Applications of HPLC-MS in compound Ilex pubescens extract study

In this paper, high performance liquid chromatography (HPLC) along with mass spectrometry (MS) and HPLC along with a diode array detector (DAD) was used to study the compound Ilex pubescens extract. Two ionization techniques: electro spray ionization (ESI) and atmospheric pressure chemical ionization (APCI) were used in this work. The liquid chromatograms obtained by DAD, total ion chromatograms (TIC) from positive-and negative-ion ESI-MS and the positive-and negative-ion APCI-MS were compared. The liquid chromatograms obtained by TIC from ESI-MS provided more information on chromatographic peaks than those obtained by DAD or TIC from APCI-MS. It is suggested that the fingerprints of the compound Ilex pubescens extract should be provided by the liquid chromatograms obtained by DAD together with TIC from the negative-ion ESI-MS. The molecular weights of the nine main components in an HPLC-DAD chromatogram were determined by the corresponding positive-and negative-ion ESI and the positive-and negative-ion APCI mass spectra information. In the liquid chromatogram obtained by TIC from the negative-ion ESI-MS, the molecular weights of 23 main components were determined based on the corresponding positive-and negative-ion ESI mass spectra information.     

Tracing novel hemostatic compounds from heating products of total flavonoids in Flos Sophorae by spectrum–effect relationships and column chromatography

Flos Sophorae and its processed product have been clinically used to treat hemorrhage. In this study, the total ion chromatographic fingerprints of the heating products of total flavonoids in Flos Sophorae were established by high‐performance liquid chromatography with tandem mass spectrometry and the hemostatic activities were studied by hemostatic screening tests in vivo. The spectrum–effect relationships between fingerprints and hemostatic activities were investigated using canonical correlation analysis to trace the peaks responsible for the hemostatic effects. The predicted active peaks in fingerprints were isolated by column chromatography and their structures were identified by NMR spectroscopy and mass spectrometry. The hemostatic activities of them were verified by platelet aggregation and procoagulation assays in vitro. Canonical correlation analysis results showed that peak 8 and peak 11 were correlated most closely, thus probably being the main hemostatic compounds. Through column chromatography separation, peak 8 (compound I) and peak 11 (compound II) were obtained with purities of 95.61 and 93.38%, respectively, and were discovered new hemostatic compounds named as huaicarbon A (I) and huaicarbon B (II), respectively. This study provides a universal model to trace the active compounds of other herbs which have bioactivity enhancement after processing by spectrum–effect relationships and column chromatography.     

Spectrum–effect relationship study between HPLC fingerprints and antioxidant of honeysuckle extract

Honeysuckle (Lonicera japonica flos) is a well‐known agent of edible and medicinal value in China and its antioxidative activity makes a major contribution to its dual use. However, the compounds responsible for its antioxidative activity are still unknown. In this study, 10 batches of honeysuckle were collected from different origins in China. The fingerprints were established by HPLC technique to investigate the compounds and a 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging activity assay was carried out to evaluate their antioxidant activity. partial least squares regression analysis was applied to set up the regression equation between DPPH radical scavenging activity and average peak area of common peaks of fingerprints. The results showed that peaks 10 (isochlorogenic acid B), 12 (isochlorogenic acid C), 11 (isochlorogenic acid A) and 9 (cynaroside) in the fingerprints were closely related to the antioxidant activity of 50% methanol extracts of honeysuckle. This study successfully established the spectrum–effect relationship between HPLC fingerprints and DPPH radical scavenging activity and provided a general model for exploring active components with a combination of chromatography and efficacy.     

Authentication of Shenqi Fuzheng Injection via UPLC-Coupled Ion Mobility—Mass Spectrometry and Chemometrics with Kendrick Mass Defect Filter Data Mining

Nearly 5% of the Shenqi Fuzheng Injection’s dry weight comes from the secondary metabolites of Radix codonopsis and Radix astragali. However, the chemical composition of these metabolites is still vague, which hinders the authentication of Shenqi Fuzheng Injection (SFI). Ultra-high performance liquid chromatography with a charged aerosol detector was used to achieve the profiling of these secondary metabolites in SFI in a single chromatogram. The chemical information in the chromatographic profile was characterized by ion mobility and high-resolution mass spectrometry. Polygonal mass defect filtering (PMDF) combined with Kendrick mass defect filtering (KMDF) was performed to screen potential secondary metabolites. A total of 223 secondary metabolites were characterized from the SFI fingerprints, including 58 flavonoids, 71 saponins, 50 alkaloids, 30 polyene and polycynes, and 14 other compounds. Among them, 106 components, mainly flavonoids and saponins, are contributed by Radix astragali, while 54 components, mainly alkaloids and polyene and polycynes, are contributed by Radix codonopsis, with 33 components coming from both herbs. There were 64 components characterized using the KMDF method, which increased the number of characterized components in SFI by 28.70%. This study provides a solid foundation for the authentification of SFIs and the analysis of its chemical composition.     

