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.
Talanta
; 198: 464-471, 2019 Jun 01.
Article
em En
| MEDLINE
| ID: mdl-30876588
ABSTRACT
Prodrug treosulfan undergoes a pH and temperature-dependent activation to the monoepoxide intermediate (EBDM) and (2S,3S)-1,23,4-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 15N-isotope labeled internal standards underwent a mild acid thermal hydrolysis and ultrafiltration (cut-off 10â¯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 0.2-200â¯pmol, HMSBG 0.2-20â¯pmol, and bis-N7G-BD 0.4-40â¯pmol. The limits of quantitation allowed to determine the adducts at concentration of 330 or 660 per 109 DNA nucleotides. The LC-MS/MS method was adequately precise (coefficient of variation ≤â¯16.7%) and accurate (relative error ≤â¯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.
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MEDLINE
Assunto principal:
Bussulfano
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DNA
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Pró-Fármacos
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Adutos de DNA
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Compostos de Epóxi
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Guanina
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article