Positive Mode Research Articles

Inquiry on Threats of Evil within the Hostile-World Scenario: Emerging Content and Mental Health Concomitants Among Holocaust Survivors

ABSTRACT Exposure to human evil, referring to malevolent deeds that deliberately inflict suffering or death, can be psychologically traumatic. This study examined self-perceived evil-related threats within the conception of hostile-world scenario (HWS) that signifies one’s mental representation of major threats in life. The study explored whether evil-related threats, along with HWS, differentiated Holocaust survivors from comparisons, and how these concepts related to mental health. Participants were Israelis aged 58–93, including 220 Holocaust survivors and 205 non-Holocaust survivors. Participants completed measures of mental health (neuroticism, positive and negative affect, life satisfaction, depressive symptoms) and HWS. Evil-related threats were assessed by a 7-item Evil Scale separated from the HWS Questionnaire. Evil-related threats contained two factors, fear of human violence and interpersonal distrust, and correlated with lower mental health. With age and gender controlled, Holocaust survivors were higher than comparisons on evil-related threats and negative engagement with HWS. Beyond associations of HWS (in its negative and positive engagement modes) with mental health, evil-related threats related to higher depressive symptoms among survivors. Results suggest that early traumatic experiences related to perceived threats in later life. Evil-related threats complemented the HWS in associating with mental health. The findings bear implications in approaching victims of evil and trauma.

Dried blood spot sampling coupled with liquid chromatography-tandem mass for simultaneous quantitative analysis of multiple cardiovascular drugs

Dried Blood Spots (DBS) revolutionize therapeutic drug monitoring using LC-MS for the precise quantification of cardiovascular drugs (CDs), enabling personalized treatment adapted to patient-specific pharmacokinetics with minimal invasiveness. This study aims to achieve simultaneous quantification of eight CDs in DBS, overcoming physicochemical challenges. A two-step protein precipitation method was used for simple and precise sample preparation. The drugs were analyzed using LC-MS/MS in ESI positive-ion mode, showing high sensitivity and linearity, with a correlation coefficient (r2) exceeding 0.999, after being separated on a reversed-phase chromatography by gradient elution of DW-acetonitrile containing 0.1 % formic acid + 2 mM ammonium formate. The validation results indicate good selectivity, with no observed matrix effect and carry-over. The intra- and inter-day accuracy and precision were within 6 % for most drugs, except for digoxin and deslanoside at low therapeutic levels where the variation was within 20 %. Stability tests confirmed suitable DBS handling and storage conditions, indicating drug stability for at least 30 days at room temperature. The analysis of whole spot has demonstrated remarkable precision and reliability in all target drugs. The analysis of 3 mm internal diameter discs, punched in and out of DBS, presumed to contain 3 µL of blood, showed acceptable accuracy for most drugs, with less polar drugs like digoxin and deslanoside showing lower accuracy, indicating a need for further correction due to non-uniform drug distribution. Consequently, the developed LC-MS/MS method enables the quantification of multiple CDs in a single DBS analysis, while suggesting the potential for accuracy-based analysis.

Chemical constituent characterization and determination of Quisqualis fructus based on UPLC-Q-TOF-MS and HPLC combined with fingerprint and chemometric analysis.

Quisqualis fructus (QF) is a traditional Chinese medicine (TCM) that it has a long history in the therapeutic field of killing parasites, eliminating accumulation, and stopping diarrhea. However, the therapeutic material basis of QF is remaining ambiguous nowadays. The geographical origin differences of QF are also usually ignored in the process of medication. In this study, the alcohol-aqueous soluble constituents in QF from different origins were systematically characterized and accurately measured by ultra-high performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and high-performance liquid chromatography (HPLC) respectively. Chemometric analysis was performed for origin differentiation and screening of potential quality marker (Q-marker). Finally, A total of 106 constituents were tentatively characterized in positive and negative ion modes, including 29 fatty acids, 26 organic acids, 11 amino acids and derivatives, 10 glycosides, 9 alkaloids and derivatives, and 21 other constituents. QF from different origins were effectively distinguished and 16 constituents were selected as the potential Q-markers subsequently. Four representative components (trigonelline, adenosine, ellagic acid, and 3,3'-di-O-methylellagic acid) in QF samples were simultaneously determined. HPLC fingerprint analysis indicated that the similarity between 16 batches of QF was in the range of 0.870-0.999. The above results provide some insights for the research on the pharmacodynamic constituents, quality control, and geographical discrimination of QF.

