Fine-Scale Characterization of Plant Diterpene Glycosides Using Energy-Resolved Untargeted LC-MS/MS Metabolomics Analysis.

Plant diterpene glycosides are essential for diverse physiological processes. Comprehensive structural characterization proved to be a challenge due to variations in glycosylation patterns, diverse aglycone structures, and the absence of comprehensive reference databases. In this study, a method for fine-scale characterization was proposed based on energy-resolved (ER) untargeted LC-MS/MS metabolomics analysis using steviol glycosides as a demonstration. Energy-dependent fragmentation patterns were unveiled by a series of model compounds. Distinct glycosylation sites were discerned by leveraging varying fragmentation energies for the precursor ions. The sugar moiety linkage at C19OOH (R1) exhibited facile and intact cleavage at low collision energies, while the sugar moiety at C13-OH (R2) demonstrated consecutive cleavage with increasing energy. Aglycone ions exhibited a higher relative intensity at NCE 50, with relative intensities ranging from 95% to 100%. Subsequently, aglycone candidates, R1 sugar composition, and R2 sugar sequence were deduced through ER-MS/MS analysis. The developed method was applied to Stevia rebaudiana leaves. A total of 91 diterpene glycosides were unambiguously identified, including 16 steviol glycosides with novel acetylglycosylation patterns. This method offers a rapid alternative for glycan analysis and the structural differentiation of isomers. The developed method enhances the understanding of diterpene glycosides in plants, providing a reliable tool for the in-depth characterization of complex metabolite profiles.

Alterations in the gut microbiome and metabolome profiles of septic mice treated with Shen FuHuang formula.

Sepsis has a high mortality rate, and treating sepsis remains a significant challenge worldwide. In former studies, our group found that traditional Chinese medicine, Shen FuHuang formula (SFH), is a promising medicine in treating coronavirus disease 2019 (COVID-19) patients with the septic syndrome. However, the underlying mechanisms remain elusive. In the present study, we first investigated the therapeutic effects of SFH on septic mice. To investigate the mechanisms of SFH-treated sepsis, we identified the gut microbiome profile and exploited untargeted metabolomics analyses. The results demonstrated that SFH significantly enhanced the mice's 7-day survival rate and hindered the release of inflammatory mediators, i.e., TNF-α, IL-6, and IL-1ß. 16S rDNA sequencing further deciphered that SFH decreased the proportion of Campylobacterota and Proteobacteria at the phylum level. LEfSe analysis revealed that the treatment of SFH enriched Blautia while decreased Escherichia_Shigella. Furthermore, serum untargeted metabolomics analysis indicated that SFH could regulate the glucagon signaling pathway, PPAR signaling pathway, galactose metabolism, and pyrimidine metabolism. Finally, we found the relative abundance of Bacteroides, Lachnospiraceae_NK4A136_group, Escherichia_Shigella, Blautia, Ruminococcus, and Prevotella were closely related to the enrichment of the metabolic signaling pathways, including L-tryptophan, uracil, glucuronic acid, protocatechuic acid, and gamma-Glutamylcysteine. In conclusion, our study demonstrated that SFH alleviated sepsis by suppressing the inflammatory response and hence reduced mortality. The mechanism of SFH for treating sepsis may be ascribed to the enrichment of beneficial gut flora and modulation in glucagon signaling pathway, PPAR signaling pathway, galactose metabolism, and pyrimidine metabolism. To sum up, these findings provide a new scientific perspective for the clinical application of SFH in treating sepsis.

Phosphorus-Containing Polybenzoxazine Aerogels with Efficient Flame Retardation and Thermal Insulation.

Bisphenol A type benzoxazine (Ba) monomers and 10-(2, 5-dihydroxyphenyl)-10- hydrogen-9- oxygen-10- phosphine-10- oxide (DOPO-HQ) were employed to prepare flame retardant and heat insulated polybenzoxazine (PBa) composite aerogels. The successful preparation of PBa composite aerogels was confirmed by Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM). The thermal degradation behavior and flame-retardant properties of the pristine PBa and PBa composite aerogels were investigated with thermogravimetric analysis (TGA) and cone calorimeter. The initial decomposition temperature of PBa decreased slightly after incorporating DOPO-HQ, increasing the char residue amount. The incorporation of 5% DOPO-HQ into PBa led to a decrease of 33.1% at the peak of the heat-release rate and a decrease of 58.7% in the TSP. The flame-retardant mechanism of PBa composite aerogels was investigated by SEM, Raman spectroscopy, and TGA coupled with infrared spectrometry (TG-FTIR). The aerogel has advantages such as a simple synthesis procedure, easy amplification, lightweight, low thermal conductivity, and good flame retardancy.

