Briefing Session
on Analysis of Sweeteners in Food
Dr. Samuel M-F LO
Chemist
Food Safety B Section
Government Laboratory, HKSAR
Contents
Background
Testing Methods –International Standard
Methods
Testing Methods – GL Methods
Findings in 2007
Proficiency Tests
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
OH
OH
OH
HO
OH
Xylitol
Sweetener
Cap 132W
FOOD AND DRUGS (COMPOSITION AND LABELLING)
REGULATIONS
Schedule 3
2. 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。
Cap 132W
FOOD AND DRUGS (COMPOSITION AND LABELLING)
REGULATIONS
 "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)
Background: Testing Scope
O
O
O
K
N
+
S
O
O
Acesulfame-K, 安賽蜜,
乙酰磺胺酸鈉, INS: 950
Relative Sweetness: 200
H
NH2
N
O
O
Cl
S O
N
O
CH2OH
O
O H
O
H
O
H
H3C
H
H
OH
CH3
O
CH2Cl
OH
H
CH3
S
N
COOH O
OH
H
OH
H
O
Sucralose, 三氯半乳蔗糖, INS:955
Relative Sweetness: 600
N
O
Aspartame, 甜味素,
天冬氨酰苯丙氨酸甲酯, INS:951
Relative Sweetness: 180
H
Saccharin, 糖精, INS:954
Relative Sweetness: 300
NH2
CH3
H
CH2Cl
H
O
CH3
CH3
N
H
O
S
O
H
Alitame, 阿力甜,
天冬氨酰丙氨酰胺, INS:956
Relative Sweetness: 2000
Cyclamate, 甜蜜素,
環己基氨基磺酸, INS:952
Relative Sweetness: 30-50
In 2007, GL has analyzed 2,010 samples for sweeteners
Background: Sample Types
Very wide range of food samples
 Diet soft drink
 Candy, chewing gum
 Preserved prune, plum etc.
 Fruits, e.g. orange, snow pear,
lemon, watermelon….
 Sauce
 Biscuit, cake
 Etc……
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 5009.972003
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 Methods
Standards
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
Testing Methods: Other Literature Methods
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 SepOct;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 Anionexchange 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 (TMSCl)  GC-FID/ GC-MS
1. Sweeteners were extracted by buffer
(0.1% formic acid pH 4.5).
2. SPE clean-up
3. Reconstitute the residues by buffer solution
4. HPLC-ELSD analysis
http://www.irmm.jrc.be/html/activities/food_additives/EUR22727EN.pdf
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
•Shake
andProcedures:
sonicate for 15 minutes
Extraction
•Carrez Solution
1: 10.6 g potassium ferrocyanide (II) (K4[Fe(CN)6].3H2O)
•Centrifuge at 2,000 rpm for 10 minutes
inHomogenise
distilled waterthe sample
•Filter and repeat the extraction twice and make up to final volume 100
•Carrez Solution 2: 22.0 g zinc acetate and 3 mL glacial acetic acid in

mLWeigh 10 g samples in a 50-mL centrifuge tube
distilled
water30and
to 100 mL.for extraction

Addthe
about
mLdilute
50% Methanol
•Filter
extraction
solution
through 0.45 mm membrance filter before
HPLC
analysis.
 Clarified
by Carrez solution 1 and 2 if necessary.
Aspartame, Acesulfame-K
Saccharin, Alitame
Sucralose
HPLC-PDA/FLU
HPLC-RI
Testing Methods: HPLC condition for Acesulfame-K, Saccharin,
Aspartame and Alitame
Column
:
C-18, 5 mm, 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 aspartame
gain 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
Mobile phase
: C-18, 5-7 µm, 250 mm in length and
4.6 mm i.d.
: 30% methanol : 70% water
Flow rate
: 1.0 mL/min.
Detector
: Refractive Index detector
Sensitivity of
detector
Injection
volume
: 256
Column
: 50 µL
Testing Methods: Chromatogram – FAPAS softdrink sample
Assigned Value: 175 ppm
Testing Methods: TLC Screening of Cyclamate
Extraction
Add 30 mL
Procedures:
HCl (v/v 1:1)
oC).
 Wash
Weigh
the
~50
aqueous
g samples
layerinwith
50050
mLmlbeaker
petroleum
and add
etherabout
(b.p 40-60
300-400mL
hot ~5ml
H2O and
untiland
the then
mixture
becomes
uniformlayer with 50
 Add
50%mix
NaOH
extract
the aqueous
 mL
Clarified
ethyl acetate
by adding 10 mL Carrez solution 1 and 2 if necessary
 Filter
Filterthe
and
ethyl
transfer
acetate
the layer
aqueous
through
extract
anhydrous
to a separating
sodium funnel
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% NH4OH/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
Softdrink
100 ppm
Ice-cream Ice-cream
100 ppm 200 ppm
Ice-cream Potato Chip Potato Chip Potato Chip
400 ppm 100 ppm 200 ppm 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.
N
H
SO3H
N
Cl
Cl
 Wash the n-heptane with sodium
carbonate solution and filter the nheptane layer through anhydrous
sodium sulphate before injection.
 HPLC-UV/PDA determination.
 Mobile Phase: 80% Methanol
 Quantitation Wavelength: 314 nm
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 5mm 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%
Ionization Mode
Acesulfame-K
Saccharin
Cyclamate
Sucralose
Aspartame
Alitame
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+HNHSC7H13]+
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 Codex
JECFA specification
http://www.fao.org/ag/agn/jecfa-additives/search.html?lang=en
Findings in 2007
 Total samples: 2,010
 No. 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 Test
FAPAS
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
QBS
QUALITY IN BEVERAGES SCHEME
•Next Dispatch Date will be on 06 May 2008
QFCS
QUALITY IN FOOD CHEMISTRY SCHEME
•2 distributions per annum
•Next Dispatch Date will be on 20 May 2008
SODAS
SOFT 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.
http://www.lgcpt.com/
Thank you