Briefing Session g on Analysis of Sweeteners in Food - Government ...
Briefing Session g on Analysis of Sweeteners in Food - Government ...
Briefing Session g on Analysis of Sweeteners in Food - Government ...
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<str<strong>on</strong>g>Brief<strong>in</strong>g</str<strong>on</strong>g> g <str<strong>on</strong>g>Sessi<strong>on</strong></str<strong>on</strong>g><br />
<strong>on</strong> <strong>Analysis</strong> <strong>of</strong> <strong>Sweeteners</strong> <strong>in</strong> <strong>Food</strong><br />
Dr. Samuel M-F LO<br />
Chemist<br />
<strong>Food</strong> Safety B Secti<strong>on</strong><br />
<strong>Government</strong> Laboratory, HKSAR
C<strong>on</strong>tents<br />
Background<br />
Test<strong>in</strong>g Test<strong>in</strong>g Methods –Internati<strong>on</strong>al Internati<strong>on</strong>al Standard<br />
Methods<br />
Test<strong>in</strong>g T ti Methods M th d – GL Methods M th d<br />
F<strong>in</strong>d<strong>in</strong>gs d gs <strong>in</strong> 2007 00<br />
Pr<strong>of</strong>iciency Tests
BBackground k d : Wh What t iis SSweetener? t ?<br />
Accord<strong>in</strong>g g to Cap p 132U <strong>Sweeteners</strong> <strong>in</strong> <strong>Food</strong><br />
Regulati<strong>on</strong>s 食物內甜味劑規例<br />
“Sweetener” means any chemical compound which is<br />
sweet to the taste, but does not <strong>in</strong>clude any sugars or<br />
other carbohydrates or polyhydric alcohols.<br />
“甜味劑” 指任何帶甜味的化合物,但不包括糖或其他碳<br />
水化合物或多羥醇<br />
HO<br />
OH OH<br />
OH<br />
Xylitol<br />
OH<br />
Sweetener
Cap 132W<br />
FOOD AND DRUGS (COMPOSITION AND LABELLING)<br />
REGULATIONS<br />
Schedule 3<br />
2. List <strong>of</strong> <strong>in</strong>gredients<br />
(1) Prepackaged food shall be legibly marked or labelled with<br />
a li list t <strong>of</strong> f <strong>in</strong>gredients, i di t hheaded d d or preceded d d bby an appropriate i t<br />
head<strong>in</strong>g c<strong>on</strong>sist<strong>in</strong>g <strong>of</strong> or <strong>in</strong>clud<strong>in</strong>g any <strong>of</strong> the words<br />
"<strong>in</strong>gredients", "compositi<strong>on</strong>", "c<strong>on</strong>tents" or words <strong>of</strong> similar<br />
mean<strong>in</strong>g mean<strong>in</strong>g.<br />
2. 配料表<br />
(1) 預先包裝食物須加上可閱的標記或標籤,用以表<br />
列食物的配料,該表並須冠以適當標題,而標題中<br />
須載有或包括任何“配料"、“成分組合"、“內<br />
含物質"的字樣或具類似意思的文字<br />
含物質 的字樣或具類似意思的文字。
Cap 132W<br />
FOOD AND DRUGS (COMPOSITION AND LABELLING)<br />
REGULATIONS<br />
"<strong>in</strong>gredient" means any substance substance, <strong>in</strong>clud<strong>in</strong>g any additive<br />
and any c<strong>on</strong>stituent <strong>of</strong> a compound <strong>in</strong>gredient, which is<br />
used <strong>in</strong> the manufacture or preparati<strong>on</strong> <strong>of</strong> a food and<br />
which hi h is i still till present t i<strong>in</strong> th the fi f<strong>in</strong>ished i h d product, d t even if i<strong>in</strong><br />
altered form<br />
“配料"指用於製造或配製食物並繼續存在於製成品中的<br />
任何物質(即使形態已有所更改),包括任何添加劑或合成<br />
配料的任何成分<br />
(6) The functi<strong>on</strong>al classes <strong>of</strong> additive for the purpose <strong>of</strong><br />
sub-paragraph (5) are<br />
(6) 就第 就第(5)節而言,添加劑的作用類別為─<br />
節而言 添加劑的作用類別為<br />
-Sweetener (甜味劑)
Background: Test<strong>in</strong>g Scope<br />
O<br />
O<br />
O<br />
S O<br />
N<br />
K +<br />
Acesulfame-K, 安賽蜜,<br />
乙酰磺胺酸鈉, INS: 950<br />
Relative Sweetness: 200<br />
H<br />
N<br />
O O O<br />
O O<br />
CH 3<br />
NH 2<br />
O<br />
O<br />
H<br />
O<br />
S<br />