Metabolomic screening of anti-inflammatory compounds in Acanthopanax sessiliflorus fruit (Ogaza) extract

This study investigated the anti-inflammatory compounds in Ogaza, Acanthopanax sessiliflorus fruit, and their extracts using metabolomic screening. Ogaza extracts were obtained in various solvents, such as 70% ethanol, 70% methanol, and water. The anti-inflammatory activity was estimated by evaluating nitric oxide production in lipopolysaccharide (LPS)-induced RAW 264.7 cells treated with the extracts. The 70% ethanol extract (EO) showed the most effective anti-inflammatory activity, inhibiting nitric oxide production by approximately 50% and downregulating iNOS expression. The 70% ethanol extract was further fractionated into three partial subfractions by preparative LC to identify the anti-inflammatory compounds. Assessment of the anti-inflammatory activity of each subfraction revealed that the third subfraction (E-F3) showed the highest inhibitory activity against nitric oxide. E-F3 effectively suppressed iNOS expression. Subsequently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) and multivariate statistical analyses were performed to identify the compounds that significantly contributed to the anti-inflammatory activity of the Ogaza extract. Fourteen and 16 compounds in the negative- and positive-ion modes, respectively, were identified as significant constituents of Ogaza. Compounds like quercetin, hyperoside, acanthoside D, oleanolic acid, and scopoletin were identified as potential anti-inflammatory components in Ogaza extract. This study characterized the functional properties of Acanthopanax sessiliflorus fruit and indicated the possibility that other compartments of Acanthopanax sessiliflorus may also serve as natural sources of nutraceuticals.

Integrated Transcriptome and Metabolome Analysis Reveals Molecular Mechanisms Underlying Resistance to Phytophthora Root Rot.

Soybean production is significantly impacted by Phytophthora root rot (PRR), which is caused by Phytophthora sojae. The nucleotide-binding leucine-rich repeat (NLR) gene family plays a crucial role in plant disease resistance. However, current understanding of the function of soybean NLR genes in resistance to PRR is limited. To address this knowledge gap, transgenic soybean plants overexpressing the NLR gene (Glyma.18g283200) were generated to elucidate the molecular mechanism of resistance. Here, transcript changes and metabolic differences were investigated at three time points (12, 24, and 36 h) after P. sojae infection in hypocotyls of two soybean lines, Dongnong 50 (susceptible line, WT) and Glyma.18g283200 overexpression line (resistant line, OE). Based on the changes in differentially expressed genes (DEGs) in response to P. sojae infection in different lines and at different time points, it was speculated that HOPZ-ACTIVATED RESISTANCE 1 (ZAR1), valine, leucine, and isoleucine degradation, and phytohormone signaling may be involved in the defense response of soybean to P. sojae at the transcriptome level by GO term and KEGG pathway enrichment analysis. Differentially accumulated metabolites (DAMs) analysis revealed that a total of 223 and 210 differential metabolites were identified in the positive ion (POS) and negative ion (NEG) modes, respectively. An integrated pathway-level analysis of transcriptomics (obtained by RNA-seq) and metabolomics data revealed that isoflavone biosynthesis was associated with disease resistance. This work provides valuable insights that can be used in breeding programs aiming to enhance soybean resistance against PRR.

Study on the impurity profiles of cloxacillin and flucloxacillin based on liquid chromatography tandem ion trap/time-of-flight mass spectrometry.

