Linear Ion Trap Quadrupole Orbitrap Research Articles

The profiles of durian (Durio zibethinus Murr.) shell phenolics and their antioxidant effects on H2O2-treated HepG2 cells as well as the metabolites and organ distribution in rats

Durian is a nutritious tropical fruit with potent antioxidant, anti-inflammatory, antibacterial and anti-cancer effects. However, the durian shell was mainly discarded as waste, while there were few studies on the characterization of its phenolic profiles, antioxidant activities, and in vivo metabolites. In the present study, a total of 17 compounds were identified in durian shell extract (DSE) by using an ultra-high-performance liquid chromatography coupled with linear ion trap quadrupole Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap-MS/MS), while 33 metabolites were found in rats' plasma, urine and organ. Moreover, DSE could effectively reduce H2O2-induced oxidative damage in HepG2 cells, reduce the expression of Reactive Oxygen Species (ROS), Malondialdehyde (MDA) and Lactate Dehydrogenase (LDH) and inhibit apoptosis by regulating the expression of Bcl-2-Associated X (BAX), B-Cell Lymphoma 2 (BCL-2), Caspase-3 and Caspase-9 genes and proteins related to mitochondrial pathway apoptosis. This is the first comprehensive report on Durian shell phenolics, their metabolic profiles and underlying mechanisms of the in vitro antioxidant activities.

Evaluation of the chemical profile from four germplasms sources of Pruni Semen using UHPLC-LTQ-Orbitrap-MS and multivariate analyses

Pruni Semen, the seed of several unique Prunus plants, is a traditional purgative herbal material. To determine the authentic sources of Pruni Semen, 46 samples from four species were collected and analyzed. Ten compounds including multiflorin A (Mul A), a notable purative compound, were isolated and identified by chemical separation and nuclear magnetic resonance spectroscopy. Seventy-six communal components were identified by ultra-high performance liquid chromatography with linear ion trap-quadrupole Orbitrap mass spectrometry, and acetyl flavonoid glycosides were recognized as characteristic constituents. The flavonoids were distributed in the seed coat and cyanogenic glycosides in the kernel. Based on this, methods for identifying Pruni Semen from different sources were established using chemical fingerprinting, quantitative analysis of the eight principal compounds, hierarchical cluster analysis, principal component analysis, and orthogonal partial least squares discriminant analysis. The results showed that the samples were divided into two categories: one is the small seeds from Prunus humilis (Ph) and Prunus japonica (Pj), and the other is the big seeds from Prunus pedunculata (Pp) and Prunus triloba (Pt). The average content of Mul A was 3.02, 6.93, 0.40, and 0.29 mg/g, while the average content of amygdalin was 18.5, 17.7, 31.5, and 30.9 mg/g in Ph, Pj, Pp, and Pt, respectively. All the above information suggests that small seeds might be superior sources of Pruni Semen. This is the first comprehensive report on the identification of chemical components in Pruni Semen from different species.

Identification of the multiple bioactive derivatives and their endogenous molecular targets that may mediate the laxative effect of rhubarb in rats

To determine the mechanism of the laxative effect of rhubarb ( Rheum tanguticum Maxim. ex Balf.) in rats using a combined bioinformatic and in vivo approach. Substances derived from rhubarb that are present in the colorectum were identified using high-performance liquid chromatography, coupled with linear ion-trap quadrupole Orbitrap high-resolution mass spectrometry. The targets with a potential laxative effect were identified from databases and the literature, then used with rhubarb-derived substances in molecular docking modeling. The expression of candidate endogenous target molecules that bound specific components of rhubarb was then measured in constipated rats that had or had not been administered rhubarb by western blotting. Finally, potentially bioactive compounds were traced back to their prototype components. We identified 17 anthraquinones and 21 anthrones derived from rhubarb in the colorectum of rats. G-scoring identified three potential mediators of the laxative effect: c-kit, 5-hydroxytryptamine receptor 4 (5-HT4), and aquaporin-3 (AQP3). In addition, 10 rhubarb-derived components (aloe-emodin, emodin, rhein, chrysophanol, physcion, sennoside A, sennoside C, physcionanthrone, aloe-emodinanthrone, and rheinanthrone), which have strong binding affinities for more than one of the three potential targets, were selected as likely active compounds. Rhubarb extract increased the expression of c-kit and 5-HT4, and reduced the expression of AQP3 in the colon of constipated rats, which might mediate its laxative effect. We also found that a single prototype component may be metabolized into several active metabolites, and a single active ingredient can also be generated from various prototype compounds. The present study demonstrates that various anthraquinones and anthrones present in rhubarb may be metabolized to form bioactive compounds that have additive or synergistic effects to promote defecation via c-kit, 5-HT4 and/or AQP3.

