Quadrupole Time-of-flight Research Articles

Rapid chemome profiling of Cistanche salsa using DI-MS/MS~(ALL)

The current study aims to rapidly and comprehensively profile the chemical composition of Cistanche salsa using direct infusion coupled with MS/MS~(ALL)(DI-MS/MS~(ALL)). The C. salsa extract was directly imported into electrospray ionization(ESI) source of quadrupole time-of-flight(Q-TOF) mass spectrometer with an infusion pump at a flow rate of 10 μL·min~(-1). Acquisition program was applied under negative ionization polarity to collect one MS~1 spectrum(m/z 50-1 200), followed by 1 150 MS~2 spectra with precursor isolation window(m/z 1) amongst mass range m/z 50-1 200. After each MS~2 spectrum was matched to its precursor ion, putative identification was conducted through matching mass spectral data with literature and database. A total of 31 components were identified from C. salsa, including 9 phenylethanoid glycosides, 2 iridoids, 4 saccharides, 9 organic acids, and 7 other compounds, similar to those from C. tubulosa and C. deserticola. In conclusion, DI-MS/MS~(ALL), a facile and reliable analytical tool, can be employed for qualitative analysis of chemical constituents in C. salsa. The research offers a promising strategy to achieve rapid chemome profiling of herbal medicine and provides an alternative source of Cistanches Herba.

A practical study of CITES wood species identification by untargeted DART/QTOF, GC/QTOF and LC/QTOF together with machine learning processes and statistical analysis

Illegal logging and trafficking of endangered timber species has attracted the world's major organized crime groups, with associated deforestation and serious social damage. The inability of traditional methodologies and DNA analysis to readily perform wood identification to the species level for monitoring has stimulated research on chemotyping techniques. In this study, simple wood extraction of endangered rosewoods (Dalbergia spp), amenable to use in the field, produced colorful hues that were suggestive of wood species. A more definitive study was conducted to develop wood species identification procedures using high-resolution quadrupole time-of-flight (QTOF) mass spectrometers interfaced with liquid chromatography (LC), gas chromatography (GC), and Direct Analysis in Real Time (DART). The time consuming process of extracting “identifying” mass spectral ions for species identification, contentious due to their ubiquitous nature, was supplanted by application of machine learning processes. The unbiased software mining of raw data from multiple analytical batches, followed by statistical Random Forest analysis, enabled discrimination between both anatomically and chemotypically similar Dalbergia species. Statistical Principal Component Analysis (PCA) scatterplots with 95% confidence ellipses were visually compelling in showing a differential clustering of Dalbergia from other commonly traded and lookalike wood species. The information rich raw data from GC or LC analyses offered a corroborative, legally defensible, and widely available confirmatory tool in the identification of timber species.

A Workflow to Perform Targeted Metabolomics at the Untargeted Scale on a Triple Quadrupole Mass Spectrometer.

The thousands of features commonly observed when performing untargeted metabolomics with quadrupole time-of-flight (QTOF) and Orbitrap mass spectrometers often correspond to only a few hundred unique metabolites of biological origin, which is in the range of what can be assayed in a single targeted metabolomics experiment by using a triple quadrupole (QqQ) mass spectrometer. A major benefit of performing targeted metabolomics with QqQ mass spectrometry is the affordability of the instruments relative to high-resolution QTOF and Orbitrap platforms. Optimizing targeted methods to profile hundreds of metabolites on a QqQ mass spectrometer, however, has historically been limited by the availability of authentic standards, particularly for “unknowns” that have yet to be structurally identified. Here, we report a strategy to develop multiple reaction monitoring (MRM) methods for QqQ instruments on the basis of high-resolution spectra, thereby enabling us to use data from untargeted metabolomics to design targeted experiments without the need for authentic standards. We demonstrate that using high-resolution fragmentation data alone to design MRM methods results in the same quantitative performance as when methods are optimized by measuring authentic standards on QqQ instruments, as is conventionally done. The approach was validated by showing that Orbitrap ID-X data can be used to establish MRM methods on a Thermo TSQ Altis and two Agilent QqQs for hundreds of metabolites, including unknowns, without a dependence on standards. Finally, we highlight an application where metabolite profiling was performed on an ID-X and a QqQ by using the strategy introduced here, with both data sets yielding the same result. The described approach therefore allows us to use QqQ instruments, which are often associated with targeted metabolomics, to profile knowns and unknowns at a comprehensive scale that is typical of untargeted metabolomics.