O<br />
N<br />
O<br />
Sacchar<strong>in</strong>, 糖精, INS:954<br />
Relative Sweetness: 300<br />
NH 2<br />
H<br />
N<br />
O<br />
Cl<br />
H H<br />
H 3<br />
C<br />
N<br />
COOH O<br />
H<br />
CH 3<br />
CH 3<br />
H<br />
S<br />
CH 2 OH<br />
OH<br />
H<br />
CH 3<br />
CH 3<br />
O<br />
OH<br />
H<br />
H O<br />
CH2Cl O<br />
H<br />
H<br />
OH<br />
OH<br />
H<br />
CH 2 Cl<br />
Sucralose, 三氯半乳蔗糖, INS:955<br />
Relative Sweetness: 600<br />
O O<br />
N<br />
H<br />
S<br />
O<br />
Aspartame, 甜味素, Alitame, 阿力甜, Cyclamate, 甜蜜素,<br />
天冬氨酰苯丙氨酸甲酯, INS:951<br />
Relative Sweetness: 180<br />
天冬氨酰丙氨酰胺, INS:956<br />
Relative Sweetness: 2000<br />
環己基氨基磺酸, INS:952<br />
Relative Sweetness: 30-50<br />
In 2007 2007, GL has analyzed 22,010 010 samples for sweeteners
Background: Sample Types<br />
Very e y wide de range a ge o<strong>of</strong> food ood sa samples p es<br />
Diet s<strong>of</strong>t dr<strong>in</strong>k<br />
Candy Candy, chew<strong>in</strong>g gum<br />
Preserved prune, plum etc.<br />
Fruits, e.g. orange, snow pear,<br />
lem<strong>on</strong>, watermel<strong>on</strong>….<br />
Sauce<br />
Biscuit Biscuit, cake<br />
Etc……
Test<strong>in</strong>g Method: Internati<strong>on</strong>al Standard Methods<br />
Standards Methods Techniques<br />
AOAC 969.27 N<strong>on</strong>-nutritive <strong>Sweeteners</strong> <strong>in</strong> N<strong>on</strong>alcoholic Beverages<br />
Qualitative Th<strong>in</strong>-layer Chromatographic Method<br />
AOAC 957.09 Cyclohexylsulfamate (Cyclamate) Salts <strong>in</strong> N<strong>on</strong>alcoholic<br />
Beverages<br />
TLC<br />
BaSO 4 precipitati<strong>on</strong> test<br />
(Qualitative)<br />
AOAC 957.109 957 109 CCyclohexylsulfamate l h l lf t (C (Cyclamate) l t ) SSalts lt i<strong>in</strong> NN<strong>on</strong>alcoholic l h li Si Similar il to t AOAC 957.09 957 09<br />
Beverages<br />
but quantified from the<br />
Gravimetric Method<br />
weight <strong>of</strong> BaSO4 AOAC 969.28 969 28 Sodium Cyclamate and Calcium Cyclamate <strong>in</strong> Canned<br />
Acid hydrolysis, hydrolysis<br />
Fruit<br />
Colourimetric Method<br />
colorimetric method<br />
NMKL 123 Cyclamate. Spectrophotometric determ<strong>in</strong>ati<strong>on</strong> <strong>in</strong> foods. Spectrometry<br />
(1998) 2 nd Ed.<br />
GB/T 5009.97-<br />
2003<br />
AOAC 941.10 Sacchar<strong>in</strong> <strong>in</strong> <strong>Food</strong><br />
Qualitative Tests<br />
食品中環己基氨基磺酸鈉的測定 GC, Colourmetric, TLC<br />
Organolepric test, Test<br />
for Salicylic acid,<br />
Phenol-sulfuric acid Test<br />
AOAC 973.29 973 29 Sacchar<strong>in</strong> <strong>in</strong> <strong>Food</strong><br />
Gravimetric Method<br />
BaSO BaSO4 precipitati<strong>on</strong>
Test<strong>in</strong>g Methods: Internati<strong>on</strong>al Standard Methods<br />
Standards Methods TTechniques h i<br />
AOAC 980.18 Sacchar<strong>in</strong> <strong>in</strong> <strong>Food</strong><br />
Differential Pulse Polarographic Method<br />
AOAC 947.10 Sacchar<strong>in</strong> <strong>in</strong> <strong>Food</strong><br />
Sublimati<strong>on</strong> Method<br />
Differential Pulse<br />
Polarographic<br />
Method<br />
Sublimati<strong>on</strong><br />
AOAC 934.04 934 04 Sacchar<strong>in</strong> <strong>in</strong> N<strong>on</strong>alcoholic Beverages Colormetric Test<br />
with NH3-free H2O and Nessler<br />
reagent<br />
NMKL 122 (199 (1997) ) S C<br />
2nd Sacchar<strong>in</strong>. Liquid chromatographic determ<strong>in</strong>ati<strong>on</strong> <strong>in</strong> HPLC<br />
Ed.<br />
beverages and sweets .<br />
