Trap-time Of Flight Mass Spectrometry Research Articles

An integrated strategy for rapid discovery and identification of the potential effective fragments of polysaccharides from Saposhnikoviae Radix

Ethnopharmacological relevanceSaposhnikoviae Radix (SR) is a traditional Chinese medicine, known as “Fangfeng”. As one of the main active components, Saposhnikoviae Radix polysaccharides (SP) demonstrated a range of biological activities, especially immunity regulation activity. Aims of the studyThis study aimed at exploring whether polysaccharides have activity after degradation, then discovering the potential effective fragments of SP. Materials and methodsHere we establish the chromatographic fingerprints method for 32 batches of 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives of oligosaccharides by HPLC, meanwhile evaluating its immunomodulatory activity in vivo. Then, the potential effective fragments of SP were screened out based on the spectrum-effect relationship analysis between fingerprints and the pharmacological results. Besides, liquid chromatography ion trap-time of flight mass spectrometry (LC-IT-TOF MS) coupled with multiple data-mining techniques was used to identify the potential effective oligosaccharides. ResultsThese findings showed that the hydrolysate of SP have significant immunomodulatory, and the immunity regulation activity varies under different hydrolysis conditions. The 4 potential effective peaks of the hydrolysate of SP were mined by spectrum-effect relationship. Finally, the chemical structure of 4 potential effective oligosaccharide fragments of SP was elucidated based on LC-IT-TOF MS. F10 was inferred tentatively to be Hex1→6Hex1→6Hex1→6Hex1→6Hex1→6Gal; F18 was confirmed to be Rhamnose; F14 was inferred tentatively to be Hex1→4Hex1→ 4Hex1→4Gal and F25 was tentatively inferred to be Ara1→6Gal. ConclusionsThis study may provide a sound experimental foundation in the exploration of the active fragments from macromolecular components with relatively complex structures such as polysaccharides.

Aspidopterys obcordata vine inulin fructan affects urolithiasis by modifying calcium oxalate crystallization

Aspidopterys obcordata vine is a Chinese Dai ethnic herb used to treat urolithiasis. However, the material basis and underlying mechanisms remain undefined. In this study, a 2.3 kD inulin-like A. obcordata fructan (AOFOS) was isolated by size exclusion column chromatography and characterized by ultrahigh-performance liquid chromatography-ion trap-time of flight mass spectrometry (UPLC-IT-TOF-MS), nuclear magnetic resonance (NMR) spectroscopy, gas chromatography mass spectrometry (GC–MS) and high-performance gel permeation chromatography (HGPC). In addition, AOFOS showed unique anti-urolithiasis activity in Drosophila kidney stone models. Mechanism study indicated that AOFOS reduced the size of calcium oxalate crystals by inhibiting the formation of large size crystals and the generation rate of calcium oxalate crystals as well as the crystal form conversion from calcium oxalate monohydrate (COM) to calcium oxalate dihydrate (COD).

Optimization and characterization of rhamnolipid production by Pseudomonas aeruginosa NY3 using waste frying oil as the sole carbon.

Yield and cost are two major factors limiting the widespread use of rhamnolipids (RLs). In the present study, waste frying oil (WFO) was used as the sole carbon source to produce environmentally friendly RLs by Pseudomonas aeruginosa NY3. The Plackett-Burman design (PBD) and Box-Behnken design (BBD) methods were used to maximize the production yield of RL. The PBD results showed that the concentrations of NaNO3 , Na2 HPO4 , and trace elements were the key factors affecting the yield of RL. Furthermore, the BBD results showed that at NaNO3 , Na2 HPO4 , and trace elements concentrations were 4.95, 0.66, and 0.64 mL/L, respectively, the average RL yield reached 9.15 ± 0.52 g/L, 1.58-fold higher than that observed before optimization. Fourier transform infrared spectroscopy (FTIR) and liquid chromatography-ion trap-time of flight mass spectrometry (LCMS-IT-TOF) were used to elucidate the diversity of RL congeners. The results showed that, after optimization, the RL congener diversity increased, and the major RL constituent was converted from di-RLs (64.04%) to mono-RLs (60.44%). These results suggested that the concentrations of the components contained in the culture medium of P. aeruginosa NY3 influenced not only the yield of RL, but also its congener distribution.