The HMG-CoA Reductase Inhibitor Activities of Soy Protein Hydrolysates from Papain Hydrolysis

The search for an HMG-CoA reductase inhibitor agent as a safe and inexpensive alternative treatment for hypercholesterolemia has been carried out using soy protein hydrolysates as one of the bioactive peptide sources. This study was conducted to explore the potency of soy protein hydrolysates as an anti hypercholesterolemia agent by an in vitro assay, through the inhibition capacity of the HMG-CoA (3-hydroxy-3-methyl glutaryl-coenzyme A) reductase enzyme as a key component of cholesterol biosynthesis. Sample preparation started with soy protein isolation through acid precipitation and separated by centrifugation. The samples were analyzed the proximate content and hydrolyzed by papain enzyme at concentration 0.2% (w/v), for 0-6 hours and at 37, 50, and 55 oC. The protein hydrolysates were subsequently evaluated for hydrolysis degree (% DH), hydrolysates profile with SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis), and anti-cholesterol assay through HMG-CoA reductase inhibition tests. The sample with the highest inhibition activity was fractionated using gel filtration chromatography (Sephadex G-10) and the molecular weight of fractions was characterized by LCMS QTOF (Liquid Chromatography-Mass Spectrometry Quadrupole Time-of-Flight) for molecular weight determination. The results indicated the optimum hydrolysis conditions of soy protein isolates were obtained at 3 hours incubation, at 50 °C with DH 33.39% and the inhibition value was 95.65% (protein concentration 39.21 μg / mL). LCMS data showed the molecular weight of fractionated peptides were 1514 and 2029 Da. We assumed that both peptides have the same affinity as previous peptides in inhibiting HMG-CoA reductase.

Recovery of Polyphenolic Fraction from Arabica Coffee Pulp and Its Antifungal Applications.

Coffee pulp is one of the most underutilised by-products from coffee processing. For coffee growers, disposing of this agro-industrial biomass has become one of the most difficult challenges. This study utilised this potential biomass as raw material for polyphenolic antifungal agents. First, the proportion of biomass was obtained from the Arabica green bean processing. The yield of by-products was recorded, and the high-potency biomass was serially extracted with organic solvents for the polyphenol fraction. Quantification of the polyphenols was performed by High Performance Liquid Chromatography (HPLC), then further confirmed by mass spectrometry modes of the liquid chromatography–quadrupole time-of-flight (QTOF). Then, the fraction was used to test antifungal activities against Alternaria brassicicola, Pestalotiopsis sp. and Paramyrothecium breviseta. The results illustrated that caffeic acid and epigallocatechin gallate represented in the polyphenol fraction actively inhibited these fungi with an inhibitory concentration (IC50) of 0.09, 0.31 and 0.14, respectively. This study is also the first report on the alternative use of natural biocontrol agent of P. breviseta, the pathogen causing leaf spot in the Arabica coffee.

