A validated LC-MS/MS method for the simultaneous quantification of iothalamate and hippuran in serum and urine for non-radioactive kidney function assessment

• Highly accurate and precise LC-MS/MS methods for quantifying non-radioactive exogenous filtration markers to assess kidney function. • Simultaneous measurement of iothalamate and hippuran in both serum and urine, with detection limits reaching the low to sub-ng/mL range. • The validated method is suitable for determining concentrations in patient samples. A novel liquid chromatography-tandem mass spectrometry method is described for the quantitative determination of the kidney function markers iothalamate and hippuran in human serum and urine. It is based on protein precipitation with methanol followed by dilution of the supernatant for serum and simple dilution for urine. The polar analytes are chromatographically separated by a 6.5-min gradient on a low-ligand density reversed-phase column; detection is performed by electrospray ionization tandem mass spectrometry in the positive ion mode against stable-isotope labeled internal standards. The results of a thorough method validation show that iothalamate and hippuran can be simultaneously quantified in the concentration ranges 0.500–30.0 ng/mL and 10.0–5000 ng/mL for serum and urine, respectively, with values for CV and absolute bias not exceeding 10 %, and with sufficient stability in all relevant matrices and solvents. The method was successfully applied for the analysis of serum and urine samples of multiple individuals who received both iothalamate and hippuran.