Simultaneous determination of various aromatic amines and metabolites of aromatic nitro compounds in urine for low level exposure using gas chromatography–mass spectrometry

A newly developed method permits the simultaneous quantitative determination of various aromatic amines (or metabolites of aromatic nitro compounds, respectively) in human urine in one analytical run. Applying this method it is possible to determine aniline, toluidines, 4-isopropylaniline, o-anisidine, 3- and 4-chloroaniline, 4-bromoaniline, aminonitrotoluenes, aminodinitrotoluenes, 3,5- and 3,4-dichloroaniline, α- and β-naphtylamine and 4-aminodiphenyl. After separation from the urinary matrix by a simple liquid–liquid extraction at pH 6.2–6.4 the analytes are converted into their pentafluoropropionic acid amides. Separation and quantitative analysis is carried out by capillary gas chromatography and mass-selective detection in the single ion monitoring mode. The limits of detection were within the range from 0.05 μg/l (4-aminobiphenyl, o-anisidine, 3,5-dichloroaniline) to 2 μg/l urine (4-amino-2,6-dinitrotoluene). The relative standard deviation of the within-series imprecision (determined at spiked concentrations of 2.0 μg/l and 10 μg/l) was between 2.9 and 13.6% depending on analyte and concentration. The relative recovery rates were in the range of 70–121%. The analytes that do not contain a nitro function showed better performance regarding the analytical reliability criteria. In order to determine the suitability of this new method for biological monitoring we analysed 20 12-h urine samples of persons without known exposure to aromatic amines, nitroaromatics or precursors in a pilot study. In these samples various aromatic amines could be clearly identified. The general population renally excretes aniline (median: 3.5 μg/l; 95th percentile: 7.9 μg/l), o- (0.12 μg/l; 2.7 μg/l), m- (0.17 μg/l; 2.2 μg/l) and p-toluidine (0.11 μg/l; 0.43 μg/l), and o-anisidine (0.22 μg/l; 0.68 μg/l). Additionally, we found that the persons investigated also excrete 3- (<0.05 μg/l; 0.55 μg/l) and 4-chloroaniline (0.11 μg/l; 0.57 μg/l) as well as 3,5-dichloroaniline (0.18 μg/l; 1.5 μg/l). 3,4-Dichloroaniline was found in some specimens (20%) in concentrations near the limit of detection (<0.05 μg/l; 0.12 μg/l). We did not detect α- or β-naphtylamine, 4-aminobiphenyl or metabolites of explosives in the samples.