Electrochemical-based ‘‘antibiotsensor’’ for the whole-cell detection of the vancomycin-susceptible bacteria

In this study, we aim to develop an antibiotic-based biosensor platform ‘Antibiotsensor’ for the specific detection of gram-positive bacteria using vancomycin modified Screen Printed Gold Electrodes (SPGEs). Through this pathway, vancomycin molecules were first functionalized with thiol groups and characterized with quadrupole time of flight (q-TOF) mass spectroscopy analysis. Immobilization of thiolated vancomycin molecules (HS-Van) onto SPGEs was carried out based on self-assembled monolayer (SAM) phenomenon. Electrochemical impedance spectroscopy (EIS) was employed to test the detection and showed a considerable change in impedance value upon the binding of HS-Van molecules onto the electrode surface. Atomic Force Microscopy analysis indicated that SPGE was successfully modified upon the treatment with HS-Van molecules based on the shift in surface roughness from 173 ± 2 nm to 301 ± 3 nm. Fourier Transform Infrared Spectroscopy (FTIR) spectroscopy proved the EIS and AFM results as well by showing characteristic peaks of immobilized HS-Van molecule. As a proof-of-concept, EIS-based susceptibility testing was performed using Escherichia coli, Staphylococcus aureus and Mycobacterium smegmatis bacteria to prove the specificity of obtained SPGE-Van. EIS data showed that the charge transfer resistance (Rct) values changed from 1.08, 1.18 to 26.5, respectively, indicating that vancomycin susceptible S. aureus was successfully attached onto SPGE-Van surface strongly, while vancomycin resistance E. coli and M. smegmatis did not show any significant attachment properties. In addition, different concentration (108-10 CFU/mL) of S. aureus was performed to investigate sensitivity of proposed sensor platform. Limit of detection and limit of quantitation was calculated as 101.58 and 104.81 CFU/mL, respectively. Scanning electron microscopy (SEM) analysis also confirmed that only S. aureus bacteria was attached to the surface in a dense monolayer distribution. We believe that the proposed approach is selective and sensitive towards the whole-cell detection of vancomycin-susceptible bacteria and can be modified for different purposes in the future.

Retention dependences support highly confident identification of lipid species in human plasma by reversed-phase UHPLC/MS.

Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method was developed with the aim to unambiguously identify a large number of lipid species from multiple lipid classes in human plasma. The optimized RP-UHPLC/MS method employed the C18 column with sub-2-μm particles with the total run time of 25 min. The chromatographic resolution was investigated with 42 standards from 18 lipid classes. The UHPLC system was coupled to high-resolution quadrupole-time-of-flight (QTOF) mass analyzer using electrospray ionization (ESI) measuring full-scan and tandem mass spectra (MS/MS) in positive- and negative-ion modes with high mass accuracy. Our identification approach was based on m/z values measured with mass accuracy within 5 ppm tolerance in the full-scan mode, characteristic fragment ions in MS/MS, and regularity in chromatographic retention dependences for individual lipid species, which provides the highest level of confidence for reported identifications of lipid species including regioisomeric and other isobaric forms. The graphs of dependences of retention times on the carbon number or on the number of double bond(s) in fatty acyl chains were constructed to support the identification of lipid species in homologous lipid series. Our list of identified lipid species is also compared with previous publications investigating human blood samples by various MS-based approaches. In total, we have reported more than 500 lipid species representing 26 polar and nonpolar lipid classes detected in NIST Standard reference material 1950 human plasma.

Mass Spectrometry Imaging of Lipids with Isomer Resolution Using High-Pressure Ozone-Induced Dissociation.

Mass spectrometry imaging (MSI) of lipids within tissues has significant potential for both biomolecular discovery and histopathological applications. Conventional MSI technologies are, however, challenged by the prevalence of phospholipid regioisomers that differ only in the location(s) of carbon–carbon double bonds and/or the relative position of fatty acyl attachment to the glycerol backbone (i.e., sn position). The inability to resolve isomeric lipids within imaging experiments masks underlying complexity, resulting in a critical loss of metabolic information. Herein, ozone-induced dissociation (OzID) is implemented on a mobility-enabled quadrupole time-of-flight (Q-TOF) mass spectrometer capable of matrix-assisted laser desorption/ionization (MALDI). Exploiting the ion mobility region in the Q-TOF, high number densities of ozone were accessed, leading to ∼1000-fold enhancement in the abundance of OzID product ions compared to earlier MALDI-OzID implementations. Translation of this uplift into imaging resulted in a 50-fold improvement in acquisition rate, facilitating large-area mapping with resolution of phospholipid isomers. Mapping isomer distributions across rat brain sections revealed distinct distributions of lipid isomer populations with region-specific associations of isomers differing in double bond and sn positions. Moreover, product ions arising from sequential ozone- and collision-induced dissociation enabled double bond assignments in unsaturated fatty acyl chains esterified at the noncanonical sn-1 position.

