Synthetic Sweeteners

Acesulfame K

It is known as acesulfame potassium, and has 200-250 times the sweetness of sugar. As a high-sweetness sweetener, it is added to sugar substitute sweeteners in combination with xylitol, soft drinks, confectionery, and pickles.

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• HPLC Conditions

  Sample	Acesulfame K Standard
  Column	Wakosil-Ⅱ5C18HG, 4.6 mmφ × 250 mm
  Eluent	0.01% Tetrabutylammonium hydrogensulfate/CH3CN = 60/40
  Flow rate	1.0 mL/min. at 40℃
  Detection	UV 227 nm
  Injection vol.	0.5 mg/mL soln. 10 μL

Aspartame

It is about 200 times as sweet as sugar and is used in foods such as diet sweeteners, soda, yogurt, and ice candies.

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• HPLC Conditions

  Sample	Aspartame Standard
  Column	Wakosil-Ⅱ 5C18HG, 4.6 mmφ × 250 mm
  Eluent	10 mmol/L Phosphate Baffer(pH4.0)/CH3CN = 85/15
  Flow rate	1.0 mL/min. at 40℃
  Detection	UV 210 nm
  Injection vol.	0.2 mg/mL soln. 10 μL

Advantame

It is a dipeptide methyl-ester derivative synthesized by reducing the alkylation of aspartame, a sweetener, with 3-hydroxy-4 methoxy-phenylpropionaldehyde. Although sweetness depends on the type of food in which it is used and blending composition, its sweetness is approximately 14000-48000 times that of sugar and 90-120 times that of aspartame.

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• HPLC Conditions

  Sample	Advantame Standard
  Column	Wakosil-Ⅱ 5C18HG, 4.6 mmφ × 250 mm
  Eluent	A:phosphate Buffer(pH 2.8) 900 mL+CH3CN 100 mL B:phosphate Buffer(pH 2.8) 400 mL+CH3CN 600 mL 0-30 min. B:15%, 30-55 min. B:15-25% 55-75 min. B:25-100%, 75-90 min. B:100%
  Flow rate	1.0 mL/min. at 50℃
  Detection	UV 210 nm
  Injection vol.	1 mg/mL soln. 20 μL

Advantamame Acid

It is a hydrolysate of advantame and is considered a major metabolite.

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• HPLC Conditions

  Sample	Advantame Acid Standard
  Column	Wakosil-Ⅱ 5C18HG, 4.6 mmφ × 250 mm
  Eluent	A:phosphate Buffer(pH 2.8) 900 mL+CH3CN 100 mL B:phosphate Buffer(pH 2.8) 400 mL+CH3CN 600 mL 0-30 min. B:15%, 30-55 min. B:15-25% 55-75 min. B:25-100%, 75-90 min. B:100%
  Flow rate	1.0 mL/min. at 40℃
  Detection	UV 280 nm
  Injection vol.	0.2 mg/mL soln. 10 μL

Dulcin

This artificial sweetener was developed in Germany in 1883. With a sweetness of about 200 - 400 times that of sucrose, it was widely distributed in Japan after the war. However, addition of the sweetener to foods was completely prohibited in January 1969 in Japan, following intoxication accidents and detection of toxicity causing hepatic dysfunction and carcinogenicity.
Currently, dulcin is not used globally. However, dulcin was detected from foods imported; therefore, inspections are performed at quarantine stations.

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• HPLC Conditions

  Sample	Dulcin Standard
  Column	Wakopak® Navi C18-5, 4.6 mmφ × 250 mm
  Eluent	H2O/CH3OH = 40/60
  Flow rate	1.0 mL/min. at 35℃
  Detection	UV 240 nm
  Injection vol.	0.1 mg/mL CH3OH 10 μL

Saccharin Sodium Dihydrate

This artificial sweetener is about 500 times as sweet as sugar in aqueous solution. Sweeteners that are currently allowed in drinks and foods include saccharin sodium dihydrate and aspartame, which was approved in August 1983 in Japan. Saccharin sodium dihydrate can also inhibit cholinesterase activity.

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• HPLC Conditions

  Sample	Saccharin Sodium Dihydrate Standard
  Column	Wakosil-Ⅱ 5C18HG, 4.6 mmφ × 150 mm
  Eluent	1 vol% H3PO4/CH3CN = 90/10
  Flow rate	1.0 mL/min. at 40℃
  Detection	UV 210 nm
  Injection vol.	0.1 mg/mL soln. 10 μL

Sucralose

Sucralose, developed in 1976, is approximately 600 times sweeter than sugar and is used as a raw material in foods and beverages.

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• HPLC Conditions

  Sample	Sucralose Standard
  Column	Wakosil-Ⅱ 5C18HG, 4.6 mmφ × 150 mm
  Eluent	H2O/CH3OH = 75/25
  Flow rate	1.0 mL/min. at 40℃
  Detection	RI
  Injection vol.	5 mg/mL soln. 10 μL

Cyclamic Acid Sodium Salt

This synthetic sweetener was permitted in the past, but in November 1969, the use of this synthetic sweetener was prohibited in Japan. However, the use of sodium cyclamate is approved in the EU, China, Taiwan, etc. and it is frequently detected at quarantine stations.

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• HPLC Conditions

  Sample	Cyclamic Acid Sodium Salt Standard
  Column	Wakosil-Ⅱ 5C18HG, 4.6 mmφ × 150 mm
  Eluent	H2O/CH3CN = 30/70
  Flow rate	1.0 mL/min. at 40℃
  Detection	UV 314 nm

  *Measured after conversion to N,N-Dichlorocyclohexylamine

Analysis of artificial sweeteners using the Ninhydrin-ethanol TS Spray

Target compound

1. Glutamyl–valyl–glycine	2. Alitame	3. Advantame

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• Analysis conditions

  Deployed solvent	Chloroform/Methanol/Acetic acid/Water = 30/20/4/6
  Concentration of mixed solution	1. 10 μg/μL, 2. 2.5 μg/μL, 3. 2.5 μg/μL
  Development distance	7 cm (saturation development)
  Injection volume	4 μL
  TLC plate	Silicagel 70 TLC Plate-Wako