Cloxacillin and flucloxacillin are prone to degradation and polymerization in humid and hot environments, and their polymers have long been recognized to trigger allergic manifestations. A series of the degradation and polymerized impurities in cloxacillin and flucloxacillin were separated and characterized to ensure safe use of these drugs by the public. By studying the chromatographic behavior of the degradation impurities and polymerized impurities in reversed-phase high-performance liquid chromatography (RP-HPLC) gradient elution, the impurities in cloxacillin and flucloxacillin were effectively separated and eluted. RP-HPLC tandem ion trap/time-of-flight mass spectrometry (MS) was applied to characterize the structures of unknown impurities eluted from the RP-HPLC methods for cloxacillin and flucloxacillin. The mechanisms of formation of the impurities in cloxacillin and flucloxacillin were also investigated. The structures of 10 unknown impurities in cloxacillin and 8 unknown impurities in flucloxacillin were elucidated based on the high-resolution MSn data at positive and negative modes, respectively. Six polymerized impurities were found and characterized, of which three were from the polymerization of cloxacillin and three were from the polymerization of flucloxacillin. The study on the impurity profiles of cloxacillin and flucloxacillin provided a scientific basis for improving their production processes and quality control.

Different formation pathways of nitrogen-containing organic compounds in aerosols and fog water in northern China

Abstract. While aqueous-phase processing is known to contribute to the formation of nitrogen-containing organic compounds (NOCs), the specific pathways involved remain poorly understood. In this study, we aimed to characterize the NOCs present in both pre-fog aerosols and fog water collected at a suburban site in northern China. Fourier-transform ion cyclotron resonance mass spectrometry was utilized to analyze the molecular composition of NOCs in both negative and positive modes of electrospray ionization (ESI− and ESI+). In both pre-fog aerosols and fog water samples, NOCs constituted a significant portion, accounting for over 60 % of all assigned formulas in ESI− and more than 80 % in ESI+. By comparing the molecular composition of NOCs originating from biomass burning, coal combustion, and vehicle emissions, we identified that 72.3 % of NOCs in pre-fog aerosols were attributed to primary anthropogenic sources (pNOCs), while the remaining NOCs were categorized as secondary NOCs formed within the aerosols (saNOCs). Unique NOCs found in fog water were classified as secondary NOCs formed within the fog water (sfNOCs). Through a comprehensive “precursor–product pair” screening involving 39 reaction pathways, we observed that the nitration reaction, the amine pathway, and the intramolecular N-heterocycle pathway of NH3 addition reactions contributed 43.6 %, 22.1 %, and 11.6 % of saNOCs, respectively. In contrast, these pathways contributed 26.8 %, 28.4 %, and 29.7 % of sfNOCs, respectively. This disparity in formation pathways is likely influenced by the diverse precursors, the aqueous acidity, and the gas-phase species partitioning. Correspondingly, saNOCs were found to contain a higher abundance of carbohydrate-like and highly oxygenated compounds with two nitrogen atoms compared to pNOCs. Conversely, sfNOCs exhibited a higher content of lipid-like compounds with fewer oxygen atoms. These results underscore the distinct secondary processes contributing to the diversity of NOCs in aerosols and fog water, which may lead to their different climate effects.

Paper-in-Tip Bipolar Electrospray Mass Spectrometry for Real-Time Chemical Reaction Monitoring.

Capturing short-lived intermediates at the molecular level is key to understanding the mechanism and dynamics of chemical reactions. Here, we have developed a paper-in-tip bipolar electrolytic electrospray mass spectrometry platform, in which a piece of triangular conductive paper incorporated into a plastic pipette tip serves not only as an electrospray emitter but also as a bipolar electrode (BPE), thus triggering both electrospray and electrolysis simultaneously upon application of a high voltage. The bipolar electrolysis induces a pair of redox reactions on both sides of BPE, enabling both electro-oxidation and electro-reduction processes regardless of the positive or negative ion mode, thus facilitating access to complementary structural information for mechanism elucidation. Our method enables real-time monitoring of transient intermediates (such as N,N-dimethylaniline radical cation, dopamine o-quinone (DAQ) and sulfenic acid with half-lives ranging from microseconds to minutes) and transient processes (such as DAQ cyclization with a rate constant of 0.15 s-1). This platform also provides key insights into electrocatalytic reactions such as Fe (III)-catalyzed dopamine oxidation to quinone species at physiological pH for neuromelanin formation.