Proteoform Differentiation using Tandem Trapped Ion Mobility, Electron Capture Dissociation, and ToF Mass Spectrometry.

Comprehensive characterization of post-translationally modified histone proteoforms is challenging due to their high isobaric and isomeric content. Trapped ion mobility spectrometry (TIMS), implemented on a quadrupole/time-of-flight (Q-ToF) mass spectrometer, has shown great promise in discriminating isomeric complete histone tails. The absence of electron activated dissociation (ExD) in the current platform prevents the comprehensive characterization of unknown histone proteoforms. In the present work, we report for the first time the use of an electromagnetostatic (EMS) cell devised for nonergodic dissociation based on electron capture dissociation (ECD), implemented within a nESI-TIMS-Q-ToF mass spectrometer for the characterization of acetylated (AcK18 and AcK27) and trimethylated (TriMetK4, TriMetK9 and TriMetK27) complete histone tails. The integration of the EMS cell in a TIMS-q-TOF MS permitted fast mobility-selected top-down ECD fragmentation with near 10% efficiency overall. The potential of this coupling was illustrated using isobaric (AcK18/TriMetK4) and isomeric (AcK18/AcK27 and TriMetK4/TriMetK9) binary H3 histone tail mixtures, and the H3.1 TriMetK27 histone tail structural diversity (e.g., three IMS bands at z = 7+). The binary isobaric and isomeric mixtures can be separated in the mobility domain with RIMS > 100 and the nonergodic ECD fragmentation permitted the PTM localization (sequence coverage of ∼86%). Differences in the ECD patterns per mobility band of the z = 7+ H3 TriMetK27 molecular ions suggested that the charge location is responsible for the structural differences observed in the mobility domain. This coupling further enhances the structural toolbox with fast, high resolution mobility separations in tandem with nonergodic fragmentation for effective proteoform differentiation.

Influence of Dietary Supplementation With a Powder Containing A.N. ProDen™ (Ascophyllum Nodosum) Algae on Dog Saliva Metabolome.

The objective of this placebo-controlled, double-blind, randomized study (designed according to evidence-based medicine standards) was to determine the effect of 30-day administration of powdered brown algae, Ascophyllum nodosum (ProDen PlaqueOff, SwedenCare AB, Sweden), on saliva metabolomes in dogs. Sixty client-owned dogs underwent professional dental cleaning and were randomly subdivided into two groups receiving daily powdered brown algae A. nodosum, or a placebo (microcrystalline cellulose in powder), adjusted to their bodyweight. After a comprehensive oral health assessment and professional dental cleaning, which were both performed under general anesthesia, clinical assessments for gingivitis, plaque, and calculus were conducted. Saliva samples were collected at Day 0 and Day 30 of supplementation. Whole saliva is a mixed fluid that is derived predominantly from the major salivary glands but it also contains numerous other constituents. Additionally, its composition varies on whether salivary secretion is basal or stimulated. Authors put efforts to avoid contamination of saliva by other constituents and character of saliva was basal. Quadrupole time-of-flight (QTOF) mass spectrometer was used to conduct analysis of the saliva samples. Metabolomic analyses identified clear changes after 30 days of supplementation, and the direction of these changes was completely different than in dogs that received a placebo treatment during the same period. The positive clinical effect of 30 days of A. nodosum supplementation on oral health status in dogs described in previous publication combined with the absence of some metabolites in the saliva of dogs on day 30 of supplementation suggest that brown algae inhibit or turn off some pathways that could enhance plaque or calculus development. The exact mechanism of A. nodosum is still unclear and warrants further study.

