Species discrimination of multiple botanical origins of Fritillaria species based on infrared spectroscopy, thin layer chromatography-image analysis and untargeted metabolomics.

BACKGROUND: Fritillaria Bulbus (FB), a precious medicinal herb renowned for its heat-clearing, lung-moistening, cough-relieving and phlegm-eliminating effects. In pursuit of profits, unscrupulous merchants have engaged in the substitution or adulteration of valuable varieties with cheaper alternatives. It is, therefore, urgent to develop effective technical approaches to identify FBs from adulterants. METHODS: This paper employed infrared spectroscopy (IR), thin layer chromatography-image analysis (TLC-IA), and untargeted metabolomics techniques to discriminate ten species of FBs. RESULTS: Five species of FBs were successfully differentiated using mid-infrared spectroscopy. Furthermore, the power of TLC-IA technology allowed the differentiation of five species of FBs and two origins of FCBs (Fritillariae Cirrhosae Bulbus). Remarkably, through the application of untargeted metabolomics technique, the precise discrimination of five species of FBs, as well as three origins of FCBs were accomplished. Moreover, a comprehensive identification of 101 markers that reliably distinguished diverse FBs was achieved through the employment of untargeted metabolomics technique. CONCLUSION: The investigation presented powerful means of detection for assuring the quality control of Fritillaria herbs.

Low-Temperature Cleanup Followed by Dispersive Solid-Phase Extraction for Determination of Nine Polar Plant Growth Regulators in Herbal Matrices Using Liquid Chromatography-Tandem Mass Spectrometry.

Polar plant growth regulators, used alone or doped in fertilizers, are most effective and widely utilized plant growth regulators (PGRs) in agriculture, which play important roles in mediating the yield and quality of crops and foodstuffs. The application scope has been extended to herbal medicines in the past 2 decades and relevant study is inadequate. The aim of this study is to establish a QuPPe-based extraction method containing low-temperature and d-SPE cleanup procedure followed by the detection on a selective multiresidue ultrahigh-performance liquid chromatography - triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS) in three herbal matrices. This simple, accurate, versatile and robust method was verified according to the validation criteria of the SANTE/12682/2019 guideline document. The analytical range was from 2.5 to 200 µg/L, and the average recoveries were in the range of 64.6-117.8% (n = 6). The optimized method was applied to 135 herbal medicines thereof. Result showed that the detection frequency of chlormequat was the highest in the investigated PGRs, with the positive rate of 15.6%. Improvement of the detection method for polar PGRs will enrich the coverage of PGRs, which is conducive to safeguard public health and ensure drug safety. Supplementary Information: The online version contains supplementary material available at 10.1007/s10337-023-04254-3.

Characteristic Malonyl Ginsenosides from the Leaves of Panax notoginseng as Potential Quality Markers for Adulteration Detection.

Due to the high price and limited supply of Panax notoginseng, a large number of samples adulterated with the leaves appear in the market. A group of new malonyl ginsenosides were exclusively detected in the P. notoginseng leaves (PNL). Targeted isolation of the malonyl ginsenosides was guided by UPLC-QDa MS. HRMS, 1D/2D NMR, and chemical methods were used for structural identification. A selected ion monitoring method was developed based on UPLC-QDa MS to detect the adulterations. In addition, the anti-inflammatory activities and the collision-induced dissociation features of the isolated saponins were studied. As a result, eight new 3-OH malonylated dammarane-type triterpene oligoglycosides (notoginsenosides L3-L10) were obtained from PNL. Adulteration with PNL can be easily detected with limit of detection as low as 0.06%. To sum up, the isolated ginsenosides can be used as quality markers for fraud detection, which will promote the quality control of the notoginseng products.

Plant metabolomics for studying the effect of two insecticides on comprehensive constituents of Lonicerae Japonicae Flos.

