Briefing Session on Analysis of Sweeteners in Food

Dr. Samuel M-F LO

Chemist

Food Safety B Section

Government Laboratory, HKSAR

BackgroundTesting Methods –International Standard

MethodsTesting Methods – GL MethodsFindings in 2007Proficiency Tests

Contents

Background : What is Sweetener?

According to Cap 132U Sweeteners in Food Regulations 食物內甜味劑規例

“Sweetener” means any chemical compound which is sweet to the taste, but does not include any sugars or other carbohydrates or polyhydric alcohols.

“甜味劑” 指任何帶甜味的化合物，但不包括糖或其他碳水化合物或多羥醇

OHOH

OH OH

OH

Sweetener

Xylitol

Cap 132W FOOD AND DRUGS (COMPOSITION AND LABELLING) REGULATIONS

Schedule 32. List of ingredients

(1) Prepackaged food shall be legibly marked or labelled with a list of ingredients, headed or preceded by an appropriate heading consisting of or including any of the words "ingredients", "composition", "contents" or words of similar meaning.

2. 配料表 (1) 預先包裝食物須加上可閱的標記或標籤，用以表列食物的配料，該表並須冠以適當標題，而標題中須載有或包括任何“配料”、“成分組合”、“內含物質”的字樣或具類似意思的文字。

"ingredient" means any substance, including any additive and any constituent of a compound ingredient, which is used in the manufacture or preparation of a food and which is still present in the finished product, even if in altered form

“配料”指用於製造或配製食物並繼續存在於製成品中的任何物質 (即使形態已有所更改 )，包括任何添加劑或合成配料的任何成分

(6) The functional classes of additive for the purpose of

sub-paragraph (5) are (6) 就第 (5)節而言，添加劑的作用類別為─ -Sweetener (甜味劑 )

Cap 132W FOOD AND DRUGS (COMPOSITION AND LABELLING) REGULATIONS

Background: Testing Scope

NH

SO

OO

SN

OO

O

H

O

CH2OH

H

Cl

H

H

OH

OH

H O

H OCH2Cl

OH

H

H

OH

CH2Cl

H

N

O O

CH3

O

NH2

O

OH

H

S

CH3CH3

NN

O

CH3O

NH2

CH3

CH3

COOH H

H

Acesulfame-K, 安賽蜜 , 乙酰磺胺酸鈉 , INS: 950 Relative Sweetness: 200

Saccharin, 糖精 , INS:954Relative Sweetness: 300

Sucralose, 三氯半乳蔗糖 , INS:955Relative Sweetness: 600

Aspartame, 甜味素 , 天冬氨酰苯丙氨酸甲酯 , INS:951Relative Sweetness: 180

Alitame, 阿力甜 ,天冬氨酰丙氨酰胺 , INS:956Relative Sweetness: 2000

Cyclamate, 甜蜜素 ,環己基氨基磺酸 , INS:952Relative Sweetness: 30-50

In 2007, GL has analyzed 2,010 samples for sweeteners

N

SO

O

O

O

K+

Background: Sample Types

Very wide range of food samplesDiet soft drinkCandy, chewing gumPreserved prune, plum etc. Fruits, e.g. orange, snow pear,

lemon, watermelon….

SauceBiscuit, cakeEtc……

Testing Method: International Standard Methods

Standards Methods Techniques

AOAC 969.27 Non-nutritive Sweeteners in Nonalcoholic Beverages

Qualitative Thin-layer Chromatographic Method

TLC

AOAC 957.09 Cyclohexylsulfamate (Cyclamate) Salts in Nonalcoholic Beverages

BaSO4 precipitation test (Qualitative)

AOAC 957.109 Cyclohexylsulfamate (Cyclamate) Salts in Nonalcoholic Beverages

Gravimetric Method

Similar to AOAC 957.09 but quantified from the weight of BaSO4

AOAC 969.28 Sodium Cyclamate and Calcium Cyclamate in Canned Fruit

Colourimetric Method

Acid hydrolysis, colorimetric method

NMKL 123 (1998) 2nd Ed.

