4 C and protect from light. Calculate the mass concentration of the stock solution and the mass fraction of the vitamin D3stand
36、ard by the procedure described in 4.12.2. This solution is stable for 6 months at - 18 C. 4.12 Standard solutions 4.12.1 Vitamin D2standard solution Pipette 1 ml of the vitamin D2stock solution (4.11.1) into a one mark 100 ml volumetric flask and dilute to the mark with ethanol (4.4). This solution
37、contains approximately 10 g/ml of vitamin D2. Prepare this solution on the day of use. NOTE The mass concentration of the standard solution can be adjusted if necessary to suit the analytical requirements. Measure the absorption of the vitamin D2standard solution in a 1 cm quartz cell at a wavelengt
38、h of 265 nm using ethanol in the reference path. Calculate the mass concentration of vitamin D2, D2, in microgram per millilitre of the standard solution using Equation (1): bMA=1000D2265D2(1) BS EN 12821:2009EN 12821:2009 (E) 7 where: A265is the absorption of the vitamin D2standard solution at 265
39、nm; MD2is the molar mass of vitamin D2(MD2= 396,7 g/mol); is the molar absorption coefficient of vitamin D2(here: = 18 843 m2/mol, calculated from the % 1cm 1E value, see 9); b is the optical path length of the quartz cell in centimetres. 4.12.2 Vitamin D3standard solution Pipette 1 ml of the vitami
40、n D3stock solution (4.11.2) into a one mark 100 ml volumetric flask and dilute to the mark with ethanol (4.4). This solution contains approximately 10 g/ml of vitamin D3. Prepare this solution on the day of use. NOTE The mass concentration of the standard solution can be adjusted if necessary to sui
41、t the analytical requirements. Measure the absorption of the vitamin D3standard solution in a 1 cm quartz cell at a wavelength of 265 nm using ethanol (4.4) in the reference path. Calculate the mass concentration of vitamin D3, D3, in microgram per millilitre of the standard solution using Equation
42、(2): bMA=1000D3265D3(2) where: A265is the absorption of the vitamin D3standard solution at 265 nm; MD3is the molar mass of vitamin D3(MD3= 384,6 g/mol); is the molar absorption coefficient of vitamin D3(here: = 18 461 m2/mol, calculated from the % 1cm 1E value, see 9); b is the optical path length o
43、f the quartz cell in centimetres. 4.13 Internal standard solutions 4.13.1 Vitamin D2internal standard solution Pipette 10 ml of the vitamin D2standard solution (4.12.1) into a one mark 100 ml volumetric flask and dilute to the mark with ethanol (4.4). Prepare this solution on the day of use. 4.13.2
44、Vitamin D3internal standard solution Pipette 10 ml of the vitamin D3standard solution (4.12.2) into a one mark 100 ml volumetric flask and dilute to the mark with ethanol (4.4). Prepare this solution on the day of use. NOTE If vitamin D3is to be determined, then vitamin D2is used as an internal stan
45、dard. If vitamin D2is to be determined, then vitamin D3is used as an internal standard. BS EN 12821:2009EN 12821:2009 (E) 8 4.14 Vitamin D2and vitamin D3semi-preparative standard solution Pipette 5 ml of the vitamin D2standard solution (4.12.1) and 5 ml of the vitamin D3standard solution (4.12.2) in
46、to a rotary evaporator flask and carefully remove the solvent (at not more than 40 C). Re-dissolve the residue in 100 ml of the semi-preparative HPLC mobile phase (4.9.1). The concentration of the semi-preparative standard may be adjusted if necessary to suit the HPLC system in use (5.4 or 5.5). 4.1
47、5 Vitamin D2and vitamin D3analytical standard solution Pipette 5 ml of the vitamin D2standard solution (4.12.1) and 5 ml of the vitamin D3standard solution (4.12.2) into a rotary evaporator flask and carefully remove the solvent (at not more than 40 C). Re-dissolve the residue in 50 ml of the analyt
48、ical HPLC mobile phase (4.9.2). 5 Apparatus 5.1 General Usual laboratory apparatus and, in particular, the following. 5.2 UV spectrometer, capable of measuring at a wavelength of 265 nm. 5.3 Rotary evaporator, with water bath and vacuum unit NOTE The use of nitrogen is recommended for releasing the
49、vacuum. 5.4 Semi-preparative HPLC system, consisting of a pump, sample injection device, UV detector, a means of collecting a defined aliquot portion of column eluent, and a recorder or integrator. 5.5 Analytical HPLC system, consisting of a pump, sample injection device, UV detector, recorder/integrator or similar data capture device. 5.6 HPLC columns 5.6.1 Semi-preparative normal phase column, e.g. silica or bonded cyano-amino, particle size 5 m, diameter 4,0 mm to 8,0 mm, length 250 mm to 300 mm. See Annex
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