Search

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. [ABSTRACT FROM AUTHOR]

An orthogonal two-dimensional solid-phase extraction strategy was established for the selective enrichment of three aminoglycosides including spectinomycin, streptomycin, and dihydrostreptomycin in milk. A reversed-phase liquid chromatography material (C18) and a weak cation-exchange material (TGA) were integrated in a single solid-phase extraction cartridge. The feasibility of two-dimensional clean-up procedure that experienced two-step adsorption, two-step rinsing, and two-step elution was systematically investigated. Based on the orthogonality of reversed-phase and weak cation-exchange procedures, the two-dimensional solid-phase extraction strategy could minimize the interference from the hydrophobic matrix existing in traditional reversed-phase solid-phase extraction. In addition, high ionic strength in the extracts could be effectively removed before the second dimension of weak cation-exchange solid-phase extraction. Combined with liquid chromatography and tandem mass spectrometry, the optimized procedure was validated according to the European Union Commission directive 2002/657/EC. A good performance was achieved in terms of linearity, recovery, precision, decision limit, and detection capability in milk. Finally, the optimized two-dimensional clean-up procedure incorporated with liquid chromatography and tandem mass spectrometry was successfully applied to the rapid monitoring of aminoglycoside residues in milk. [ABSTRACT FROM AUTHOR]

The separation of basic macrolide antibiotics suffers from peak tailing and poor efficiency on traditional silica-based reversed-phase liquid chromatography columns. In this work, a C18HCE column with positively charged surface was applied to the separation of macrolides. Compared with an Acquity BEH C18 column, the C18HCE column exhibited superior performance in the aspect of peak shape and separation efficiency. The screening of mobile phase additives including formic acid, acetic acid and ammonium formate indicated that formic acid was preferable for providing symmetrical peak shapes. Moreover, the influence of formic acid content was investigated. Analysis speed and mass spectrometry compatibility were also taken into account when optimizing the separation conditions for liquid chromatography coupled with tandem mass spectrometry. The developed method was successfully utilized for the determination of macrolide residues in a honey sample. Azithromycin was chosen as the internal standard for the quantitation of spiramycin and tilmicosin, while roxithromycin was used for erythromycin, tylosin, clarithromycin, josamycin and acetylisovaleryltylosin. Good correlation coefficients ( r2 > 0.9938) for all macrolides were obtained. The intra-day and inter-day recoveries were 73.7-134.7% and 80.7-119.7% with relative standard deviations of 2.5-8.0% and 3.9-16.1%, respectively. Outstanding sensitivity with limits of quantitation (S/N ≥ 10) of 0.02-1 μg/kg and limits of detection (S/N ≥ 3) of 0.01-0.5 μg/kg were achieved. [ABSTRACT FROM AUTHOR]

Hydrophilic interaction liquid chromatography has emerged as a valuable alternative approach to ion-pair chromatography for the separation of aminoglycoside antibiotics in recent years. However, the resolution of structurally related aminoglycosides is a great challenge owing to the limited selectivity. In this work, a cysteine-based zwitterionic stationary phase (named Click TE-Cys) was utilized and compared with five commonly used hydrophilic interaction liquid chromatography columns. Click TE-Cys displayed much better selectivity for structurally similar aminoglycosides. The retention behaviors of aminoglycosides were investigated in detail, revealing that low pH (2.7 or 3.0) and high buffer concentration (≥50 mM) were preferable for achieving good peak shape and selectivity. Effective resolution of ten aminoglycosides including spectinomycin, dihydrostreptomycin, streptomycin, gentamicin C1, gentamicin C2/C2a, gentamicin C1a, kanamycin, paromonycin, tobramycin, and neomycin was realized at optimized conditions. Additionally, spectinomycin and its related impurities were successfully resolved. The results indicated the great potential of the Click TE-Cys column in the separation of aminoglycoside mixtures and related impurities. [ABSTRACT FROM AUTHOR]

Highlights: [•] A novel HILIC/CEX stationary phase was developed based on glutathione (Click TE-GSH). [•] The material was facilely synthesized via copper-free thiol-ene click chemistry. [•] Click TE-GSH showed good hydrophilicity and adjustable cation-exchange property. [•] Click TE-GSH exhibited good selectivity in separating carbohydrates and peptides. [•] The novel material has good potential in glycomics and proteomics. [ABSTRACT FROM AUTHOR]

