Development and validation of an HPLC-DAD analysis for pharmacopoeial qualification of industrial Capsicum extracts

A reverse-phase high-performance liquid chromatography method was developed to quantify capsaicin (trans-8-methyl-N-vanillyl-6-nonenamid), dihydrocapsaicin (8-methyl-N-vanillylnonanamide) and the main capsaicinoid contents of Capsicum extracts. The chromatographic separation was carried out on a C8 column using isocratic mobile phase consisting of 40% (v/v) acetonitrile and 60% (v/v) orthophosphoric acid solution with flow rate of 1.5 mL/min. The concentration of the eluting compounds was monitored by a diode-array detector at wavelength of 281 nm. The method was evaluated for number of validation characteristics (selectivity, accuracy (confidence intervals <1%), repeatability and intermediate precision (RSD% < 2.5%), limit of detection (LOD), limit of quantification (LOQ) and calibration range). The LOD was 0.25 µg/mL and the LOQ was 0.5 µg/mL. Using methanolic solutions of United States Pharmacopoeia (USP) Capsaicin and Dihydrocapsaicin Reference Standards, the method was linear over the concentration range 0.0005-0.5000 mg/mL for both capsaicinoids. The method was applied to qualify capsaicinoid content of two industrial capsicum extracts according to the USP 29.