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Realised by ALMS™
developer of the AIDS-HIV Reference project
Abstract No.: ThP-066
Session: Biomarkers
Presentation date: Thu, Aug 31, 2006
Presentation time: 14:30 – 16:00

An LC-MS/MS Affinity-based Assay for Total Nitrotyrosine Quantitation in Biological Fluids

Melissa Renee Radabaugh1, Olga V. Nemirovskiy1, Poonam Aggarwal1, Hideji Fujiwara1, Thomas P. Misko1, W. Rodney Mathews1

1 Pfizer, St. Louis, United States

Correspondence address: Melissa Renee Radabaugh, Pfizer, Molecular Pharmacology - Biomarkers, 700 Chesterfield Parkway West, Chesterfield, MO, 63017 United States.

Keywords: Affinity, Chromatography; Biomarkers; MS/MS, Liquid Chromatography; Quantitative Analysis.

Novel aspect: A novel affinity-based method for determination of total nitrotyrosine in biological fluids was developed. This method offers increased sensitivity over previously reported methods, with the lowest limit of detection being 5 pg/mL.


Nitrotyrosine is widely recognized as a surrogate marker of increased expression of inducible NO synthase at the sites of inflammation. It is formed in the presence of peroxynitrite (ONOO-), a highly reactive oxidant species produced by the reaction of the superoxide (O2·-) and nitric oxide (NO·) free radicals. The inducible form of nitric oxide synthase is known to be responsible for the overproduction of NO·. The expression of iNOS in many cells and tissues is reported to be rapidly up-regulated after challenge with bacterial lipopolysaccharide (LPS) and proinflammatory cytokines such as interleukin 1 (IL-1), tumor necrosis factor &alpha(TNF-α), or interferon gamma (IFN-γ). This modulation is presumably via the activation of NF-κB, a transcription factor involved in the regulation of many proinflammatory cytokines and enzymes. The formation of NO, especially peroxynitrite, has been implicated in the pathophysiology of numerous inflammatory diseases, including rheumatoid arthritis and osteoarthritis. The identification of iNOS in human rheumatoid synovium and osteoarthritic cartilage suggests that regulation of the NO pathway in these diseases may offer a route of therapeutic intervention.

In an effort to further study and elucidate the role of the NO pathway in these disease states, an immuno-affinity based liquid chromatographic method with tandem mass spectrometry detection was developed and validated to quantify free and total nitrotyrosine levels in biological fluids from pre-clinical and clinical models (plasma, urine, serum, and synovial fluid). Analysis is by LC-MS/MS in the negative ion mode using multiple reaction monitoring. The range of quantification for total nitrotyrosine is 5 to 500 pg/mL. This nitrotyrosine assay requires a 0.050 mL aliquot of plasma and utilizes a protein digestion step to allow for measurement of total nitrotyrosine within the sample. Plasma was collected in EDTA collection tubes, which was determined to have a negative effect on the efficiency of the pronase digestion. In order to overcome the interference of the EDTA, three metal ions are added to the samples. Levels of detection for this assay were greatly increased over previously reported levels, with the lower level of detection being 5 pg/mL.