Simultaneous Estimation of Scopoletin, Bacopaside-II, Bacopasaponin-C, Withanolide-A, and Withanoside-IV in a Clinically Proven Polyherbal Formulation for Treatment of Insomnia

The present work is aimed to develop and validate a reverse phase high performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of Scopoletin, Bacopaside-II, Bacopasaponin-C, Withanolide-A, and Withanoside-IV in a proprietary polyherbal formulation containing Bacopa monnieri (Brahmi), Convolvulus pluricaulis (Shankhapushpi), Withania somnifera (Ashwagandha), Nardostachys jatamansi (Jatamansi), Myristica fragrans (Jatiphal) and Valeriana wallichii (Tagar) extracts intended for the treatment of insomnia. The HPLC analysis was performed on a Inertsil ODS, 3V, 250 x 4.6 mm x 5µm, C18 column using 0.1% orthophosphoric acid buffer as the mobile phase (solvent A) and acetonitrile (solvent B) with the gradient: 0-5 min, 10-20% B; 5-10 min, 20-30% B; 10-25 min, 30% B; 25-30 min, 30-40% B; 30-40 min, 40% B; 40-45 min, 40-60% B; 45-48 min, 60% B; 48-50 min, 60-30% B; 50-52 min, 30-10% B and 52-55 min, 10% B at a flow rate of 0.8 ml/min. The detection wavelength was chosen at 227 nm for Withanoside-IV and Withanolide-A, for Scopoletin, Bacopaside-II and Bacopasaponin C, it was 205 nm. The HPLC method was validated as per ICH guidelines for linearity, LOD and LOQ. The calibration curve of all the five phytomarkers showed excellent linear correlation coefficients with values (r2=0.996) for Scopoletin, (r2=0.995) for Withanoside-IV, (r2=0.996) for Withanolide-A, (r2=0.996) for Bacopaside-II and (r2=0.999) for Bacopasaponin-C. Limits of detection (LOD) were 0.04, 0.43, 0.35, 0.39 and 0.18 μg/ml and limits of quantification (LOQ) were 0.12, 1.29, 1.06, 1.18 and 0.54 μg/ml for Scopoletin, Withanoside-IV, Withanolide-A, Bacopaside-II and Bacopasaponin-C respectively. The developed HPLC method showed good separation of all the five constituents, enabling efficient analysis of Scopoletin, Withanoside-IV, Withanolide-A, Bacopaside-II, and Bacopasaponin-C in the polyherbal formulation