SIST EN 15911:2011
(Main)Foodstuffs - Simultaneous determination of nine sweeteners by high performance liquid chromatography and evaporative light scattering detection
Foodstuffs - Simultaneous determination of nine sweeteners by high performance liquid chromatography and evaporative light scattering detection
This European Standard specifies a method for the simultaneous determination of nine sweeteners in beverages and canned or bottled fruits by high performance liquid chromatography (HPLC) with evaporative light scattering detection (HPLC-ELSD). This method has been validated in an interlaboratory study via the analysis of spiked samples on the following matrices:
- acesulfame-K (ACS-K) (from 38,3 mg/l to 383,5 mg/l) in beverages and (from 38,4 mg/kg to 391,3 mg/kg) in canned fruits;
- alitame (ALI) (from 31,1 mg/l to 114,5 mg/l) in beverages and (from 36 mg/kg to 175,2 mg/kg) in canned fruits;
- aspartame (ASP) (from 38,1 mg/l to 702 mg/l) in beverages and (from 37,2 mg/kg to 1 120,2 mg/kg) in canned fruits;
- cyclamic acid (CYC) (from 28,3 mg/l to 307,2 mg/l) in beverages and (from 27,5 mg/kg to 1 100,6 mg/kg) in canned fruits;
- dulcin (DUL) (from 55,0 mg/l to 115,1 mg/l) in beverages and (from 49,8 mg/kg to 172,6 mg/kg) in canned fruits;
- neotame (NEO) (from 37,6 mg/l to 115,3 mg/l) in beverages and (from 37,3 mg/kg to 173,7 mg/kg) in canned fruits;
- neohesperidine dihydrochalcone (NHDC) (from 31,4 mg/l to 59,3 mg/l) in beverages and (from
35,3 mg/kg to 59,3 mg/kg) in canned fruits;
- saccharin (SAC) (from 36,2 mg/l to 87,6 mg/l) in beverages and (from 44,3 mg/kg to 235,3 mg/kg) in canned fruits;
- sucralose (SCL) (from 36,8 mg/l to 346,8 mg/l) in beverages and (from 35,3 mg/kg to 462,4 mg/kg) in canned fruits. For further information on the validation see Clause 8 and Annex C.
Lebensmittel - Gleichzeitige Bestimmung von neun Süßungsmitteln mit Hochleistungs-Flüssigchromatographie und Verdampfungs-Lichtstreu-Detektion
Diese Europäische Norm legt ein Verfahren zur gleichzeitigen Bestimmung von neun Intensivsüßungsmitteln in
Getränken und in Dosen konserviertem oder eingemachtem Obst durch Hochleistungs-Flüssigchromatographie
mit Verdampfungs-Lichtstreu-Detektion (HPLC-ELSD) fest. Das Verfahren wurde in einem Ringversuch durch
die Analyse von aufgestockten Proben an den folgenden Matrices validiert:
⎯ Acesulfam-K (ACS-K) (von 38,3 mg/l bis 383,5 mg/l) in Getränken und (von 38,4 mg/kg bis 391,3 mg/kg)
in Dosenobst;
⎯ Alitam (ALI) (von 31,1 mg/l bis 114,5 mg/l) in Getränken und (von 36 mg/kg bis 175,2 mg/kg) in
Dosenobst;
⎯ Aspartam (ASP) (von 38,1 mg/l bis 702 mg/l) in Getränken und (von 37,2 mg/kg bis 1 120,2 mg/kg) in
Dosenobst;
⎯ Cyclohexansulfamidsäure (CYC) (von 28,3 mg/l bis 307,2 mg/l) in Getränken und (von 27,5 mg/kg bis
1 100,6 mg/kg) in Dosenobst;
⎯ Dulcin (DUL) (von 55,0 mg/l bis 115,1 mg/l) in Getränken und (von 49,8 mg/kg bis 172,6 mg/kg) in
Dosenobst;
⎯ Neotam (NEO) (von 37,6 mg/l bis 115,3 mg/l) in Getränken und (von 37,3 mg/kg bis 173,7 mg/kg) in
Dosenobst;
⎯ Neohesperidin-Dihydrochalcon (NHDC) (von 31,4 mg/l bis 59,3 mg/l) in Getränken und (von 35,3 mg/kg
bis 59,3 mg/kg) in Dosenobst;
⎯ Saccharin (SAC) (von 36,2 mg/l bis 87,6 mg/l) in Getränken und (von 44,3 mg/kg bis 235,3 mg/kg) in
Dosenobst;
⎯ Sucralose (SCL) (von 36,8 mg/l bis 346,8 mg/l) in Getränken und (von 35,3 mg/kg bis 462,4 mg/kg) in
Dosenobst.
