Development and validation of an HPLC-UV method for determination of iohexol in human plasma

An HPLC-UV analytical method for estimation of iohexol in human plasma was developed and validated. Protein precipitation and iohexol extraction from plasma (100 microl) was carried out by adding 800 microl perchloric acid (5%, v/v in water) containing iohexol related compound B as the internal standard followed by vortex mixing and centrifugation. The supernatant (90 microl) was then injected onto a microBondapak C(18) column (150 mm x 3.9 mm, 10 microm) maintained at 30 degrees C. The mobile phase comprised of various proportions of acetonitrile and water with a total run time of 12 min and the wavelength of the UV detector was set at 254 nm. The extraction recovery of iohexol from plasma was >95% and the calibration curve was linear (r(2)=0.99) over iohexol concentrations ranging from 10 to 750 microg/ml (n=8). The method had an accuracy of >92% and intra- and inter-day CV of <3.7% and <3.6%, respectively. The method reported is simple, reliable, precise, accurate and has the capability of being used for determination of iohexol in clinical settings.