Simultaneous quantitative determination of 5-aza-2'-deoxycytidine genomic incorporation and DNA demethylation by liquid chromatography tandem mass spectrometry as exposure-response measures of nucleoside analog DNA methyltransferase inhibitors

Nicole M. Anders, Jianyong Liu, Teresia Wanjiku, Hugh Giovinazzo, Jianya Zhou, Ajay Vaghasia, William G. Nelson, Srinivasan Yegnasubramanian, Michelle A. Rudek

Research output: Contribution to journalArticlepeer-review

Abstract

The epigenetic and anti-cancer activities of the nucleoside analog DNA methyltransferase (DNMT) inhibitors decitabine (5-aza-2'-deoxycytidine, DAC), azacitidine, and guadecitabine are thought to require cellular uptake, metabolism to 5-aza-2'-deoxycytidine triphosphate, and incorporation into DNA. This genomic incorporation can then lead to trapping and degradation of DNMT enzymes, and ultimately, passive loss of DNA methylation. To facilitate measurement of critical exposure-response relationships of nucleoside analog DNMT inhibitors, a sensitive and reliable method was developed to simultaneously quantitate 5-aza-2'-deoxycytidine genomic incorporation and genomic 5-methylcytosine content using LC-MS/MS. Genomic DNA was extracted and digested into single nucleosides. Chromatographic separation was achieved with a Thermo Hyperpcarb porous graphite column (100 mm × 2.1 mm, 5 μm) and isocratic elution with a 10 mM ammonium acetate:acetonitrile with 0.1% formic acid (70:30, v/v) mobile phase over a 5 min total analytical run time. An AB Sciex 5500 triple quadrupole mass spectrometer operated in positive electrospray ionization mode was used for the detection of 5-aza-2'-deoxycytidine, 2'-deoxycytidine, and 5-methyl-2'-deoxycytidine. The assay range was 2-400 ng/mL for 5-aza-2'-deoxycytidine, 50-10,000 ng/mL for 2'-deoxycytidine, and was 5-1000 ng/mL for 5-methyl-2'-deoxycytidine. The assay proved to be accurate (93.0-102.2%) and precise (CV ≤ 6.3%) across all analytes. All analytes exhibited long-term frozen digest matrix stability at -70 °C for at least 117 days. The method was applied for the measurement of genomic 5-aza-2'-deoxycytidine and 5-methyl-2'-deoxycytidine content following exposure of in vitro cell culture and in vivo animal models to decitabine.

Original languageEnglish (US)
Pages (from-to)38-45
Number of pages8
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume1022
DOIs
StatePublished - Jun 1 2016

Keywords

  • 5-Aza-2'-deoxycytidine
  • 5-Methyl-2'-deoxycytidine
  • 5-Methylcytosine
  • DNA methyltransferase inhibitor
  • Decitabine
  • Genomic DNA
  • Global DNA demethylation
  • LC/MS/MS

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry
  • Cell Biology

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