Weitere Informationen zu den Validierungsergebnissen siehe Abschnitt 8 und Anhang C.
ANMERKUNG Das Verfahren wurde durch einen Ringversuch [1] nach dem IUPAC-Harmonisierungsprotokoll [2] an
Analyt-Matrix-Kombinationen aller neun Süßungsmittel in Getränken und in Dosen konserviertem oder eingemachtem
Obst vollständig validiert.
Denrées alimentaires - Dosage simultané de neuf édulcorants par chromatographie liquide haute performance et détection à diffusion de lumière par évaporation
Cette Norme européenne spécifie une méthode de chromatographie liquide haute performance avec détection à diffusion de lumière par évaporation (CLHP-ELSD) pour le dosage simultané de neuf édulcorants intenses, à savoir l’acésulfame-K (ACS-K), l’alitame (ALI), l’aspartame (ASP), l’acide cyclamique (CYC), la dulcine (DUL), le néotame (NEO), la néohespéridine dihydrochalcone (NHDC), la saccharine (SAC) et le sucralose (SCL) dans des denrées alimentaires.
NOTE La méthode a été intégralement validée [1] dans le cadre d’une étude interlaboratoires, conformément au Protocole harmonisé IUPAC [2], sur des combinaisons analyte/matrice des neuf édulcorants dans des boissons et des fruits en conserve ou en bouteille.
Živila - Simultano določevanje devetih sladil z metodo tekočinske kromatografije visoke ločljivosti in z detekcijo disperzije svetlobe (ELS)
Ta evropski standard določa metodo za simultano določevanje devetih sladil v pijačah in konzerviranega ali stekleničenega sadja s tekočinsko kromatografijo visoke ločljivosti (HPLC) z detekcijo disperzije svetlobe (HPLC-ELSD). Ta metoda je bila potrjena v medlaboratorijski študiji z analizo vzorcev z internimi dodatki na naslednjih matricah:
– acusulfam-K (ACS-K) (od 38,3 mg/l do 383,5 mg/l) v pijačah in (od 38,4 mg/kg do 391,3 mg/kg) v konzerviranem sadju;
– alitam (ALI) (od 31,1 mg/l do 114,5 mg/l) v pijačah in (od 36 mg/kg do 175,2 mg/kg) v konzerviranem sadju;
– aspartam (ASP) (od 38,1 mg/l do 702 mg/l) v pijačah in (od 37,2 mg/kg do 1.120,2 mg/kg) v konzerviranem sadju;
– ciklaminska kislina (CYC) (od 28,3 mg/l do 307,2 mg/l) v pijačah in (od 27,5 mg/kg do 1.100,6 mg/kg) v konzerviranem sadju;
– dulcin (DUL) (od 55,0 mg/l do 115,1 mg/l) v pijačah in (od 49,8 mg/kg do 172,6 mg/kg) v konzerviranem sadju;
– neotam (NEO) (od 37,6 mg/l do 115,3 mg/l) v pijačah in (od 37,3 mg/kg do 173,7 mg/kg) v konzerviranem sadju;
– neohesperidin dihidrokalcon (NHDC) (od 31,4 mg/l do 59,3 mg/l) v pijačah in (od
35,5 mg/kg do 59,3 mg/kg) v konzerviranem sadju;
– saharin (SAC) (od 36,2 mg/l do 87,6 mg/l) v pijačah in (od 44,3 mg/kg do 235,3 mg/kg) v konzerviranem sadju;
– sukraloza (SCL) (od 36,8 mg/l do 346,8 mg/l) v pijačah in (od 35,3 mg/kg do 462,4 mg/kg) v konzerviranem sadju. Za dodatne informacije o potrjevanju glej klavzulo 8 in dodatek C.
General Information
Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Gleichzeitige Bestimmung von neun Süßungsmitteln mit Hochleistungs-Flüssigchromatographie und Verdampfungs-Lichtstreu-DetektionDenrées alimentaires - Dosage simultané de neuf édulcorants par chromatographie liquide haute performance et détection à diffusion de lumière par évaporationFoodstuffs - Simultaneous determination of nine sweeteners by high performance liquid chromatography and evaporative light scattering detection67.180.10Sladkor in sladkorni izdelkiSugar and sugar productsICS:Ta slovenski standard je istoveten z:EN 15911:2010SIST EN 15911:2011en,fr,de01-marec-2011SIST EN 15911:2011SLOVENSKI
STANDARD
SIST EN 15911:2011
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 15911
October 2010 ICS 67.180.10 English Version
Foodstuffs - Simultaneous determination of nine sweeteners by high performance liquid chromatography and evaporative light scattering detection
Denrées alimentaires - Détermination simultanée de neuf édulcorants par chromatographie liquide haute performance et détection à diffusion de lumière
Lebensmittel - Gleichzeitige Bestimmung von neun Süßungsmitteln mit Hochleistungs-Flüssigchromatographie und Verdampfungs-Lichtstreu-Detektion This European Standard was approved by CEN on 18 September 2010.