Effect of Freeze Drying and Simulated Gastrointestinal Digestion on Phenolic Metabolites and Antioxidant Property of the Natal Plum (Carissa macrocarpa).

Natal plums (Carissa macrocarpa) are a natural source of bioactive compounds, particularly anthocyanins, and can be consumed as a snack. This study characterized the impact of freeze drying and in vitro gastrointestinal digestion on the phenolic profile, antioxidant capacity, and α-glucosidase activity of the Natal plum (Carissa macrocarpa). The phenolic compounds were quantified using high performance liquid chromatography coupled to a diode-array detector HPLC-DAD and an ultra-performance liquid chromatograph (UPLC) with a Waters Acquity photodiode array detector (PDA) coupled to a Synapt G2 quadrupole time-of-flight (QTOF) mass spectrometer. Cyanidin-3-O-β-sambubioside (Cy-3-Sa) and cyanidin-3-O-glucoside (Cy-3-G) were the dominant anthocyanins in the fresh and freeze-dried Natal plum powder. Freeze drying did not affect the concentrations of both cyanidin compounds compared to the fresh fruit. Both cyanidin compounds, ellagic acid, catechin, epicatechin syringic acid, caffeic acid, luteolin, and quercetin O-glycoside from the ingested freeze-dried Natal plum powder was quite stable in the gastric phase compared to the small intestinal phase. Cyanidin-3-O-β-sambubioside from the ingested Natal plum powder showed bioaccessibility of 32.2% compared to cyanidin-3-O-glucoside (16.3%). The degradation of anthocyanins increased the bioaccessibility of gallic acid, protocatechuic acid, coumaric acid, and ferulic acid significantly, in the small intestinal digesta. The ferric reducing antioxidant power (FRAP), 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) activities, and inhibitory effect of α-glucosidase activity decreased in the small intestinal phase. Indigenous fruits or freeze-dried powders with Cy-3-Sa can be a better source of anthocyanin than Cy-3-G due to higher bioaccessibility in the small intestinal phase.

Dissociation of Biomolecules by an Intense Low-Energy Electron Beam in a High Sensitivity Time-of-Flight Mass Spectrometer

We report the progress on an electron-activated dissociation (EAD) device coupled to a quadrupole TOF mass spectrometer (QqTOF MS) developed in our group. This device features a new electron beam optics design allowing up to 100 times stronger electron currents in the reaction cell. The electron beam current reached the space-charge limit of 0.5 μA at near-zero electron kinetic energies. These advances enable fast and efficient dissociation of various analytes ranging from singly charged small molecules to multiply protonated proteins. Tunable electron energy provides access to different fragmentation regimes: ECD, hot ECD, and electron-impact excitation of ions from organics (EIEIO). The efficiency of the device was tested on a wide range of precursor charge states. The EAD device was installed in a QqTOF MS employing a novel trap-and-release strategy facilitating spatial mass focusing of ions at the center of the TOF accelerator. This technique increased the sensitivity 6-10 times and allows for the first time comprehensive structural lipidomics on an LC time scale. The system was evaluated for other compound classes such as intact proteins and glycopeptides. Application of hot ECD for the analysis of glycopeptides resulted in rich fragmentation with predominantly peptide backbone fragments; however, glycan fragments attributed to the ECD process were also observed. A standard small protein ubiquitin (8.6 kDa) was sequenced with 90% cleavage coverage at spectrum accumulation times of 100 ms and 98% at 800 ms. Comparable cleavage coverage for a medium-size protein (carbonic anhydrase: 29 kDa) could be achieved, albeit with longer accumulation times.

