Automated spectrophotometric assay for urine p-aminophenol by an oxidative coupling reaction

Urine p-aminophenol (PAP) concentration serves as a biological marker for occupational exposures to aniline. We report the development of a rapid, simple spectrophotometric method for quantification of urine PAP concentration using a chemical autoanalyzer (Olympus Reply). The method involves oxidative coupling of PAP with an aromatic compound, xylenol, that contains an electron-donating group, based on an electrophilic aromatic substitution reaction catalyzed by sodium periodate. A calibration curve is constructed in the same matrix, urine, as the unknown samples to be analyzed. In this way, potential matrix interferences are largely avoided. The linearity range of the method is 20 to 400 mg/L. Time-course studies show that the color formation by reaction of PAP with xylenol is rapid and essentially complete within 5 min. Within-run and day-to-day reproducibility data at medium (50 mg/L) and high (200 mg/L) concentrations yield CV's <5.0%. Several prescription drugs and drugs of abuse, as well as related compounds, gave negative tests for interference in the procedure. Clinical applications of the method are illustrated by data for (a) PAP concentrations in 255 urine samples from workers at a rubber plant, and (b) PAP elimination in serial urine samples from 5 volunteers after an oral dose (500 mg) of acetaminophen. In summary, the new method has the advantages of automation, operational simplicity, and suitability for monitoring workers for exposures to aniline.