核苷的气相色谱法。衍生化与色谱分析。

Gas-liquid chromatography of nucleosides. Derivatization and chromatography.

作者信息

Gehrke C W, Patel A B

出版信息

J Chromatogr. 1976 Aug 4;123(2):335-45. doi: 10.1016/s0021-9673(00)82202-5.

Abstract

The aims of this investigation were to establish the optimum reaction conditions for silylation of nucleotides with bis(trimethylsilyl)trifluoroacetamide (BSTFA) and to investigate the chromatographic properties of the following nucleosides: adenosine, guanosine, cytidine, thymine, inosine, xanthosine, and uridine. Closed tube silylations were performed with a 1000 molar excess of BSTFA at 25, 75, 120, 150, and 175 degrees for 15, 30, 60, 120 and 240 min. The optimal time and temperature for derivatization were found to be 150 degrees and 15 min. Using these reaction conditions, samples were then silylated with 50, 100, 200, 500, and 1000 molar excess of BSTFA; a molar excess of 225 was best. The stability of the nucleoside derivatives on standing at room temperature for 1-7 days was investigated. Quantitative gas-liquid chromatography of trimethylsilyl (TMS) nucleosides can be performed if samples are analyzed within 48 h, after which time the relative weight response for the nucleosides decreased somewhat. Chromatographic column studies were made using various liquid phases and supports. With methylsiloxanes as the liquid phase, Supelcoport as support was found to be superior. Resolution of TMS guanosine from TMS cytidine was attempted at different column lengths using 3% (w/w) SE-30 on Supelcoport, different loadings (%, w/w) of SE-30 on Supelcoport, and different polarity liquid phases, 4% (w/w) OV-11 or 3% (w/w) OV-17 or 4% (w/w) Dexsil-300 on Supelcoport. A complete separation of the six ribonucleosides including guanosine and cytidine was obtained with 1 m x 4 mm I.D. glass columns of 4% (w/w) OV-11 and 3% (w/w) OV-17 on 100-120 mesh Supelcoport. It was observed that with 2'-deoxycytidine, one obtains the TMS cytosine peak (retention temperature 150 degrees) plus another peak with a retention temperature of 120 degrees, under all derivatization conditions with BSTFA and bis(trimethylsilyl)-acetamide. Similar formation of bases from other 2'-deoxyribonucleosides occurred when the molar excess of BSTFA was greater than 500. The minimal detectable amounts obtained for all the nucleosides ranged from 5 to 10 ng injected with a signal-to-noise ratio of 3. The relative standard deviations for all nucleosides and deoxynucleosides ranged from 1.2 to 4.8% on different methylsiloxanes on Supelcoport columns.

摘要

本研究的目的是确定用双（三甲基硅基）三氟乙酰胺（BSTFA）对核苷酸进行硅烷化的最佳反应条件，并研究以下核苷的色谱性质：腺苷、鸟苷、胞苷、胸腺嘧啶、次黄嘌呤核苷、黄苷和尿苷。在封闭管中，用1000摩尔过量的BSTFA在25、75、120、150和175摄氏度下进行硅烷化反应15、30、60、120和240分钟。发现衍生化的最佳时间和温度分别为150摄氏度和15分钟。使用这些反应条件，然后用50、100、200、500和1000摩尔过量的BSTFA对样品进行硅烷化；225摩尔过量效果最佳。研究了核苷衍生物在室温下放置1 - 7天的稳定性。如果在48小时内对样品进行分析，可进行三甲基硅基（TMS）核苷的定量气相色谱分析，此后核苷的相对重量响应会有所下降。使用各种液相和载体进行了色谱柱研究。发现以甲基硅氧烷为液相、Supelcoport为载体效果更佳。使用Supelcoport上3%（w/w）的SE - 30、Supelcoport上不同负载量（%，w/w）的SE - 30以及Supelcoport上不同极性的液相4%（w/w）的OV - 11或3%（w/w）的OV - 17或4%（w/w）的Dexsil - 300，在不同柱长下尝试分离TMS鸟苷和TMS胞苷。使用100 - 120目Supelcoport上4%（w/w）的OV - 11和3%（w/w）的OV - 17的1米×4毫米内径玻璃柱，实现了包括鸟苷和胞苷在内的六种核糖核苷的完全分离。观察到在所有用BSTFA和双（三甲基硅基）乙酰胺进行衍生化的条件下，对于2'-脱氧胞苷，会得到TMS胞嘧啶峰（保留温度150摄氏度）以及另一个保留温度为120摄氏度的峰。当BSTFA的摩尔过量大于500时，其他2'-脱氧核糖核苷也会类似地形成碱基。所有核苷的最小检测量范围为进样5至10纳克，信噪比为3。在Supelcoport柱上不同甲基硅氧烷上，所有核苷和脱氧核苷的相对标准偏差范围为1.2%至4.8%。