Chromatographic conditions for the elution of aspartame as optimized by Arora, Shendurse, Sharma, Wadhwa, and Singh (2013) was used. However, chromatographic conditions for the elution of neotame were optimized as following
Three different mobile phases were chosen for the elution of neotame viz. 0.09% TFA:water:acetonitrile (70:30, 80:20, 60:40). 0.09% TFA:water:acetonitrile (60:40) gave the best resolution as compared to the other two mobile phases. Three flow rates (0.6, 0.8 and 1.0 ml/min) of the mobile phase were assayed for the esti-mation of neotame. The neotame peak merged with the product matrix peak at a flow rate of 0.8 and 1.0 ml/min, hence, a flow rate 0.6 ml/min was selected for the analysis. At this flow rate the retention time increased and better resolution was obtained as compared to the flow rate of 0.8 and 1.0 ml/min.
In order to select the wavelength for analysis of neotame, a ref-erence standard solution of neotame was injected and UV spectra were scanned in the UV range (200–380 nm). Neotame showed kmax at 210 nm.