DETECTING LOW-LEVEL SYNTHESIS IMPURITIES IN MODIFIED PHOSPHOROTHIOATE OLIGONUCLEOTIDES USING LIQUID CHROMATOGRAPHY - HIGH RESOLUTION MASS SPECTROMETRY.
DETECTING LOW-LEVEL SYNTHESIS IMPURITIES IN MODIFIED PHOSPHOROTHIOATE OLIGONUCLEOTIDES USING LIQUID CHROMATOGRAPHY - HIGH RESOLUTION MASS SPECTROMETRY.
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DOI：
10.1016/j.ijms.2010.06.001
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发表时间：
2011-07-01
影响因子：
1.8
通讯作者：
Limbach PA
中科院分区：
文献类型：
--
作者：
Nikcevic I;Wyrzykiewicz TK;Limbach PA
描述了一种基于使用高分辨率傅里叶变换离子回旋共振质谱（FTIRCMS）分析寡核苷酸合成杂质的LC-MS方法。寡核苷酸硫代磷酸二酯（硫代磷酸寡核苷酸），其中每个磷中心的一个非桥接氧原子被硫原子取代，由于其易于化学合成和有利的药代动力学特性，现在是最流行的寡核苷酸修饰之一。尽管用于生产这些寡核苷酸的固相寡聚化化学取得了重大进展，但合成寡核苷酸始终伴随着多种低水平杂质。液相色谱-质谱联用已成为鉴定这些合成杂质的有力技术。然而，杂质谱分析，其中低水平合成杂质的整个补充在一个单一的分析，是更具挑战性的。在这里，我们提出了一种LC-MS方法的基础上使用高分辨率质谱，特别是傅立叶变换离子回旋共振质谱（FTIRCMS或FTMS）。确定了最佳的LC-FTMS条件，包括固定相和移动的相，用于硫代寡核苷酸的分离和鉴定。然后，电荷状态信息使得能够对检测到的同位素分布进行更准确的建模，以识别检测到的杂质的化学组成。使用这种方法，可以通过LC-FTMS检测许多硫代磷酸酯杂质，包括携带3′-末端磷酸单酯和3′-末端硫代磷酸酯单酯的失效序列、不完全骨架硫化和脱硫产物、高分子量杂质以及全长产物的三氯乙醛、异丁酰基和N3（2-氰乙基）加合物。与低分辨率LC-MS相比，当电荷状态和同位素分布信息可用并用于杂质分析时，可以识别出约60%的杂质。
An LC-MS method based on the use of high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTIRCMS) for profiling oligonucleotides synthesis impurities is described. Oligonucleotide phosphorothioatediesters (phosphorothioate oligonucleotides), in which one of the non-bridging oxygen atoms at each phosphorus center is replaced by a sulfur atom, are now one of the most popular oligonucleotide modifications due to their ease of chemical synthesis and advantageous pharmacokinetic properties. Despite significant progress in the solid-phase oligomerization chemistry used in the manufacturing of these oligonucleotides, multiple classes of low-level impurities always accompany synthetic oligonucleotides. Liquid chromatography-mass spectrometry has emerged as a powerful technique for the identification of these synthesis impurities. However, impurity profiling, where the entire complement of low-level synthetic impurities is identified in a single analysis, is more challenging. Here we present an LC-MS method based the use of high resolution-mass spectrometry, specifically Fourier transform ion cyclotron resonance mass spectrometry (FTIRCMS or FTMS). The optimal LC-FTMS conditions, including the stationary phase and mobile phases for the separation and identification of phosphorothioate oligonucleotides, were found. The characteristics of FTMS enable charge state determination from single m/z values of low-level impurities. Charge state information then enables more accurate modeling of the detected isotopic distribution for identification of the chemical composition of the detected impurity. Using this approach, a number of phosphorothioate impurities can be detected by LC-FTMS including failure sequences carrying 3′-terminal phosphate monoester and 3′-terminal phosphorothioate monoester, incomplete backbone sulfurization and desulfurization products, high molecular weight impurities, and chloral, isobutyryl, and N3 (2-cyanoethyl) adducts of the full length product. When compared with low resolution LC-MS, ~60% more impurities can be identified when charge state and isotopic distribution information is available and used for impurity profiling.
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DOI：
10.1016/s1570-0232(02)00694-3
发表时间：
2002-12-25
影响因子：
3
作者：
Berger, B;Hölzl, G;Parson, W
通讯作者：
Parson, W
影响因子：
2.9
作者：
Cui, Zhengfang;Theruvathu, Jacob A.;Sowers, Lawrence C.
通讯作者：
Sowers, Lawrence C.
影响因子：
2.7
作者：
Kurata, C;Bradley, K;Capaldi, DC
通讯作者：
Capaldi, DC
影响因子：
14.9
作者：
Gilar, M;Belenky, A;Cohen, AS
通讯作者：
Cohen, AS
影响因子：
2.3
作者：
Zhang, Haiying;Yang, Yanou
通讯作者：
Yang, Yanou