Abstract: A hydrophilic interaction/weak cation-exchange (HILIC/WCX) mixed-mode chromatography was developed for separation of chitooligosaccharides (COS) using a weak cation-exchange column. For better understanding of the retention characteristics of the column, organic solvent content effect was investigated. WCX mode, HILIC mode, and mixed-mode could be performed on this column according to investigating results. COS were separated on the column in mixed-mode. The usage of volatile buffer salt made it favorable for evaporative light-scattering detector (ELSD). Mild elution conditions were economical and environment-friendly relative to other conditions used in early reports. This method may offer a possibility for large-scale COS preparation. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: A cysteine-bonded zwitterionic hydrophilic interaction chromatography (HILIC) stationary phase (Click TE-Cys) was prepared based on the “thiol-ene” click chemistry. The Click TE-Cys material was characterized by solid state 13C cross polarization/magic-angle spinning (CP/MAS) NMR and elemental analysis. The dynamic evaluation for cytosine, cytidine and orotic acid was performed using Van Deemter plots. The plate height values were no more than 24μm for the flow rate between 0.5 and 5.4mms−1 (0.3–3.5mLmin−1), which proved the excellent separation efficiency of Click TE-Cys stationary phase. The influences of the content of water, concentration of salt and pH of the buffer solution on the retention of model compounds were investigated. The results demonstrated that the separation of polar analytes was dominated by the partitioning mechanism, while the contribution of electrostatic interaction was minor. The thermodynamic characteristic of Click TE-Cys stationary phase was also studied according to van’t Hoff plot. An exothermic process for transferring analytes from the mobile phase to the stationary phase was observed and a linear relationship for ln k and 1/T was achieved, indicating no change of retention mechanism within the measured temperature range. Besides, the zwitterionic stationary phase exhibited good stability. Considering the high hydrophilicity of Click TE-Cys stationary phase, the application in the separation of protein tryptic digests was carried out using hydrophilic interaction chromatography–electrospray ionization mass spectrometry (HILIC–ESI-MS). More peaks were adequately resolved on the Click TE-Cys column comparing with that on the TSK Amide-80 column. In addition, the orthogonality between HILIC and RPLC system was investigated utilizing geometric approach. The XTerra MS C18 and Click TE-Cys column displayed great difference in separation selectivity, with the orthogonality reaching 88.0%. On the other hand, the orthogonality between Click TE-Cys and TSK Amide-80 system was 21.4%, i.e. the selectivity was similar but slightly different from each other. The successful separation of protein digests indicated the great potential of Click TE-Cys stationary phase in the separation of complex samples and applicability in two-dimensional liquid chromatography (2D-LC). [Copyright &y& Elsevier]

A controlled thiol-initiated surface polymerization strategy has been successfully developed and employed to prepare hydrophilic polymer stationary phases, which exhibited excellent chromatographic performance and protein non-fouling properties. [ABSTRACT FROM AUTHOR]

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. 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. 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. Seventy compounds were isolated and twelve showed GPCR activity. Three compounds showed potent GPR35 agonistic activity (EC 50 < 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. 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. [Display omitted] • Jinqiancao granules could be a potential treatment of prostatitis and BPH. • Compounds showed activity on four disease-related GPCRs and NO inhibition. • PAL-21 (Scutellarein) was a GPR35 agonist with an EC50 of 1.50 ± 0.18 μM. [ABSTRACT FROM AUTHOR]

A novel zwitterionic stationary phase with high hydrophilicity was facilely synthesized based on the “thiol-ene” click reaction between cysteine and vinyl silica, which exhibited great potential in the separation of oligosaccharides, peptides and basic compounds, as well as in the enrichment of glycopeptides. [ABSTRACT FROM AUTHOR]

• It is the first time that thiol-Michael addition reaction was used for chemical modification on silica particle herein. • A new phenyl vinyl sulfone stationary phase was synthesized and characterized. • This stationary phase provided multiple interactions. 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 C 18 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• An offline NPLC × RPLC system was developed for the separation of E.kansui extract. • 240 compounds including 141 new diterpenoids were identified by 2D-LC Q-TOF-MS/MS. • The fragmentation behaviors of different types of diterpenoids were summarized. 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. [ABSTRACT FROM AUTHOR]

