Information-dependent Acquisition Research Articles

Determination of 35 Free Amino Acids in Tea Using Ultra-Performance Liquid Chromatography Coupled With Quadrupole Time-of-Flight Mass Spectrometry.

The purpose of this research was to develop a simple, sensitive, and accurate method for simultaneous determination of 35 free amino acids using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Tea samples were extracted with boiling water bath, and then separated by XBridge BEH Amide column by gradient elution. The exact mass and MS/MS spectra of the target compound was detected under the TOF–MS and Information dependent acquisition (IDA)–MS/MS mode. The results demonstrated good linearity (R2 > 0.9980) in the range of 0.5–1,000 ng/mL. The limits of detection (LODs) were 0.13–25.00 mg/kg and the limits of quantitation (LOQs) were 0.25–50.00 mg/kg. The recovery rate ranged from 70.1 to 105.1% with relative standard deviations (RSDs) <11% (n = 6). This research provides a targeted strategy for developing an analysis method for amino acids in tea.

Ion scanning or ion trapping: Why not both?

The present contribution describes analogies and differences between the quadrupolar ion trap (QIT) and the quadrupole mass analyzers, shows the potentialities of their combination in a single instrument and presents a review of applications of such a technology in different fields. The first section describes the quadrupole mass filter (QMF), outlining its principles of operation and the ion sorting procedure according to the use of oscillating electric fields inducing stable trajectories to the ions allowing them to reach the detector. Multiple quadrupole systems (normally triple quadrupoles) are then explained, showing their use in tandem mass spectrometry in space experiments (MS/MS-in-space). QIT principles of operation are then examined, pointing out that in this case the use of the same combination of oscillating electric fields takes advantage of unstable ion trajectories for their sorting. Substantially, analogies and differences between QMF and QIT come out, which consist in the fact that QMF is a scanning mass analyzer, whereas QIT is a sequential mass analyzer. In addition, the section underlines that QIT is capable to perform tandem mass spectrometry in time experiments (MS/MS-in-time). Later, the possibility to use a quadrupole as a trapping system with a prevailing dimension (linear ion trap [LIT]) is taken into consideration, and the possibility to combine both QMF and LIT in a single instrument, a QTrap mass spectrometer, is illustrated. In this frame, a lot more experiment types are possible with respect to both standalone triple quadrupoles and LIT, and they are described as well. Several combinations of these QTrap features can be used in information dependent acquisition (IDA) mode, allowing the high versatility of this instrumental configuration. The second section deals with a review of applications in different fields. These are organized by kind of QTrap and IDA features and cover different topics in biological, medical, agrochemical, nutritional and environmental fields.

Impurity profiling of bisoprolol fumarate by liquid chromatography-high-resolution mass spectrometry: A combination of targeted and untargeted approaches using a synthesis reaction matrix and general unknown comparative screening

Several examples of the emergence of unexpected impurities in medicinal products in the past, e.g. the 2018 valsartan scandal, disclosed the need for sophisticated approaches in impurity profiling. Advanced techniques in mass spectrometry offer the possibility to detect impurities in untargeted approaches complementing the targeted search for potential impurities. In this study, a combination of targeted and untargeted approaches using LC-MS/HRMS was applied in creating an impurity profile of bisoprolol fumarate. In the targeted approach, a reaction matrix was used to predict potential impurities, which were searched for in addition to the related substances stated in the European Pharmacopoeia. For the untargeted analytics, general unknown comparative screening was performed to detect unexpected impurities. To cover a maximum range of detectable analytes, two complementary mobile phases, i.e. buffered to acidic and basic pH, were combined with four mass spectrometric ionization conditions, i.e. electrospray ionization and atmospheric pressure chemical ionization in both positive and negative mode. Information-dependent acquisition was used to generate MS and MS/MS data in a single run. The targeted and the untargeted approach revealed the presence of 18 and 17 impurities, respectively. The plausibility of proposed/elucidated structural formulae was checked by in silico fragmentation and assignment of characteristic fragments/neutral losses. Quantification of the impurities was performed with respect to the internal standard metoprolol. The analyzed batches showed contents of up to 0.05% of single impurities. A thorough procedure for the development of a complete impurity profile for drug substances was demonstrated. To ensure a maximum of patient safety, the described approach is prototypical and should be implemented during drug development and after relevant changes in manufacturing processes.

