One-pot stable isotope dilution- and LC-MS/MS-based quantitation of guanine, O6-methylguanine, and N7-methylguanine.

Nitrosamines (NAs) are a class of compounds designated as probable human carcinogens by the IARC, acting DNA mutagens, participating in alkylating DNA at both N⁷- and O⁶- positions of guanine, particular for N-nitrosodimethylamine (NDMA). In the present study, we report the development of an LC-MS/MS-based assay to simultaneously measure guanine (Gua), O⁶-methylguanine (6MGua), and N⁷-methylguanine (7MGua) in DNA hydrolysates. With the use of three stable isotope internal standards (6-CD₃-Gua, 7-CD₃-Gua, and Gua-¹³C₂,¹⁵N), validation, i.e. determination of selectivity, precision, accuracy, extraction recovery, matrix effect, stability, and feasibility was evaluated by use of the approach developed. This method was used for simultaneous determination of the levels of Gua, 6MGua, and 7MGua in DNA acidic hydrolytes present in a series of samples from human and rat primary hepatocyte treated with NDMA. The stable isotope dilution-based approach was proven to be selective, sensitive, accurate, and convenient, which is a good and convincing in vitro assay for the quantitative analysis of DNA methylation such as 6MGua and 7MGua in Gua.