Determination of iohexol in human serum by a semi-automated liquid chromatography tandem mass spectrometry method

Objectives: Measured glomerular filtration rate (mGFR) is the best indicator of renal function in children and adolescents. GFR determination using iohexol clearance has been increasingly accepted and applied in clinical practice because it is accurate, readily available, non-radioactive, safe and is used intravenously even in the presence of renal disease. This study describes the development and evaluation of a semi-automated method for determination of iohexol in human serum using liquid chromatography coupled with electrospray ionization (ESI) tandem mass spectrometry (LC-MS/MS). Design and methods: Iohexol was extracted from serum using a MICROLAB® NIMBUS4 automation robot and supernatant was dried under nitrogen gas and reconstituted in mobile phase. Ioversol was used as the internal standard. Chromatography was performed using a C-8 analytical column (Phenomenex, 3. μm, 50 × 3.0. mm I.D.) at room temperature and a gradient LC method on a Waters 2795 Alliance HT HPLC system. The flow rate was 0.5. mL/min and the retention times were 2.36. min and 2.14. min for iohexol and ioversol, respectively. Detection by MS/MS was achieved using a (Micromass Quattro Micro) tandem mass spectrometer operated in the ESI-positive mode. The multiple-reaction monitoring (MRM) method used ion transitions m/z 821.9 to 803.7 for iohexol and m/z 807.9 to 588.7 for ioversol. Method validation studies were conducted to determine the linearity, accuracy, precision, matrix effects and stability. A method comparison of blinded, residual patient samples was conducted with a well-established method. Results: The method was linear from 7.7 μg/mL to 2000.0 μ g/mL. The low limit of quantification and the detection limit were established at 7.7 and 3.0μg/mL, respectively. Within-run and between-run precisions were found to be <6% CV and measured values deviated no more than 5% from target concentrations. Carryover and matrix effects were not significant. Comparison to a well-established method showed very good agreement with correlation coefficient of 0.996 for iohexol and 0.993 for GFR/1.73m². Conclusions: This method accurately and precisely quantifies iohexol in 50. μL of serum, enabling determination of mGFR by iohexol clearance. The method is highly correlated to a reference method. Use of an automated liquid handler reduces labor-intensive, manual sample preparation steps. The stability of this analyte and the robustness of this assay fit well within our clinical workflow and we have successfully applied this method to determine mGFR in pediatric patients.