Paper‐in‐Tip Bipolar Electrospray Mass Spectrometry for Real‐Time Chemical Reaction Monitoring

AbstractCapturing short‐lived intermediates at the molecular level is key to understanding the mechanism and dynamics of chemical reactions. Here, we have developed a paper‐in‐tip bipolar electrolytic electrospray mass spectrometry platform, in which a piece of triangular conductive paper incorporated into a plastic pipette tip serves not only as an electrospray emitter but also as a bipolar electrode (BPE), thus triggering both electrospray and electrolysis simultaneously upon application of a high voltage. The bipolar electrolysis induces a pair of redox reactions on both sides of BPE, enabling both electro‐oxidation and electro‐reduction processes regardless of the positive or negative ion mode, thus facilitating access to complementary structural information for mechanism elucidation. Our method enables real‐time monitoring of transient intermediates (such as N,N‐dimethylaniline radical cation, dopamine o‐quinone (DAQ) and sulfenic acid with half‐lives ranging from microseconds to minutes) and transient processes (such as DAQ cyclization with a rate constant of 0.15 s−1). This platform also provides key insights into electrocatalytic reactions such as Fe (III)‐catalyzed dopamine oxidation to quinone species at physiological pH for neuromelanin formation.

Study on the Mass Spectrometry Cleavage Pattern of Quinolone Antibiotics.

Quinolone antibiotics are extensively used clinically for human treatment and in agriculture. However, improper and excessive use can lead to the persistence of quinolone residues in animal tissues, potentially accumulating in the human body and posing health risks. Investigating the correlation between mass spectrometry cleavage patterns and molecular structural features enhances the analytical framework for detecting trace or unknown impurities in quinolones. To collect data, we employed triple quadrupole linear ion trap mass spectrometry in electrospray positive ion mode. Primary mass spectrometry scanning was utilized to confirm parent ions, while secondary mass spectrometry scanning enabled the observation of fragment ions. The cleavage characteristics and pathways of the compounds were inferred from accurate mass-to-charge ratios obtained from both primary and secondary mass spectrometry. Under soft ionization conditions, the compounds generally exhibited characteristic fragment ions of [M+H-H2O]+, [M+H-CO]+, and [M+H-H2O-CO]+. Additionally, subtle variations were observed in each compound due to differences in modifying groups. For instance, upon deacidification, the piperazine ring structure underwent breakage and rearrangement, yielding fragment ion peaks devoid of neutral molecules such as C2H5N, C3H7N, or C4H8N. Notably, compounds featuring a cyclopropyl substituent group at the N-1 position typically exhibited characteristic fragments resulting from the loss of the cyclopropyl radical (⋅C3H5). Moreover, substituents at the N-1 and C-8 positions, when linked to form a six-membered carbocyclic ring, were prone to cleavage, releasing the neutral C3H6 molecule. Quinolone antibiotics share structural similarities in their parent nuclei, leading to partially similar cleavage pathways. Nevertheless, distinct cleavage patterns emerge due to variations in functional groups. According to the difference of mass spectrometry cleavage patterns, it can provide an identification basis for the measured detection of antibiotics.

Dielectric barrier discharge ionization procures characteristic adducts of polyfluoroalkyl substances (PFAS) amenable by gas chromatography-mass spectrometry

Fluorotelomer alcohols (FTOHs) and acrylates (FTACs) have been reported as precursors of perfluoroalkyl acids and are known to contaminate biotic and abiotic compartments. Here, a detailed study was carried out for assessing the capability of dielectric barrier discharge ionization (DBDI) to be used as soft ionization technique, as alternative to the more energetic electron ionization (EI), for the quantitative determination of four FTOHs and two FTACs in environmental, biological or food samples by gas chromatography-mass spectrometry. While gas chromatography-EI-mass spectrometry (GC-EI-MS) shows significant fragmentation of the compounds, the ionization obtained by gas chromatography-DBDI-high resolution mass spectrometry (GC-DBDI-HRMS) affords dominant molecular adducts on the spectra. In the positive ion mode, the ion [M + H3O]+ and, of lower intensity, [M + H]+ are observed in the spectra of FTOHs, while the ion [M + H]+ is the most abundant in the spectra of FTACs and accompanied by a lower signal attributed to the ion [M + NO]+. In the negative ion mode, the FTOHs give rise to carbon dioxide and oxygen adducts observed with ions [M − H + CO2]- and [M + O2]-. Structure assignments were confirmed with stable isotope-labelled FTOH analogs and 13CO2. Gas chromatographic separation of the six compounds reveals room for development of methods applicable to soils, plants, water, food and biological materials.