Novel Method for Comprehensive Annotation of Plant Glycosides Based on Untargeted LC-HRMS/MS Metabolomics.

Glycosides are a large family of secondary metabolites in plants, which play a critical role in plant growth and development. Due to the complexity and diversity in structures and the limited availability of authentic standards, comprehensive annotation of the glycosides remains a great challenge. In this study, using maize as an example, a deep annotation method of glycosides was proposed based on untargeted liquid chromatography-high-resolution tandem mass spectrometry metabolomics analysis. First, knowledge-based in silico aglycone and glycosyl/acyl-glycosyl libraries were built. A total of 1240 known and potential aglycones from databases and literature were recorded. Next, the MS parameters beneficial to aglycone ion-rich MS/MS were explored using 1782 high-resolution MS/MS spectra of glycosides from the MassBank of North America (MoNA) and confirmed by 52 authentic glycoside standards. Then, screening rules for aglycon ions in MS/MS were recommended. Glycoside candidates were further filtered by MS/MS-based chemical classification and MS/MS similarity of aglycon-glycoside pairs. Finally, the glycosylation sites of flavonoid mono-O-glycosides were recommended by characteristic fragmentation patterns. The developed method was validated using glycosides and nonglycosides from the MoNA library. The annotation accuracy rates were 96.8, 94.9, and 98.0% in negative ion mode (ESI-), positive ion mode (ESI+), and the combined ESI- & ESI+, respectively. The annotation specificity was 99.6% (ESI-), 99.6% (ESI+), and 99.2% (ESI- & ESI+). A total of 274 glycosides (including 34 acyl-glycosides) were tentatively annotated in maize by the developed method. The method enables effective and reliable annotation for plant glycosides.

Qin-Qiao-Xiao-Du formula alleviate influenza virus infectious pneumonia through regulation gut microbiota and metabolomics.

Qin-Qiao-Xiao-Du (QQXD), a traditional Chinese medicine (TCM) formula, has been used in the clinical treatment of influenza virus pneumonia. However, the effects and mechanisms of QQXD on influenza virus pneumonia remain unknown. Therefore, this study explores the mechanisms of QQXD in the treatment of influenza virus pneumonia from the point of view of intestinal flora and metabolism. The results showed that QQXD was able to reduce mortality, weight loss, lung viral load, lung index, and lung injury in influenza virus mice. A cytokine array found that the QQXD attenuated the expression of serum IL-1α, IL-4, IL-12(P70), and TNF-α. Subsequently, 16s rRNA gene sequencing showed that QQXD could increase the relative abundances of Gemmiger, Anaerofustis, Adlercreutzia, and Streptococcus and decrease those of Dehalobacteriu, Burkholderia, Prevotella, Butyrimimonas, Delftia, and others. Meanwhile, targeted metabolic profiling analysis showed that QQXD could regulate nitrogen metabolism, phenylalanine metabolism, valine, leucine, and isoleucine biosynthesis. Correlation analysis demonstrated that the regulatory effect of QQXD on the cyanoamino acid metabolism pathway was associated with changes in the abundance of Parabacteroides, Pediococcus, and Clostridium in influenza mice. In conclusion, our study revealed that QQXD can inhibit influenza virus replication, suppress cytokine storms, and protect mice from influenza virus infection pneumonia. The mechanisms are likely to be related to improved gut microbiota dysbiosis, increased intestinal carbohydrate metabolism, and up-regulated cyanoamino acid metabolism pathways.

[Systematic review and Meta-analysis of Lianhua Qingwen preparations combined with Oseltamivir in treatment of influenza].

