N-desmethyldauricine from Menispermum dauricum DC suppresses triple-negative breast cancer growth in 2D and 3D models by downregulating the NF-κB signaling pathway.

Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, for which targeted therapy regimens are lacking. The traditional Chinese medicine Menispermum dauricum DC (M. dauricum) and its compounds have been reported to have antitumor activity against various cancers; however, their anti-TNBC activity is unknown. In this work, dauricine and N-desmethyldauricine from M. dauricum were separated and identified to have anti-TNBC via a multi-component bioactivity and structure-guided method. The cell counting kit 8 assay showed that dauricine and N-desmethyldauricine inhibited the proliferation of four tested TNBC cell lines, with half maximal inhibitory concentration values ranging from 5.01 µM to 13.16 µM. Further research suggested that N-desmethyldauricine induced cell apoptosis, arrested cell cycle progression in the G0/G1 phase, and inhibited cell migration. Western blot analysis revealed that the proapoptotic protein cleaved-poly-ADP-ribose polymerase 1 was upregulated, and the G0/G1 phase-related proteins cyclin-dependent kinase 2 and cyclin D1 and the migration-related protein matrix metallopeptidase 9 were downregulated. Furthermore, N-desmethyldauricine decreased the protein expression of p65, an important subunit of nuclear factor kappa-beta (NF-κB). Moreover, an antiproliferation assay of three-dimensional (3D) tumor spheroids showed that N-desmethyldauricine diminished cell‒cell adhesion and suppressed the growth of TNBC 3D spheroids. Taken together, these findings indicate that N-desmethyldauricine inhibited the proliferation of TNBC cells and decreased the expression of p65 in the NF-κB pathway.

Elucidation of active components and target mechanism in Jinqiancao granules for the treatment of prostatitis and benign prostatic hyperplasia.

ETHNOPHARMACOLOGICAL RELEVANCE: Prostatitis and benign prostatic hyperplasia (BPH) are inflammations of the prostate gland, which surrounds the urethra in males. Jinqiancao granules are a traditional Chinese medicine used to treat kidney stones and this medicine consists of four herbs: Desmodium styracifolium (Osbeck) Merr., Pyrrosia calvata (Baker) Ching, Plantago asiatica L. and stigma of Zea mays L. AIM OF THE STUDY: We hypothesized that Jinqiancao granules could be a potential therapy for prostatitis and BPH, and this work aimed to elucidate active compounds in Jinqiancao granules and their target mechanisms for the potential treatment of the two diseases. MATERIALS AND METHODS: Jinqiancao granules were commercially available and purchased. Database-driven data mining and networking were utilized to establish a general correlation between Jinqiancao granules and the two diseases above. Ultra-performance liquid chromatography-mass spectrometry was used for compound separation and characterization. The characterized compounds were evaluated on four G-protein coupled receptors (GPCRs: GPR35, muscarinic acetylcholine receptor M3, alpha-1A adrenergic receptor α1A and cannabinoid receptor CB2). A dynamic mass redistribution technique was applied to evaluate compounds on four GPCRs. Nitric acid (NO) inhibition was tested on the macrophage cell line RAW264.7. Molecular docking was conducted on GPR35-active compounds and GPR35 crystal structure. Statistical analysis using GEO datasets was conducted. RESULTS: Seventy compounds were isolated and twelve showed GPCR activity. Three compounds showed potent GPR35 agonistic activity (EC50 < 10 µM) and the GPR35 agonism action of PAL-21 (Scutellarein) was reported for the first time. Docking results revealed that the GPR35-targeting compounds interacted at the key residues for the agonist-initiated activation of GPR35. Five compounds showed weak antagonistic activity on M3, which was confirmed to be a disease target by statistical analysis. Seventeen compounds showed NO inhibitory activity. Several compounds showed multi-target properties. An experiment-based network reflected a pharmacological relationship between Jinqiancao granules and the two diseases. CONCLUSIONS: This study identified active compounds in Jinqiancao granules that have synergistic mechanisms, contributing to anti-inflammatory effects. The findings provide scientific evidence for the potential use of Jinqiancao granules as a treatment for prostatitis and BPH.

