title: 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.
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 (100mm×2.1mm, 5μm) and isocratic elution with a 10mM ammonium acetate:acetonitrile with 0.1% formic acid (70:30, v/v) mobile phase over a 5min 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 2-400ng/mL for 5-aza-2'-deoxycytidine, 50-10,000ng/mL for 2'-deoxycytidine, and was 5-1000ng/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.
authors.affiliation: The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University, Baltimore, MD, USA; Department of Oncology, School of Medicine, Johns Hopkins University, 1650 Orleans Street, Baltimore, MD, 21231 USA.
authors.forename: Nicole M
authors.initials: NM
authors.lastname: Anders
authors.affiliation: The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University, Baltimore, MD, USA; Department of Oncology, School of Medicine, Johns Hopkins University, 1650 Orleans Street, Baltimore, MD, 21231 USA.
authors.forename: Jianyong
authors.initials: J
authors.lastname: Liu
authors.affiliation: The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University, Baltimore, MD, USA; Department of Oncology, School of Medicine, Johns Hopkins University, 1650 Orleans Street, Baltimore, MD, 21231 USA.
authors.forename: Teresia
authors.initials: T
authors.lastname: Wanjiku
authors.affiliation: Department of Pharmacology and Molecular Sciences, School of Medicine, Johns Hopkins University, 1650 Orleans Street, Baltimore, MD, 21231 USA.
authors.forename: Hugh
authors.initials: H
authors.lastname: Giovinazzo
authors.affiliation: The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University, Baltimore, MD, USA; Department of Oncology, School of Medicine, Johns Hopkins University, 1650 Orleans Street, Baltimore, MD, 21231 USA; Department of Respiratory Disease, Thoracic Disease Center, The First Affiliated Hospital College of Medicine, Zhejiang University, Hangzhou, PR China.
authors.forename: Jianya
authors.initials: J
authors.lastname: Zhou
authors.affiliation: The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University, Baltimore, MD, USA; Department of Oncology, School of Medicine, Johns Hopkins University, 1650 Orleans Street, Baltimore, MD, 21231 USA.
authors.forename: Ajay
authors.initials: A
authors.lastname: Vaghasia
authors.affiliation: The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University, Baltimore, MD, USA; Department of Oncology, School of Medicine, Johns Hopkins University, 1650 Orleans Street, Baltimore, MD, 21231 USA; Department of Pharmacology and Molecular Sciences, School of Medicine, Johns Hopkins University, 1650 Orleans Street, Baltimore, MD, 21231 USA; Department of Pathology, School of Medicine, Johns Hopkins University, 1650 Orleans Street, Baltimore, MD, 21231 USA; Brady Urological Institute, School of Medicine, Johns Hopkins University, 1650 Orleans Street, Baltimore, MD, 21231 USA.
authors.forename: William G
authors.initials: WG
authors.lastname: Nelson
authors.affiliation: The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University, Baltimore, MD, USA; Department of Oncology, School of Medicine, Johns Hopkins University, 1650 Orleans Street, Baltimore, MD, 21231 USA; Brady Urological Institute, School of Medicine, Johns Hopkins University, 1650 Orleans Street, Baltimore, MD, 21231 USA. Electronic address: syegnasu@jhmi.edu.
authors.forename: Srinivasan
authors.initials: S
authors.lastname: Yegnasubramanian
authors.affiliation: The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University, Baltimore, MD, USA; Department of Oncology, School of Medicine, Johns Hopkins University, 1650 Orleans Street, Baltimore, MD, 21231 USA; Division of Clinical Pharmacology, Department of Medicine, School of Medicine, Johns Hopkins University, 1650 Orleans Street, Baltimore, MD, 21231 USA. Electronic address: mrudek2@jhmi.edu.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Rudek
mesh_terms: D044503:5-Methylcytosine / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods*; D004247:DNA / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D019175:DNA Methylation / Q000502:physiology*; D000077209:Decitabine; D006801:Humans; D057230:Limit of Detection; D016014:Linear Models; D008780:Methyltransferases / Q000037:antagonists & inhibitors*; D051379:Mice; D008819:Mice, Nude; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D044503:5-Methylcytosine; D000077209:Decitabine; D004247:DNA; D008780:Methyltransferases; D001374:Azacitidine
keywords: 5-Aza-2′-deoxycytidine; 5-Methyl-2′-deoxycytidine; 5-Methylcytosine; DNA methyltransferase inhibitor; Decitabine; Genomic DNA; Global DNA demethylation; LC/MS/MS