CEN members are bound to comply 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-CENELEC 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 same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, 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 STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre:
Avenue Marnix 17,
B-1000 Brussels © 2010 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 15911:2010: ESIST EN 15911:2011
EN 15911:2010 (E) 2 Contents Page Foreword .31Scope .42Normative references .43Principle .44Reagents .55Apparatus and equipment .76Procedure .87Calculation of results . 118Precision . 129Test report . 14Annex A (informative)
Table A.1
Suitable method conditions . 16Annex B (informative)
Examples of chromatograms . 17Annex C (informative)
Precision data . 19Annex D (informative)
Present EU limits for the nine sweeteners . 29Bibliography . 30 SIST EN 15911:2011
EN 15911:2010 (E) 3 Foreword This document (EN 15911:2010) 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 April 2011, and conflicting national standards shall be withdrawn at the latest by April 2011. 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. 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, Croatia, 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 15911:2011
EN 15911:2010 (E) 4 1 Scope This European Standard specifies a method for the simultaneous determination of nine sweeteners in beverages and canned or bottled fruits by high performance liquid chromatography (HPLC) with evaporative light scattering detection (HPLC-ELSD). This method has been validated in an interlaboratory study via the analysis of spiked samples on the following matrices: acesulfame-K (ACS-K) (from 38,3 mg/l to 383,5 mg/l) in beverages and (from 38,4 mg/kg to 391,3 mg/kg) in canned fruits; alitame (ALI) (from 31,1 mg/l to 114,5 mg/l) in beverages and (from 36 mg/kg to 175,2 mg/kg) in canned fruits; aspartame (ASP) (from 38,1 mg/l to 702 mg/l) in beverages and (from 37,2 mg/kg to 1 120,2 mg/kg) in canned fruits; cyclamic acid (CYC) (from 28,3 mg/l to 307,2 mg/l) in beverages and (from 27,5 mg/kg to 1 100,6 mg/kg) in canned fruits; dulcin (DUL) (from 55,0 mg/l to 115,1 mg/l) in beverages and (from 49,8 mg/kg to 172,6 mg/kg) in canned fruits; neotame (NEO) (from 37,6 mg/l to 115,3 mg/l) in beverages and (from 37,3 mg/kg to 173,7 mg/kg) in canned fruits; neohesperidine dihydrochalcone (NHDC) (from 31,4 mg/l to 59,3 mg/l) in beverages and (from 35,3 mg/kg to 59,3 mg/kg) in canned fruits; saccharin (SAC) (from 36,2 mg/l to 87,6 mg/l) in beverages and (from 44,3 mg/kg to 235,3 mg/kg) in canned fruits; sucralose (SCL) (from 36,8 mg/l to 346,8 mg/l) in beverages and (from 35,3 mg/kg to 462,4 mg/kg) in canned fruits. For further information on the validation see Clause 8 and Annex C. NOTE The method has been fully validated [1] through collaborative trial, according to the IUPAC Harmonised Protocol [2], on analyte-matrix combinations of all nine sweeteners in beverages and canned or bottled fruits. 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 The procedure involves extraction of the nine sweeteners with a buffer solution, sample clean-up using solid-phase extraction cartridges followed by HPLC-ELSD analysis. SIST EN 15911:2011
EN 15911:2010 (E) 5 4 Reagents During the analysis, unless otherwise stated, use only reagents of recognised analytical grade for HPLC analysis and water of at least grade 1 as defined in EN ISO 3696:1995. When preparing solutions, the purity of the substances shall be taken into account. 4.1 Acesulfame-K, with a mass fraction w of at least 99,0 %. 4.2 Alitame, w ≥ 99,0 %. 4.3 Aspartame, w ≥ 99,0 %. 4.4 Dulcin, for HPLC. 4.5 Neotame, w ≥ 99,0 %. 4.6 Neohesperidine dihydrochalcone, w ≥ 95,0 %. 4.7 Saccharin, sodium salt dihydrate, w ≥
98,0 %. 4.8 Sodium cyclamate, w ≥
