基于液质联用技术的中药植化成分研究方法与策略

中药是中医基础理论指导下用以防病治病的药物,为中华民族的繁荣昌盛做出了巨大的贡献。然而,中药植化成分极为复杂,各成分的含量差异较大,药效物质基础不明已经成为制约中药现代化的瓶颈。液质联用技术拥有色谱的高分离性能和质谱高灵敏度的特点,已被广泛用于天然药物的分析,极大地推动了中药研究的发展。许多研究围绕植化成分的全面表征,通过质谱裂解规律的研究及不同质谱技术的整合,建立了快速、准确、系统的分析中药化学成分的新方法和新策略,为中药的质量控制和资源合理利用奠定了研究基础。     

基于UPLC-Q/TOF-MS的甘草酸单铵和甘草酸二铵在大鼠体内移行成分表征

目的 采用超高效液相色谱-四极杆/飞行时间高分辨质谱技术(UPLC-Q/TOF-MS)对甘草酸单铵和甘草酸二铵在大鼠体内的代谢轮廓进行表征，探讨两者可能的代谢途径。方法 大鼠分别灌胃给予甘草酸单铵和甘草酸二铵后，收集不同时间点大鼠脑组织、血浆、尿液、粪样等生物样本。经沉淀蛋白后，采用UPLC-Q/TOF-MS技术，以含0.1%甲酸的水-乙腈为流动相进行梯度洗脱；在正离子模式下采集生物样本的质谱数据。采用质量残差亏损过滤技术并结合原型成分的质谱裂解规律，预测代谢物类型，对两者相关的体内代谢产物进行鉴定。结果 甘草酸单铵和甘草酸二铵在大鼠体内主要以代谢物形式存在，共鉴定得到20个代谢产物，两者主要通过肠道排泄，在体内原型化合物脱糖基形成的甘草次酸基础上发生一系列的Ⅰ相氧化还原反应是主要代谢类型；甘草次酸是在生物样本中主要暴露成分，提示了其可能是甘草酸单铵及甘草酸二铵在体内发挥药效的关键药效成分。结论 首次阐明了甘草酸单铵及甘草酸二铵在大鼠体内的代谢轮廓，为进一步对两者发挥功效作用的物质基础及药效作用机制揭示和开发利用提供参考。     

基于UHPLC-Q-Orbitrap HRMS技术的天麻首乌胶囊化学成分研究

目的：为了系统阐明复方中药制剂天麻首乌胶囊中的化学成分组成，同时建立一种精准、高效的中药复杂成分鉴定分析方法。方法：本研究采用超高效液相色谱-四级杆/静电场轨道阱高分辨质谱（UHPLC-Q-Orbitrap HRMS），通过分析获得的化合物精确分子量、保留时间和多级碎片离子信息，结合对照品相关质谱数据、质谱谱图数据库及参考文献，实现对天麻首乌胶囊中化合物的准确定性。结果：本研究共鉴定出化合物51个，涉及黄酮类、酚酸类、苯酞类、蒽醌类、其他类等5大类物质。结论：本研究可系统、准确、高效地鉴定天麻首乌胶囊中的多种化学成分，并为研究其药效物质基础及指导临床合理用药提供了科学的理论依据。     

基于UPLC-Q-Orbitrap-HRMS技术分析半夏白术天麻汤的化学成分及入血成分

目的:采用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱(UPLC-Q-Orbitrap-HRMS)技术分析半夏白术天麻汤(Banxia Baizhu Tianma Decoction,BBTD)化学成分及入血成分,阐明其可能的药效物质基础。方法:色谱柱为Waters ACQUITY UPLC HSS T3 C₁₈(2.1mm×100mm,1.8μm),流动相为0.1%甲酸乙腈(A)-0.1%甲酸水溶液(B)梯度洗脱,流速0.2mL·min^-1。采用电喷雾离子源ESI,正、负离子模式扫描。根据准分子离子与二级碎片离子,结合对照品及文献数据鉴定BBTD化学成分、入血成分。结果:BBTD表征出133个成分,含药大鼠血浆样品分析出48个化合物,包括39个原型成分和9个代谢产物。结论:通过UPLC-Q-Orbitrap-HRMS技术对BBTD化学成分及入血成分进行表征,为其药效物质基础研究和质量控制奠定基础。     

