Determination of Sodium (2-(2, 6-dichloroanilino) phenyl) Acetic Acid in Human Plasma by Rapid and Sensitive HPLC Method and UV-Spectrophotometry: Its Comparative Evaluation

Abstract

Aim: The objectives of current study were to develop and validate a simple, rapid, and sensitive HPLC method for determination of sodium (2-(2, 6- dichloroanilino) phenyl) acetic acid (SDPAA) in human plasma according to US-FDA guidelines and determination of SDPAA by UV- Spectrophotometry. Methodology: In case of HPLC method, the mobile phase composed a mixture of acetonitrile (ACN) and phosphate buffer (pH 6.8) in a ratio of 40:60 ml (pH 3.5). A Shimadzu HPLC machine (HPLC 10 ATVP) with a column Chromolith-R high resolution RP–18 end caped and a column length of 100mm to 4.6 mm, with a UV detector was used. The peak was observed at wavelength of 281 nm. The sample was injected at a flow rate of 1.5 ml per min. The solvent run time was 8 minutes and the average retention time of the six sample observed was 5.66 minutes. In case of UV-spectrophotometric method, Shimadzu UV-1800 spectrophotometer was used. Mixture of phosphate buffer of pH 6.8:ACN was selected as a solvent for determination of SDPAA at 281 nm. Beer law was obeyed in the range of 2-22 µg/ml. The results of both UV-spectrophotometric and HPLC methods in determination SDPAA, were compared. Results: SDPAA was detected at 281nm and eluted at 5.669 min (without plasma) while the samples extracted from the plasma eluted at 5.667 min. The average % RSD was less than 2%. Accuracy was confirmed with the recovery studies and by three test assays. Accuracy was tested at three %age level that is within 95–99%. The linear range of 0.5-20 µg/ml (for HPLC) showed the regression coefficient (R2) 0.999 and linear range of 2-22 µg/ml (for UV method) showed the regression coefficient (R2) 0.998 which were in acceptable range. Conclusion: The developed HPLC method was simple, rapid, reliable, specific and sensitive with ability to determine drug concentrations from human plasma.