Graphite Sheet-Assisted Laser Desorption Ionization-Mass Spectrometry for Small Organic Compound Analysis.

Carbon-based nanopowders have been used as ionization materials for laser desorption ionization-mass spectrometry (LDI-MS) and are very efficient at detection in low m/z regions. In this study, we aimed to develop a new sheet-type graphite material that possessed a randomly grooved nanostructured surface consisting of developed sp2-conjugated atomic carbon to facilitate the desorption/ionization of small compounds in LDI-MS. The graphite sheet exhibited higher UV absorption and provided higher ionization efficiency and survival yield in the LDI-MS detection of a thermometer ion, 4-chloro-benzopyridinium, than those of highly oriented graphite plates. These properties demonstrate that the present graphite sheet is suited for use as an LDI-MS material. Graphite sheet-assisted LDI-MS successfully detected various substances, including amino acids, peptides, and polyethylene glycol polymers, with higher ion intensities and less noise than those associated with conventional organic matrix-assisted LDI-MS (MALDI-MS). Furthermore, graphite sheet-assisted LDI-MS analysis provided more peaks (252 peaks) derived from soy sauce than those obtained by MALDI-MS (36 peaks) and required fewer preparation processes (dilution and air-dried) compared with previously established graphite carbon black-assisted LDI-MS (171 peaks) in the positive mode. This study demonstrates that graphite sheet-assisted LDI-MS has the potential for small organic compound analyses in the biomedical and food science fields.

Insights into Metabolites Profiling and Pharmacological Investigation of Aconitum heterophyllum wall ex. Royle Stem through Experimental and Bioinformatics Techniques.

The Aconitum genus is a leading source of a wide range of structurally diverse metabolites with significant pharmacological implications. The present study investigated metabolite profiling, pharmacological investigation, anticancer potential, and molecular docking analysis of the stem part of Aconitum heterophyllum (AHS). The metabolite profiling of the AHS extract was experimentally examined using LC-MS/MS-orbitrap in both modes (ESI+/ESI-) and GC-MS in EI mode. The in vitro MTT model was used to study the anticancer potential, while the in vivo animal model was used to study the anti-inflammatory and antinociceptive activities. The MOE software was used for the molecular docking study. A total of 118 novel and previously known metabolites, among 44 metabolites (26 in ESI+ positive mode and 18 in ESI- negative mode) in the MeOH extract, while 74 metabolites (46 in ESI+ and 28 in ESI- mode) were identified in the n-hexane extract via LCMS/MS. The identified metabolites include 24 phenolic compounds, 18 alkaloids, 10 flavonoids, 24 terpenoids, 2 coumarins, 2 lignans, and 38 other fatty acids and organic compounds. The major bioactive metabolites identified were hordenine, hernagine, formononetin, chrysin, N-methylhernagine, guineesine, shogaol, kauralexin, colneleate, zerumbone, medicarpin, boldine, miraxinthin-v, and lariciresinol-4-O-glucoside. Furthermore, the GC-MS study helped in the identification of volatile and nonvolatile chemical constituents based on the mass spectrum and retention indices. The methanol extract significantly inhibited tumor progression in H9c2 and MDCK cancer cells with IC50 values of 186.39 and 199.63 μg/mL. In comparison, the positive control aconitine exhibited potent IC50 values (132.32 and 141.58 μg/mL) against H9c2 and MDCK cell lines. The anti-inflammatory (carrageenan-induced hind paw edema) and antinociceptive (acetic acid-induced writhing) effects were significantly dose-dependent, (p < 0.001) and (p < 0.05), respectively. In addition, a molecular docking study was conducted on identified ligands against the anti-inflammatory enzyme (COX-2) (PDB ID: 5JVZ) and the cancer enzyme ADAM10 (PDB ID: 6BDZ) which confirmed the anti-inflammatory and anticancer effects in an in silico model. Among all ligands, L2, L3, and L7 exhibit the most potent potential for inhibiting COX-2 inflammation with binding energies of -7.3424, -7.0427, and -8.3562 kcal/mol. Conversely, against ADAM10 cancer protein, ligands L1, L4, L6, and L7, with binding energies of -8.0650, -7.7276, -7.0454, and -7.2080 kcal/mol, demonstrated notable effectiveness. Overall, the identified metabolites revealed in this AHS research study hold promise for discovering novel possibilities in the disciplines of chemotaxonomy and pharmacology.

