Application of a LC-MS/MS method developed for the determination of p-phenylenediamine, N-acetyl-p-phenylenediamine and N,N-diacetyl-p-phenylenediamine in human urine specimens

Abstract

Cases of poisoning by p-phenylenediamine (PPD) are detected sporadically. Recently an article on the development and validation of a LC-MS/MS method for the detection of PPD and its metabolites, N-acetyl-p-phenylenediamine (MAPPD) and N,N-diacetyl-p-phenylenediamine (DAPPD) in blood was published. In the current study this method for detection of these compounds was validated and applied to urine samples. The analytes were extracted from urine samples with methylene chloride and ammonium hydroxide as alkaline medium. Detection was performed by LC-MS/MS using electrospray positive ionization under multiple reaction-monitoring mode. Calibration curves were linear in the range 5–2000 ng/mL for all analytes. Intra- and inter-assay imprecisions were within 1.58–9.52% and 5.43–9.45% respectively, for PPD, MAPPD and DAPPD. Inter-assay accuracies were within -7.43 and 7.36 for all compounds. Lower limit of quantification was 5 ng/mL for all analytes. The method, which complies with the validation criteria, was successfully applied to the analysis of PPD, MAPPD and DAPPD in human urine samples collected from clinical and postmortem cases.