SIST-TS CEN/TS 15606:2009

Foodstuffs - Determination of acesulfame-K, aspartame, neohesperidine-dihydrochalcone and saccharin - High performance liquid chromatographic method

This Technical Specification (prCEN/TS 15606:2009) specifies a high performance liquid chromatographic (HPLC) method for the determination of acesulfame-K, aspartame, neohesperidine-dihydrochalcone and saccharin in foodstuffs. The method has been fully validated [1] for the dialysis procedure through collaborative trial, according to the IUPAC Harmonised Protocol [2], on the following analyte matrix combinations:
   acesulfame-K and aspartame in water-based drink, fruit-based drink, cheesecake with biscuit base, canned soup and instant chocolate drink;
   saccharin in water-based drink, fruit-based drink, cheesecake with biscuit base and canned soup;
   neohesperidine-dihydrochalcone in water-based drink, fruit-based drink and canned soup.

Lebensmittel - Bestimmung von Acesulfam-K, Aspartam, Neohesperidin-Dihydrochalcon und Saccharin - Hochleistungs-flüssigchromatographisches Verfahren

Diese Technische Spezifikation (prCEN/TS 15606:2009) legt ein hochleistungs-flüssigchromatographisches (en: high performance liquid chromatographic, (HPLC)) Verfahren zur Bestimmung von Acesulfam-K, Aspartam, Neohesperidin-Dihydrochalcon und Saccharin in Lebensmitteln fest. Das Verfahren ist für das Dialyseverfahren vollständig in einem Ringversuch nach dem IUPAC Harmonized Protocol [2] für die folgenden Analyt/Matrix-Kombinationen validiert worden [1]:
- Acesulfam-K und Aspartam in Getränken auf Wasserbasis, Fruchtsaftgetränken, Käsekuchen mit Biskuit-boden, Dosensuppen und Schokoladengetränkepulver;
- Saccharin in Getränken auf Wasserbasis, Fruchtsaftgetränken, Käsekuchen mit Biskuitboden und Dosen-suppen;
- Neohesperidin-Dihydrochalcon in Getränken auf Wasserbasis, Fruchtsaftgetränken und Dosensuppen.

Produits alimentaires - Dosage de l'acésulfame-K, de l'aspartame, de la saccharine et de la néohespéridine dihydrochalcone - Méthode par chromatographie liquide haute performance

La présente Spécification Technique (prCEN/TS 15606:2009) spécifie une méthode par chromatographie liquide haute performance (CLHP) pour le dosage de l'acésulfame-K, de l'aspartame, de la néohespéridine dihydrochalcone et de la saccharine dans les produits alimentaires. La méthode a été entièrement validée [1] pour la technique de dialyse au moyen d'un essai interlaboratoire réalisé, conformément au Protocole Harmonisé [2] de l'UICPA, sur les combinaisons analyte-matrice suivantes :
   acésulfame-K et aspartame dans une boisson à base d'eau, une boisson à base de fruits, un gâteau au fromage blanc à base de biscuit, un potage en boîte et une boisson chocolatée instantanée ;
   saccharine dans une boisson à base d'eau, une boisson à base de fruits, un gâteau au fromage blanc à base de biscuit et un potage en boîte ;
   néohespéridine dihydrochalcone dans une boisson à base d'eau, une boisson à base de fruits et un potage en boîte.

Živila - Določevanje K-acesulfama, aspartama, neohesperidin-dihidrokalcona in saharina - Metoda tekočinske kromatografije visoke ločljivosti

General Information

• Status: Published
• Publication Date: 15-Nov-2009
• ICS: 67.050 - General methods of tests and analysis for food products
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 03-Nov-2009
• Due Date: 08-Jan-2010
• Completion Date: 16-Nov-2009
• Ref Project: CEN/TS 15606:2009 - Foodstuffs - Determination of acesulfame-K, aspartame, neohesperidine-dihydrochalcone and saccharin - High performance liquid chromatographic method

Standards Content (Sample)

