生物材料中核苷的定量高效液相色谱法。

Quantitative high-performance liquid chromatography of nucleosides in biological materials.

作者信息

Gehrke C W, Kuo K C, Davis G E, Suits R D, Waalkes T P, Borek E

出版信息

J Chromatogr. 1978 Mar 21;150(2):455-76. doi: 10.1016/s0021-9673(00)88205-9.

Abstract

A rigorous, comprehensive, and reliable reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the analysis of ribonucleosides in urine (psi, m1A, m1I, m2G, A, m2(2)G). An initial isolation of ribonucleosides with an affinity gel containing an immobilized phenylboronic acid was used to improve selectivity and sensitivity. Response for all nucleosides was linear from 0.1 to 50 nmoles injected and good quantitation was obtained for 25 microliter or less of sample placed on the HPLC column. Excellent precision of analysis for urinary nucleosides was achieved on matrix dependent and independent samples, and the high resolution of the reversed-phase column allowed the complete separation of 9 nucleosides from other unidentified UV absorbing components at the 1-ng level. Supporting experimental data are presented on precision, recovery, chromatographic methods, minimum detection limit, retention time, relative molar response, sample clean-up, stability of nucleosides, boronate gel capacity, and application to analysis of urine from patients with leukemia and breast cancer. This method is now being used routinely for the determination of the concentration and ratios of nucleosides in urine from patients with different types of cancer and in chemotherapy response studies.

摘要

已开发出一种严谨、全面且可靠的反相高效液相色谱（HPLC）方法，用于分析尿液中的核糖核苷（假尿苷、1-甲基腺苷、1-甲基肌苷、N2,N2-二甲基鸟苷、腺苷、N2,N2,O2'-三甲基鸟苷）。首先使用含有固定化苯硼酸的亲和凝胶对核糖核苷进行分离，以提高选择性和灵敏度。所有核苷的响应在进样量为0.1至50纳摩尔时呈线性，对于注入HPLC柱的25微升或更少样品可实现良好的定量。在依赖基质和不依赖基质的样品上均实现了对尿核苷分析的极高精密度，反相柱的高分辨率使得能够在1纳克水平将9种核苷与其他未鉴定的紫外吸收成分完全分离。文中给出了关于精密度、回收率、色谱方法、最低检测限、保留时间、相对摩尔响应、样品净化、核苷稳定性、硼酸凝胶容量以及在白血病和乳腺癌患者尿液分析中的应用等方面的支持性实验数据。该方法目前正常规用于测定不同类型癌症患者尿液中核苷的浓度和比例，以及化疗反应研究。