Quality by Design-Steered Chromatographic Separation and Identification of the Geometric Isomers of Capsiate by Reversed-Phase HPLC and LC-MS.

An efficient reverse-phase high-performance liquid chromatographic method, based on the design of the experiment approach, was developed for the simultaneous determination of capsiate isomers. Critical method parameters, i.e., flow rate and mobile phase composition, demarcated during preliminary screening were optimized using central composite design. Chromatographic separation was achieved on a Nucleodur C18 column (250 × 4.6 mm, 5 μm), with the mobile phase consisting of water-acetonitrile (40:60), both acidified with 0.1% v/v formic acid, passed at a flow rate of 1 mL/min. E- and Z-capsiates were eluted from the column at the retention times of 18.56 ± 0.09 and 17.30 ± 0.08 min, respectively, with a resolution factor of 1.693 ± 0.046. The method was found to be linear within the concentration range of 1.054-5.270 and 8.623-43.115 μg/mL for Z- and E-isomers, respectively, with an R2 of >0.99. Recovery of the individual values was in range of 96.15-101.92%, with a relative standard deviation of <2%. The developed method was used to quantify capsiate isomers extracted from sweet pepper fruits. Therefore, the proposed method is presented for optimum isomeric resolution of these closely related E- and Z-isomers with convenient sample preparation, acceptable run-time, cost effectiveness and use of conventional instruments.