Amendment to the Enforcement Ordinance of the Food Sanitation Law and the 
Standards and Specifications fo r Foods and Food Additives.  
 
The government of Japan will designate Adva ntame as an authorized food additive. 
 Summary 
Under Article 10 of the Food Sanitation La w, food additives shall not be used or 
marketed without authorization by the Mi nister of Health, Labour and Welfare 
(hereinafter referred as “the Minister”). In addition, when specifications or standards are 
established for food additives based on Article 11 of the act and stipulated in the Ministry of 
Health, Labour and Welfare Notification (Ministry of Health and Welfare Notification No. 370, 
1959), those additives shall not be used or ma rketed unless they meet the standards or 
specifications.   
In response to a request from the Minister, the Committee on Food Additives of the 
Food Sanitation Council that is establishe d under the Pharmaceutical Affairs and Food 
Sanitation Council has discussed the adequacy  of the designation of Advantame as a 
food additive. The conclusion of the committee is outlined below.   Outline of conclusion  
The Minister of Health, Labour and Welfare, based on Article 10 of the Food 
Sanitation Law, should designate Advantame as a food additive unlikely to harm human health, and establish standard for use and compositional specifications, based 
on Article 11 of the law (see Attachment).     Attachment  
 
Advantame 
アドバンテーム  
 
 
 
