Quantitative monitoring of biologically active methylations
of
guanines in samples exposed to temozolomide (TMZ) would be useful
in glioblastoma research for preclinical TMZ experiments, for clinical
pharmacology questions regarding appropriate exposure, and ultimately
for precision oncology. The known biologically active alkylation of
DNA induced by TMZ takes place on O6 position of guanines. However,
when developing mass spectrometric (MS) assays, the possible signal
overlap of O6-methyl-2′-deoxyguanosine (O6-m2dGO) with other
methylated 2′-deoxyguanosine species in DNA and methylated
guanosines in RNA must be considered. Liquid chromatography–tandem
MS (LC–MS/MS) offers the analytical requirements for such assays
in terms of specificity and sensitivity, especially when multiple
reaction monitoring (MRM) is available. In preclinical research, cancer
cell lines are still the gold standard model for in vitro drug screening.
Here, we present the development of ultra-performance LC-MRM-MS assays
for the quantification of O6-m2dGO in a TMZ-treated glioblastoma cell
line. Furthermore, we propose adapted parameters for method validation
relevant to the quantification of drug-induced DNA modifications.