Tapping into the realm of underutilised green leafy vegetables: Using LC-IT-Tof-MS based methods to explore phytochemical richness of Sonchus oleraceus (L.) L.

Sonchus oleraceus (L.) L. (sow thistle) is an underutilised functional leafy vegetable with both high nutritional and pharmacological values. In the current study, high performance liquid chromatography ion trap time-of flight mass spectrometry (HPLC-IT-Tof-MS)-based metabolomics in combination with principal component analysis (PCA) were used to provide an overall phytochemical composition of different tissues (leaf, stem and roots) of this underutilised plant. A total of 32 phytochemicals, comprising hydroxycinnamic acids, flavonoids and terpene classes were identified across the three different tissues of S. oleraceus. The study used the new approach of colour coded PCA models to info-graphically depict the metabolic profiles across different parts of the plant. The results of the current study indicate S. oleraceus as a potential source of metabolites with highly claimed nutraceutical properties. As such, the results herein can be used as a template reference for further biological studies aimed at the evaluation of nutraceutical properties of the S. oleraceus plant.

Screening of Phosphodiesterase-5 Inhibitors and Their Analogs in Dietary Supplements by Liquid Chromatography-Hybrid Ion Trap-Time of Flight Mass Spectrometry.

An accurate and reliable method based on ion trap–time of flight mass spectrometry (IT–TOF MS) was developed for screening phosphodiesterase-5 inhibitors, including sildenafil, vardenafil, and tadalafil, and their analogs in dietary supplements. Various parameters affecting liquid chromatographic separation and IT–TOF detection were investigated, and the optimal conditions were determined. The separation was achieved on a reversed-phase column under gradient elution using acetonitrile and water containing 0.2% acetic acid at a flow rate of 0.2 mL/min. The chromatographic eluents were directly ionized in the IT–TOF system equipped with an electrospray ion source operating in the positive ion mode. The proposed screening method was validated by assessing its linearity, precision, and accuracy. Sequential tandem MS was conducted to obtain structural information of the references, and the fragmentation mechanism of each reference was proposed for providing spectral insight for newly synthesized analogs. Structural information, including accurate masses of both parent and fragment ions, was incorporated into the MSn spectral library. The developed method was successfully applied for screening adulterated dietary supplement samples.

Laser desorption ionization time-of-flight mass spectrometry of GexSe1-x chalcogenide glasses, their thin films, and Ge:Se mixtures

The aim of this work is to generate and study the clusters formed as a result of the interaction of laser pulses with solid state materials such as chalcogenide glasses. The other target is to determine the stoichiometry of the clusters generated from studied chalcogenide glasses and their thin films in order to get the information about structural fragments presents in the plasma plume. The aims were achieved by exploiting laser desorption ionization (LDI) with quadrupole ion trap time-of-flight mass spectrometry (TOFMS) examination of the GexSe1-x (x = 0.1, 0.2, 0.3, 0.33) chalcogenide glasses and Ge0.2Se0.8 and Ge0.33Se0.67 thin films prepared by magnetron sputtering. The interaction of laser pulses with GexSe1-x chalcogenide glasses and their thin films produces many positively and negatively charged unary and binary (Gea+/−, Seb+/−, and GeaSeb+/−) clusters. About ~50 different clusters were identified for each glass sample. We assume that some species indicate local structure of the studied materials. However, other clusters are formed either by a fragmentation of original glass structure or by laser ablation synthesis. When using parafilm, many new, higher mass clusters were produced. The results were compared with LDI TOFMS analysis of Ge:Se elemental mixtures. Concluding, LDI TOFMS is useful technique not only for the generation and study of the clusters but also it gives partial structural information about glasses and other materials as well as information about processes proceeding in the plasma.