This study aims to explore the efficacy and safety of Lianhua Qingwen preparations combined with Oseltamivir in the treatment of influenza patients. PubMed, Cochrane Library, EMbase, SinoMed, CNKI, Wanfang, and VIP were searched for the randomized controlled trials(RCTs) involving the comparison between the influenza patients treated with Lianhua Qingwen preparations combined with Oseltamivir and those treated with Oseltamivir alone. Fever clearance time was taken as the primary outcome indicator. Clinical effective rate(markedly effective and effective), time to muscle pain relief, time to sore throat relief, time to cough relief, time to nasal congestion and runny nose relief, time to negative result of viral nucleic acid test, and adverse reactions were taken as the secondary outcome indicators. The data were extracted based on the outcome indicators and then combined. The Cochrane collaboration's tool for assessing risk of bias was used to evaluate the quality of a single RCT, and the grading of recommendations assessment, development and evaluations(GRADE) system to assess the quality of a single outcome indicator. RevMan 5.3 was employed to analyze data and test heterogeneity. Finally, 16 RCTs involving 1 629 patients were included for analysis. The Meta-analysis showed that Lianhua Qingwen preparations combined with Oseltamivir was superior to Oseltamivir alone in the treatment of influenza in terms of clinical effective rate(RR=1.16, 95%CI [1.12, 1.20], P<0.000 01), fever clearance time(SMD=-2.02, 95%CI [-2.62,-1.41], P<0.000 01), time to muscle pain relief(SMD=-2.50, 95%CI [-3.84,-1.16], P=0.000 2), time to sore throat relief(SMD=-1.40, 95%CI [-1.93,-0.85], P<0.000 01), time to cough relief(SMD=-1.81, 95%CI [-2.44,-1.19], P<0.000 01), time to nasal congestion and runny nose(SMD=-2.31, 95%CI [-3.61,-1.01], P=0.000 5), and time to negative result of viral nucleic acid test(SMD=-0.68, 95%CI [-1.19,-0.16], P=0.01). However, due to the low quality of the trials, the above conclusions need to be proved by more high-quality clinical studies. In addition, we still need to attach importance to the adverse reactions of the integrated application of Chinese and western medicines.

In-depth characterization of nitrogen heterocycles of petroleum by liquid chromatography-energy-resolved high resolution tandem mass spectrometry.

With the increased attention to processing heavy crude oils, a detailed description of chemical composition is critical for the petroleum refining industry. The current analytical technique such as ultrahigh resolution mass spectrometry has been successfully applied for the molecular level characterization of complex petroleum fractions. But the structural characterization of heavy petroleum feedstock is still a great challenge. In this study, a novel in-depth characterization method of nitrogen heterocycles (N-heterocycles) in heavy petroleum mixtures was proposed by online liquid chromatography coupled with electrospray ionization high resolution energy-resolved mass spectrometry. A series of typical basic aromatic, neutral aromatic and naphtheno-aromatic nitrogen heterocyclic model compounds were synthesized to investigate energy-resolved fragmentation behaviors in high energy collision-induced dissociation at 10-100 eV. Energy-dependent fragmentation pathways were elucidated. Notably, characteristic double bond equivalent (DBE) versus carbon number distributions of N1 ions and all CH ions were discovered, which were closely related to their core structure. Then a workflow to assign core structures of alkyl-substituted N-heterocycles in petroleum was proposed and validated. The developed method was applied to investigate the structural isomers in feed and product vacuum gas oil (VGO) fractions. Core structural differences in feed VGO and subtle structural variations between feed and product VGOs were recognized. This work can distinguish structural isomers of N-heterocycles with the subtle difference in their core structure in heavy petroleum fractions based on global energy dimensional fragmentation characteristics.

A data processing pipeline for petroleomics based on liquid chromatography-high resolution mass spectrometry.

Online liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) has attracted much attention in the molecular characterization of crude oil. Neither open access nor commercially available petroleomics tools were developed specifically to process LC-HRMS data. Here, a novel data processing pipeline was specifically designed for LC-HRMS-based petroleomics data. A customizable formula database was established deriving from the detected sample, which could avoid the interference caused by a large number of redundant molecules in a conventionally theoretical molecular database. Molecular formula candidates were assigned by the formula database using a low noise threshold, and false-positive assignments were eliminated by the chromatographic retention behaviors. Multi-dimensional information was obtained, including heteroatom class, double bond equivalent (DBE), carbon number, retention time, and MS/MS spectra. The developed method was compared with a popular petroleomics software, similar relative abundance class distribution was obtained, and much more formulas of low abundant components were uniquely extracted by the developed method. Finally, it was applied to reveal variation between feed and product oils in hydrodenitrogenation. Significantly compositional and structural differences were revealed. The developed method provides a useful pipeline for molecular data mining of petroleum samples.

