Ultraperformance Liquid Chromatography–Tandem Mass Spectrometry Assay for Iohexol in Human Serum

Abstract

Iohexol is an iodinated contrast dye that has been shown to be useful in the estimation of glomerular filtration rate (GFR) in patients with suspected renal insufficiency. We developed and validated an ultraperformance liquid chromatography (UPLC)-triple quadrupole mass spectrometry (MS/MS) assay for quantifying iohexol in human serum. Sample preparation involved dilution of 50 microL serum with 400 microL water, followed by protein precipitation with zinc sulfate and methanol containing the structural analog ioversol as the internal standard. After 1:20 dilution of the supernatant with water, 5 microL was injected into the UPLC-MS/MS system. Chromatography was performed using a Waters Oasis HLB 5-microm particle size, 2.1 x 20 mm column maintained at 50 degrees C. We used a 1-step acetonitrile/0.1% formic acid gradient to elute the compounds of interest at a common retention time of 0.96 min. The multiple reaction monitoring transitions used for integration and quantification were m/z 821.7-->803.7 for iohexol and m/z 807.9-->589.0 for ioversol in the electrospray positive ionization mode. The assay was linear from 2.5 mg/L (lower limit of quantification) to 1500 mg/L iohexol, with a mean extraction efficiency of >99%. Recovery of nominal target concentrations was 99%-102%. Interassay imprecision ranged from 7.9% at a concentration of 2.5 mg/L to 4.1% at 1000 mg/L. Ion suppression studies showed no matrix effects on the ionization of the 2 compounds. This rapid UPLC-MS/MS method can be successfully used for quantifying iohexol in human serum.