99,0 %. 4.9 Sucralose, w ≥ 99,0 %. 4.10 Formic acid, HCOOH for HPLC. 4.11 Triethylamine, (C2H5)3N, w ≥ 99,5 %. 4.12 Methanol, for HPLC. 4.13 Acetone, for HPLC. 4.14 Buffer solution (pH = 4,5). Dissolve 4 ml of formic acid (4.10) in 5 l of water. Adjust to pH 4,5 with approximately 12,5 ml triethylamine (4.11). 4.15 HPLC mobile phase A, methanol/buffer solution/acetone 69:24:7 (v/v/v). Mix 690 ml of methanol (4.12) with 240 ml of buffer solution (4.14) and with 70 ml of acetone (4.13). Degas by sonication for 10 min. 4.16 HPLC Mobile phase B, methanol/buffer solution/acetone 11:82:7 (v/v/v). Mix 110 ml of methanol (4.12) with 820 ml of buffer solution (4.14) and with 70 ml of acetone (4.13). Degas by sonication for 10 min. 4.17 Mixed stock solution, containing ACS-K, ALI, ASP, CYC-Na, DUL, NEO, NHDC, SAC-Na and SCL; mass concentration ρ(sweetener i) = 30 µg/ml to 250 µg/ml. Prepare a mixed stock solution of all nine sweeteners by weighing the given masses of the individual sweetener standards (Table 1) first into a 100 ml beaker and dissolving them in 50 ml of methanol/water (1:1) until complete dissolution. Then transfer the obtained solution quantitatively into a 500 ml volumetric flask and make up to the mark with the buffer solution (4.14). Mix thoroughly by sonication until complete dissolution. SIST EN 15911:2011
EN 15911:2010 (E) 6 Table 1 — Masses of individual standards for preparation of mixed stock solution Standard Mass weighed into 500 ml volumetric flask c mg Final mass concentration of sweetener i in mixed stock standard µg/ml Acesulfame-K (ACS-K) 45 90 Alitame (ALI) 25 50 Aspartame (ASP) 125 250 Sodium cyclamate (CYC-Na) 140 a – Cyclamic acid (CYC) (free acid) – 249,42 Dulcin (DUL) 25 50 Neotame (NEO) 25 50 Neohesperidine dihydrochalcone (NHDC) 15 30 Saccharin, sodium salt dihydrate (SAC-Na·2H2O) 35 b – Saccharin (SAC) (free imide) – 53,17 Sucralose (SCL) 50 100 a Equivalent to 124,71 mg free cyclamic acid; conversion factor to calculate mass of free cyclamic acid = 0,890 8; mCYC = 0,890 8 × mCYC-Na. b Equivalent to 26,58 mg free saccharin; conversion factor to calculate mass of free saccharin = 0,759 5; mSAC = 0,759 5 × mSAC-Na·2H2O. c First weigh into 100 ml beaker, dissolve in 50 ml of a methanol:water (1:1) mixture and then transfer quantitatively into 500 ml volumetric flask.
NOTE In case of cyclamic acid and saccharin, their sodium salts are used, since they are either not available in free form or poorly soluble. The final concentrations of the individual sweeteners in micrograms per millilitre in the mixed stock solution have to be calculated by using the actually weighed masses. 4.18 Standard solutions. From the mixed stock solution (4.17) prepare a series of standard solutions containing the sweeteners at levels fitting appropriate limits, e.g. the highest concentration of the calibration shall be at least equivalent to 125 % of the given limits, such as those in Commission Directives [3], [4], [5] (see Table D.1), whilst taking the dilution steps within the procedure into account (see Table 2). For sweeteners not authorised by the current EU legislation (ALI, DUL and NEO) fictitious maximum usable dosages (MUD) are assumed at approximately 200 mg/l or 200 mg/kg. The user of the standard has to check whether the limits in Table D.1 are still valid. If not, the mass concentration of the standard substance in the calibration solution shall be adjusted to meet the current MUDs. NOTE The present procedure is simplified by preparing one calibration series for both food matrices. The described calibration series is fitted to canned fruits as the MUDs for canned fruits are in some cases higher than the MUDs for beverages. In case only the latter matrix is analysed the calibration series can be fitted to the MUDs of beverages. Pipette the following volumes (see Table 2) from the mixed stock solution (4.17) into appropriate volumetric flasks (10 ml to 50 ml) and make up to the mark with buffer solution (4.14) and shake thoroughly. SIST EN 15911:2011
EN 15911:2010 (E) 7 Table 2 — Preparation of series of standard solutions Calibration solution Volume of volumetric flask ml Volume taken from mixed stock solution (4.17) ml Volume taken from buffer solution (4.14) ml 1a 10 1002 10 8 2 3 10 6 4 4 10 4 6 5 10 2 8 6 25 3 22 7 50 3 47 8 50 1,5 48,5 a Undiluted mixed stock solution (4.17).
