RESUMEN
A sensitive and efficient ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry(UHPLC-Q-TOF-MS) approach was established. Based on the self-developed information library, the chemical components from Euodiae Fructus were systematically characterized and identified. The chromatographic separation conditions(e. g., stationary phase,mobile phase, column temperature, and elution gradient) and MS detection conditions(nozzle voltage, capillary voltage, fragmentor,and collision energy) were optimized. Ultimately, an HSS T3 column(2. 1 mm×100 mm, 1. 8 µm) maintained at 35 â was used,and 0. 1% formic acid water-acetonitrile at the flow rate of 0. 4 m L·min~(-1) was used as the mobile phase. Electrospray ionization was adopted to collect the positive and negative ion mass spectrometry data in Auto MS/MS mode. According to the reference compound comparison, fragment ion information interpretation, literature, and retrieval in the self-developed information library, 92 compounds were characterized or derived from the decoction of Euodiae Fructus, including 33 alkaloids, 23 flavonoids, 12 terpenoids, 12phenylpropanoids, and 12 others. Among them, 17 compounds were identified by comparison with the reference compounds, and 11compounds were unreported from Euodiae Fructus. This study realizes the rapid characterization and identification of multi-class chemical components in the decoction of Euodiae Fructus and provides a reference for the studies regarding its effective substances and quality control.
Asunto(s)
Medicamentos Herbarios Chinos , Evodia , Frutas , Cromatografía Líquida de Alta Presión/métodos , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/análisis , Frutas/química , Evodia/química , Espectrometría de Masas/métodos , Espectrometría de Masas en Tándem/métodos , Estructura Molecular , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
This study was designed to comprehensively characterize and identify the chemical components in traditional Chinese medicine Psoraleae Fructus by establishing an ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry(UHPLC-Q-TOF-MS) method in combination with in-house library. The chromatographic separation conditions(stationary phase, column temperature, mobile phase, and elution gradient) and key MS monitoring parameters(capillary voltage, nozzle voltage, and fragmentor) were sequentially optimized via single-factor experiments. A BEH C_(18) column(2.1 mm×100 mm, 1.7 µm) was finally adopted, with the mobile phase consisting of 0.1% formic acid in water(A) and acetonitrile(B) at the flow rate of 0.4 mL·min~(-1) and column temperature of 30 â. Auto MS/MS was utilized for data acquisition in both positive and negative ion modes. By comparison with reference compounds, analysis of the MS~2 fragments, in-house library retrieval and literature research, 83 compounds were identified or tentatively characterized from Psoraleae Fructus, including 58 flavonoids, 11 coumarins, 4 terpenoid phenols, and 10 others. Sixteen of them were identified by comparison with reference compounds, and ten compounds may have not been reported from Psoraleae Fructus. This study achieved a rapid qualitative analysis on the chemical components in Psoraleae Fructus, which provided useful reference for elucidating its material basis and promoting the quality control.
Asunto(s)
Medicina Tradicional China , Espectrometría de Masas en Tándem , Cromatografía Líquida de Alta Presión , Ciclo Celular , CumarinasRESUMEN
Wenxin granule (WXG) is a popular traditional Chinese medicine (TCM) preparation for the treatment of arrhythmia disease. Potent analytical technologies are needed to elucidate its chemical composition and assess the quality differences among multibatch samples. In this work, both a multicomponent characterization and quantitative assay of WXG were conducted using two liquid chromatography-mass spectrometry (LC-MS) approaches. An ultra-high performance liquid chromatography-ion mobility quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS) approach combined with intelligent peak annotation workflows was developed to characterize the multicomponents of WXG. A hybrid scan approach enabling alternative data-independent and data-dependent acquisitions was established. We characterized 205 components, including 92 ginsenosides, 53 steroidal saponins, 14 alkaloids, and 46 others. Moreover, an optimized scheduled multiple reaction monitoring (sMRM) method was elaborated, targeting 24 compounds of WXG via ultra-high performance liquid chromatography-triple quadrupole linear ion trap mass spectrometry (UHPLC/QTrap-MS), which was validated based on its selectivity, precision, stability, repeatability, linearity, sensitivity, recovery, and matrix effect. By applying this method to 27 batches of WXG samples, the content variations of multiple markers from Notoginseng Radix et Rhizoma (21) and Codonopsis Radix (3) were depicted. Conclusively, we achieved the comprehensive multicomponent characterization and holistic quality assessment of WXG by targeting the non-volatile components.