SLOVENSKI STANDARD
SIST-TS CEN/TS 15606:2009
01-december-2009
äLYLOD'RORþHYDQMH.DFHVXOIDPDDVSDUWDPDQHRKHVSHULGLQGLKLGURNDOFRQDLQ
VDKDULQD0HWRGDWHNRþLQVNHNURPDWRJUDILMHYLVRNHORþOMLYRVWL
Foodstuffs - Determination of acesulfame-K, aspartame, neohesperidine-
dihydrochalcone and saccharin - High performance liquid chromatographic method
Lebensmittel - Bestimmung von Acesulfam-K, Aspartam, Neohesperidin-Dihydrochalcon
und Saccharin - Hochleistungs-flüssigchromatographisches Verfahren
Produits alimentaires - Dosage de l'acésulfame-K, de l'aspartame, de la saccharine et de
la néohespéridine dihydrochalcone - Méthode par chromatographie liquide haute
performance
Ta slovenski standard je istoveten z: CEN/TS 15606:2009
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
SIST-TS CEN/TS 15606:2009 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST-TS CEN/TS 15606:2009

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SIST-TS CEN/TS 15606:2009


TECHNICAL SPECIFICATION
CEN/TS 15606

SPÉCIFICATION TECHNIQUE

TECHNISCHE SPEZIFIKATION
September 2009
ICS 67.050
English Version
Foodstuffs - Determination of acesulfame-K, aspartame,
neohesperidine-dihydrochalcone and saccharin - High
performance liquid chromatographic method
Produits alimentaires - Dosage de l'acésulfame-K, de Lebensmittel - Bestimmung von Acesulfam-K, Aspartam,
l'aspartame, de la saccharine et de la néohespéridine Neohesperidin-Dihydrochalcon und Saccharin -
dihydrochalcone - Méthode par chromatographie liquide Hochleistungsflüssigchromatographisches Verfahren
haute performance
This Technical Specification (CEN/TS) was approved by CEN on 20 June 2009 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit their
comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available
promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS)
until the final decision about the possible conversion of the CEN/TS into an EN is reached.

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 STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2009 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 15606:2009: E
worldwide for CEN national Members.

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Contents Page
Foreword .3
1 Scope .4
2 Normative references .4
3 Principle .4
4 Reagents .4
5 Apparatus and equipment .7
6 Procedure .8
7 Calculations . 11
8 Precision . 12
9 Test report . 14
Annex A (normative) Example for chromatographic conditions which have been proven to lead to
satisfactory results . 15
Annex B (informative) Figures . 16
Annex C (informative) Precision data . 18
Bibliography . 21

2

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Foreword
This document (CEN/TS 15606:2009) has been prepared by Technical Committee CEN/TC 275 “Food
analysis - Horizontal methods”, the secretariat of which is held by DIN.
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 announce this Technical Specification: 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.
3

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1 Scope
This Technical Specification (CEN/TS 15606:2009) specifies a high performance liquid chromatographic
(HPLC) method with UV-detection for the determination of acesulfame-K, aspartame, neohesperidine-
dihydrochalcone and saccharin in foodstuffs. The method has been fully validated [1] for the dialysis
procedure through collaborative trial (see 8.2, 8.3 and Annex C), according to the IUPAC Harmonised
Protocol [2], on the following analyte matrix combinations:
 acesulfame-K (from 86 mg/l to 331 mg/l) and aspartame (from 97 mg/kg to 610 mg/l)in water-based drink,
fruit-based drink, cheesecake with biscuit base, canned soup and instant chocolate drink
 saccharin (from 70 mg/l to 97 mg/kg) in water-based drink, fruit-based drink, cheesecake with biscuit
base and canned soup;
 neohesperidine-dihydrochalcone (from 27 mg/l to 57 mg/kg)in water-based drink, fruit-based drink and
canned soup.
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, Water for analytical laboratory use — Specification and test methods (ISO 3696:1987)
3 Principle
The sample is extracted or diluted with water. If necessary, the sample solution with the intense sweeteners is
purified with Carrez reagents. Alternatively, solid samples are slurried in NaCl/HCl solution and the
sweeteners extracted by dialysis using cellulose acetate (molecular weight cut-off of approximately 12 000).
The purified extract is analysed by ion-pair reversed-phase HPLC with UV detection.
4 Reagents
4.1 General
During the analysis, unless otherwise stated, use only reagents of recognised analytical grade for HPLC
analysis and water suitable for HPLC or of at least grade 1 as defined in EN ISO 3696. When preparing
solutions, the purity of the substances shall be taken into account.
4.2 Methanol, HPLC grade.
1
4.3 Hydrochloric acid, w(HCl) ≈ 36 % .
2
4.4 Phosphoric acid, c(H PO ) = 5 mol/l .
3 4