整合动态PK-PD相关性分析在中药药效物质基础发现中的研究策略

中药药效物质基础研究是解释中医药作用机制的基石,然而中药复方作为一个复杂体系,作用机制与体内效应过程不明确等问题长期桎梏中医药事业的发展。药动学-药效学（PK-PD）相关性研究是动态表征和定量描述活性成分与药理作用之间相关性的有效方法,将时间、浓度和效应结合起来,已经成为阐明中药药效物质基础的技术策略,但是目前研究大都集中在结构明确的化学药物,缺乏符合中医药发展模式的相关策略。因此,非常有必要深入挖掘新的研究策略,建立符合中医药特色的PK-PD模型,通过进行方法开发与技术重组,利用从化学物质基础、关键成分的筛选、整合PK-PD相关性的多维整合的研究思路,引入代谢组学等现代技术手段,全方位、及时灵敏地表征药效特点,评价药动学考察药物机制研究,旨在构建更易于发现药效物质的技术体系,为阐明中药复方的药效物质基础提供参考。     

质谱成像技术在中药研究中的应用现状

质谱成像技术能够将质谱数据转变为可视化的离子成像图,具有前处理简单及结果清晰直观的优势。近年来,质谱成像被应用于揭示中药中天然成分的空间分布特征,探索中药药理作用机制,研究中药质量评价新方法,为中药研究带来了全新的视角。本综述概述了几种常见的质谱成像技术的原理、应用及发展史,重点介绍了基质辅助激光电离解吸附质谱成像技术、解吸电喷雾电离质谱成像技术及二次离子质谱成像技术;随后,概括了利用质谱成像探索中药成分的常见操作流程,列举了质谱成像技术在中药研究中的多个应用实例,详细说明质谱成像在中药研究中的独特贡献;最后,探讨了质谱成像应用于中药研究中的不足之处,期望能在未来得到补足。     

质谱技术在中药成分鉴定中的应用

中药现代化必须提高中药质量研究。质谱技术具有强大的结构鉴定能力,在中药质量研究中具有十分重要的地位。飞行时间质谱具有测定精确分子量的能力,离子阱质谱则能测定多级质谱,将两者结合起来,能够在中药化学成分鉴定中发挥巨大的威力。本文就这两种质谱技术在中药成分鉴定中的应用作一综述。     

基于UPLC-QE-Orbitrap-MS技术分析鉴定中药三棱入血成分

目的:利用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱法(UPLC-QE-Orbitrap-MS)分析三棱提取物在大鼠体内的入血成分。方法:采用ACQUITY UPLC HSS T3色谱柱(2.1 mm×100 mm, 1.8μm),以0.1%甲酸乙腈溶液(A)-0.1%甲酸水溶液(B)为流动相进行梯度洗脱，流速0.2 mL·min^-1,进样量5μL。质谱分析采用电喷雾离子源，在正、负离子模式下采集数据，扫描范围m/z 100～1 000。采用Compound Discoverer 3.0软件获取特征碎片离子信息，结合数据库(mzCloud, mzVault、OTCML数据库及文献确定入血成分。结果:综合质谱检测信息，从三棱药材提取物中解析出70个化学成分，大鼠口服给药后，含药血浆中分析出40个成分，包括20种原型成分和20个代谢产物，其主要代谢途径分别为甲基化、去甲基化、还原反应、葡萄糖醛酸化等。结论:本文采用UPLC-QE-Orbitrap-MS技术分析鉴定了三棱主要入血成分及部分代谢产物，为阐明三棱的药效物质基础和质量控制奠定基础。     

超高压液相/电喷雾-LTQ-Orbitrap质谱联用技术分析三七根中皂苷类成分

建立三七药材的UPLC-ESI-HRMS定性分析方法。采用Kinetics色谱柱,梯度洗脱,经DAD检测器后采用ESI-LTQ-Orbitrap质谱负离子模式采集,三七成分的一级全扫描离子在离子阱内进行CID多级质谱,FT检测高分辨质谱数据进行分析。结果表明三七皂苷成分分离良好,根据多级质谱碎片信息和精确相对分子质量信息,结合文献,从三七中鉴定分析了43个成分,并首次从三七中检测到新型的三七皂苷母核和乙酰取代皂苷成分。本方法分离度良好、灵敏度高,可用于三七药材的定性分析,并有助于对三七进行进一步的活性成分研究和质量控制。     