Comprehensive chemical profiling with UHPLC-MS, in-vitro, in-silico, and in-vivo antidiabetic potential of Typha domingensis Pers; A novel source of bioactive compounds

Aim of studyTypha is one of the valuable plant genera in folklore use in diabetes, and it can be a good source of bioactive chemicals. The current project was planned to investigate the pharmaceutical potential of n-butanol fraction of Typha domingensis Pers. (TDBF), a less explored medicinal plant. Materials and methodsThe phytochemical composition of the TDBF was assessed through a comprehensive analysis, encompassing the quantification of total bioactive contents (including total tannins and HPLC-PDA quantification) and a thorough exploration of secondary metabolites through scanning with UHPLC-MS. The pharmacological assessment included the determination of antioxidant activity (Total Antioxidant Capacity and Metal Chelating Assay), hemolytic potential, in-vitro antidiabetic effects (α-glucosidase and α-amylase inhibition, and antiglycation activity), as well as in-vivo antidiabetic activities. Molecular docking and ADMET studies were also performed for UHPLC-MS identified compounds. ResultsThe TDBF showed 134.76 ± 8.23 mg TAE/g total tannin contents. With HPLC-PDA, p-Hydroxy benzoic acid and p-Coumaric acid were quantified. During UHPLC-MS scanning, a total of five bioactive metabolites were identified in the negative mode, while 54 were detected in the positive mode. The in-vivo assessment of antidiabetic activity revealed a notable reduction in blood glucose levels in diabetic rats following treatment with the extract, demonstrating its efficacy compared to the control groups. Furthermore, the extract exhibited a positive impact on renal and liver function, evidenced by lowered levels of serum urea, ALT, ALP, creatinine, and a decrease in serum cholesterol levels. ConclusionThe results of this study showed that Typha domingensis Pers. possesses a variety of bioactive compounds and demonstrates notable potential in regulating diabetes. The study recommends further investigation into isolating these bioactive phytochemicals for their potential therapeutic applications across various diseases.

Quantification of AICAR and study of metabolic markers after administration.

Objectives: AICAR (5-amino-4-imidazolecarboxyamide ribonucleoside) was reported as the first pharmacological AMPK (adenosine 5'-monophosphate (AMP)-activated protein kinase) activator, and it has been confirmed to exhibit a significant endurance enhancement effect and prohibited for doping by the World Anti-Doping Agency. Due to the fact that the human body can produce such substances, in order to ensure fairness in sports competition, methods for rapid detection and multi-type identification of AICAR drugs taken orally should be established. Methods: to assess AICAR levels, a new rapid, sensitive, efficient, and selective method was reported for the quantitative detection of AICAR in urine using LC-MS/MS. The method was validated for quantitative purposes based on the elemental selectivity, intra- (1.0-15.6%) and inter-day precision (1.3-16.3%), accuracy (99.9-112.8%), matrix effects (88.9-103.6%), recovery (87.4-106.5%), and stability at four different concentrations. The calibration curve was linear over a wide concentration range of 10-10,000 ng mL-1 with a high coefficient of determination (R 2 > 0.998). The limit of detection (LOD) and limit of quantification (LOQ) for the experiment were determined to be 1 and 10 ng mL-1, respectively. Simultaneously, metabolomics analysis was used to obtain the metabolic fingerprint of different populations and biomarkers to distinguish administration cases through partial least squares discriminant analysis (PLS-DA) and a receiver operating characteristic (ROC) curve. Results: the method enables easy quantitation for LC-MS/MS analysis with the best recovery yield maintained, and the method was applied to 122 Asian biological samples with an average concentration of 1310.5 ± 1031.4 ng mL-1. Through drug metabolism research, 734 and 294 variables were extracted for data analysis respectively in the positive and negative ion modes, and more than 100 metabolites with significant up- and down-regulation were found after the test. Conclusions: this research developed a fast, precise, effective, and specific approach for the qualitative and quantitative identification of AICAR in urine. Meanwhile, administration metabolism studies found that there were significant changes in AICAR levels and other compounds, such as PC types PC(18:1/16:0), PC(16:0/18:0), and PC(16:0/16:0), PE types PE(18:0/20:4), and LPE-type 18:1, which could better distinguish samples before and after AICAR administration. The analysis provides a multi-perspective reference for WADA to determine a positive criterion.

