Simultaneous Determination of Phyllanthin and Hypophyllanthin in Herbal formulation by Derivative Spectrophotometry and Liquid Chromatography

Two simple and accurate methods to determine Phyllanthin (PTN) and Hypophyllanthin (HTN) in herbal dosage forms containing Phyllanthus niruri extract, were developed using Zero order first derivative spectrophotometry and reversed–phase liquid chromatography (LC). PTN and HTN in herbal preparations (tablets) were quantitated using the Zero order first derivative responses at 259.2 nm for PTN and 252.4 nm for HTN in spectra of their solution in methanol. The calibration curves were linear with correlation coefficient, r = 0.9983 for PTN and 0.9977 for HTN in the concentration range of 10 to 50 µg/mL for PTN and 4 to 20 µg/mL for HTN. In the LC method, analysis was performed on a Phenomenex C-18 column (250 mm × 4.6 mm ID, 5 µm particle size), with isocratic elution using a mixture of tetrahydrofuran: water: acetonitrile in the ratio of 10:50:40 v/v/v at flow rate of 1 mL/min with UV detection at 230 nm. Both drugs were well resolved on the stationary phase, and the retention times were 16.05 minutes for PTN and 17.61 minutes for HTN. The calibration curves were linear (r = 0.9978 for PTN and 0.9996 for HTN) in the concentration range 10-100µg/mL for PTN and 5-50µg/mL for HTN. Both methods were validated, and the results were compared statistically. They were found to be accurate, precise, and specific. The methods were successfully applied to the estimation of PTN and HTN in herbal formulation containing Phyllanthus niruri extract.