4种秦艽中化学成分的UPLC-LTQ-Orbitrap-MS快速分析研究

目的利用UPLC-LTQ-Orbitrap-MS技术对4种秦艽药材的化学成分进行定性分析。方法采用Waters Acquity UPLC HSS T3色谱柱(2.1 mm×50 mm,1.7μm),在电喷雾正负离子模式下采集数据。将母离子列表(PIL)-MS2和高分辨质谱结合,用于秦艽药材中化学成分的鉴别。结果结合质谱数据和文献数据共鉴定、推断出66个成分,其中包括环烯醚萜类36个、木脂素类5个、黄酮类5个、三萜类12个、其他类8个。其中19个成分为首次报道。结论 4种秦艽成分大致相同,利用UPLC-LTQOrbitrap-MS技术结合母离子列表的方法可以快速筛选《中国药典》规定的4种秦艽中的化学成分,为秦艽化学成分的提取分离及其药效物质基础研究提供参考。     

Recent advances in metabolite identification and quantitative bioanalysis by LC-Q-TOF MS

The need for rapid, sensitive and effective identification and quantitation of drugs and metabolites to accelerate drug discovery and development has given MS its central position in drug metabolism and pharmacokinetic research. This review attempts to orient the readers with respect to hybrid Q-TOF MS, which enables accurate mass measurement and generates information-rich datasets. The key properties of the Q-TOF MS system, including mass accuracy, resolution, scan speed and dynamic range, are herein discussed. Developments on tandem separation techniques (e.g., UHPLC(?) and ion mobility spectrometry), data acquisition and data-mining methods (e.g., mass defect, product/neutral loss, isotope pattern filters and background subtraction) that facilitate qualitative and quantitative analysis are then examined. The performance and versatility of LC-Q-TOF MS are thoroughly illustrated by its applications in metabolite identification and quantitative bioanalysis. Future perspectives are also discussed.     

大鼠灌胃四逆散提取物后血浆、尿液、粪便、胆汁中主要代谢产物的鉴定

采用UPLC-Q-TOF/MS研究四逆散提取物在大鼠体内的代谢产物,利用碰撞能量梯度(MSE)和质量亏损过滤(MDF)技术进行分析,鉴定大鼠灌胃四逆散提取物后血浆、尿液、粪便、胆汁中的代谢产物。四逆散提取物中柚皮苷、柚皮素、橙皮苷、新橙皮苷、甘草苷、甘草素、甘草酸、甘草次酸、柴胡皂苷a、柴胡皂苷d在大鼠不同代谢途径中推测出原形、羟基化、葡糖醛酸化、硫酸化、葡糖醛酸化与硫酸化结合、羟化葡糖醛酸化等共41个代谢产物。     

Metabolism studies on hydroxygenkwanin and genkwanin in human liver microsomes by UHPLC-Q-TOF-MS

Hydroxygenkwanin (HYGN) and genkwanin (GN) are major constituents of Genkwa Flos for the treatment of edema, ascites, cough, asthma and cancer. This is a report about the investigation of the metabolic fate of HYGN and GN in human liver microsomes and the recombinant UDP-glucuronosyltransferase (UGT) enzymes by using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). An on-line data acquisition method multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS) was developed to trace all probable metabolites. Based on this analytical strategy, three phase I metabolites and seven glucuronide conjugation metabolites of HYGN, seven phase I metabolites and 12 glucuronide conjugation metabolites of GN were identified in the incubation samples of human liver microsomes. The results indicated that demethylation, hydroxylation and o-glucuronidation were main metabolic pathways of HYGN and GN. The specific UGT enzymes responsible for HYGN and GN glucuronidation metabolites were identified using recombinant UGT enzymes. The results indicated that UGT1A1, UGT1A3, UGT1A9, UGT1A10 and UGT2B7 might play major roles in the glucuronidation reactions. Overall, this study may be useful for the investigation of metabolic mechanism of HYGN and GN, and it can provide reference and evidence for further experiments.     

