1、BSI Standards PublicationBS EN 14164:2014Foodstuffs Determinationof vitamin B6 by highperformance chromatographyBS EN 14164:2014 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 14164:2014. It supersedes BS EN 14164:2008 which is withdrawn.The UK participation in
2、 its preparation was entrusted to Technical Committee AW/275, Food analysis - Horizontal methods.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are respon
3、sible for its correct application. The British Standards Institution 2014. Published by BSI Standards Limited 2014ISBN 978 0 580 77941 1ICS 67.050Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards
4、Policy and Strategy Committee on 30 June 2014.Amendments/corrigenda issued since publicationDate Text affectedBS EN 14164:2014EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 14164 June 2014 ICS 67.050 Supersedes EN 14164:2008English Version Foodstuffs - Determination of vitamin B6 by high perfo
5、rmance chromatography Produits alimentaires - Dtermination de la teneur en vitamine B6 par chromatographie liquide haute performanceLebensmittel - Bestimmung von Vitamin B6 mit Hochleistungs-Flssigchromatographie This European Standard was approved by CEN on 17 April 2014. CEN members are bound to c
6、omply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENE
7、LEC Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the
8、 same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembo
9、urg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
10、 2014 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 14164:2014 EBS EN 14164:2014EN 14164:2014 (E) 2 Contents Page Foreword 3 1 Scope 4 2 Normative references 4 3 Principle 4 4 Reagents .4 5 Apparatus .8 6 Procedure .8 7 Calculati
11、on 9 8 Test report . 10 Annex A (informative) Example of a chromatogram . 11 Annex B (informative) Precision data . 12 Annex C (informative) Sample treatment option without acid hydrolysis 14 Annex D (informative) Examples for molar absorption coefficients . 15 Bibliography . 16 BS EN 14164:2014EN 1
12、4164:2014 (E) 3 Foreword This document (EN 14164:2014) 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 tex
13、t or by endorsement, at the latest by December 2014 and conflicting national standards shall be withdrawn at the latest by December 2014. 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 respons
14、ible for identifying any or all such patent rights. This document supersedes EN 14164:2008. The Annexes A, B, C and D are informative. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Aus
15、tria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Swed
16、en, Switzerland, Turkey and the United Kingdom. WARNING The use of this European Standard can involve hazardous materials, operations and equipment. This European Standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this Europe
17、an Standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. BS EN 14164:2014EN 14164:2014 (E) 4 1 Scope This European Standard specifies a method for the determination of vitamin B6in foodstuffs by high performance liquid c
18、hromatography (HPLC). Vitamin B6is the mass fraction of the sum of pyridoxine, pyridoxal, pyridoxamine including their phosphorylated derivatives determined as pyridoxine. The -glycosylated forms are not taken into account. These can be determined with the method given in EN 14663 1 by which the dif
19、ferent vitamins of vitamin B6(pyridoxal, pyridoxamine and pyridoxine) are separated and individually quantified. A third European Standard, EN 14166 2, determines the total vitamin B6by microbiological assay. 2 Normative references The following documents, in whole or in part, are normatively refere
20、nced in this document and are indispensable for its application. 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 analytical laboratory use - Specification and test m
21、ethods (ISO 3696) 3 Principle Pyridoxal, pyridoxamine and pyridoxine are extracted from food by acid hydrolysis and dephosphorylated enzymatically using acid phosphatase. By reaction with glyoxylic acid in the presence of Fe2+as a catalyst, pyridoxamine is transformed into pyridoxal, which is then r
