Analysis of N-benzoyl-L-tyrosyl-p-aminobenzoic acid (bentiromide) metabolites in urine by ion-pair high-performance liquid chromatography.

A high-performance liquid chromatographic method is described for the analysis of bentiromide metabolites in urine. The procedure involves no more than direct injection of the diluted urine sample, obviating the need for an extraction step or an internal standard. A mu Bondapak C18 column is used with a mobile phase of 0.01 M tetrabutylammonium chloride (pH 7.4)--methanol (9:1). A flow-rate of 1.4 ml/min, detection at 254 nm and column temperature of 40 degrees C are employed. These conditions were achieved by investigating the effects of mobile phase pH, and concentrations and types of organic modifiers, buffers and ion-pairing agents on the resolution of the metabolites. The analysis time is 18 min per sample and the coefficient of variation on replicate assays is less than 10% for most concentrations studied. Analytical recoveries were between 95 and 100% throughout the appropriate concentration ranges and no interferences were obtained with the exception of p-acetamidobenzoyl glucuronide which could be eliminated by treatment of the samples with beta-glucuronidase. Concentration profiles of the metabolites were studied in normal subjects, and the method was found to be potentially useful for clinical situations in which the existing bentiromide test leads to ambiguous results because of small bowel and hepatic dysfunctions.