Determination of lipophilic marine biotoxins in shellfish foods by ultra performance liquid chromatography-hybrid triple quadrupole linear ion trap-mass spectrometry

An ultra performance liquid chromatography-hybrid triple quadrupole linear ion trap-mass spectrometry(UPLC-QqLIT-MS) was established for determination of lipophilic marine biotoxins in shellfish. And the 12 lipophilic marine biotoxins in shellfish were surveyed. The lipophilic marine biotoxins in homogenized shellfish were ultrasonically extracted by methanol in super-sonic instrument, and cleaned up by solid phase extraction of Strata-X column, and eluted with methanol(containing 0.3% ammonia water). The elution was diluted with water, and cleaned by 0.22 μm millipore filter. The filtrate was separated on a Waters ACQUITY UPLC BEH C_(18) column(150 mm×2.1 mm, 1.7 μm)by gradient elution in 12 minutes with acetronitrile-water(containing 0.01%(V/V) ammonia and 2 mmol/L ammonium formate) as mobile phase, and detected by ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS), identified by electrospray ionization(ESI) in simultaneous scanning mode of positive and negative ions using multiple reaction monitoring, and quantified with external standards. Information dependent acquisition scan function(IDA) combined with enhanced production scan(EPI) was used to confirm the 12 lipophilic marine biotoxins. The calibration curves of 12 lipophilic marine biotoxins showed good linearity in the range of 0.5-50 μg/L with correlation coefficients were 0.9984-0.9999.The detection limits of the method were 0.15-0.29 μg/kg. The recoveries of three spiking levels ranged from 80.0% to 116.0%, and the relative standard deviation(RSD) were 0.6%-6.4%(n=7). The method for determination of 12 lipophilic marine biotoxins in shellfish by UPLC-QqLIT-MS was of operation convenience, less interference from impurities and good accuracy, which could meet the requirements for the determination of 12 lipophilic marine biotoxins residues in sea foods.

Protein Targets of Acetaminophen Covalent Binding in Rat and Mouse Liver Studied by LC-MS/MS.

Acetaminophen (APAP) is a mild analgesic and antipyretic used commonly worldwide. Although considered a safe and effective over-the-counter medication, it is also the leading cause of drug-induced acute liver failure. Its hepatotoxicity has been linked to the covalent binding of its reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI), to proteins. The aim of this study was to identify APAP-protein targets in both rat and mouse liver, and to compare the results from both species, using bottom-up proteomics with data-dependent high resolution mass spectrometry and targeted multiple reaction monitoring (MRM) experiments. Livers from rats and mice, treated with APAP, were homogenized and digested by trypsin. Digests were then fractionated by mixed-mode solid-phase extraction prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Targeted LC-MRM assays were optimized based on high-resolution MS/MS data from information-dependent acquisition (IDA) using control liver homogenates treated with a custom alkylating reagent yielding an isomeric modification to APAP on cysteine residues, to build a modified peptide database. A list of putative in vivo targets of APAP were screened from data-dependent high-resolution MS/MS analyses of liver digests, previous in vitro studies, as well as selected proteins from the target protein database (TPDB), an online resource compiling previous reports of APAP targets. Multiple protein targets in each species were found, while confirming modification sites. Several proteins were modified in both species, including ATP-citrate synthase, betaine-homocysteine S-methyltransferase 1, cytochrome P450 2C6/29, mitochondrial glutamine amidotransferase-like protein/ES1 protein homolog, glutamine synthetase, microsomal glutathione S-transferase 1, mitochondrial-processing peptidase, methanethiol oxidase, protein/nucleic acid deglycase DJ-1, triosephosphate isomerase and thioredoxin. The targeted method afforded better reproducibility for analysing these low-abundant modified peptides in highly complex samples compared to traditional data-dependent experiments.