Glycyrrhizin (GA), a major bioactive compound in licorice, has been extensively used throughout the world as a medicine to treat chronic viral hepatitis and allergic dermatitis. In this study, a new method based on hydrophilic interaction solid phase extraction (HILIC-SPE) and preparative reversed-phase liquid chromatography (prep-RPLC) was developed to purify GA with high purity from the complex licorice extract. Via evaluation of retention behavior of GA and flavonoids in different commercially available columns, a hydrophilic column——Click XIon was finally chosen for the purification due to its excellent resolution toward GA and flavonoids under HILIC mode. To optimize the SPE elution conditions, relative factors including water content, pH and ionic strength had been investigated in chromatographic condition. The result indicated that the most appropriate water content was 30% and pH at 4.00, as well as salt concentration should be controlled at 5 mM. In addition, the optimization revealed that GA experiences both hydrophilic interaction and ion-exchange interaction on the Click XIon material. According to the chromatographic evaluation, the optimized conditions were applied to HILIC-SPE to enrich GA from licorice, which leads to an increased content of GA from 13.67% to 64.22%. Finally, prep-RPLC was performed to obtain GA with purity higher than 99.00%，which demonstrating great prospect in large-scale preparation of GA. [ABSTRACT FROM AUTHOR]

Zwitterionic polymer stationary phases have attracted increasing attention in hydrophilic interaction chromatography (HILIC). In this work, a zwitterionic sulfobetaine functionalized polyacrylamide stationary phase (named TENS) based on porous silica particles was prepared via controlled surface initiated reversible addition-fragmentation transfer (RAFT) polymerization. Instead of traditional methacrylate type sulfobetaine monomer, acrylamide type sulfobetaine monomer, which has higher chemical stability and hydrophicility, was employed in this work. The characterization of elemental analysis and solid-state 13 C cross polarization/magic-angle-spinning nuclear magnetic resonance indicated the successful preparation of TENS stationary phase. Meanwhile, scanning electron microscope (SEM), nitrogen adsorption experiment and study of size exclusion performance were conducted, revealing that the surface initiated polymerization was well controlled. For better understanding of TENS material under HILIC mode, chromatographic evaluation of TENS material was performed, among which, TENS material exhibited good hydrophilicity and chemical stability. To further study the applicability of TENS material, saccharides which were considered as challenging targets in HILIC, were chosen as tested analytes. Various saccharide samples, including fructooligosaccharide, trisaccharide isomers and ginsenosides, were well separated on TENS material. Moreover, TENS material displayed good selectivity for the enrichment of glycopeptides. These results demonstrated the capability of TENS as a promising material in glycomics and glycoproteomics. [ABSTRACT FROM AUTHOR]

Nowadays more and more hydrophilic interaction liquid chromatography (HILIC) columns with diverse functional groups have become commercially available, which pose a challenge to select an appropriate one. However, there is no universal model to provide guidance for selecting HILIC columns. To handle this problem, a retention model named “hydrophilic-subtraction model” was developed to characterize and compare HILIC columns. The hydrophilic-subtraction model, which was designed based on the widely recognized HILIC retention mechanisms including hydrophilic partitioning, hydrogen-bonding and electrostatic interactions, was established by the retention of 41 solutes with various properties on 8 representative HILIC columns. High correlation coefficients ( R 2 ≥ 0.990) and small standard deviations ( SD ≤ 0.041) indicated that this model correlated effectively the retention with solute descriptors and column parameters. To evaluate reliability of the model, the model was further applied to characterize 15 additional HILIC columns using 41 solutes. The results of multiple linear regression confirmed the significance of the model. The regression coefficients of the model were used to investigate retention mechanisms occurring in different chromatographic systems. Based on these regression coefficients, selectivities of HILIC stationary phases were exhibited intuitively by an angle graph and a spider diagram, which could be used as guidance for researchers to select appropriate columns for HILIC separation. Additionally, a rapid and convenient procedure was proposed for characterizing HILIC columns. [ABSTRACT FROM AUTHOR]

Highlights: [•] Two classes of bufadienolides were separated by HILIC-SPE. [•] This prevented their co-elution in RP-HPLC. [•] A total of 12 compounds from the two classes were purified by this method. [Copyright &y& Elsevier]

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. [Display omitted] • A new online HC RPLC × RPLC system was developed for analysis of alkaloids from Gelsemium elegans Benth. • 18 Gelsemium standards were used to construct the RPLC × RPLC system with different pH. • A heart-cutting valve combined with a comprehensive valve was used to remove principal components. • 256 alkaloids were tentatively characterized and 156 of them were not reported before. [ABSTRACT FROM AUTHOR]