Identification of the impurity in auramine O by high performance liquid chromatography-ion trap-time of flight mass spectrometry and preparation of the auramine O reference standard by preparative high performance liquid chromatography

A novel and effective method was established for the qualitative analysis of impurities in auramine O samples of illegal food additives using high performance liquid chromatography-ion trap-time of flight mass spectrometry (HPLC-IT-TOF-MS). An impurity was identified in the auramine O sample using the optimized HPLC-IT-TOF-MS method. According to the exact mass of each fragment ion measured by multistage MS, the structure of the impurity was determined to be that of 4-(imino (4-(methylamino) phenyl) methyl)-N,N-dimethylaniline hydrochloride. The synthetic route of the auramine O and the source of the identified impurity were proposed. Simultaneously, a preparative high performance liquid chromatography (prep-HPLC) technique was successfully applied for the purification of the auramine O from complex samples. Prep-HPLC columns with particle sizes of 10 μm and 5 μm were used for the separation and purification with injection volumes of 1 mL and 500 μL, respectively. Finally an auramine O reference standard with 99.52% purity was obtained by secondarily purification and determined by the analytical HPLC area normalization method. Deducting the 0.34% moisture content and 0.13% ash content, the final purity of the sample was 99.05%, as determined by mass balance method. The chemical structure was examined using UV, IR, LC-MS, and NMR. The developed method is simple and efficient, and can be applied for the preparation of reference standard materials for other illegal food additives.

Metabolic profiling putatively identifies plasma biomarkers of male infertility using UPLC-ESI-IT-TOFMS.

Male infertility has become a global health problem. Currently, the diagnosis of male infertility depends on the results of semen quality or requires invasive surgical intervention. The process is complex and time-consuming. Metabolomics is an emerging platform with unique advantages in disease diagnosis and pathological mechanism research. In this study, ultra-performance liquid chromatography-electrospray ionization-ion trap-time of flight mass spectrometry (UPLC-ESI-IT-TOFMS) combined with chemometrics methods was used to discover potential biomarkers of male infertility based on non-targeted plasma metabolomics. Plasma samples from healthy controls (HC, n = 43) and various types of infertile patients, i.e., patients having oligozoospermia (OS, n = 36), asthenospermia (AS, n = 56) and erectile dysfunction (ED, n = 45) were analyzed by UPLC-ESI-IT-TOFMS. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed. The results of OPLS-DA showed that HCs could be discriminated from infertile patients including OS (R2 = 0.903, Q2 = 0.617, AUC = 0.992), AS (R2 = 0.985, Q2 = 0.658, AUC = 0.999) or ED (R2 = 0.942, Q2 = 0.500, AUC = 0.998). Some potential biomarkers were successfully discovered by variable selection methods and variable important in the projection (VIP) in combination with the T-test. Statistical significance was set at p < 0.05; the Benjamini–Hochberg false discovery rate was used to reduce type 1 errors resulting from multiple comparisons. The identified biomarkers were associated with energy consumption, hormone regulation and antioxidant defenses in spermatogenesis. To elucidate the pathophysiology of male infertility, relative metabolic pathways were studied. It was found that male infertility is closely related to disturbed phospholipid metabolism, amino acid metabolism, steroid hormone biosynthesis metabolism, metabolism of fatty acids and products of carnitine acylation, and purine and pyrimidine metabolisms. Plasma metabolomics provides a novel strategy for the diagnosis of male infertility and offers a new insight to study pathogenesis mechanism.

Comparison of chemical constituents among licorice, roasted licorice, and roasted licorice with honey.

Licorice (root and rhizome of Glycyrrhiza uralensis Fisch.) is sometimes used as crude drug after processing. In this report, we prepared roasted licorice with or without honey using 3 lots of crude drug samples derived from wild G. uralensis, and analyzed the constituents in unprocessed, roasted, and honey-roasted licorice samples by high performance liquid chromatography-electrospray ionization-ion trap-time of flight mass spectrometry (HPLC-ESI-IT-TOF-MSn) with principal component analysis. We found that the areas of 41 peaks were noticeably changed by processing. Among them, the areas of 12 peaks, viz. isoliquiritin, isoliquiritigenin, glucoisoliquiritin, 6″-O-acetylisoliquiritin, 6″-O-acetylisoliquiritin apioside, glycyrrhetinic acid 3-O-glucuronide, 5 kinds of sugar-derivatives and one compound whose molecular weight was 386Da were increased by roasting in all 3 lots, and those peak areas were increased by higher heating temperatures. Among the increased peaks, 3 kinds of sugar-derivatives had larger areas, and 6″-O-acetylisoliquiritin had lower areas than those in honey-roasted licorice. Those sugar-derivatives were the only characteristics differing between honey-roasted licorice and roasted licorice. Meanwhile, the areas of 9 peaks, four of them identified as 6″-O-acetylliquiritin, 6″-O-acetylliquiritin apioside, formononetin and gancaonin L, were decreased by roasting in all 3 lots, but there were no differences between roasted licorice with or without honey. Those compounds whose amounts were changed by processing could be used as markers for the quality control of roasted and honey-roasted licorice.

