{"title":"使用不含离子对的苯基键合固定相的反相超高效液相色谱法分离和鉴定寡核苷酸杂质和降解产物--向可持续发展迈出的一步","authors":"","doi":"10.1016/j.chroma.2024.465380","DOIUrl":null,"url":null,"abstract":"<div><p>This manuscript discusses the development of a reversed-phase ultra high-performance liquid chromatography (RP UHPLC) method based on phenyl-bonded stationary phases without ion-pairs for the separation and identification of oligonucleotides. The elimination of ion-pair reagents makes the proposed protocol as more compliant to the principles of green chemistry, compared to the traditional ion-pair reversed-phase liquid chromatography methods (IP RP LC). In detail, three phenyl-based stationary phases were tested, namely a C18/AR (a C18 stationary phase with the addition of aromatic groups), a Phenyl-hexyl, and a Diphenyl. Generally, the retention of oligonucleotides increases with the increase of salt concentration and the decrease of the pH, thus confirming the significant impact of van der Waals interactions, salting-out effect, and π-electrons interactions in the retention mechanism. The highest retention and best peak symmetry were observed for the C18/AR stationary phase, while the lowest retention for the Phenyl-hexyl, with retention influenced by the type of salt in the mobile phase. The obtained methods using C18/AR stationary phases allow for the effective separations of positional isomers and for identifying impurities and degradation products using RP UHPLC Q-TOF-MS in a comparatively short time. The application of RP UHPLC Q-TOF-MS provides reasonable selectivity for the resolution of 33 impurities and two degradation products. Both groups of compounds are mainly 3′N and 5′N-shortmers, but in the case of impurities, modifications of cyclic phosphate and phosphate groups were also identified. Nevertheless, Diphenyl and Phenyl-Hexyl may be applied to separate modified oligonucleotides with higher salt concentrations. The proposed separation methods without ion-pair reagents contribute to a more sustainable approach in oligonucleotide analysis.</p></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":null,"pages":null},"PeriodicalIF":3.8000,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Separation and identification of oligonucleotides impurities and degradation products by reversed phase ultra-high performance liquid chromatography using phenyl-bonded stationary phases without ion pairs - A step towards sustainability\",\"authors\":\"\",\"doi\":\"10.1016/j.chroma.2024.465380\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>This manuscript discusses the development of a reversed-phase ultra high-performance liquid chromatography (RP UHPLC) method based on phenyl-bonded stationary phases without ion-pairs for the separation and identification of oligonucleotides. The elimination of ion-pair reagents makes the proposed protocol as more compliant to the principles of green chemistry, compared to the traditional ion-pair reversed-phase liquid chromatography methods (IP RP LC). In detail, three phenyl-based stationary phases were tested, namely a C18/AR (a C18 stationary phase with the addition of aromatic groups), a Phenyl-hexyl, and a Diphenyl. Generally, the retention of oligonucleotides increases with the increase of salt concentration and the decrease of the pH, thus confirming the significant impact of van der Waals interactions, salting-out effect, and π-electrons interactions in the retention mechanism. The highest retention and best peak symmetry were observed for the C18/AR stationary phase, while the lowest retention for the Phenyl-hexyl, with retention influenced by the type of salt in the mobile phase. The obtained methods using C18/AR stationary phases allow for the effective separations of positional isomers and for identifying impurities and degradation products using RP UHPLC Q-TOF-MS in a comparatively short time. The application of RP UHPLC Q-TOF-MS provides reasonable selectivity for the resolution of 33 impurities and two degradation products. Both groups of compounds are mainly 3′N and 5′N-shortmers, but in the case of impurities, modifications of cyclic phosphate and phosphate groups were also identified. Nevertheless, Diphenyl and Phenyl-Hexyl may be applied to separate modified oligonucleotides with higher salt concentrations. 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引用次数: 0
摘要
本手稿讨论了基于无离子对苯键固定相的反相超高效液相色谱(RP UHPLC)方法的开发,用于寡核苷酸的分离和鉴定。与传统的离子对反相液相色谱法(IP RP LC)相比,不使用离子对试剂使该方法更符合绿色化学原理。具体而言,测试了三种苯基固定相,即 C18/AR(添加芳香基团的 C18 固定相)、苯基己基和二苯基固定相。一般来说,寡核苷酸的保留率随着盐浓度的增加和 pH 值的降低而增加,从而证实了范德华相互作用、盐析效应和 π 电子相互作用在保留机制中的重要影响。C18/AR 固定相的保留率最高,峰对称性最好,而苯基己基固定相的保留率最低,保留率受流动相中盐类的影响。利用 C18/AR 固定相获得的方法可以在较短时间内有效分离位置异构体,并利用 RP UHPLC Q-TOF-MS 鉴定杂质和降解产物。应用 RP UHPLC Q-TOF-MS 对 33 种杂质和两种降解产物的分辨具有合理的选择性。这两类化合物主要是 3′N和 5′N-shortmers,但在杂质中也发现了环磷酸基和磷酸基的修饰。不过,二苯基和苯基己基可用于分离盐浓度较高的修饰寡核苷酸。建议的不使用离子对试剂的分离方法有助于在寡核苷酸分析中采用更可持续的方法。
Separation and identification of oligonucleotides impurities and degradation products by reversed phase ultra-high performance liquid chromatography using phenyl-bonded stationary phases without ion pairs - A step towards sustainability
This manuscript discusses the development of a reversed-phase ultra high-performance liquid chromatography (RP UHPLC) method based on phenyl-bonded stationary phases without ion-pairs for the separation and identification of oligonucleotides. The elimination of ion-pair reagents makes the proposed protocol as more compliant to the principles of green chemistry, compared to the traditional ion-pair reversed-phase liquid chromatography methods (IP RP LC). In detail, three phenyl-based stationary phases were tested, namely a C18/AR (a C18 stationary phase with the addition of aromatic groups), a Phenyl-hexyl, and a Diphenyl. Generally, the retention of oligonucleotides increases with the increase of salt concentration and the decrease of the pH, thus confirming the significant impact of van der Waals interactions, salting-out effect, and π-electrons interactions in the retention mechanism. The highest retention and best peak symmetry were observed for the C18/AR stationary phase, while the lowest retention for the Phenyl-hexyl, with retention influenced by the type of salt in the mobile phase. The obtained methods using C18/AR stationary phases allow for the effective separations of positional isomers and for identifying impurities and degradation products using RP UHPLC Q-TOF-MS in a comparatively short time. The application of RP UHPLC Q-TOF-MS provides reasonable selectivity for the resolution of 33 impurities and two degradation products. Both groups of compounds are mainly 3′N and 5′N-shortmers, but in the case of impurities, modifications of cyclic phosphate and phosphate groups were also identified. Nevertheless, Diphenyl and Phenyl-Hexyl may be applied to separate modified oligonucleotides with higher salt concentrations. The proposed separation methods without ion-pair reagents contribute to a more sustainable approach in oligonucleotide analysis.
期刊介绍:
The Journal of Chromatography A provides a forum for the publication of original research and critical reviews on all aspects of fundamental and applied separation science. The scope of the journal includes chromatography and related techniques, electromigration techniques (e.g. electrophoresis, electrochromatography), hyphenated and other multi-dimensional techniques, sample preparation, and detection methods such as mass spectrometry. Contributions consist mainly of research papers dealing with the theory of separation methods, instrumental developments and analytical and preparative applications of general interest.