Phytochemistry and Antioxidant Activity of Aerial Parts of Phagnalon sordidum L.

One new natural monoterpene, 5-O-β-d-glucopyranosyl-2-hydroxy-p-cymene (1: ), and 11 known compounds were isolated through a biologically oriented approach from the aerial parts of Phagnalon sordidum L. The most active extract and fractions were selected using 3 complementary antioxidant activity assays. Results and the different methods were compared by relative antioxidant capacity index. In addition, the most active extract of P. sordidum was subjected to liquid chromatography coupled with electrospray ionization hybrid linear ion trap quadrupole Orbitrap mass spectrometry to quantify secondary metabolites. Antioxidant activities of ethyl acetate extract, and purified 3,4-dihydroxyacetophenone (3: ) and nebrodenside A (7: ) were demonstrated by in vitro cell free model assays, and their protective effect against H2O2-induced oxidative stress in a HepG2 (human hepatocellular carcinoma) cell line was established.

Rapid characterization of chemical constituents and rat metabolites of Fufang Gancao Tablets by UHPLC-LTQ-Orbitrap mass spectrometer

Ultra-high-performance liquid chromatography coupled with tandem high-resolution mass spectrometry (UHPLC-HR-MSn ) method was used to analyze the constituents of Fufang Gancao Tablets and its main metabolites in rat plasma. Rat plasma was collected both before and after oral administration of Fufang Gancao Tablets. After solid phase extraction, ACQUITY UHPLC BEH C₁₈ (2.1 mm×100 mm, 1.7 μm) was used with 0.1% formic acid-acetonitrile solution as the mobile phase for gradient elution. The chemical components in Fufang Gancao Tablets and their prototypes and metabolites in plasma samples were analyzed by LTQ-Orbitrap equipped with an ESI ion source in a positive ion mode. Based on the accurate mass measurements, the retention time and mass fragmentation patterns, a total of 55 compounds were tentatively identified from Fufang Gancao Tablets, including 42 flavonoids, 9 triterpenes and 4 alkaloids. Furthermore, metabolites in rat plasma after oral administration of Fufang Gancao Tablets were also analyzed. A total of 26 compounds were identified, including 20 prototypes and 6 metabolites mainly through metabolic pathways of hydroxylation, glucuronide conjugation, and sulfate conjugation, etc. Our results showed that the ultra-high-performance liquid chromatography coupled with linear ion trap quadrupole Orbitrap high resolution mass spectrometry (UHPLC-LTQ-Orbitrap) could comprehensively elucidate the chemical constituents of Fufang Gancao Tablets and their migrating components in rat plasma, providing scientific basis for further studying the metabolism process and pharmacodynamic substance of Fufang Gancao Tablets.

Application of High-Performance Liquid Chromatography Coupled with Linear Ion Trap Quadrupole Orbitrap Mass Spectrometry for Qualitative and Quantitative Assessment of Shejin-Liyan Granule Supplements.

A method for high-performance liquid chromatography coupled with linear ion trap quadrupole Orbitrap high-resolution mass spectrometry (HPLC-LTQ-Orbitrap MS) was developed and validated for the qualitative and quantitative assessment of Shejin-liyan Granule. According to the fragmentation mechanism and high-resolution MS data, 54 compounds, including fourteen isoflavones, eleven ligands, eight flavonoids, six physalins, six organic acids, four triterpenoid saponins, two xanthones, two alkaloids, and one licorice coumarin, were identified or tentatively characterized. In addition, ten of the representative compounds (matrine, galuteolin, tectoridin, iridin, arctiin, tectorigenin, glycyrrhizic acid, irigenin, arctigenin, and irisflorentin) were quantified using the validated HPLC-LTQ-Orbitrap MS method. The method validation showed a good linearity with coefficients of determination (r2) above 0.9914 for all analytes. The accuracy of the intra- and inter-day variation of the investigated compounds was 95.0–105.0%, and the precision values were less than 4.89%. The mean recoveries and reproducibilities of each analyte were 95.1–104.8%, with relative standard deviations below 4.91%. The method successfully quantified the ten compounds in Shejin-liyan Granule, and the results show that the method is accurate, sensitive, and reliable.