A complete proteomic profile of human and bovine milk exosomes by liquid chromatography mass spectrometry

Background The current study investigates the proteomic content of milk-derived exosomes. A detailed description of the content of milk exosomes is essential to improve our understanding of the various components of milk and their role in nutrition. Methods The exosomes used in this study were isolated as previously described and characterized by their morphology, particle concentration, and the presence of exosomal markers. Human and bovine milk exosomes were evaluated by Information Dependent Acquisition (IDA) Mass Spectrometry. A direct comparison was made between their proteomic profiles. Results IDA analyses revealed similarities and differences in protein content. 229 and 239 proteins were identified in the human and bovine milk exosome proteome respectively, of which 176 and 186 were unique to each species. Fifty-three proteins were common to both groups. These include proteins associated with specific biological processes and molecular functions. Most notably, the 4 abundant milk proteins lactadherin, butyrophillin, perilipin-2 and xanthine dehydrogenase/oxidase were present in the top 20 list for both human and bovine milk exosomes. Conclusion The milk exosome protein profiles we have provided are crucial new information for the field of infant nutrition. They provide new insight into the components of milk from both humans and bovines.

Determination and structural characterization of ravidasvir metabolites by LC coupled to triple quadrupole linear ion trap MS: Application to pharmacokinetics and phase I metabolism in rats.

Hepatitis C virus (HCV) is an infectious disease that has become a global clinical issue because of its significant morbidity and mortality. Novel anti-hepatitis C drugs are continuously developed to decrease the pervasiveness of the infection globally. A synthetic ravidasvir, benzimidazole-naphthylene-imidazole derivatives, has been used as an anti-HCV drug. This study determined the metabolites of ravidasvir and its pharmacokinetics in rats using information-dependent acquisition and multiple reaction monitoring scanning modes in linear ion trap LC-MS/MS instrument, respectively. Two time-programming linear-gradient chromatographic methods were employed using a Kinetex C18 column (50 × 3 mm, 2.6 μm) and a Luna HILIC column (100 × 4.6 mm, 3 μm) for the qualitative and quantitative determination of ravidasvir and its metabolites, respectively. In silico prediction where sites in a molecule are susceptible to metabolism by cytochrome P450 was implemented, which helped in proposing the metabolic pathway of ravidasvir. The most dominant metabolite in rat liver microsomal samples was oxidative ravidasvir, where one O-demethylated metabolite and eight isomers of the oxidative ravidasvir metabolites were identified. The study provides essential data for proposing the metabolic pathway and successfully applied it to determine the pharmacokinetics of ravidasvir in rat plasma.

Rapid analysis and identification of dianthrone glycosides in Polygoni Multiflori Caulis based on enrichment of macroporous absorbent resin and UPLC-Q-TOF-MS/MS.

Polygoni Multiflori Caulis (PMC) has been used as a traditional Chinese medicine for a long time in China. However, hepatotoxic events of PMC have been reported in recent years, but the potential toxic compounds have remained unclear. Dianthrones as the secondary plant metabolites were revealed to potential hepatotoxicity in a previous study. However, no reports focused on dianthrones in PMC. In the quest for exploring potential hepatotoxic compounds in PMC, the aim of this work was to undertake a comprehensive characterisation of dianthrones in PMC. A simple and effective macroporous absorbent resin column chromatography method was established in this study to enrich the minor dianthrones from PMC extracts. Exploration and characterisation of dianthrones in PMC was conducted by an ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS) method and information dependent acquisition (IDA) mode. The aglycones of dianthrone glycosides were further verified by acid hydrolysis method. Seventy-two dianthrone glycosides and their five aglycones were discovered and tentatively characterised in PMC for the first time, of which 29 dianthrones were identified as potential new compounds. Dianthrone glycosides could be classified into three types according to their aglycone structures, and their fragmentation pathway rules and diagnosed ions were also summarised comprehensively. This was the first comprehensive investigation on dianthrones in PMC. The result would help to fully understand the phytochemical constituents and toxic components in PMC, and highlight the need for further toxicological investigations of the dianthrones in PMC due to their potential hepatotoxicity correlation.