Pesticides' overuse and misuse have been reported to induce ingredient variations in herbal medicine, which is now gaining attention in the medicinal field as a form of alternative medicine. To date, available studies on pesticide-induced ingredient variations of herbal medicine are limited only on a few compounds and remain most others unexamined. In this study, a plant metabolomics-based strategy was performed to systematically explore the effects of two frequently used insecticides on the comprehensive constituents of Lonicerae Japonicae Flos (LJF), the flower buds of Lonicera japonica Thunb. Field trials were designed on a cultivating plot of L. japonica with controls and treatments of imidacloprid (IMI) and compound flonicamid and acetamiprid (CFA). Unbiased metabolite profiling was conducted by ultra-high performance liquid chromatography/quadrupole-Orbitrap mass spectrometer. After data pretreatment by automatic extraction and screening, a data matrix of metabolite features was submitted for statistical analyses. Consequently, 29 metabolic markers, including chlorogenic acids, iridoids and organic acid-glucosides were obtained and characterized. The relative quantitative assay was subsequently performed to monitor their variations across flowering developments. This is the first study that systematically explored the insecticide-induced metabolite variations of LJF while taking into account the inherent variability of flowering development. The results were beneficial for holistic quality assessment of LJF and significant for guiding scientific use of pesticides in the large-scale cultivation.

Isolation and identification of a new sildenafil analogue, hydroxycarbodenafil, found as an adulterant in a health supplement.

During routine screening of illegal adulterants in health supplements, a novel sildenafil analogue was discovered, and subsequently isolated by recrystallization. Its structure was elucidated by extensive analyses of high resolution mass spectrometry (HRMS), one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) data. The analogue was finally determined as hydroxycarbodenafil, featuring a hydroxyethyl group instead of an ethyl group on piperazine ring in comparison with carbodenafil.

A metabolomics strategy for authentication of plant medicines with multiple botanical origins, a case study of Uncariae Rammulus Cum Uncis.

Source authentication of herbal medicines was essential for ensuring their safety, efficacy and quality consistency, especially those with multiple botanical origins. This study proposed a metabolomics strategy for species discrimination and source recognition. Uncariae Rammulus Cum Uncis, officially stipulating the stems with hooks of five Uncaria species as its origins, was taken as a case study. Firstly, an untargeted MSE method was developed by ultra-high performance liquid chromatography hyphenated with quadrupole time-of-flight mass spectrometry for global metabolite characterization. Subsequently, data pretreatment was conducted by using Progenesis QI software and screening rules. The obtained metabolite features were defined as variables for statistical analyses. Principal component analysis and chemical fingerprinting spectra suggested that five official species were differentiated from each other except for Uncaria hirsuta and Uncaria sinensis. Furthermore, orthogonal partial least squares discrimination analysis was performed to discriminate confused two species, and resulted in the discovery of nine contributing markers. Ultimately, a Support Vector Machine model was developed to recognize five species and predict origins of commercial materials. The study demonstrated that the developed strategy was effective in discrimination and recognition of confused species, and promising in tracking botanical origins of commercial materials.

An integrated approach for global profiling of multi-type constituents: Comprehensive chemical characterization of Lonicerae Japonicae Flos as a case study.

To fully capture the chemo-diversity of medicinal plants is very essential for understanding of their pharmacological activities and guiding scientific quality control. Aiming to facilitate chemical characterization and novel natural products discovery, the present study proposed an integrated approach based on two-dimensional liquid chromatography coupled with quadrupole-Orbitrap mass spectrometer (2D LC/Q-Orbitrap MS). An offline comprehensive two-dimensional (2D) LC system was constructed to cover and separate multi-type constituents by combining hydrophilic interaction chromatography (HILIC) and conventional reversed phase C18. A two-step mass defect filtering-induced exclusion list-data dependent acquisition was developed to increase MS/MS coverage and selectivity. Additionally, an efficient interpretation strategy, combining an automatic matching algorithm and molecular networking (MN), was introduced for rapid recognition of known compounds and efficient elucidation of unreported ones. As a case study, the integrated approach was tentatively applied for comprehensive characterization of complex multi-type components in Lonicerae Japonicae Flos (LJF), a traditional Chinese medicine. Consequently, a total of 537 compounds were characterized from LJF, including a large number of potential novel structures. It was demonstrated that the integrated approach is powerful in deep investigation on chemical diversity of medicinal plants and discovery of novel structures. Its application could also be extended for global profiling of other complicated chemical systems, such as Chinese medicinal formulas.

