Stock solutions (1000gml−1) of dodeca-2(E), 4(E)-dienoic acid
isobutylamide (a reference standard for spilanthol) was prepared
by dissolving 5mg of thecompoundin 5ml ofHPLCgrade 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 250–15gml−1. Each
concentration of standard was filtered through a 0.22m nylon
membrane filter (Millipore, USA) before HPLC analysis.