Determination of exposure multiples of human metabolites for MIST assessment in preclinical safety species without using reference standards or radiolabeled compounds

A simple, reliable, and accurate method was developed for quantitative assessment of metabolite coverage in preclinical safety species by mixing equal volumes of human plasma with blank plasma of animal species and vice versa followed by an analysis using high-resolution full-scan accurate mass spectrometry. This approach provided comparable results (within (±15%) to those obtained from regulated bioanalysis and did not require synthetic standards or radiolabeled compounds. In addition, both qualitative and quantitative data were obtained from a single LC-MS analysis on all metabolites and, therefore, the coverage of any metabolite of interest can be obtained.     

Application of stable isotope-labeled compounds in metabolism and in metabolism-mediated toxicity studies

Stable isotope-labeled compounds have been synthesized and utilized by scientists from various areas of biomedical research during the last several decades. Compounds labeled with stable isotopes, such as deuterium and carbon-13, have been used effectively by drug metabolism scientists and toxicologists to gain better understanding of drugs' disposition and their potential role in target organ toxicities. The combination of stable isotope-labeling techniques with mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy, which allows rapid acquisition and interpretation of data, has promoted greater use of these stable isotope-labeled compounds in absorption, distribution, metabolism, and excretion (ADME) studies. Examples of the use of stable isotope-labeled compounds in elucidating structures of metabolites and delineating complex metabolic pathways are presented in this review. The application of labeled compounds in mechanistic toxicity studies will be discussed by providing an example of how strategic placement of a deuterium atom in a drug molecule mitigated specific-specific renal toxicity. Other examples from the literature demonstrating the application of stable isotope-labeled compounds in understanding metabolism-mediated toxicities are presented. Furthermore, an example of how a stable isotope-labeled compound was utilized to better understand some of the gene changes in toxicogenomic studies is discussed. The interpretation of large sets of data produced from toxicogenomics studies can be a challenge. One approach that could be used to simplify interpretation of the data, especially from studies designed to link gene changes with the formation of reactive metabolites thought to be responsible for toxicities, is through the use of stable isotope-labeled compounds. This is a relatively unexplored territory and needs to be further investigated. The employment of analytical techniques, especially mass spectrometry and NMR, used in conjunction with stable isotope-labeled compounds to establish and understand mechanistic link between reactive metabolite formation, genomic, and proteomic changes and onset of toxicity is proposed. The use of stable isotope-labeled compounds in early human ADME studies as a way of identifying and possibly quantifying all drug-related components present in systemic circulation is suggested.     

Insight into chemical basis of traditional Chinese medicine based on the state-of-the-art techniques of liquid chromatography−mass spectrometry

Traditional Chinese medicine (TCM) has been an indispensable source of drugs for curing various human diseases. However, the inherent chemical diversity and complexity of TCM restricted the safety and efficacy of its usage. Over the past few decades, the combination of liquid chromatography with mass spectrometry has contributed greatly to the TCM qualitative analysis. And novel approaches have been continuously introduced to improve the analytical performance, including both the data acquisition methods to generate a large and informative dataset, and the data post-processing tools to extract the structure-related MS information. Furthermore, the fast-developing computer techniques and big data analytics have markedly enriched the data processing tools, bringing benefits of high efficiency and accuracy. To provide an up-to-date review of the latest techniques on the TCM qualitative analysis, multiple data-independent acquisition methods and data-dependent acquisition methods (precursor ion list, dynamic exclusion, mass tag, precursor ion scan, neutral loss scan, and multiple reaction monitoring) and post-processing techniques (mass defect filtering, diagnostic ion filtering, neutral loss filtering, mass spectral trees similarity filter, molecular networking, statistical analysis, database matching, etc.) were summarized and categorized. Applications of each technique and integrated analytical strategies were highlighted, discussion and future perspectives were proposed as well.KEY WORDS: Liquid chromatography−mass spectrometry, Qualitative analysis, Traditional Chinese medicine, Data acquisition, Data post-processing     

Detection and characterization of metabolites in biological matrices using mass defect filtering of liquid chromatography/high resolution mass spectrometry data.

