Comparing and optimizing precise methods for measuring capsaicin content in dried peppers

This study evaluated the efficacy of two distinct Enzyme-Linked Immunosorbent Assay (ELISA) kits for quantifying total capsaicinoids in dried peppers, benchmarked against High-Performance Liquid Chromatography (HPLC) as the reference method. Our analysis revealed a high correlation between the results from the ELISA-1 kit and HPLC (R² = 0.9335, p < 0.0001), confirming its accuracy and reliability. In contrast, the ELISA-2 kit proved unsuitable, yielding inconsistent data. To maximize the utility of the more cost-effective ELISA-1 assay, we systematically optimized its extraction protocol. The optimal conditions were established as using 80-mesh sieved pericarp powder, 95% ethanol as the extraction solvent, and an ultrasonic treatment of 15 minutes, which significantly enhanced capsaicinoid recovery. Consequently, this research establishes an optimized protocol, validated through its strong correlation with HPLC, that offers an efficient and economical workflow for capsaicinoid analysis. This provides a valuable tool for high-throughput screening in pepper breeding programs and for industrial quality control. • Compared the adaptability and accuracy of three widely used capsaicin determination methods. • ELISA has been optimized to extract higher levels of capsaicin. • ELISA is a cost-effective alternative comparable to HPLC.