An enhanced strategy integrating offline two-dimensional separation and step-wise precursor ion list-based raster-mass defect filter: Characterization of indole alkaloids in five botanical origins of Uncariae Ramulus Cum Unicis as an exemplary application.

Comprehensive chemical profiling is of great significance for understanding the therapeutic material basis and quality control of herbal medicines, which is challenging due to its inherent chemical diversity and complexity, as well as wide concentration range. In this study, we introduced an enhanced strategy integrating offline two-dimensional (2D) separation and the step-wise precursor ion list-based raster-mass defect filter (step-wise PIL-based raster-MDF) scan by tandem LTQ-Orbitrap mass spectrometer. A comprehensive analysis of indole alkaloids in five botanical origins of Uncariae Ramulus Cum Unicis (Gou-Teng) was used as an exemplary application. A positively charged reversed phase (PR) × conventional RP LC system in different pH conditions was constructed with the orthogonality of 74%. A theoretical step-wise PIL among 310-950 Da with the step-size of 2 Da was developed to selectively trigger fragmentations and extend the coverage of potential indole alkaloids. Simultaneously, by defining parent mass width (PMW) of the step-wise PIL to ±55 mDa, a raster-MDF screening was achieved in the acquisition process. Additionally, subtype classification and structural elucidation were facilitated by a four-step interpretation strategy. As a result, a total of 1227 indole alkaloids were efficiently exposed and characterized from five botanical origins of Gou-Teng, which showed high chemical diversity. A systematic comparison among five species was first performed and only 66 indole alkaloids were common. For method validation, three new alkaloid N-oxides were isolated and unambiguously identified by NMR. The present study provides a novel data-dependent acquisition method with improved target coverage and high selectivity. The integrated strategy is practical to efficiently expose and comprehensively characterize complex components in herbal medicines.

Global profiling combined with predicted metabolites screening for discovery of natural compounds: Characterization of ginsenosides in the leaves of Panax notoginseng as a case study.

Liquid chromatography-mass spectrometry (LC-MS) guided isolation is a favored strategy to quickly and efficiently explore the chemical diversity of herbal medicines. In this study, two methods were adopted to improve the performance of the strategy, including offline two-dimensional (2D) LC to extend the peak capacities and predicted metabolites screening (PMS) to automatically screen the targets with expanded databases. Ginsenosides in the leaves of Panax notoginseng (PNL) were taken as a case. An offline 2D LC system was constructed with an orthogonality of 0.69 and peak capacity of 8925. Quadrupole time-of-flight mass spectrometry-fast data directed analysis (QTOF-Fast DDA) was employed for detection of the ginsenosides in the fractioned samples. Four modified groups, including glucose, xylose, rhamnose and malonyl, were adopted and markedly extended the screening coverage. The combined strategy showed about 7.5 times improvement in the screening capability. PMS is conveniently and automatically implemented in UNIFI. Using this strategy, 945 ginsenosides were discovered from PNL, including 662 potentially novel ginsenosides. Furthermore, two new ginsenosides were purified, and unambiguously identified by NMR analysis, partially demonstrating the LC-MS guided isolation. The combined strategy can also be applied in characterizing and discovering new bioactive constituents from other herbal medicines.

Geographic impact evaluation of the quality of Alismatis Rhizoma by untargeted metabolomics and quantitative assay.