Liu Shen Capsule Alters Airway Microbiota Composition and Metabolite Profiles in Healthy Humans.

Alteration in airway microbiota composition and perturbations in microbe-metabolites interactions have been proposed as markers of many diseases. Liu Shen (LS) capsule, a traditional Chinese medicine, was proved as favorable in treating respiratory diseases. However, the effects of the LS capsule in terms of regulating human microorganisms and metabolite profiles are not well known. This study aimed to define and compare the respiratory microbiota composition and circulating and fecal metabolite profiles before and after LS capsule administration. A total of 30 healthy volunteers were recruited. The pharyngeal swab samples were collected for 16S rRNA gene sequencing. The serum and fecal samples were collected to analyze the non-targeted ultra-performance liquid chromatography-tandem mass spectrometry metabolomics. The airway microbial compositions were profoundly altered after LS capsule administration, as evidenced by increased microbial diversity and altered microbial taxa distribution. The increasing abundance of bacterial Bifidobacteria, and Lactobacillus characterized the after-administration groups, and the increasing of abundance bacterial Proteobacteria, Veillonella, Prevotella, Neisseria, and Actinomyces characterized the before-administration groups. Significant discriminations were observed in both serum and fecal metabolic profiles between the before- and after-administration groups. A total number of 134 and 71 significant HMDB taxonomic metabolites including glycerophospholipids, fatty acyls, and prenol lipids in the serum and fecal samples were identified respectively between the before- and after-administration groups. The integrated analysis showed that some altered airway microbiota phylum, such as Bacteroidetes and Proteobacteria, significantly correlated with metabolites in serum and fecal. Hence, our study reported the alternations in the composition and functions of the airway microbial community and the changes in circulating and fecal metabolite profiles after LS capsule administration in healthy humans, thus providing a novel insight into the mechanisms underlying the role of LS capsule treating and preventing related diseases.

Omega-3 fatty acid prevents the development of heart failure by changing fatty acid composition in the heart.

Some clinical trials showed that omega-3 fatty acid (FA) reduced cardiovascular events, but it remains unknown whether omega-3 FA supplementation changes the composition of FAs and their metabolites in the heart and how the changes, if any, exert beneficial effects on cardiac structure and function. To clarify these issues, we supplied omega-3 FA to mice exposed to pressure overload, and examined cardiac structure and function by echocardiography and a proportion of FAs and their metabolites by gas chromatography and liquid chromatography-tandem mass spectrometry, respectively. Pressure overload induced cardiac hypertrophy and dysfunction, and reduced concentration of all FAs' components and increased free form arachidonic acid and its metabolites, precursors of pro-inflammatory mediators in the heart. Omega-3 FA supplementation increased both total and free form of eicosapentaenoic acid, a precursor of pro-resolution mediators and reduced free form arachidonic acid in the heart. Omega-3 FA supplementation suppressed expressions of pro-inflammatory cytokines and the infiltration of inflammatory cells into the heart and ameliorated cardiac dysfunction and fibrosis. These results suggest that omega-3 FA-induced changes of FAs composition in the heart have beneficial effects on cardiac function via regulating inflammation.

Zero-fluoroscopy approach for ablation of supraventricular tachycardia using the Ensite NavX system: a multicenter experience.