Identification of cytochrome P450s involved in the metabolism of 6-benzyl-1-benzyloxymethyl-5-iodouracil (W-1) using human recombinant enzymes and rat liver microsomes in vitro

1. The aim of this study was to identify the hepatic metabolic enzymes, which involved in the biotransformation of 6-benzyl-1-benzyloxymethyl-5-iodouracil (W-1), a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) in rat and human in vitro.2. The parent drug of W-1 was incubated with rat liver microsomes (RLMs) or recombinant CYPs (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5, respectively) in the presence or absence of nicotinamide adeninedinucleotide phosphate (NADPH)-regenerating system. The metabolites of W-1 were analyzed with liquid chromatography-ion trap-time of flight-mass spectrometry (LC-IT-TOF-MS).3. The parent drug of W-1 was metabolized in a NADPH-dependent manner in RLMs. The kinetic parameters of prototype W-1 including Km, Vmax, and CLint were 2.3 μM, 3.3 nmol/min/mg protein, and 1.4 mL/min/mg protein, respectively. Two metabolites M1 and M2 were observed in shorter retention times (2.988 and 3.188 min) with a higher molecular ion at m/z 463.0160 (both M1 and M2) than that of the W-1 parent drug (6.158 min with m/z 447.0218). The CYP selective inhibition and recombinant enzymes also showed that two hydroxyl metabolites M1 and M2 are mainly mediated by CYP2C19 and CYP3A4.4. The identification of CYPs involved in W-1 biotransformation is important to understand and minimize, if possible, the potential of drug–drug interactions.

Effects of elevated carbon dioxide on contraction force and proteome composition of sea urchin tube feet

This study examined how contraction force and protein profiles of the tube feet of the sea urchin (Pseudocentrotus depressus) were affected when acclimated to 400 (control), 2000 and 10,000μatm CO2 for 48days. Acclimation to higher CO2 conditions significantly reduced contraction force of the tube feet. Two-dimensional gel electrophoresis showed that eight spots changed in protein volume: six up-regulated and two down-regulated. Using matrix-assisted laser desorption/ionization-quadrupole ion trap-time of flight mass spectrometry, three up-regulated spots (tubulin beta chain, tropomyosin fragment, and actin N-terminal fragment) and two down-regulated spots (actin C-terminal fragment and myosin light chain) were identified. One possible interpretation of the results is that elevated CO2 weakened contraction of the tube feet muscle through an alteration of proteome composition, mainly associated with post-translational processing/proteolysis of muscle-related proteins.

An Orally Active Cannabis Extract with High Content in Cannabidiol attenuates Chemically-induced Intestinal Inflammation and Hypermotility in the Mouse.

