Based on the Association of Official Analytical Chemists official Method 970.64 , two grams of hot pepper sample were ground using a mortar and pestle and transferred to a 100-mL flask covered with a stopper. The sample was blended for one minute with a mixture of 30 mL hexane:acetone:ethanol:toluene (10:7:6:7). For hot saponification, 2 mL of 40% methanolic KOH were pipetted into the flask, swirled for one minute and placed in a 56 °C water bath for 20 min. The sample was cooled for one hour in the dark, and then, 30 mL of hexane were pipetted into the flask, dried over anhydrous sodium sulfate made up to volume and shaken vigorously for one minute. Upper phase was 50 mL. Absorbance was measured at 436 nm using the IRMECO UV-Vis spectrophotometer Model U2020 with β-carotene (Sigma-Aldrich, St. Louis, MO, USA) as the standard.