Student Research: Michael Paulsen

MS, , 2007
Faculty Advisor: Christopher D. Simpson

Measurement of Diesel Exhaust Biomarkers of Exposure by HPLC/MS/MS and GC/MS


Diesel exhaust (DE) contains numerous known and suspected carcinogenic and mutagenic components, including polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs. As a mixture, DE has been classified by the US Environmental Protection Agency (EPA) and the International Agency for Research on Cancer (IARC) as a probably human carcinogen. Epidemiological studies report associations between DE and lung cancer, asthma, and other diseases. However, a major limiting factor in studying disease causality is the lack of sensitive and specific chemical markers of DE, both atmospheric markers and exposure biomarkers. Effective markers of DE would provide researchers with tools to study health effects resulting from exposure to DE and to evaluate changes to exposures resulting from environmental protection efforts or workplace exposure control mechanisms.

1-Nitropyrene (!-NP) has been proposed as an atmospheric marker of DE because it is formed almost exclusively in diesel engines and because 1-NP is the most abundant nitro-arene compound in diesel exhaust particulate matter (DPM). Metabolites of 1-NP have been proposed as DE biomarkers of exposure in humans and have been reported in one article to date.

The present study developed two highly sensitive and selective assays for assessing human exposures to DE by measuring urinary metabolites of 1-NP using LC/MS/MS and GC/MS. Sample preparation involved enzymatic deconjugation and solid phase extraction using blue rayon followed by extract cleanup using alumina. Samples analyzed by GC/MS were further treated with zinc for nitro-reduction and with heptafluorobutyrylimidazole (HFBI) for derivatization. isotopically-labeled internal standards of each analyte were added to urine to correct for losses during sample preparation.

Method performance was evaluated with human urine spiked at levels representing occupational exposures or high environmental exposures. LC/MS/MS results were superior to those from the GC/MS assay. Average internal standard-corrected recovery of NP metabolites were approximately 86% for both low and high spiked urine samples when measured by LC/MS/MS. Precision was 21% and 7% CV for the low and high spike samples, respectively. Based on method performance and ease of sample preparation, LC/MS/MS is the preferred instrument for analysis of NP metabolites in human urine.