Determination of capsaicinoids in different genotypes of Capsicum by validated HPLC method

Capsaicin is a commonly used phytochemical, well-known by its pharmacological properties as analgesic, antidiabetic, hypolipidemic and antitumor agent. Development of a simple method for extraction and quantification of capsaicin from hot pepper fruits (Capsicum annuum L.) gives a chances for effective exploitation of this highly represented agro culture in Republic of Macedonia, and brings an opportunity for further investigations on its pharmacological activity. Therefore, the aim of this study was to determine capsaicinoids (capsaicin, dihydrocapsaicin and nordihydrocapsaicin) in ethanolic extracts of 11 different genotypes of hot pepper fruits from Republic of Macedonia with a validated simple and sensitive HPLC method. Materials and methods: Fruits, dried and grounded, were used as a plant material for Soxlet extraction by using a 96 % (V/V) ethanol as a solvent (70˚C, for 5 hours). Quantification of a capsaicin and dihydrocapsaicin was performed on a RP-HPLC (reverse phase-high performance liquid chromatography) system by using a Zorbax SB-C18 column, (5 μm, 250 × 4.6 mm), mobile phase: H2O/CH3CN, 50:50 (V/V), flow rate: 1.5 mL/min and UV detection at 220 nm. Results: The analytical method was validated by using the protocols set out in the International Conference on Harmonization (ICH) guidelines. The required validation parameters, specificity, linearity, accuracy, precision, limit of detection, and limit of quantification, were studied for capsaicin and dihydrocapsaicin. The linearity range was found to be 1.52 – 380.00 µg/mL for capsaicin, and 1.12 – 279.00 µg/mL for dihydrocapsaicin, respectively. Limit of detection for capsaicin and dihydrocapsaicin was 0.075 and 0.109 µg/mL, and limit of quantification was 0.230 and 0.331 µg/mL for capsaicin and dihydrocapsaicin, consequently. The highest concentration of capsaicin, 2835 µg/g and for dihydrocapsaicin 2443 µg/g was found in the extract obtained from genotype Feferona. Conclusions: The results showed that this method can be employed as quantification method for determination of capsaicinoids in the Capsicum oleoresins