Quantification and Confirmation of Zearalenone Using a LC-MS/MS QTRAP System in Multiple Reaction Monitoring and Enhanced Product Ion Scan Modes

A simple, rapid, and efficient liquid chromatography tandem mass spectrometry (LC–MS/MS) method, operated in electrospray ionization (ESI) and quadrupole linear ion trap modes, has been developed for the identification and structural characterization of zearalenone (ZEN) in corn oil. Samples (5 g) were extracted with acetonitrile/water (80:20, v/v). After centrifugation and dilution, the extracts were separated on a C18 analytical column by gradient elution (acetonitrile/water) and analyzed by UPLC–MS/MS. The developed multiple reaction monitoring–information-dependent acquisition–enhanced product ion method enabled quantification and confirmation of the analyte in a single run. Enhanced product ion mode was used for qualitative analysis, while multiple reaction monitoring mode was used for quantitative analysis. An in-house library was constructed for identification. Calibration curve showed good linearity with correlation coefficients (r) higher than 0.995. Limit of detection was determined to be below 0.20 μg kg−1 for ZEN. The recovery for ZEN was in the acceptable range of 86.6 to 97.2%. 82.4 % of the samples were found to contain ZEN among the 51 samples.

Analytical Methods for Quantification and Identification of Intact Glucosinolates in Arabidopsis Roots Using LC-QqQ(LIT)-MS/MS.

Glucosinolates are biologically active secondary metabolites in Brassicaceae plants that play a critical role in positive and negative interactions between plants and root-associated microbial communities. The aim of this study was to develop a reversed-phase liquid chromatography method to quantify and identify intact glucosinolates in the root of Arabidopsis thaliana (Arabidopsis) grown in non-sterile natural soil by using liquid chromatography-hybrid triple quadruple-linear ion trap (LC-QqQ(LIT)) mass spectrometry. The Synergi Fusion C18-based column was found to be effective for sufficient retention and separation of nine intact glucosinolates without the need for time-consuming desulfation or ion-pairing steps. Method validation results showed satisfactory inter-day and intra-day precision for all glucosinolates except for 4-hydroxyglucobrassicin. Good inter-day and intra-day accuracy and recovery results were observed for glucoiberin, gluconapin, glucobrassicin, 4-methoxyglucobrassicin and neoglucobrassicin. However, for 4-hydroxyglucobrassicin, glucoraphanin and glucoerucin corrections to quantification results might be necessary since the recovery and accuracy results were not optimal. Matrix effects were shown to have a negligible effect on the ionization of all target analytes. The established liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was applied to quantify target intact glucosinolates in Arabidopsis root crude extract of four different wild-type accessions where differences in terms of concentration and composition of intact glucosinolates were observed. Employment of sensitive and selective precursor ion survey scan of m/z 97 in combination with the information-dependent acquisition (IDA) of the enhanced product ion (EPI) dependent scan (Prec97-IDA-EPI) using LC-QqQ(LIT) provided high confidence in structural characterization of diverse intact glucosinolate profiles in complex Arabidopsis root crude extract.

Rapid screening and determination of fentanyl and its analogues in drugs by liquid chromatography- quadrupole time-of-flight mass spectrometry

A method based on liquid chromatography coupled with high-resolution quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) was developed for the simultaneous screening and determination of fentanyl and its 26 analogs in liquid and solid powder drugs. The established method involves successive extraction by 5 mL 75% (v/v) acetonitrile aqueous solution and 5 mL acetonitrile, followed by clean-up using the hydrophilic lipophilic balance (HLB) solid-phase extraction method. Detection was achieved by electrospray ionization (ESI) in the positive mode, TOF-MS, and information-dependent acquisition (IDA)-MS/MS acquisition; the external standard method was adopted for quantification. Two databases of accurate mass and fragment ions were created. The standard matrix-matched calibration curves of the 27 target compounds were linear in the range of 5.00-100 μg/L, with the correlation coefficients (r2)>0. 99. The limits of quantification for the 27 target compounds were 10.0 μg/kg. The recoveries for all the target compounds in vitamin C tablet, headache powder, cough syrup, and transdermal patch samples were in the ranges of 82.9%-106%, 84.8%-106%, 86.9%-109%, and 83.1%-106%, respectively, with relative standard deviations ranging from 0.38% to 8.71% (n=6). The results demonstrated that the developed method is rapid and sensitive for the simultaneous monitoring and determination of fentanyl and 26 its analogs in liquid and solid powder drugs.

