Mass Spectrometry System Research Articles

(Invited) A Novel Dems Approach for Studying Gas Evolution in Pouch Cells

In this study, we designed a novel differential electrochemical mass spectrometry (DEMS) system to in-situ quantify the gas generation in batteries. While a lot of battery gas analyses were carried out by DEMS previously, most of those studies were based on small coin cells or EL cells. The fluid electrolyte and limited amount of active material may make it challenging to quantify the gas generation. The DEMS we designed here can continually sample gas from a small 2.5Ah pouch cell, and it has a similar electrolyte to capacity ratio, 1.8g/Ah, with our production cell. To the best of our knowledge, this is the first DEMS design based on a pouch cell.Moreover, by using flowrate-controlled Ar carrier gas and a reasonable residence time setup, our DEMS design not only can identify volatile electrolyte degradation products throughout battery cycling but also quantify the gaseous species production rates as a function of the state of charge. We found that cell chemistry and operation protocol greatly impact gas product species, the production profile of different gases during cycling, and the total gas quantity. For example, the gas evolution of oxidation products, such as CO2 and CO, is exacerbated by the elevated upper cutoff voltage, while reduction products are only minimally affected. Furthermore, changing the electrolyte SEI former, from EC to FEC, or adding different electrolyte additives can affect the gas product species generation dramatically as well.Therefore, we successfully demonstrated DEMS as a powerful tool to probe the gas evolution behavior in different applications-based batteries, provided valuable data for battery reactivity simulation, and most importantly, provided guidance for electrolyte optimization.

Analysis of Essential Oil of Salix babylonica Collected in Vietnam: Phytochemical Components and Antibacterial and Anticancer Activity

This study investigated the chemical compositions and inhibitory activities of essential oils (EOs) of Salix babylonica from Vietnam. The gas chromatography–mass spectrometry (GC/MS) system was used to analyze the chemical compositions of Salix babylonica essential oils. A total of twenty-eight and thirty-one compounds were identified in essential oils of the leaves and bark, among which many chemical compositions were identified for the first time in this plant. Salix babylonica essential oils demonstrated antibacterial activities against Gram-negative strains such as Pseudomonas aeruginosa (PA) and Escherichia coli and Gram-positive strains such as Staphylococcus aureus (SA), and demonstrated anti-cancer activities against three cancer cell lines (HepG2, MCF-7 cell, and A549). The evaluation of the ability to inhibit three strains of microorganisms and inhibit the growth of three cancer cell lines was first conducted using essential oils extracted from the plant species S. babylonica collected in Asia, which will be the basis for using essential oils of this plant in medicine.

SFC-MS/MS for orthogonal separation of hydroxylated 17α-methyltestosterone isomers.

Because of their performance-enhancing effect, anabolic androgenic steroids (AAS) are often misused in sports. Nearly half of the adverse analytical findings (AAF) in 2022 doping controls are correlated to AAS misuse. Metabolites play a crucial role in the bioanalysis of endogenous and exogenous steroids. Therefore, one important field in antidoping research is the investigation on drug metabolizing and steroidogenic enzymes. The introduction of a hydroxy group is the most common reaction, which is catalyzed by cytochrome P450 (CYP) enzymes in phase-I metabolism. Analysis of AAS metabolites is commonly performed using gas chromatography mass spectrometry (GC-MS) systems. Laborious sample preparation and extended run times compared to liquid chromatography (tandem) mass spectrometry (LC-MS/MS)methods are usually correlated with this type of analysis. On the other hand, liquid chromatography (tandem) mass spectrometry (LC-MS[/MS]) methods have a lower separation efficiency than GC-MS methods. Both techniques lack selectivity for hydroxylated 17α-methyltestosterone metabolites. Therefore, as an orthogonal analytical approach, a supercritical fluid chromatography tandem mass spectrometrymethod was developed to separate four hydroxy metabolites of 17α-methyltestosterone (2α-/2β-/4-/6β-hydroxy-17α-methyltestosterone). This project aimed to get a more in-depth look at the metabolization and analysis of 17α-methyltestosterone and its hydroxylated metabolites. The developed method revealed lower limits of quantitation between 0.6 and 6ng/ml at an accuracy of 85-115% using a matrix matched calibration. An in vitro study with human liver microsomes shows 6β-hydroxy-17α-methyltestosterone as main metabolite (15.9%) as well as the metabolite 2β-hydroxy-17α-methyltestosterone (0.5%). The results show that the developed method is sensitive and robust. In addition, the method allows a previously missing discrimination of the hydroxylated metabolites in a short analysis time without prior, complex derivatizations.

