Figure 1.
Chemical structures and respective multiple reaction monitoring (MRM) transitions of (A) PPD and its metabolites (MAPPD and DAPPD) and (B) the internal standard (IS) 2-amino-5-nitropyridine.
Figure 2.
TOF spectrum (pos. mode) for the identification of two PPD metabolites MAPPD (m/z [M+H]+ 151) and DAPPD (m/z [M+H]+ 193) in patient urine.
Comparative control urine did not show any signals at the corresponding m/z values.
Figure 3.
Wet chemistry assay for spiked blank and clinical urine samples.
(A) Left row bottom to top: urine blank and 5 different spiked concentrations (25, 50, 100, 250 and 500 µmol/L). Right row top three vials: three clinical samples, which show a positive test result for PPD. (B) Concentration dependent transmittance (%) from 25–250 µmol/L on a spectrophotometer at 450 nm.
Table 1.
Validation results of PPD in human urine.
Figure 4.
Calibration curve of PPD (A) on the MALDI-MS/MS system (50–1000 µmol/L) and (B) on the HPLC-UV system (150–1000 µmol/L).
Graphs shows the mean values of (A) 5 and (B) 3 independent measurements and the corresponding r2 value. Mean +/− SEM.
Table 2.
Validation results of PPD in human urine.
Figure 5.
MALDI-MS/MS measurements of urine samples from patients with suspected PPD intoxication and controls.
(A) Measured PPD concentrations in clinical urine samples (n = 15); dotted lines show the LLOQ of the MALDI-MS/MS and the HPLC-UV application, resp. (B) Box-and-whisker blot with the 5–95 percentiles for the comparison of PPD, MAPPD and DAPPD peak areas in drug free control urine (n = 5) and clinical samples of intoxication (n = 15). (C) Data correlation (r2 = 0.7618) of PPD and DAPPD in patient urine (n = 15). Respective p-values are given in the graphs.