[Monoterpenoids from Paeoniae Radix Rubra based on UPLC-Q-TOF-MS].

The ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was used to conduct the qualitative analysis of the monoterpene chemical components from Paeoniae Radix Rubra. Gradient elution was performed on C_(18) HD(2.1 mm×100 mm, 2.5 µm) column with a mobile phase of 0.1% formic acid(A) and acetonitrile(B). The flow rate was 0.4 mL·min~(-1) and the column temperature was 30 ℃. MS analysis was conducted in both positive and negative ionization modes using electrospray ionization(ESI) source. Qualitative Analysis 10.0 was used for data processing. The identification of chemical components was realized by the combination of standard compounds, fragmentation patterns, and mass spectra data reported in the literature. Forty-one monoterpenoids in Paeoniae Radix Rubra extract were identified. Among them, 8 compounds were reported in Paeoniae Radix Rubra for the first time and 1 was presumed to be the new compound 5″-O-methyl-galloylpaeoniflorin or its positional isomer. The method in this study realizes the rapid identification of monoterpenoids from Paeoniae Radix Rubra and provides a material and scientific basis for quality control and further study on the pharmaceutical effect of Paeoniae Radix Rubra.

[Investigation of the chemical components of Ciwujia injection using ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry].

Ciwujia injection is commonly used to treat cerebrovascular and central nervous system diseases in clinical practice. It can significantly improve blood lipid levels and endothelial cell function in patients with acute cerebral infarction and promote the proliferation of neural stem cells in cerebral ischemic brain tissues. The injection has also been reported to have good curative effects on cerebrovascular diseases, such as hypertension and cerebral infarction. At present, the material basis of Ciwujia injection remains incompletely understood, and only two studies have reported dozens of components, which were determined using high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF MS). Unfortunately, the lack of research on this injection restricts the in-depth study of its therapeutic mechanism.In the present study, a qualitative method based on ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry (UHPLC-Q/Orbitrap HRMS) was developed to analyze the chemical components of Ciwujia injection. Separation was performed on a BEH Shield RP18 column (100 mm×2.1 mm, 1.7 µm) using 0.1% formic acid aqueous solution (A) and acetonitrile (B) as the mobile phases, and gradient elution was performed as follows: 0-2 min, 0%B; 2-4 min, 0%B-5%B; 4-15 min, 5%B-20%B; 15-15.1 min, 20%B-90%B; 15.1-17 min, 90%B. The flow rate and column temperature were set to 0.4 mL/min and 30 ℃ respectively. MS1 and MS2 data were acquired in both positive- and negative-ion modes using a mass spectrometer equipped with an HESI source. For data post-processing, a self-built library including component names, molecular formulas, and chemical structures was established by collecting information on the isolated chemical compounds of Acanthopanax senticosus. The chemical components of the injection were identified by comparison with standard compounds or MS2 data in commercial databases or literature based on precise relative molecular mass and fragment ion information. The fragmentation patterns were also considered. For example, the MS2 data of 3-caffeoylquinic acid (chlorogenic acid), 4-caffeoylquinic acid (cryptochlorogenic acid), and 5-caffeoylquinic acid (neochlorogenic acid) were first analyzed. The results indicated that these compounds possessed similar fragmentation behaviors, yielding product ions at m/z 173 and m/z 179 simultaneously. However, the abundance of the product ion at m/z 173 was much higher in 4-caffeoylquinic acid than in 5-caffeoylquinic acid or 3-caffeoylquinic acid, and the fragment signal at m/z 179 was much stronger for 5-caffeoylquinic acid than for 3-caffeoylquinic acid. Four caffeoylquinic acids were identified using a combination of abundance information and retention times. MS2 data in commercial database and literature were also used to identify unknown constituents. For example, compound 88 was successfully identified as possessing a relative molecular mass and neutral losses similar to those of sinapaldehyde using the database, and compound 80 was identified as salvadoraside because its molecular and fragmentation behaviors were consistent with those reported in the literature. A total of 102 constituents, including 62 phenylpropanoids, 23 organic acids, 7 nucleosides, 1 iridoid, and 9 other compounds, were identified. The phenylpropanoids can be further classified as phenylpropionic acids, phenylpropanols, benzenepropanals, coumarins, and lignans. Among the detected compounds, 16 compounds were confirmed using reference compounds and 65 compounds were identified in Ciwujia injection for the first time. This study is the first to report the feasibility of using the UHPLC-Q/Orbitrap HRMS method to quickly and comprehensively analyze the chemical components of Ciwujia injection. The 27 newly discovered phenylpropanoids provide further material basis for the clinical treatment of neurological diseases and new research targets for the in-depth elucidation of the pharmacodynamic mechanism of Ciwujia injection and its related preparations.

