Development of simplified method for the detection of capsaicin using high performance liquid chromatography

• A new method was developed for extracting capsaicin from wet and dry chili pepper. • A rapid method for quantifying capsaicin was developed for determining capsaicin using HPLC. • The method was able to detect capsaicin in both types of chili pepper (wet and dry). • Capsaicin extraction was greater when extracted from dry chili pepper indicating moisture interference in extraction yield. Peppers are widely used for their pungency, and Scoville Heat Unit (SHU) is often used to measure the pungency of the peppers caused by compounds called capsaicinoids. Liquid Chromatography (LC) methods have emerged as effective tools for determining capsaicinoids. However, accurate extraction and quantification of capsaicin remain challenging. Here, we developed a simple, rapid, and robust high-performance liquid chromatography-ultraviolet detector (HPLC-UV) method for quantifying capsaicin in green Thai pepper (Bird’s eye chili). Capsaicin separation was achieved using a SiELC Newcrom R1 column (100 × 3.2 mm, 3µm) under isocratic mobile phase consisting of 0.5% formic acid in water and acetonitrile (30%:70%) at a flow rate of 0.4 mL/min. Detection of capsaicin was done at 220 nm and 280 nm wavelengths. Capsaicin extraction from pepper samples was evaluated at 20 min, 60 min, and 120 min intervals. Results showed that a 60-min extraction at 60°C with orbital mixing 150 rpm was sufficient to recover the majority of the capsaicin from green Thai pepper. The method showed excellent linearity across 11 concentration levels (0.5 - 600 ppm) with an R 2 value of 0.99. While capsaicin analysis has been investigated previously, this streamlined HPLC-UV method provides an accessible and effective alternative for improving capsaicin extraction and detection in green chili peppers.