BIOANALYTICAL HPLC METHOD DEVELOPMENT AND VALIDATION FOR QUANTIFICATION OF ASIATIC ACID FROM CENTELLA ASIATICA LINN. Research Article

Centella asiatica Linn. (Family Apiaceae) is widely used in Indian system of medicine. Early there in vivo study had developed but oral administration of extract in rat was not developed. The objective of present study is to develop and validation of the bioanalytical method using a HPLC of Asiatic acid. A HPLC system with UV-vis. detector for the quantitative determination of total concentration of Asiatic acid albino wistar rat whole blood. Asiatic acid was extracted with n-hexane:dichloromethane:2-propanol (20:10:1,v/v/v) from whole blood. Chromatographic separation was used RP-C18 column with solvent system consisting of watermethanol in gradient mode and detection at 205nm using PDA detector. For validation of method by standardizing the parameters like LOD, LOQ, sensitivity, selectivity, accuracy and precision, extraction recovery and stability were validated. A calibration curve ranging from 0.25-50µg/ml was shown to be linear and the lower limit of quantification was 0.25µg/ml. The intra-day and inter-day precisions which were determined by five different concentrations ranged from 4.25%-1.06% and 4.03%-1.50% respectively. Extraction recoveries were not less than 73.72%. Whole blood samples containing Asiatic acid were stable for 14 days at -20oC. The method successfully applied to a Pharmacokinetic study in rat after oral administration of Centella asiatica extract for three different doses liked 600, 800 and 1000mg/kg. The parameters of Pharmacokinetic obtained were T1/2: 12.1, 12.65, 12.59hrs; Tmax: 4hrs; Cmax: 1.8, 2.35, 2.9µg/ml; AUC0-t and AUC0-∞:17.2 and 23.4µg/ml, 3.23 and 24.82µg/ml, 29.13 and 30.79µg/ml respectively. The developed method was simple, reproducible, sensitive and specific for determination of Asiatic acid from whole blood of rat.