GB/T 5009.28-2003 食品中糖精鈉的測定 HPLC-UV, TLC,<br />
i<strong>on</strong> selective<br />
electrode<br />
EN 1379 : 1997 <strong>Food</strong>stuffs - Determ<strong>in</strong>ati<strong>on</strong> <strong>of</strong> cyclamate and sacchar<strong>in</strong> <strong>in</strong><br />
liquid table top sweetener preparati<strong>on</strong>s –<br />
Method by high performance liquid chromatography<br />
EN 12857:1999 <strong>Food</strong>stuffs - Determ<strong>in</strong>ati<strong>on</strong> <strong>of</strong> cyclamate –<br />
High performance liquid chromatographic method<br />
EN 12856:1999 <strong>Food</strong>stuffs- Determ<strong>in</strong>ati<strong>on</strong> <strong>of</strong> acesulfame-K, aspartame<br />
and sacchar<strong>in</strong> – High performance liquid chromatographic<br />
method<br />
HPLC-UV<br />
Derivatizati<strong>on</strong>,<br />
HPLC-UV<br />
HPLC-UV/PDA
Test<strong>in</strong>g Methods: Other Literature Methods<br />
Determ<strong>in</strong>ati<strong>on</strong> <strong>of</strong> Sucralose <strong>in</strong> <strong>Food</strong>s<br />
LGC Sucralose SOP<br />
Summary<br />
SPE HPLC-RI<br />
Determ<strong>in</strong>ati<strong>on</strong> <strong>of</strong> seven artificial sweeteners <strong>in</strong> diet food preparati<strong>on</strong>s Extracti<strong>on</strong> HPLC-UV.<br />
by reverse-phase liquid chromatography with absorbance detecti<strong>on</strong> Applicable to aspartame,<br />
Lawrence JF, Charb<strong>on</strong>neau CF, J Assoc Off Anal Chem. 1988 Sep- sacchar<strong>in</strong>, cyclamate, alitame,<br />
Oct;71(5):934-7<br />
Oct;71(5):934-7. acesulfam-K acesulfam-K, sucralose and<br />
dulc<strong>in</strong>.<br />
Simultaneous Determ<strong>in</strong>ati<strong>on</strong> <strong>of</strong> N<strong>in</strong>e <strong>in</strong>tense <strong>Sweeteners</strong> <strong>in</strong> foodstuffs SPE HPLC-ELSD<br />
by High Performance Liquid Chromatography and Evaporative Light<br />
Scatter<strong>in</strong>g Detecti<strong>on</strong> – Development and S<strong>in</strong>gle-laboratory Validati<strong>on</strong><br />
Andrzek Wasik, Joseph<strong>in</strong>e McCourt, Manuela Buchgraber, Journal <strong>of</strong><br />
Chromatography A 2007, 1157, 187-196.<br />
( IRMM Validated Method)<br />
Determ<strong>in</strong>ati<strong>on</strong> <strong>of</strong> Sucralose <strong>in</strong> Splenda and a Sugar-free beverage<br />
us<strong>in</strong>g High-Performance Ani<strong>on</strong>-Exchange Chromatography with<br />
Pulsed Amperometric Detecti<strong>on</strong>. Detecti<strong>on</strong><br />
Valoran O. Hanko, Jeffrey S. Rohrer, J Agric <strong>Food</strong> Chem., 2004, 52,<br />
4375-4379.<br />
GC-MS GC MS Determ<strong>in</strong>ati<strong>on</strong> <strong>of</strong> Sucralose <strong>in</strong> Splenda<br />
Derivatized by<br />
Wenl<strong>on</strong>g Qiu, Zh<strong>on</strong>gfu Wang, Wanli Nie, Yuna Guo, L<strong>in</strong>juan Huang,<br />
Chromatographia 2007, 66, 935-939.<br />
Applicable to aspartame,<br />
sacchar<strong>in</strong>, cyclamate, alitame,<br />
acesulfam-K, sucralose, dulc<strong>in</strong>,<br />
neotame and neohesperid<strong>in</strong>e<br />
dihydrochalc<strong>on</strong>e.<br />
High-performance Ani<strong>on</strong>exchange<br />
Chromatography –<br />
Pulsed Amperometric Detecti<strong>on</strong>. Detecti<strong>on</strong><br />
hexamethyldisilazane (HMDS)<br />
and trimethylchlorosilane (TMS-<br />
Cl) GC-FID/ GC-MS
1. <strong>Sweeteners</strong> were extracted by buffer<br />
(0.1% formic acid pH 4.5).<br />
2. SPE clean-up clean up<br />