Dynamic binning peak detection and assessment of various lipidomics liquid chromatography-mass spectrometry pre-processing platforms

Liquid chromatography-mass spectrometry (LC-MS)-based lipidomics generates large datasets that need to be interpreted using high-performance data pre-processing tools such as XCMS, mzMine, and Progenesis. These pre-processing tools rely heavily on accurate peak detection, which depends on proper setting of the peak detection mass tolerance (PDMT). The PDMT is usually set with a fixed value in either ppm or Da units. However, this fixed value may result in duplicates or missed peak detection and inaccurate peak quantification. To improve the accuracy of peak detection, we developed the dynamic binning method, which considers peak broadening described by the physics of ion separation and sets the PDMT dynamically in function of m/z. In our method, the PDMT is proportional to (mz)2 for Fourier-transform ion cyclotron resonance (FTICR), to (mz)1.5 for Orbitrap and to m/z for Quadrupole time-of-flight (Q-TOF), and is a constant for Quadrupole mass analyzer. The dynamic binning method was implemented in XCMS [1,2], and the adopted source code is available in GitHub at https://github.com/xiaodfeng/DynamicXCMS. We have compared the performance of the XCMS implemented dynamic binning with different popular lipidomics pre-processing tools to find differential compounds. We generated set samples with 43 lipid internal standards that were differentially spiked to aliquots of one human plasma lipid sample using Orbitrap LC-MS/MS. The performance of various pipelines using matched parameter sets was quantified by a quality score system that reflects the ability of a pre-processing pipeline to detect differential peaks spiked at various concentrations. The quality score indicated that our dynamic binning method improves the quantification performance of XCMS (maximum p-value 9.8·10−3 of two-sample Wilcoxon test) over its original implementation. We also showed that the XCMS with dynamic binning found differential spiked-in lipids better or with similar performance as mzMine and Progenesis do.

Inter-platform assessment of performance of high-throughput desorption electrospray ionization mass spectrometry

High throughput analysis is increasingly relevant in both industry and academia, with applications reaching from enzymology and drug discovery to organic reaction optimization and diagnostics. Currently, chromatography-free mass spectrometry (MS) techniques have acquired an important role in this field due to their inherent speed, versatility, and chemical specificity. Desorption electrospray ionization (DESI) MS is one of these approaches, which allows for direct analysis of complex samples in the ambient environment, with throughputs better than 1 sample per second and no need for sample treatment. Here we assess the evolution of the high throughput DESI-MS analytical performance from the early DESI source developed by Prosolia Inc. to the recently commercialized version of Waters Corporation. The evaluation was carried out through both quantitative and qualitative analysis of biologically relevant compounds including metabolites, peptides, lipids and pharmaceuticals. Our results indicate that both platforms are successful at the direct analysis of these species even from complex matrices such as bioassay buffers. However, the newest iteration of the DESI stage, when combined with a quadrupole time-of-flight (Q-ToF) instrument, provides higher sensitivity (1-3 orders of magnitude lower limits of detection) and reproducibility (ca. 10% average reduction in coefficients of variation for quantitation using an internal standard). The cases explored in this study also showcase the broad applicability of high throughput DESI-MS for bioanalysis and quality control.

Salt-Tolerant Metabolomics for Exometabolomic Measurements of Marine Bacterial Isolates.

Identifying and quantifying metabolites secreted by microbial isolates can aid in understanding the physiological traits of diverse species and their interaction with the environment. Mass spectrometry-based metabolomics has potential to provide a holistic view of the exometabolism of marine isolates, but the high salt content of such samples interferes with chromatography and ionization during the measurement of polar exometabolites. The most common desalting methods are faced with major limitations, including limited separation of small polar metabolites from salts, the use of organic solvents that cannot accommodate large salt quantities, and sample throughput. Here, we utilize a cyano stationary phase to develop a high-throughput, isocratic liquid chromatography-mass spectrometry (LC-MS) desalting method that mitigates these shortcomings. We demonstrate that counterions present in a common marine growth medium experience distinct elution times, which prevents their coelution with 73 physiologically relevant polar metabolites, effectively minimizing the effects of salt content on ion suppression. We determined optimal salt concentrations for quadrupole time-of-flight (QTOF) MS measurements and limits of quantification in the low micromolar range in the salty matrix. The efficacy of this method was demonstrated through the measurement of exometabolites secreted by three marine bacterial isolates originating from a carrageenan degrading microbial community. This method provides a simple, versatile desalting method for measuring exometabolites of environmental isolates and other biological matrices.