The geographic impact on the quality of Alismatis Rhizoma (derived from the tuber of Alisma orientale), a reputable diuretic traditional Chinese medicine, has seldom been evaluated. Here a metabolomics-driven approach targeting the bioactive protostane triterpenes was developed, by incorporating UHPLC with quadrupole time-of-flight mass spectrometry-based untargeted metabolite profiling and multiple reaction monitoring quantitative assay, to probe the triterpene differences between Alismatis Rhizoma samples collected from Sichuan, Fujian, and Jiangxi Provinces. Following the metabolomics workflows, the samples from Sichuan and Jiangxi displayed distinct differences in their triterpene profiles, whereas those from Fujian showed remarkable intra-class variation. Twenty-three triterpenes were identified to contribute most to the differentiated clustering. A sensitive, precise, repeatable, and accurate quantitative assay method was established on a hybrid triple quadrupole-linear ion trap mass spectrometer to quantify the contents of eight triterpene compounds. Taking into account the metabolomics and quantitation results, alisol B 23-acetate and alisol A are significantly different in Alismatis Rhizoma from Sichuan and Jiangxi Provinces, and they may have the potential for geographic discrimination. These results illustrate how geographic difference impacts the triterpene chemistry of Alismatis Rhizoma. Metabolomics-driven chemical comparison is suitable for the quality evaluation of traditional Chinese medicine.

Characteristic Chromatogram: A Method of Discriminate and Quantitative Analysis for Quality Evaluation of Uncaria Stem with Hooks.

It remains a challenge to establish new monographs for herbal drugs derived from multiple botanical sources. Specifically, the difficulty involves discriminating and quantifying these herbs with components whose levels vary markedly among different samples. Using Uncaria stem with hooks as an example, a characteristic chromatogram was proposed to discriminate its five botanical origins and to quantify its characteristic components in the chromatogram. The characteristic chromatogram with respect to the components of Uncaria stem with hooks with the five botanical origins was established using 0.02% diethylamine and acetonitrile as the mobile phase. The total analysis time was 50 min and the detection wavelength was 245 nm. Using the same chromatogram parameters, the single standard to determine multicomponents method was validated to simultaneously quantify nine indole alkaloids, including vincosamide, 3α-dihydrocadambine, isocorynoxeine, corynoxeine, isorhynchophylline, rhynchophylline, hirsuteine, hirsutine, and geissoschizine methyl ether. The results showed that only the Uncaria stem with hooks from Uncaria rhynchophylla, the most widely used in the herbal market, showed the presence of these nine alkaloids. The conversion factors were 1.27, 2.32, 0.98, 1.04, 1.00, 1.02, 1.26, 1.33, and 1.25, respectively. The limits of quantitation were lower than 700 ng/mL. The total contents of 31 batches of Uncaria stem with hooks were in the range of 0.1 - 0.6%, except for Uncaria hirsuta Havil and Uncaria sinensis (Oliv.) Havil. The results also showed that the total content of indole alkaloids tended to decrease with an increase in the hook diameter. This showed that the characteristic chromatogram is practical for controlling the quality of traditional Chinese medicines with multiple botanical origins.

Mass defect filtering-oriented classification and precursor ions list-triggered high-resolution mass spectrometry analysis for the discovery of indole alkaloids from Uncaria sinensis.

Discovery of new natural compounds is becoming increasingly challenging because of the interference from those known and abundant components. The aim of this study is to report a dereplication strategy, by integrating mass defect filtering (MDF)-oriented novelty classification and precursor ions list (PIL)-triggered high-resolution mass spectrometry analysis, and to validate it by discovering new indole alkaloids from the medicinal herb Uncaria sinensis. Rapid chromatographic separation was achieved on a Kinetex® EVO C18 column (<16min). An in-house MDF algorithm, developed based on the informed phytochemistry information and molecular design, could more exactly screen the target alkaloids and divide them into three novelty levels: Known (KN), Unknown-but-Predicted (UP), and Unexpected (UN). A hybrid data acquisition method, namely PIL-triggered collision-induced dissociation-MS2 and high-energy C-trap dissociation-MS3 with dynamic exclusion on a linear ion trap/Orbitrap mass spectrometer, facilitated the acquisition of diverse product ions sufficient for the structural elucidation of both indole alkaloids and the N-oxides. Ultimately, 158 potentially new alkaloids, including 10 UP and 108 UN, were rapidly characterized from the stem, leaf, and flower of U. sinensis. Two new alkaloid compounds thereof were successfully isolated and identified by 1D and 2D NMR analyses. The varied ring E and novel alkaloid-acylquinic acid conjugates were first reported from the whole Uncaria genus. Conclusively, it is a practical chemical dereplication strategy that can enhance the efficiency and has the potential to be a routine approach for the discovery of new natural compounds.

