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.
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.
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DOI：
10.1016/j.jchromb.2016.03.029
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发表时间：
2016-06-01
期刊：
影响因子：
--
通讯作者：
Rudek MA
中科院分区：
文献类型：
--
作者：
Anders NM;Liu J;Wanjiku T;Giovinazzo H;Zhou J;Vaghasia A;Nelson WG;Yegnasubramanian S;Rudek MA
核苷类似物DNA甲基转移酶(DNMT)抑制剂Decitabine(5-aza-2‘-deoxcytidine，DAC)、azacitidine和guadecitabine的表观遗传和抗癌活性被认为需要细胞摄取，代谢到5-aza-2’-脱氧胞苷三磷酸，并整合到DNA中。这种基因组的整合可以导致DNMT酶的捕获和降解，最终导致DNA甲基化的被动丧失。为了便于测定核苷类似物DNMT抑制剂的临界暴露-反应关系，建立了一种灵敏可靠的LC-MS/MS同时定量5-氮杂-2‘-脱氧胞苷基因组掺入和基因组5-甲基胞嘧啶含量的方法。色谱柱为Thermo Hyperpcarb多孔石墨柱(10 0 mm×2.1 mm，5μm)，流动相为10 mm醋酸铵：乙腈-0.1%甲酸(70：30，v/v)，洗脱时间为5min。采用AB Sciex 5500三极四极质谱计，在正电喷雾电离模式下对5-氮杂-2‘-脱氧胞苷、2’-脱氧胞苷和5-甲基-2‘-脱氧胞苷进行了检测。5-氮杂-2‘-脱氧胞苷、2’-脱氧胞苷、5-甲基-2‘-脱氧胞苷的检测范围分别为2~400 ng/m L、50~10,000 ng/m L、5-1,000 ng/m L。在所有分析物中，该方法被证明是准确的(93.0-102.2%)和精密度(CV≤6.3%)。所有分析物在−70°C下显示出至少117天的长期冷冻消化基质稳定性。该方法用于体外细胞培养和体内动物模型中5-氮杂-2‘-脱氧胞苷和5-甲基-2’-脱氧胞苷含量的测定。
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, 5μm) and isocratic elution with a 10 mM ammonium acetate:acetonitrile with 0.1% formic acid (70:30, v/v) mobile phase over a 5 minute 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 – 1,000 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.