1
w is the mass fraction.
2
c is the substance concentration.
4

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4.5 Sodium hydroxide, c(NaOH) = 5 ol l.
m /
4.6 Saccharin, w(C H NO S): 98,0 % to 101,0 %. or w(C H NNaO S x 2H O): 99,0 % to 101,0 %.
7 5 3 7 4 3 2
4.7 Neohesperidine-dihydrochalcone, w(C H O ) ≥ 99,0 %.
28 34 15
4.8 Acesulfame-K, w(C H KNO S) ≥ 99,0 %.
4 4 4
4.9 Aspartame, w(C H N O ) ≥ 98 %.
14 18 2 5
4.10 Sodium chloride
4.11 Tetrapropylammonium hydrogensulphate (TPrA-HSO )
4
4.12 HPLC mobile phases
4.12.1 Mobile phase 1 (5 % methanol in water)
Pipette (5.4) 50 ml of methanol (4.2) into a 1 l volumetric flask (5.2). Make up to the mark with water. Add
0,24 g ± 0,01 g of TPrA-HSO (4.11) and shake. Adjust to pH 3,5 with drop-wise addition of either 5 mol/l
4
sodium hydroxide (4.5) or 5 mol/l phosphoric acid (4.4).
4.12.2 Mobile phase 2 (75 % methanol in water)
Transfer 750 ml methanol (4.2) from a measuring cylinder into a 1 l volumetric flask (5.2) and make up to the
mark with water. Add 0,24 g ± 0,01 g TPrA-HSO (4.11) and shake. Adjust to pH 3,5 with drop-wise addition of
4
either a 5 mol/l sodium hydroxide (4.5) or 5 mol/l phosphoric acid (4.4).
4.12.3 Isocratic elution mobile phase (15 % methanol in water)
Transfer 150 ml of methanol (4.2) from a measuring cylinder into a 1 l volumetric flask (5.2) and make up to
the mark with water. Add 0,24 g ± 0,01 g of TPrA-HSO (4.11) and shake. Adjust to pH 3,5 with drop-wise
4
addition of either 5 mol/l of sodium hydroxide (4.5) or 5 mol/l of phosphoric acid (4.4).
Filter (5.5) the solutions and sonicate (5.9) for 30 min.
4.13 Dialysing solution, 1,71 mol/l NaCl and 0,01 mol/l HCl, optional.
Dissolve 100 g of NaCl (4.10) and 1 ml of concentrated HCl (4.3) to 1 l in a volumetric flask (5.2) with water.
4.14 Dialysate solution, 0,01 mol/l HCl, optional.
Dissolve 1 ml of concentrated HCl (4.3) to 1 l in a volumetric flask (5.2) with water.
4.15 Stock solutions of acesulfame-K, aspartame, saccharin and neohesperidine-
dihydrochalcone
Prepare 1 000 mg/l solutions of acesulfame-K (4.8) and aspartame (4.9) in water. Prepare a solution of
saccharin (4.6) equivalent to 1 000 mg/l free imide (e.g. 1 126 mg/l of anhydrous sodium salt) in water and a
1 000 mg/l solution of neohesperidine-dihydrochalcone (4.7) in a mixture of 50 % methanol (4.2) and 50 %
water.
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4.16 Mixed calibration solutions containing acesulfame-K, aspartame, saccharin and
neohesperidine-dihydrochalcone
From the stock solutions (4.15) prepare a series of mixed standards containing the sweeteners at
concentrations equivalent to a maximum level of 150 % of the appropriate limit (for example, those given in
Commission Directives 94/35/EC and 96/83/EC, as amended) whilst taking the dilution steps within the
procedure into account.
EXAMPLE Calibration standards for measurement of sweeteners in a dairy-based dessert.
The present EU limits for the four sweeteners in dairy-based desserts are:
− acesulfame-K 350 mg/kg;
− saccharin 100 mg/kg (free imide);
− aspartame 1 000 mg/kg;
− neohesperidine dihydrochalcone 50 mg/kg.
150 % of the limits are:
− acesulfame K 525 mg/kg;
− saccharin 150 mg/kg (free imide);
− aspartame 1 500 mg/kg;
− neohesperidine-dihydrochalcone 75 mg/kg.
Sweeteners from 20 g of sample are extracted in 200 ml of dialysate (10:1 dilution). Thus, calibration should
be carried out for extracts containing up to the following concentrations of sweetener:
− acesulfame-K 50 mg/l;
− saccharin 15 mg/l (free imide);
− aspartame 150 mg/l;
− neohesperidine-dihydrochalcone 7 mg/l.
For a seven point calibration, take the volumes as given in Table 1 from the 1 000 mg/l stock solutions (4.15)
and make up to 50 ml with water in volumetric flasks (5.2).
Table 1 — Preparation of the calibration graph
Acesulfame-K Saccharin Aspartame Neohesperidine-

