Briefing Session g on Analysis of Sweeteners in Food - Government ...

<strong>Briefing</strong> g <strong>Session</strong>
on Analysis of Sweeteners in Food
Dr. Samuel M-F LO
Chemist
Food Safety B Section
Government Laboratory, HKSAR

Contents
Background
Testing Testing Methods –International International Standard
Methods
Testing T ti Methods M th d – GL Methods M th d
Findings d gs in 2007 00
Proficiency Tests

BBackground k d : Wh What t iis SSweetener? t ?
According g to Cap p 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
HO
OH OH
OH
Xylitol
OH
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 li list t of f ingredients, i di t hheaded d d or preceded d d bby an appropriate i t
heading consisting of or including any of the words
"ingredients", "composition", "contents" or words of similar
meaning 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
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 substance, including any additive
and any constituent of a compound ingredient, which is
used in the manufacture or preparation of a food and
which hi h is i still till present t iin th the fi finished i h d product, d t even if iin
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 就第(5)節而言,添加劑的作用類別為─
節而言 添加劑的作用類別為
-Sweetener (甜味劑)

Background: Testing Scope
O
O
O
S O
N
K +
Acesulfame-K, 安賽蜜,
乙酰磺胺酸鈉, INS: 950
Relative Sweetness: 200
H
N
O O O
O O
CH 3
NH 2
O
O
H
O
S
O
N
O
Saccharin, 糖精, INS:954
Relative Sweetness: 300
NH 2
H
N
O
Cl
H H
H 3
C
N
COOH O
H
CH 3
CH 3
H
S
CH 2 OH
OH
H
CH 3
CH 3
O
OH
H
H O
CH2Cl O
H
H
OH
OH
H
CH 2 Cl
Sucralose, 三氯半乳蔗糖, INS:955
Relative Sweetness: 600
O O
N
H
S
O
Aspartame, 甜味素, Alitame, 阿力甜, Cyclamate, 甜蜜素,
天冬氨酰苯丙氨酸甲酯, INS:951
Relative Sweetness: 180
天冬氨酰丙氨酰胺, INS:956
Relative Sweetness: 2000
環己基氨基磺酸, INS:952
Relative Sweetness: 30-50
In 2007 2007, GL has analyzed 22,010 010 samples for sweeteners

Background: Sample Types
Very e y wide de range a ge oof food ood sa samples p es
Diet soft drink
Candy Candy, chewing gum
Preserved prune, plum etc.
Fruits, e.g. orange, snow pear,
lemon, watermelon….
Sauce
Biscuit 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
AOAC 957.09 Cyclohexylsulfamate (Cyclamate) Salts in Nonalcoholic
Beverages
TLC
BaSO 4 precipitation test
(Qualitative)
AOAC 957.109 957 109 CCyclohexylsulfamate l h l lf t (C (Cyclamate) l t ) SSalts lt iin NNonalcoholic l h li Si Similar il to t AOAC 957.09 957 09
Beverages
but quantified from the
Gravimetric Method
weight of BaSO4 AOAC 969.28 969 28 Sodium Cyclamate and Calcium Cyclamate in Canned
Acid hydrolysis, hydrolysis
Fruit
Colourimetric Method
colorimetric method
NMKL 123 Cyclamate. Spectrophotometric determination in foods. Spectrometry
(1998) 2 nd Ed.
GB/T 5009.97-
2003
AOAC 941.10 Saccharin in Food
Qualitative Tests
食品中環己基氨基磺酸鈉的測定 GC, Colourmetric, TLC
Organolepric test, Test
for Salicylic acid,
Phenol-sulfuric acid Test
AOAC 973.29 973 29 Saccharin in Food
Gravimetric Method
BaSO BaSO4 precipitation

Testing Methods: International Standard Methods
Standards Methods TTechniques h i
AOAC 980.18 Saccharin in Food
Differential Pulse Polarographic Method
AOAC 947.10 Saccharin in Food
Sublimation Method
Differential Pulse
Polarographic
Method
Sublimation
AOAC 934.04 934 04 Saccharin in Nonalcoholic Beverages Colormetric Test
with NH3-free H2O and Nessler
reagent
NMKL 122 (199 (1997) ) S C
2nd Saccharin. Liquid chromatographic determination in HPLC
Ed.
beverages and sweets .
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
EN 12857:1999 Foodstuffs - Determination of cyclamate –
High performance liquid chromatographic method
EN 12856:1999 Foodstuffs- Determination of acesulfame-K, aspartame
and saccharin – High performance liquid chromatographic
method
HPLC-UV
Derivatization,
HPLC-UV
HPLC-UV/PDA

