Method optimization and validation for the determination of major phenolic compound in daphne sericea and daphne gnidioides species by HIGH performance liquid chromatography and determination of biochemical content of these species in different development stages

In this study, optimum extraction parameters for herba Daphne sericea subsp sericea Vahl. collected in the flowering period were determined according to the results of total phenolic content analysis. Biochemical characterization of different (leaf, flower and herba) parts of D. sericea and D. gnidioides collected in pre-flowering, flowering and post-flowering stages was carried out. In order to investigate biochemical contents of two species, the total phenolic content, total flavonoid content, amounts of antioxidant activity by DPPH and FRAP methods, total chlorophyll content, total carotenoid content, total anthocyanin content, vitamin C content, amounts of phenolic and flavonoid components, amounts of organic acid components, amounts of sugar components, total oil yield and amounts of fatty components analysis were performed. Additionally, method optimization and method validation were carried out by High Performance Liquid Chromatography (HPLC) method for the determination of rosmarinic acid substance found in D. sericea and D. gnidioides. The optimum extraction solvent was determined as 70 % (V/V) methanol, the optimum temperature was obtained as 65 °C and the optimum extraction time was found to be 24 hours. Total amounts of phenolic compounds in D. sericea and D. gnidioides species were obtained in the range of 26.89-38.90 mg GAE/g and 39.33-74.94 mg GAE/g, respectively. For these species, total amounts of flavonoid substance were determined to be 22.21-30.57 mg CE/g and 11.10-31.0 mg CE/g, respectively. In D. sericea and D. gnidioides species, the amounts of vitamin C were in the range of 21.08-27.50 mg/100 g and 28.13-50.83 mg/100 g, respectively. Catechin, syringic acid, sinapic acid, rosmarinic acid, chlorogenic acid and epicatechin phenolic components were detected in D. sericea plant specie. Nonacosane, 1-eicosanol, hexacosane, linoleic acid, palmitic acid and oleic acid were determined as the main fatty components in D. gnidioides plant specie. The different mobile phase compositions were tested to optimize the method for the determination of rosmarinic acid content in D. sericea and D. gnidioides species and the optimum mobile phase composition was found to be acetonitrile/methanol/formic acid mixture (10/90/0.5, V/V/V). Optimum flow rate was obtained as 0.4 mL/min. Optimum column temperature was detected to be 30 °C and optimum injection volume was found as 10 µL. In order to perform method validation, linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ) and selectivity parameters were obtained. Recovery (%) value was determined as 101.36 ± 7.19. LOD and LOQ values were calculated as 0.37 mg/L and 0.95 mg/L, respectively.