Liquid chromatography for iothalamate in biological samples

We have previously reported an iothalamate assay for the assessment of the glomerular filtration rate (GFR) that required a long column equilibration time and 22 min run time per sample. We now report a simpler assay that requires a run time of only 5.5 min and is more precise and accurate than the earlier technique. The mobile phase consisted of methanol-acetonitrile-50 mM sodium monobasic phosphate (10:5:85, v/v) at pH 4.4, pumped at a rate of 1.5 ml/min on a C(18) reversed-phase column. Samples of plasma and urine were deproteinized with 1 volume of 4% perchloric acid or 9 volumes of 2% perchloric acid, respectively. No internal standard was used. The diode array detection system collected absorbance at 240 nm and the peak height areas of iothalamate were determined. The iothalamate peak appeared at 3.5 min. Detector response was linear over the range tested (10-2000 microg/ml). Within-run precision was <3% for both plasma and urine and accuracy was 96-102%. Between-day precision for plasma and urine analyses were <7%. The recovery of iothalamate in urine and plasma were 102% and 91%, respectively. There was excellent thermal and pH stability of iothalamate. No interference was found with para-amino hippuric acid (PAH) or N-acetyl PAH, which can be simultaneously assayed, if desired.