Testing Methods: Other Literature Methods
Determination of Sucralose in Foods
LGC Sucralose SOP
Summary
SPE HPLC-RI
Determination of seven artificial sweeteners in diet food preparations Extraction HPLC-UV.
by reverse-phase liquid chromatography with absorbance detection Applicable to aspartame,
Lawrence JF, Charbonneau CF, J Assoc Off Anal Chem. 1988 Sep- saccharin, cyclamate, alitame,
Oct;71(5):934-7
Oct;71(5):934-7. acesulfam-K acesulfam-K, sucralose and
dulcin.
Simultaneous Determination of Nine intense Sweeteners in foodstuffs SPE HPLC-ELSD
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)
Determination of Sucralose in Splenda and a Sugar-free beverage
using High-Performance Anion-Exchange Chromatography with
Pulsed Amperometric Detection. Detection
Valoran O. Hanko, Jeffrey S. Rohrer, J Agric Food Chem., 2004, 52,
4375-4379.
GC-MS GC MS Determination of Sucralose in Splenda
Derivatized by
Wenlong Qiu, Zhongfu Wang, Wanli Nie, Yuna Guo, Linjuan Huang,
Chromatographia 2007, 66, 935-939.
Applicable to aspartame,
saccharin, cyclamate, alitame,
acesulfam-K, sucralose, dulcin,
neotame and neohesperidine
dihydrochalcone.
High-performance Anionexchange
Chromatography –
Pulsed Amperometric Detection. Detection
hexamethyldisilazane (HMDS)
and trimethylchlorosilane (TMS-
Cl) GC-FID/ GC-MS

1. Sweeteners were extracted by buffer
(0.1% formic acid pH 4.5).
2. SPE clean-up 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
SSucralose l – HPLC method th d uses RI as ddetector. t t
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
ddone bby LC LC-MS MS /LC /LC-MS/MS.
MS/MS

Testing Methods: HPLC method for Acesulfame-K,
SSaccharin, h i AAspartame, t SSucralose l and d Alit Alitame
Extraction Procedures:
Homogenise the sample

Testing Methods: HPLC method for Acesulfame-K,
SSaccharin, h i AAspartame, t SSucralose l and d Alit Alitame
Extraction Procedures:
Homogenise the sample
Weigh 10 g samples in a 50-mL centrifuge tube

Testing Methods: HPLC method for Acesulfame-K,
SSaccharin, h i AAspartame, t SSucralose l and d Alit Alitame
Extraction Procedures:
Homogenise the sample
Weigh 10 g samples in a 50-mL centrifuge tube
Add about 30 mL 50% Methanol for extraction

Testing Methods: HPLC method for Acesulfame-K,
SSaccharin, h i AAspartame, t SSucralose l and d Alit Alitame
•Carrez Solution 1: 10.6 g potassium ferrocyanide (II) (K 4[Fe(CN) 6].3H 2O)
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.

Testing Methods: HPLC method for Acesulfame-K,
SSaccharin, h i AAspartame, t SSucralose l and d Alit Alitame
•Shake and sonicate for 15 minutes

Testing Methods: HPLC method for Acesulfame-K,
SSaccharin, h i AAspartame, t SSucralose l and d Alit Alitame
•Shake and sonicate for 15 minutes
•Centrifuge at 22,000 000 rpm for 10 minutes

Testing Methods: HPLC method for Acesulfame-K,
SSaccharin, h i AAspartame, t SSucralose l and d Alit Alitame
•Shake and sonicate for 15 minutes
•Centrifuge at 22,000 000 rpm for 10 minutes
•Filter and repeat the extraction twice and make up to final volume 100
mL

Testing Methods: HPLC method for Acesulfame-K,
SSaccharin, h i AAspartame, t SSucralose l and d Alit Alitame
•Shake and sonicate for 15 minutes
•Centrifuge at 22,000 000 rpm for 10 minutes
•Filter and repeat the extraction twice and make up to final volume 100
mL
•Filter Filt the th extraction t ti solution l ti through th h 00.45 45 μm membrance b filt filter bbefore f
HPLC analysis.