Cyclamate. Spectrophotometric determination in foods. Spectrometry

GB/T 5009.97-2003

食品中環己基氨基磺酸鈉的測定 GC, Colourmetric, TLC

AOAC 941.10 Saccharin in Food

Qualitative Tests

Organoleptic test, Test for Salicylic acid, Phenol-sulfuric acid Test

AOAC 973.29 Saccharin in Food

Gravimetric Method

BaSO4 precipitation

Testing Methods: International Standard MethodsStandards Methods Techniques

AOAC 980.18 Saccharin in Food

Differential Pulse Polarographic Method

Differential Pulse Polarographic Method

AOAC 947.10 Saccharin in Food

Sublimation Method

Sublimation

AOAC 934.04 Saccharin in Nonalcoholic Beverages Colormetric Test with NH3-free H2O and Nessler reagent

NMKL 122 (1997) 2nd Ed.

Saccharin. Liquid chromatographic determination in beverages and sweets .

HPLC

GB/T 5009.28-2003 食品中糖精鈉的測定 HPLC-UV, TLC, ion selective electrode

EN 1379 : 1997 Foodstuffs - Determination of cyclamate and saccharin in liquid table top sweetener preparations –

Method by high performance liquid chromatography

HPLC-UV

EN 12857:1999 Foodstuffs - Determination of cyclamate –

High performance liquid chromatographic method

Derivatization, HPLC-UV

EN 12856:1999 Foodstuffs- Determination of acesulfame-K, aspartame and saccharin – High performance liquid chromatographic method

HPLC-UV/PDA

Summary

Determination of Sucralose in Foods

LGC Sucralose SOP

SPE HPLC-RI

Determination of seven artificial sweeteners in diet food preparations by reverse-phase liquid chromatography with absorbance detection

Lawrence JF, Charbonneau CF, J Assoc Off Anal Chem. 1988 Sep-Oct;71(5):934-7.

Extraction HPLC-UV.

Applicable to aspartame, saccharin, cyclamate, alitame, acesulfam-K, sucralose and dulcin.

Simultaneous Determination of Nine intense Sweeteners in foodstuffs by High Performance Liquid Chromatography and Evaporative Light Scattering Detection – Development and Single-laboratory Validation

Andrzek Wasik, Josephine McCourt, Manuela Buchgraber, Journal of Chromatography A 2007, 1157, 187-196.

( IRMM Validated Method)

SPE HPLC-ELSD

Applicable to aspartame, saccharin, cyclamate, alitame, acesulfam-K, sucralose, dulcin, neotame and neohesperidine dihydrochalcone.

Determination of Sucralose in Splenda and a Sugar-free beverage using High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection.

Valoran O. Hanko, Jeffrey S. Rohrer, J Agric Food Chem., 2004, 52, 4375-4379.

High-performance Anion-exchange Chromatography – Pulsed Amperometric Detection.

GC-MS Determination of Sucralose in Splenda

Wenlong Qiu, Zhongfu Wang, Wanli Nie, Yuna Guo, Linjuan Huang, Chromatographia 2007, 66, 935-939.

Derivatized by hexamethyldisilazane (HMDS) and trimethylchlorosilane (TMS-Cl) GC-FID/ GC-MS

Testing Methods: Other Literature Methods

http://www.irmm.jrc.be/html/activities/food_additives/EUR22727EN.pdf

1. Sweeteners were extracted by buffer (0.1% formic acid pH 4.5).2. SPE clean-up3. Reconstitute the residues by buffer solution4. HPLC-ELSD analysis

Testing Methods: GL Methods

Acesulfame-K, Saccharin, Aspartame, Alitame – HPLC method similar to EN 12856:1999 method

Sucralose – HPLC method uses RI as detector.Cyclamate – TLC screening based on AOAC 969.27

Cyclamate – Quantified by HPLC method similar to EN12857:1999

Structural confirmation for Acesulfame-K, Saccharin, Cyclamate, Aspartame, Sucralose and Alitame are done by LC-MS /LC-MS/MS.