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. • Dauricine and N -desmethyldauricine from traditional Chinese medicine M. dauricum. display good bioactivity on anti -TNBC. • N -desmethyldauricine exhibits anti -TNBC activity both in 2D and 3D models in vitro. • N -desmethyldauricine affects TNBC cell cycle, apoptosis, migration and downregulates the expression of NF-κB. [ABSTRACT FROM AUTHOR]

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). 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. 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 β2-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. 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 β2-adrenoceptor agonists. Pathway deconvolution assays suggested activation of GPR35 triggered PI3K, MEK, JNK pathways and EGFR transactivation, and the activation of β2-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. This study elucidates the active ingredients, targets and pathways of LCDD. This is useful for elucidating multitarget synergistic action for its clinical therapeutic efficacy. [Display omitted] [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A novel online comprehensive two-dimensional liquid chromatography (2DLC) coupled with quadrupole time-of-flight (Q-TOF) mass spectrometry (MS) method is developed for the analysis of Curcuma kwangsiensis ( C. kwangsiensis ) extract. In this system, a newly developed phenyl/tetrazole sulfoether (PTAS) bonded stationary phase was introduced to construct RPLC×RPLC combined with C18. The unique structure endowed PTAS with very different selectivity from C18, reaching a high orthogonality of 93.2%. Moreover, such a combination settled compatibility issues because of the weaker hydrophobic retaining property of PTAS, thus allowing direct interfacing in online configuration. As a result of coupling with the mass spectrometry, a four-dimensional (4D) data plot was presented, in which 439 peaks (containing positive mode and negative mode) were counted, and 105 compounds were grouped and tentatively identified in C. kwangsiensis extract, including 73 unreported ones. Some novel types of compounds with masses exceeding 500 were discovered for the first time. Besides, compared to one-dimensional liquid chromatography (1DLC), the great resolution power of this system allowed separation of more isomers. These results provide supplementary to the material basis of C. Kwangsiensis and in-depth research should be conducted. The configuration of RPLC×RPLC-Q-TOF MS can be a powerful and efficient tool for separation and characterization of chemical substances in complicated herbal extracts. [ABSTRACT FROM AUTHOR]

In this study, “two dimensional” molecularly imprinted solid-phase extraction (2D-MIP-SPE) of semi-preparative grade was constructed to fast purify ellagitannins in pomegranate husk extract with the help of crystallization and reverse-phase liquid chromatgoraphy (RPLC). Ellagic acid and punicalagin imprinted polymers were synthesized in batch mode and two semi-preparative MIP-SPE columns were individually packed. After investigaing “functional complementation”, 2D-MIP-SPE was constructed using ellagic acid MIP and punicalagin MIP-SPE as the first and second dimension, respectively. Then, pomegranate husk extract was fast divided into four fractions individually enriching in ellagic acid, granatin A, punicalagin and ellagic acid glucoside by 2D-MIP-SPE. With the aid of crystallization and RPLC, ellagic acid (13.5 mg) and punicalagin (53.4 mg) were fast obtained in 30 min. Ellagic acid glucoside was purified to the purity near 100% with a recovery of 86.1%. Granatin A (92%) was directly obtained by 2D-MIP-SPE with the recovery of 81.8%. All above indicated that 2D-MIP-SPE was highly efficient in natural product purification. The concept of “functional complementation” was expected to be a useful tool in the construction of 2D-MIP-SPE. [ABSTRACT FROM AUTHOR]

A practical method was established for the definition of chromatographic parameters in preparative liquid chromatography. The parameters contained both the peak broadening level under different amounts of sample loading and the concentration distribution of the target compound in the elution. The parameters of the peak broadening level were defined and expressed as a matrix, which consisted of sample loading, the forward broadening and the backward broadening levels. The concentration distribution of the target compound was described by the heat map of the elution profile. The most suitable stationary phase should exhibit the narrower peak broadening and it was best to broaden to both sides to compare to the peak under analytical conditions. Besides, the concentration distribution of the target compounds should be focused on the middle of the elution. The guiding principles were validated by purification of amitriptyline from the mixture of desipramine and amitriptyline. On the selected column, when the content of the impurity desipramine was lower than 0.1%, the recovery of target compound was much higher than the other columns even when the sample loading was as high as 8.03 mg/cm3. The parameters and methods could be used for the evaluation and selection of stationary phases in preparative chromatography. [ABSTRACT FROM AUTHOR]