Application of ultra-high-performance liquid chromatography coupled with LTQ-Orbitrap mass spectrometry for identification, confirmation and quantitation of illegal adulterated weight-loss drugs in plant dietary supplements.

In this paper, an ultra-high-performance liquid chromatography coupled with linear ion trap quadrupole Orbitrap high resolution mass spectrometry (UHPLC-LTQ-Orbitrap HRMS) method was developed and validated for identification, confirmation and quantitation of illegal adulterated weight-loss drugs in plant dietary supplements. 13 wt-loss drugs were well separated by the gradient elution of 10mmol/L ammonium acetate - 0.05% formic acid H2O and acetonitrile at a flow rate of 0.2mL/min within 12min. The MS analysis was operated under the positive ion and in full MS/dd-MS2 (data-dependent MS2) mode. The full MS scan with resolution at 60 000 FWHM and narrow mass windows at 5ppm acquired data for identification and quantitation, and dd-MS2 scan with resolution at 15 000 FWHM obtained product ions for confirmation. The method validation showed good linearity with coefficients of determination (r2) higher than 0.9951 for all analytes. Meantime, all the LOD and LLOQ values were in the respective range of 0.3-2 and 1-9ng/g. The accuracy, intra- and inter-day precision were in the ranges of -1.7 to 3.4%, 1.7-5.0% and 1.9-4.4%, respectively. The mean recoveries ranged from 85.4 to 107.1%, while the absolute and relative matrix effect were in the corresponding range of 98.2-108.6% and 2.6-8.7%. Among 120 batches of weight loss plant dietary supplements, sibutramine and fluoxertine or both were positive in 29 samples. In general, LTQ-Orbitrap HRMS technology was a powerful tool for the analysis of illegal ingredients in dietary supplements.

Polyphenolic profile of persimmon leaves by high resolution mass spectrometry (LC-ESI-LTQ-Orbitrap-MS)

Persimmon leaves have played an important role in Chinese medicine. Persimmon extracts and formulations have been shown to possess a wide range of pharmacological activities, including antioxidant, hypolipidaemic and antidiabetic, and they have been used to treat cardiovascular disease, improve homeostasis, as antibacterial and anti-inflammatory agents, and as a beauty treatment. In this work, liquid chromatography coupled to hybrid linear ion trap quadrupole Orbitrap mass spectrometry was used to accurately identify persimmon leaf polyphenols. Forty-one phenolic compounds, including simple phenolic acids, hydroxybenzoic acids, hydroxycinnamic acids, flavanols, flavonols, flavanones, flavone-chalcones, tyrosols and their conjugated derivatives, were identified and quantified using high mass accuracy data and confirmed by MS2 experiments. To the best of our knowledge, this is the most extensive study of persimmon leaf polyphenols performed so far, since 33 phenolic compounds are reported for the first time.

Systematic Analysis of Absorbed Anti-Inflammatory Constituents and Metabolites of Sarcandra glabra in Rat Plasma Using Ultra-High-Pressure Liquid Chromatography Coupled with Linear Trap Quadrupole Orbitrap Mass Spectrometry.

Ultra-high-pressure liquid chromatography (UHPLC) was coupled with linear ion trap quadrupole Orbitrap mass spectrometry (LTQ-Orbitrap) and was used for the first time to systematically analyze the absorbed components and metabolites in rat plasma after oral administration of the water extract of Sarcandra glabra. This extract is a well-known Chinese herbal medicine for the treatment of inflammation and immunity related diseases. The anti-inflammatory activities of the absorbed components were evaluated by measuring nitric oxide (NO) production and proinflammatory genes expression in lipopolysaccharide (LPS)-stimulated murine RAW 264.7 macrophages. As a result, 54 components in Sarcandra glabra were detected in dosed rat plasma, and 36 of them were positively identified. Moreover, 23 metabolites were characterized and their originations were traced. Furthermore, 20 of the 24 studied components showed anti-inflammatory activities. These results provide evidence that this method efficiency detected constituents in plasma based on the anti-inflammatory mechanism of multiple components and would be a useful technique for screening multiple targets for natural medicine research.

