Artifact-free quantification of free 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine in human plasma by electron capture-negative chemical ionization gas chromatography mass spectrometry and liquid chromatography-electrospray ionization tandem mass spectrometry

Halogenation and nitration of biomolecules have been proposed as key mechanisms of host defense against bacteria, fungi, and viruses. Reactive oxidants also have the potential to damage host tissue, and they have been implicated in disease. In the current studies, we describe specific, sensitive, and quantitative methods for detecting three stable markers of oxidative damage: 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine. Our results indicate that electron capture-negative chemical ionization-gas chromatography/mass spectrometry (EC-NCI GC/MS) is 100-fold more sensitive than liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS) for analyzing authentic 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine. Using an isotopomer of tyrosine to evaluate artifactual production of the analytes during sample preparation and analysis, we found that artifact generation was negligible with either technique. However, LC-MS/MS proved cumbersome for analyzing multiple samples because it required 1.5 h of run and equilibration time per analysis. In contrast, EC-NCI GC/MS required only 5 min of run time per analysis. Using EC-NCI GC/MS, we were able to detect and quantify attomole levels of free 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine in human plasma. Our results indicate that EC-NCI GC/MS is a sensitive and specific method for quantifying free 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine in biological fluids in a single, rapid analysis and that it avoids generating any of the analytes ex vivo.