22、educed to pyridoxine by the action of sodium borohydride in alkaline medium. Pyridoxine is then quantified in the sample solution by HPLC with a fluorometric detection 3, 4. 4 Reagents During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and water of at leas
23、t grade 1 according to EN ISO 3696, or double distilled water. 4.1 Acid phosphatase, (CAS 9001-77-8), from potatoes, enzyme activity is 33 nkat/mg1)with substrate p-nitrophenyl phosphate at pH = 4,8 and T = 37 C, for example from Boehringer or Sigma2). 33 nkat/mg corresponds to 2 U/mg. 4.1.1 Acid ph
24、osphatase solution Prepare a solution of 20 mg/ml acid phosphatase in sodium acetate solution (4.14). It is necessary to use acid phosphatase rather than Taka-diastase to obtain a complete hydrolysis of phosphorylated forms of vitamin B6. Where 300 mg of Taka-diastase is needed to obtain good dephos
25、phorylation, only 0,5 mg of acid phosphatase is needed, see 5. 1)Katal (symbol kat) is a derived SI unit of enzyme activity. One katal is that catalytic activity which will raise the rate of reaction by one mol/s in a specified assay system. 2)This information is given for the convenience of users o
26、f this European standard and does not constitute an endorsement by CEN of the supplier. Equivalent products may be used if they can be shown to lead to the same results. BS EN 14164:2014EN 14164:2014 (E) 5 4.1.2 Activity check of acid phosphatase The activity of acid phosphatase can be checked as pr
27、oposed below. Prepare a stock solution of approximately 0,1 mg/ml of pyridoxal phosphate (4.9) in water. Mix 3,0 ml of the PLP stock solution and 10 ml of hydrochloric acid (4.21) in a 20 ml volumetric flask and fill up to the mark with water. Check the concentration of PLP by measuring the absorban
28、ce at 293 nm in a 1 cm cell using a UV-spectrometer (5.1) against a hydrochloric acid solution (4.20) as reference. The molar absorption coefficient () of PLP in 0,1 mol/l HCl is 7 200. Calculate the mass concentration PLPof the stock solution, in milligram per millilitre, according to Formula (1):
29、FMA=PLP293PLP(1) where A293is the absorption value of the solution at 293 nm; MPLPis the molar mass of vitamin B6standard substance, in gram per mol (MPLP= 247,14); F is the dilution factor (here F = 20/3); is the molar absorption coefficient of PLP in 0,1 mol/l of hydrochloric acid at 293 nm, in l
30、mol1cm1, (here: = 7 200), see 6. Take 1,0 ml of the PLP stock solution for extraction and proceed with 6.2.1, 6.2.2, 6.2.3 and 6.2.4. Calculate the pyridoxine (PN) conversion rate from the dephosphorylated pyridoxal phosphate solution according to Formula (2): PNPLPSTPLPSHClPN0001100822,01002(%) rat
31、e ConversionMAMA=(2) where PN HCLis the mass concentration of pyridoxine hydrochloride in the standard test solution, in micrograms per millilitre; ASis the peak area or peak height for pyridoxine obtained with the sample test solution, in units of area or height; 2 is the factor of dilution of the
32、reaction with sodium borohydride if acetic acid is added, otherwise the dilution factor is 1,9; 100 is the total volume of the sample test solution, in millilitre; 0,822 is the factor to convert pyridoxine hydrochloride to pyridoxine; 100 is the conversion factor for %; MPLPis the molar mass of pyri
33、doxal phosphate (PLP), in gram per mol (MPLP= 247,14); ASTis the peak area or peak height for pyridoxine obtained with the standard test solution, in units of area or height; 1 000 is the factor to convert microgram to milligram; PLPis the mass concentration of pyridoxal phosphate (PLP) in the stock
34、 solution, in milligrams per millilitre; MPNis the molar mass of pyridoxine (PN), in gram per mol (MPN= 169,1). BS EN 14164:2014EN 14164:2014 (E) 6 4.2 Sodium acetate, trihydrate, mass fraction w(CH3COONa 3H2O) 99,0 %. 4.3 Glacial acetic acid, w(CH3COOH) 99,8 %. 4.4 Glyoxylic acid, w(C2H2O3 H2O) 97,
35、0 %. 4.5 Ferrous sulfate II, heptahydrate, w(FeSO4 7H2O) 99,5 %. 4.6 Sodium hydroxide, w(NaOH) 99,0 %. 4.7 Sodium borohydride, w(NaBH4) 97,0 %. 4.8 Potassium dihydrogen phosphate, w(KH2PO4) 99,0 %. 4.9 Pyridoxal phosphate (PLP), w 99,0 %. 4.10 Orthophosphoric acid, w(H3PO4) 84,0 %. 4.11 Sodium octan