沉香的高效液相色谱-四极杆-飞行时间质谱数字化指纹图谱研究

采用高效液相色谱-四极杆-飞行时间质谱联用(HPLC-Q-TOF-MS)技术,研究构建了一种沉香数字化色谱-质谱指纹图谱的新方法。沉香药材经乙醇提取后,采用HPLC-Q-TOF-MS测定,并同时采集HPLC-Q-TOF-MS及液相色谱-紫外数据,得到液相色谱-紫外检测(HPLC-UV)色谱图和高分辨飞行时间质谱(TOF-MS)总离子流色谱图。对色谱图中的各个色谱峰进行精确质量数识别,建立数字化指纹图谱,以精确质量数结合保留时间表征沉香中的化学成分,即为每个色谱峰给出具有唯一性的数字信息,以数字化的形式反映其化学成分,并根据精确质量及同位素推算出分子式,结合二级质谱及文献资料共鉴定出30个化学成分。该方法对沉香的每种化学成分给出了类似于身份认定的数字化信息,具有唯一性,能全面反映沉香的物质成分,可为沉香的药理、药效及质量标准研究提供科学的数据。     

复方丹参片的高效液相色谱/质谱特征图谱研究

研究了复方丹参片乙醇提取物电喷雾离子阱质谱(ESI-MS)和(HPLC-MS)特征图谱,并对特征图谱中各主要峰进行了初步定性分析,为复方丹参片的快速指纹鉴别提供了新方法。     

Potential antioxidant compounds in Mallotus species fingerprints. Part II: Fingerprint alignment,data analysis and peak identification

Some Mallotus species are commonly used as traditional medicine (TM) ingredients in Vietnam and China, but only a few are studied for their activities. In Part I, high-performance liquid chromatography (HPLC) fingerprints of 39 Mallotus samples (17 species) were developed and, because of the complexity of and the large differences between the samples, it was chosen to analyse the unaligned fingerprints. The peaks, potentially responsible for the antioxidant activity in given Mallotus species, were indicated by the regression coefficients from an orthogonal projections to latent structures (O-PLS) model. In the present study, an in depth discussion on the need for alignment of the Mallotus fingerprints for the indication of the potentially active compounds is made, as well as an experimental analysis and identification of the previously indicated peaks by HPLC–mass spectrometry (HPLC–MS). Additionally, to thoroughly study and discuss the alignment problem, the modelling and prediction of the antioxidant activity of green tea samples based on HPLC fingerprints were also considered.     

Simultaneous analysis of caffeic acid derivatives and alkamides in roots and extracts of Echinacea purpurea by high-performance liquid chromatography-photodiode array detection-electrospray mass spectrometry

High-performance liquid chromatography (HPLC) coupled with UV photodiode-array detection and electrospray ionization mass spectrometry was developed for the simultaneous analysis of caffeic acid derivatives and alkamides in the roots and extracts of Echinacea purpurea. Caffeic acid derivatives and alkamides produced very abundant peaks in the total ion current chromatogram during negative and positive cone voltage switching. Cichoric acid and the isomer pair, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide, were used as a standard for quantification of caffeic acid derivatives and alkamides in E. purpurea. This novel method surpasses previously published ones in product quality control and providing the HPLC chromatographic fingerprints of biological active components in E. purpurea.     

