SIST EN 14148:2003
(Main)Foodstuffs - Determination of vitamin K1 by HPLC
Foodstuffs - Determination of vitamin K1 by HPLC
This draft European Standard specifies a method for the determination of vitamin K1 in foodstuffs by high performance liquid chromatography (HPLC). The determination of Vitamin K1 content is carried out by measurement of reduced phylloquinone. The method has been validated for milk and milk products, however laboratory experiences exist which show that the method is also applicable to other type of foodstuffs.
Lebensmittel - Bestimmung von Vitamin K1 mit HPLC
Diese Europäische Norm legt ein Verfahren zur Bestimmung von Vitamin K1 in Lebensmitteln mit Hochleistungsflüssigchromatographie (HPLC) fest. Der Gehalt an Vitamin K1 wird durch die Messung von reduziertem Phyllochinon bestimmt. Das Verfahren wurde für Milch und Milchprodukte validiert, es gibt jedoch experimentelle Labor-Erfahrungen, die zeigen, dass das Verfahren auch auf andere Lebensmittel anwendbar ist.
Produits alimentaires - Dosage de la vitamine K1 par CLHP
La présente Norme européenne spécifie une méthode de dosage de la vitamine K1 présente dans les produits alimentaires par chromatographie liquide haute performance (CLHP). La détermination de la teneur en vitamine K1 est effectuée par mesurage de la forme réduite de la phylloquinone. Cette méthode a été validée pour le lait et les produits laitiers pour nourrissons. Toutefois, des expériences en laboratoire ont montré que cette méthode est également applicable aux autres catégories de produits alimentaires [10].
Živila – Določevanje vitamina K1 s HPLC
General Information
Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von Vitamin K1 mit HPLCProduits alimentaires - Dosage de la vitamine K1 par CLHPFoodstuffs - Determination of vitamin K1 by HPLC67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 14148:2003SIST EN 14148:2003en01-november-2003SIST EN 14148:2003SLOVENSKI
STANDARD
SIST EN 14148:2003
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14148July 2003ICS 67.050English versionFoodstuffs - Determination of vitamin K1 by HPLCProduits alimentaires - Dosage de la vitamine K1 par CLHPLebensmittel - Bestimmung von Vitamin K1 mit HPLCThis European Standard was approved by CEN on 2 May 2003.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 and bibliographical references concerning such nationalstandards may be obtained on application to the 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 translationunder the responsibility of a CEN member into its own language and notified to the Management Centre has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Slovakia, Spain, Sweden, Switzerland and UnitedKingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2003 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14148:2003 ESIST EN 14148:2003
EN 14148:2003 (E)2ContentspageForeword.31Scope.42Normative references.43Principle.44Reagents.45Apparatus.66Procedure.77Calculation.88Precision.99Test report.10Annex A (informative)
Figures.11Annex B (informative)
Precision data.13Annex C (informative)
Alternative HPLC-Systems.14Bibliography.15SIST EN 14148:2003
EN 14148:2003 (E)3ForewordThis document (EN 14148:2003) 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 orby endorsement, at the latest by January 2004, and conflicting national standards shall be withdrawn at the latestby January 2004.Annexes A, B and C are informative.WARNING — The use of this standard can involve hazardous materials, operations and equipment. Thisstandard does not purport to address all the safety problems associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety and health practices and determine theapplicability of regulatory limitations prior to use.According to the CEN/CENELEC Internal Regulations, the national standards organizations of the followingcountries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal,Slovakia, Spain, Sweden, Switzerland and the United Kingdom.SIST EN 14148:2003
