1,6-hexamethylene diisocyanate (HDI) is extensively used in the automotive repair industry and is a commonly reported cause of occupational asthma in industrialized populations. However, the exact pathological mechanism remains uncertain. Characterization and quantification of biomarkers resulting from HDI exposure can fill important knowledge gaps between exposure, susceptibility, and the rise of immunological reactions and sensitization leading to asthma. Here, we discuss existing challenges in HDI biomarker analysis including the quantification of N-acetyl-1,6-hexamethylene diamine (monoacetyl-HDA) and N,N′-diacetyl-1,6-hexamethylene diamine (diacetyl-HDA) in urine samples based on previously established methods for HDA analysis. In addition, we describe the optimization of reaction conditions for the synthesis of monoacetyl-HDA and diacetyl-HDA, and utilize these standards for the quantification of these metabolites in the urine of three occupationally exposed workers. Diacetyl-HDA was present in untreated urine at 0.015 – 0.060 μg/l. Using base hydrolysis, the concentration range of monoacetyl-HDA in urine was 0.19 – 2.2 μg/l, 60-fold higher than in the untreated samples on average. HDA was detected only in one sample after base hydrolysis (0.026 μg/l). In contrast, acid hydrolysis yielded HDA concentrations ranging from 0.36 to 10.1 μg/l in these three samples. These findings demonstrate HDI metabolism via N-acetylation metabolic pathway and protein adduct formation resulting from occupational exposure to HDI