Offline two-dimensional normal-phase × reversed-phase liquid chromatography coupled with high-resolution mass spectrometry for comprehensive analysis of chemical constituents in Euphorbia kansui.

Euphorbia kansui is the dried root of Euphorbia kansui T. N. Liou ex T.P. Wang. Its main chemical components are diterpenoids, triterpenes, and volatile oil. In this study, an offline two-dimensional (2D) normal-phase × reversed-phase liquid chromatography method coupled with quadrupole time-of-flight mass spectrometry was established to comprehensively analyze the chemical constituents in E. kansui. A total of 240 compounds were identified from the E. kansui extract, including 218 diterpenoids (77 known, 141 new), 16 known volatile oils, and six known triterpenes. The relationship between the structural characteristics and tandem mass spectroscopy fragments of diterpenoids was further analyzed. Based on the characteristic fragment ions, 141 new diterpenoids were determined as 118 ingenane diterpenoids and 23 jatrophane diterpenoids. The newly identified diterpenoids may provide lead compounds for drug discovery, improving the medicinal value of E. kansui.

Combining multidimensional chromatography-mass spectrometry and feature-based molecular networking methods for the systematic characterization of compounds in the supercritical fluid extract of Tripterygium wilfordii Hook F.

Tripterygium wilfordii Hook F from the family Celastraceae is a traditional Chinese medicine (TCM) whose principal chemical constituents are terpenoids, including sesquiterpene alkaloids and diterpenoids, which have unique and diverse structures and remarkable biological activities. In order to advance pharmacological research and guide the preparation of monomer compounds derived from T. wilfordii, a systematic approach to efficiently discover new compounds or their derivatives is needed. Herein, compound separation and identification were performed by offline reversed-phase × supercritical fluid chromatography coupled mass spectrometry (RP × SFC-Q-TOF-MS/MS) and Global Natural Product Social (GNPS) molecular networking. The 2D chromatography system exhibited a high degree of orthogonality and significant peak capacity, and SFC has an advantage during the separation of sesquiterpene alkaloid isomers. Feature-based molecular networking offers the great advantage of quickly detecting and clustering unknown compounds, which greatly assists in intuitively judging the type of compound, and this networking technique has the potential to dramatically accelerate the identification and characterization of compounds from natural sources. A total of 324 compounds were identified and quantitated, including 284 alkaloids, 22 diterpenoids and 18 triterpenoids, which means that there are numerous potential new compounds with novel structures to be further explored. Overall, feature-based molecular networking provides an effective method for discovering and characterizing novel compounds and guides the separation and preparation of targeted natural products.

Integrated strategy for identifying isoflavones in Belamcandae Rhizoma based on the combination of mass defect filtering and neutral-loss-triggered multistage fragmentation.

