在线能量分辨质谱结合量子化学计算解析牛膝中甾酮类同分异构体

Analysis of Sterones Isomers in Achyranthis Bidentatae Radix by Online Energy-Resolved Mass Spectrometry Combined with Quantum Structural Calculation

甾酮是牛膝的活性成分之一,其同分异构体的难以区分给牛膝化学成分的精准鉴定带来了巨大挑战。在线能量分辨质谱(online energy-resolved mass spectrometry,Online ER-MS)被证明是一种区分同分异构体的有效方法。本研究首先采用超高效液相色谱-四极杆-串联飞行时间质谱(UHPLC-Q-TOF-MS/MS)技术初步鉴定牛膝中的甾酮类化合物,为了进一步区分甾酮类同分异构体,利用超高效液相色谱-四极杆线性离子阱-串联质谱(UHPLC-Q-trap-MS/MS)的多反应监测模式采集各化合物拟离子对(pseudo-ion transitions,PITs)的碰撞能量(collision energy,CE)及相对离子强度,通过逐级改变CE绘制裂解曲线,定义裂解曲线顶点为最佳碰撞能(optimal collision energy,OCE),分别计算各化合物不同离子对的OCE。同时,结合量子化学计算各候选分子化学结构断裂的键能,建立化学结构与OCE的关系。结果表明,从牛膝中共鉴定到包括3组同分异构体的12个甾酮类化合物,并通过与标准品比对验证了该方法的可行性。在线能量分辨质谱与量子化学的结合能够实现同分异构体的高效区分,为中药中同分异构体的精准鉴定提供了新思路。

Sterones are one of the active components of Achyranthes bidentata,and the difficulty in distinguishing sterone isomers poses a great challenge to the accurate identification of the chemical components of Achyranthes bidentata,while online energy-resolved mass spectrometry(Online ER-MS)has been proved to be an effective method for distinguishing the isomers due to its ability to provide rich MS/MS fragment information.In this study,mass spectra of sterones were collected by ultra-high performance liquid chromatography-quadrupole tandem time-of-flight mass spectrometry(UHPLC-Q-TOF-MS/MS),and then the structures of the compounds were matched with MS/MS fragment ions to initially identify the sterone compounds in Achyranthes bidentata.In order to further distinguish the sterones isomers,the fragment ions with higher abundance detected in UHPLC-Q-TOF-MS/MS were selected to construct pseudo-ion transitions(PITs),and the collision energy(CE)and relative ion intensity of PITs were collected by using the multi-reaction monitoring mode of ultra-high performance liquid chromatography tandem quadrupole linear ion trap mass/mass spectrometry(UHPLC-Q-trap-MS/MS).Further,the relative ion intensity of each PIT under different CE was recorded,and the dataset was imported into GraphPad Prism software to draw the fragmentation curve for each ion transition via Gaussian fitting.The vertex of the fragmentation curve was defined as the optimal collision energy(OCE),and the OCE of different ion transitions of each compound was calculated respectively.At the same time,combined with quantum structural calculation,the thermal enthalpies of precursor ions and product ions in each candidate structure were calculated using Gaussian program based on density functional theory,and the bond energy required to break the corresponding chemical bond was calculated according to the difference between the precursor ion and the two product ions.The OCE and bond energies of each group of isomers were analyzed to establish the relationship between the chemical structure and OCE.The results showed that the energy required to break the same chemical bond was not the same due to the different substituent positions of isomers,which made the OCE of different ion transitions of isomers also differ.Finally,12 sterones including three groups of the isomers were identified from Achyranthes bidentata,and the feasibility of the method was verified by comparing with the standards.Therefore,the combination of online energy-resolved mass spectrometry and quantum structural calculation can achieve efficient differentiation of isomers,providing a new solution for accurate identification of isomers in traditional Chinese medicines.