dihydrochalcone
ml ml
µl
µl
1 0,25 75 0,75 50
2 0,50 150 1,50 100
3 0,75 225 2,25 150
4 1,00 300 3,00 200
5 1,50 450 4,50 250
6 2,00 600 6,00 300
7 2,50 750 7,50 350
Table 2 details the concentration of sweeteners in each calibration standard following dilution:
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Table 2 — Concentration of sweeteners in each calibration solution
Acesulfame-K Saccharin Aspartame Neohesperidine-
(free imide) dihydrochalcone
mg/l mg/l
mg/l mg/l
1 5 1,5 15 1
2 10 3 30 2
3 15 4,5 45 3
4 20 6 60 4
5 30 9 90 5
6 40 12 120 6
7 50 15 150 7
4.17 Blank and fortified samples for determination of recovery
If the sample preparation is carried out by dialysis, a homogenised sample of the same type as the test
sample should be prepared. Divide the sample into two portions. Add a known quantity of sweeteners to one
portion (gravimetrically as solid or volumetrically in solution as appropriate).
4.18 Carrez solution No 1
Dissolve 15 g potassium hexacyanoferrate (II) (K [Fe(CN) ] · 3 H O) in water and dilute to 100 ml.
4 6 2
4.19 Carrez solution No 2
Dissolve 30 g zinc sulfate (ZnSO · 7 H O) in water and dilute to 100 ml.
4 2
5 Apparatus and equipment
5.1 General
Usual laboratory equipment and glassware. The following items are also required.
5.2 Volumetric flasks, e.g. 50 ml and smaller.
5.3 HPLC vials, clear, 2 ml.
5.4 Pipette, 50 ml and smaller.
5.5 Mobile phase glass filter equipment
5.6 Screw cap bottles, 250 ml.
5.7 Glass funnels
5.8 Homogeniser, Ultra Turrax ® or equivalent.
5.9 Ultrasonic bath
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5.10 Magnetic stirrer, multi-place preferred (or roller mixer, or some other means of mixing the dialysate).
5.11 Mini-magnets
5.12 Membrane syringe filters, 13 mm, 0,45 µm (PVDF or equivalent).
5.13 Mobile phase filters, diameter 4,8 cm, pore size 0,45 µm.
5.14 Dialysis tubing, cellulose acetate, planar width 4,8 cm; molecular weight cut-off approximately
12 000, optional.
5.15 pH meter
5.16 High performance liquid chromatograph, equipped with an analytical column (5.17), a pump
system (gradient capability), an autosampler, an ultraviolet (UV) detector (capable of operating at a
wavelength of 220 nm and 280 nm, preferably a diode array detector) and equipped with a recorder and/or
integrator which allows the measurement of peak heights and peak areas.
5.17 HPLC-reversed phase column, e.g. ODS3 10 µm; 250 mm x 3,2 mm or equivalent (10 µm
particle size 250 mm x 3,2 mm).
NOTE Column dimensions other than those specified in this Technical Specification can be used (e.g. of diameter
3,0 mm to 4,6 mm, length 100 mm to 250 mm). Separation parameters (flow, injection volume) should be adapted to
guarantee equivalent results.
5.18 HPLC System suitability
Sweeteners acesulfame K, saccharin, aspartame and neohesperidine-dihydrochalcone should be resolved (R
s
= 1,3) from each other and from other components of the matrix. Two commonly encountered 'critical pairs'