3. Rec<strong>on</strong>stitute the residues by buffer soluti<strong>on</strong><br />
4. HPLC-ELSD analysis<br />
http://www.irmm.jrc.be/html/activities/food_additives/EUR22727EN.pdf
Test<strong>in</strong>g Methods: GL Methods<br />
Acesulfame-K, Sacchar<strong>in</strong>, Aspartame, Alitame –<br />
HPLC method similar to EN 12856:1999 method<br />
SSucralose l – HPLC method th d uses RI as ddetector. t t<br />
Cyclamate – TLC screen<strong>in</strong>g based <strong>on</strong> AOAC 969.27<br />
Cyclamate – Quantified by HPLC method similar to<br />
EN12857:1999<br />
Structural c<strong>on</strong>firmati<strong>on</strong> for Acesulfame-K, Sacchar<strong>in</strong>,<br />
Cyclamate, Aspartame, Sucralose and Alitame are<br />
dd<strong>on</strong>e bby LC LC-MS MS /LC /LC-MS/MS.<br />
MS/MS
Test<strong>in</strong>g Methods: HPLC method for Acesulfame-K,<br />
SSacchar<strong>in</strong>, h i AAspartame, t SSucralose l and d Alit Alitame<br />
Extracti<strong>on</strong> Procedures:<br />
Homogenise the sample
Test<strong>in</strong>g Methods: HPLC method for Acesulfame-K,<br />
SSacchar<strong>in</strong>, h i AAspartame, t SSucralose l and d Alit Alitame<br />
Extracti<strong>on</strong> Procedures:<br />
Homogenise the sample<br />
Weigh 10 g samples <strong>in</strong> a 50-mL centrifuge tube
Test<strong>in</strong>g Methods: HPLC method for Acesulfame-K,<br />
SSacchar<strong>in</strong>, h i AAspartame, t SSucralose l and d Alit Alitame<br />
Extracti<strong>on</strong> Procedures:<br />
Homogenise the sample<br />
Weigh 10 g samples <strong>in</strong> a 50-mL centrifuge tube<br />
Add about 30 mL 50% Methanol for extracti<strong>on</strong>
Test<strong>in</strong>g Methods: HPLC method for Acesulfame-K,<br />
SSacchar<strong>in</strong>, h i AAspartame, t SSucralose l and d Alit Alitame<br />
•Carrez Soluti<strong>on</strong> 1: 10.6 g potassium ferrocyanide (II) (K 4[Fe(CN) 6].3H 2O)<br />
<strong>in</strong> distilled water<br />
•Carrez Soluti<strong>on</strong> 2: 22.0 g z<strong>in</strong>c acetate and 3 mL glacial acetic acid <strong>in</strong><br />
distilled water and dilute to 100 mL.
Test<strong>in</strong>g Methods: HPLC method for Acesulfame-K,<br />
SSacchar<strong>in</strong>, h i AAspartame, t SSucralose l and d Alit Alitame<br />
•Shake and s<strong>on</strong>icate for 15 m<strong>in</strong>utes
Test<strong>in</strong>g Methods: HPLC method for Acesulfame-K,<br />
SSacchar<strong>in</strong>, h i AAspartame, t SSucralose l and d Alit Alitame<br />
•Shake and s<strong>on</strong>icate for 15 m<strong>in</strong>utes<br />
•Centrifuge at 22,000 000 rpm for 10 m<strong>in</strong>utes
Test<strong>in</strong>g Methods: HPLC method for Acesulfame-K,<br />
SSacchar<strong>in</strong>, h i AAspartame, t SSucralose l and d Alit Alitame<br />
•Shake and s<strong>on</strong>icate for 15 m<strong>in</strong>utes<br />
•Centrifuge at 22,000 000 rpm for 10 m<strong>in</strong>utes<br />
•Filter and repeat the extracti<strong>on</strong> twice and make up to f<strong>in</strong>al volume 100<br />
mL
Test<strong>in</strong>g Methods: HPLC method for Acesulfame-K,<br />
SSacchar<strong>in</strong>, h i AAspartame, t SSucralose l and d Alit Alitame<br />
•Shake and s<strong>on</strong>icate for 15 m<strong>in</strong>utes<br />
•Centrifuge at 22,000 000 rpm for 10 m<strong>in</strong>utes<br />
•Filter and repeat the extracti<strong>on</strong> twice and make up to f<strong>in</strong>al volume 100<br />
mL<br />
•Filter Filt the th extracti<strong>on</strong> t ti soluti<strong>on</strong> l ti through th h 00.45 45 μm membrance b filt filter bbefore f<br />
HPLC analysis.