 Standards for use 
Not established.  Compositional specifications 
Substance name  Advantame 
Molecular formula  C24H30N2O7･H2O   
Molecular weight   467.52 
Chemical name [CAS number]    
Methyl N-[3-(3-Hydroxy-4-methoxyphenyl)propyl]- L--aspartyl- L-phenylalaninate 
monohydrate  [714229-20-6] 
Content  Adantame, when calculated on the anhydrous basis, contains 97.0–102.0% of 
advantame (C 24H30N2O7＝458.50). 
Description   Advantame is a white to yellowish white powder.   
Identification  Determine the infrared absorption spectrum of Advantame as directed in 
the Potassium Bromide Disk Method under Infrared Spectrophotometry, and compare with the Reference Spectrum. Both spectra ex hibit absorptions having about the same 
intensity at the same wavenumbers.  
Purity  
(1) Specific rotation ሿ
஽ଶ଴: –39 to –46  (0.2 g, ethanol (99.5), 100 ml, on the anhydrous basis). 
(2) Lead  Not more than 1.0 g/g as Pb. 
Test Solution  Weigh 4.0 g of Advantame in a plat inum, quartz, or porcelain crucible 
or a quartz beaker. Moisten the sample entire ly with diluted sulfuric acid (1 in 4), and 
heat by increasing the temperature gradually until it is carbonized and white fumes are 
no longer evolved. If necessary, add diluted sulf uric acid (1 in 4) again, and heat until it 
is almost carbonized. Loosely lid the crucible if  necessary, heat in an electric furnace by 
increasing the temperature grad ually, and incinerate at 450–600 C. If carbonized 
matter still remains, crush it with a glass rod,  moisten with 1 ml of diluted hydrochloric 
acid (1 in 4) and 1 ml of nitric acid, heat  until white fumes are no longer evolved, and 
ignite in the electric furnace to incineration.  To the residue, add 10 ml of diluted hydrochloric acid (1 in 4), and evaporate on a water bath to dryness. To the residue, add a little amount of diluted nitric acid (1 in 100), and dissolve it while heating. After 
cooling, add diluted nitric acid (1 in 100) again to make exactly 10 ml. When the 
incineration is done at a temperature of 500 C or less, a heat-resistant glass beaker 
may be used.  
Control Solution  Add water to exactly measured 1 ml of Lead Standard Stock 
Solution to make exactly 100 ml. To exactly me asured 4 ml of this solution, add diluted 
nitric acid (1 in 100) to make exactly 10 ml. 
Procedure  Proceed as directed under Method 1 in the Lead Limit Test.     
(3) Advantame-acid  Not more than 1.0%.  
Test Solution  Weigh accurately about 0.1 g of Advantame, and add a 7:3 mixture of 
water/acetonitrile to dissolv e and make exactly 100 ml.  
Standard Solution  Weigh accuretaly about 0.1 g of advantame-acid, and add a 7:3 
mixture of water/ acetonitrile to dissolve and make exactly 100 ml. Measure exactely 2 
ml of this solution, and add a 7:3 mixture of  water/acetonitrile to make exactly 20 ml. 
Measure exactely 2 ml of the second  solution, and add a 7:3 mixture of 
water/acetonitrile to make exactly 20 ml.    
Procedure  Analyze 20 l potions of the test solution and the standard solution by 
liquid chromatography using the op erating conditions given below.  Measure the peak 
areas (A T and A S) of advantame-acid for the test solution and the standard solution, and 
determine its amount by the formula: 
Amount (%) of advantame-acid   
ൌ		W	
Weight	ሺgሻ	of		the	sampleൈA୘
Aୗ W= weight (g) of advantame-acid taken   
Operating Conditions  
Detector: Ultraviolet spectrophotometer (wavelength 210 nm).  
Column: A stainless steel tube of about 4. 6 mm internal diameter and about 25 cm 
length.   
Column packing material: 5- m octadecylsilanized silica gel for liquid 
chromatography. 
Column temperature: A cons tant temperature around 50 C.  
Mobile phase  
A: Dissolve 13.6 g of potassium dihydrog en phosphate in 1000 ml of water, and 
adjust the pH to 2.8 with phosphoric ac id. To 900 ml of this solution, add 100 
ml of acetonitrile.     
B: Dissolve 13.6 g of potassium dihydrog en phosphate in 1000 ml of water, and 
adjust the pH to 2.8 with phosphoric ac id. To 400 ml of this solution, add 600 
ml of acetonitrile. 
Concentration gradient (A/B): Maintain  (85/15) for 30 minutes, and run a linear 
gradient from 85/15 to 75/25 over 25 mi nutes, then 75/25 to 0/100 over 20 
minutes, and maintain 0/100 for 15 minutes.    
Flow rate: 1.0 ml/minute. 
(4) Related substances other than adva ntame-acid  Not mo re than 1.5%.  
Test Solution and Standard Solution  Use the test solution and the standard 
solution prepatred in Purity (3). Analyze 20 l potions of these solutions by liquid 
chromatography using the operating co nditions given below. Continues the 
chromatography for about thr ee times the retention time of  advantame-acid. Measure 
the sum (A sum) of peak areas of all components, other than advantame and  
advantame-acid, in the test soluti on. Also measure the peak area (A S) of 
advantame-acid in the standard solution . Determine the amo unt of the related 
substances other than advantame-acid by the formula: 
Amount (%) of related substances = ୛	
୛ୣ୧୥୦୲	ሺ୥ሻ	୭୤		୲୦ୣ	ୱୟ୫୮୪ୣൈ୅౩౫ౣ
୅౏ 
W= weight (g) of advantame-acid taken.   
Operating Conditions   Follow the operating conditions  specified in Purity (3). 
Water   Not more than 5.0% (0.1g, Direct Titration).     
Residue on Ignition   Not more than 0.2% ( 550 C, 3 hours) 
Assay   
Test Solution   Weigh accurately about 0.04 g of  Advantame, and add a 7:3 mixture of water/acetonitrile to dissolve and make ex actely 50 ml. To exactly measured 10 ml of 
this solution, add exactly 5 ml of the internal standard and a 7:3 mixture of water/acetonitrile to make exactly 50 ml.   
Standard Solution  Weigh accurately about 0.04 g of advantame for assay, and 
proceed as directed for the test solution. 
Internal Standard  To 0.04 g of benzoic acid, exactl y weighed, add a 7:3 mixture of 
water/acetonitrile to make 50 ml. 
   Procedure  Analyze 20 l potions of the test solution and the standard solution by 
liquid chromatography using the operating co nditions given below. Determine the peak 
area ratios (Q T and Q S) of advantame to benzoic acid for both solutions to calculate the 
Content of advantame by the formula:   
				Content	 ሺ%ሻ	of	advantame	 ሺCଶସHଷ଴NଶO଻ሻ
ൌ		Anhydrous	based	weight		 ሺgሻ	of	advantame	for	assay
Anhydrous	based	weight	 ሺgሻ	of		the	sampleൈQ୘
Qୗൈ100  
Operating Conditions  
Detector: Ultraviolet spectrophotometer (wavelength 280 nm).  
Column: A stainless steel tube of about 4. 6 mm internal diameter and about 25 cm 
length.   
Column packing material: 5- m octadecylsilanized silica gel for liquid 
chromatography. 
Column temperature: A cons tant temperature around 40 C.  
Mobile phase  
A: Dissolve 13.6 g of potassium dihydrog en phosphate in 1000 ml of water, and 
adjust the pH to 2.8 with phosphoric ac id. To 750 ml of this solution, add 250 
ml of acetonitrile.     
B: Dissolve13.6 g of potassium dihydrogen phosphate in 1000 ml of water, and 
adjust the pH to 2.8 with phosphoric ac id. To 500 ml of this solution, add 500 
ml of acetonitrile. 
Concentration gradient (A /B): Maintain 100/0 for 20 minutes, and run a linear 
gradient from 100/0 to 0/100 over 5 mi nutes, then 0/100 for 15 minutes.    
Flow rate: 1.0 ml/minute.  
 