BACKGROUND: Three-dimensional electroanatomic mapping systems have demonstrated a significant reduction in radiation exposure during radiofrequency catheter ablation procedures. We aimed to investigate the safety, feasibility and efficacy of a completely zero-fluoroscopy approach for catheter ablation of supraventricular tachycardia using the Ensite NavX navigation system compared with a conventional fluoroscopy approach. METHODS: A multicenter prospective non-randomized registry study was performed in seven centers from January 2013 to February 2018. Consecutive patients referred for catheter ablation of supraventricular tachycardia were assigned either to a completely zero-fluoroscopic approach (ZF) or conventional fluoroscopy approach (CF) according to the operator's preference. Patients with atrial tachycardia were excluded. RESULTS: Totally, 1020 patients were enrolled in ZF group; 2040 patients ablated by CF approach were selected for controls. There was no significant difference between the zero-fluoroscopy group and conventional fluoroscopy group as to procedure time (60.3 ± 20.3 vs. 59.7 ± 22.6 min, P = 0.90), immediate success rate of procedure (98.8% vs. 99.2%, P = 0.22), arrhythmia recurrence (0.4% vs. 0.5%, P = 0.85), total success rate of procedure (98.4% vs. 98.8%, P = 0.39) or complications (1.1% vs. 1.5%, P = 0.41). Compared with the conventional fluoroscopy approach, the zero-fluoroscopy approach provided similar outcomes without compromising the safety or efficacy of the procedure. CONCLUSION: The completely zero-fluoroscopy approach demonstrated safety and efficacy comparable to a conventional fluoroscopy approach for catheter ablation of supraventricular tachycardia, and mitigated radiation exposure to both patients and operators. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT03042078; first registered February 3, 2017; retrospectively registered.

A Strategy for Selecting "Q-Markers" of Chinese Medical Preparation via Components Transfer Process Analysis with Application to the Quality Control of Shengmai Injection.

Chinese medical preparation has complicated chemical constituents. Consequently, the proper quality control methods for these Chinese medical preparations have been great challenges to the traditional Chinese medicine modernization and internationalization. What components should be chosen for quality control is a big challenge in the development of traditional Chinese medicine. A new concept of "Quality Marker" was proposed by Liu et al. to solve this problem and established a new research paradigm for traditional Chinese medicine quality study. Several strategies were proposed by the researchers in traditional Chinese medicine, here, we used Shengmai injection as an example to discuss a strategy for selecting "Quality Markers" of Chinese medical preparation by the components transfer process analysis in the Shengmai injection manufacturing process. Firstly, a total of 87 compounds were identified or partially characterized in shengmai injection. Secondly, referenced to the quality control method in China pharmacopeia and considered the biomarkers in the original medicines and representative components in the manufacturing process, four ginsenosides in Panax ginseng (Hongshen), two compounds in Schisandra chinensis (Wuweizi), and a sugar from Ophiopogon japonicas (Maidong) were quantified. As a result, these seven representative compounds exhibited an acceptable transitivity throughout the Shengmai injection manufacturing process. Finally, combined with the active ingredients, components transfer process analysis, and comprehensive evaluation by "Spider-web" analysis, six compounds were selected as the quality markers for the quality control of Shengmai injection. Through this strategy of optimization for quality markers of Shengmai injection, we found that these six compounds could represent the main bioactive substances and be easily detected in the whole process of production. Furthermore, the quality control method was developed for quality assessment and control of these six quality markers in the Shengmai injection. The total content range of the selected quality markers in the 10 batches of the Shengmai injection is 13.844-22.557 mg/mL.

Microfluidic formation of core-shell alginate microparticles for protein encapsulation and controlled release.

Alginate hydrogel particles are promising delivery systems for protein encapsulation and controlled release because of their excellent biocompatibility, biodegradability, and mild gelation process. In this study, a facile microfluidic approach is developed for making uniform core-shell hydrogel microparticles. To address the challenge of protein retention within the alginate gel matrix, poly(ethyleneimine) (PEI)- and chitosan-coated alginate microparticles were fabricated demonstrating improved protein retention as well as controlled release. Furthermore, a model protein ovalbumin was loaded along with delta inulin microparticulate adjuvant into the water-core of the alginate microparticles. Compared to those microparticles with only antigen loaded, the antigen + adjuvant loaded microparticles showed a delayed and sustained release of antigen. This microfluidic approach provides a convenient method for making well-controlled alginate microgel particles with uniform size and controlled properties, and demonstrates the ability to tune the release profiles of proteins by engineering microparticle structure and properties.

A partially purified outer membrane protein VirB9-1 for low-cost nanovaccines against Anaplasma marginale.

