Stock solutions (1000 gml−1) of dodeca-2(E), 4(E)-dienoic acid isobutylamide (a reference standard for spilanthol) was prepared by dissolving 5 mg of the compound in 5 ml of HPLC grade methanol. The solution was then stored at −20 ◦C. Quantification was carried out using 5 levels of external standards obtained by serial dilutions of stock solutions at a concentration range of 250–15 gml−1. Each concentration of standard was filtered through a 0.22 m nylon membrane filter (Millipore, USA) before HPLC analysis.