Large-Scale Screening of Pharmaceutical Compounds to Explore the Application Space of On-Tissue MALDI and MALDI-2 Mass Spectrometry.

The successful application of matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) in pharmaceutical research is strongly dependent on the detection of the drug of interest at physiologically relevant concentrations. Here we explored how insufficient sensitivity due to low ionization efficiency and/or the interaction of the drug molecule with the local biochemical environment of the tissue can be mitigated for many compound classes using the recently introduced MALDI-MSI coupled with laser-induced postionization, known as MALDI-2-MSI. Leveraging a MALDI-MSI screen of about 1,200 medicines/drug-like compounds from a broad range of medicinal application areas, we demonstrate a significant improvement in drug detection and the degree of sensitivity uplift by using MALDI-2 versus traditional MALDI. Our evaluation was made under simulated imaging conditions using liver homogenate sections as substrate, onto which the compounds were spotted to mimic biological conditions to the first order. To enable an evaluable detection by both MALDI and MALDI-2 for the majority of employed compounds, we spotted 1 μL of a 10 mM solution using a spotting robot and performed our experiments with a Bruker timsTOF fleX MALDI-2 instrument in both positive and negative ion modes. Specifically, we demonstrate using a large cohort of drug-like compounds that ∼60% of the tested compounds showed a more than 10-fold increase in signal intensity and ∼16% showed a more than 100-fold increase upon use of MALDI-2 postionization. Such increases in sensitivity could help advance pharmaceutical MALDI-MSI applications toward the single-cell level.

Determination of 24 Benzimidazoles in meat and meat products by modified QuEChERS coupled with UPLC-MS/MS

In this study, an effective method for the simultaneous determination of 24 Benzimidazoles in meat and meat products based on a modified QuEChERS sample preparation technique coupled with ultra-performance liquid chromatography–tandem mass spectrometry was established. Then, BZDs in meat and meat products were extracted by 0.1 % ammonium hydroxide in acetonitrile, followed by cleanup with 0.1 g C18 and 0.3 g MgSO4, ionized in electrospray positive ionization source mode, and scanned for determination of the precursor ion and product ion by mass spectrometry in multiple reaction monitoring mode. The results showed that 24 BZDs had good linearity in the range of 0.2–100 μg/kg with correlation coefficients >0.999, and the limits of detection ranged from 0.001 to 0.244 µg/kg and the limits of quantification were between 0.002 and 0.738 µg/kg. The average recoveries of BZDs at three different spike levels of 2, 20, and 50 µg/kg were 74.6 % −113.9 %, with intra-day and inter-day relative standard deviations less than 9.86 % and 11.03 %, respectively. This method has been successfully applied to the determination of BZDs in real samples, providing a powerful tool for the monitoring of BZDs residues in animal-derived foods.