Performance assessment of the Bruker Biotyper MALDI-TOF MS for the identification of difficult-to-identify viridans group streptococci.

Differentiating Streptococcus pneumoniae among nonpneumococcal viridans group streptococci (VGS) is challenging in conventional laboratories. Therefore, we aimed to evaluate the performance of the latest Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system in identifying VGS by comparing the results to those of the specific gene sequencing approach. Clinical isolates were initially identified using the BD Phoenix system to identify Streptococcus species. The optochin test was used to distinguish nonpneumococcal VGS from S. pneumoniae. The species of individual reference strains and clinical isolates were determined by comparing the sequences of the 16S rDNA, gyrB, sodA, groESL, or coaE genes with those in the GenBank sequence databases. We evaluated the performance of the Bruker Biotyper MALDI-TOF MS in VGS identification using two different machines with three databases. We collected a total of 103 nonpneumococcal VGS and 29 S. pneumoniae blood isolates at a medical center in northern Taiwan. Among these isolates, only seven could not be identified at the species level by the specific gene sequencing approach. We found that none of the nonpneumococcal VGS isolates were misidentified as pneumococci by the latest Biotyper system, and vice versa. However, certain strains, especially those in the mitis and bovis groups, could still not be correctly identified. The latest Bruker Biotyper 4.1 (DB_10833) showed significant improvement in identifying VGS strains. However, a specific gene sequencing test is still needed to precisely differentiate the species of strains in the mitis and bovis groups.

Development of an analytical method for the determination of pesticides in tropical fruits by LC-QTOF-MS/MS after QuEChERS extraction sample cleanup and DLLME preconcentration

In this study, QuEChERS extraction was combined with dispersive liquid–liquid microextraction (DLLME) to extract pesticides from tropical fruits for determination by a highly accurate and sensitive liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS/MS) system. The QuEChERS method served as a matrix clean-up tool and the DLLME method preconcentrated the analytes for their determination at trace levels. All parameter variables of the DLLME method were optimized to improve the extraction output and lower the limits of detection and quantification (LOD and LOQ) for all the analytes. Under the optimum experimental conditions, the LOD and LOQ values were found in the range of 0.004–0.013 and 0.27–0.61 μg l−1, respectively. The detection limits achieved by direct LC-QTOF-MS/MS analysis were increased by about 10–260 folds using the optimized DLLME method. To assess the accuracy and applicability of the developed method, spike recovery experiments on tropical fruits were carried out. The matrix matching calibration method was used to enhance the quantification accuracy of the analytes in kiwi, pineapple, and mango matrices, with percent recoveries ranging between 89 and 117%.

OpenASAP: An affordable 3D printed atmospheric solids analysis probe (ASAP) mass spectrometry system for direct analysis of solid and liquid samples

Atmospheric Solids Analysis Probe (ASAP) mass spectrometry is a versatile technique allowing direct sampling of solid and liquid samples, but its adoption is limited due to the high cost of commercial ASAP systems. To address this, we present OpenASAP, an open-source ASAP system for mass spectrometers that can be fabricated for $20 or less using 3D-printing. Our design is readily adaptable to instruments from different manufacturers and can be produced with a variety of additive manufacturing techniques on consumer-grade 3D-printers. The probe allows for rapid sampling of solid and liquid samples without sample preparation, making it useful for high throughput screening, investigating spatial localization and function of analytes in biological samples, and incorporating mass spectrometry in instructional settings. We demonstrate its effectiveness by obtaining mass spectra of three natural product standards at levels as low as 10 ng/ml in liquid samples, and detecting these metabolites in microbial cultures that are difficult to analyze due to complex sample matrices or analyte properties. Furthermore, we demonstrate direct sampling of thin layer chromatography (TLC) spots of these cultures.