Metabolic profile of naringenin in the stomach and colon using liquid chromatography/electrospray ionization linear ion trap quadrupole-Orbitrap-mass spectrometry (LC-ESI-LTQ-Orbitrap-MS) and LC-ESI-MS/MS

Several biological activities (antioxidant, anti-inflammatory, anticarcinogenic) are attributed to naringenin (NAR)—a predominant flavonoid of citrus fruit and tomato—despite its low bioavailability after ingestion. NAR undergoes extensive metabolism when crossing the gastrointestinal tract, resulting in enteric, hepatic and microbial metabolites, some of them with recognized beneficial effects on human health. This study sought to provide new insights into the metabolism of NAR in regions of the gastrointestinal tract where it has been less studied: the stomach and colon. With this purpose, liquid chromatography coupled with an electrospray ionization hybrid linear ion trap quadrupole Orbitrap mass spectrometry technique (LC-ESI-LTQ-Orbitrap-MS) was used for an accurate identification of NAR metabolites, and liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) on a triple quadrupole was used for their identification and quantification. The combination of both analytical techniques provided a broader metabolic profile of NAR. As far as we know, this is the first in-depth metabolic profiling study of NAR in the stomach of mice. Three of the metabolites determined using the LC-LTQ-Orbitrap could not be identified by LC-ESI-MS/MS in stomach perfusion samples: apigenin, 3-(4-hydroxyphenyl) propionic acid and phloroglucinol. The number of colonic metabolites determined using the LTQ-Orbitrap-MS was more than twice the number identified by LC-ESI-MS/MS.

A comprehensive characterisation of beer polyphenols by high resolution mass spectrometry (LC–ESI-LTQ-Orbitrap-MS)

Beer is the second most consumed alcoholic beverage in Europe and shown by the European Prospective Investigation into Cancer and Nutrition cohort study to be the main food contributor to hydroxybenzoic acid intake. About 70–80% of the total polyphenol content in beer comes from malt, and the remaining 30–20% from hops. In this work, liquid chromatography coupled with an electrospray ionization hybrid linear ion trap quadrupole Orbitrap mass spectrometry technique has been used for an accurate identification of beer polyphenols. 47 phenolic compounds were identified using high mass accuracy and confirmed by MS2 experiments, including simple phenolic acids, hydroxycinnamoylquinics, flavanols, flavonols, flavones, alkylmethoxyphenols, alpha- and iso-alpha-acids, hydroxyphenylacetic acids and prenylflavonoids. As far as we know, 7 of these compounds have been recognised in beer for the first time: feruloylquinic acid, caffeic acid-O-hexoside, coumaric acid-O-hexoside, sinapic acid-O-hexoside, catechin-O-dihexoside, kaempferol-O-hexoside, and apigenin-C-hexoside-pentoside.

The QuEChERS approach in a novel application for the identification of antifungal compounds produced by lactic acid bacteria cultures

Lactic Acid Bacteria (LAB) play an important role as natural food preservatives in many fermented food systems. To-date, characterisation of their diverse range of metabolites has been limited. Improved quantitation of low, medium and high concentration antifungal compounds is required, ensuring that both known and unknowns compounds are identified. This manuscript reports the first application of QuEChERS (quick, easy, cheap, effective, rugged and safe) for the extraction of natural antifungal metabolites in LAB cultures. The method provides improved individual recoveries (>78%) for 15 known antifungal compounds, an improvement of 26% compared to previously reported techniques (>52%). A protocol was developed that allowed LAB cultures to be easily assessed on a fully validated high performance liquid chromatography with ultra violet/diode array detection (HPLC-UV/DAD) method. Previously reported methods involving direct injection of filtered extracts and SPE clean-up, suffered from a rise in chromatographic baseline due to interfering matrix components, limiting accurate quantitation. This QuEChERS method removed these interfering matrix components to deliver clean chromatograms with greater recoveries (78.2–127.4%) and lower RSD values (2.5–10.8%) of all 15 antifungal compounds. The validated method was applied to LAB strains showing particularly strong antifungal activity and provided an increase in the number of compounds detected (both known and unknown) compared to previous techniques for the same strains, due to the improved recoveries now possible by this method. Confirmation of the compounds identified was performed by analysis on a liquid chromatography linear ion trap quadrupole Orbitrap hybrid Fourier transform mass spectrometer (LC-FTMS). This first application of QuEChERS to LAB cultures has significantly improved the analytical capabilities of antifungal compound profiling especially where the synergy of numerous compounds is suspected as producing the observed activity. LAB cultures can now be easily integrated into various food matrices, as natural food preservatives, now that a complete analyte profile is achievable.