Differentiation of Cultivars of Flos Chrysanthemum with the Use of High-Performance Liquid Chromatography Fingerprints and Chemometrics

Flos Chrysanthemum is a generic name for a particular group of edible plants, which also have medicinal properties. There are, in fact, twenty to thirty different cultivars, which are commonly used in beverages and for medicinal purposes. In this work, four Flos Chrysanthemum cultivars, Hangju, Taiju, Gongju, and Boju, were collected and chromatographic fingerprints were used to distinguish and assess these cultivars for quality control purposes. Chromatography fingerprints contain chemical information but also often have baseline drifts and peak shifts, which complicate data processing, and adaptive iteratively reweighted, penalized least squares, and correlation optimized warping were applied to correct the fingerprint peaks. The adjusted data were submitted to unsupervised and supervised pattern recognition methods. Principal component analysis was used to qualitatively differentiate the Flos Chrysanthemum cultivars. Partial least squares, continuum power regression, and K-nearest neighbors were used to predict the unknown samples. Finally, the elliptic joint confidence region method was used to evaluate the prediction ability of these models. The partial least squares and continuum power regression methods were shown to best represent the experimental results.     

Qualitative and quantitative evaluation of Oroxylum indicum (L.) Kurz by HPLC and LC‐qTOF‐MS/MS

Oroxylum indicum, as a popular functional Chinese herbal medicine for reducing hyperactivity, relieving sore throat, smoothing the liver and adjusting stomach, mainly contains flavonoids. In this study, we aimed to establish a fast and sensitive method that enables to analyze the chemical components in O. indicum qualitatively and quantitatively. First, a total of 42 components were characterized by LC‐quadrupole time‐of‐flight (qTOF)‐tandem mass spectrometry (MS/MS), including 23 flavonoid glycosides, 13 flavonoids and six other types of compounds. Then, 17 characteristic components of the 19 common peaks in the chromatographic fingerprints of O. indicum were confirmed. Fifty samples were classified into two groups by hierarchical clustering analysis and orthogonal partial least squares‐discriminant analysis, which also identified the 10 main chemical markers responsible for differences between samples. Last, the quantitative analysis of multiple components with a single marker method was established for simultaneous determination of six main active components in O. indicum by LC‐UV with oroxin B was chosen as internal reference substance. Finally, a rapid and efficient method integrating HPLC with LC‐electrospray ionization‐qTOF‐MS/MS analysis was established to comprehensively discriminate and assess the quality of O. indicum samples.     

Characterization of ginsenoside compound K metabolites in rat urine and feces by ultra‐performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Ginsenoside compound K (CK) is an active metabolite of ginsenoside and has been shown to have ameliorative property in various diseases. However, the detailed in vivo metabolism of this compound has rarely been reported. In the present study, a method using liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry together with multiple data processing techniques, including extracted ion chromatogram, multiple mass defect filter and MS/MS scanning, was developed to detect and characterize the metabolites of CK in rat urine and feces. After oral administration of CK at a dose of 50 mg/kg, urine and feces were collected for a period of time and subjected to a series of pretreatment. A total of 12 metabolites were tentatively or conclusively identified, comprising 11 phase I metabolites and a phase II metabolite. Metabolic pathways of CK has been proposed, including oxidation, deglycosylation, deglycosylation with sequential oxidation and dehydrogenation and deglycosylation with sequential glucuronidation. Relative quantitative analyses suggested that deglycosylation was the main metabolic pathway. The result could offer insights for better understanding of the mechanism of its pharmacological activities.     

Improved liquid chromatography fingerprint of fat-soluble Radix isatidis extract using multi-wavelength combination technique

We first investigated liquid chromatography (LC) fingerprint method using multi-wavelength combination technique, and successfully used this method for the analysis of a fat-soluble extract from Radix isatidis. LC fingerprints of fat-soluble R. isatidis extracts from 11 origins were established using the Origin software and Similarity Evaluation System for Chromatographic Fingerprints of traditional Chinese medicine (TCM). The typical LC fingerprints of fat-soluble extracts from R. isatidis were first established, and the reference chromatogram was also generated with 24 common peaks showing large peak areas and good separation from adjacent peaks. Seven common characteristic peaks were identified for the first time: anthranilic acid, syringic acid, benzoic acid, salicylic acid, tryptanthrin, indigo and indirubin. The total peak areas of 24 common peaks were more than 80% of the total peak areas. Hierarchical clustering analysis (HCA) of 11 R. isatidis samples was performed, and the results show that the differences between 11 origin R. isatidis were large. Principal component analysis (PCA) on 24 common peaks was obtained to find the possible chemical markers for the discrimination of different samples. The loading plot indicated that peaks 8, 11, 13 and 14 may have more influence on the discrimination of the samples. All these were useful for evaluating and controlling the quality of R. isatidis. Our work provides a general model of chromatogram combination at multi-wavelength detection to study the complex or the undeveloped materials, which can be used to scientifically ensure the quality of such samples and deeply do qualitative, quantitative and multicomponent pharmacodynamic research combined with modern advanced chromatographic technique.     

