Abstract

DEVELOPMENT AND VALIDATION OF HPLC-UV METHOD FOR THE DETERMINATION OF E AND Z GUGGLESTERONE IN RAT PLASMA: APPLICATION TO PLASMA PROTEIN BINDING STUDY

Shriyans Jain*, Vikas Kumar, Aina and Rajvir Singh

Abstract

A simple, sensitive, precise and accurate reversed phase liquid chromatographic method has been developed for the estimation of E and Z guggulsterone in rat plasma. The method was developed using a Phenomenex C-18 Column (150 x 4.6mm, 5μ) with a mobile phase consisting of Methanol : TDW (0.5% Formic Acid) – 75 : 25, at a flow rate of 1.0 mL min-1. With prednisone as internal standard, plasma samples were extracted by manual solid-phase extraction method using C-18 SPE cartridges. Detection was performed by HPLC-UV detector. The UV detection was achieved at 240 nm, over a wide dynamic range of 0.62 to 20 μg mL-1. The retention time of E & Z guggulsterone and internal standard was 7.2, 9.2 and 11 minute respectively. The method was successfully validated in accordance to FDA guidelines acceptance criteria for specificity, linearity, accuracy, precision,. Calibration curve was linear over the concentration range of 0.62 to 20 μg mL-1 (r2  0.998) for both analytes. The intra- and inter-day accuracy and precision method were within -4.05 to 5.92 (% bias) and 3.97 to 12.98 (% RSD) respectively. The analytes were stable after three freeze-thaw cycles. The method was successfully applied to protein binding study of E and Z guggulsterone in rat plasma.

Keywords: Guggulstrone, HPLC, Rat Plasma.