Test<strong>in</strong>g Methods: HPLC method for Acesulfame-K,<br />
SSacchar<strong>in</strong>, h i AAspartame, t SSucralose l and d Alit Alitame<br />
•Shake and s<strong>on</strong>icate for 15 m<strong>in</strong>utes<br />
•Centrifuge at 22,000 000 rpm for 10 m<strong>in</strong>utes<br />
•Filter and repeat the extracti<strong>on</strong> twice and make up to f<strong>in</strong>al volume 100<br />
mL<br />
•Filter Filt the th extracti<strong>on</strong> t ti soluti<strong>on</strong> l ti through th h 00.45 45 μm membrance b filt filter bbefore f<br />
HPLC analysis.<br />
Aspartame Aspartame, Acesulfame-K<br />
Sucralose<br />
Sacchar<strong>in</strong>, Alitame<br />
HPLC-PDA/FLU<br />
HPLC PDA/FLU HPLC-RI HPLC RI
Test<strong>in</strong>g Methods: HPLC c<strong>on</strong>diti<strong>on</strong> for Acesulfame-K, Sacchar<strong>in</strong>,<br />
Aspartame and Alitame<br />
Cl Column : CC-18, 18 5 μm, 250 mm i<strong>in</strong> llength th and d 4.6mm 4 6 i.d. i d<br />
Mobile phase Sol A : 0.02 M KH2PO4 (pH 4.0) <strong>in</strong> water<br />
Sol B CH 3 3CN<br />
Gradient program : A:B (9:1) to A:B (8:2) <strong>in</strong> 10 m<strong>in</strong>utes<br />
Fl Flow rate : 11 1.1 mL/m<strong>in</strong>. L/ i<br />
PDA wavelength : 210 nm – 500 nm<br />
Quantitati<strong>on</strong> Wavelength <strong>of</strong> PDA<br />
Sacchar<strong>in</strong>, Acesulfame-K : 225 nm<br />
Alitame, Aspartame : 210 nm<br />
Fluorescence Wavelength : excitati<strong>on</strong> 205 nm and emissi<strong>on</strong> 284 nm for aspartame<br />
ga<strong>in</strong> 100, atten 1 and bandwidth 18 nm<br />
Injecti<strong>on</strong> volume : 20 µL
Test<strong>in</strong>g Methods: Chromatogram – 50 ppm blank spiked sample<br />
(S<strong>of</strong>t dr<strong>in</strong>k)<br />
225 nm<br />
210 nm<br />
FLU
Test<strong>in</strong>g Methods: HPLC c<strong>on</strong>diti<strong>on</strong> for Sucralose<br />
Column : C-18, 5-7 µm, 250 mm <strong>in</strong> length and<br />
4.6 mm i.d.<br />
Mobile phase : 30% methanol : 70% water<br />
Flow rate : 1.0 mL/m<strong>in</strong>.<br />
Dt Detector t : Rf Refractive ti Index Id detector dt t<br />
Sensitivity <strong>of</strong><br />
detector<br />
: 256<br />
Injecti<strong>on</strong><br />
volume<br />
: 50 µL
Test<strong>in</strong>g Methods: Chromatogram – FAPAS s<strong>of</strong>tdr<strong>in</strong>k sample<br />
Assigned Value: 175 ppm
Test<strong>in</strong>g Methods: TLC Screen<strong>in</strong>g <strong>of</strong> Cyclamate<br />
EExtracti<strong>on</strong> t ti Procedures:<br />
P d<br />
Weigh ~50 g samples <strong>in</strong> 500 mL beaker and add about 300-400mL<br />
hot H2O and mix until the mixture becomes uniform
Test<strong>in</strong>g Methods: TLC Screen<strong>in</strong>g <strong>of</strong> Cyclamate<br />
EExtracti<strong>on</strong> t ti Procedures:<br />
P d<br />
Weigh ~50 g samples <strong>in</strong> 500 mL beaker and add about 300-400mL<br />
hot H2O and mix until the mixture becomes uniform<br />
Cl Clarified ifi d bby add<strong>in</strong>g ddi 10 mL L CCarrez soluti<strong>on</strong> l ti 1 and d 2 if necessary
Test<strong>in</strong>g Methods: TLC Screen<strong>in</strong>g <strong>of</strong> Cyclamate<br />
EExtracti<strong>on</strong> t ti Procedures:<br />
P d<br />
Weigh ~50 g samples <strong>in</strong> 500 mL beaker and add about 300-400mL<br />
hot H2O and mix until the mixture becomes uniform<br />
Cl Clarified ifi d bby add<strong>in</strong>g ddi 10 mL L CCarrez soluti<strong>on</strong> l ti 1 and d 2 if necessary<br />
Filter and transfer the aqueous extract to a separat<strong>in</strong>g funnel
Test<strong>in</strong>g Methods: TLC Screen<strong>in</strong>g <strong>of</strong> Cyclamate<br />
Add 30 mL L HCl ( (v/v / 11:1) 1)<br />
Wash the aqueous layer with 50 ml petroleum ether (b.p 40-60oC).
