Development and validation of RP-HPLC-PDA method for identification and quantification of major active constituent in Cynodon dactylon (L.) pers.

In this research, two primary bioactive components were isolated from Cynodon dactylon (L) Pers, commonly known as Durva grass. A gradient reverse phase High performance liquid chromatography coupled to Photodiode array detector (RP-HPLC-PDA) technique was established to identify and quantify these major bioactive constituents in 70% hydroalcoholic extracts of Cynodon dactylon (L) Pers. The chromatographic separation was achieved using an RP-C18 column (250 mm × 4.6 mm, 5 µm), Alliance (Waters) Empower3 liquid chromatography system, and a gradient mobile phase consisting of 5% acetic acid and acetonitrile. The flow rate was set at 1.2 mL/min, and a PDA detector at 320 nm was employed to analyze column effluents, targeting the active compounds' isotactic point. The retention times for the compounds p-coumeric and ferulic acid were determined as 24.11 and 31.52 min. The method exhibited linearity within the concentration range of 2–10 µg/mL, with regression coefficients of 0.998 and 0.9967 for the respective compounds. The mean recovery of p-coumeric and ferulic acid were found to between 102.64 and 109.44%. The RP-HPLC method was validated in accordance with ICH guidelines. KEY WORDS: 4-Hydroxycinnamic acid, Durva grass, Bioactive, Liquid-liquid extraction, RP-HPLC Bull. Chem. Soc. Ethiop. 2024, 38(6), 1533-1542. DOI: https://dx.doi.org/10.4314/bcse.v38i6.3