High-resolution mass spectrometry applied to the identification of transformation products of quinolones from stability studies and new metabolites of enrofloxacin in chicken muscle tissues

The aim of this work was the identification of new metabolites and transformation products (TPs) in chicken muscle from enrofloxacin (ENR), ciprofloxacin (CIP), difloxacin (DIF) and sarafloxacin (SAR), which are antibiotics that belong to the fluoroquinolones family. The stability of ENR, CIP, DIF and SAR standard solutions versus pH degradation process (from pH 1.5 to 8.0, simulating the pH since the drug is administered until its excretion) and freeze–thawing (F/T) cycles was tested. In addition, chicken muscle samples from medicated animals with ENR were analyzed in order to identify new metabolites and TPs.The identification of the different metabolites and TPs was accomplished by comparison of mass spectral data from samples and blanks, using liquid chromatography coupled to quadrupole time-of-flight (LC-QqToF) and multiple mass defect filter (MMDF) technique as a pre-filter to remove most of the background noise and endogenous components. Confirmation and structure elucidation was performed by liquid chromatography coupled to linear ion trap quadrupole Orbitrap (LC-LTQ-Orbitrap), due to its mass accuracy and MS/MS capacity for elemental composition determination.As a result, 21 TPs from ENR, 6 TPs from CIP, 14 TPs from DIF and 12 TPs from SAR were identified due to the pH shock and F/T cycles. On the other hand, 14 metabolites were identified from the medicated chicken muscle samples. Formation of CIP and SAR, from ENR and DIF, respectively, and the formation of desethylene–quinolone were the most remarkable identified compounds.

The proline‐rich tetramerization peptides in equine serum butyrylcholinesterase

Soluble, tetrameric, plasma butyrylcholinesterase from horse has previously been shown to include a non-covalently attached polyproline peptide in its structure. The polyproline peptide matched the polyproline-rich region of human lamellipodin. Our goal was to examine the tetramer-organizing peptides of horse butyrylcholinesterase in more detail. Horse butyrylcholinesterase was denatured by boiling, thus releasing a set of polyproline peptides ranging in mass from 1173 to 2098 Da. The peptide sequences were determined by fragmentation in MALDI-TOF-TOF and linear ion trap quadrupole Orbitrap mass spectrometers. Twenty-seven polyproline peptides grouped into 13 families were identified. Peptides contained a minimum of 11 consecutive proline residues and as many as 21. Many of the peptides had a non-proline amino acid at the N-terminus. A search of the protein databanks matched peptides to nine proteins, although not all peptides matched a known protein. It is concluded that polyproline peptides of various lengths and sequences are included in the tetramer structure of horse butyrylcholinesterase. The function of these polyproline peptides is to serve as tetramer-organizing peptides.

Determination of sedatives and adrenergic blockers in blood meal using accelerated solvent extraction and Orbitrap mass spectrometry

The detection of veterinary drugs in blood meal is needed since it is used as an environment-friendly agricultural material despite its origination from animal blood. A method using accelerated solvent extraction and liquid chromatographic linear ion trap quadrupole Orbitrap mass spectrometry was developed to determine sedatives and adrenergic blockers in blood meal. The determination method was established following optimizations of accelerated solvent extraction, dispersive solid-phase extraction and high resolution mass spectrometric detection. Linearity, sensitivity, accuracy, repeatability and reproducibility of the method were fully validated. The method was applied to commercial blood meal products.