Anaplasma marginale is a devastating tick-borne pathogen causing anaplasmosis in cattle and results in significant economic loss to the cattle industry worldwide. Currently, there is no widely accepted vaccine against A. marginale. New generation subunit vaccines against A. marginale, which are much safer, more efficient and cost-effective, are in great need. The A. marginale outer membrane protein VirB9-1 is a promising antigen for vaccination. We previously have shown that soluble recombinant VirB9-1 protein can be expressed and purified from Escherichia coli and induce a high level of humoral and cellular immunity in mice. In this study, we re-formulated the nanovaccines using the partially-purified VirB9-1 protein as the antigen and hollow nano-size silica vesicles (SV-100) as the adjuvant. We simplified the purification method to obtain the partially-purified antigen VirB9-1 with a six-fold higher yield. The new formulations using the partially-purified VirB9-1 protein achieved higher antibody and cell-mediated immune responses compared to the purified ones. This finding suggests that the partially-purified VirB9-1 protein performs better than the purified ones in the vaccination against A. marginale, and a certain level of contaminants in the protein antigen can be self-adjuvant and boost immunogenicity together with the nanoparticle adjuvant. This may lead to finding a "Goldilocks" level of contaminants. The new nanovaccine formulation using partially-purified antigens along with nanoparticle adjuvants offers an alternative strategy for making cheaper veterinary vaccines.

Toxicity evaluation of wastewater collected at different treatment stages from a pharmaceutical industrial park wastewater treatment plant.

The toxicity of water-receiving bodies, the effluent and other treatment stages in wastewater treatment plants has recently been of interest to the public due to the lack of a regulated toxicity-based index for wastewater discharge in China. This study aimed to evaluate the conventional pollution parameters and toxicities of wastewaters collected at different treatment stages from a pharmaceutical industrial park wastewater treatment plant through dehydrogenase activity (DHA) and bioluminescent bacteria (Vibrio qinghaiensis) tests. The results of an analysis of conventional parameters indicated that the total suspended solids (TSS), chemical oxygen demand (COD), total nitrogen (TN), ammonia nitrogen (NH3N), and total phosphorus (TP) were largely removed after various treatments. However, the TN, NH3N and COD still exceeded the regulated standards. The tested pharmaceutical park effluents were mainly polluted with organic pollutants and nitrogenous. The toxicity test results indicated that the toxicities could be markedly reduced after treatment, with the toxicities of two out of the six effluent samples at different treatment stages being greater than the influent toxicity. Spearman's rank correlation coefficients indicated a significantly positive correlation between the toxicity values obtained using the DHA and Vibrio qinghaiensis tests. Compared with the DHA measurement, the Vibrio qinghaiensis test was faster and more sensitive. Meanwhile, the toxicity indicators were significantly and positively correlated with the TSS, TN, TP and COD concentrations. These results may aid the understanding of the toxicity of pharmaceutical industrial park wastewaters and toxicity removal using the treatment techniques that are currently utilized in China.

Immunogenicity of Outer Membrane Proteins VirB9-1 and VirB9-2, a Novel Nanovaccine against Anaplasma marginale.

Anaplasma marginale is the most prevalent tick-borne livestock pathogen and poses a significant threat to cattle industry. In contrast to currently available live blood-derived vaccines against A. marginale, alternative safer and better-defined subunit vaccines will be of great significance. Two proteins (VirB9-1 and VirB9-2) from the Type IV secretion system of A. marginale have been shown to induce humoral and cellular immunity. In this study, Escherichia coli were used to express VirB9-1 and VirB9-2 proteins. Silica vesicles having a thin wall of 6 nm and pore size of 5.8 nm were used as the carrier and adjuvant to deliver these two antigens both as individual or mixed nano-formulations. High loading capacity was achieved for both proteins, and the mouse immunisation trial with individual as well as mixed nano-formulations showed high levels of antibody titres over 107 and strong T-cell responses. The mixed nano-formulation also stimulated high-level recall responses in bovine T-cell proliferation assays. These results open a promising path towards the development of efficient A. marginale vaccines and provide better understanding on the role of silica vesicles to deliver multivalent vaccines as mixed nano-formulations able to activate both B-cell and T-cell immunity, for improved animal health.

[Metabolic profiling analysis of amino metabolites in plant extract based on pre-column derivatization-ultra high performance liquid chromatography-mass spectrometry].