Simultaneous Determination of Multi-Class Mushroom Toxins in Mushroom and Biological Liquid Samples Using LC-MS/MS

A comprehensive analytical method based on liquid chromatography–tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous detection of 12 mushroom toxins (ibotenic acid, muscimol, muscarine, β-amanitin, α-amanitin, desoxoviroidin, γ-amanitin, phallisacin, illudin S, phallacidin, phalloidin and illudin M) in mushrooms, serum, urine and simulated gastric fluid. The samples were extracted with water or acetonitrile solution, and the serum sample was further purified with PSA sorbent. Chromatographic separation was performed on an ACQUITY UPLC HSS T3 column with gradient elution using methanol and water containing 1 mM ammonia fluoride as a mobile phase. Mass spectrometric acquisition was performed in electrospray positive ionization mode. Good linearities (R2 &gt; 0.994) were obtained for 12 toxins over the range of 0.05~200 µg/L. Matrix-matched calibration curves were used for quantification. The method limits of quantification were 0.01~0.2 mg/kg for mushrooms and 0.15~2.0 µg/L for three biological liquid samples. The mean recoveries of 12 target toxins (spiked at three concentration levels) ranged from 73.0% to 110.3%, with relative standard deviations not exceeding 19.4%, which meets the requirements for the determination of trace compounds in a biological matrix. This method was applied to the analysis of mushroom samples from Yunnan Province. As a result, 11 toxins, not including illudin M, were detected with a concentration range of 0.61~2143 mg/kg.

Development of a high-throughput dual-stream liquid chromatography-tandem mass spectrometry method to screen for inhibitors of glutamate carboxypeptidase II.

Glutamate carboxypeptidase II (GCPII) catalyzes the hydrolysis of N-acetylaspartylglutamate (NAAG) to yield glutamate (Glu) and N-acetylaspartate (NAA). Inhibition of GCPII has been shown to remediate the neurotoxicity of excess Glu in a variety of cell and animal disease models. A robust high-throughput liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was needed to quantify GCPII enzymatic activity in a biochemical high-throughput screening assay. A dual-stream LC/MS/MS method was developed. Two parallel eluent streams ran identical HILIC gradient methods on BEH-Amide (2×30 mm) columns. Each LC channel was run independently, and the cycle time was 2min per channel. Overall throughput was 1min per sample for the dual-channel integrated system. Multiply injected acquisition files were split during data review, and batch metadata were automatically paired with raw data during the review process. Two LC sorbents, BEH-Amide and Penta-HILIC, were tested to separate the NAAG cleavage product Glu from isobaric interference and ion suppressants in the bioassay matrix. Early elution of NAAG and NAA on BEH-Amide allowed interfering species to be diverted to waste. The limit of quantification was 0.1pmol for Glu. The Z-factor of this assay averaged 0.85. Over 36 000 compounds were screened using this method. A fast gradient dual-stream LC/MS/MS method for Glu quantification in GCPII biochemical screening assay samples was developed and validated. HILIC separation chemistry offers robust performance and unique selectivity for targeted positive mode quantification of Glu, NAA, and NAAG.

Assessing the matrix effects on MALDI-MS in the positive and negative ion mode detection for protein-protected metal nanoclusters

Protein-protected metal nanoclusters (MNCs) represent a new class of highly photoluminescent nanomaterials that have wide applications. Suitable reaction conditions combining protein and metal precursors can produce a vast range of different NC sizes (i.e. different number of metal atoms). The average number of metal atoms per protein can be determined by mass spectrometry (MS). MS coupled with matrix-assisted laser desorption ionization (MALDI) presents a number of advantages such as detection with high sensitivity of nanoclusters with high molecular weights. Although many protein-protected MNCs have been characterized by MALDI-MS, a large dispersion in the number of metal atoms has been reported mainly due to sample preparation. In this work, we optimized the protocols for negative and positive ion detection mode as a general MALDI-MS sample preparation method for protein-protected MNCs (bovine serum albumin and lysozyme and with gold and silver). Negative and positive ion mode detection was compared, showing that negative ion mode detection in MALDI-MS can also be used with acidic matrices. Obvious matrix effects on ion signals and peak positions by MALDI-MS were observed. The average metal numbers of MNCs embedded in proteins are different depending on the MALDI matrix. The matrix effects give a warning for more serious consideration on MALDI-MS measurement and spectra analysis of MNCs.