Table 3 details the concentration of sweetener i in each calibration standard following preparation described in Table 2. If not all of the sweeteners covered by this standard are the subject of analysis in routine use of the method, when applied to a particular set of samples consideration may be given to reduce the levels of the calibration solutions used for those samples. Table 3 — Concentration of sweetener i in the individual standard solutions Sweetener Calibration solution mg/ml
1 2 3 4 5 6 7 8 ACS-K 90,0 72,0 54,0 36,0 18,0 10,8 5,4 2,7 a ALI 50,0 40,0 30,0 20,0 10,0 6,0 3,0 a 1,5 a ASP 250,0 200,0 150,0 100,0 50,0 30,0 15,0 7,5 CYC 249,4 199,5 149,7 99,8 49,9 29,9 15,0 7,5 DUL 50,0 40,0 30,0 20,0 10,0 6,0 a 3,0 a 1,5 a NEO 50,0 40,0 30,0 20,0 10,0 6,0 3,0 a 1,5 a NHDC 30,0 24,0 18,0 12,0 6,0 3,6 a 1,8 a 0,9 a SAC 53,2 42,5 31,9 21,3 10,6 6,4 3,2 a 1,6 a SCL 100,0 80,0 60,0 40,0 20,0 12,0 6,0 3,0 a a The concentration level might be below the limit of quantification (LOQ). If yes, the result obtained by HPLC analysis is not included in the construction of the calibration graph, e.g. in case of ACS-K a seven point calibration is performed, ignoring the result obtained for calibration solution 8. 5 Apparatus and equipment Usual laboratory apparatus and, in particular, the following: 5.1 Common laboratory glassware, such as graduated cylinders, volumetric pipettes, glass beakers. 5.2 Analytical balance, capable of weighing to 0,01 mg. 5.3 Laboratory balance, capable of weighing to 0,01 g. 5.4 Positive displacement pipette, or equivalent, capable of delivering 1 ml to 10 ml (variable volume). SIST EN 15911:2011
EN 15911:2010 (E) 8 5.5 Volumetric flasks, of suitable capacity, e.g. 10 ml, 25 ml, 50 ml, 100 ml and 500 ml. 5.6 Centrifuge tubes, made of polypropylene, of suitable capacity, e.g. 50 ml. 5.7 Graduated test tubes, of suitable capacity, e.g. 5 ml. 5.8 Food blender, suitable for homogenisation of food samples. 5.9 Ultrasonic bath. 5.10 Centrifuge, capable of maintaining 4 000 min-1. 5.11 SPE Vacuum system, or equivalent. 5.12 Equipment for solvent evaporation. 5.13 pH meter. 5.14 C18 SPE cartridges. 5.15 Analytical reverse phase column, fully end-capped, allowing sufficient separation of all nine sweeteners. E.g. with: an RP C 18 stationary phase of 5 µm; a length of 250 mm; internal diameter of 3 mm. 5.16 HPLC system, equipped with a binary pump capable of maintaining a flow rate of 0,5 ml/min, preferably an automatic injection system, and an evaporative light scattering detector. Other detection systems such as MS as substitute for ELSD or UV and DAD when substances do absorb in the UV region can also be used provided that the equivalent performance characteristics can be obtained. 5.17 Data acquisition and analysis software. 6 Procedure 6.1 General Comminute the entire test sample to give a homogenous suspension (5.8). Liquid samples can be subjected directly to the extraction procedure. 6.2 Preparation of test sample 6.2.1 Step 1 Weigh approximately 5 g (M1, recorded to two decimal places) of the homogenised test sample (6.1) into a volumetric flask of 50 ml (V1). Make up to the mark with buffer solution (4.14), mix thoroughly by hand to obtain a homogeneous suspension and sonicate (5.9) for 15 min. SIST EN 15911:2011
EN 15911:2010 (E) 9 6.2.2 Step 2 Transfer the obtained suspension to a 50 ml centrifuge tube. Centrifuge at 4 000 min-1 for 10 min. NOTE In case the test sample gives a clear solution (e.g. some beverages), this step can be ignored. 6.3 Solid phase extraction 6.3.1 Step 1 Condition the cartridges (5.14) by applying 3 ml of methanol (4.12) and let it pass through using a slight vacuum resulting in a flow rate of 1 ml/min to 2 ml/min. Make sure that a small portion of methanol remains above the sorbent bed (1 mm). 6.3.2 Step 2 Equilibrate the cartridges (5.14) by applying 2 ml of buffer solution (4.14) and let it pass through using a slight vacuum resulting in a flow rate of 1 ml/min to 2 ml/min. Make sure that a small portion of buffer solution remains above the sorbent bed (1 mm). Repeat the procedure two times. 6.3.3 Step 3 Load the cartridges (5.14) with 5 ml of sample extract (V2 first loading), i.e. the supernatant from (6.2.2), and let it pass through using a slight vacuum resulting in a flow rate of 1 ml/min to 2 ml/min. Make sure that a small portion remains above the sorbent bed (1 mm). Repeat the procedure once more (V2 in total 10 ml). 6.3.4 Step 4 Wash the cartridges (5.14) with 3 ml of buffer solution (4.14) and let it pass through using a slight vacuum resulting in a flow rate of 1 ml/min to 2 ml/min. Make sure that a small portion of buffer solution remains above the sorbent bed (1 mm).