Testing Methods: HPLC method for Acesulfame-K, Saccharin, Aspartame, Sucralose and Alitame

Aspartame, Acesulfame-KSaccharin, Alitame

HPLC-PDA/FLU

Sucralose

HPLC-RI

Extraction Procedures: Homogenise the sample Weigh 10 g samples in a 50-mL centrifuge tube Add about 30 mL 50% Methanol for extraction Clarified by Carrez solution 1 and 2 if necessary.

•Carrez Solution 1: 10.6 g potassium ferrocyanide (II) (K4[Fe(CN)6].3H2O) in distilled water •Carrez Solution 2: 22.0 g zinc acetate and 3 mL glacial acetic acid in distilled water and dilute to 100 mL.

•Shake and sonicate for 15 minutes•Centrifuge at 2,000 rpm for 10 minutes•Filter and repeat the extraction twice and make up to final volume 100 mL•Filter the extraction solution through 0.45 m membrance filter before HPLC analysis.

Testing Methods: HPLC condition for Acesulfame-K, Saccharin, Aspartame and Alitame

Column : C-18, 5 m, 250 mm in length and 4.6mm i.d.

Mobile phase Sol A : 0.02 M KH2PO4 (pH 4.0) in water

Sol B CH3CN

Gradient program : A:B (9:1) to A:B (8:2) in 10 minutes

Flow rate : 1.1 mL/min.

PDA wavelength : 210 nm – 500 nm

Quantitation Wavelength of PDA

Saccharin, Acesulfame-K : 225 nm

Alitame, Aspartame : 210 nm

Fluorescence Wavelength : excitation 205 nm and emission 284 nm for aspartamegain 100, atten 1 and bandwidth 18 nm

Injection volume : 20 µL

Testing Methods: Chromatogram – 50 ppm blank spiked sample (Soft drink)

225 nm

210 nm

FLU

Testing Methods: HPLC condition for Sucralose

Column : C-18, 5-7 µm, 250 mm in length and 4.6 mm i.d.

Mobile phase : 30% methanol : 70% water

Flow rate : 1.0 mL/min.

Detector : Refractive Index detector

Sensitivity of detector

: 256

Injection volume

: 50 µL

Testing Methods: Chromatogram – FAPAS softdrink sample

Assigned Value: 175 ppm

Testing Methods: TLC Screening of CyclamateExtraction Procedures: Weigh ~50 g samples in 500 mL beaker and add about 300-

400mL hot H2O and mix until the mixture becomes uniform Clarified by adding 10 mL Carrez solution 1 and 2 if necessary Filter and transfer the aqueous extract to a separating funnel

Add 30 mL HCl (v/v 1:1) Wash the aqueous layer with 50 ml petroleum ether (b.p 40-60oC). Add ~5ml 50% NaOH and then extract the aqueous layer with 50

mL ethyl acetate Filter the ethyl acetate layer through anhydrous sodium sulphate

and evaporate the solvent to dryness. Dissolve the residue in 0.5mL NH4OH-H2O-EtOH (5+5+10) and then

spot on TLC plate.

Mobile Phase: n-Butanol/95% Ethanol/28% NH44OH/Water (40:4:1:9).Spray Reagents:

1. 5% Bromine in DCM (v/v)2. 0.25% fluorescein in DMF-ethanol (1+1)

Testing Methods: TLC plate

Softdrink100 ppm

Ice-cream100 ppm

Ice-cream200 ppm

Ice-cream400 ppm

Potato Chip

100 ppmPotato Chip

200 ppmPotato Chip

400 ppm

Testing Methods: Confirmation of Cyclamate

Method is based on EN 12857:1999

Cyclamate is extracted by water. Add sulfuric acid, n-heptane and sodium

hypochlorite to derivate cyclamate for HPLC detection.

Wash the n-heptane with sodium carbonate solution and filter the n-heptane layer through anhydrous sodium sulphate before injection.

