An improved high-performance liquid chromatography (HPLC) method for analysis of capsaicinoids in dried Capsicum fruit powder, involving changes in extraction, mobile phase, flow rate, and excitation and emission spectra and resulting in reduced analysis time, increased sensitivity, and safety, is reported. Extraction of Capsicum fruit powder using acetonitrile proved to be the best capsaicinoid extractor in the shortest time interval. Solvents used for HPLC separation and quantification of capsaicinoids include methanol and water at 1 ml·min–1 flow rate. Instrument sensitivity is enhanced by altering the fluorescence detector excitation and emission wavelengths. Two analytical methods have been developed. One method determines total amount of heat units in 7 minutes, while the other provides total amount of heat units as well as separation of all present major and minor capsaicinoids in 20 minutes. These improved techniques provide inexpensive and rapid methods for quantitative and qualitative analysis of capsaicinoids in Capsicum fruit samples along with good sensitivity and no interference or confounding peaks.

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