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STANDARD
SIST EN 15652:2009
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 15652May 2009ICS 67.050 English VersionFoodstuffs - Determination of niacin by HPLCProduits alimentaires - Dosage de la niacine par CLHPLebensmittel - Bestimmung von Niacin mit HPLCThis European Standard was approved by CEN on 23 April 2009.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 CEN 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 CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, 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 United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre:
Avenue Marnix 17,
B-1000 Brussels© 2009 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 15652:2009: ESIST EN 15652:2009
EN 15652:2009 (E) 2 Contents Page Foreword .31Scope .42Normative references .43Principle .44Reagents .45Apparatus .76Procedure . 107Calculation . 118Precision . 129Test report . 14Annex A (informative)
Typical chromatogram . 15Annex B (informative)
Precision data for acid-, enzymatic- and acid/alkaline hydrolysis . 16Annex C (informative)
Comparison between three different ways of hydrolysis . 19Bibliography . 21 SIST EN 15652:2009
EN 15652:2009 (E) 3 Foreword This document (EN 15652:2009) 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of an identical text or by endorsement, at the latest by November 2009, and conflicting national standards shall be withdrawn at the latest by November 2009.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 responsible for identifying any or all such patent rights. WARNING —The use of this standard may involve hazardous materials, operations and equipment. This standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. 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 Republic, 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. SIST EN 15652:2009
EN 15652:2009 (E) 4 1 Scope This European Standard specifies a method for the determination of the mass fraction of niacin in foodstuffs by high performance liquid chromatography (HPLC) by three different ways of hydrolysis, acid hydrolysis (A), enzymatic hydrolysis (B) or acid/alkaline hydrolysis (C). The method has been validated in interlaboratory tests on fortified and non-fortified samples such as breakfast cereal powder, chocolate cereals, cooked ham, green peas, lyophilized green peas with ham, lyophilized soup, nutritive orange juice, milk powder and wheat flour, at levels from 0,5 mg/100 g to 24 mg/100 g. For further information on the validation data, see Annex B. A and B give similar results for niacin. In options A and B niacin is calculated as the sum of nicotinamide and nicotinic acid, and expressed as nicotinic acid [1]. Option C gives higher results than A and B for niacin with non-supplemented cereals, but similar results for other products. In option C, niacin is calculated and expressed as nicotinic acid after transformation of nicotinamide into nicotinic acid [2]. Option A is faster and cheaper than B and C. Option B is used if an exact quantification of nicotinamide and nicotinic acid is needed. This cannot be done with option A, because there is a slight transformation of nicotinamide into nicotinic acid during the acid hydrolysis. Option C quantifies total niacin. The alkaline hydrolysis is able to liberate other forms giving higher results for niacin, which in some foods such as maize and cereals are not normally biologically available, see [3], [4] and [5]. Information on a comparison between the three different ways of hydrolysis is given in Annex C. 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 amendments) applies. EN ISO 3696:1995, Water for analytical laboratory use – Specification and test methods (ISO 3696:1987) 3 Principle Niacin vitamers are extracted from food by an acid (option A), an enzymatic (option B) or an acid/alkaline (option C) treatment and quantified by HPLC with a fluorimetric detection after a post-column derivatization with UV irradiation, see [1] and [2]. For option A and option B, niacin is determined as the sum of nicotinamide and nicotinic acid. Niacin is expressed as nicotinic acid after correction of the molecular weights. For option C, niacin is determined and expressed as nicotinic acid. The alkaline treatment transforms all nicotinamide into nicotinic acid. 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 3696:1995.