Belamcandae Rhizoma is a widely used traditional Chinese herbal medicine with isoflavones as the main active ingredient. In this paper, an integrated strategy was developed to discover and identify new isoflavones in Belamcandae Rhizoma by an ultra-high-performance liquid chromatography coupled with high resolution multistage mass spectrometry. Different characterization methods were used based on structural features of isoflavone aglycones and glycosides. On one hand, we adopted a data-dependent acquisition mode incorporated into intelligent AcquireX deep scan algorithms to analyze crude extract, and used a mass defect filtering technique to filter out two kinds of isoflavone aglycones from the extract. On the other hand, neutral-loss-triggered MSn was used to analyze isoflavone glycosides, and under this acquisition mode, MSn scan only took place when chemical components exhibited specific neutral losses. Identification of isoflavones was achieved either by comparison with reference compounds or analysis of characteristic product ions based on MS2 or MSn fragmentation patterns. UV absorbance spectra also contributed to the confirmation of isoflavones. As a result, a total of 65 isoflavone aglycones (42 new aglycones) and 142 isoflavone glycosides (122 new glycosides) were discovered, including a number of trace components. Meanwhile, modifications of new sugar moieties, such as xylose, rhamnose and 6-O-(4­hydroxy-3,5-dimethoxybenzoyl)-ß-D-glucose, were discovered in Belamcandae Rhizoma for the first time. These results indicated the feasibility of this established strategy for in-depth identification of new isoflavone aglycones and glycosides.

Comprehensive characterization of ingredients in Crocus sativus L. from different origins based on the combination of targeted and nontargeted strategies.

As a valuable medicinal and edible plant, Crocus sativus L. has had wide applications since ancient times. Herein, a comprehensive approach for characterization of constituents in saffron was established based on the combination of targeted and non-targeted strategies. A targeted UPLC-ESI/MSn strategy was applied for in-depth identification of crocins, and a non-targeted UPLC-ESI/MS2 approach characterized other components. This integration strategy was used to analyze ingredients in 21 batches of saffrons from 6 origins. Forty-seven crocins belonging to 8 types were identified including 32 new crocins. Among them, 6 new compounds with specific structures were reported for the first time, i.e. trans-6(G, 2G), trans-4(GT, g), trans-3(GT), cis-3(GT), methyl ester-trans-2(G) and methyl ester-cis-2(G). Besides, 91 non-crocin components were identified including 43 new compounds. Based on systematic investigation of crocins and non-crocins, we found that crocins were the critical components to distinguish saffrons from different origins, especially between domestic and foreign samples.

Chemical profiling of Qingfei Paidu Decoction by triplex off-line two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Qingfei Paidu Decoction is a Chinese medicine formula that has been proved effective in the treatment of coronavirus disease 2019. However, the comprehensive separation and characterization of Qingfei Paidu Decoction are of a great challenge due to the diversity of chemical components in a wide range of polarity. In this study, a triplex off-line two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry is developed for the analysis of Qingfei Paidu Decoction. One reversed-phase liquid chromatography×hydrophilic interaction liquid chromatography system and two reversed-phase liquid chromatography×reversed phase liquid chromatography systems were constructed to separate polar components and weak-polar components in Qingfei Paidu Decoction, respectively. Benefiting from the good orthogonality of two-dimensional liquid chromatography and high sensitivity of quadrupole time-of-flight MS, chemical components with different polarities and content were discovered. A total of 749 peaks were detected in positive and negative ionization mode and presented as a four-dimensional data plot. Meanwhile, 498 compounds belonging to 14 categories were tentatively identified. These results provide good supplementary to elucidate the material basis of Qingfei Paidu Decoction. The triplex off-line two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry strategy can be a powerful and efficient tool for the separation and characterization of chemical substances in traditional Chinese medicine formulas.