EN 14148:2003 (E)41 ScopeThis European Standard specifies a method for the determination of vitamin K1 in foodstuffs by high performanceliquid chromatography (HPLC). The determination of Vitamin K1 content is carried out by measurement of reducedphylloquinone. The method has been validated for milk and infant formula, however laboratory experiences existwhich show that the method is also applicable to other type of foodstuffs [10].2 Normative referencesThis European Standard incorporates by dated or undated reference, provisions from other publications. Thesenormative references are cited at the appropriate places in the text and the publications are listed hereafter. Fordated references, subsequent amendments to or revisions of any of these publications apply to this EuropeanStandard only when incorporated in it by amendment or revision. For undated references the latest edition of thepublication referred to applies (including amendments).EN ISO 3696, Water for analytical laboratory use — Specification and test methods (ISO 3696:1987).3 PrincipleAfter enzymatic removal of fat from the sample vitamin K1 is determined in an appropriate sample solution by highperformance liquid chromatographic separation coupled with post-column reduction and subsequent fluorometricdetection. Vitamin K1 isomers are quantified as a single unresolved peak with a C18 column [1] to [4].4 Reagents4.1 GeneralDuring the analysis, unless otherwise stated, use only reagents of recognised analytical grade and water of at leastgrade 1 according to EN ISO 3696 or use distilled water.4.2 Chemicals and solutions4.2.1 Methanol, mass fraction w(CH3OH) ³ 99,8 %4.2.2 Ethanol, volume fraction (C2H5OH) ³ 99,8 %4.2.3 Reagent alcohol, (C2H5OH) = 95 %Mix 950 ml of ethanol (4.2.2) with 50 ml of methanol (4.2.1).4.2.4 Dichloromethane, w(CH2Cl2) ³ 99,5 %4.2.5 n-Hexane, w(C6H14)
%4.2.6 Light petroleum, bp. 35 °C to 60 °C, p.a.4.2.7 Potassium hydroxide, w(KOH)
%4.2.8 Potassium hydroxide solution, substance concentration c(KOH) = 10 mol/lSIST EN 14148:2003
EN 14148:2003 (E)54.2.9 Potassium dihydrogen phosphate, w(KH2PO4) ³ 99,5 %4.2.10 Potassium carbonate, w(K2CO3) ³ 99,9 %4.2.11 Sodium acetate, anhydrous, w(CH3COONa) ³ 99,5 %4.2.12 Acetic acid, w(CH3COOH) ³ 99,8 %4.2.13 Zinc chloride, w(ZnCl2) ³ 98 %4.2.14 Zinc, powder, particle size < 63 mm, w(Zn)
%4.2.15 Phosphate buffer pH 7,9 to 8,0Dissolve 54,0 g of potassium dihydrogen phosphate (4.2.9) in approximately 350 ml of water, adjust the pH to 7,9to 8,0 with potassium hydroxide solution (4.2.8) and dilute to 500 ml with water.4.2.16 Zinc chloride-acetate solutionWeigh 13,7 g of zinc chloride (4.2.13), 4,1 g of anhydrous sodium acetate (4.2.11) and 3,0 g of acetic acid (4.2.12)in a 50 ml volumetric flask, dissolve in methanol (4.2.1) and dilute to 50 ml with methanol.4.2.17 Lipase type VIIe.g. from Candida rugosa, activity ca. 1000 U/mg or suitable alternative1); other enzyme sources fromPseudomonas and Rhizopus species can also be used considering the different activity profile.4.2.18 HPLC Mobile phaseMix 100 ml of dichloromethane (4.2.4), 900 ml of methanol (4.2.1) and 5 ml of zinc chloride-acetate solution(4.2.16). Filter through a 0,45 mm filter.4.3 Vitamin K1 standard substance (Phyllochinone, 3-Phythylmenadione), w(C31H46O2) ³ 99%Vitamin K1 can be obtained from various suppliers. The purity of the phylloquinone standard may vary. It istherefore necessary to determine the concentration of the calibration solution by UV-spectrometry (seeconcentration test 4.4.4)4.4 Stock solutions4.4.1 PrecautionsVitamin K1 is very sensitive to light. Measures have to be taken to protect the standard and the correspondingsolutions during the whole procedure e.g. by using generally brown glass ware.4.4.2 Vitamin K1 stock solution I, mass concentration r(C31H46O2) » 1,0 mg/mlWeigh accurately approximately 100 mg of vitamin K1 standard substance (4.3) into a 100 ml volumetric flaskdissolve in methanol (4.2.1) and dilute to 100 ml. This solution can be stored under nitrogen for 3 months at -20 °Cin the dark.NOTEThe amount of vitamin K1 can be difficult to dissolve in methanol.
