质谱技术在中药研究中的应用进展

中药是中华传统医学的瑰宝,在我国已广泛应用于疾病预防和治疗中,但其存在化学成分复杂、作用机制不明确等问题,制约了中药现代化及国际化的发展进程。质谱技术具有高灵敏度、高选择性、高稳定性及高通量的特点,特别适合于中药复杂成分及其代谢物的定性定量分析。该文综述了近年来质谱技术在中药成分鉴定及质量控制、中药代谢组学及中药药代动力学研究方面的典型应用,并对存在的问题及改进方法进行了展望。     

Modern analytical techniques in metabolomics analysis

Metabolomics is the comprehensive assessment of endogenous metabolites and attempts to systematically identify and quantify metabolites from a biological sample. Small-molecule metabolites have an important role in biological systems and represent attractive candidates to understand disease phenotypes. Metabolites represent a diverse group of low-molecular-weight structures including lipids, amino acids, peptides, nucleic acids, organic acids, vitamins, thiols and carbohydrates, which makes global analysis a difficult challenge. The recent rapid development of a range of analytical platforms, including GC, HPLC, UPLC, CE coupled to MS and NMR spectroscopy, could enable separation, detection, characterization and quantification of such metabolites and related metabolic pathways. Owing to the complexity of the metabolome and the diverse properties of metabolites, no single analytical platform can be applied to detect all metabolites in a biological sample. The combined use of modern instrumental analytical approaches has unravelled the ideal outcomes in metabolomics, and is beneficial to increase the coverage of detected metabolites that can not be achieved by single-analysis techniques. Integrated platforms have been frequently used to provide sensitive and reliable detection of thousands of metabolites in a biofluid sample. Continued development of these analytical platforms will accelerate widespread use and integration of metabolomics into systems biology. Here, the application of each hyphenated technique is discussed and its strengths and limitations are discussed with selected illustrative examples; furthermore, this review comprehensively highlights the role of integrated tools in metabolomic research.     

基于质谱分析的代谢组学研究进展

质谱分析技术是代谢组学研究的重要技术之一。该文通过近5年来的文献分析,对基于质谱分析的代谢组学研究方法的新进展,包括样品前处理方法、分析检测方法、数据处理方法等,以及近年来代谢组学在疾病诊断、药物研发、营养学、毒理学、运动医学等领域的应用进展,进行了较全面的综述,并对未来的发展趋势进行了展望。     

Characterization of metabolites in rats after intravenous administration of salvianolic acid for injection by ultra‐performance liquid chromatography coupled with quadrupole‐time‐of‐flight mass spectrometry

It is an essential requirement to clarify the metabolites of traditional Chinese medicine (TCM) injections, which contain numerous ingredients, to assess their safe and effective use in clinic. Salvianolic acid for injection (SAFI), made from hydrophilic phenolic acids in Salvia miltiorrhiza Bunge, has been widely used for the treatment of cerebrovascular diseases, but information on its metabolites in vivo is still lacking. In the present study, we aimed to holistically characterize the metabolites of the main active ingredients in rat plasma, bile, urine and feces following intravenous administration of SAFI. An ultra‐performance liquid chromatography coupled with quadrupole‐time‐of‐flight mass spectrometry (UPLC/Q‐TOF‐MS) method was developed. Combining information on retention behaviors, multistage mass spectra and literature data, a total of eight prototypes and 52 metabolites were tentatively characterized. Metabolites originated from rosmarinic acid and salvianolic acid B comprised the majority of identified compounds. Meanwhile, four metabolites derived from salvianolic acid D and five from salvianolic acid B are reported for the first time. This study revealed that methylation, sulfation and glucuronidation were the major metabolic pathways of phenolic acids in SAFI in vivo. Furthermore, the developed UPLC/Q‐TOF‐MS method could also benefit the metabolic investigation of extracts and preparations in TCM with hydrophilic ingredients. Copyright © 2016 John Wiley & Sons, Ltd.     

基于液相色谱-质谱联用技术的代谢组学分析方法研究进展

代谢组学是研究小分子代谢物的有用工具,能够直接反映生命体终端和表型信息,在精准医学和转化医学中发挥着重要作用。色谱-质谱联用技术具有灵敏度高、选择性好、动态范围宽、信息丰富等优点,已成为代谢组学研究的主要技术平台。代谢组学分析方法的创新与进展是代谢组学在各领域广泛应用的重要前提。该文综述了近5年来基于液相色谱-质谱联用技术的代谢组学分析方法取得的成果,并对目前存在的问题及发展前景给予展望。综述引用文献81篇。     

Ultra-performance liquid chromatography/time-of-flight mass spectrometry based metabolomics of raw and steamed Panax notoginseng

At present, metabolite profiling is of growing importance in herbal medicine fields such as breeding, formulation, quality control and clinical trials. This preliminary study indicated that ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOFMS)-based metabolomics allows direct detection of down-stream derivatives of metabolites, arising from the herbal formulation process. This analytical approach allows the discrimination and tentative authentication of unique biomarkers related to different herbal extracts using unsupervised multivariate principal component analysis (PCA). The tentative identification of biomarkers is complemented significantly by the accurate mass measurement of TOFMS and the high resolution and high retention time reproducibility rendered by UPLC. The application of this approach in herbal extract discrimination and ginsenoside biomarker discovery of raw and steamed Panax notoginseng (Burk.) F.H. Chen is demonstrated and discussed.     