Analysis of alkaloids in Gelsemium elegans Benth using an online heart-cutting + comprehensive RPLC×RPLC system tandem mass spectrometry.

Characterization of alkaloids and new compound discovery become increasing challenging for Gelsemium elegans Benth. (G. elegans), due to the lack of an effective separation method. In this study, we developed a new online heart-cutting + comprehensive (HC) RPLC × RPLC system with pH difference, which was coupled to a mass detector to realize the separation and characterization of alkaloids from G. elegans. 18 Gelsemium standards were used to construct the RPLC × RPLC system with pH difference (pH 3 and 11), and good orthogonality (correlation coefficient 0.3) was obtained. A heart-cutting valve was introduced into the traditional online comprehensive RPLC × RPLC system to remove principal components and improve detection of minor components. The online HC RPLC × RPLC system achieved good resolving power (effective peak capacity 687) in condition of optimized practical factors, like the first- and second-dimension flow rates, modulation period and elution gradient et al. Finally, a total of 256 alkaloids were grouped and tentatively identified, among which 156 were unreported, including a new alkaloid type in G. elegans and many dimeric indole alkaloids, which was an important supplement to the study on chemical constituents of G. elegans.

Isoquinoline alkaloid dimers with dopamine D1 receptor activities from Menispermum dauricum DC.

A phytochemical investigation on chemical constituents from the rhizomes of Menispermum dauricum DC. identified eight undescribed dimeric alkaloids with structurally diverse monomeric isoquinoline. Alkaloid structures were elucidated by a combination of spectroscopic data analyses and time-dependent density functional theory (TDDFT) ECD calculation. The isolates were evaluated for inhibitory effect on dopamine D1 receptor and compound 1 exhibited potent D1 receptor antagonistic activity with an IC50 value of 8.4 ± 2.0 µM.

Mechanism deconvolution of Qing Fei Pai Du decoction for treatment of Coronavirus Disease 2019 (COVID-19) by label-free integrative pharmacology assays.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine (TCM) has a long history in the prevention and treatment of pandemics. The TCM formula Lung Cleansing and Detoxifying Decoction (LCDD), also known as Qing Fei Pai Du Decoction, has been demonstrated effective against Coronavirus Disease 2019 (COVID-19). AIM OF THE STUDY: This work aimed to elucidate the active ingredients, targets and pathway mechanism of LCDD related to suppression of inflammatory, immunity regulation and relaxation of airway smooth muscle for the treatment of COVID-19. MATERIALS AND METHODS: Mining chemical ingredients reported in LCDD, 144 compounds covering all herbs were selected and screened against inflammatory-, immunity- and respiratory-related GPCRs including GPR35, H1, CB2, B2, M3 and ß2-adrenoceptor receptor using a label-free integrative pharmacology method. Further, all active compounds were detected using liquid chromatography-tandem mass spectrometry, and an herb-compound-target network based on potency and content of compounds was constructed to elucidate the multi-target and synergistic effect. RESULTS: Thirteen compounds were identified as GPR35 agonists, including licochalcone B, isoliquiritigenin, etc. Licochalcone B, isoliquiritigenin and alisol A exhibited bradykinin receptor B2 antagonism activities. Atractyline and shogaol showed as a cannabinoid receptor CB2 agonist and a histamine receptor H1 antagonist, respectively. Tectorigenin and aristofone acted as muscarinic receptor M3 antagonists, while synephrine, ephedrine and pseudoephedrine were ß2-adrenoceptor agonists. Pathway deconvolution assays suggested activation of GPR35 triggered PI3K, MEK, JNK pathways and EGFR transactivation, and the activation of ß2-adrenoceptor mediated MEK and Ca2+. The herb-compound-target network analysis found that some compounds such as licochalcone B acted on multiple targets, and multiple components interacted with the same target such as GPR35, reflecting the synergistic mechanism of Chinese medicine. At the same time, some low-abundance compounds displayed high target activity, meaning its important role in LCDD for anti-COVID-19. CONCLUSIONS: This study elucidates the active ingredients, targets and pathways of LCDD. This is useful for elucidating multitarget synergistic action for its clinical therapeutic efficacy.