Testing Methods: HPLC method for Acesulfame-K,
SSaccharin, h i AAspartame, t SSucralose l and d Alit Alitame
•Shake and sonicate for 15 minutes
•Centrifuge at 22,000 000 rpm for 10 minutes
•Filter and repeat the extraction twice and make up to final volume 100
mL
•Filter Filt the th extraction t ti solution l ti through th h 00.45 45 μm membrance b filt filter bbefore f
HPLC analysis.
Aspartame Aspartame, Acesulfame-K
Sucralose
Saccharin, Alitame
HPLC-PDA/FLU
HPLC PDA/FLU HPLC-RI HPLC RI

Testing Methods: HPLC condition for Acesulfame-K, Saccharin,
Aspartame and Alitame
Cl Column : CC-18, 18 5 μm, 250 mm iin llength th and d 4.6mm 4 6 i.d. i d
Mobile phase Sol A : 0.02 M KH2PO4 (pH 4.0) in water
Sol B CH 3 3CN
Gradient program : A:B (9:1) to A:B (8:2) in 10 minutes
Fl Flow rate : 11 1.1 mL/min. L/ i
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
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.
Dt Detector t : Rf Refractive ti Index Id detector dt t
Sensitivity of
detector
: 256
Injection
volume
: 50 µL

Testing Methods: Chromatogram – FAPAS softdrink sample
Assigned Value: 175 ppm

Testing Methods: TLC Screening of Cyclamate
EExtraction t ti Procedures:
P d
Weigh ~50 g samples in 500 mL beaker and add about 300-400mL
hot H2O and mix until the mixture becomes uniform

Testing Methods: TLC Screening of Cyclamate
EExtraction t ti Procedures:
P d
Weigh ~50 g samples in 500 mL beaker and add about 300-400mL
hot H2O and mix until the mixture becomes uniform
Cl Clarified ifi d bby adding ddi 10 mL L CCarrez solution l ti 1 and d 2 if necessary

Testing Methods: TLC Screening of Cyclamate
EExtraction t ti Procedures:
P d
Weigh ~50 g samples in 500 mL beaker and add about 300-400mL
hot H2O and mix until the mixture becomes uniform
Cl Clarified ifi d bby adding ddi 10 mL L CCarrez solution l ti 1 and d 2 if necessary
Filter and transfer the aqueous extract to a separating funnel

Testing Methods: TLC Screening of Cyclamate
Add 30 mL L HCl ( (v/v / 11:1) 1)
Wash the aqueous layer with 50 ml petroleum ether (b.p 40-60oC).

Testing Methods: TLC Screening of Cyclamate
Add 30 mL L HCl ( (v/v / 11:1) 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 Lethyl th l acetate
t t

Testing Methods: TLC Screening of Cyclamate
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 00.5mL 5mL NH NH4OH-H OH-H2O-EtOH O-EtOH (5+5+10) and then
spot on TLC plate.

Testing Methods: TLC Screening of Cyclamate
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 00.5mL 5mL NH NH4OH-H OH-H2O-EtOH O-EtOH (5+5+10) and then
spot on TLC plate.
Mobile Phase: n-Butanol/95% Ethanol/28% NH 4OH/Water OH/Water
(40:4:1:9).
Spray Reagents:
11. 5% Bromine in DCM (v/v)
2. 0.25% fluorescein in DMF-ethanol (1+1)

Testing Methods: TLC plate
Softdrink
100 ppm
Ice-cream
100 ppm
Ice-cream
200 ppm
Ice-cream
400 ppm
Potato Chip
100 ppm
Potato Chip
200 ppm
Potato 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.
N SO 3 H
H
N Cl
Wash the n-heptane with sodium
carbonate solution and filter the nheptane
layer through anhydrous
sodium sulphate before injection injection.
HPLC-UV/PDA determination.
Mobile Phase: 80% Methanol
Quantitation Wavelength: 314 nm
Cl

Testing g Methods: Confirmation of Cyclamate y
10 ppm standard
100 ppm blank spike
(Chocolate)