Test<strong>in</strong>g Methods: TLC Screen<strong>in</strong>g <strong>of</strong> Cyclamate<br />
Add 30 mL L HCl ( (v/v / 11:1) 1)<br />
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<br />
mL Lethyl th l acetate<br />
t t
Test<strong>in</strong>g Methods: TLC Screen<strong>in</strong>g <strong>of</strong> Cyclamate<br />
Add 30 mL HCl (v/v 1:1)<br />
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<br />
mL ethyl acetate<br />
Filter the ethyl acetate layer through anhydrous sodium sulphate<br />
and evaporate the solvent to dryness.<br />
Dissolve the residue <strong>in</strong> 00.5mL 5mL NH NH4OH-H OH-H2O-EtOH O-EtOH (5+5+10) and then<br />
spot <strong>on</strong> TLC plate.
Test<strong>in</strong>g Methods: TLC Screen<strong>in</strong>g <strong>of</strong> Cyclamate<br />
Add 30 mL HCl (v/v 1:1)<br />
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<br />
mL ethyl acetate<br />
Filter the ethyl acetate layer through anhydrous sodium sulphate<br />
and evaporate the solvent to dryness.<br />
Dissolve the residue <strong>in</strong> 00.5mL 5mL NH NH4OH-H OH-H2O-EtOH O-EtOH (5+5+10) and then<br />
spot <strong>on</strong> TLC plate.<br />
Mobile Phase: n-Butanol/95% Ethanol/28% NH 4OH/Water OH/Water<br />
(40:4:1:9).<br />
Spray Reagents:<br />
11. 5% Brom<strong>in</strong>e <strong>in</strong> DCM (v/v)<br />
2. 0.25% fluoresce<strong>in</strong> <strong>in</strong> DMF-ethanol (1+1)
Test<strong>in</strong>g Methods: TLC plate<br />
S<strong>of</strong>tdr<strong>in</strong>k<br />
100 ppm<br />
Ice-cream<br />
100 ppm<br />
Ice-cream<br />
200 ppm<br />
Ice-cream<br />
400 ppm<br />
Potato Chip<br />
100 ppm<br />
Potato Chip<br />
200 ppm<br />
Potato Chip<br />
400 ppm
Test<strong>in</strong>g Methods: C<strong>on</strong>firmati<strong>on</strong> <strong>of</strong> Cyclamate<br />
Method is based <strong>on</strong> EN 12857:1999<br />
Cyclamate is extracted by water.<br />
Add sulfuric acid, n-heptane and<br />
sodium hypochlorite to derivate<br />
cyclamate for HPLC detecti<strong>on</strong>.<br />
N SO 3 H<br />
H<br />
N Cl<br />
Wash the n-heptane with sodium<br />
carb<strong>on</strong>ate soluti<strong>on</strong> and filter the nheptane<br />
layer through anhydrous<br />
sodium sulphate before <strong>in</strong>jecti<strong>on</strong> <strong>in</strong>jecti<strong>on</strong>.<br />
HPLC-UV/PDA determ<strong>in</strong>ati<strong>on</strong>.<br />
Mobile Phase: 80% Methanol<br />
Quantitati<strong>on</strong> Wavelength: 314 nm<br />
Cl
Test<strong>in</strong>g g Methods: C<strong>on</strong>firmati<strong>on</strong> <strong>of</strong> Cyclamate y<br />
10 ppm standard<br />
100 ppm blank spike<br />
(Chocolate)
Alternative C<strong>on</strong>firmati<strong>on</strong> Technique:<br />
LC LC-MS MS or LC LC-MS/MS MS/MS c<strong>on</strong>firmati<strong>on</strong><br />
Column: : Agilent Eclipase XDB-C18 5μm 4.6<br />
x 100mm<br />
MS/MS system : F<strong>in</strong>nigan LCQ Advantage Max with<br />
I<strong>on</strong> Max source or equivalent<br />
Sh Shealth lth gas fl flow : 50 arb b<br />
Auxillary/ sweep gas flow : 40 arb<br />
Capillary Temperature : 350o Capillary Temperature : 350 C<br />
