DEVELOPMENT AND VALIDATION OF ANALYTICAL METHODS OF INDOMETHACIN IN RABBIT BLOOD PLASMA BY RP-HPLC TECHNIQUE

Abstract

The objective of the present work involved development of simple, rapid, sensitive, and cost effective RP-HPLC method for the estimation and quantification of indomethacin in rabbit plasma to evaluate their linearity, accuracy, precision, and recovery studies. The spiked plasma serum samples used for the calibration curves were prepared by adding required volumes of standard indomethacin solutions and volume made up to 2 ml by pooled blank rabbit plasma serum to yield final concentrations of 250-5000 ng mL^-1. Each sample was run one by one by injecting 20 μL into the injecting port; after washing the HPLC machine, initially with methanol followed by mobile phase, in a flow rate of 1.5 ml/min, detection was carried out at 240 nm and washing time with mobile phase itself was given to 30 min. Good linearity was obtained for calibration curve. By plotting the peak height vs. the indomethacin concentration (ng ml^-1), the following regression equations were found: Y=39.72X-563.29 (detected at 240 nm; concentration range: 250-5000 ng ml^-1; r² =0.9998). The precision of the method was estimated by calculating the RSD values for the results obtained at two different plasma concentrations. At 1000 & 250 ng ml^-1, the within day reproducibility was 2.18 & 3.34% (n = 3) respectively, while the between day reproducibility was 2.37 & 3.75% (n = 3) respectively. Inter-day precision ranged from 1.38% to 3.75%. The percentage of intra and inter-day accuracy was in the range of 100.1-100.54% and 99.85-99.97%, respectively. The analyte was found to be stable in rabbit plasma when stored at -20^oC. The method was simple, rapid, highly sensitive, accurate, precise, and cost effective for indomethacin after rabbit plasma was simply extracted by liquid-liquid extraction method. Also, this method was extended for determination of indomethacin presence in blood plasma serum.