36、esulfonate, w(C8H17NaO3S) 98,0 %, or sodium heptanesulfonate, w (C7H15NaO3S) 98,0 %. 4.12 Acetonitrile (HPLC grade), w(C2H3N) 99,8 %. 4.13 Sodium acetate solution, substance concentration c(CH3COONa 3H2O) = 2,5 mol/l. Dissolve 170,1 g of sodium acetate, trihydrate (4.2) in 500 ml of water. 4.14 Sodi
37、um acetate solution, c(CH3COONa 3H2O) = 0,05 mol/l (pH = 4,5). Dissolve 6,8 g of sodium acetate, trihydrate (4.2) in 1 l of water. Adjust the pH to 4,5 with glacial acetic acid (4.3). 4.15 Ferrous sulfate solution, c(FeSO4 7H2O) = 0,0132 mol/l. Dissolve 36,6 mg of ferrous sulfate II, heptahydrate (4
38、.5) in 10 ml of sodium acetate solution (4.14). Prepare fresh each day of use. NOTE In a study described by Mann et al., see 7, a ferrous sulfate solution of 10 g/l was used. This concentration was based on the completion of the conversion of pyridoxamine to pyridoxal at pyridoxamine levels up to 8
39、times the minimum level of vitamin B6required by the infant formula Act in the US, see Mann et al. 8. This concentration seems not to be necessary for the European situation. 4.16 Sodium hydroxide solution, c(NaOH) = 0,2 mol/l. Dissolve 800 mg of sodium hydroxide (4.6) in 100 ml of water. 4.17 Sodiu
40、m hydroxide solution, c(NaOH) = 6,0 mol/l. Dissolve 24 g of sodium hydroxide (4.6) in 100 ml of water. 4.18 Sodium borohydride solution, c(NaBH4) = 0,1 mol/l. Dissolve 378 mg of sodium borohydride (4.7) in 100 ml of sodium hydroxide solution (4.16). Prepare fresh on day of use. 4.19 Glyoxylic acid s
41、olution, c(C2H2O3 H2O) = 1 mol/l (pH = 4,5). BS EN 14164:2014EN 14164:2014 (E) 7 Dissolve 4,7 g of glyoxylic acid monohydrate (4.4) in 30 ml of sodium acetate solution (4.13). Adjust the pH to 4,5 with the sodium hydroxide solution (4.17) and dilute to 50 ml with water in a volumetric flask. Prepare
42、 fresh on day of use. 4.20 Hydrochloric acid, c(HCl) = 0,1 mol/l. 4.21 Hydrochloric acid, c(HCl) = 0,2 mol/l. 4.22 HPLC mobile phase In a beaker add 940 ml of water, 40 ml of acetonitrile (4.12), 160 mg of sodium octanesulfonate or sodium heptanesulfonate (4.11) and 6,8 g of potassium dihydrogen pho
43、sphate (4.8). After dissolving sodium octanesulfonate or sodium heptanesulfonate and potassium dihydrogen phosphate by stirring, adjust the pH to 2,5 with orthophosphoric acid (4.10). Transfer the solution in a 1 l volumetric flask. Adjust to the mark with water. Filter through a 0,45 m filter. 4.23
44、 Pyridoxine hydrochloride (vitamin B6standard substance), w(C8H11NO3HCI) 99 %. 4.24 Pyridoxine hydrochloride stock solution, mass concentration 0,5 mg/ml. Dissolve an accurately weighed amount of pyridoxine hydrochloride (4.23), e.g. 50 mg, in a defined volume, e.g. 100 ml, of water. The stock solut
45、ion is stable for 4 weeks if stored at 4 C in the dark. For the concentration test, dilute 0,5 ml of pyridoxine hydrochloride stock solution (4.24) to 20 ml with 0,1 mol/l HCI (4.20) and measure the absorbance at 290 nm in a 1 cm cell using a UV-spectrometer (5.1) against 0,1 mol/l HCl solution as r
46、eference. Calculate the mass concentration , in microgram per millilitre of the stock solution according to Formula (3): FMA=0001PNHCl290PNHCl(3) where A290is the absorption of the value of the solution at 290 nm; MPNHClis the molar mass of vitamin B6standard substance, in gram per mol (MPNHCl= 205,
47、64); F is the dilution factor (here F = 40); is the molar absorption coefficient of pyridoxine hydrochloride in 0,1 mol/l of hydrochloric acid at 291 nm, in l mol1cm1(here = 8 600), see 9. Further information on molar absorption coefficients in other solutions than 0,1 mol/l HCl (pH 1) is given in A
48、nnex D. 4.25 Standard solutions 4.25.1 Pyridoxine hydrochloride intermediate standard solution, (C8H11NO3 HCI) 10 g/ml. Pipette 1 ml of the vitamin B6stock solution (4.24) into a 50 ml volumetric flask and dilute to the mark with water. Prepare this solution each day of analysis. 4.25.2 Pyridoxine h
49、ydrochloride standard test solution for HPLC, (C8H11NO3 HCI) 0,1 g/ml to 1 g/ml. BS EN 14164:2014EN 14164:2014 (E) 8 Prepare a series of appropriate test standard solutions of concentrations ranging from e.g. 0,1 g/ml to 1 g/ml of pyridoxine hydrochloride by using the pyridoxine intermediate standard solution (4.25.1). Prepare these solutions fresh on day of use. Perform a system suitability test by injecting a mixed standard test solution for HPLC of pyridoxi
