UPLC-UV and UPLC-MS/MS method development for the quantification of iohexol in plasma.

Abstract

The kidneys main role is to maintain homeostasis in electrolyte and fluid balance and blood pressure. Glomerular filtration rate (GFR) is considered the ideal marker for evaluation of the overall health of the kidneys. Measuring the GFR gives an estimate of the complex function of the kidney and can predict how diseases are progressing and gives an opportunity for early diagnosis of renal disease. An accurate measure of GFR in patients with diagnosed kidney disease is at upmost importance for a followup and to determine course of treatment.Iohexol is a stable compound that has been used as a GFR marker for 30 years. Iohexol is a non- ionic contrast agent and is well suited as the marker of choice for GFR determination. Iohexol is filtered in the glomeruli and has a low extra renal excretion, low protein binding and is neither secreted nor absorbed by the kidney and is non-toxic, carries a low cost and is stable in plasma. Several different methods have been described for measuring GFR using iohexol including LC-MS/MS.This method was optimized using Design of Experiments and will be validated according to the FDA guidelines for bioanalytical method validation, EMA (Guideline Bioanalytical Method Validation – European Medicines Agency) and CLSI (C62-A LC-MS methods; Approved Guidelines). Sample preparation performed with protein precipitation using methanol.A test, to show the current status of the method was performed by establishing a calibration curve which represents the reference range and the corresponding quality coltrols (QCs). Baseline separation of the peaks was achieved with the desirable delta retention time (RT). The method will then be fully validated in the upcoming months at Landspítali, department of Clinical Biochemistry.