Time<br />
(m<strong>in</strong>)<br />
Flow<br />
(mL/m<strong>in</strong>)<br />
A:<br />
1% Acetic Acid<br />
B:<br />
ACN<br />
0.00 0.5 95% 5%<br />
6.00 0.5 95% 5%<br />
18 18.00 00 05 0.5 80% 20%<br />
21.00 0.5 80% 20%<br />
22.00 0.5 95% 5%<br />
30.00 0.5 95% 5%
Acesulfame-K Sacchar<strong>in</strong> Cyclamate Sucralose Aspartame Alitame<br />
I<strong>on</strong>izati<strong>on</strong> Mode Negative Negative Negative Negative Positive Positive<br />
I spray voltage<br />
(kV)<br />
Capillary<br />
Voltage (V)<br />
4.5 5.0 4.5<br />
-13 -20 6<br />
Tube lens <strong>of</strong>fset -23 -60 55<br />
(V)<br />
Parent I<strong>on</strong>s<br />
(m/z)<br />
[M-H] -<br />
162<br />
[M-H] -<br />
182<br />
[M-H] -<br />
178<br />
[M+OAc] - [M+H] + [M+H] +<br />
455 (100),<br />
456 (25),<br />
457 (100)<br />
295 332<br />
D ht I NA NA NA NA [M H H O] + [M H H O] +<br />
Daughter I<strong>on</strong>s<br />
(m/z)<br />
NA NA NA NA [M+H-H2O] +<br />
277 (100%),<br />
[M-H-OAc] +<br />
235 (70%),<br />
[M+H-aspartyl] +<br />
[M+H-H2O] +<br />
314 (40%),<br />
[M+H-SC7H13 ] +<br />
204 (50%),<br />
[ p y] [M+H- [<br />
180 (20%) NHSC7H13 ] +<br />
187 (100%)<br />
Normalized<br />
CCollisi<strong>on</strong><br />
Energy<br />
NA NA NA NA 30% 30%
Test<strong>in</strong>g Methods: LC-MS and LC-MS/MS Chromatograms <strong>of</strong><br />
mixed sweeteners standard<br />
RT: 0.00 - 30.01<br />
100<br />
50<br />
0<br />
100<br />
50<br />
0<br />
100<br />
50<br />
0<br />
100<br />
50<br />
0<br />
100<br />
50<br />
0<br />
100<br />
50<br />
0<br />
RT: 5.67<br />
MA: 2870161<br />
0.13 2.48 4.38 7.45 9.90<br />
RT: 9.49<br />
MA: 3383414<br />
11.81 13.99 15.64 17.20 17.51<br />
2.49 2.60 3.07 5.56 6.54 9.20 9.97 11.52 13.13 14.65 16.64 17.11<br />
RT: 11.37<br />
MA: 4978085<br />
2.46<br />
2.00 2.57 8.95<br />
12.16<br />
17.20<br />
12.86<br />
6.24 7.02 9.02<br />
15.71<br />
17.55<br />
Sucralose (0.6 ppm)<br />
AAspartame t (0.3 (0 3 ppm) )<br />
Alitame (0 (0.6 6 ppm)<br />
Acesulfame-K (1 ppm)<br />
Sacchar<strong>in</strong> (1 ppm)<br />
Cyclamate (1 ppm)<br />
RT: 21.14<br />
MA: 4625829<br />
17.89 20.60 21.47<br />
R T: 2 1 .8 0<br />
MA: 5074805<br />
22.48 25.38 26.79 29.33<br />
R T: 2 4 .3 2<br />
MA: 7597789<br />
23.91<br />
22.14 25.12 26.41 28.99<br />
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30<br />
Tim e (m <strong>in</strong>)<br />
NL: 3.24E5<br />
m/z= 161.50 161 50-162 162.50 50 F: - c<br />
ESI Full m s [<br />
150.00-500.00] MS<br />
AS080206W2_08022911<br />
0034<br />
NL: 2.51E5<br />
m/z= 181.50-182.50 F: - c<br />
ESI Full ms [<br />
150.00-500.00] MS<br />
AS080206W2_08022911<br />
0034<br />
NL: 3.27E5<br />
m/z= 177.50-178.50 F: - c<br />
ESI Full m s [<br />
150.00-500.00] MS<br />
AS080206W2_08022911<br />
0034<br />
NL: 5.52E5<br />
T IC F: - p ESI SIM m s [<br />
453.00-459.00] MS<br />
A S080206W2_ 08022911<br />
0034<br />
NL: 6.32E5<br />
T IC F: + c ESI Full m s2<br />
295.00@35.00 [<br />
100.00-350.00] MS<br />
AS080206W2 AS080206W2_ 08022911<br />
0034<br />
NL: 9.37E5<br />
T IC F: + c ESI Full m s2<br />
332.00@30.00 [<br />
100.00-350.00] MS<br />
AS080206W2_08022911<br />
0034