A new strategy for the discovery of epimedium metabolites using high-performance liquid chromatography with high resolution mass spectrometry

In this paper, a new strategy of drug metabolite discovery and identification was established using high-performance liquid chromatography with high resolution mass spectrometry (HPLC–HRMS) and a mass spectral trees similarity filter (MTSF) technique. The MTSF technique was developed as a means to rapidly discover comprehensive metabolites from multiple active components in a complicated biological matrix. Using full-scan mass spectra as the stem and data-dependent subsequent stage mass spectra to form branches, the HRMS and multiple-stage mass spectrometric data from detected compounds were converted to mass spectral trees data. Potential metabolites were discovered based on the similarity between their mass spectral trees and that known compounds or metabolites in a mass spectra trees library. The threshold value for match similarity scores was set at above 200, allowing approximately 80% of interference to be filtered out. A total of 115 metabolites of five flavonoid monomers (epimedin A, epimedin B, epimedin C, icariin, and baohuoside I) and herbal extract of epimedium were discovered and identified in rats via this new strategy. As a result, a metabolic profile for epimedium was obtained and a metabolic pathway was proposed. In addition, comparing to the widely used neutral loss filter (NLF), product ion filter (PIF), and mass defect filter (MDF) techniques, the MTSF technique was shown superior efficiency and selectivity for discovering and identifying metabolites in traditional Chinese medicine (TCM).     

True ion pick (TIPick): a denoising and peak picking algorithm to extract ion signals from liquid chromatography/mass spectrometry data

Liquid Chromatography ‐ Time of Flight Mass Spectrometry has become an important technique for toxicological screening and metabolomics. We describe TIPick a novel algorithm that accurately and sensitively detects target compounds in biological samples. TIPick comprises two main steps: background subtraction and peak picking. By subtracting a blank chromatogram, TIPick eliminates chemical signals of blank injections and reduces false positive results. TIPick detects peaks by calculating the S(CC_INI) values of extracted ion chromatograms (EICs) without considering peak shapes, and it is able to detect tailing and fronting peaks. TIPick also uses duplicate injections to enhance the signals of the peaks and thus improve the peak detection power. Commonly seen split peaks caused by either saturation of the mass spectrometer detector or a mathematical background subtraction algorithm can be resolved by adjusting the mass error tolerance of the EICs and by comparing the EICs before and after background subtraction. The performance of TIPick was tested in a data set containing 297 standard mixtures; the recall, precision and F‐score were 0.99, 0.97 and 0.98, respectively. TIPick was successfully used to construct and analyze the NTU MetaCore metabolomics chemical standards library, and it was applied for toxicological screening and metabolomics studies. Copyright © 2013 John Wiley & Sons, Ltd.     

Rapid profiling of polymeric phenolic acids in Salvia miltiorrhiza by hybrid data‐dependent/targeted multistage mass spectrometry acquisition based on expected compounds prediction and fragment ion searching

Phenolic acids are the major water‐soluble components in Salvia miltiorrhiza (>5%). According to previous studies, many of them contribute to the cardiovascular effects and antioxidant effects of S. miltiorrhiza. Polymeric phenolic acids can be considered as the tanshinol derived metabolites, e.g., dimmers, trimers, and tetramers. A strategy combined with tanshinol‐based expected compounds prediction, total ion chromatogram filtering, fragment ion searching, and parent list‐based multistage mass spectrometry acquisition by linear trap quadropole‐orbitrap Velos mass spectrometry was proposed to rapid profile polymeric phenolic acids in S. miltiorrhiza. More than 480 potential polymeric phenolic acids could be screened out by this strategy. Based on the fragment information obtained by parent list‐activated data dependent multistage mass spectrometry acquisition, 190 polymeric phenolic acids were characterized by comparing their mass information with literature data, and 18 of them were firstly detected from S. miltiorrhiza. Seven potential compounds were tentatively characterized as new polymeric phenolic acids from S. miltiorrhiza. This strategy facilitates identification of polymeric phenolic acids in complex matrix with both selectivity and sensitivity, which could be expanded for rapid discovery and identification of compounds from complex matrix.     

