Development of a simple HPLC-UV method for determination of GFR by serum iohexol clearance

The glomerular filtration rate (GFR) is considered the best indicator of kidney function. Iohexol, a contrast agent, is currently considered to be a reference marker since it meets all the requirements of an ideal GFR marker. The aim of this study was to develop and validate a method for iohexol measurement using high performance liquid chromatography with ultraviolet detection (HPLCUV). The method developed includes a mobile phase with water and acetonitrile with a gradient of 5%-30% organic, a C18 analytical column (100 × 4.6 mm, 2.7 µm particle size) at a temperature of 40 °C and a flow rate of 0.5 ml/min. Serum samples were deproteinized by addition of perchloric acid (5%). The assay met the sensitivity cut-off with a mean signal-to-noise ratio of 17.2 at a level of 10 µg/mL iohexol. Mean recovery was 103.7% (CV=4.4%). The dilution experiment allowed for 5-times dilution up to iohexol levels of 500 µg/mL with an accuracy of 103.1% (CV=1.3%) For selectivity, no interfering endogenous compounds at the retention time of iohexol were observed. The matrix effect experiment showed a clinically acceptable variation at all concentration levels with a CV of the slopes of 2.7%. Stability of the stock solution was proven for at least 9 months at -80°C. Sample post-extraction stability was adequate at 84 hours. Interference testing yielded between -7% and -12% difference at 2% haemolysis and -9% to -13% difference at 10% lipaemia. With regards to these findings, this method is simple, specific, linear, precise and robust, which allows its application for the measurement of GFR in serum.

Grantor

Department of Clinical Laboratory Sciences

Year dc:date.issued

2022

Author dc:creator

• van Der Westhuizen, Diederick J

Advisors dc:contributor.advisor

• Pillay, Joanne
• van der Watt, George

• Medicine

Handle dc:identifier.uri

http://hdl.handle.net/11427/37046

OAI identifier oai:identifier

oai:open.uct.ac.za:11427/37046