6.3.5 Step 5 Elute the sweeteners from the cartridges (5.14) by applying 2 ml of methanol (4.12) and collecting the eluate in a graduated 5 ml test tube. Use a slight vacuum to obtain a flow rate of 1 ml/min. Make sure that a small portion of methanol remains above the sorbent bed (1 mm). Wait 10 min before applying a second portion of 2 ml of methanol and elute it subsequently to the same 5 ml test tube using the same vacuum conditions but this time letting the cartridges (5.14) run dry. Avoid in all steps (6.2.1 to 6.3.5) that the sorbent bed runs dry with the only exception of the last step, i.e. second elution of analytes (6.3.5).
6.3.6 Step 6 Evaporate the solvent from the methanolic SPE extract to 3 ml under a stream of nitrogen at ambient temperature. Temperatures above 40 °C have to be avoided, since aspartame can degrade.
6.3.7 Step 7 Fill the graduated test tube containing the SPE extract (6.3.6) up to the 5 ml mark with buffer solution (4.14) (V3). Mix thoroughly and transfer the content into a suitable HPLC vial and analyse by HPLC. SIST EN 15911:2011
EN 15911:2010 (E) 10 6.4 HPLC conditions Establish suitable HPLC conditions to meet the predefined performance criteria (6.5). The separation and quantification have proven to be satisfactory using the following experimental conditions and HPLC gradient conditions as outlined in Table 4: Column:
see 5.15; Column temperature:
ambient temperature; Injection volume:
10 µl; Mobile phase:
see 4.15 and 4.16; Separation mode:
gradient; Detector:
evaporative light scattering detector (ELSD); ELSD drift tube temperature: 85 °C; ELSD nitrogen flow:
2,5 l/min; ELSD gain:
1; ELSD impactor:
Off. Table 4 — gradient analysis by HPLC, Flow rate 0,5 ml/min Time min Mobile phase A % Mobile phase B % 0 0 100 4 0 100 11 53 47 23 100 0 24 100 0 26 0 100 36 0 100
NOTE The given detector parameters are applicable to the Alltech ELSD 2000ES system1). Alternative ELSD systems and experimental conditions, used in an inter-laboratory study, are listed in Annex A. HPLC and ELSD operating conditions can be changed to obtain optimum separation. 6.5 System suitability test – Resolution of separation system The details of the chromatographic procedure depend, among other factors, on equipment, type, age, and supplier of the column, sample size and detector. Different columns can be used, and injection volumes can be varied, if the requirements of the system suitability tests are met.
1) This system is an example of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of this product. SIST EN 15911:2011
EN 15911:2010 (E) 11 The HPLC-ELSD system shall be capable of separating all nine sweeteners from each other with at least baseline separation. This requirement can be proven by using calibration solution 1 (4.18) as shown in
Figure B.1. Moreover, the system shall be capable of separating all nine sweeteners from other components of the matrix. Many matrix components, such as sodium benzoate, sorbic acid, citric acid, phosphoric acid, malic acid, ascorbic acid, glutamic acid, sucrose, glucose, fructose, lactose, caffeine, taurine, D-glucurono--lactone and sorbitol, etc., are removed throughout the SPE clean-up. A commonly encountered critical pair is alitame (sweetener not authorised by the current EU legislation) and quinine, which is not removed by the SPE clean-up [7]. In case of failure, the chromatographic conditions (e.g. sample volume injected, mobile phase rate, gradient program, etc.) or the ELSD conditions (e.g. drift tube temperature, nitrogen/air flow) have to be optimized. 6.6 Construction of calibration graph Analyse all standard solutions (4.18, Table 2) using HPLC conditions (6.4) identical to those used for the test samples, i.e. inject 10 µl of each solution into the HPLC system. Construct a calibration chart for each sweetener i from the results of the analysis of the standard solutions. Plot the obtained peak area as log10(Peak area i) (y-axis) against the log10(Concentration i) (x-axis). Fit a straight line (y = a + bx) to the results, where b is the value of the slope of the linear function and a is the value where the calibration function intercepts the y-axis. If the results of the analyses of the standard solutions are linear, the calibration line can be used to calculate the concentration of sweetener i in the sample extract.