Test<strong>in</strong>g g Methods: MS spectrums p<br />
Sucralose<br />
Aspartame Alitame
Report<strong>in</strong>g Limits<br />
Aces Acesulfame-K, lfame K Sacchar<strong>in</strong> and 20 mg/kg<br />
Alitame<br />
Aspartame p 40 mg/kg g g<br />
Sucralose 50 mg/kg<br />
Cyclamate (as cyclamic acid) 100 mg/kg
Standards validati<strong>on</strong><br />
Id Identificati<strong>on</strong> tifi ti and d assay<br />
determ<strong>in</strong>ati<strong>on</strong><br />
Reference Procedures<br />
can be obta<strong>in</strong>ed from<br />
<strong>Food</strong> Chemcials Codex<br />
JECFA specificati<strong>on</strong><br />
http://www.fao.org/ag/agn/jecfa-additives/search.html?lang=en
F<strong>in</strong>d<strong>in</strong>gs <strong>in</strong> 2007<br />
Total samples: 2,010<br />
No. <strong>of</strong> positive samples: 82<br />
Analytes No. <strong>of</strong> C<strong>on</strong>centrati<strong>on</strong> range found Types <strong>of</strong> samples<br />
Positive samples<br />
Acesulfame-K 38 36 ppm to 1,900 ppm Diet s<strong>of</strong>t dr<strong>in</strong>k, candy,<br />
話梅<br />
Sacchar<strong>in</strong> 26 20 ppm to 2,700 ppm Diet s<strong>of</strong>t dr<strong>in</strong>k, soy<br />
sauce, v<strong>in</strong>egar,話梅<br />
Aspartame 34 46 ppm to 1,800 ppm Diet s<strong>of</strong>t dr<strong>in</strong>k, candy,<br />
話梅<br />
Sucralose 9 50 ppm to 580 ppm Diet s<strong>of</strong>t dr<strong>in</strong>k, candy,<br />
Yoghurt g<br />
Cyclamate 24 110 ppm to 9,000 ppm Diet s<strong>of</strong>t dr<strong>in</strong>k, 甜蕎<br />
頭<br />
Alitame 0 Nil Nil
Pr<strong>of</strong>iciency Test<br />
FAPAS<br />
Pr<strong>of</strong>iciency Test Start Date Matrix Analytes<br />
0374 22/02/2008 T<strong>on</strong>ic Water -Acesulfame-K<br />
-Aspartame Aspartame<br />
-Benzoic Acid<br />
-Qu<strong>in</strong><strong>in</strong>e<br />
0376 15/04/2008 Cola Dr<strong>in</strong>k -Acesulfame-K<br />
-Benzoic Acid<br />
-Caffe<strong>in</strong>e<br />
-Sacchar<strong>in</strong><br />
0379 15/08/2008 S<strong>of</strong>t Dr<strong>in</strong>k -Sucralose<br />
0381 23/10/2008 S<strong>of</strong>t Dr<strong>in</strong>k -Brix<br />
-pH<br />
-Citric Acid<br />
-Sorbic Acid<br />
-Cyclamate<br />
-Sacchar<strong>in</strong> S h i<br />
0383 26/02/2009 T<strong>on</strong>ic Water -Acesulfame-K<br />
-Aspartame<br />
-Benzoic Acid<br />
-Qu<strong>in</strong><strong>in</strong>e Qu<strong>in</strong><strong>in</strong>e<br />
http://www.fapas.com
Pr<strong>of</strong>iciency Test<br />
LGC Standards Pr<strong>of</strong>iciency Test<strong>in</strong>g Group<br />
QBS<br />
QUALITY IN BEVERAGES SCHEME Scheme<br />
•Next Dispatch Date will be <strong>on</strong> 06 May 2008<br />
QFCS<br />
QUALITY IN FOOD CHEMISTRY SCHEME<br />
•2 distributi<strong>on</strong>s per annum<br />
•Next Dispatch Date will be <strong>on</strong> 20 May 2008<br />
SODAS<br />
SOFT DRINKS PROFICIENCY TESTING SCHEME<br />
•Scheme operates from October to September<br />
each h year and d test t t materials t i l are distributed<br />
di t ib t d<br />
quarterly .<br />
•Next Dispatch Date will be <strong>on</strong> 07 Apr 2008.<br />
http://www.lgcpt.com/
Thank you