Benefits of Ion Mobility Separation and Parallel Accumulation-Serial Fragmentation Technology on timsTOF Pro for the Needs of Fast Photochemical Oxidation of Protein Analysis.

Fast photochemical oxidation of proteins (FPOP) is a recently developed technique for studying protein folding, conformations, interactions, etc. In this method, hydroxyl radicals, usually generated by KrF laser photolysis of H2O2, are used for irreversible labeling of solvent-exposed side chains of amino acids. Mapping of the oxidized residues to the protein’s structure requires pinpointing of modifications using a bottom-up proteomic approach. In this work, a quadrupole time-of-flight (QTOF) mass spectrometer coupled with trapped ion mobility spectrometry (timsTOF Pro) was used for identification of oxidative modifications in a model protein. Multiple modifications on the same residues, including six modifications of histidine, were successfully resolved. Moreover, parallel accumulation–serial fragmentation (PASEF) technology allows successful sequencing of even minor populations of modified peptides. The data obtained indicate a clear improvement of the quality of the FPOP analysis from the viewpoint of the number of identified peptides bearing oxidative modifications and their precise localization. Data are available via ProteomeXchange with identifier PXD020509.

Intra-laboratory comparison of four analytical platforms for lipidomic quantitation using hydrophilic interaction liquid chromatography or supercritical fluid chromatography coupled to quadrupole - time-of-flight mass spectrometry

The lipidomic research is currently devoting considerable effort to the harmonization that should enable the generation of comparable and accurate quantitative lipidomic data across different laboratories and regardless of the mass spectrometry-based platform used. In the present study, we systematically investigate the effects of the experimental setup on quantitative lipidomics data obtained by two lipid class separation approaches, hydrophilic interaction liquid chromatography (HILIC) and ultrahigh-performance supercritical fluid chromatography (UHPSFC), coupled to two different quadrupole – time of flight (QTOF) mass spectrometers from the same vendor. This approach is applied for measurements of 268 human plasma samples of healthy volunteers and renal cell carcinoma patients resulting in four data sets. We investigate and visualize differences among these data sets by multivariate data analysis methods, such as principal component analysis (PCA), orthogonal partial least square discriminant analysis (OPLS-DA), box plots, and logarithmic correlations of molar concentrations of individual lipid species. The results indicate that even measurements in the same laboratory for the same samples using different analytical platforms may yield slight variations in the molar concentrations determined. The normalization to a reference sample with defined lipid concentrations can further diminish these small differences, resulting in highly homogenous molar concentrations of individual lipid species. This strategy indicates a potential approach towards the reporting of comparable quantitative results independent from the quantitative approach and mass spectrometer used, which is important for a wider acceptance of lipidomics data in various biomarker inter-laboratory studies and ring trials.

Metabolomic profile associated with obstructive sleep apnoea severity in obese pregnant women with gestational diabetes mellitus: A pilot study.

