Simple HPLC–UV method for determination of iohexol, iothalamate, p-aminohippuric acid and n-acetyl-p-aminohippuric acid in human plasma and urine with ERPF, GFR and ERPF/GFR ratio determination using colorimetric analysis

A simple high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of iohexol, iothalamate, p-aminohippuric acid (PAH) and n-acetyl-p-aminohippuric acid (n-acetyl-PAH) in human plasma and urine. A C18 column at a flow rate of 1 ml/min with an aqueous mobile phase of trifluoroacetic acid (0.1% TFA in deionized water (pH 2.2), v/v) and methanol gradient was used for component separation. The plasma and urine assay demonstrated linearity from 10 to 50 μg/ml for iohexol and iothalamate, 5 to 40 μg/ml for PAH and 2.5 to 40 μg/ml for n-acetyl-PAH. The HPLC plasma and urine results obtained for PAH were used to calculate the subject kidney effective renal plasma flow (ERPF) and the iohexol results were used to calculate the subject kidney glomerular filtration rate (GFR). The HPLC results for PAH were then compared to an alternative colorimetric method for analyzing PAH to determine if subject metabolism (acetylation) of PAH affected the ERPF results obtained using the colorimetric method, the subsequent ERPF/GFR ratio and clinical impression of subject patient kidney function. The method was utilized in several different clinical studies evaluating the effect of kidney function from medications (phase IV evaluations) marketed for patients with cardiovascular disease.