If detectors other than ELSD are used, other calibration functions may be used. 6.7 HPLC analysis of sample test solution Analyse 10 µl of the sample test solution obtained from step in 6.3.7. 6.8 Interpretation of chromatographic data Identify the individual sweeteners in the test samples by comparison of the retention time of sweeteners observed during the analysis of standard solutions analysed in the same batch as samples with the retention time of compounds eluted during the analysis of the test samples. The elution order of the individual sweeteners together with the retention times are given in an example chromatogram in Figure B.1. Measure the peak area response (Ri) observed for sweetener i in each solution. In case the peak area of sweetener i in the chromatogram of the test sample solution exceeds the area of the respective sweetener peak in the chromatogram obtained for the standard solution with the highest concentration, the test sample solution is diluted with buffer solution (4.14) and the diluted extract re-analysed. 7 Calculation of results Quantify sweetener i by integration of the peak area i (Ri) obtained from the analysis of the injected SPE extract. Use the resulting calibration function, i.e. y = a + bx (6.6) to calculate the mass concentration of sweetener i (ρ1i) in the measured sample extract solution using Equations (1) and (2). iiiibaR−=)(loglog10110ρ (1) ()ii110log110ρρ= (2) where SIST EN 15911:2011
EN 15911:2010 (E) 12 Ri is the peak area response (6.8) for sweetener i; ai is the intercept of the calibration line (6.6) for sweetener i; bi is the slope of the calibration line (6.6) for sweetener i; ρ1i is the mass concentration of sweetener i in the SPE extract in micrograms per millilitre. Calculate the concentration/mass fraction iw2/ρof sweetener i in the test sample according to Equation (3). 213112/VMVVwii×××=ρρ (3) where ρ1i is the mass concentration of sweetener i in the SPE extract in micrograms per millilitre (as determined in Equation (2)); M1 is the mass of the sample taken for extraction in grams, i.e. 5 g (6.2.1); V1 is the total volume of the sample solution in millilitres, i.e. 50 ml (6.2.1); V2 is the volume of the sample solution loaded onto the SPE cartridge in millilitres, i.e. 10 ml (6.3.3); V3 is the final volume of the SPE extract in millilitres, i.e. 5 ml (6.3.7). 8 Precision 8.1 General Details of the methods used by the individual laboratories in the inter-laboratory test are listed in Table A.1. Details of the inter-laboratory test of the precision of the method are summarized in Annex C. The values derived from the inter-laboratory test may not be applicable to analyte concentration ranges and matrices other than those detailed in Annex C. 8.2 Repeatability and reproducibility The absolute difference between two single test results determined on identical test material by one operator using the same apparatus within the shortest feasible time interval will exceed the repeatability limit r in not more than 5 % of cases. The absolute difference between two single test results on identical test material reported by two laboratories will exceed the reproducibility limit R in not more than 5 % of the cases. The values r, R are summarized below (see Table 5 and Table 6) and further data from the validation study are given in Annex C and [1]. SIST EN 15911:2011
EN 15911:2010 (E) 13 Table 5 — Values for the nine sweeteners in beverages according to Tables C.1 to C.9