Obstructive sleep apnoea (OSA) is prevalent in obese women with gestational diabetes mellitus (GDM). The present pilot study explored associations between OSA severity and metabolites in women with GDM. A total of 81 obese women with diet-controlled GDM had OSA assessment (median gestational age [GA] 29weeks). The metabolic profile was assayed from fasting serum samples via liquid chromatography-mass spectrometry (LC-MS) using an untargeted approach. Metabolites were extracted and subjected to an Agilent 1,290 UPLC coupled to an Agilent 6,545 quadrupole time-of-flight (Q-TOF) MS. Data were acquired using electrospray ionisation in positive and negative ion modes. The raw LC-MS data were processed using the OpenMS toolkit to detect and quantify features, and these features were annotated using the Human Metabolite Database. The feature data were compared with OSA status, apnea-hypopnea index (AHI), body mass index (BMI) and GA using "limma" in R. Correlation analyses of the continuous covariates were performed using Kendall's Tau test. The p values were adjusted for multiple testing using the Benjamini-Hochberg false discovery rate correction. A total of 42 women (51.8%) had OSA, with a median AHI of 9.1events/hr. There were no significant differences in metabolomics profiles between those with and without OSA. However, differential analyses modelling in GA and BMI found 12 features that significantly associated with the AHI. These features could be annotated to oestradiols, lysophospholipids, and fatty acids, with higher levels related to higher AHI. Metabolites including oestradiols and phospholipids may be involved in pathogenesis of OSA in pregnant women with GDM. A targeted approach may help elucidate our understanding of their role in OSA in this population.

Identification of chloramination disinfection by-products from phenylalanine in tap drinking water

Phenylalanine (Phe) is widely present in natural water and serves as a precursor of disinfection by-products (DBPs). We reported the identification of chloramination DBPs from Phe in drinking water using ultra-high performance liquid chromatography (UHPLC) coupled with complementary high-resolution quadrupole time-of-flight (QTOF) and triple quadrupole (tQ) tandem mass spectrometry (MS/MS). In the chloraminated Phe water solution, sixteen new DBPs in a total of seventeen were identified based on their accurate mass, MS/MS spectra and 35Cl/37Cl isotopic patterns. Three of these DBPs were verified as benzamide, phenylacetamide, and p-hydroxyphenylacetamide with their standards, while the others were chlorinated derivatives of Phe, hydrazone, amidine, amide and peroxide, in which the unique structures of these DBPs were rarely reported. Their stability and formation process were investigated as well. Furthermore, a method consisting of solid phase extraction (SPE) and UHPLC-MS/MS using dynamic multiple reaction monitoring (dMRM) was developed to investigate these DBPs in authentic waters. Phe, benzamide, phenylacetamide, and N–Cl-2-phenylacetimidamide were detected in chlorinated tap water. Compared with the other identified DBPs, these three DBPs were exceptionally stable and could be formed in wide formation conditions. Our work not only provided ideas for the identification of new chloramination DBPs, but also demonstrated that some DBPs usually generated in the chloramination disinfection process could also be found in the chlorinated drinking water.

Hydrolysis Kinetics of a Novel 3,4-Dichloroisothiazole Plant-Activator Candidate LY5-24-2 by UPLC-Q-TOF

Hydrolysis characteristics of a novel 3,4-dichloroisothiazole based fungicide with activating plant defense responses as a candidate plant-activator LY5-24-2 were investigated under different conditions (pH and temperature) using ultra-performance liquid chromatography (UPLC) and quadrupole Time-of-Flight (Q-TOF). The hydrolysis case complied with the first-order kinetic model, with half-lives ranging from 4.8h to 3.2days at pH 4, 7, 9 and temperature at 25 and 50℃. One of the hydrolysis metabolite 3,4-dichloroisothiazole-5-carboxylic acid (metabolite 1, M1) was determined and quantified using authentic standard. The other hydrolysate 3-chloro-5-(trifluoromethyl) pyridin-2-amine (metabolite 2, M2) was determined and identified according to accurate mass information, fragmentation patterns and principle component analysis (PCA). By utilizing high-resolution mass spectrometry and multivariate statistical analysis, hydrolysis dynamic of the metabolites was characterized and figured out. This research provided a non-target screening method to analyze hydrolysis metabolites of a new plant-activator and to find its degradation products in aqueous solution.