 
 Reagents and Solutions 
Advantame-acid  C 28H28N2O7  
N-[3-(3-Hydroxy-4-meth oxyphenyl)propyl]- L--aspartyl- L-phenyl Alanine  
A white to yellow powder. 
Content   Not less than 94% of advantame-acidon anhydrous basis. 
Purity  (1) Chlorides  Not more than 1.0% as Cl. 
   Prepare a test solution by adding a 7:3 mixture of water/acetonitrile to about 0.01 g 
of Advantame-acid, accurately  weighed, to dissolve and ma ke exactely 100 ml. Prepare 
two standard solutions. First, add water to about 0.016 g of sodium chloride, accurately 
weighed, to disslove and make exactely 100 ml . Designate this as standard solution A. 
Prepare standard solution B by dilutuing exac tely measured 2 ml of standard solution A 
with water to make exactely 20 ml. Anlyze 30- l potions of the test solution and the 
standard solutions by liquid chromatography using the operating conditions given below. 
Measure the peak areas of cloride anion for standard solutions A and B to make a calibration curve. Measure the peak area of cloride anion for the test solution. Determine the concentration of chlorides in the test solution from the clibration curve and calculate the amount of chlorides by the formula: 
Amount	ሺ%ሻof	chlorides	 ൌConcentration	ሺg/mlሻ	of	chlorides	in	the	test	solution
Weight	ሺgሻ	of		the	sampleൈ10,000  
Operating conditions  
Detector: Electric conductivity detector.  
Column: A polyether ketone tube of about  4.6 mm internal diameter and about 15 
cm length.   
Column packing material: 6- m strongly basic anion-exchange resin. 
Column temperature: A cons tant temperature around 40 C.  
Mobile phase: A mixture prepared by dissolving 201.62 mg of sodium hydrogen 
carbonate and 264.98 mg of anhydrous sodium carbonate in 1000 ml of water. 
Flow rate: Adjust so that the retention time of chloride anion is about 7 minutes.  
(2)  Sodium  Not more  than 5.0% as Na. 
  Prepare a test solution by adding a 7:3 mixture of water/acetonitrile to about 0.01 g 
ofAdvantame-acid, accurately weighed, to dissolve and make exactely 100 ml. Prepare 
two standard solutions. First, add water to about 0.006 g of sodium chloride, accurately 
weighed, to disslove and make exactely 100 ml . Designate this as standard solution A. 
Prepare standard solution B by dilutuing exac tely measured 2 ml of standard solution A 
with water to make exactely 20 ml. Anlyze 30- l potions of the test solution and the 
standard solutions by liquid chromatography using the operating conditions given below. Measure the peak areas of sodium for standa rd solutions A and B to make a calibration 
curve. Measure the peak area of sodium ca tion for the test so lution. Determine the 
concentration of sodium cation in the test  solution from the clibration curve and 
calculate the amount of sodium by the formula: 
Amount	ሺ%ሻ	of	sodium	 ൌConcentration	 ሺg/mlሻ	of	sodium	in	the	test	solution
Weight	ሺgሻ	of		the	sampleൈ10,000  
Operating conditions  
Detector: Electric conductivity detector.  
Column: A polyether ketone tube of about 4.6 mm internal diameter and about 15 
cm length.   
Column packing material: 3- m weakly acidic cation-exchange resin for liquid 
chromatography. 
Column temperature: A cons tant temperature around 40 C.  
Mobile phase: A mixture prepared by dissolving 77.58 mg of L-histidine in 1.25 ml 
of 2 mol/L methanesulfonic acid and adding 1000 ml of water. 
Flow rate: Adjust so that the retention ti me of sodium cation is about 4 minutes.  
Water  Not more that 1.0% (0.1  g, Direct Titration) 
Assay  Prepare a test solution by adding a 7:3 mixture of water/acetonitrile to 0.01 g 
of Advantame-acid to dissolve an d make exactely 100 ml. Anlyze 20- l potions of the 
test solution by liquid chromatography us ing the operating conditions given below. 
Continue chromatography for about six time s the retention time of advantame-acid. 
Measure the peak areas, and de termine the area percentage, C (%), of the main peak by 
normalizing the sum of the peak areas of all components in the tets solution to 100. Calculate the content by the formula: 
Content (%) of advantame-acid 
 = ሺ100െchlorides	amount	െsodium	amountെwater ሻൈ஼ሺ%ሻ
ଵ଴଴ 
Operating conditions  Follow the conditio ns given in the Assay for Advantame in 
the Monographs. 
 