SIST EN 15652:2009
EN 15652:2009 (E) 5 4.2 Chemicals and solutions 4.2.1 Sodium acetate, mass fraction w(C2H3NaO2) > 99 % 4.2.2 Potassium monohydrogen phosphate, w(K2HPO4) > 99,5 % 4.2.3 Potassium dihydrogen phosphate, w(KH2PO4) > 99,5 % 4.2.4 Non stabilized hydrogen peroxyde solution, w(H2O2) = 30 % 4.2.5 Copper sulfate, w(Cu(II)SO4·5H2O) > 99 % 4.2.6 Acetic acid, w(CH3COOH) > 99,8 % 4.2.7 Concentrated hydrochloric acid solution (option A and C), w(HCl) = 37,0 % 4.2.8 NADase from Neurospora crassa (option B), enzyme activity 0,55 U/mg of protein. Store below 0 oC. NOTE For the interlaboratory study, NADase from Neurospora crassa from Sigma Chemicals with reference N 9629, lyophilised powder, 0,5 U/mg to 3,0 U/mg protein (biuret) has been used1. 4.2.9 Acetic acid solution, substance concentration c(CH3COOH) = 5 mol/l 4.2.10 Sodium acetate solution, c(C2H3NaO2)= 2,5 mol/l 4.2.11 Copper sulfate solution, c(Cu(II)SO4.5H2O) = 0,005 mol/l 4.2.12 Sodium acetate solution (option B), c(C2H3NaO2)= 0,05 mol/l, pH = 4,5 Dissolve 4,10 g of sodium acetate (4.2.1) in 900 ml of water. Adjust the solution to pH = 4,5 with acid acetic (4.2.6), and then dilute to 1000 ml with water. 4.2.13 Phosphate buffer solution (option B), c(K2HPO4) = 0,05 mol/l and c(KH2PO4) = 0,05 mol/l, pH = 6,8 Mix 1 part per volume of K2HPO4 solution (0,05 mol/l) and 1 part per volume of KH2PO4 solution (0,05 mol/l). Adjust pH to 6,8 with sodium acetate solution (4.2.10) if necessary. 4.2.14 NADase solution (option B) Dissolve 2,9 mg of NADase (4.2.8) in 5 ml of phosphate buffer (4.2.13). This solution is stable for 1 week at
-18 °C. 4.2.15 Hydrochloric acid solution (options A and C), c(HCl) = 0,1 mol/l
1 This information is given for the convenience of users of this European Standard 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. SIST EN 15652:2009
EN 15652:2009 (E) 6 4.2.16 HPLC mobile phase Dissolve 4,77 g of potassium dihydrogen phosphate (4.2.3) in 400 ml of water. Add 3,8 ml of hydrogen peroxide solution (4.2.4) and 0,5 ml of copper sulfate solution (4.2.11). Dilute to 500 ml. The pH is about 4,5. Filter through a membrane filter (5.7). This solution is stable for one day. 4.2.17 Sodium hydroxide (option C), w(NaOH) ≥ 99 % 4.2.18 Sodium hydroxide solution (option C), c(NaOH) = 5 mol/l Dissolve 20 g of sodium hydroxide (4.2.17) in 80 ml of water. After cooling dilute to 100 ml. 4.2.19 Hydrochloric acid solution (option C), w(HCl) = 3,7 % Dilute 5 ml of the concentrated hydrochloric acid solution (4.2.7) to 50 ml with water. 4.3 Standard substances 4.3.1 General Nicotinic acid and nicotinamide can be obtained from various suppliers. The purity may vary and it is therefore necessary to determine the concentration of the calibration solution by a spectrometric determination (see 4.4.3). 4.3.2 Nicotinic acid, w(C6H5NO2) ≥ 99,5 % 4.3.3 Nicotinamide (options A and B), w(C6H6N2O) ≥ 99,5 % 4.4 Stock solutions 4.4.1 Nicotinic acid stock solution, mass concentration (C6H5NO2) = 1 mg/ml Dissolve an amount of the nicotinic acid standard substance (4.3.2), e.g. approximately 100 mg to the nearest 1 mg in 100 ml of water. This solution is stable for 1 week at -18 °C. 4.4.2 Nicotinamide stock solution, (options A and B), (C6H6N2O) = 1 mg/ml Dissolve an amount of the nicotinamide standard substance (4.3.3), e.g. approximately 100 mg to the nearest 1 mg in 100 ml of water. This solution is stable for 1 week at -18 °C. 4.4.3 Concentration tests 4.4.3.1 Nicotinic acid solution, (C6H5NO2) = 1 mg/ml Dilute 1 ml of the nicotinic acid stock solution (4.4.1) in 100 ml of hydrochloric acid solution (4.2.15) and measure the absorbance at 260 nm in a 1 cm cell using a UV spectrometer (5.2) against hydrochloric acid solution (4.2.15) as reference. Calculate the mass concentration, , in milligram per millilitre of the stock solution, using Equation (1): 4201000260×=Aρ (1) where SIST EN 15652:2009
