Simultaneous analysis of urinary 4,4'-methylenebis(2-chloroaniline) and N-acetyl 4,4'-methylenebis(2-chloroaniline) using solid-phase extraction and liquid chromatography/tandem mass spectrometry

Analysis of 4,4'-methylenebis(2-cholroaniline) (MOCA) or its metabolites in urine has been considered as the appropriate method to assess MOCA exposures through inhalation and skin absorption. MOCA and its metabolite, N-acetyl 4,4'-methylenebis(2-chloroaniline) (acetyl-MOCA), are analyzed using methods either limited by sensitivity or sample preparation. Therefore, a solid-phase extraction (SPE) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed to simultaneously analyze MOCA and acetyl-MOCA in urine to serve as biomarkers for MOCA exposure. Protein was precipitated by using acetonitrile, and SPE were applied to clean up samples to eliminate the matrix effect and to improve the recovery. The limit of quantitation of this method was at 1.0 ng/mL for MOCA and 0.03 ng/mL for acetyl-MOCA (signal-to-noise (S/N) ratio = 10). Urinary MOCA and acetyl-MOCA levels in MOCA-exposed workers were analyzed and quantitated to be 191.9 +/- 373.2 (mean +/- standard deviation (SD)) and 11.79 +/- 23.8 ng/mL (N = 54) with the median values 38.6 and 1.8 ng/mL, respectively. MOCA concentrations are significantly correlated with their corresponding acetyl-MOCA levels in urine (Spearman correlation coefficient r = 0.916, p < 0.001). These results show that this method has been successfully developed and provides high-throughput potential to analyze MOCA and acetyl-MOCA to serve as exposure biomarkers for future study of the potential health effects associated with MOCA exposures.