Alternative Confirmation Technique:
LC LC-MS MS or LC LC-MS/MS MS/MS confirmation
Column: : Agilent Eclipase XDB-C18 5μm 4.6
x 100mm
MS/MS system : Finnigan LCQ Advantage Max with
Ion Max source or equivalent
Sh Shealth lth gas fl flow : 50 arb b
Auxillary/ sweep gas flow : 40 arb
Capillary Temperature : 350o Capillary Temperature : 350 C
Time
(min)
Flow
(mL/min)
A:
1% Acetic Acid
B:
ACN
0.00 0.5 95% 5%
6.00 0.5 95% 5%
18 18.00 00 05 0.5 80% 20%
21.00 0.5 80% 20%
22.00 0.5 95% 5%
30.00 0.5 95% 5%

Acesulfame-K Saccharin Cyclamate Sucralose Aspartame Alitame
Ionization Mode Negative Negative Negative Negative Positive Positive
I spray voltage
(kV)
Capillary
Voltage (V)
4.5 5.0 4.5
-13 -20 6
Tube lens offset -23 -60 55
(V)
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
D ht I NA NA NA NA [M H H O] + [M H H O] +
Daughter Ions
(m/z)
NA NA NA NA [M+H-H2O] +
277 (100%),
[M-H-OAc] +
235 (70%),
[M+H-aspartyl] +
[M+H-H2O] +
314 (40%),
[M+H-SC7H13 ] +
204 (50%),
[ p y] [M+H- [
180 (20%) NHSC7H13 ] +
187 (100%)
Normalized
CCollision
Energy
NA NA NA NA 30% 30%

Testing Methods: LC-MS and LC-MS/MS Chromatograms of
mixed sweeteners standard
RT: 0.00 - 30.01
100
50
0
100
50
0
100
50
0
100
50
0
100
50
0
100
50
0
RT: 5.67
MA: 2870161
0.13 2.48 4.38 7.45 9.90
RT: 9.49
MA: 3383414
11.81 13.99 15.64 17.20 17.51
2.49 2.60 3.07 5.56 6.54 9.20 9.97 11.52 13.13 14.65 16.64 17.11
RT: 11.37
MA: 4978085
2.46
2.00 2.57 8.95
12.16
17.20
12.86
6.24 7.02 9.02
15.71
17.55
Sucralose (0.6 ppm)
AAspartame t (0.3 (0 3 ppm) )
Alitame (0 (0.6 6 ppm)
Acesulfame-K (1 ppm)
Saccharin (1 ppm)
Cyclamate (1 ppm)
RT: 21.14
MA: 4625829
17.89 20.60 21.47
R T: 2 1 .8 0
MA: 5074805
22.48 25.38 26.79 29.33
R T: 2 4 .3 2
MA: 7597789
23.91
22.14 25.12 26.41 28.99
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Tim e (m in)
NL: 3.24E5
m/z= 161.50 161 50-162 162.50 50 F: - c
ESI Full m s [
150.00-500.00] MS
AS080206W2_08022911
0034
NL: 2.51E5
m/z= 181.50-182.50 F: - c
ESI Full ms [
150.00-500.00] MS
AS080206W2_08022911
0034
NL: 3.27E5
m/z= 177.50-178.50 F: - c
ESI Full m s [
150.00-500.00] MS
AS080206W2_08022911
0034
NL: 5.52E5
T IC F: - p ESI SIM m s [
453.00-459.00] MS
A S080206W2_ 08022911
0034
NL: 6.32E5
T IC F: + c ESI Full m s2
295.00@35.00 [
100.00-350.00] MS
AS080206W2 AS080206W2_ 08022911
0034
NL: 9.37E5
T IC F: + c ESI Full m s2
332.00@30.00 [
100.00-350.00] MS
AS080206W2_08022911
0034

Testing g Methods: MS spectrums p
Sucralose
Aspartame Alitame

Reporting Limits
Aces Acesulfame-K, lfame K Saccharin and 20 mg/kg
Alitame
Aspartame p 40 mg/kg g g
Sucralose 50 mg/kg
Cyclamate (as cyclamic acid) 100 mg/kg

Standards validation
Id Identification tifi ti and d 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 Concentration range found Types of samples
Positive 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 g
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 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 S h i
0383 26/02/2009 Tonic Water -Acesulfame-K
-Aspartame
-Benzoic Acid
-Quinine Quinine
http://www.fapas.com

Proficiency Test
LGC Standards Proficiency Testing Group
QBS
QUALITY IN BEVERAGES SCHEME 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 h year and d test t t materials t i l are distributed
di t ib t d
quarterly .
•Next Dispatch Date will be on 07 Apr 2008.
http://www.lgcpt.com/

Thank you