Abstract
Aromatic amines are widely used in personal care products and human exposure to this class of chemicals is widespread. Bioanalytical methods to determine trace levels of aromatic amines in human urine are scarce. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to determine 39 primary aromatic amines (AAs) along with nicotine and cotinine in human urine. Chromatographic separation of the 41 analytes was achieved on an Ultra Biphenyl (100 mm × 2.1 mm, 5 µm) column. Mass spectrometry was operated in electrospray ionization positive ion multiple reaction monitoring (MRM) mode. The method exhibited excellent linear dynamic range (0.1-50 ng/mL) with correlation coefficients (r) > 0.999 for all analytes. Urine samples (2 mL) were hydrolyzed using 10 M NaOH at 95 °C for 15 h and target analytes were extracted using methyl-tert-butyl ether (MTBE). Addition of 15 µL of 0.25 M HCl to the sample extracts improved the recoveries of several target analytes. The method was validated through the analysis of fortified quality control (QC) samples and a certified standard reference material (SRM). Relative recoveries (%) of target analytes fortified in QC samples were in the range of 75-114% for 37 of the 41 analytes while the other analytes exhibited lower recoveries (16-74%). The limits of detection (LOD) and limits of quantification (LOQ) of target analytes were in the range of 0.025-0.20 ng/mL and 0.1-1.0 ng/mL, respectively. Intra-day and inter-day precision of the method assessed through the analysis of fortified urine QC samples at three different concentrations were < 11.7% and < 15.9% (measured as RSD), respectively. The method was applied in the analysis of urine samples from the general population and known smokers; aniline, para-anisidine, para-toluidine, ortho/meta-toluidine, 3-chloroaniline, 4-chloroaniline, 3,4-dichloroaniline, and 4,4'-methylenedianiline were found in all smoker's urine at sum concentrations ranging from 0.04 to 9.16 ng/mL.