B-171 Iohexol Mass Spectrometry Method Validation and Comparison in Clinical Pediatric Laboratory
Authors: Li X , Kyanna K 
Published: October 2024

Abstract

Background
Accurate assessment of kidney function is important yet challenging in pediatric patients due to changing muscle mass affecting serum creatinine. There are several key methods to assess kidney function in this population, including estimated glomerular filtration rate (eGFR) from serum creatinine, urine creatinine clearance, and eGFR from serum iohexol clearance, which often provides a more reliable estimate. Iohexol is a non-ionic iodinated contrast agent filtered freely through the glomeruli without tubular secretion, making it an ideal GFR marker.In this study, we validated the LC-MS/MS method to quantitatively measure iohexol in clinical pediatric plasma and serum samples. The clinical performance was compared with the novel Verispray method.

Methods
A series of iohexol standards (Caymen Chemical) were prepared at 10, 100, 500, 1000 µg/mL in serum (UTAK). The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and Verispray method were developed to quantitatively measure iohexol concentrations in spiked pediatric specimens. The internal standard (IS) solution was prepared with Iohexol-D5 (Cayman Chemical) at 9 µg/mL in UPLC grade methanol. Specimen or calibration/QC (50 µL) were mixed with IS solution (450 µL), and then centrifuged at 14,000xg for 3 minutes. The supernatants were analyzed. For LC-MS/MS, 3 µL supernatants were chromatographed on a AccucoreTM Vanquish C18 50 x 2.1mm (Thermo Scientific) using a gradient of water with 0.1% formic acid (FA) against methanol with 0.1% FA. Iohexol and IS were measured by multiple reaction monitoring using a TSQ Quantis Plus Mass Spectrometer (ThermoFisher). For Verispray, 10 µL supernatants were loaded on the Verispray sample plate (ThermoFisher) without LC portion. The samples were extracted from the plate paper using 95% acetonitrile with 5% water and 0.1% FA before directly injected into the mass spectrometer. The LC-MS/MS method was evaluated for linearity, patient comparison, carryover, matrix effect, and stability. 40 patients were analyzed between methods.

Results
Linearity was assessed over 5 days at 4 calibrator levels, the coefficient of variation (CV) ranged from 3.46 - 5.99%. The patient comparison of 40, 20 serum and plasma respectively, was conducted with Mayo Clinics, and good concordance was observed. Matrix effects were evaluated with spiked samples prepared with 50% mobile phase A and B, the ion enhancement was at 10.7%, within the allowable limits. No significant carryover was seen. The samples were stable at room temperature for 48 hours and 4oC for 7 days. Freezing is not recommended. The comparison between Verispray and LC-MS/MS showed significant bias between and within the performing personnel, the deviation can be up to 40% comparing to LC-MS/MS method.

Conclusions
The LC-MS/MS method demonstrated stable and consistent analytical performance for iohexol measurement. However, the Verispray method showed significant inter- and intra-user variabilities. As the Verispray sample spot can only be used once, respotting is required for any additional measurement, reducing reproductivity. It is highly recommended to duplicate all samples when using Verispray, to limit preparation to one user per patient run. Overall, our validation indicates the LC-MS/MS method provides reliable performance as a useful alternative to other complex gold standard pediatric tests of kidney function.