Anecdotal and scientific evidence suggests that Cannabis use may be beneficial in inflammatory bowel disease (IBD) patients. Here, we have investigated the effect of a standardized Cannabis sativa extract with high content of cannabidiol (CBD), here named CBD BDS for “CBD botanical drug substance,” on mucosal inflammation and hypermotility in mouse models of intestinal inflammation. Colitis was induced in mice by intracolonic administration of dinitrobenzenesulfonic acid (DNBS). Motility was evaluated in the experimental model of intestinal hypermotility induced by irritant croton oil. CBD BDS or pure CBD were given - either intraperitoneally or by oral gavage – after the inflammatory insult (curative protocol). The amounts of CBD in the colon, brain, and liver after the oral treatments were measured by high-performance liquid chromatography coupled to ion trap-time of flight mass spectrometry. CBD BDS, both when given intraperitoneally and by oral gavage, decreased the extent of the damage (as revealed by the decrease in the colon weight/length ratio and myeloperoxidase activity) in the DNBS model of colitis. It also reduced intestinal hypermotility (at doses lower than those required to affect transit in healthy mice) in the croton oil model of intestinal hypermotility. Under the same experimental conditions, pure CBD did not ameliorate colitis while it normalized croton oil-induced hypermotility when given intraperitoneally (in a dose-related fashion) or orally (only at one dose). In conclusion, CBD BDS, given after the inflammatory insult, attenuates injury and motility in intestinal models of inflammation. These findings sustain the rationale of combining CBD with other minor Cannabis constituents and support the clinical development of CBD BDS for IBD treatment.

Identification, toxicity and control of iodinated disinfection byproducts in cooking with simulated chlor(am)inated tap water and iodized table salt

Chlorine/chloramine residuals are maintained in drinking water distribution systems to prevent microbial contamination and microorganism regrowth. During household cooking processes (e.g., soup making), the residual chlorine/chloramines in tap water may react with the iodide in iodized table salt to form hypoiodous acid, which could react with remaining natural organic matter in tap water and organic matter in food to generate iodinated disinfection byproducts (I-DBPs). However, I-DBPs formed during cooking with chloraminated/chlorinated tap water are almost completely new to researchers. In this work, by adopting precursor ion scan of m/z 127 using ultra performance liquid chromatography/electrospray ionization-triple quadrupole mass spectrometry, many new polar I-DBPs formed during cooking with chloraminated/chlorinated tap water were detected and proposed with structures, of which 3-iodo-4-hydroxybenzaldehyde, 3-iodo-4-hydroxybenzoic acid, 3-iodo-4-hydroxy-5-methylbenzoic acid, diiodoacetic acid, 3,5-diiodo-4-hydroxybenzaldehyde, 3,5-diiodo-4-hydroxybenzoic acid, 2,6-diiodo-4-nitrophenol, 2,4-diiodo-6-nitrophenol, and 2,4,6-triiodophenol were confirmed with standard compounds. With the aid of ultra fast liquid chromatography/ion trap-time of flight-mass spectrometry, molecular formula identification of five new I-DBPs (C8H5O4I, C7H4NO4I, C8H5O5I, C7H4NO5I, and C8H6O3I2) was achieved. A developmental toxicity with a recently developed sensitive bioassay was conducted for the newly identified I-DBPs, suggesting that phenolic I-DBPs (except for iodinated carboxyphenols) were about 50–200 times more developmentally toxic than aliphatic I-DBPs. The major I-DBPs in a baseline simulated cooking water sample were determined to be from 0.72 to 7.63 μg/L. Polar I-DBPs formed under various disinfection and cooking conditions were compared, and suggestions for controlling their formation were provided.

Metabonomic Study of Biochemical Changes in Human Hair of Heroin Abusers by Liquid Chromatography Coupled with Ion Trap-Time of Flight Mass Spectrometry.

Hair analysis is with the advantage of non-invasive collection and long surveillance window. The present study employed a sensitive and reliable liquid chromatography coupled with ion trap-time of flight mass spectrometry method to study the metabonomic characters in the hair of 58 heroin abusers and 72 non-heroin abusers. Results indicated that certain endogenous metabolites, such as sorbitol and cortisol, were accelerated, and the level of arachidonic acid, glutathione, linoleic acid, and myristic acid was decreased in hair of heroin abusers. The metabonomic study is helpful for further understanding of heroin addiction and clinical diagnosis.