中药质量控制的研究策略和色谱技术

综述了色谱技术作为现代高效分离分析技术在中药药效物质基础研究和中药质量控制中的重要作用。提出中药药效物质组学研究策略,以实现中药药效物质基础与作用机理研究的一体化,进而从深层次揭示和全面控制中药的内在质量。论述结果表明,现代色谱及其联用技术是中药质量研究的重要技术平台之一。     

Ultra-performance liquid chromatography coupled to quadrupole-orthogonal time-of-flight mass spectrometry

Ultra-performance liquid chromatography (UPLC) utilizes sub-2 microm particles with high linear solvent velocities to effect dramatic increases in resolution, sensitivity and speed of analysis. The reduction in particle size to below 2 microm requires instrumentation that can operate at pressures in the 6000-15,000 psi range. The typical peak widths generated by the UPLC system are in the order of 1-2 s for a 10-min separation. In the present work this technology has been applied to the study of in vivo drug metabolism, in particular the analysis of drug metabolites in bile. The reduction in peak width significantly increases analytical sensitivity by three- to five-fold, and the reduction in peak width, and concomitant increase in peak capacity, significantly reduces spectral overlap resulting in superior spectral quality in both MS and MS/MS modes. The application of UPLC/MS resulted in the detection of additional drug metabolites, superior separation and improved spectral quality.     

Untargeted Tumor Metabolomics with Liquid Chromatography–Surface‐Enhanced Raman Spectroscopy

Metabolomics is a powerful systems biology approach that monitors changes in biomolecule concentrations to diagnose and monitor health and disease. However, leading metabolomics technologies, such as NMR and mass spectrometry (MS), access only a small portion of the metabolome. Now an approach is presented that uses the high sensitivity and chemical specificity of surface‐enhanced Raman scattering (SERS) for online detection of metabolites from tumor lysates following liquid chromatography (LC). The results demonstrate that this LC‐SERS approach has metabolite detection capabilities comparable to the state‐of‐art LC‐MS but suggest a selectivity for the detection of a different subset of metabolites. Analysis of replicate LC‐SERS experiments exhibit reproducible metabolite patterns that can be converted into barcodes, which can differentiate different tumor models. Our work demonstrates the potential of LC‐SERS technology for metabolomics‐based diagnosis and treatment of cancer.     

Analysis of alkaloids in Coptis chinensis Franch by accelerated solvent extraction combined with ultra performance liquid chromatographic analysis with photodiode array and tandem mass spectrometry detections

A new method based on accelerated solvent extraction (ASE) followed by ultra performance liquid chromatography (UPLC) analysis has been developed for the identification and quantification of major alkaloids in extracts of Coptis chinensis Franch. The UPLC system consisted of a dual detection system of photodiode array detector (PDA) and positive ion electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in sequential configuration. The operational parameters of ASE including extraction solvent, extraction temperature, static extraction time and extraction cycles were optimized. UPLC analysis was performed on an ACQUITY UPLC BEH C₁₈ column eluted by a mobile phase of acetonitrile spiked with a buffer solution consisting of 0.50% acetic acid and 20 mmol L⁻¹ ammonium acetate. A tandem quadrupole spectrometer operating in either full scan mode or in MS/MS mode for multiple reaction monitoring (MRM) was used for the identification and quantitative analysis of eight major alkaloids in C. chinensis Franch extracts. The samples were also analyzed on a high-performance liquid chromatography-electrospray ionization-time-of-flight mass spectrometry (HPLC-ESI-TOF-MS) system to confirm the identification results. Three of the eight major alkaloids, berberine, palmatine and jatrorrhizine were quantified by UPLC-PDA and UPLC-MS/MS. The results indicated that both UPLC-PDA and UPLC-MS/MS methods were simple, sensitive and reliable for the determination of alkaloids in C. chinensis Franch. Seven Huanglian samples from different locations were analyzed using the established methods. UPLC fingerprints based on the distribution of the eight major alkaloids can serve as a rapid and reliable method for the authentication and quality evaluation of traditional Chinese medicine (TCM) herbs.     

