Fast determination of capsaicinoids from peppers by high-performance liquid chromatography using a reversed phase monolithic column

This article reports the development of a rapid and reproducible method of HPLC with fluorescence detection for the determination and quantification of the main capsaicinoids (nordihydrocapsaicin, capsaicin, dihydrocapsaicin, homocapsaicin and homodihydro-capsaicin) present in hot peppers by employing a monolithic column. The type of column employed is a RP-18e (100 mm × 4.6 mm) monolithic column. A gradient method was utilised for the chromatographic separation: solvent A: water (0.1% acetic acid) and solvent B: methanol (0.1% acetic acid). A study was also made of the robustness of the method in respect of the conditions of temperature in the separation column (15–40 °C), the solvent flowrate (4–7 mL min−1), the injection volume (10–50 μL), and the percentage of methanol in the sample (25–100%). The repeatability and reproducibility of the method showed relative standard deviations of less than 2%. The robustness of the method was determined by utilising different injection volumes and different percentages of methanol in the extracts. The method developed has then been utilised for the quantification of the major capsaicinoids present in different varieties of hot peppers grown in Spain. The capsaicinoids have been separated in a time of less than 8 min.