Sample 2 mg/l Sample 3 mg/l Sample 4 mg/l Sample 5 mg/l acesulfame-K x 38,3 266,6 324,1 383,5 r 7,4 16,9 29,7 25,7 R 11,6 43,8 56,2 54,0 alitame x 31,1 69,1 96,4 114,5 r 6,2 7,7 6,3 4,3 R 8,3 21,1 7,2 11,0 aspartame x 38,1 485,1 584,8 702 r 5,2 26,5 14,1 16,2 R 17,1 93,3 86,6 65,9 cyclamate x 28,3 248,9 256,8 307,2 r 3,5 18,4 10,2 16,5 R 16,3 43,1 39.2 43,4 dulcin x 55,0 79,6 95,7 115,1 r 3,8 8,2 2,8 4,3 R 9,4 10,9 14,7 14,7 neotame x 37,6 77,9 97,2 115,3 r 2,4 5,2 6,7 7,7 R 6,8 12,9 13,5 14,4 neohesperidine dihydrochalcone x 31,4 42,8 51,0 59,3 r 9,3 4,7 4,9 7,3 R 25,1 18,7 12,4 14,5 saccharin x 36,2 60,1 74,1 87,6 r 3,9 4,7 8,3 2,7 R 11,3 7,7 13,6 14,5 sucralose x 36,8 245,1 282,9 346,8 r 3,8 10,6 7,4 22,9 R 14,7 28,2 45,3 37,4
SIST EN 15911:2011
EN 15911:2010 (E) 14 Table 6 — Values for nine sweeteners in canned fruits according to Tables C.1 to C.9
Sample 7 mg/kg Sample 8 mg/kg Sample 9 mg/kg Sample 10 mg/kg acesulfame-K x 38,4 259,2 323 391,3 r 7,4 25,6 11,5 32
R 15,9 35,5 44,8 49,1 alitame x 36 113,7 142,5 175,2 r 9,7 6,9 8,8 18 R 9,7 10,6 12,3 21,1 aspartame x 37,2 739,8 951,9 1 120,2 r 10,1 46,3 12,5 37,8 R 10,1 82,0 77,1 88,8 cyclamate x 27,5 749,7 924,7 1 100,6 r 12,4
19,6 40,5 35,6 R 13,7 86,5
124,2 104,3 dulcin x 49,8 111 141,7 172,6 r 10,3 8,4 10,1 8,6 R 12,0 13,4 13,1 15,2 neotame x 37,3 116,2 140,6 173,7 r 3,6 10,1 6,2 13,5 R 6,2 17,6 21,1 21,7 neohesperidine dihydrochalcone x 35,3 40,5 49,8 59,3 r 6,1 2,8 5,6 6,5 R 12,2 13,0 9,2 15,3 saccharin x 44,3 151,9 193,4 235,3 r 6,8 11,3 12,0 18,8 R 23,6 29,6 37,7
42,0 sucralose x 35,3 306,1 380,2 462,4 r 6,3 20,6 23,8 27,1 R 10,8 24,4 29,1 27,1 9 Test report The test report shall contain at least the following data: all information necessary for the identification of the sample; a reference to this European Standard (i.e. EN 15911) or to the method used; the date and time of sampling procedure (if known); SIST EN 15911:2011
EN 15911:2010 (E) 15 the date of receipt; the date of test; the results and the units in which the results have been expressed; any particular points observed in the course of the test; any operations not specified in the method or regarded as optional which might have affected the results.
SIST EN 15911:2011
EN 15911:2010 (E) 16 Annex A (informative)
Table A.1
Suitable method conditions SPE characteristics - brand name Chromabond® Chromabond® Bakerbond spe® Chromabond® Chromabond® Chromabond® Chromabond® - stationary phase C18ec C18ec C18 C18ec C18ec C18ec C18ec - capacity [ml/mg] 6/1 000 6/1 000 3/500 6/1 000 6/1 000 6/1 000 6/1 000 HPLC column characteristics - brand name Purospher® Star Purospher® Star Purospher® Star Nucleodur® Purospher® Star Purospher® Star Purospher® Star - stationary phase RP-C18 endcapped RP-C18 endcapped RP-C18 endcapped C-18ec Pyramid RP-C18 endcapped RP-C18 endcapped RP-C18 endcapped - length [mm] 250 250 250 250 250 250 250 - i.d. [mm] 3 3 3 3 3 3 3 - particle size [µm] 5 5 5 5 5 5 5 HPLC mobile phase - mobile phase A composition [v/v/v] Methanol:Buffer solution:Acetone; 69:24:7 Methanol:Buffer solution:Acetone; 69:24:7 Methanol:Buffer solution:Acetone; 69:24:7 Methanol:Buffer solution:Acetone; 69:24:7 Methanol:Buffer solution:Acetone; 69:24:7 Methanol:Buffer solution:Acetone; 69:24:7 Methanol:Buffer solution:Acetone; 69:24:7 - mobile phase B composition [v/v/v] Methanol:Buffer solution:Acetone; 11:82:7 Methanol:Buffer solution:Acetone; 11:82:7 Methanol:Buffer solution:Acetone; 11:82:7 Methanol:Buffer solution:Acetone; 11:82:7 Methanol:Buffer solution:Acetone; 11:82:7 Methanol:Buffer solution:Acetone; 11:82:7 Methanol:Buffer solution:Acetone; 11:82:7 - flow rate [ml/min] 0,5 0,5 0,5 0,5 0,6 0,55 0,5 HPLC separation mode - gradient program [min - mobile phase A %] 0 min - 100 % A; 4 min - 100 % A; 11 min - 47 % A; 23 min - 2 % A;
24 min -2 % A;
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