Astragalus polysaccharide attenuates nonalcoholic fatty liver disease through THDCA in high-fat diet-fed mice

Ethnopharmacological relevanceAstragalus polysaccharide (APS) extracted from Astragalus membranaceus (Fisch.) Bunge was proven to be effective in preventing high-fat diet (HFD) induced nonalcoholic fatty liver disease (NAFLD). However, the exact mechanisms were not completely elucidated. Aim of the studyThe aim was to reveal the mechanisms of APS on preventing NAFLD from the aspects of regulating bile acids (BAs) homeostasis. Materials and methodsSerum and liver BAs in HFD fed mice with or without APS intervention were quantified with an ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) system. The effect of APS on hepatic proteins involved in BAs synthesis were analyzed with Western blot. Finally, the effect of identified taurohyodeoxycholic acid (THDCA) that was significantly increased by APS on hepatic triglyceride (TG) accumulation was explored in vivo and in vitro. ResultsAPS regulated serum and liver BA profiles in HFD fed mice, especially increased serum THDCA. The levels of hepatic cholesterol 7a-hydroxylase (CYP7A1) and sterol 12a-hydroxylase (CYP8B1) which catalyzed the classical BAs synthesis pathway were significantly decreased by APS, while oxysterol 7a-hydroxylase (CYP7B1) which catalyzed the alternative BAs synthesis pathway was significantly increased by APS. THDCA reduced HFD-induced hepatic lipid accumulation and improved glucose homeostasis in mice, and decreased TG level in palmitic acid/oleic acid treated alpha mouse liver 12 (AML-12) cells. THDCA significantly downregulated the protein level of cluster of differentiation 36 (CD36) involved in fatty acid transport into the liver. Importantly, THDCA showed similar effect with APS in upregulating hepatic CYP7B1 and downregulating CYP7A1. ConclusionThis study revealed the protective effect of APS on NAFLD was associated with the regulation on BA profiles, and proved the potential anti-NAFLD effect of THDCA, highlighting the involvement of BA metabolism in efficacy of herb-derived polysaccharides on metabolism.

Long-term performance of simultaneous measurement of stable isotopes of oxygen and carbon in cellulose with a high-temperature pyrolysis/gas chromatography/isotope ratio mass spectrometry system at the Institute for Geological and Geochemical Research

AbstractA high-temperature pyrolysis/gas chromatography/isotope ratio mass spectrometry system was established at the Institute for Geological and Geochemical Research in 2013. A dedicated field of application of the system is the simultaneous measurement of stable carbon and oxygen isotope ratios in the cellulose of modern, relict and subfossil plant tissues and sediments. The measurement protocol was fine-tuned during the first year of operation and documented in detail in this report. To quantify the long-term reproducibility of the simultaneous measurement of stable isotopes of oxygen and carbon in cellulose, a 2σ range inferred from repeated measurements of a Quality Assurance standard can be recommended: 0.16 and 0.20‰, for δ13C and δ18O, respectively. An extensive set of samples with known pyrolysis-based δ13C data was analyzed in combustion mode and the paired results were used to assess the necessity of adjustment of the pyrolysis-based δ13C measurements. The variances of the two datasets were not significantly different; the slope (intercept) of the regression was indistinguishable from unity (zero), suggesting that probably owing to the relatively frequent cleaning of the pyrolysis furnace, pyrolysis-based δ13C data neither suffer from a variance bias nor require a specific adjustment.

A region-resolved proteomic map of the human brain enabled by high-throughput proteomics.

