NEOTAME New specifications prepared at the 61st JECFA (2003) and published in FNP 52 Add 11(2003). An ADI of 0 – 2 mg/kg bw was established at the 61st JECFA (2003). INS No. 961 SYNONYMS Neotame is manufactured in single process in which aspartame and 3,3-DEFINITION dimethylbutyraldehyde are reacted together in a methanol solution in the presence of hydrogen. Neotame is isolated by removal of methanol, followed by washing and drying. Chemical names N-[N-(3,3-Dimethylbutyl)-L-α-aspartyl]- L-phenylalanine 1-methyl ester . number 165450-17-9 Chemical formula CHNO 203025 Structural formula COOHOHNNOCH3HO Formula weight Assay Not less than % and not more than % on the anhydrous basis White to off-white powder DESCRIPTION Sweetener, flavour enhancer FUNCTIONAL USES CHARACTERISTICS IDENTIFICATION Solubility (Vol. 4) Sparingly soluble in water, very soluble in ethanol Infrared spectrum The infrared spectrum of a potassium bromide dispersion of the sample corresponds to the standard infrared spectrum in Appendix A. PURITY pH (Vol. 4) – ( % solution) Melting range (Vol. 4) 81° - 84° Water (Vol. 4) Not more than % in a sample size of 25±5 mg (Karl Fischer)
N-[N-(3,3-Dimethylbutyl)-α-Not more than % aspartyl]-L-phenylalanine See under METHOD OF ASSAY Other related substances Not more than % based on the results of the Method of Assay using the following formula: 100 x A/(A+B) where A = the sum of the peak areas for all secondary peaks other than those for neotame and N-[N-(3,3-dimethylbutyl)-L-α-aspartyl]-L-phenylalanine and B = the sum of the peak areas for neotame and N-[N-(3,3-dimethylbutyl)-L-α-aspartyl]-L-phenylalanine. Sulfated ash (Vol. 4) Not more than % 20Specific rotation (Vol. 4) [α]: Between –° and –° ( % solution) calculated on the Danhydrous basis Lead (Vol. 4) Not more than 1 mg/kg Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles described in Volume 4, “Instrumental Methods”. Neotame METHOD OF ASSAY Determine by HPLC using the following conditions: Mobile phase: 25% acetonitrile and 75% buffer (final pH of ). The buffer is composed of M heptanesulfonic acid sodium salt and % v/v triethylamine at pH . Preparation of sample: Dissolve the sample in mobile phase solution to a concentration of 1 mg/ml. Preparation of standard: Dissolve the neotame standard (NutraSweet Kelco) in mobile phase solution to a concentration of 1 mg/ml HPLC Conditions: Column: Partisil 5 ODS3 ( x 100 mm length) or equivalent. Column temperature: 45° Pump:IsocraticSolvent: 25% acetonitrile and 75% buffer adjusted to a pH of ). Flow rate: ml/min Injection:25µlDetection: UV 210 nm Run Time: approximately 18 min. Calculation: Compare the area of the neotame peak in the sample (A) to that in the standard (A). Calculate the percentage samplestandardcontent of the sample, on the dry basis, from the formula % neotame = (A/A) x 100 x F samplestandard
Where F= 100/(100 - % water in sample) N-[N-(3,3-Dimethylbutyl)-α-aspartyl]-L-phenylalanine This is determined using the same HPLC method: Preparation of sample: Dissolve the sample in mobile phase solution to a concentration of 2 mg/ml. Preparation of standard: Dissolve the N-[N-(3,3-dimethylbutyl)-α-aspartyl]-L-phenylalanine standard (NutraSweet Kelco) in mobile phase solution to concentrations of 75, 45, 15, 3 and µg/ml. Calculation: The retention time for N-[N-(3,3-dimethylbutyl)-α-aspartyl]-L-phenylalanine is approximately min compared with approximately min for neotame. Determine the area response of N-[N-(3,3-dimethylbutyl)-L-α-aspartyl]-L-phenylalanine from the sample preparation. Prepare a full fit linear regression standard curve by plotting the area response of N-[N-(3,3-dimethylbutyl)-L-α-aspartyl]-L-phenylalanine in the standard solution on the ordinate scale versus its respective concentration in µg/ml. From the slope and intercept of the standard curve, calculate the concentration, C 1(µg/ml), of N-[N-(3,3-dimethylbutyl)-L-α-aspartyl]-L-phenylalanine in the sample using the equation: C= (A – intercept)/slope of curve 1 sample Calculate the percentage of N-[N-(3,3-dimethylbutyl)-L-α-aspartyl]-L-phenylalanine in the sample using the equation % N-[N-(3,3-dimethylbutyl)-L-α-aspartyl]-L-phenylalanine = (C/C) x 100 12where Cis the concentration of the sample. 2 See Appendix B for examples of chromatograms obtained using the method. Appendix A IR Spectrum of neotame standard
Appendix B Chromatograms for neotame and N-[N-(3,3-dimethylbutyl)-L-α-aspartyl]-L-phenylalanine (Std E1)
