Simultaneous determination of sugars and picrosides in Picrorhiza species using ultrasonic extraction and high-performance liquid chromatography with evaporative light scattering detection

Sugars play a critical role in regulating overall cellular metabolism in high altitude growing plants. These plants are shown to have high levels of sugars to enhance their tolerance to abiotic stresses such as drought and freezing temperature. In the present study, a simple, sensitive, selective and reliable HPLC method based on ultrasonic extraction and evaporative light scattering detection (ELSD) has been developed and validated for the simultaneous determination of important sugars (xylose, xylitol, mannitol, glucose and sucrose) and picrosides (picroside-I and picroside-II) in two species Picrorhiza kurroa and P. scrophulariiflora. The analysis was carried out on a Zorbax amino column (250 mm × 4.6 mm i.d., 5 μm) with isocratic elution of acetonitrile:water (78:22, v/v). The method was validated for accuracy, precision, limit of detection and quantification according to International Conference on Harmonization (ICH) guidelines. The drift tube temperature of the ELSD system was set to 81 °C and nitrogen flow rate was 2.0 standard liter per minute (SLM). The regression equation revealed a good linear relationship (r2 = 0.9997 ± 0.0012) within test ranges. The limit of detection and quantification for seven analytes in ELSD were less than 0.98 and 2.95 μg, respectively. The method showed good reproducibility for the quantification of seven analytes in Picrorhiza species with intra- and inter-day variation of less than 2.0%.