A strategy for efficient enrichment of steroidal alkaloids from Fritillaria based on fluorinated reversed-phase stationary phase.

Plant-derived alkaloids are bioactive natural ingredients, but their contents are relatively low in plants. Therefore, the efficient enrichment of alkaloids is a prerequisite for purification and further pharmacological research. In this study, an efficient and simple strategy for enrichment of steroidal alkaloids in Fritillaria was developed for the first time based on the fluorinated reverse-phase stationary phase (FC8HL). Superior selectivity between alkaloids and non-alkaloids was achieved in a non-aqueous system, and a simple solvent system containing low-content additives was applied to elute alkaloids. Key parameters that affected the elution were investigated, including different types of buffer salts and optimized concentrations. The optimized elution system was then applied to selectively enrich alkaloids from five species of Fritillaria. Its practicability was further demonstrated by enrichment of alkaloids from Fritillaria cirrhosa D.Don at a preparative level. This developed method has great potential for other types of hydrophobic alkaloids.

Correction to: Combination of multi-model statistical analysis and quantitative fingerprinting in quality evaluation of Shuang-huang-lian oral liquid.

The authors would like to call the reader's attention to the fact that unfortunately there was a mistake in Table 1 of this contribution.

Combination of multi-model statistical analysis and quantitative fingerprinting in quality evaluation of Shuang-huang-lian oral liquid.

A model consisting of quantitative fingerprinting integrated with fundamental statistical analysis was established to carry out quality control analysis of Shuang-huang-lian (SHL) oral liquid. The quantitative fingerprinting approach was developed by systematic investigation of the chromatographic condition and optimization of a gradient using a complex sample analysis software system (CSASS). Five pivotal components from three traditional Chinese medicines of SHL oral liquid were determined at dual wavelengths, including phillyrin, baicalin, chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid. Among them, neochlorogenic acid and cryptochlorogenic acid were quantified by quantitative analysis of multi-components with a single marker (QAMS) method. Further, the developed quantitative fingerprinting approach was validated. Good linearity with correlation coefficients (R2) higher than 0.9999 were achieved for phillyrin, baicalin and chlorogenic acid. Recoveries of the three analytes were between 96% and 108%. Relative standard deviation (RSD) values were below 3% regarding the stability and intra-day and inter-day precision. Besides, the feasibility of the QAMS method was verified by an external standard method (ESM) using 18 batches of SHL oral liquid. Fifty-nine batches of SHL oral liquid from nine manufacturers were then analyzed. Effective distinction was realized based on a linear principal component analysis (linear-PCA) model by the combination of the quantitative data and chromatographic fingerprinting. The linear-PCA model based on quantitative chromatographic fingerprinting exhibited great advantage over conventional similarity analysis to distinguish between different samples. The strategy provided a particularly simple and effective approach for quality evaluation of SHL oral liquid from various manufacturers. Graphical abstract.

Synthesis and chromatographic evaluation of a new stationary phase based on mild thiol-Michael addition reaction.

Click chemistry has attracted increasing attention for the synthesis of novel stationary phases. Considering the advantage of click chemistry, a strategy based on thiol-Michael addition was developed for the preparation of a new stationary phase herein, and a phenyl vinyl sulfone stationary phase (M-PVS) was prepared. The resulting M-PVS bonded silica was characterized by elemental analysis, solid-state 13C cross-polarization/magic-angle spinning NMR and infrared spectroscopy, confirming the successful immobilization of phenyl vinyl sulfone on the silica support. The retention properties of M-PVS were investigated and exhibited unambiguous reversed phase retention characteristics. Moreover, shape selectivity and silanol activity were studied to reveal the diverse interactions of M-PVS, including hydrophobic, π-π, hydrogen bonding, and ion-exchange interactions. In addition, de-wetting tolerance and hydrophilic properties were evaluated and a pronounced "U" retention curves were obtained, indicating enhanced retention for polar analytes and transitions of different interaction modes. Selectivity differences between M-PVS column, phenyl column and conventional C18 column were examined using series natural standards. The diverse interactions of M-PVS demonstrated its improved selectivity for the compounds with similar hydrophobic skeleton but different polar substituents.