1)e.g. L-1754; Sigma Chemical Co, P.O. 14508, St. Louis, MO 63178 USA. This product was used in the interlaboratorystudy. This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN ofthe product named. Equivalent products may be used if they can be shown to lead to the same results.SIST EN 14148:2003
EN 14148:2003 (E)64.4.3 Vitamin K1 stock solution II, r(C31H46O2) » 50,0 mg/mlPipette 5,0 ml of vitamin K1 stock solution I (4.4.2), into a 100 ml volumetric flask and dilute to volume withmethanol (4.2.1). This solution can be stored under nitrogen for 1 month at –20 °C in the dark.4.4.4 Concentration testEvaporate 5,0 ml of vitamin K1 stock solution II (4.4.3) by means of a rotary evaporator under partial vacuum orunder a stream of nitrogen. Redissolve the residue in 25,0 ml of n-hexane (4.2.5) or light petroleum (4.2.6).Measure the absorbance of this solution in a 1-cm-cell against n-hexane or light petroleum as reference at themaximum wavelength of about 248 nm with a spectrometer (5.1). Calculate the vitamin K1 mass concentration r, inmicrograms per millilitre, of the vitamin K1 stock solution II (4.4.3) according to equation (1):419510A4248´´=r(1)whereA248is the absorption value of the solution at the maximum wave length of about 248 nm;419is the 1%1cmAvalue of vitamin K1 in n-hexane (4.2.5) or light petroleum (4.2.6) at 248 nm[5];104is the conversion of 1%1cmA to microgramme per milliliter;5is the dilution step during solvent change from methanol to n-hexane.4.5 Standard solutions4.5.1 Intermediate standard solution, vitamin K1, r (C31H46O2) » 2,5 mg/mlPipette 5,0 ml of vitamin K1 stock solution II (4.4.3), into a 100 ml volumetric flask and dilute to volume withmethanol (4.2.1).4.5.2 Standard test solution for HPLC, vitamin K1, r (C31H46O2) » 25,0 ng/mlPipette appropriate volumes e.g. 1 ml of the vitamin K1 intermediate standard solution (4.5.1) into brown volumetricflasks e.g. 100 ml and add methanol (4.2.1) to dilute to volume. Prepare this solution fresh every day.5 ApparatusUse laboratory apparatus and, in particular, the following:5.1 UV SpectrometerUV spectrometer capable of measuring absorptions at defined wavelengths, with appropriate cells, e.g. of 1 cmlength.5.2 HPLC systemHPLC system, consisting of a pump, a sample injecting device, a fluorescence detector with an excitationwavelength set at e.g. 243 nm and an emission wavelength set at e.g. 430 nm and an evaluating system such asan integrator.SIST EN 14148:2003
EN 14148:2003 (E)75.3 HPLC columnAnalytical reversed phase column, e.g. of diameter 3,0 mm to 4,6 mm, length 100 mm to 250 mm, filled withparticle size 3 mm to 10 mm.Particle sizes and column dimensions other than those specified in this European Standard may be used.Separation parameters have to be adapted to such materials to guarantee equivalent results.Other systems (see annex C) can be used providing that a satisfactory separation of phylloquinone from other co-extractives is achieved.5.4 Post-column reductorA stainless steel or glass column placed between analytical column and fluorescence detector, e.g. of diameter2,0 mm to 6,0 mm, length 10 mm to 150 mm, filled with zinc powder (4.2.14).5.5 Filter deviceMembrane filter with pore size of, e.g. 0,45 mm are appropriate.NOTEFiltering of the mobile phase as well as of the sample solution through a membrane filter prior to use or injection issupposed to increase longevity of the columns.6 Procedure6.1 PrecautionsVitamin K1 is very sensitive to light. Measures have to be taken to protect the sample and the correspondingsolutions during the whole procedure e.g. by using genera
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