1、BRITISH STANDARDBS EN 14663:2005Foodstuffs Determination of vitamin B6 (including its glycosylated forms) by HPLCThe European Standard EN 14663:2005 has the status of a British StandardICS 67.050g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g3
2、8g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58Licensed Copy: Wang Bin, na, Tue Apr 18 01:38:56 BST 2006, Uncontrolled Copy, (c) BSIBS EN 14663:2005This British Standard was published under the authority of the Standards Policy and Strategy Committee o
3、n 16 January 2006 BSI 16 January 2006ISBN 0 580 46982 4National forewordThis British Standard is the official English language version of EN 14663:2005.The UK participation in its preparation was entrusted to Technical Committee AW/-/3, Food analysis Horizontal methods, which has the responsibility
4、to: aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep UK interests informed; monitor related international and European developments and promulgate them in the UK.A list of organiza
5、tions represented on this committee can be obtained on request to its secretary.Cross-referencesThe British Standards which implement international or European publications referred to in this document may be found in the BSI Catalogue under the section entitled “International Standards Corresponden
6、ce Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Standards Online.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard does not of itsel
7、f confer immunity from legal obligations.Summary of pagesThis document comprises a front cover, an inside front cover, the EN title page, pages 2 to 22, an inside back cover and a back cover.The BSI copyright notice displayed in this document indicates when the document was last issued.Amendments is
8、sued since publicationAmd. No. Date CommentsLicensed Copy: Wang Bin, na, Tue Apr 18 01:38:56 BST 2006, Uncontrolled Copy, (c) BSIEUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 14663December 2005ICS 67.050English VersionFoodstuffs - Determination of vitamin B6 (including itsglycosylated forms) by
9、HPLCProduits alimentaires - Dosage de la vitamine B6 (ycompris ses formes glycosyles) par CLHPLebensmittel - Bestimmung von Vitamin B6 (einschlielichglucosidisch gebundener Verbindungen) mit HPLCThis European Standard was approved by CEN on 26 October 2005.CEN members are bound to comply with the CE
10、N/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the Central Secretariat or to a
11、ny CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the officialversions.CEN
12、members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,Slovenia, Spain, Sweden, Switzerland and U
13、nited Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 2005 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14663:2005: E
14、Licensed Copy: Wang Bin, na, Tue Apr 18 01:38:56 BST 2006, Uncontrolled Copy, (c) BSIEN 14663:2005 (E) 2 Contents Page Foreword 3 1 Scope .4 2 Normative references .4 3 Principle.4 4 Reagents4 5 Apparatus 9 6 Procedure 9 7 Calculation.11 8 Precision12 9 Test report .14 Annex A (informative) Precisio
15、n data 15 Annex B (informative) Examples for suitable HPLC-conditions for the determination of vitamin B6compounds19 Annex C (informative) Examples for molar extinction coefficients .20 Annex D (informative) Figures.21 Bibliography.22 Figure Figure D.1 Standard substances and sample potato puree.21
16、Tables Table 1 Examples for molecular extinction coefficients of vitamin B6 compounds7 Table A.1 Precision data for Semolina with milk, powder15 Table A.2 Precision data for Potato puree,powder.16 Table A.3 Precision data for vegetables with ham (baby food) .17 Table A.4 Precision data for multi vit
17、amin drink.18 Table B.1 Examples for suitable HPLC-conditions for the determination of vitamin B6 compounds 19 Table C.1 Examples for molar extinction coefficients (E) of vitamin B6 compounds 3, 4 20 Licensed Copy: Wang Bin, na, Tue Apr 18 01:38:56 BST 2006, Uncontrolled Copy, (c) BSIEN 14663:2005 (
18、E) 3 Foreword This document (EN 14663:2005) 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 or by end
19、orsement, at the latest by June 2006, and conflicting national standards shall be withdrawn at the latest by June 2006. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium,
20、Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. Licensed Copy: Wang Bin, na, Tue Apr 18 01:38:
21、56 BST 2006, Uncontrolled Copy, (c) BSIEN 14663:2005 (E) 4 1 Scope This document specifies a method for the determination of vitamin B6in foodstuffs by high performance liquid chromatography (HPLC). Vitamin B6is the mass fraction of the sum of pyridoxine, pyridoxal, pyridoxamine including their phos
22、phorylated derivatives as well as the -glycosylated forms, calculated as pyridoxine. This method has been successfully validated with semolina with milk (infant food), potato puree, vegetables with ham (convenient products) and a multi vitamin drink at levels from 0,034 mg/100 g to 1,21 mg/100 g. 2
23、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, Water for analy
24、tical laboratory use Specification and test methods (ISO 3696:1987). 3 Principle Pyridoxal, pyridoxamine and pyridoxine are extracted from food by acid hydrolysis and dephosphorylated and deglycosilated enzymatically using acid phosphatase and -glucosidase. The different derivatives of vitamin B6(py
25、ridoxal, pyridoxamine and pyridoxine) are separated by HPLC and quantified by fluorometric detection 1, 2. 4 Reagents 4.1 General During the analysis, unless otherwise stated, use only reagents of recognised analytical grade and water of at least grade 1 according to EN ISO 3696, or double distilled
26、 water. 4.2 Di-potassium hydrogen phosphate, mass fraction w(K2HPO4 3 H2O) 99,9 % 4.3 Sodium acetate, without crystal water, w(CH3COONa) 99,0 % 4.4 Trichloroacetic acid (TCA), w(Cl3CCOOH) 99,0 % 4.5 Sodium acetate solution, substance concentration c(CH3COONa) = 2,5 mol/l Dissolve 205 g of sodium ace
27、tate (4.3) in 1 l of water. 4.6 Post-column reagent (optional), K2HPO4solution c(K2HPO4) = 0,15 mol/l Dissolve 34,2 g of di-potassium hydrogen phosphate (4.2) in water, dilute to 1 000 ml, mix and degas. Licensed Copy: Wang Bin, na, Tue Apr 18 01:38:56 BST 2006, Uncontrolled Copy, (c) BSIEN 14663:20
28、05 (E) 5 4.7 Hydrochloric acid, c(HCl) = 1 mol/l 4.8 Hydrochloric acid, c(HCl) = 0,1 mol/l 4.9 Hydrochloric acid, c(HCl) = 0,2 mol/l 4.10 Sulfuric acid, c(H2SO4)= 1 mol/l 4.11 Light petroleum, boiling range of 40 C to 60 C 4.12 Acid phosphatase, from potatoes. Enzymatic activity approximately 5,3 U/
29、mg1). It is important that the enzyme used complies with the activity check 4.13.2, for further information see 2, 7. 4.13 Acid phosphatase solution 4.13.1 General Dissolve/solubilise 60 mg of acidic phosphatase (4.12) in 10 ml of water in a beaker by stirring for 2 min. Prepare this solution on the
30、 day of analysis. 4.13.2 Activity check of Acid Phosphatase Weigh 10 g of pork, 5 g of potato puree or 5 g of whole meal into a beaker, and extract with acid as described in 6.2.1. Add 1 ml of acid phosphatase solution (4.13.1) and optional 1 ml of -glucosidase solution (4.15) to 12,5 ml of the extr
31、acted sample solution and mix. Incubate the solution at least 12 h or overnight at 37 C with continuous stirring. Repeat this step with the double amount of acid phosphatase solution. Determine the mass concentration of vitamins according to 6.6. The activity of the enzyme used is sufficient, if the
32、 resulting mass concentrations of vitamin B6compounds in both sample solutions are equivalent. The chromatogram shall not show a peak arising from pyridoxamin phosphate. NOTE For the interlaboratory test, the acid phosphatase from Sigma Nr P 37522)has been used. 4.14 -Glucosidase, from almonds. Enzy
33、matic activity of approximately 3,2 U/mg. It is important that the enzyme used complies with the activity check 4.15.2, for further information see 2, 7. 4.15 -Glucosidase solution 4.15.1 General Dissolve/solubilise 100 mg of -glucosidase (4.14) in 10 ml of water in a beaker by stirring for 2 min. P
34、repare this solution on the day of analysis. 1)U, this unit (often called the International unit or standard unit) is defined as the amount of enzyme which catalyses the transformation of 1 mol substrate per minute under standard conditions. 2) This information is given for the convenience of users
35、of this document and does not constitute an endorsement by CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results. Licensed Copy: Wang Bin, na, Tue Apr 18 01:38:56 BST 2006, Uncontrolled Copy, (c) BSIEN 14663:2005 (E) 6 4.15.2 Activity check of -gl