Substantial efforts are underway to deepen our understanding of human brain morphology, structure, and function using high-resolution imaging as well as high-content molecular profiling technologies. The current work adds to these approaches by providing a comprehensive and quantitative protein expression map of 13 anatomically distinct brain regions covering more than 11,000 proteins. This was enabled by the optimization, characterization, and implementation of a high-sensitivity and high-throughput microflow liquid chromatography timsTOF tandem mass spectrometry system (LC-MS/MS) capable of analyzing more than 2,000 consecutive samples prepared from formalin-fixed paraffin embedded (FFPE) material. Analysis of this proteomic resource highlighted brain region-enriched protein expression patterns and functional protein classes, protein localization differences between brain regions and individual markers for specific areas. To facilitate access to and ease further mining of the data by the scientific community, all data can be explored online in a purpose-built R Shiny app (https://brain-region-atlas.proteomics.ls.tum.de).

A high-throughput quality control method for assessing the serial dilution performance of dose–response plates with acoustic ejection mass spectrometry

This study aimed to develop a streamlined method for evaluating the dilution ratio of drug dose–response plates created by automated liquid handlers in the early stages of drug discovery. The quantitative techniques commonly used for this purpose have restrictions due to their limited linear dynamic range and inaccuracies in assessing serial dilution performance. To address this challenge, we describe a method based on acoustic ejection mass spectrometry (AEMS). The method involves using standard compounds and an internal standard to evaluate each dilution point in quality control (QC) plates. The samples are transferred to a chromatography-free tandem mass spectrometry system through an acoustic source, enabling the analysis of one sample per three seconds from a microtiter plate. This approach provides precise, accurate, label-free, and rapid data acquisition to support high-throughput screening efforts.

Determination of pesticide residue in marginal lagoons of natural parks in Brazil using an improved calibrate passive sampler

Passive sampling is a sensitive and efficient method for analyzing pesticides in water. This article describes a hollow fiber liquid-phase microextraction (HF-LPME) device that was improved using polypropylene membranes grafted with nanocellulose for the passive sampling of pesticides in water. A comprehensive gas chromatography time-of-flight mass spectrometry (GCxGC/Q-TOFMS) system was used to separate, identify, and quantify pesticides. The sampling rates of 38 moderately hydrophobic to hydrophobic agricultural pesticides (2.18 < log Kow < 6.89) from different chemical classes, including the main triazine, organochlorine and organophosphate compounds, were calculated. A calibration process was applied to evaluate the role of flow velocity and select potential candidates for a possible performance reference compound (PRC). Sampling rates varied between 0.17 mL d−1 and 23.15 mL d−1. The accumulation curves identified linear periods ranging from 3 to 18 days. The new passive sampler device was applied for 8 days in rivers and marginal lagoons of natural parks of the São Francisco basin in Minas Gerais, Brazil and identified 10 target pesticides. Furthermore, 10 non-targeted pesticides were detected by the GCxGC/Q-TOFMS method.

Rapid Droplet Sampling Interface for Low-Volume, High-Throughput Mass Spectrometry Analysis.

Here, we present a rapid droplet sampling interface (RDSI) electrospray ionization mass spectrometry (ESI-MS) system as a high-throughput, low-volume, noncontact, and minimal-carryover approach for characterization of liquids. Liquid characterization was achieved by combining droplet ejection with an open-face microflow capillary with a 2.5 μL/min continuous flow of carrier solvent. Through this implementation, single 0.3 nL droplets containing the analyte effectively mix with 4-8 nL of carrier solvent and create a combined electrospray plume. The carrier solvent continuously cleaned the system, eliminating carryover. A sampling rate of 5 Hz was achieved for droplets containing 1 μM propranolol or 5 μM leu-enkephalin with each droplet fully baseline-resolved (138 ± 32 ms baseline-to-baseline). Using a SCIEX API4000 mass spectrometer, a lower limit of quantification (LLOQ) of propranolol was 15 nM, corresponding to 1.16 fg of propranolol in the droplet, and was linear across 3 orders of magnitude. Quantitation could be achieved by adding an isotopically labeled internal standard, as done in conventional ESI. Signal transients were faster than the acquisition speed of the mass spectrometer, resulting in artificially high reproducibility of 15-30% RSD droplet-to-droplet. Analyte-solvent mixing ratios could be controlled by adjusting droplet positioning along the open-face capillary with an optimal position about 0.4 mm from the tip end. The range of analyte coverage was exemplified by measures of peptides and drugs in methanol, water, and buffer solutions. In a comparison to the Open Port Sampling Interface (OPSI) implemented on the same system, the RDSI had 78× greater sensitivity, 6× greater throughput and used significantly less carrier solvent.

