Development and validation of HPLC assay for estimation of Irbesartan in Human Plasma

A new simple and selective high performance liquid chromatography method with UV detection at 245 nm was developed for quantification of irbesartan in human plasma using valsartan as internal standard (IS). A simple liquidliquid extraction technique with high recovery of irbesartan was developed using ethyl acetate as extracting solvent. Chromatographic separation was performed on Agilent Eclipse C₁₈ column (5μ, 4.6 mm x 150 mm) using acetonitrile-ammonium acetate buffer (pH 4.0, 20mM) (40:60, v/v), pumped at flow rate of 1 mL/min. The run time was kept at 15 minutes. The method was validated according to recent EMEA guideline on bioanalytical method validation. The lower limit of quantification of irbesartan in plasma was 250 ng/mL. The calibration curve was linear over range of 250 ng/mL to 4000 ng/mL. Within-run and between run accuracy and precision of method were within the acceptable limits. Short-term, freeze-thaw and long term stabilities in plasma were successfully evaluated. The described method can be applicable to give accurate measurements of irbesartan in real clinical samples.