Amino metabolites are important compounds that play a key role in plant growth and development. A metabolic profiling analysis method of amino metabolites in plant extract was developed based on pre-column derivatization-ultra high performance liquid chromatography- mass spectrometry. Using the tobacco leaf as an example, a total of 87 amino metabolites, including amino acids, amines, peptides, alkaloids etc. were detected. The repeatability of the method was good with RSDs of 85 amino metabolites between 1. 5% and 18. 8%. Forty-three amino metabolites validated by standard samples showed good linearity with the correlation coefficients of 0.993-0.999, covered linear range of four orders of magnitude. The limits of detection were 0.03-6.58 ng/mL. The intra-day and inter-day precisions were 0.7%-15.6% and 0.8%-22.9%, respectively. The recoveries were 74.4%-122.7%. The influence of topping on metabolic profiling of amino metabolites in fresh tobacco was investigated using the developed method. The results showed that the amino metabolites in the upper tobacco leaves were most affected than those in the middle and lower leaves. Metabolism of amino metabolites in the upper leaves after topping was mainly towards the alkaloid synthesis. The method integrated the advantages of triple quadrupole mass spectrometry and high resolution quadrupole-time of flight mass spectrometry. It can be used for metabolic profiling analysis of amino metabolites in plant extract with high sensitivity and selectivity.

Non-targeted metabolomics study for the analysis of chemical compositions in three types of tea by using gas chromatograph-mass spectrometry and liquid chromatography-mass spectrometry.

Tea is one of the most widely consumed beverages in the world for its benefits to daily life and health. To discover the difference and correlation of chemical compositions in the three typical types of tea, a non-targeted metabolomics method was developed. After the optimization of extraction methods, gas chromatography-time-of-flight mass spectrometry and liquid chromatography-quadrupole time-of-flight mass spectrometry were applied for metabolomics analysis, 1,812 and 2,608 features were obtained, respectively. By comparing with the known compounds in public and/or commercial databases, 173 compounds were tentatively identified, and 109 of them were experimentally confirmed by standards. Totally, 33 tea samples including 12, 12 and 9 samples of green, oolong and black tea, respectively, were analyzed by using the above two methods. Multivatiate analysis, Mann-Whitney U test and hierarchical cluster analysis were used to find and visualize the differential components in the three types of tea. Finally, 90 compounds, which contain catechins, amino acids, organic acids, flavonol glycosides, alkaloids, carbohydrates, lipids, etc, were found with a significant difference among them. This study demonstrates the potentials and power of metabolomics methods to understand the chemical secrets of tea. This should help a lot to optimize the processes of agriculture, storage, preparation and consumption.

Metabolomics study of cured tobacco using liquid chromatography with mass spectrometry: method development and its application in investigating the chemical differences of tobacco from three growing regions.

Cured tobacco is an important plant material. Component studies are a big challenge for its significantly diverse chemical properties and vastly different concentrations. In this work, liquid chromatography with quadrupole time-of-flight mass spectrometry was used to perform a metabolomics study of cured tobacco owing to its efficient separation and detection of semipolar metabolites. A solvent of methanol/water (8:2, v/v) and 30 min of ultrasound time were found to be optimal to perform extraction. 95, 92, and 93% of metabolite features had within 20% of coefficient of variation for repeatability, intraday and interday precision analysis, respectively, indicating a good stability of the method developed. 113 metabolites were identified in cured tobacco based on accurate mass, retention time, and MS/MS fragments. The developed method was applied to a metabolomics study of cured tobacco from three growing regions. Forty three metabolites were found to be contributed to the classification. It is shown that the developed method can be applied to metabolomics analysis of plant materials.

A comparative study of volatile components in green, oolong and black teas by using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry and multivariate data analysis.

The difference of volatile components in green, oolong and black teas was studied by using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS). Simultaneous distillation extraction was proved to be a suitable technique to extract the analytes with interest. A total of 450 compounds were tentatively identified with comparison to the standard mass spectra in available databases, retention index on the first dimension and structured chromatogram. 33 tea samples, including 12, 12 and 9 samples of green, oolong and black tea were analyzed by using GC×GC-TOFMS. After peak alignment, around 3600 peaks were detected. Partial least squares - discriminant analysis and hierarchical cluster analysis were used to classify these samples, then non-parametric hypothesis test (Mann-Whitney U test) and the variable importance in the projection (VIP) were applied to discover the key components to distinguish the three types of tea with significant difference amongst them. 74 differential compounds are defined to interpret the chemical differences of 3 types of tea. This study shows the power of GC×GC-TOFMS method combined with multivariate data analysis to investigate natural products with high complexity for information extraction.