[Establishment of chromatographic fingerprint of Squama Manis and its applications in animal source identification and quality grade discrimination].

Squama Manis, or "Chuanshanjia" in Chinese, is a traditional Chinese medicine (TCM) for promoting blood circulation and reducing swelling and discharge; the only animal source used in TCM is the scales of Manis pentadactyla. However, in today's pharmaceutical market, there are many scales from other species of the same genus that are difficult to distinguish from Squama Manis. High-quality and low-quality scales are also severely confused. To solve the above problems, various analytical methods have been developed, such as thin-layer chromatography, mass spectrometry and DNA detection. Owing to their low resolving ability, high equipment cost, and inconvenient operation, none of these methods are appropriate for routine identification of Squama Manis. A chromatographic fingerprint can comprehensively reflect the synergic action of multiple chemical compositions in TCM and has been widely used for the quality control of TCM. In the present study, we established a fingerprint of Squama Manis and explored its feasibility in identifying the origin and quality grade of scales. First, Squama Manis powder was hydrolyzed by hydrochloric acid (1 mol/L). Next, the extract was analyzed on a Symmetry 300 C18 column by linear gradient elution, using 0.1% trifluoroacetic acid (v/v) in water and 0.1% trifluoroacetic acid (v/v) in acetonitrile as the mobile phase and 280 nm as the detection wavelength. The established method was systematically validated, demonstrating good precision, repeatability and sample stability (relative standard deviation (RSD)<5%). Subsequently, samples of different sources and quality grades were distinguished by similarity evaluation and discrimination analysis based on the fingerprint data. In the similarity evaluation, the reference fingerprint was defined as the average fingerprint of twelve first-class samples, and seventeen chromatographic peaks were identified as common peaks. Similarities between the reference fingerprint and fingerprints with different base sources and quality grades were calculated using the absolute area of common peaks as original data. The similarities between Squama Manis and scales from other animals were all less than 0.776, while the similarities between Squama Manis of different grades overlapped significantly, varying from 0.988 to 0.996 for first-class samples and 0.950 to 0.995 for general samples. The results reflected the feasibility of similarity evaluation for discriminating base source and its limitation in the distinguishing between quality grades. Nonetheless, first-class scales showed higher average similarity and lower RSD than general scales, which indicates some level of revelation between fingerprint similarity and quality grade. Thus, a better algorithm or discriminant model is required to distinguish between quality grades. Therefore, a supervised chemometric technique, kernel-based support vector machine (SVM), was applied to construct predictive models. The SVM is a common discriminant model that classifies samples by constructing a separate hyperplane in n-dimensional space, maximizing the margin between classes. Combination with a kernel function can effectively avoid "dimension disaster" when dealing with nonlinear data. In the model, the quality grade was defined as a sample label, and the absolute peak areas constituted the data matrix. Verified by 10-fold cross-validation, the unbiased prediction accuracy was up to 95.83%. The predicted results were highly consistent with the actual classifications. The results indicate the high feasibility of the established model for determining quality grade, as it performed significantly better than the similarity evaluation. Samples from batches A and B were completely discriminated and only two samples from batch S were incorrectly classified. Given the batch bias, we believe that model error may have been caused by man-made tag errors rather than the model itself. In conclusion, we established a chromatographic fingerprint for Squama Manis quality analysis and demonstrated its feasibility in animal source identification and quality determination by combining different data analysis methods. The established strategy may provide a new method for improving the the validity and accuracy of Squama Manis in clinical use.

