Development and Validation of RP-HPLC Method for Simultaneous Determination of Apigenin and Luteolin in Ethanol Extract of Clerodendrum serratum (Linn.) Leaves

Shanmuga Sundaram Rajagopal, Babitha K Vazhayil, Liz Varghese, Mahadevan Nanjaian


A simple, specific, precise, accurate, and sensitive method for separation and quantification of two flavonoids mainly apigenin (API) and luteolin (LUT) by reverse phase high performance liquid chromatography (RP-HPLC) was developed and validated. Flavonoids present in the leaves of Clerodendrum serratum L. (C. serratum) were analyzed and quantified. Analysis was carried out on enable C18G column (250 mm × 4.6 mm i.d, 5 µm) as stationary phase, mobile phase consisting of methanol-acetonitrile-acetic acid-orthophosphoric acid-water (40:20:0.05:0.05:40) at a flow rate of 0.6 mL min-1 and detection wavelength at 352 nm. The proposed method was validated by ICH Harmonized Tripartite Guidelines, Validation of Analytical Procedures: Text and Methodology Q2 (R1). In this study, an excellent linearity was obtained with correlation coefficient (r2) higher than 0.999. Besides, the chromatographic peaks also showed good resolution. Other validation parameters including precision, specificity, accuracy, and robustness demonstrated good reliability in the quantification of apigenin and luteolin. Thus the newly developed and validated method can be conveniently used for the quantification of API and LUT in leaves of C. serratum L. leaves and also be applied to standardization of multicomponent herbal remedies containing C. Serratum.



Clerodendrum serratum, Apigenin, Luteolin, RP-HPLC

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