Iohexol assay for direct determination of glomerular filtration rate: optimization and development of an HPLC-UV method for measurement in serum and urine.

Determination of glomerular filtration rate (GFR) is considered the best indicator of renal function. Iohexol, a non-ionic contrast agent, is currently considered to be a reference marker since it meets all the requirements of an ideal GFR marker. The aim of our work is to develop and optimize a method for iohexol measurement by HPLC-UV. The results showing the following optimal conditions : a mobile phase with water and 5% of acetonitrile, a C18 analytical column (250 × 4 mm, 5 μm particle size) with a temperature of 40 °C and a flow rate of 1 ml/min. Serum samples are deproteinized by addition of perchloric acid (6%), while urine samples are only diluted. For both matrices, the method is linear (r > 0.99) and the recovery is > 98%. For selectivity, no interfering endogenous components at the retention time of iohexol was observed. The results of the matrix effect showed a clinically acceptable variation in most concentration levels except for 100 μg/mL, in urine, where the effect was slightly present. Iohexol stability in urine decreases significantly only after 3 freeze-thaw cycles and after freezing at -80 °C for two months. According to those findings results this method is simple, specific, linear, precise and robust, which allows its application for the direct measurement of the GFR after its analytical validation.