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Briefing Session on Analysis of Sweeteners in Food

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.

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Briefing Session on Analysis of Sweeteners in Food

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  1. Briefing Session on Analysis of Sweeteners in Food Dr. Samuel M-F LO Chemist Food Safety B Section Government Laboratory, HKSAR

  2. Contents • Background • Testing Methods –International Standard Methods • Testing Methods – GL Methods • Findings in 2007 • Proficiency Tests

  3. 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 Sweetener Xylitol

  4. 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。

  5. 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)

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

  7. 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……

  8. Testing Method: International Standard Methods

  9. Testing Methods: International Standard Methods

  10. Testing Methods: Other Literature Methods

  11. 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

  12. 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.

  13. Testing Methods: HPLC method for Acesulfame-K, Saccharin, Aspartame, Sucralose and Alitame • 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 mm membrance filter before HPLC analysis. • 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. 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. Aspartame, Acesulfame-K Saccharin, Alitame HPLC-PDA/FLU Sucralose HPLC-RI

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

  15. Testing Methods: Chromatogram – 50 ppm blank spiked sample (Soft drink) 225 nm 210 nm FLU

  16. Testing Methods: HPLC condition for Sucralose

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

  18. 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 0.5mL NH4OH-H2O-EtOH (5+5+10) and then spot on TLC plate. Extraction 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 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)

  19. 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

  20. 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

  21. Testing Methods: Confirmation of Cyclamate 10 ppm standard 100 ppm blank spike (Chocolate)

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

  23. 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)

  24. Testing Methods: MS spectrums Sucralose Aspartame Alitame

  25. Reporting Limits

  26. 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

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

  28. Proficiency Test FAPAS http://www.fapas.com

  29. 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/

  30. Thank you

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