超高效液相色谱/飞行时间质谱用于人参皂甙Rg3作用后大鼠尿液代谢物指纹图谱分析及标记物的鉴定

超高效液相色谱技术(UPLC)采用1.7 μm的色谱柱填料,有更好的分离效率、峰容量以及灵敏度;高分辨的时间飞行质谱(TOF-MS)能够测定化合物准确的分子质量并具有MS/MS功能。 两者的联用适合于复杂体系的分离分析和未知物的结构鉴定。 因此建立了一种基于UPLC/TOF-MS测定人参皂甙Rg3给药后大鼠尿液代谢物变化的方法,对其中2种发生显著变化的代谢物分别通过准确的质量测定得到其元素组成,通过MS/MS技术得到其结构信息,并通过检索数据库最终分别鉴定为4,8-二羟喹啉甲酸和4-羟基-2-喹啉酸。     

High-throughput bioanalysis with simultaneous acquisition of metabolic route data using ultra performance liquid chromatography coupled with time-of-flight mass spectrometry

The capability of ultra performance liquid chromatography coupled with time-of-flight mass spectrometry (UPLC/TOFMS) in the high-throughput quantitative analysis of a drug candidate in plasma has been investigated. Data obtained were compared with results from conventional analysis by high-performance liquid chromatography with tandem mass spectrometric detection on a triple quadrupole instrument (HPLC/MS/MS). The accuracies and precisions of the two approaches were comparable. The UPLC/TOFMS system displayed excellent robustness over the course of 276 injections of protein-precipitated plasma samples. With the instrumentation used, the limits of detection and quantification were approximately five-fold higher with UPLC/TOFMS than for HPLC/MS/MS. Nevertheless, the UPLC/TOFMS system proved adequate to quantify plasma concentrations of a drug molecule administered orally to rats at a pharmacologically relevant dose of 4 mg/kg. As well as providing quantitative data on the test compound, it was also possible to extract data for eight different metabolites, including several isomeric species (three +O and three +2O) from the UPLC/TOFMS data sets, using an analytical method with a 2.5-minute run time. Selectivity for the test compound and its metabolites was derived from the accurate mass capabilities of the TOF instrument, and no MS method development was required.     

Nontargeted urine metabolomics analysis of the protective and therapeutic effects of Citri Reticulatae Chachiensis Pericarpium on high-fat feed-induced hyperlipidemia in rats

In this study, we focused on studying the changes in urine metabolites in hyperlipidemic rats using ultra-performance liquid chromatography coupled with quadrupole time-of-fight mass spectrometry (UPLC–Q-TOF/MS) and metabolomics, as well as the effect of Citri Reticulatae Chachiensis Pericarpium (CRCP) on hyperlipidemia. These urine samples were examined by UPLC–Q-TOF/MS to obtain MS data. The MS data were analyzed by principal component analysis and partial least squares-discriminant analysis to identify the differential metabolites. CRCP reduced the body weight and levels of triglycerides, total cholesterol and low-density lipoprotein cholesterol and abnormally decreased high-density lipoprotein cholesterol in hyperlipidemic rats, which were significantly raised by a high-fat diet. Twenty-seven potential biomarkers were identified within the complex sample matrix of urine. Fourteen biomarkers increased in the hyperlipidemia rats compared with normal rats. Meanwhile, 13 biomarkers decreased. CRCP reversed abnormal changes in biomarkers, including 5-l -glutamyl-taurine, 5-aminopentanoic acid, cis-4-octenedioic acid and 2-octenedioic acid. These biomarkers show that hyperlipidemia is related to the metabolic pathways of taurine and hypotaurine metabolism, fatty acid biosynthesis , and arginine and proline metabolism . CRCP mainly prevents hyperlipidemia by intervening in these metabolic pathways.     

Identification of Metabolites of Epimedin A in Rats Using UPLC/Q–TOF–MS

Herba Epimedii (Epimedium) is a kind of tonic herb, widely used in China. Epimedin A is a major component of Herba Epimedii with bioactivities. Analysis of the metabolic profile in vivo plays a pivotal role in understanding how traditional Chinese medicine works. And the metabolites of epimedin A might influence the effects of Herba Epimedii. Moreover, the metabolic routes of epimedin A provide an important basis for safety evaluation. Until now, little has been known about the metabolism of epimedin A. The current study was designed to characterize the metabolic pathways of epimedin A in vivo. The metabolites in rat plasma, bile, feces, and urine were identified by UPLC/Q–TOF–MS analysis. A total of 27 metabolites from epimedin A were detected or tentatively identified. The major metabolic processes were hydrolysis, hydrogenation, hydroxylation, dehydrogenation, demethylation, and conjugation with glucuronic acid and different sugars. The present study revealed the metabolic pathways of epimedin A in rat for the first time, and epimedin A could undergo extensive phase I and phase II metabolism in rat. These findings would provide an important basis for the further study and clinical application of epimedin A. In addition, the results of this work have shown the feasibility of the UPLC/Q–TOF–MS approach for rapid and reliable characterization of metabolites.