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、8g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS EN 14663:2005This British Standard was published under the authority of the Standards Policy and Strategy Committee on 16 January 2006 BSI 16 January 2006ISBN 0 580 46982 4National forewordThis British
3、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 to: aid enquirers to understand the text; present to the responsible international/Eu
4、ropean 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 organizations represented on this committee can be obtained on request to its secretary.Cross
5、-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 Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of Br
6、itish 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 itself confer immunity from legal obligations.Summary of pagesThis document comprises a fr
7、ont 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 issued since publicationAmd. No. Date CommentsEUROPEAN STANDARDNORME EUROPENNEEUROPISCH
8、E NORMEN 14663December 2005ICS 67.050English VersionFoodstuffs - Determination of vitamin B6 (including itsglycosylated forms) by HPLCProduits alimentaires - Dosage de la vitamine B6 (ycompris ses formes glycosyles) par CLHPLebensmittel - Bestimmung von Vitamin B6 (einschlielichglucosidisch gebunden
9、er Verbindungen) mit HPLCThis European Standard was approved by CEN on 26 October 2005.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists
10、and bibliographical references concerning such nationalstandards may be obtained on application to the Central Secretariat or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibil
11、ity of a CEN member into its own language and notified to the Central Secretariat has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Germany, Greece, Hungary, Iceland, Ireland, Italy
12、, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 200
13、5 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14663:2005: EEN 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 T
14、est report .14 Annex A (informative) Precision 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 St
15、andard substances and sample potato puree.21 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 foo
16、d) .17 Table A.4 Precision data for multi vitamin 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 EN 14663:2005 (E) 3 Foreword This document (EN 14663:20
17、05) 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 endorsement, at the latest by June 2006, an
18、d 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, Cyprus, Czech Republic, Denmark, Estonia
19、, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EN 14663:2005 (E) 4 1 Scope This document specifies a method for the determination of
20、vitamin B6in foodstuffs by high performance liquid chromatography (HPLC). Vitamin B6is the mass fraction of the sum of pyridoxine, pyridoxal, pyridoxamine including their phosphorylated derivatives as well as the -glycosylated forms, calculated as pyridoxine. This method has been successfully valida
21、ted 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 Normative references The following referenced documents are indispensable for the application of this document. For dated ref
22、erences, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN ISO 3696, Water for analytical laboratory use Specification and test methods (ISO 3696:1987). 3 Principle Pyridoxal, pyridoxamine and pyridoxine are e
23、xtracted from food by acid hydrolysis and dephosphorylated and deglycosilated enzymatically using acid phosphatase and -glucosidase. The different derivatives of vitamin B6(pyridoxal, pyridoxamine and pyridoxine) are separated by HPLC and quantified by fluorometric detection 1, 2. 4 Reagents 4.1 Gen
24、eral 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 water. 4.2 Di-potassium hydrogen phosphate, mass fraction w(K2HPO4 3 H2O) 99,9 % 4.3 Sodium acetate, without crystal water,
25、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 acetate (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-p
26、otassium hydrogen phosphate (4.2) in water, dilute to 1 000 ml, mix and degas. EN 14663:2005 (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
27、40 C to 60 C 4.12 Acid phosphatase, from potatoes. Enzymatic activity approximately 5,3 U/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 phosphatas
28、e (4.12) in 10 ml of water in a beaker by stirring for 2 min. Prepare this solution on the 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
29、solution (4.13.1) and optional 1 ml of -glucosidase solution (4.15) to 12,5 ml of the extracted 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 concent
30、ration of vitamins according to 6.6. The activity of the enzyme used is sufficient, if the 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 a
31、cid phosphatase from Sigma Nr P 37522)has been used. 4.14 -Glucosidase, from almonds. Enzymatic 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/solub
32、ilise 100 mg of -glucosidase (4.14) in 10 ml of water in a beaker by stirring for 2 min. Prepare 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 mi
33、nute under standard conditions. 2) 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 products may be used if they can be shown to lead to the same results. EN 14663:2005 (E) 6 4.15.2 Activity check o
34、f -glucosidase 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
35、 at least 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 B6comp
36、ounds 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
37、TCA) 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 pyr
38、idoxine (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
39、 HCl) 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
40、 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. 4.19.2 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
41、(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. 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. Equival
42、ent products may be used if they can be shown to lead to the same results. EN 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 100 ml volumetric flask in 0,1 mol/l HCl (4.8) and dilute to the mark with 0,1
43、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 pyridoxine (4.19.3) respectively in a 50 ml volumetric flask and dilute to the m
44、ark 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 vitamin B6compound, i, using the molar extinction coefficient as given in equation
45、 (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 solutions at the maximum wavelength max(see table 1); iis the molecular absorban
46、ce 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; F is the calculation factor of HCl free vitamin B6compounds. Use these mass co
47、ncentrations 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
48、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-dihydrochloride (4.18.2) bPL .HCl = Pyridoxal-hydrochloride (4.18.3) cPN .HCl = Pyridoxine-hydrochloride (4.18.4) 4.20 Standard solutions 4.20.1 Pyridoxamine (PM) standard solution I, (PM) approximately 10 g/m
49、l Dilute 2 ml of pyridoxamine stock solution (4.19.1) with 0,1 mol/l HCl (4.8) to 100 ml. Prepare freshly every day EN 14663:2005 (E) 8 4.20.2 Pyridoxal (PL) standard solution I, (PL) approximately 10 g/ml Dilute 2 ml of PL stock solution (4.19.2) with 0,1 mol/l HCl (4.8) to 100 ml. Prepare freshly every day 4.20.3 Pyridoxine (PN) standard solution I, (PN) approximately 10 g/ml Dilute 2 ml of pyridoxine stock solution (4.19.3) with 0,1 mol/l HCl (4.8) to 100 ml. Prepare