36、ucosidase Weigh 10 g of pork, 5 g of potato puree or 5 g of whole meal into a beaker, and extract with acid as described in 6.2.1. Add 1 ml of acid phosphatase solution (4.13.1) and 1 ml of -glucosidase solution (4.15.1) to 12,5 ml of the extracted sample solution and mix. Incubate the solution at l
37、east 12 h or overnight at 37 C with continuous stirring. Repeat this step with the double amount -glucosidase solution. Determine the mass concentration of vitamin B6compounds according to 6.6. The activity of the enzyme used is sufficient, if the resulting mass concentrations of vitamin B6compounds
38、 in both sample solutions are equivalent. The chromatogram shall not show a peak arising from pyridoxamin phosphate. NOTE For the interlaboratory test, the -glucosidase from Sigma Nr G-0395 1)has been used. 4.16 Mobile phase for HPLC (Sulfuric acid, c(H2SO4)= 0,015 mol/l containing 0,005 mol/l TCA)
39、Dissolve 817 mg 5 mg of trichloroacetic acid (4.4) in 15 ml of 1 mol/l sulfuric acid (4.10), transfer into a 1 000 ml volumetric flask, dilute to the mark with water, mix and degas. 4.17 Silicon oil, for defoaming 4.18 Standard substances 4.18.1 General Pyridoxamine (PM), Pyridoxal (PL) and pyridoxi
40、ne (PN) can be obtained from various suppliers. The purity of the standards may vary, and it is therefore necessary to determine the concentration and purity (see 4.19.4 and 4.20.7). 4.18.2 Pyridoxamine (PM) dihydrochloride, w(C 8H12N2O2 2HCl) 98 % 4.18.3 Pyridoxal (PL) hydrochloride, w(C8H9NO3 HCl)
41、 98 % 4.18.4 Pyridoxine (PN) hydrochloride, w(C8H11NO3 HCl ) 98 % 4.19 Stock solutions 4.19.1 Pyridoxamine (PM) stock solution, mass concentration (PM) approximately 500 g/ml Dissolve 71,7 mg of pyridoxamine dihydrochloride (4.18.2) in a 100 ml volumetric flask in 0,1 mol/l HCl (4.8) and dilute to t
42、he mark with 0,1 mol/l HCl. The solution can be stored without any losses for up to one week at 4 C or up to two months at -18 C. 4.19.2 Pyridoxal (PL) stock solution, (PL) approximately 500 g/ml Dissolve 60,9 mg of pyridoxal hydrochloride (4.18.3) in a 100 ml volumetric flask in 0,1 mol/l HCl (4.8)
43、 and dilute to the mark with 0,1 mol/l HCl. The solution can be stored without any losses for up to one week at 4 C or up to two months at -18 C. 1)This information is given for the convenience of users of this document and does not constitute an endorsement by CEN of the product named. Equivalent p
44、roducts may be used if they can be shown to lead to the same results. Licensed Copy: Wang Bin, na, Tue Apr 18 01:38:56 BST 2006, Uncontrolled Copy, (c) BSIEN 14663:2005 (E) 7 4.19.3 Pyridoxine (PN) stock solution, (PN) approximately 500 g/ml Dissolve 60,8 mg of pyridoxine hydrochloride (4.18.4) in a
45、 100 ml volumetric flask in 0,1 mol/l HCl (4.8) and dilute to the mark with 0,1 mol/l HCl. The solution can be stored without any losses for up to one week at 4 C or up to two months at 18 C. 4.19.4 Concentration tests Pipette 1 ml of stock solutions of pyridoxamine (4.19.1), pyridoxal (4.19.2) and
46、pyridoxine (4.19.3) respectively in a 50 ml volumetric flask and dilute to the mark with 0,1 mol/l HCl (4.8). Measure the absorbance of the solutions in a 1 cm quartz-cell against 0,1 mol/l HCl at the maximum wavelength using UV-spectrometry (see Table 1). Calculate the mass concentration of each vi
47、tamin B6compound, i, using the molar extinction coefficient as given in equation (1): FVMAiii= (1) where: iis the mass concentration of pyridoxamine, pyridoxal and pyridoxine respectively in microgram per millilitre stock solution; A is the absorbance value of pyridoxamine, pyridoxal and pyridoxine
48、solutions at the maximum wavelength max(see table 1); iis the molecular absorbance coefficient of PM, PL or PN at the appropriate pH as defined in table 1; Miis the molecular weight of PM, PL and PN respectively standard substances as defined in table 1; V is the dilution factor, in this case V = 50
49、; F is the calculation factor of HCl free vitamin B6compounds. Use these mass concentrations to calculate the exact concentrations of 4.19.1 to 4.19.3 and 4.20.1 to 4.20.6. Table 1 Examples for molecular extinction coefficients of vitamin B6compounds Compounds Solvent maxi mmol-1cm-1Mi g mol 1F PM .2 HCla 0,1 mol/l HCl, pH 1 292 8,2 241,1 0,698 PL .HClb 0,1 mol/l HCl, pH 1 288 9,0 203,6 0,821 PN .HClc 0,1 mol/l HCl, pH 1 291 8,6 205,6 0,823 aPM .2 HCl = Pyridoxamine-dihydroch
