Optimized Extraction of Oleoresin Capsicum and Analytical Method Validation for Capsaicin using HPLC

Background: Capsaicin, the main active constituent of oleoresin capsicum present in capsicum fruits, is responsible for their pungency and color. Capsaicinoids is an important phytoconstituents and hence exported for various benefits. Hypothesis of work: The process of extraction of oleoresin capsicum by reflux method was to be optimized using in terms of their percentage yield. Further ICH complicent analytical method for quantitative analysis of caipsaicin using HPLC was developed and validated in present study. Methods: Critical extraction parameters like extraction time, temperature and solvent were optimized to develop an efficient method for extraction of oleoresin from capsicum fruits. A simple, rapid and stable HPLC method of capsaicin was developed using C₁₈ (2) HPLC column, 250x4.5 (mm), particle size 5 μm and validated using various parameters viz., linearity, accuracy, precision and LOD and LOQ using methanol and HPLC grade water (65:35v/v) as mobile phase. The elution was performed at 280 nm with run time of 10 min and flow rate of 1 mL/min. Results: The optimized conditions for oleoresin extraction were found to be 40°C (temperature), 5 hr (extraction time) and acetone as a solvent with highest percentage yield of (3.7% w/w). Furthermore, the developed HPLC method showed linear response over a concentration range of 1-9 μg/mL with standard regression equation (y=4614.9x+5344.1.9) with a coefficient of correlation value, of R² 0.9974. The validation parameter evaluated using this method showed effective and satisfactory results as per ICH Q2 (R1) guidelines and percentage recovery ranged from (98%-99.71%) indicated good accuracy. The inter and intraday precision with relative standard deviations of capsaicin < 1%, while the LOD and LOQ 1.04 to 3.03 μg/mL respectively. The oleoresin capsicum extract was analyzed for the content of capsaicin using HPLC showing the same retention time with highly pungency value. The percentage of capsaicin in the extract was 7.30%. Conclusion: The developed extraction and analytical method for capsaicin and oleoresin capsicum was accurate, precise, stable and may be useful for routine analysis of capsaicin content in capsicum fruits as well as various pharmaceutical and food preparations.