Asunto(s)
Ginsenósidos , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Liquida , Medicamentos Herbarios Chinos , Ginsenósidos/análisis , Espectrometría de Masas/métodosRESUMEN
Comprehensive characterization of the chemical constituents of Chinese patent medicine poses a great challenge due to the frustrating complexity resulting from superposition of multiple drugs. Lanqin Oral Liquid is a five-component Chinese patent medicine widely applied to treat pharyngeal inflammation in clinic. Here, we streamline a universal three-dimensional separation approach to efficiently identify the multicomponents from Lanqin Oral Liquid by ultra-high-performance liquid chromatography/ion mobility quadrupole time-of-flight mass spectrometry and UNIFI/in-house library-driven automatic peak annotation. Because of the systematic optimization, the use of an HSS T3 column enabled good separation of the multiple components within 42 min, while high-definition MSE in both the negative and positive modes could characterize more classes of herbal components, thus providing the retention, collision cross-section, and MS information for each component. Benefiting from the ion mobility separation, cleaner MS1 and MS2 spectra were acquired. Aided by comparison and analysis of the fragmentation pathways of 49 reference compounds, we could characterize 175 compounds from Lanqin Oral Liquid. A validated high-performance liquid chromatography fingerprinting approach unveiled good similarity (0.985-1.000) among 22 batches of commercial samples. Conclusively, we demonstrated a practical solution to elucidating the chemical composition of Chinese patent medicines, with the potential of popularization.
Asunto(s)
Medicamentos Herbarios Chinos/química , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Movilidad Iónica/métodos , Medicina Tradicional China , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
INTRODUCTION: Post-stroke cognitive impairment is one of the major causes of disability due to cerebral ischemia. MAD2B is an inhibitor of Cdh1/APC, and loss of Cdh1/APC function in mature neurons increases ROCK2 activity, leading to changes in synaptic plasticity and memory loss in mouse neurons. Whether MAD2B regulates learning memory capacity through ROCK2 in cerebral ischemia is not known. OBJECTIVES: We investigated the role and mechanism of MAD2B in cerebral ischemia-induced cognitive dysfunction. METHODS: The expression of MAD2B and its downstream related molecules was detected by immunoblotting and intervened with neuroprotectants after middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation/reoxygenation (OGD/R). We constructed MAD2B-cKO-specific knockout mice, knocked down and overexpressed MAD2B in mouse hippocampus by lentiviral injection in brain stereotaxis, modeled cerebral ischemia by using MCAO, and explored the role of MAD2B in post-stroke cognitive impairment (PSCI) by animal behaviors such as Y-maze and Novel object recognition test. Then the expression of MAD2B/ROCK2, downstream molecules and apoptosis-related molecules was detected. Finally, ROCK2 expression was intervened using its inhibitor and shRNA-ROCK2 lentivirus. RESULTS: The expression of MAD2B and its downstream molecules increased after MCAO and OGD/R. Nonetheless, this expression underwent a decline post-therapy with neuroprotective agents. Deletion of MAD2B in the hippocampus ameliorated memory and learning deficits and improved motor coordination in MCAO mice. Conversely, the overexpression of MAD2B in the hippocampus exacerbated learning and memory deficits. Deletion of MAD2B resulted in the downregulation of ROCK2/LIMK1/cofilin. It effectively reduced ischemia-induced upregulation of BAX and cleaved caspase-3, which could be reversed by MAD2B overexpression. Inhibition or knockdown of ROCK2 expression in primary cultured neurons led to the downregulation of LIMK1/cofilin expression and reduced the expression of apoptosis-associated molecules induced by ischemia. CONCLUSIONS: Our findings suggest that MAD2B affects neuronal apoptosis via Rock2, which affects neurological function and cerebral infarction.
RESUMEN
Challenges encountered in plant metabolites characterization by liquid chromatography/mass spectrometry can arise from the insufficient chromatography separation, the lack of specific database, and low reliability of identification because of the ubiquitous isomerism. Herein, we present an integral approach, by combining comprehensive off-line two-dimensional liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (2D-LC/IM-QTOF-MS), automatic peak annotation, molecular networking, and collision cross section (CCS) prediction, aimed to improve the resolution and reliability in MS-oriented metabolites characterization. Using the seeds of Cuscuta chinensis as a case, the configuration of an XBridge Amide column of hydrophilic interaction chromatography (HILIC) and a Zorbax SB-Aq column of reversed-phase chromatography (RPC), in an off-line mode, showed the orthogonality of 0.73 and effective peak capacity of 4361. Data-independent high-definition MSE (HDMSE) in the negative mode could enable high-coverage MS2 data acquisition and enhance the ions resolution, while computational peak annotation workflows facilitated by UNIFITM and Global Natural Products Social Molecular Network (GNPS) could efficiently characterize the targeted and untargeted compound analogs. A total of 302 compounds were identified or tentatively characterized, and 109 thereof were unreported. Moreover, CCS prediction (www.allccs.zhulab.cn) provided more possibilities to distinguish 12 pairs of isomers in the lack of reference standards. The 2D-LC/IM-QTOF-MS approach enabled the collection of five dimension of data related to each component (tR by HILIC and RPC, CCS, m/z in MS1 and MS2), and the intelligent metabolites characterization with more reliable MS data. Conclusively, the established integral strategy can be utilized in metabolome analysis to support the quality control of herbal medicines.
