AMBER GOVERT
Development of an Analytical Method for the Determination of 3-Nitrotyrosine in Human Samples by HPLC-MS/MS
Industrial Hygiene and Safety, MS
Preceptor: Christopher Simpson, PhD
Diesel engines are an efficient source of power and are increasingly being used worldwide in heave machinery, emergency power generation, and mass transit. However, diesel exhaust (DE) is believed to be an important occupational and environmental toxicant. Increased evidence of lung cancer, exacerbation of asthma symptoms, and decreased lung function have been associated with DE exposure. Unfortunately, relationships in vivo between acute or chronic DE exposures and human symptoms remain difficult to determine, due in part to the lack of appropriate sensitive pre-clinical markers of adverse heath effects. It is believed that one mechanism by which DE can act on the body is by inducing oxidative stress. Diesel exhaust contains a variety of chemicals which can induce oxidative and nitrative stress. 3-Nitrotyrosine (3-NT) has been shown to be a useful biomarker for the in vivo measurement of oxidative stress brought on by reactive nitrogen species (RNS).
Development of an analytical method that will be sensitive enough to detect and quantify baseline concentrations of a 3-NT and its metabolite 4-hydroxy-3-nitrophenylacetic acid (NHPA) by using liquid chromatography tandem mass spectrometry (HPLC-MS/MS) is described. Isotopically labeled analogs of 3-NT and NHPA were used to monitor analyte recoveries, where as isotopically labeled analogs of tyrosine (Tyr) and hydroxlphenylacetic asdic (HPA) were used to monitor artifactual generation of analytes during sample preparation.
Method validation results showed that the solid phase extraction (SPE) methods used to enrich the analytes from biological matrices and remove extraneous material all had recoveries of greater than 80% for the analytes of interest from urine, plasma, and digested protein samples. The limit of detection (LOD) for 3-NT as determined from analysis of calibration solutions was found to be less than 2 pg, while the LOD for NHPA was found to be less than 100 pg. Limited artifact formation was observed during the cleanup process for urine and plasma. Preliminary results analyzing both urine and plasma samples showed quantifiable levels of 3-NT to be present. However, matrix effects do appear to significantly reduce the response of the analytes in biological samples. Additional sample purification approaches will be required to ensure adequate sensitivity and robust performance for the application of this assay to human biological samples.