An enhanced targeted identification strategy for the selective identification of flavonoid O-glycosides from Carthamus tinctorius by integrating offline two-dimensional liquid chromatography/linear ion-trap-Orbitrap mass spectrometry, high-resolution diagnostic product ions/neutral loss filtering and liquid chromatography-solid phase extraction-nuclear magnetic resonance.

Targeted identification of potentially bioactive molecules from herbal medicines is often stymied by the insufficient chromatographic separation, ubiquitous matrix interference, and pervasive isomerism. An enhanced targeted identification strategy is presented and validated by the selective identification of flavonoid O-glycosides (FOGs) from Carthamus tinctorius. It consists of four steps: (i) enhanced separation and detection by offline two-dimensional liquid chromatography/LTQ-Orbitrap MS (offline 2D-LC/LTQ-Orbitrap MS) using collision-induced dissociation (CID) and high-energy C-trap dissociation (HCD); (ii) improved identification of the major aglycones by acid hydrolysis and LC-SPE-NMR; (iii) simplified spectral elucidation by high-resolution diagnostic product ions/neutral loss filtering; and (iv) more convincing structural identification by matching an in-house library. An offline 2D-LC system configuring an Acchrom XAmide column and a BEH Shield RP-18 UPLC® column enabled much better separation of the easily co-eluting components. Combined use of CID and HCD could produce complementary fragmentation information. The intensity ratios of the aglycone ion species ([Y0-H]-/Y0- and [Y0-2H]-/Y0-) in the HCD-MS2 spectra were found diagnostic for discriminating the aglycone subtypes and characterizing the glycosylation patterns. Five aglycone structures (kaempferol, 6-hydroxykaempferol, 6-methoxykaempferol, carthamidin, and isocarthamidin) were identified based on the 1H-NMR data recorded by LC-SPE-NMR. Of the 107 characterized flavonoids, 80 FOGs were first reported from C. tinctorius. Unknown aglycones, pentose, and novel acyl substituents were discovered. A new compound thereof was isolated and fully identified, which could partially validate the MS-oriented identification. This integral strategy can improve the potency, efficiency, and accuracy in the detection of new compounds from medicinal herbs and other natural sources.

Supercritical fluid chromatography for separation and preparation of tautomeric 7-epimeric spiro oxindole alkaloids from Uncaria macrophylla.

Increasing challenge arising from configurational interconversion in aqueous solvent renders it rather difficult to isolate high-purity tautomeric reference standards and thus largely hinders the holistic quality control of traditional Chinese medicine (TCM). Spiro oxindole alkaloids (SOAs), as the markers for the medicinal Uncaria herbs, can easily isomerize in polar or aqueous solvent via a retro-Mannich reaction. In the present study, supercritical fluid chromatography (SFC) is utilized to separate and isolate two pairs of 7-epimeric SOAs, including rhynchophylline (R) and isorhynchophylline (IR), corynoxine (C) and corynoxine B (CB), from Uncaria macrophylla. Initially, the solvent that can stabilize SOA epimers was systematically screened, and acetonitrile was used to dissolve and as the modifier in SFC. Then, key parameters of ultra-high performance SFC (ultra-performance convergence chromatography, UPC2), comprising stationary phase, additive in modifier, column temperature, ABPR pressure, and flow rate, were optimized in sequence. Two isocratic UPC2 methods were developed on the achiral Torus 1-AA and Torus Diol columns, suitable for UV and MS detection, respectively. MCI gel column chromatography fractionated the U. macrophylla extract into two mixtures (R/IR and C/CB). Preparative SFC, using a Viridis Prep Silica 2-EP OBD column and acetonitrile-0.2% diethylamine in CO2 as the mobile phase, was finally employed for compound purification. As a result, the purity of four SOA compounds was all higher than 95%. Different from reversed-phase HPLC, SFC, by use of water-free mobile phase (inert CO2 and aprotic modifier), provides a solution to rapid analysis and isolation of tautomeric reference standards for quality control of TCM.