Determination of 8 Endogenous Alkaloid Components inBoletusUsing Ultrahigh-Performance Liquid Chromatography Combined with Quadrupole-Time of Flight Mass Spectrometry

A ultrahigh performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UPLC-Q-TOF/MS) method was developed for simultaneous determination of 8 endogenous alkaloid compounds inBoletus.Boletussamples were extracted by 50% (V/V) methanol-water solution, then separated by CORTECS UPLC HILIC column using a binary solvent system by gradient elution. The analytes were determined by Q-TOF/MS in TOF MS and information dependent acquisition (IDA)-MS/MS mode. The results showed that mass accuracy error of the 8 endogenous alkaloids were lower than 5.0 × 10−6, good linear relationship was got in range of 0.2–500 μg/L, and correlation coefficient was higher than 0.9990. The limit of detection was in the range of 0.002–0.100 mg/kg and the limit of quantification was in the range of 0.004–0.200 mg/kg. Recovery of the method was in range of 80.1%–101.5% with spike levels of 0.004–2.00 mg/kg, relative standard deviations were lower than 10%. The method was simple, specific, and reliable. It could be used for the rapid screening and quantitative analysis of 8 endogenous alkaloids inBoletus.

Use of information dependent acquisition mass spectra and sequential window acquisition of all theoretical fragment-ion mass spectra for fruit juices metabolomics and authentication.

LC-MS based untargeted metabolomics are the main untargeted methods used for juice metabolomics to solve the authentication problem faced in fruit juice industry. To evaluate the performances of different untargeted metabolomics methods on fruit juices metabolomics and authentication, orange and apple fruit juices were selected for this study. IDA-MS and SWATH-MS based on UHPLC-QTOF were used for the metabolomics and authenticity determination of apple and orange juices, including the lab-made samples of oranges (Citrus sinensis Osb.) from Jiangxi Province, apples (Malus domestica Borkh) from Shandong Province, and different brands of commercial orange and apple juice samples from markets. IDA-MS and SWATH-MS could both acquire numerous MS1 features and MS2 information of juice components, while SWATH-MS excels at the acquisition rate of MS2. Distinctive separation between authentic orange juice and not authentic orange juice could be seen from principal component analysis and hierarchical clustering analysis based on both IDA-MS and SWATH-MS. After analysis of variance, fold change analysis and orthogonal projection to latent structures discriminant mode, 53 and 46 potential markers were defined by IDA-MS and SWATH-MS (with 77.4% and 100% MS2 acquisition rate) separately. Subsequently, these potential markers were putatively annotated using general chemical databases with 6 more annotated by SWATH-MS. Furthermore, 7 of the potential markers, l-asparagine, umbelliferone, glucosamine, phlorin, epicatechin, phytosphingosine and chlorogenic acid, were identified with standards. For the consideration of model simplicity, two determined makers (umbelliferone and chlorogenic acid) were selected to construct the DD-SIMCA model in commercial samples because of their good correlation with apple adulteration proportion, and the sensitivity and specificity of the model were 100% and 95%. SWATH-MS excels at the MS2 acquisition of juice components and potential markers. This study provides an overall performance comparison between IDA-MS and SWATH-MS, and guidance for the method selection on fruit juice metabolomics and juice authenticity determination. Two of the potential markers determined, umbelliferone and chlorogenic acid, could be used as apple juice indicators in orange juice.

Comprehensive proteomics data on whole rice grain of selected pigmented and non-pigmented rice varieties using SWATH-MS approach.

The proteome data of whole rice grain is considerably limited particularly for rice with pigmentations such as black and red rice. Hence, we performed proteome analysis of two black rice varieties (BALI and Pulut Hitam 9), two red rice varieties (MRM16 and MRQ100) and two white rice varieties (MR297 and MRQ76) using label-free liquid chromatography Triple TOF 6600 tandem mass spectrometry (LC-MS/MS). Our aim was to profile and identify proteins related to nutritional (i.e. antioxidant, folate and low glycaemic index) and quality (i.e. aromatic) traits based on peptide-centric scoring from the Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) approach. Both information dependent acquisition (IDA) and SWATH-MS run were performed in this analysis. Raw data was then processed using ProteinPilot software to identify and compare proteins from the six different varieties. In future, this proteomics data will be integrated with previously obtained genomics [1] and transcriptomics [2] data focusing on the above nutritional and quality traits, with an ultimate aim to develop a panel of functional biomarkers related to those traits for future rice breeding programme. The raw MS data of the pigmented and non-pigmented rice varieties have been deposited to ProteomeXchange database with accession number PXD018338.