[Display omitted] • A comprehensive approach for characterization of saffron samples was developed. • In-depth identification of crocins was achieved via a targeted UPLC-ESI/MS n method. • Non-crocin components were identified based on a non-targeted UPLC-ESI/MS2 method. • 32 new crocins were identified and 6 structures were reported for the first time. • 91 non-crocin components were discovered, including 43 new compounds. As a valuable medicinal and edible plant, C rocus 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/MS n 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. [ABSTRACT FROM AUTHOR]

• A strategy combines AcquireX, neutral-loss-triggered MSn and mass defect filtering. • A total of 42 new isoflavone aglycones and 122 new glycosides were identified. • Modification of several new sugar moieties was discovered for the first time. • The material basis of Belamcandae Rhizoma was significantly enriched. • An effective strategy for the discovery of new isoflavones in TCM. 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-(4‑hydroxy-3,5-dimethoxybenzoyl)-β-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. [ABSTRACT FROM AUTHOR]

An interesting and novel method for the selective and sequential enrichment of singly- and multiply-phosphorylated peptides with a zwitterionic material “Click TE-Cys” is presented. Retention mechanisms between phosphopeptides and Click TE-Cys are systematically investigated by checking the influence of acetonitrile content, pH value, and buffer concentration on the retention of phosphopeptides. Both hydrophilic interaction and electrostatic interaction are involved in retention between phosphopeptides and Click TE-Cys. Based on these results, an optimized method is established for selective enrichment of phosphopeptides using Click TE-Cys. This method not only exhibits high selectivity for phosphopeptides, but also fractionates singly- and multiply-phosphorylated peptides into two fractions. This method was evaluated using relatively complex samples, including peptide mixtures of α-casein and bovine serum albumin (BSA) at a molar ratio of 1:10 and skim milk. This efficient and optimized protocol has great potential for enriching multiply-phosphorylated peptides and could be a valuable tool for specific enrichment of phosphopeptides in phosphoproteome analysis. [ABSTRACT FROM AUTHOR]

Expression levels of N-linked glycans derived from human serum glycoproteins have been shown to change during the progression of many diseases. Generally, N-glycans released from human serum proteins co-exist with endogenous serum peptides, salts, and other contaminants. Effective removal of these contaminants is essential to obtain the glycan profile of human serum proteins. Here, we developed a sample preparation method for mass spectrometry (MS) analysis of N-linked glycans derived from human serum glycoproteins based on a zwitterionic hydrophilic material named Click TE-Cys. The high hydrophilicity of Click TE-Cys, resulting from its unique surface structure and charge distribution, facilitated removal of co-existing salts and endogenous serum peptides. Furthermore, the present enrichment approach was handled in parallel, thus saving time. Using this method, a total of 47 unique N-glycans released from human serum proteins were identified. The intrabatch and interbatch coefficients of variation for the 47 N-linked glycans were 8.57% ± 0.96% and 9.22% ± 1.03%, respectively. These results demonstrate that the present method is suitable for fast purification of N-linked glycans derived from human serum glycoproteins, and has potential for clinical application. [ABSTRACT FROM AUTHOR]

Highlights: [•] A novel method was firstly developed for the extraction and cleanup of triptorelin from plasma. [•] The performance of the new sorbent Click XIon was excellent and satisfactory in terms of the matrix effect, recovery and overall process efficiency at the low, middle and high concentration levels of triptorelin. [•] The method was validated and successfully applied to determine triptorelin in rat plasma. [Copyright &y& Elsevier]

Highlights: [•] It is a new way to quickly separate and purify bufadienolides from toad skin. [•] A distinct separation selectivity was obtained by the positively charged C18 column. [•] The peak tailing problem was resolved and two kinds of bufadienolides were purified. [ABSTRACT FROM AUTHOR]

Highlights: [•] An efficient HILIC method was developed to separate isomeric saponins. [•] A Click XIon column with improved hydrophilicity and polar selectivity was used. [•] Methanol was the weak eluent, and symmetrical peak in high sample loading was obtained. [•] Eleven saponins were isolated and identified. [•] A new saponin has been isolated and identified. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Image 1 • An offline SFC × RPLC two-dimensional system was constructed for GLS analysis for the first time. • A GLS fast screening and automatic identification strategy was established by in-house library with self-developed software. • 79 gangliosides were tentatively identified in swine brain extract and 20 of them were new. [ABSTRACT FROM AUTHOR]