Dihydrochalcones and homoisoflavanes from the red resin of Dracaena cochinchinensis (Chinese dragon's blood)

Two new dihydrochalcones, 4-hydroxy-2,4′-dimethoxydihydrochalcone (1) and 3,4′-dihydroxy-2,4,6-trimethoxydihydrochalcone (2), and a new homoisoflavane, 7,3′-dihydroxy-8,4′-dimethoxyhomoisoflavane (3), along with 12 known compounds (4–15), were isolated from the red resin of Dracaena cochinchinensis (Chinese dragon's blood). Their structures were assigned by a variety of spectroscopic techniques. Diversity of cleavage pathways were proposed for dihydrochalcones and homoisoflavanes based on the mass spectroscopic behaviors of those identified compounds using hybrid ion trap-time of flight mass spectrometry. All the compounds were evaluated for their inhibitory effects on nitric oxide production in lipopolysaccharide-induced RAW 264.7 macrophages, and compound 9 exhibited mild inhibition of NO production in this assay with IC50 value of 50.3μM.

Efficient preparative isolation and identification of walnut bioactive components using high-speed counter-current chromatography and LC-ESI-IT-TOF-MS

Preparative isolation of complex mixtures of compounds from walnut polar extracts was established by a combination of high-speed counter-current chromatography (HSCCC) and electrospray ionization-ion trap-time of flight mass spectrometry (ESI-IT-TOF-MS). Compounds were isolated after a solvent optimisation selection based on solute distribution in a biphasic solvent system. Isolation was achieved through one or two successive HSCCC runs, and final purification on Sephadex LH-20. Isolated compounds included ellagitannins (1–11), gallic acid (12), dicarboxylic acid glucosides (13–15), hydrojuglone glucoside (16), catechin (17), procyanidin B2 (18), and megasterone glucosides (19–20). Praecoxin D (4) was isolated for the first time from walnut, while praecoxin A methyl ester (5) and glansreginin A n-butyl ester (14) are newly identified compounds. The purity and identity of isolated compounds were confirmed by NMR and HPLC-ESI-MS/MS. These results provided a foundation for in depth characterisation of walnut compounds and offered an efficient strategy for isolation of potentially health-relevant phytochemicals from walnuts.

Characterization of the herb-derived components in rats following oral administration of Carthamus tinctorius extract by extracting diagnostic fragment ions (DFIs) in the MS(n) chromatograms.

In this study, a new strategy named extracting diagnostic fragment ions (DFIs) in the MS(n) chromatograms [E(DFI)MS(n)Cs] was proposed to rapidly detect and identify the in vivo components derived from the extract of Carthamus tinctorius (ECT), using high performance liquid chromatography coupled with hybrid ion trap-time of flight mass spectrometry. In order to comprehensively summarize the DFIs for the global identification of in vivo constituents of ECT, chemical profiling was carried out, and then the typical metabolic pathways of the primary components were proposed according to their chemical categories, by orally administering representative reference compounds. Based on the proposed metabolic pathways and the fragmentation rules, a list of DFIs was constructed and adopted to differentiate and identify the metabolites from the endogenous substances in the MS(n) chromatograms of ECT-treated biological samples, in combination with the neutral loss scan mode as a supplement. As a result, a total of 156 compounds were tentatively assigned in vivo, including 63, 73, 50, and 17 components from rat plasma, urine, bile, and feces, respectively, following oral administration of ECT. Deglycosylation, oxidation, methylation, sulfonation, and glucuronidation were observed as the major metabolic pathways for the chemical constituents of ECT, and dehydroxylation was detected at the A-ring of flavones for the first time. The findings suggested that the E(DFI)MS(n)Cs-based strategy which integrated ideas from single compounds to herbal extracts and from extract chemical profiling to in vivo metabolite profiling, could be used as a reliable tool for rapidly discovering and identifying herb-related constituents in vivo.

Effect of Allium macrostemon on a rat model of depression studied by using plasma lipid and acylcarnitine profiles from liquid chromatography/mass spectrometry

