Direct and accurate purity evaluation for isovanillin conversion to HMCA through aldol condensation by using RID and its comparison with UV detection

As an important intermediate of Advantame, 3-hydroxy-4-methoxy-cinnamaldehyde (HMCA) is derived from isovanillin by Aldol reaction. The HPLC-UV method was initially established in acetonitrile/water for the separation and quantification of two compounds. UV-chromophores rapidly reach plateauing effect and limit linear regression range below 1.00 mg/mL at UV 210,280 and 340 nm. HPLC-RID with wider range manner was established. The solubility assay of HMCA and isovanillin was investigated for working range limit for the RID under different mobile phase acetonitrile ratios. RID are sensitive enough as UV at micrograms level in methodology comparison. Wider working range and universal response of RID facilitate the purity evaluation. RID detection showed reliable and precise performance as UV in method precision, stability (as all RSDs < 2 %), and similar recovery for accuracy (99.99–101.94 %). The proposed RID method was applied for purity evaluation by using area for direct calculation. The actual purity values had a high correlation with the RID area response directly, and the wavelength complexity resulted in diverse purity evaluations in UV method.