Evolution of Relaxor‐Type High‐k Dielectric in Bulk Pristine Cu2+ and Li+ Co‐Substituted Wurtzite ZnO

AbstractDielectric constants determine the level of miniaturization in capacitive components involved in electronic devices. Cu‐Li co‐doping is attempted to introduce the multiferroic relaxor nature of dielectricity and dilute magnetism in the bulk material simultaneously as Li+ ion substitution in ZnO lattice is known to form acceptor states within the band gap of ZnO and paramagnetic Cu2+ present in ZnO lattice can introduce dilute magnetism. Phase purity and substitution of Cu2+ and Li+ ions in ZnO lattice along with oxide‐ion vacancy formation was confirmed using Powder x‐ray diffraction (XRD), Scanning Electron Microscope (SEM), and Energy Dispersive X‐ray analysis (EDX), X‐ray Photoelectron Spectroscopy (XPS), Inductively Coupled Plasma Mass Spectrometry (ICP‐MS) and Magnetic property measurement system (MPMS) studies. The dielectric constant (ϵr′) of bulk pristine Zn0.92Cu0.05Li0.03O and Zn0.9Cu0.05Li0.05O is found to be 18000 and 20000 respectively at 1000 Hz frequency at 600 °C. Both dielectric constant and dielectric loss were decreasing with increasing frequencies. The increase in dielectric constant (ϵr′) with increasing temperatures at different frequencies for Zn0.92Cu0.05Li0.03O1‐δ and Zn0.9Cu0.05Li0.05O1‐δ is likely due to the localized nature of hopping charge carriers in addition to interfacial polarization due to space charge. Tm was found to increase with increasing frequencies suggesting the relaxor nature of dielectricity in the Zn0.9Cu0.05Li0.05O1‐δ. Bulk pristine Zn0.9Cu0.05Li0.05O1‐δ also exhibits ferroelectricity at room temperature with remnant polarization Pr and Vc equal to 4.20e–02 μC/cm2 and 4.1e+03 V/cm at (30KV, 500 Hz) respectively. Zn0.9Cu0.05Li0.05O also shows the paramagnetic behaviour.

An Integrated Molecular Networking and Docking Approach to Characterize the Metabolome of Helichrysum splendidum and Its Pharmaceutical Potentials.

South Africa is rich in diverse medicinal plants, and it is reported to have over 35% of the global Helichrysum species, many of which are utilized in traditional medicine. Various phytochemical studies have offered valuable insights into the chemistry of Helichrysum plants, hinting at bioactive components that define the medicinal properties of the plant. However, there are still knowledge gaps regarding the size and diversity of the Helichrysum chemical space. As such, continuous efforts are needed to comprehensively characterize the phytochemistry of Helichrysum, which will subsequently contribute to the discovery and exploration of Helichrysum-derived natural products for drug discovery. Thus, reported herein is a computational metabolomics work to comprehensively characterize the metabolic landscape of the medicinal herb Helichrysum splendidum, which is less studied. Metabolites were methanol-extracted and analyzed on a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. Spectral data were mined using molecular networking (MN) strategies. The results revealed that the metabolic map of H. splendidum is chemically diverse, with chemical superclasses that include organic polymers, benzenoids, lipid and lipid-like molecules, alkaloids, and derivatives, phenylpropanoids and polyketides. These results point to a vastly rich chemistry with potential bioactivities, and the latter was demonstrated through computationally assessing the binding of selected metabolites with CDK-2 and CCNB1 anti-cancer targets. Molecular docking results showed that flavonoids (luteolin, dihydroquercetin, and isorhamnetin) and terpenoids (tiliroside and silybin) interact strongly with the CDK-2 and CCNB1 targets. Thus, this work suggests that these flavonoid and terpenoid compounds from H. splendidum are potentially anti-cancer agents through their ability to interact with these proteins involved in cancer pathways and progression. As such, these actionable insights are a necessary step for further exploration and translational studies for H. splendidum-derived compounds for drug discovery.

