Quantitation of iohexol, a glomerular filtration marker, in human plasma by LC–MS/MS

Abstract

We developed a high-performance liquid chromatography mass spectrometry method for quantitating iohexol in 50 μL human plasma. After acetonitrile protein precipitation, chromatographic separation was achieved with a Shodex Asahipak NH2P-50 2D (5 μm, 2 × 150 mm) column and a gradient of 0.1 % formic acid in acetonitrile and 0.1 % formic acid in water over a 10 min run time. Mass spectrometric detection was performed on a Micromass Quatromicro triple-stage bench-top mass spectrometer with electrospray, positive-mode ionization. The assay was linear from 1 to 500 μg/mL for iohexol, proved to be accurate (101.3–102.1 %) and precise (<3.4 %CV), and fulfilled Food and Drug Administration (FDA) criteria for bioanalytical method validation. Recovery from plasma was 53.1–64.2 % and matrix effect was trivial (−3.4 to −1.3 %). Plasma freeze thaw stability (97.4–99.4 %), stability for 5 months at −80 °C (95.5–103.3 %), and stability for 4 h at room temperature (100.6–103.3 %) were all acceptable. This validated assay using a deuterated internal standard will be an important tool in measuring iohexol clearance and determining glomerular filtration rate (GFR) in patients.