Determination of Picrosides, Phenolics and Cucurbitacins by Ultra-High Performance Liquid Chromatography-Diode Array Detection in Picrorhiza kurroa Royle Ex Benth

Picrorhiza kurroa in the Indian system of medicine is used to treat jaundice, fatty liver, diabetes and respiratory disorders. Quality control methods have been reported earlier for picrosides-I and II, but extended coverage of markers is required for reliable authenticity. Therefore, the present study is aimed to develop and validate a UHPLC-DAD method for concurrent estimation of picrosides (I-III), p-hydroxyacetophenone glucoside, cucurbitacin B hydrate, gallic, caffeic, syringic and cinnamic acids. The method was developed and validated as per the International Council for Harmonisation guidelines for linearity, limits of detection and quantification, precision (inter- and intra-day), reproducibility, stability and recovery. This method was applied to estimate picrosides, phenolics and cucurbitacins in leaves and rhizomes. The separation of molecules was achieved at 260 nm in 20 min on a C-18 BEH column (2.1 x 100 mm, particle size of 1.7 mu m) with gradient elution using 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Linearity (r(2)) of standards was found to be 0.999, limits of detection and quantification were between 0.06-1.4 and 0.19-4 mu g/mL, respectively. The recoveries of molecules were in the range of 86.6-102.7%. The method was found to be reproducible, uniform and specific. The contents of identified analytes in rhizomes were the highest in ethanol, followed by methanol and water extracts, whereas water extract of leaves contained the highest content of analytes followed by ethanol and methanol. The UHPLC-DAD method was validated for the first time to estimate simultaneously nine analytes. This method will help in quality control of Picrorhiza based material and agriculture interventions.