Simple and micro high-performance liquid chromatographic method for simultaneous determination of p-aminohippuric acid and iothalamate in biological fluids.
A simple, rapid and micro high-performance liquid chromatographic method was developed for separate or simultaneous determination of p-aminohippuric acid and iothalamate in plasma and urine using p-aminobenzoic acid as an internal standard. The method involved deproteinizing samples with two volumes of acetonitrile followed by injection of 5 microliters of deproteinized supernatant onto a C18 reversed-phase column. The mobile phase contained 3.5% acetonitrile in 0.04% phosphoric acid and flowed at a rate of 1.5 ml/min. The column effluent was monitored by an ultraviolet detector at 254 nm. Retention times for p-aminohippuric acid, iothalamate and p-aminobenzoic acid were approximately 4.5, 6 and 8 min, respectively. This method requires as little as 5 microliters of sample and can be used to measure accurately down to 1 microgram/ml p-aminohippuric acid and 0.5 microgram/ml iothalamate in plasma samples. The coefficients of variation of the assay with or without the use of internal standard were generally low (below 7%). No interferences from endogenous substances or any drugs tested were found.
D010129:4-Aminobenzoic Acid / Q000032:analysis; D000618:Aminohippuric Acids / Q000032:analysis*; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004285:Dogs; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007483:Iothalamic Acid / Q000032:analysis* / Q000097:blood / Q000652:urine; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D010130:p-Aminohippuric Acid / Q000032:analysis* / Q000097:blood / Q000652:urine
D000618:Aminohippuric Acids; D007483:Iothalamic Acid; D010129:4-Aminobenzoic Acid; D010130:p-Aminohippuric Acid