A new MAS-related G protein-coupled receptor X2 cell membrane chromatography analysis model based on HALO-tag technology and its applications

Cell membrane chromatography (CMC) is an effective method for studying receptors with multiple transmembrane structure such as MAS-related G protein-coupled receptor X2 (MrgX2). CMC relies on the maintenance of the complete biological structure of a membrane receptor; however, it needs to be further improved to obtain a more convenient and stable CMC model. In the present study, the haloalkane dehalogenase protein tag (HALO-tag) technology was used to construct a new MrgX2/CMC model. The fusion receptors of MrgX2 with HALO-tag at the C terminus were expressed in HEK293 cells. The silica gel was modified with a substrate of HALO-tag (chloroalkanes) via one-step acylation for the rapid capture of fusion receptors. The new CMC model (MrgX2-HALO-tag/CMC model) was not only quicker to prepare but also more stable and had a longer lifespan than a previous MrgX2-SNAP-tag/CMC model. In combination with the high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) system, the MrgX2-HALO-tag/CMC model was used to screen and identify bioactive components in traditional Chinese medicine. Using this combination, sanggenon C and morusin were identified from Mori Cortex as anti-pseudo-allergic components. The MrgX2-HALO-tag/CMC model alone was also applied to analyze ligand–receptor interaction. The affinity order of four ligands to MrgX2 was as follows: desipramine < imipramine < amitriptyline < clomipramine. This was consistent with the results obtained using the MrgX2-SNAP-tag/CMC model. The MrgX2-HALO-tag/CMC model provides ideas and application prospects for the immobilization of cell membrane that contains receptors with more transmembrane structures.

A side-by-side comparison of the new VITEK MS PRIME and the MALDI Biotyper sirius in the clinical microbiology laboratory

PurposeThis study aims to evaluate the performance of two latest generation matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems in routine laboratory settings, focusing on turnaround time (TAT), time to results (TTR), hands-on time, and identification rate.MethodsWe conducted a time and motion study on three workflow scenarios to simulate different laboratory settings. Overall, 618 bacterial isolates from a tertiary hospital’s laboratory were processed using the VITEK MS PRIME (bioMérieux) and the MALDI Biotyper sirius (Bruker Daltonics) and their corresponding databases VITEK IVD Database 3.2 and MBT reference library 12.ResultsThe target preparation process showed no significant difference in TAT, but the Biotyper workflow had a shorter hands-on time by 3 to 6 min. In the measurement process, TTR was three to five times shorter for the Biotyper sirius while hands-on time was significantly shorter for VITEK MS PRIME (approximately 1.5 min per target). The identification rate without retesting was 97.9% for VITEK MS PRIME and 98.9% for Biotyper sirius. Both systems achieved 100% agreement at genus and 96.2% at species level.ConclusionBoth systems exhibited excellent identification rates for routine bacterial isolates. Due to its high speed, the Biotyper sirius is suited for laboratories with high sample throughput and a workflow designed for processing larger batches. The VITEK MS PRIME, with its “load and go” system accommodating up to 16 targets, reduces hands-on time, making it a reasonable choice for laboratories with fewer identifications overall but a higher number of targets and a workflow designed for parallel processing on different workstations.