New monoterpenoid oxindole alkaloid derivatives from the stems of Uncaria hirsuta Havil. and their cytotoxicity and tandem mass spectrometric fragmentation.

Four new alkaloids, comprising three 3-oxo-3,7-seco-oxindole alkaloids (hirsutanine D-F, 1-3) and one oxindole alkaloid N-oxide (uncarine B N-oxide, 4), together with four known heteroyohimbine-type oxindole alkaloids, were isolated from the stems of Uncaria hirsuta Havil. Structures of 1-4 were elucidated by extensive NMR and HR-ESIMS data analyses. Compound 3 is the first 3-oxo-3,7-seco-oxindole alkaloid with ring B opened and degraded isolated from the Uncaria genus. Compounds 1-3 exhibited slight inhibition effect on the proliferation of the breast cancer cell MDA-MB-231. The positive mode collision-induced dissociation of the 3-oxo-3,7-seco-oxindole alkaloids (1-3) was featured by the ß-cleavage and α-cleavage of the amido bond, while the N-oxide (4) showed characteristic neutral eliminations of ·OH and H2O.

UHPLC-Q-TOF-MS-based metabolomics approach to compare the saponin compositions of Xueshuantong injection and Xuesaitong injection.

Various traditional Chinese medicine preparations developed from Notoginseng total saponins, including Xueshuantong injection and Xuesaitong injection, are extensively used in China to treat cardiocerebrovascular diseases. However, the difference of their saponin compositions remains unknown. An ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry based metabolomics approach was developed to probe the saponin discrimination between Xueshuantong and Xuesaitong and the related factors by large sample analysis. A highly efficient chromatographic separation was achieved on an HSS T3 column within 20 min with the holistic metabolites information recorded in the negative MSE mode. A six-step data pretreatment procedure mainly based on Progenesis QI and mass defect filtering was established. Pattern recognition chemometrics was used to discover the potential saponin markers. The saponin composition of Wuzhou Xueshuantong showed distinct discrimination from the other products. Wuzhou Xueshuantong contains more abundant protopanaxatriol-type noto-R1 , Rg1 , Re, and protopanaxadiol-type Rb1 , but less Rd and other low-polarity protopanaxadiol-type ginsenosides. These differences could not directly correlate to the use of different parts of Panax notoginseng, but possibly to the different preparation techniques employed by different manufacturers. These results are beneficial to the establishment of pharmacopoeia standards and the assessment of the efficacy and adverse drug reactions for these homologous products.

An in-source multiple collision-neutral loss filtering based nontargeted metabolomics approach for the comprehensive analysis of malonyl-ginsenosides from Panax ginseng, P. quinquefolius, and P. notoginseng.

The simultaneous identification and quantification of target metabolites from herbal medicines are difficult to implement by the full-scan MS based nontargeted metabolomics approaches. Here an in-source multiple collision-neutral loss filtering (IMC-NLF) based nontargeted metabolomics approach is developed and applied to identify and quantify the variations of malonyl-ginsenosides, a common group of acyl saponins with potential anti-diabetic activity, among Panax ginseng, P. quinquefolius, and P. notoginseng. The key steps of the IMC-NLF strategy are the acquisition of specific high-resolution neutral loss data and the efficient filtering of target precursor ions from the full-scan spectra. Using a hybrid LTQ-Orbitrap mass spectrometer after UHPLC separation, abundant in-source product ions, [M-H-CO2]- (due to the vulnerability of the carboxyl group) and [M-H-Mal.]-, were generated at the energies of 70 V and 90 V, respectively. After spectral deconvolution, the generated peak list was screened by dual NLF using a Neutral Loss MS Finder software (NL of 43.9898 Da for CO2 and 86.0004 Da for the malonyl substituent). By combining the precursor ions list-triggered HCD-MS/MS and basic hydrolysis, a total of 101 malonyl-ginsenosides (including 69 from P. ginseng, 52 from P. quinquefolius, and 44 from P. notoginseng) were identified or tentatively characterized. The variations of 81 characterized malonyl-ginsenosides among 45 batches of Ginseng samples were statistically analyzed disclosing ten potential markers. It is the first systematic analysis of malonyl-ginsenosides. The IMC-NLF approach by a single analytical platform is promising in targeted analyses of modification-specific metabolites in metabolomics and drug metabolism.

