Liquid chromatography–tandem mass spectrometry method for simultaneous determination of three N-7-guanine adducts of the active epoxides of prodrug treosulfan in DNA in vitro

Abstract Prodrug treosulfan undergoes a pH and temperature-dependent activation to the monoepoxide intermediate (EBDM) and (2 S ,3 S )-1,2:3,4-diepoxybutane (DEB). The latter DNA cross-linker is presently believed to mainly account for the pharmacological action of treosulfan. However, neither respective monoadducts nor cross-links have been isolated from treosulfan-treated DNA, and the exact alkylation mechanism of the treosulfan epoxides is unclear. In this paper, liquid chromatography method with tandem mass spectrometry detection (LC-MS/MS) for simultaneous determination of the N -7-guanine adducts of EBDM and DEB − (2′ S ,3′ S )- N -7-(2′3′-dihydroxy-4′-methylsulfonyloxybut-1′-yl)guanine (HMSBG), N -7-(2′,3′,4′-trihydroxybut-1′-yl)guanine (THBG), and 1,4-bis( N -7-guanyl)butane-2,3-diol cross-link (bis-N7G-BD) − in calf-thymus DNA has been developed and validated for the first time. The mixture of drug-free nucleic acid with the analytes and 15 N-isotope labeled internal standards underwent a mild acid thermal hydrolysis and ultrafiltration (cut-off 10 kDa). Following offline LC purification, the analytes and internal standards were determined in the LC-MS/MS system with an electrospray interface. Complete resolution of THBG, HMSBG, and bis-N7G-BD was accomplished on a Zorbax Eclipse C18 column using gradient elution with a mobile phase composed of 0.1% formic acid and acetonitrile. Calibration curves were linear in the ranges: THBG 0.2–200 pmol, HMSBG 0.2–20 pmol, and bis-N7G-BD 0.4–40 pmol. The limits of quantitation allowed to determine the adducts at concentration of 330 or 660 per 10 9 DNA nucleotides. The LC-MS/MS method was adequately precise (coefficient of variation ≤ 16.7%) and accurate (relative error ≤ 17.7%). Calibration standards were stable for 14 days at –25 °C. The validated method enabled determination of THBG, HMSBG, and bis-N7G-BD in calf thymus DNA treated with treosulfan at pH 7.2 and 37 °C, which constitutes a novel bioanalytical application. To the authors’ best knowledge, the quantification of THBG and bis-N7G-BD in one analytical run is also reported for the first time.