A-012 Novel Method for Determining the Concentration of Total N-terminal Pro-hormone BNP (NT-proBNP) in a Sample with Mass Spectrometric Detection

Abstract Background NT-proBNP, the N-terminal fragment of the hormone B-type natriuretic peptide (BNP), is a useful clinical biomarker for diagnostic and prognostic evaluation of heart failure (HF). Recent studies show that commercially available NT-proBNP assays underestimate concentrations of NT-proBNP due to the usage of antibodies targeting the glycosylated central region of the NT-proBNP molecule. With this, a clinical need arises to establish a robust and reliable assay method which can measure the total amount of NT-proBNP, independent of the glycosylation status of the molecule. Methods To determine the concentration of NT-proBNP in patient samples, a known quantity of labelled NT-pro-BNP was added to each sample as standard. The capture of the analyte (native and standard) was performed by Dynabeads™ M-270 Epoxy beads coated with polyclonal goat antibodies, targeting non-glycosylated sites of the NT-proBNP molecule. Magnetic beads were added to the sample and incubated overnight. The isolated analytes were trypsinated directly on the beads after treatment for denaturation by unfolding and oxidation/alkylation. The reaction was stopped by addition of formic acid and the tryptic peptides were cleaned on C18 SPE columns, dried by SpeedVac and resuspended in acetonitrile for separation and analysis on LC-MS/MS (Agilent 1200 Series LC/MS system coupled to an Agilent G6490 triple quadrupole mass spectrometer). Results The Gentian LC-MS/MS validation was conducted including five studies such as Linearity, LoQ, Trueness, Precision and Carry-Over. All studies passed our defined acceptance criteria. To elucidate how glycosylation influences measurements of NT-proBNP, 81 plasma and serum samples covering a concentration range from 100 ng/L to 25 000 ng/L were measured by the Gentian LC-MS/MS method and the Roche Elecsys® proBNP I assay. Within the clinically relevant range of 100–2000 ng/L, the results between both methods exhibit a linear correlation of R2 = 0.97 and show that the commercial assay consistently underestimates the concentration of NT-proBNP by an average factor of 4.3 (3.5–5.8). Furthermore, results reveal that the concentration measurements between the commercial assay and the LC-MS/MS method converge at higher NT-proBNP concentrations. Method comparison was repeated using the Siemens ADVIA Centaur® and the Siemens IMMULITE Immunoassay systems, confirming the results of underestimation of NT-proBNP in clinically relevant areas. For method robustness investigations, the LC-MS/MS method was set up and used at two geographically different sites by different operators, using two different mass spectrometry systems (Agilent 1200 with triple quadrupole and the nano-LC-ESI-Orbitrap from Thermo Fisher Scientific), resulting in CVs from 13% to 2% for NT-proBNP concentrations ranging from 220 ng/L to 9700 ng/L. Conclusion Gentian AS presents a robust and reliable mass spectrometry method, capable of detecting the total amount of NT-proBNP in a sample, independent of its degree of glycosylation. This method is suitable for serving either as a reference method for calibrating and ensuring the accuracy of existing methods, harmonization of existing methods, or as a novel method in clinical chemistry for accurately determining the concentration of total amount of NT-proBNP.

A head-to-head comparison of three MALDI-TOF mass spectrometry systems with 16S rRNA gene sequencing.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized diagnostics in culture-based microbiology. Commonly used MALDI-TOF MS systems in clinical microbiology laboratories are MALDI Biotyper (Bruker Daltonics) and Vitek MS (bioMérieux), but recently the new EXS2600 (Zybio) has been launched. This study aimed to evaluate the performance of the three devices by comparing the results to 16S rRNA gene sequencing. A set of 356 previously collected difficult-to-identify bacteria was tested in parallel with the three systems. Only the direct smear method and simple formic acid extraction were applied. Valid results were achieved for 98.6%, 94.4%, and 93.3% of all isolates by MALDI Biotyper, EXS2600, and Vitek MS, respectively. Of all valid results, agreement with sequencing data was achieved in 98.9%, 98.5%, and 99.7% by MALDI Biotyper, EXS2600, and Vitek MS, respectively. Considering only the isolates with valid measurements at the single-species level, misidentification rates were 0%, 2.6%, and 1.1% for MALDI Biotyper, EXS2600, and Vitek MS, respectively. Apart from minor performance differences, our data demonstrate that the three systems provide comparable results and are suitable for use in medical diagnostic laboratories.