Fragmentation mass spectra dataset of linear cyanopeptides - microginins.

Microginins are the less common class of bioactive linear cyanobacterial peptides. Recently, an investigation for their presence in cyanobacteria from Greek freshwaters and strain cultures was carried out. The present dataset is related to the research article “New microginins from cyanobacteria of Greek freshwaters” [1]. Cyanobacterial biomass from bloom samples and cultured strains were extracted with aqueous methanol. Extracts were analysed by liquid chromatography coupled to hybrid triple quadrupole/linear ion trap mass spectrometer (LC-qTRAP MS/MS) in information dependent acquisition (IDA) mode. Enhanced ion product (EIP) mode was applied for the collection of ion fragmentation spectra. Identification of microginins was based on the characteristic fragment ions of the unique microginin amino acid 3-amino-2-hydroxy-decanoic acid (Ahda) and its modified forms. The analysis of fragmentation spectra revealed 51 microginin structures, including 36 new variants. This article provides the dataset of fragmentation mass spectra of the microginins detected in cyanobacteria from Greek freshwaters. As this class of cyanopeptides is produced by cyanobacteria from different geographical regions, the aim of this dataset is to enable the identification of microginins in future studies and therefore to contribute to a better evaluation of their presence in freshwater bodies worldwide.

A Method for the Isolation of Exosomes from Human and Bovine Milk.

Scope Milk provides a natural means of nutrient supply to infants. Exosomes are an important component of milk that are not only being studied for their promise in translational medicine but also in infant nutrition. They also play important roles in intercellular communication and immune function in mammary glands and are able to transfer their materials to the recipient. Therefore, the isolation of high-quality exosomes is an important aspect of exosome research. Methods and Results This study is a technical study, which provides a detailed methodology for the isolation and enrichment of exosomes from milk. In this study, we evaluate the suitability of using the exosome enrichment method that we have recently published for bovine milk, on human milk. We initially isolated extracellular vesicles from human and bovine milk on a fresh set of samples, using ultracentrifugation, and then exosomes were subsequently enriched via size exclusion chromatography (SEC). Following isolation and enrichment, exosomes from both species were characterized by particle concentration (nanoparticle tracking analysis, NTA), morphology (transmission electron microscopy, TEM), and the presence of exosomal markers (immunoblotting and mass spectrometry using information dependant acquisition (IDA)). The key exosomal characteristics of spherical/donut-shaped morphology, the presence of exosomal markers, e.g., FLOT-1 and the tetraspanins, CD9 and CD81), and particle concentration were confirmed in both human and bovine milk exosomes. Conclusion We conclude that our robust exosome enrichment method, previously published for bovine milk, is suitable for use on human milk.

Comparative analysis of constitutes and metabolites for traditional Chinese medicine using IDA and SWATH data acquisition modes on LC-Q-TOF MS

Identification of components and metabolites of traditional Chinese medicines (TCMs) employing liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF MS) techniques with information-dependent acquisition (IDA) approaches is increasingly frequent. A current drawback of IDA-MS is that the complexity of a sample might prevent important compounds from being triggered in IDA settings. Sequential window acquisition of all theoretical fragment-ion spectra (SWATH) is a data-independent acquisition (DIA) method where the instrument deterministically fragments all precursor ions within the predefined m/z range in a systematic and unbiased fashion. Herein, the superiority of SWATH on the detection of TCMs’ components was firstly investigated by comparing the detection efficiency of SWATH-MS and IDA-MS data acquisition modes, and sanguisorbin extract was used as a mode TCM. After optimizing the setting parameters of SWATH, rolling collision energy (CE) and variable Q1 isolation windows were found to be more efficient for sanguisorbin identification than the fixed CE and fixed Q1 isolation window. More importantly, the qualitative efficiency of SWATH-MS on sanguisorbins was found significantly higher than that of IDA-MS data acquisition. In IDA mode, 18 kinds of sanguisorbins were detected in sanguisorbin extract. A total of 47 sanguisorbins were detected when SWATH-MS was used under rolling CE and flexible Q1 isolation window modes. Besides, 26 metabolites of sanguisorbins were identified in rat plasma, and their metabolic pathways could be deduced as decarbonylation, oxidization, reduction, methylation, and glucuronidation according to their fragmental ions acquired in SWATH-MS mode. Thus, SWATH-MS data acquisition could provide more comprehensive information for the component and metabolite identification for TCMs than IDA-MS.