Method development and application of offline two-dimensional liquid chromatography/quadrupole time-of-flight mass spectrometry-fast data directed analysis for comprehensive characterization of the saponins from Xueshuantong Injection.

Xueshuantong Injection (XSTI), derived from Notoginseng total saponins, is a popular traditional Chinese medicine injection for the treatment of thrombus-resultant diseases. Current knowledge on its therapeutic basis is limited to five major saponins, whereas those minor ones are rarely investigated. We herein develop an offline two-dimensional liquid chromatography/quadrupole time-of-flight mass spectrometry-fast data directed analysis (offline 2D LC/QTOF-Fast DDA) approach to systematically characterize the saponins contained in XSTI. Key parameters affecting chromatographic separation in 2D LC (including stationary phase, mobile phase, column temperature, and gradient elution program) and the detection by QTOF MS (involving spray voltage, cone voltage, and ramp collision energy) were optimized in sequence. The configured offline 2D LC system showed an orthogonality of 0.84 and a theoretical peak capacity of 8976. Total saponins in XSTI were fractionated into eleven samples by the first-dimensional hydrophilic interaction chromatography, which were further analyzed by reversed-phase UHPLC/QTOF-Fast DDA in negative ion mode. The fragmentation features evidenced from 36 saponin reference standards, high-accuracy MS and Fast-DDA-MS(2) data, elemental composition (C<80, H<120, O<50), double-bond equivalent (DBE 5-15), and searching an in-house library of Panax notoginseng, were simultaneously utilized for structural elucidation. Ultimately, 148 saponins were separated and characterized, and 80 have not been isolated from P. notoginseng. An in-depth depiction of the chemical composition of XSTI was achieved. The results obtained would benefit better understanding of the therapeutic basis and significant promotion on the quality standard of XSTI as well as other homologous products.

An efficient and target-oriented sample enrichment method for preparative separation of minor alkaloids by pH-zone-refining counter-current chromatography.

An efficient and target-oriented sample enrichment method was established to increase the content of the minor alkaloids in crude extract by using the corresponding two-phase solvent system applied in pH-zone-refining counter-current chromatography. The enrichment and separation of seven minor indole alkaloids from Uncaria rhynchophylla (Miq.) Miq. ex Havil(UR) were selected as an example to show the advantage of this method. An optimized two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (3:7:1:9, v/v) was used in this study, where triethylamine (TEA) as the retainer and hydrochloric acid (HCl) as the eluter were added at the equimolar of 10mM. Crude alkaloids of UR dissolved in the corresponding upper phase (containing 10mM TEA) were extracted twice with lower phase (containing 10mM TEA) and lower phase (containing 10mM HCl), respectively, the second lower phase extract was subjected to pH-zone-refining CCC separation after alkalization and desalination. Finally, from 10g of crude alkaloids, 4g of refined alkaloids was obtained and the total content of seven target indole alkaloids was increased from 4.64% to 15.78%. Seven indole alkaloids, including 54mg isocorynoxeine, 21mg corynoxeine, 46mg isorhynchophylline, 35mg rhynchophylline, 65mg hirsutine, 51mg hirsuteine and 27mg geissoschizine methylether were all simultaneously separated from 2.5g of refined alkaloids, with the purity of 86.4%, 97.5%, 90.3%, 92.1%, 98.5%, 92.3%, and 92.8%, respectively. The total content and purities of the seven minor indole alkaloids were tested by HPLC and their chemical structures were elucidated by ESI-HRMS and (1)H NMR.