An improved mass defect filter (MDF) method employing both drug and core structure filter templates was applied to the processing of high resolution liquid chromatography/mass spectrometry (LC/MS) data for the detection and structural characterization of oxidative metabolites with mass defects similar to or significantly different from those of the parent drugs. The effectiveness of this approach was investigated using nefazodone as a model compound, which is known to undergo multiple common and uncommon oxidative reactions. Through the selective removal of all ions that fall outside of the preset filter windows, the MDF process facilitated the detection of all 14 nefazodone metabolites presented in human liver microsomes in the MDF-filtered chromatograms. The capability of the MDF approach to remove endogenous interferences from more complex biological matrices was examined by analyzing omeprazole metabolites in human plasma. The unprocessed mass chromatogram showed no distinct indication of metabolite peaks; however, after MDF processing, the metabolite peaks were easily identified in the chromatogram. Compared with precursor ion scan and neutral loss scan techniques, the MDF approach was shown to be more effective for the detection of metabolites in a complex matrix. The comprehensive metabolite detection capability of the MDF approach, together with accurate mass determination, makes high resolution LC/MS a useful tool for the screening and identification of both common and uncommon drug metabolites.     

Rapid Screening for 67 Drugs and Metabolites in Serum or Plasma by Accurate-Mass LC-TOF-MS

Sixty-seven drugs and metabolites were detected in serum or plasma using a fast (7.5 min) liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) method. This method was developed as a blood drug screen, with emphasis on the detection of common drugs of abuse and drugs used to manage chronic pain. Qualitative drug detection may identify a drug exposure, assure patient adherence with prescribed therapy and document abstinence from non-prescribed medications. Compound identification is based on chromatographic retention time, mass, isotope spacing and isotope abundance. Data analysis software (Agilent) generates a compound score based on how well these observed criteria matched theoretical and empirical values. The method was validated using fortified samples and 299 residual patient specimens (920 positive results). All results were confirmed by gas chromatography-MS or LC-tandem MS. The accuracy of positive results (samples meeting all qualitative criteria for retention time, mass and compound score) was >90% for drugs and/or metabolites, except for two benzodiazepines. There were 35 false positive results (seven compounds, 3.8%) that could be distinguished by retention time and/or absence of metabolites. The most frequent was 6-acetylmorphine in the absence of morphine. The LC-TOF-MS targeted screening method presented represents a sensitive and specific technology for drug screening of serum or plasma.     

High-Resolution Mass Spectrometry Elucidates Metabonate (False Metabolite) Formation from Alkylamine Drugs during In Vitro Metabolite Profiling

In vitro metabolite profiling and characterization experiments are widely employed in early drug development to support safety studies. Samples from incubations of investigational drugs with liver microsomes or hepatocytes are commonly analyzed by liquid chromatography/mass spectrometry for detection and structural elucidation of metabolites. Advanced mass spectrometers with accurate mass capabilities are becoming increasingly popular for characterization of drugs and metabolites, spurring changes in the routine workflows applied. In the present study, using a generic full-scan high-resolution data acquisition approach with a time-of-flight mass spectrometer combined with postacquisition data mining, we detected and characterized metabonates (false metabolites) in microsomal incubations of several alkylamine drugs. If a targeted approach to mass spectrometric detection (without full-scan acquisition and appropriate data mining) were employed, the metabonates may not have been detected, hence their formation underappreciated. In the absence of accurate mass data, the metabonate formation would have been incorrectly characterized because the detected metabonates manifested as direct cyanide-trapped conjugates or as cyanide-trapped metabolites formed from the parent drugs by the addition of 14 Da, the mass shift commonly associated with oxidation to yield a carbonyl. This study demonstrates that high-resolution mass spectrometry and the associated workflow is very useful for the detection and characterization of unpredicted sample components and that accurate mass data were critical to assignment of the correct metabonate structures. In addition, for drugs containing an alkylamine moiety, the results suggest that multiple negative controls and chemical trapping agents may be necessary to correctly interpret the results of in vitro experiments.