Asunto(s)
Cuscuta , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Liquida , Espectrometría de Masas/métodos , Reproducibilidad de los ResultadosRESUMEN
Liquid chromatography/mass spectrometry (LC/MS) is extensively applied for the untargeted/targeted analyses of the herbal components, utilizing data-dependent acquisition (DDA) or data-independent acquisition (DIA) to record the fragmentation information useful for the structural elucidation. A new trend recently has emerged by integrating DDA and DIA to render the hybrid scan, which, unfortunately, has rarely been reported. Herein, by using the Vion™ ion-mobility quadrupole time-of-flight mass spectrometer, a hybrid scan strategy (HDMSE-HDDDA) was presented and validated by the untargeted characterization of the multicomponents from Carthamus tinctorius (safflower), in combination with reversed-phase ultra-high performance liquid chromatography (RP-UHPLC). Good chromatographic separation was achieved on an HSS T3 column within 26 min, while HDMSE-MS/MS was used to acquire the collision-induced dissociation MS2 data in the negative mode. Automatic workflows (e.g., data correction, precursors/product ions matching, and peak annotation) were well established on UNIFI™ (incorporating an in-house library recording 261 known compounds) to process the obtained MS2 data. Compared with single DDA or DIA, the hybrid approach of HDMSE-HDDDA better balanced between the coverage and reliability, led to high-definition MS spectra, offered useful collision cross section (CCS) information, and showed satisfactory identification performance comparable to MSE. A total of 141 components (involving 41 quinochalcones, 66 flavanols/flavones, 11 flavanones, 6 organic acids, 1 polyacetylene, and 16 others) were characterized from safflower. Moreover, CCS prediction could assist isomers characterization, to some extent. Conclusively, this hybrid scan approach enables a dimension-enhanced MS data acquisition strategy providing the complementary structural information, which more suits the chemical characterization of complex samples.
Asunto(s)
Carthamus tinctorius , Cromatografía Líquida de Alta Presión/métodos , Iones , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodosRESUMEN
Hypochlorous acid (HClO) is a special kind of reactive oxygen species, which plays an important role in resisting pathogen invasion and maintaining cell redox balance and other physiological processes. In addition, HClO is commonly used in daily life as a bleaching and disinfectant agent. Its excessive use can also lead to death of water animals and serious respiratory and skin diseases in humans. Therefore, it is of great significance to develop a quick and convenient tool for detecting HClO in the environment and organisms. In this paper, we utilize the specific reaction of HClO with dimethylthiocarbamate to develop a novel naphthalene derivative fluorescent probe (BNA-HClO), it was designed and synthesized by using 6-(2-benzothiazolyl)-2-naphthol as the fluorophore and N,N-dimethylthiocarbamate as the recognition group. BNA-HClO shows large fluorescence enhancement (374-fold), high sensitivity (a detection limit of 37.56 nM), rapid response (<30 s), strong anti-interference ability and good specificity in vitro. Based on the outstanding in vitro sensing capability of BNA-HClO, it has been successfully used to detect spiked HClO in tap water, medical wastewater and fetal bovine serum with good recovery. BNA-HClO has also been successfully used as a portable test strip for the in situ semi-quantitative detection of HClO in tap water solutions. In addition, BNA-HClO can successfully enable the detection and imaging of exogenous and endogenous HClO in living cells. This work provides a simple and effective tool for the detection and imaging of HClO in environmental and biological systems, and provides some theoretical guidance for future exploration of biological and pathological studies related to HClO.
RESUMEN
Selenocysteine (Sec) is recognized as the 21st amino acid employing as an essential building block for selenoproteins (SePs), which plays a significant role in various physiological processes. Therefore, there is an urgent need to reasonably develop some reliable and rapid methods for Sec detection in biological systems. In this work, we reported a new two-photon (TP) fluorescent probe BNT-Sec for Sec detection and imaging in living cells and zebrafish with two part: (1) a D-π-A-structured naphthalene derivative as a TP fluorophore; (2) a well-know Sec responsive site with strong intromolecular charge transfer effect (ICT) to selectively detect endogenous and exogenous. In the presence of Sec, probe BNT-Sec can initiate a Se-dependent specific aromatic nucleophilic substitution reaction, which exhibited BNT-Sec had a large fluorescence intensity enhancement with ~18.9-fold at 510 nm, a high sensitivity low LOD value' 10.6 nM, good light stability, strong specificity, pH stability and low cytotoxicity. In addition, BNT-Sec can be conveniently used to detect Sec in living cells and zebrafish for TP imaging due to the great TP measurement properties of fluorophore, exhibiting it has the potential to reveal the role of selenocysteine in physiological and pathological processes in further biological applications.