The present study aimed to investigate the anti-depressive effect of the traditional Chinese medicine Allium macrostemon in a rat model of depression induced by exposure to chronic immobilization stress. Lipid and acylcarnitine metabolism were set into the focus of this study due to their key role in the pathogenesis of depression. Plasma lipid profiling was performed by ultra fast liquid chromatography/ion trap-time of flight mass spectrometry. Ultra-high performance liquid chromatography/triple quadrupole mass spectrometry was used to characterize the plasma acylcarnitine profile. Principal component analysis (PCA) revealed distinct differences in plasma lipid and acylcarnitine profiles of depressed rats from those in the control rats, which were also validated by univariate analysis. Several lysophosphatidylcholines (LPC (18:1→:2), LPC (20:1), LPC (O-16:2), and LPC (O-18:3)) as well as most medium- and long-chain acylcarnitines were elevated, while some phosphatidylcholines (PC (32:1), PC (36:4→:5), PC (37:4), PC (38:4→:6), PC (40:6), PC (O-36:4), and PC (O-38:5)) and triglycerides (TG (58:12), TG (60:12), and TG (62:13→:14)) were decreased in the plasma of depressed rats. These changes indicate that depressed rats were associated with inflammatory conditions and an incomplete β-oxidation of fatty acids. Most of these dysregulated metabolites were returned to their normal levels after treatment with A. macrostemon according to PCA and univariate analysis, highlighting the anti-depressive effect of this traditional Chinese medicine. These results show that liquid chromatography/mass spectrometry-based quantitative metabolic profiling method is a useful tool to investigate the metabolic changes in depression and the anti-depressive effect of traditional Chinese medicine.

Thermally Accelerated Oxidative Degradation of Quercetin Using Continuous Flow Kinetic Electrospray-Ion Trap-Time of Flight Mass Spectrometry

Thermally accelerated oxidative degradation of aqueous quercetin at pH5.9 and 7.4 was kinetically measured using an in-house built online continuous flow device made of concentric capillary tubes, modified to fit to the inlet of an electrospray ionization-ion trap-time-of-flight-mass spectrometer (ESI-IT-TOF-MS). Time-resolved mass spectral measurements ranging from 2 to 21min were performed in the negative mode to track intermediate degradation products and to evaluate the degradation rate of the deprotonated quercetin ion, [Q-H](-). Upon heating solutions in the presence of dissolved oxygen, degradation of [Q-H](-) was observed and was accelerated by an increase in pH and temperature. Regardless of the condition, the same degradation pathways were observed. Degradation mechanisms and structures were determined using higher order tandem mass spectrometry (up to MS(3)) and high mass accuracy. The observed degradation mechanisms included oxidation, hydroxylation, and ring-cleavage by nucleophilic attack. A chalcan-trione structure formed by C-ring opening after hydroxylation at C2 was believed to be a precursor for other degradation products, formed by hydroxylation at the C2, C3, and C4 carbons from attack by nucleophilic species. This resulted in A-type and B-type ions after cross-ring cleavage of the C-ring. Based on time of appearance and signal intensity, nucleophilic attack at C3 was the preferred degradation pathway, which generated 2,4,6-trihydroxymandelate and 2,4,6-trihydroxyphenylglyoxylate ions. Overall, 23 quercetin-related ions were observed.

Expression and secretion of functional recombinant μO-conotoxin MrVIB-His-tag in Escherichia coli

μO-conotoxin MrVIB is a 31-amino acid peptide containing three disulfide bonds isolated from the venom of Conus marmoreus, which is a selective antagonist of voltage-gated sodium channel (VGSC) Nav1.8 and has a long-lasting analgesic activity. Drug development of MrVIB has long been hindered over 15 years by difficult chemical synthesis and oxidative folding. Herein we describe a different approach based on the recombinant expression of gene MrVIB in Escherichia coli. A secretion vector pET22b(+)-MrVIB fused with pelB leader signal peptide and His-tag was constructed, which was transformed into BL21 (DE3) strain of E. coli. The recombinant conotoxin MrVIB-His-tag (rMrVIB-His) was successfully expressed and secreted into the periplasmic space of BL21 (DE3) cells. The pelB leader signal peptide was properly cleaved and three disulfide bonds were also formed properly to yield biological active rMrVIB-His. Folded rMrVIB-His in the periplasmic fraction was isolated with a Ni-NTA affinity column, which was further purified using reverse-phase high-performance liquid chromatography (RP-HPLC) and identified by liquid chromatography/mass spectrometry-ion trap-time of flight mass spectrometry (LC/MS-IT-TOF). Biological activity assay of rMrVIB-His showed it had good analgesic effects in three pain models.