Advantame for Assay  C24H30N2O7･H2O  A white to yellowish white powder.   
Content  Not less than  99.0% of advantame (C 24H30N2O7) when calculated on the 
anhydrous basis.  
Identification  Determine the infrared absorption spectrum of Advantame for Assay 
as directed in the Potassium Bromide Disk Method under Infrared Spectrophotometry. It exhibits absorption bands at about 3405 cm
-1, 3320 cm-1, 2945 cm-1, 1717 cm-1, 1661 cm-1, 1582 cm-1, 1376 cm-1, 1242 cm-1, 1131 cm-1, and 703 cm-1.  
Purity (1)  Specific rotation  ሿ஽ଶ଴: –39 to –46  (0.2 g, ethanol (99.5), 100 ml, on the 
anhydrous basis).  
(2) Related substances   Not more than 1.0% as advantame-acid. 
Prepare a test solution by  adding a 7:3 mixture of water/acetonitrile to about 0.1 g of 
Advantame for Assay, accurately weighed, to  dissolve and make exactly 100 ml. Prepare 
a standard solution as follows:  Add a 7:3 mixture of water/acetonitrile to about 0.1 g of 
advantame-acid, accuretaly weighed, to di ssolve and make exactly 100 ml. To exactly 
measured 2 ml of this solution, add a 7:3 mixture of water/acetonitrile to make exactly 20 ml. To exactly measured 2 ml of the second solution, add a 7:3 mixture of 
water/acetonitrile to make exactly 20 ml. Analyze 20 l potions of the test solution and 
the standard solution by liquid chromatogr aphy using the operating conditions given 
below. Continues the chromatography for about three times the retention time of 
advantame-acid. Me asure the sum (A
T) of the peak areas of all components, other than 
advantame, in the test solution and the peak area (A S) of advantame-acid in the 
standard solution.  Calculate the amount of the related substances by the formula: 
				Amount	ሺ%ሻ	of	related	substances ൌW	
Weight	ሺgሻ	of		the	sampleൈA୘
Aୗ 
W= weight (g) of advantame-acid taken.   
Operating Conditions  Follow the operating conditio ns given in Purity (3) for 
Advantame in the Monographs.  
Water   Not more than 5.0% (0.1g, Direct Titration). 
Residue on Ignition  Not more than 0.2% (550 C, 3 hours) 
Assay  To about 0.5 g of Advantame for Assa y, accurately weighed, add 100 ml of 
ethanol to dissolve, and titrate with 0. 1 mol/L sodium hydroxide. Usually, a 
potentiometer is used to confir m the end point. Perform a blan k test to make correction.  
Each mL of 0.1 mol/L sodium hydroxide = 45.85 mg of C 24H30N2O7 
L-Histidine   C 6H9N3O2  White crystals or powder.  
Content  Not less than 98.0% of  L-histidine. 
Purity  Specific rotation  ሿ஽ଶ଴: +12.0 to +13.0  (1 g, hydrochloric acid, 10 ml). 
Assay   Dissolve about 0.15 g of L-Histidine, accurately weig hed, in 2 ml of formic 
acid, add 50 ml of acetic acid, and titr ate with 0.1 mol/L perchloric acid. Usually, a 
potentiometer is used to conf irm the end point. Perform a bl ank test to make correction. 
Each ml of 0.1 mol/L perchloric acid = 15.52 mg of C 6H9N3O2 Methanesulfonic Acid  CH 4O3S  A colorless to light yellow-brown, clear liquid.  
Content  Not less than 98.0% of  methanesulfonic acid. 
Assay   Mix about 2 g of Methanesulfonic Acid , accurately weighed, with 40 ml of 
water. Titrate with 1 mol/L sodium hydroxide using 2 drops of bromothymol blue TS as an indicator. Perform a blank test to make correcction. 
Each ml of 1 mol/L sodium hydroxide = 96.11 mg of CH
4O3S 
Strongly Basic Anion-exchange Re sin for Liquid Chromatography  Use a product 
prepared for liquid chromatography.   
Weakly Acidic Cation-exchange Re sin for Liquid Chromatography Use a product 
prepared for liquid chromatography. 
 
Infrared spectrum 
 Advantame