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

doi:10.1016/j.jchromb.2016.03.029

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

J Chromatogr B Analyt Technol Biomed Life Sci. 2016 Jun 1:1022:38-45. doi: 10.1016/j.jchromb.2016.03.029. Epub 2016 Mar 21.

Authors

Nicole M Anders¹, Jianyong Liu¹, Teresia Wanjiku¹, Hugh Giovinazzo², Jianya Zhou³, Ajay Vaghasia¹, William G Nelson⁴, Srinivasan Yegnasubramanian⁵, Michelle A Rudek⁶

Affiliations

¹ 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.
² Department of Pharmacology and Molecular Sciences, School of Medicine, Johns Hopkins University, 1650 Orleans Street, Baltimore, MD, 21231 USA.
³ 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.
⁴ 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.
⁵ 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.
⁶ 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.

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.

Keywords: 5-Aza-2′-deoxycytidine; 5-Methyl-2′-deoxycytidine; 5-Methylcytosine; DNA methyltransferase inhibitor; Decitabine; Genomic DNA; Global DNA demethylation; LC/MS/MS.

MeSH terms

5-Methylcytosine / chemistry
5-Methylcytosine / metabolism
Animals
Azacitidine / analogs & derivatives*
Azacitidine / analysis
Azacitidine / chemistry
Azacitidine / metabolism
Cell Line, Tumor
Chromatography, Liquid / methods*
DNA / analysis
DNA / chemistry*
DNA / metabolism
DNA Methylation / physiology*
Decitabine
Humans
Limit of Detection
Linear Models
Methyltransferases / antagonists & inhibitors*
Mice
Mice, Nude
Reproducibility of Results
Tandem Mass Spectrometry / methods*

Substances

5-Methylcytosine
Decitabine
DNA
Methyltransferases
Azacitidine

Abstract

MeSH terms

Substances

Grants and funding