Identification of metabolites in rat plasma,bile,urine and feces after oral administration of Cinnamomi Cortex aqueous extract by UPLC-Qtrap-MS

An ultra-performance liquid chromatography hybrid triple quadrupole-linear ion trap mass spectrometry(UPLC-QtrapMS) method was established to identify the metabolites in rat plasma,bile,urine and feces after oral administration of Cinnamomi Cortex(CC) aqueous extract. Several survey experiments,such as enhanced mass spectrum scan(EMS),precursor ion scan(PI),neutral loss scan(NL) and multiple ions monitoring(MIM) were applied to search target components,and two separate enhanced product ion(EPI) scans were triggered via information-dependent acquisition(IDA) method to generate the MS/MS spectra. According to the mass spectrometric data collected from reference standards and reported literature,the structures of metabolites were deduced. A total of76 metabolites and 5 original compounds were tentatively identified in rats after oral administration of CC aqueous extract. Deglycosylation,methylation,sulfonation,and glucuronidation were observed as the primary metabolic pathways for the chemical constituents of CC. These data are able to benefit the clarification of the therapeutic material basis,the clinical usage and further R&D of CC.

Application of a multiclass screening method for veterinary drugs and pesticides using HPLC-QTOF-MS in egg samples

A multi-class screening method for the determination of veterinary drugs and pesticides in eggs was developed by high performance liquid chromatography quadrupole-time-of-flight mass spectrometry (HPLC-QTOF-MS). A single-injection workflow was developed for qualitative analysis, and method validation was conducted in terms of retention time deviation (ΔRt), exact mass error (Δm), selectivity, specificity, precision and trueness. The characteristic fragment ions in information dependent acquisition (IDA) mode were summarized for retrospective and non-target screening. Five of 70 samples were found positive with sulfachloropyridazine, sulfamonomethoxine and fipronil sulfone. Compared with triple-quadrupole (QqQ) MS method, the obtained results showed that QTOF-MS gave comparable results to QqQ-MS with residue concentrations higher than 10 μg·kg−1, whereas QqQ-MS was more powerful at lower concentrations (<1 μg·kg−1). Moreover, the amount of fragment ions acquired by HPLC-QTOF-MS decreased substantially at trace levels, which may be a potential cause of reduced reliability of identification.

Enhancing signal and mitigating up-front peptide fragmentation using controlled clustering by gas-phase modifiers.

Up-front CID fragmentation is a phenomenon where molecular ions are activated and fragment as they enter the atmosphere-to-vacuum region of the mass spectrometer, and consequently can complicate the mass spectra and their analysis. This phenomenon can be minimized by controlling the voltages on lens/optic elements where ions are sampled from the atmospheric region, but this approach can also have a negative effect on overall ion sensitivity. In this study, we introduce gas-phase modifiers (acetonitrile, acetone, cyclohexane, water, and methanol) to the curtain gas to mitigate up-front CID fragmentation. These modifiers cluster with incoming ions, increasing the energy barrier to fragmentation and consequently reducing the complexity of mass spectra. The clustering is monitored by differential mobility spectrometry-mass spectrometry (DMS-MS) and precursor mass spectrum-scanning. Unlike typical singly charged species, peptide ion-modifier clusters were found to survive through the atmosphere-to-vacuum interface of the mass spectrometer, showing that highly charged peptides cluster most strongly with acetonitrile and acetone. In addition, when peptides cluster with acetonitrile, they produce a large increase in signal intensity for the most highly charged and fragile ions. This results in a significant reduction, up to 90% with some modifiers, in up-front CID fragmentation for these fragile highly charged peptides, increasing the overall analytical sensitivity and decreasing the limits of detection by up to 82% depending on the analyte. The proposed technique has no significant detrimental effect on the peptide mass fingerprinting of a BSA or mAb protein digest, but it does reduce the amount of redundant and data-deficient spectra needed to produce adequate sequence coverage using information-dependent acquisition methods by ~ 40%. We propose that this technique could have a benefit in the fields of proteomics and peptidomics where up-front CID fragmentation and chemical noise routinely mask targets of biological importance. Graphical abstract.