Development and validation of an LC–MS/MS method for determination of p-phenylenediamine and its metabolites in blood samples

•LC–MS/MS method was developed to determinate PPD and its metabolites in human blood.•The method was fully validated and successfully applied to postmortem human blood.•PPD was not stable in blood and extracted samples. In some developing countries, p-phenylenediamine (PPD) is used in combination with Henna as hair dye or skin decoration. A sensitive LC–MS/MS method was developed and validated for the simultaneous determination of p-phenylenediamine (PPD) and its metabolites N-acetyl-p-phenylenediamine (MAPPD) and N,N-diacetyl-p-phenylenediamine (DAPPD) in human blood. Acetanilide was used as an internal standard (IS). The LC–MS/MS was operated under multiple reaction-monitoring mode using the electrospray positive ionization technique. The transition ions m/z 109→92, m/z 151→92, m/z 193→92, and m/z 136→77 were selected for the quantification of PPD, MAPPD, DAPPD, and IS, respectively. The linear range was 10–2000ng/mL for all the compounds. The absolute recoveries were 51.94, 56.20 and 54.88% for PPD, MAPPD and DAPPD, respectively. Intra- and inter-assay imprecision were lower than 14% (RSD), and the bias of the assay was lower than 15% for all the compounds. The stability studies demonstrated that critical degradation for PPD in blood samples and autosampler occurred after 6h, while MAPPD and DAPPD were stable in blood samples and the autosampler up to 48h and 24h, respectively. This newly developed method allows for the detection of PPD and its metabolites in blood samples in the clinical and forensic setting.