Asunto(s)
Colorantes Fluorescentes , Selenocisteína , Animales , Células HeLa , Humanos , Imagen Óptica , Fotones , Pez CebraRESUMEN
Herbs represent complex chemical systems involving various primary and secondary metabolites that are featured by large spans of acid-base property, polarity, molecular mass, and content, etc., which thus poses great challenges to characterize the metabolites contained. Here, the combination of multiple-mechanism chromatography coupled with improved data-dependent-MS2 acquisition (DDA-MS2) is presented as a strategy to support the deep metabolites characterization. Targeting Uncaria sessilifructus, a reputable medicinal herb containing alkaloids and triterpenic acids (TAs) as the main pharmacologically bioactive ingredients, a three-dimensional liquid chromatography (3D-LC) system was established by integrating ion exchange chromatography, hydrophilic interaction chromatography, and reversed-phase chromatography (IEC-HILIC-RPC). The first-dimensional chromatography, configuring a PhenoSphere SCX column eluted by methanol/20 mM ammonium acetate-0.05% formic acid in water, could well fractionate the total extract into two fractions (unretained ingredients and alkaloids). The subsequent HILIC using an XAmide column and RPC by a CSH Phenyl-Hexyl column achieved the sufficient resolution of the total TAs and total alkaloids, respectively. A polarity-switching precursor ions list-including DDA approach by Q-Orbitrap-MS enabled the high-efficiency, coverage-enhanced identification of alkaloids and TAs. This 3D-LC/Q-Orbitrap-MS system was validated as precise (RSD < 5% for intra-day/inter-day precision), Up to 308 components were separated from U. sessilifructus, and 128 thereof (including 85 alkaloids, 29 TAs, and 14 others) were identified or tentatively characterized, exhibiting superiority over the conventional one-dimensional LC/MS. Conclusively, 3D-LC/MS in an off-line mode can facilitate the flexible configuration of multiple chromatography to accomplish the fit-for-purpose characterization of the metabolites from an herbal extract or a biosample.
Asunto(s)
Cromatografía Liquida/métodos , Medicamentos Herbarios Chinos/química , Espectrometría de Masas/métodos , Uncaria/química , Alcaloides/análisis , Interacciones Hidrofóbicas e Hidrofílicas , Peso Molecular , Triterpenos/análisisRESUMEN
Despite the extensive consumption of ginseng, precise quality control of different ginseng products is highly challenging due to the containing of ginsenosides in common for different Panax species or different parts (e.g. root, leaf, and flower) of a same species. Herein we performed a comparative investigation of diverse ginseng products by simultaneously assaying 15 saponins (notoginsenoside R1, ginsenosides Rg1, -Re, -Rf, -Ra2, -Rb1, -Rc, -Ro, -Rb2, -Rb3, -Rd, 20(R)-ginsenoside Rg3, 24(R)-pseudoginsenoside F11, chikusetsusaponins IV, and -IVa) using an ultra-high-performance liquid chromatography/charged aerosol detector (UHPLC-CAD) approach. Twelve Panax-derived ginseng products (involving P. ginseng root, P. quinquefolius root, P. notoginseng root, Red ginseng, P. ginseng leaf, P. quinquefolius leaf, P. notoginseng leaf, P. ginseng flower, P. quinquefolius flower, P. notoginseng flower, P. japonicus root, and P. japonicus var. major root) were considered. Benefiting from the condition optimization, the baseline resolution of 15 ginsenosides was achieved on a CORTECS UPLC Shield RP18 column. This method was validated as specific, precise (0.81-1.94% intra-day variation; 0.86-2.35% inter-day variation), and accurate (recovery: 90.73-107.5%), with good linearity (R2 > 0.999), high sensitivity (limit of detection: 0.02-0.21 µg; limit of quantitation: 0.04-0.42 µg) and sample stability (1.49-4.74%). Its application to 119 batches of ginseng samples unveiled vital information enabling the authentication of these different ginseng products. Detection of ginsenosides by CAD exhibited superiority over UV in sensitivity and the ability to monitor chromophore-free structures. Large-scale comparative studies by quantifying multiple markers provide methodological reference to the precise quality control of herbal medicine.