HPLC-UV/PDA determination. Mobile Phase: 80% Methanol Quantitation Wavelength: 314 nm

NSO3H

H

NCl

Cl

Testing Methods: Confirmation of Cyclamate

10 ppm standard

100 ppm blank spike(Chocolate)

Alternative Confirmation Technique: LC-MS or LC-MS/MS confirmation

Column: : Agilent Eclipase XDB-C18 5m 4.6 x 100mm

MS/MS system : Finnigan LCQ Advantage Max with Ion Max source or equivalent

Shealth gas flow : 40 arb

Auxillary/ sweep gas flow : 30 arb

Capillary Temperature : 350oC

Time (min)

Flow (mL/min)

A: 1% Acetic Acid

B: ACN

0.00 0.4 95% 5%

10.00 0.4 100% 0%

14.00 0.4 70% 30%

21.00 0.4 70% 30%

Acesulfame-K Saccharin Cyclamate Sucralose Aspartame Alitame

Ionization Mode Negative Negative Negative Negative Positive Positive

I spray voltage (kV)

4.5 5.0 4.5

Capillary Voltage (V)

-13 -20 6

Tube lens offset (V)

-23 -60 55

Parent Ions (m/z)

[M-H]-

162[M-H]-

182[M-H]-

178[M+OAc]- [M+H]+ [M+H]+

455 (100), 456 (25), 457 (100)

295 332

Daughter Ions (m/z)

NA NA NA NA [M+H-H2O]+

277 (100%), [M-H-OAc]+ 235 (70%),

[M+H-aspartyl]+ 180 (20%)

[M+H-H2O]+

314 (40%), [M+H-SC7H13]

+

204 (50%),[M+H-

NHSC7H13]+

187 (100%)

Normalized Collision Energy

NA NA NA NA 30% 30%

Testing Methods: LC-MS and LC-MS/MS Chromatograms of mixed sweeteners standard

Acesulfame-K (1 ppm)

Saccharin (1 ppm)

Cyclamate (1 ppm)

Sucralose (1 ppm)

Aspartame (0.3 ppm)

Alitame (0.3 ppm)

Testing Methods: MS spectrums

Sucralose

Aspartame Alitame

Reporting Limits

Acesulfame-K, Saccharin and Alitame

20 mg/kg

Aspartame 40 mg/kg

Sucralose 50 mg/kg

Cyclamate (as cyclamic acid) 100 mg/kg

Standards validation

Identification and assay determination

Reference Procedures can be obtained from

Food Chemcials CodexJECFA specification

http://www.fao.org/ag/agn/jecfa-additives/search.html?lang=en

Findings in 2007Total samples: 2,010No. of positive samples: 82

Analytes No. of

Positive samples

Concentration range found Types of samples

Acesulfame-K 38 36 ppm to 1,900 ppm Diet soft drink, candy, 話梅

Saccharin 26 20 ppm to 2,700 ppm Diet soft drink, soy sauce, vinegar,話梅

Aspartame 34 46 ppm to 1,800 ppm Diet soft drink, candy, 話梅

Sucralose 9 50 ppm to 580 ppm Diet soft drink, candy, Yoghurt

Cyclamate 24 110 ppm to 9,000 ppm Diet soft drink, 甜蕎頭

Alitame 0 Nil Nil

Proficiency TestFAPAS

Proficiency Test Start Date Matrix Analytes

0374 22/02/2008 Tonic Water -Acesulfame-K-Aspartame-Benzoic Acid-Quinine

0376 15/04/2008 Cola Drink -Acesulfame-K-Benzoic Acid-Caffeine-Saccharin

0379 15/08/2008 Soft Drink -Sucralose

0381 23/10/2008 Soft Drink -Brix-pH-Citric Acid-Sorbic Acid-Cyclamate-Saccharin

0383 26/02/2009 Tonic Water -Acesulfame-K-Aspartame-Benzoic Acid-Quinine

http://www.fapas.com

Proficiency Test

LGC Standards Proficiency Testing Group

SODASSOFT DRINKS PROFICIENCY TESTING SCHEME•Scheme operates from October to September each year and test materials are distributed quarterly .•Next Dispatch Date will be on 07 Apr 2008.

QFCSQUALITY IN FOOD CHEMISTRY SCHEME•2 distributions per annum •Next Dispatch Date will be on 20 May 2008

http://www.lgcpt.com/

QBS QUALITY IN BEVERAGES SCHEME•Next Dispatch Date will be on 06 May 2008

Thank you