Application of two-dimensional liquid chromatography in the separation of traditional Chinese medicine.

Traditional Chinese medicines have been widely used to prevent and cure diseases for thousands of years. For the purpose of better understanding the extremely complicated traditional Chinese medicines, powerful separation techniques are essential. Two-dimensional liquid chromatography has been proven to be more powerful for the separation of complex traditional Chinese medicines due to its enhanced peak capacity and resolution compared with one-dimensional liquid chromatography. Enormous efforts have been made on the coupling of independent separation mechanisms to improve the resolving power for complex traditional Chinese medicine samples, including the development and introduction of novel stationary phases. This review aims to give an overview on the applications of two-dimensional liquid chromatography in traditional Chinese medicine research since 2008, including comprehensive two-dimensional liquid chromatography, heart-cutting two-dimensional liquid chromatography both in on-line, and off-line mode. Different couplings of separation modes were respectively discussed based on specific studies, with emphasis on the applications of novel stationary phases in the two-dimensional liquid chromatography.

An offline two-dimensional supercritical fluid chromatography × reversed phase liquid chromatography tandem quadrupole time-of-flight mass spectrometry system for comprehensive gangliosides profiling in swine brain extract.

Gangliosides, widely distributed in tissues and body fluid, have been connected to the therapy of cancer and brain related diseases. The complexity of the gangliosides structures with different polar moieties coexisting, a carbohydrate moiety and a ceramide chain, make it a great challenge in separation and analysis science. This study aimed to develop a strategy on the basis of high-accuracy data collected by an offline two-dimensional (2D) supercritical fluid chromatography (SFC) × reversed phase liquid chromatography (RPLC)/quadrupole time-of-flight (Q-ToF) system, and to integrate an in-house library with self-developed software for fast screening and identification of gangliosides from a complex sample (swine brain extract). Subsequent positive-mode MS/MS was used to validate the identified gangliosides. Finally, 153 gangliosides were separated and 79 of them were identified by the in-house library and self-developed software, 4-fold more than those by manual identification (18 gangliosides). Among the identified ones, 20 were detected in swine brain for the first time. This study established an offline 2D SFC × RPLC system and provided a new method for fast screening and automatic identification of gangliosides in complex mixtures. It will be conducive to further study of biological functions of gangliosides.

Preparation of glutathione-functionalized zwitterionic silica material for efficient enrichment of sialylated N-glycopeptides.

A glutathione (GSH)-functionalized silica material was prepared using divinyl sulfone activation chemistry (named SiO2-DVS-GSH). The successful synthesis of the SiO2-DVS-GSH material was confirmed by FT-IR, elemental analysis, and zeta potential analysis. The effects of water content, pH value, and salt concentration in the mobile phase on the model compound (uracil, uridine, cytosine, cytidine, guanosine, xanthosine, orotic acid) retention was studied, and a hydrophilic interaction liquid chromatography (HILIC) retention feature together with electrostatic interaction of the SiO2-DVS-GSH material was observed. The prepared stationary phase was further applied for the separation of oligosaccharide. In addition, the SiO2-DVS-GSH material displayed remarkable selectivity and specificity for the sialylated N-glycopeptides' enrichment from bovine fetuin tryptic digests, even at a mass ratio of 1:1000 (w/w) to bovine serum albumin (BSA, non-glycosylated protein), showing superior performance compared to commercial ZIC-HILIC material. Moreover, the SiO2-DVS-GSH material behaved well in the N-glycopeptides' enrichment from human serum, demonstrating its promising potential for glycoproteomics of complex biological samples. Graphical Abstract A glutathione (GSH)-functionalized silica material was prepared using divinyl sulfone activation chemistry, and it shows remarkable selectivity and specificity for the sialylated N-glycopeptides' enrichment.