Development and validation of a stability indicative HPLC–PDA method for kaurenoic acid in spray dried extracts of Sphagneticola trilobata (L.) Pruski, Asteraceae

A gradient stability indicative HPLC–UV method was developed and validated for assay of the marker kaurenoic acid (KA) in spray dried extract of Sphagneticola trilobata (L.) Pruski. The marker, and another unidentified polar component, were separated on a Luna Phenomenex C₁₈ column (250×4.6mm, 5μm) with mobile phase composed of acetonitrile:acidified water pH 3.0 with phosphoric acid, in a gradient run of 40min; at a flow rate of 1.0mLmin⁻¹, 35°C, using wavelengths of 210 and 338nm. The method was linear over a KA concentration range of 4.5–30.0μgmL⁻¹, without interference of the herbal matrix on the linearity of the method. The RSD% values for the intra- and inter-day precision studies were <2.0 and <8.0% for inter-laboratorial study. The method showed excellent KA recovery (99.0%). The LOQ value was found to be 1.13μgmL⁻¹ and the method proved to be robust for small, deliberate changes in temperature and pH of the mobile phase with RSD%<2.5% for the KA assay. A forced degradation study of S. trilobata dried extract was conducted under conditions of visible light (1.200.000 l×h⁻¹) and UV (200Whm⁻²) irradiation, acid (0.5molL⁻¹ HCl, 30min), basic (1mol L⁻¹ NaOH, 2h) and oxidative (30% H₂O₂, 4h) hydrolysis, in order to develop a gradient stability-indicating LC–UV method for KA quantification, the selected marker, and also to detect the major polar components of the extract, under investigation. The KA contents remaining after these stress conditions were 72.3, 70.0, 97.6, 65.8 and 87.0%, respectively. The alkaline conditions resulted in higher degradation for the unknown polar components of the extract, without interference of supplementary peaks at the retention time of the KA. This method can be used for the KA assay and qualitative analysis of polar components in stability study of spray dried extracts of S. trilobata, for subsequent use in the quality control of dosage forms.