固相萃取-高效液相色谱-电喷雾串联质谱法分析芍药和酒芍药的水溶性化学成分

利用固相萃取-高效液相色谱-电喷雾串联质谱法(SPE-HPLC-DAD-ESI-MS2)分析了芍药(Paeonia lactiflora Pall.)及其炮制品酒芍药(Paeonia lactiflora Pall.preparated with wine)水煎液中化学成分的种类和结构,并对其主要有效成分芍药苷进行了定量测定.用此方法得到了芍药和酒芍药水煎液的紫外色谱(UV)图、总离子流色谱(TIC)图和萃取离子色谱(EIC)图,以及相应色谱峰的紫外光谱图和EIC/MS2的质谱图.对其进行解析,发现芍药和酒芍药水煎液中所含化学成分基本一致,但酒芍药水煎液中除主要活性成分芍药苷含量基本未变外,其它酚酸性化学成分含量相对于芍药水煎液均有不同程度的下降.同时鉴别出芍药和酒芍药水煎液中共同含有的16种成分,它们分别是:蔗糖、(+)-儿茶素、Mudanpioside E、芍药新苷、芍药内酯苷、氧化芍药苷、芍药苷、羟基芍药苷、没食子酰芍药苷*、牡丹皮苷Ⅰ、苯甲酰芍药苷、三没食子酰葡萄糖、四没食子酰葡萄糖、五没食子酰葡萄糖.以上分析说明,酒炙可保留芍药的药效,同时"缓和其酸寒伐肝之性".     

Medium-Pressure Liquid Chromatography Coupled to Electrospray Ionization Mass Spectrometry for Separation and On-Line Characterization of Flavonoids from Asparagus officinalis

A novel method for separation and on-line characterization of flavonoids from Asparagus officinalis by medium-pressure liquid chromatography coupled to electrospray ionization multi-stage mass spectrometry (MPLC-ESI-MSⁿ) was successfully established. The hyphenation between MPLC and ESI-MSⁿ was designed to keep the split ratio exactly in the range from 1:100 to 1:300. The separation procedure was guided by the chromatogram of ion current of MSⁿ and the structures of compounds were characterized by fragments information at the same time. Consequently, it was proved that MPLC coupled with ESI-MSⁿ was an effective method for separation of compounds from multi-component mixtures with high purity and desired amounts and simultaneous elucidation of chemical structures.

     

On-line identification of phytochemical constituents in botanical extracts by combined high-performance liquid chromatographic-diode array detection-mass spectrometric techniques

It is necessary to determine all of the phytochemical constituents of botanical extracts in order to ensure the reliability and repeatability of pharmacological and clinical research, to understand their bioactivities and possible side effects of active compounds and to enhance product quality control. HPLC chromatographic fingerprints can be applied for this kind of documentation. Combined HPLC-diode array detection–MS techniques can provide on-line UV and MS information for each peak in a chromatogram. In most cases, direct identification of the peaks is possible, based on comparison with published data or with standard compounds. This review will primarily focus on electrospray and thermospray ionization MS and their applications for the qualitative analyses of phenolic compounds, saponins, alkaloids and other classes of natural products in botanical extracts. Twenty-one of the most commonly used herbal examples, their phytochemical analyses and characteristics of their mass spectra are described.     

Rapid annotation and structural characterization of saponins in the active fraction of Albizia julibrissin by high‐performance liquid chromatography coupled with quadrupole time of flight mass spectrometry based on accurate mass database

The purified active fraction of Albizia julibrissin saponin was proved to be a promising adjuvant candidate for vaccine. In this study, a simple, convenient, and practical strategy was established for characterizing the saponins in this purified active fraction. The personal accurate mass database including chemical structure, molecular formula, and theoretical mass was first constructed by collecting 110 reported known saponins from genus Albizia species. The raw data was obtained by high‐performance liquid chromatography coupled with quadrupole time of flight mass spectrometry. The potential compounds were extracted from raw data, and matched with the accurate mass databases. A series of saponin compounds were predicted and their chemical structures were characterized by interpreting the tandem mass spectrometry data. A total of 29 saponins including 10 new compounds and 5 first found saponins from A. julibrissin were successfully characterized in this purified active fraction using this new strategy.