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Prepared at the 46th JECFA (1996), published in FNP 52 Add 4 (1996) superseding specifications prepared at the 44th JECFA (1995), published in FNP 52 Add 3 (1995) |
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SYNONYMS |
INS No. 956 |
DEFINITION | |
Chemical names |
L-alpha-Aspartyl-N-(2,2,4,4-tetramethyl-3-thietanyl)-D-alaninamide, hydrated |
C.A.S. number |
99016-42-9 (hydrated form) 80863-62-3 (anhydrous form) |
Chemical formula |
C14H25N3O4S · 2.5 H2O |
Structural formula |  |
Formula weight |
376.5 (hydrated form) |
Assay |
Not less than 98.0% and not more than 101.0% on the anhydrous basis |
DESCRIPTION |
White, crystalline powder, odourless or having a slight characteristic odour. Approximately 2000 times sweeter than sucrose |
FUNCTIONAL USES |
Sweetener |
CHARACTERISTICS | |
IDENTIFICATION | |
Solubility |
Freely soluble in water and in ethanol |
Infrared absorption |
The infrared spectrum of a potassium bromide dispersion of the sample corresponds with the reference infrared spectrum below |
pH |
Between 5.0 and 6.0 (5 in 100 soln) |
Colour reactions |
To 5 ml of a solution of 300 mg of ninhydrin in 100 ml of n-butanol and 2 ml of glacial acetic acid, add 10 mg of the sample, and heat to gentle reflux. An intense blue-violet colour is formed.
To 5 ml of a freshly prepared 0.001 mol/l potassium permanganate solution add 10 mg of the sample and mix thoroughly. The purple solution changes to brown. |
PURITY | |
Beta isomer |
Not more than 0.3%, calculated on the anhydrous basis See description under METHOD OF ASSAY |
Alanine amide |
Not more than 0.2%, calculated on the anhydrous basis See description under METHOD OF ASSAY |
Water |
Between 11 and 13% (Karl Fischer Method) |
Specific rotation |
[alpha] 25, D: Between +40º and +50º, 1% (w/v) in water |
Sulfated ash |
Not more than 1.0% |
Heavy metals |
Not more than 10 mg/kg Test 2 g of the sample as directed in the Limit Test (Method II) |
METHOD OF ASSAY |
Principle:Alitame and its constituents, the beta isomer and alanine amide, are measured by reverse-phase ion-pair HPLC.
Chromatographic System:Fit a high pressure liquid chromatograph, operated at room temperature, with a constant flow pump and a 15 x 0.4 cm NovaPak C18 reverse phase ion-pair column (Waters, or equivalent). The mobile phase is maintained at a pressure and flow rate (typically 1.0 ml/min) capable of giving the required elution time (see System Suitability Test). An ultraviolet detector that monitors absorption at 217 nm is used.
Mobile Phase:To make the buffer solution, add 0.69 g of sodium phosphate, monobasic, monohydrate and 4.32 g sodium 1-octanesulfonate, reagent grade (obtainable from Regis Chemical Co.) to a 1000-ml volumetric flask. Add 200 ml of water, stir to dissolve the salts and adjust the pH to 2.5 with phosphoric acid (85%, reagent grade). Add water to volume. Filter through 0.22 µm Millipore filter or equivalent. Accurately measure one part by volume of acetonitrile (LC grade) and three parts by volume of buffer solution, and combine. De-gas under aspirator vacuum.
Standard SolutionsStandard Solution A1: Weigh accurately about 25 mg each of the beta isomer and alanine amide, and transfer quantitatively to a 500 ml volumetric flask. Add 50 ml methanol to aid dissolution, and dilute with water to volume. Store in a refrigerator.
Standard Solution A2: Transfer 15.0 ml of Standard Solution A1 into a 50 ml volumetric flask and dilute with water to volume.
Working Standard W1: Weigh accurately about 50 mg Alitame Reference Standard (obtainable from Pfizer Central Research, Eastern Point Road, Groton, Connecticut, USA), transfer quantitatively to a 10 ml volumetric flask, add 5 ml of Standard Solution A2, and dilute with water to volume.
Working Standard W2: Transfer 5.0 ml of Working Standard W1 to a 50 ml volumetric flask and dilute with water to volume.
Test SolutionsTest Solution S1: Weigh accurately about 50 mg of the sample, transfer quantitatively to a 10 ml volumetric flask and dilute with water to volume.
Test Solution S2: Transfer 5.0 ml of Test Solution S1 to a 50 ml volumetric flask and dilute with water to volume.
System Suitability TestInject triplicate 100 µl portions of Working Standards W1 and W2 into the chromatograph. The retention times for the beta isomer, alitame, and alanine amide should be approximately 6, 10, and 15 min respectively. (Note: The retention time quoted is appropriate for a 15 x 0.4 cm NovaPak column. If a column of a different make or length is used, it may be necessary to adjust the proportion of acetonitrile in the eluent to obtain the required retention time). The coefficient of variation (100 x standard deviation divided by mean peak area) for the peak areas should not exceed 2%.
ProcedureEquilibrate the column by pumping mobile phase through it until a drift-free baseline is obtained. Analyze the Standard Solutions and Test Solutions under the conditions described above. Inject three replicate samples of Working Standard W1, and calculate the average peak areas for the beta isomer and for alanine amide. Inject three replicate samples of Working Standard W2 and calculate the average peak area for alitame. Inject three replicate samples of Test Solution S1 and calculate the average peak areas for the beta isomer and alanine amide. Inject three replicate samples of Test Solution S2 and calculate the average peak area for alitame.
Calculate the percentage purity of alitame by the formula:
where RA = the response of the analyte peak in Test Solution S2 WS = the weight of the alitame Reference Standard corrected for water content, in g PS = the percent purity of the Reference Standard, i.e., 100.00 - sum of impurities RS = the response of the analyte peak in Working Standard W2 WA = the weight of the sample corrected for the water content, in g.
Calculate the percentage content of the beta isomer and alanine amide by the following formula:
where RA = the response of the analyte peak in Test Solution S1 WS = the weight of the beta isomer or alanine amide standards, uncorrected for water content, in g PS = the percentage purity of the beta isomer or alanine amide standard, i.e. 100.00 - sum of impurities RS = the response of the analyte peak in Working Standard W1 WA = the weight of the sample, uncorrected for water content, in g DF = the dilution factor, i.e. 0.003. |
| Infrared spectrum |
Alitame |