EN 15652:2009 (E) 7 A260 is the absorbance value of the solution at 260 nm; 420 is the % 1cm 1E value for nicotinic acid in 0,1 mol/l HCl, see [6]. 4.4.3.2 Nicotinamide solution, (C6H6N2O) = 1 mg/ml Dilute 1 ml of the nicotinamide stock solution (4.4.2) in 100 ml of hydrochloric acid solution (4.2.15) and measure the absorbance at 260 nm in a 1 cm cell using a UV spectrometer (5.2) against hydrochloric acid solution (4.2.15) as reference. Calculate the mass concentration, , in milligram per millilitre of the stock solution, using Equation (2): 4101000260×=Aρ (2) where A260 is the absorbance value of the solution at 260 nm; 410 is the % 1cm 1E value for nicotinamide in 0,1 mol/l HCl, see [6]. 4.5 Nicotinic acid and nicotinamide standard solutions, (C6H5N02) = (C6H6N2O) = 0,05 µg/ml to 5 µg/ml Prepare e.g. a first solution with 1 ml of each stock solution (4.4.1) or (4.4.2) in 100 ml of water. From this solution prepare four standard solutions (0,5 ml, 2,5 ml, 10 ml and 50 ml) in 100 ml of water. These solutions are stable for one day at room temperature. 5 Apparatus 5.1 General Usual laboratory apparatus and glassware, and the following. 5.2 UV spectrometer Capable of measurement of absorbance at defined wavelenghts 5.3 Oven, capable of maintaining a temperature of 37 °C 5.4 Autoclave, capable of maintaining a temperature of 120 °C 5.5 HPLC system Consisting of a pump, sample injecting device, fluorescence detector with excitation and emission wavelengths set at 322 nm and 380 nm, and an evaluation system such as an integrator. SIST EN 15652:2009
EN 15652:2009 (E) 8 5.6 Analytical reverse phase separating column, e.g. LiChrospher 60 RP-18 Select B endcapped2 The column, with the following characteristics, shall ensure a baseline resolution of the analytes concerned: a) a length of 25 cm; b) an inner diameter of 4,0 mm; c) a particle size of 5 µm. Other particle sizes or column dimensions than specified in this European Standard may be used. Separation parameters shall be adapted to such other materials to guarantee equivalent results. 5.7 Filter device Membrane filter with a pore size of for example 0,45 µm. 5.8 Post-column derivatization tube and UV lamp A polytetrafluoroethylene (PTFE) tube (length of 5 m, inner diameter of 0,5 mm, external diameter of 1,6 mm) surrounding a UV black-light-blue (BLB) lamp with low-pressure tube (VL-120 BLB, 20 W, 365 nm, intensity is 55 µW/cm2 from Vilber Lourmat)2), see Figure 1 and Figure 2 and also [7].
WARNING 1
— Harmful UV light could come out of the metal box containing the lamp. WARNING 2 — If bubble formation occurs in the tube due to overheating, the tube should be efficiently cooled by air circulation, for example by lifting the box.
2 LiChrospher 60 RP-18 Select B endcapped and VL-120 BLB are examples of suitable products available commercially. This information is given for the convenience of users of ths European Standard 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. SIST EN 15652:2009
EN 15652:2009 (E) 9
Key 1 lamp tube 2 from column 3 to detector Figure 1 — Schematic representation and dimensions (mm) of the lamp, lamp housing (in upside down position) and placement of the lamp housing on bench (in operating position)
Dimensions in millimetres
Key 1 reflector 2 lamp tube Figure 2 — Cross section of the lamp housing (in upside down position) with tube lamp and dimensions SIST EN 15652:2009
EN 15652:2009 (E) 10 6 Procedure 6.1 Sample preparation Homogenize the test sample. Grind coarse material with an appropriate mill and mix again. Measures such as pre-cooling shall be taken to avoid exposing to high temperature for long periods of time. 6.2 Extraction 6.2.1 Extraction option A, acid hydrolysis Similar results are obtained as option B for niacin, but with a slight transformation of nicotinamide to nicotinic acid during the hydrolysis step. Weigh an appropriate amount of the test sample to the nearest mg, e.g. 1 g to 5 g in a conical flask. Add 50 ml of the hydrochloric acid solution (4.2.15). Let the conical flask in a boiling water bath for 1 h. After cooling add sodium acetate (4.2.10) solution to reach a pH of 4,5. Transfer to a volumetric flask. Make up to 100 ml with water. Shake the solution and filter through a paper filter. Filter again through a membrane filter (5
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