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LC-MS/MS Measurement of Nanomaterial Induced Genotoxicity in Isolated DNA



Bryant C. Nelson, Elijah J. Petersen, Pawel Jaruga, M Miral Dizdar


Making accurate measurements of the environmental fate and environmental and biological effects of engineered nanomaterials (ENMs) is critical for reliable risk assessment of these materials. However, the unique behaviors of ENMs may cause artifacts in measurements of their potential effects. For example, results from several DNA damage studies using the Comet assay have reported artifacts from the presence of ENMs. Potential explanations for this phenomenon are that ENMs may induce DNA damage during processing after the exposure period has concluded or that the ENMs may be in the Comet tail and mistaken for DNA. In addition, accurate measurements of DNA damage are needed to clarify potential mechanisms of ENM toxicity. In this document, we describe a protocol to quantitatively measure a range of modified 2’- deoxynucleosides in DNA using liquid chromatrography/tandem mass spectrometry (LC-MSMS) with isotope-dilution. This approach utilizes NIST standard reference material (SRM) 2396 (Oxidative DNA Damage Mass Spectrometry Standards) for the measurement of 8-hydroxy-2- deoxyguanosine (9-OH-dGuo); the other four analytes that can be measured (described below) must utilize stable isotope-labeled internal standards that are synthesized separately. This method circumvents many of the artifacts observed in the Comet assay in nanogenotoxicity tests by directly quantifying the DNA lesion levels for a range of oxidatively induced damage products rather than making a non-specific measurement of DNA damage (i.e., Comet tail length). Isotope-dilution mass spectrometry methods have been recently utilized by our laboratory to successfully measure DNA damage both in vitro and in vivo caused by gold nanoparticles, copper oxide nanoparticles, single-wall carbon nanotubes, iron oxide nanoparticles, titanium dioxide nanoparticles and silver nanoparticles.
Special Publication (NIST SP) - 1200-20
Report Number


Mass spectrometry, Nanomaterial, Toxicity, Genotoxicity, DNA damage


Nelson, B. , Petersen, E. , Jaruga, P. and , M. (2015), LC-MS/MS Measurement of Nanomaterial Induced Genotoxicity in Isolated DNA, Special Publication (NIST SP), National Institute of Standards and Technology, Gaithersburg, MD, [online], (Accessed April 22, 2024)
Created November 24, 2015, Updated November 10, 2018