Comprehensive quality control of Qingjin Yiqi granule based on UHPLC-Q-Orbitrap-MS and UPLC-QQQ-MS.

Qingjin Yiqi granule (QYG) is a prescription medicine of traditional Chinese medicine which is widely used clinically for the recovery of coronavirus patients. However, there is currently limited research on the quality control of QYG. To evaluate the quality of QYG qualitatively and quantitatively by making full use of advanced chromatography-mass spectrometry techniques. Firstly, a multicomponent characterisation of QYG was performed by ultrahigh-performance liquid chromatography coupled with a Q Exactive™ hybrid quadrupole-Orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS) system using a rapid negative/positive switching mode. Secondly, the co-condition fingerprint analysis of constituted herbal medicines of QYG was performed to unveil active ingredients as the quality markers of QYG. Thirdly, the marker compounds in 10 batches of QYG were quantified by ultrahigh-performance liquid chromatography coupled with a Waters Xevo TQ-S triple quadrupole mass spectrometry (UPLC-QQQ-MS) system. A comprehensive method that combined the inclusion list and data-dependent acquisition (DDA) to achieve a systematic characterisation of QYG was established by UHPLC-Q-Orbitrap-MS. After analysis based on Compound Discoverer software and Global Natural Products Social (GNPS) platform, a total of 332 compounds were detected. Eleven Q-markers were determined for the quality evaluation of QYG by comparison with the fingerprint of nine constituted herbal medicines. An adjusted multiple reaction monitoring (MRM) quantification method was further established to simultaneously determine the 11 Q-markers for holistic quality evaluation of QYG. This is the first study to report comprehensive multicomponent characterisation, identification, and quality assessment of QYG, which could be used for effective guarantee of the quality of QYG.

Altered gut microbiota and metabolites in untreated myasthenia gravis patients.

The homeostasis of the immune system is influenced by the gut microbiota. Previous studies have reported dysbiosis in the gut microbiota of myasthenia gravis (MG) patients. To investigate potential alterations in gut microbiota and metabolites in newly diagnosed and untreated MG patients, we conducted a case-control study. Fecal samples were collected from 11 newly diagnosed and untreated MG patients as well as 11 age-and sex-matched healthy controls. These samples underwent analysis for gut microbiota using 16S ribosomal RNA (rRNA) gene sequencing, while fecal metabolome was analyzed using liquid chromatography-electrospray tandem mass spectrometry system (LC-ESI-MS/MS). The microbial community richness (observed species) and diversity (Shannon and Simpson indices) were significantly lower in the MG group compared to the control group. Microbiota composition analysis revealed significant differences between the MG and control groups at phylum, family, and genus levels. Linear discriminant analysis effect size (LEfSe) analysis showed a substantial decrease in abundance of the genus Faecalibacterium within the MG group. Fecal metabolome analysis identified three up-regulated metabolites involved in amino acid metabolism (taurine, creatinine, L-carnitine), one up-regulated metabolite involved in lipid metabolism (oleic acid), with correlation analysis indicating a positive association between Faecalibacterium abundance and creatinine levels. Our findings suggest that dysbiosis already exists in newly diagnosed and untreated MG patients, implying that dysbiosis within the gut microbiota may be an initiating factor contributing to MG pathogenesis. Furthermore, F. prausnitzii may hold promise as a probiotic for treating MG.