are (a) acesulfame-K and theobromine; and (b) aspartame and caffeine.
6 Procedure
6.1 Sample preparation without dialysis
6.1.1 Clear liquid products such as lemonades, cola or beverages
Dilute 20 ml of the sample in a 100 ml volumetric flask with water. Filter the solution through a membrane filter
of pore size 0,45 µm before injection.
6.1.2 Cloudy liquid products such as juices or flavoured milk drinks
Dilute 20 ml of the homogenised sample in a 100 ml volumetric flask with 50 ml water, add 2 ml of Carrez
solution No 1 (4.18), mix and add 2 ml of Carrez solution No 2 (4.19). Shake vigorously and allow the solution
to stand at room temperature for 10 min. Dilute to the mark with water. Filter through a fluted filter paper
discarding the first 10 ml of the filtrate. Pass the filtrate through a membrane filter of pore size 0,45 µm before
injecting the sample test solution into the HPLC system.
To make allowance for the volume of any precipitate, if the fat free insoluble matter in the sample volume
(here 20 ml) exceeds approximately 3 g, it is advisable to centrifuge the clarified sample mixture for 10 min at
at least 1 400 g before filtering it quantitatively into the 100 ml volumetric flask. Wash the settled matter twice
with water and centrifuge again, collect each of the supernatants in the 100 ml volumetric flask and then dilute
the solution to the mark with water. This procedure may also be followed when the amount of insoluble matter
is less than 3 g.
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6.1.3 Jams, preserves, marmalades and related products except fruit curds
Weigh, to the nearest 1 mg, about 20 g of homogenised sample into a 100 ml volumetric flask. Add about
60 ml of water and place the flask in an ultrasonic bath at 40 °C for 20 min. The temperature shall not exceed
40 °C since aspartame can be degraded.
Cool the solution to room temperature. Add 2 ml of Carrez solution No 1 (4.18), mix and then add 2 ml of
Carrez solution No 2 (4.19). Shake vigorously and allow the solution to stand at room temperature for 10 min.
Dilute to the mark with water. Filter the solution through a fluted filter paper discarding the first 10 ml of the
filtrate. Pass the filtrate through a membrane filter of pore size 0,45 µm before injecting the sample test
solution into the HPLC system.
To make allowance for the volume of any precipitate, if the fat free insoluble matter in the initial sample mass
exceeds approximately 3 g, it is advisable to centrifuge the clarified sample mixture for 10 min at a centrifugal
acceleration of at least 1 400 g before filtering it quantitatively into the 100 ml volumetric flask. Wash the
settled matter twice with water and centrifuge again, collect each of the supernatants in the 100 ml volumetric
flask and then dilute the solution to the mark with water. This procedure may also be followed when the
amount of insoluble matter is l
...