Negative Mode Research Articles

Advancing Targeted Metabolomics Using Cyanopropyl-Based Liquid Chromatography Tandem Mass Spectrometry.

The change of metabolic pathways is recognized as the key to disease discovery prompting the development of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)-based quantitative platforms to explore the dynamic metabolite profiles of organisms. In this study, a liquid chromatography method based on cyanopropyl (CN) was developed. By adjusting the pH environment of the column, we achieved the elution of 51 metabolites spanning the most comprehensive set of biological pathways currently known. Offering rapid chromatography, efficient separation, and green chemistry benefits, the method encompasses nucleosides and nucleotides, the oxidative-redox metabolome, the glycolysis pathway, the pentose phosphate pathway, the purine de novo pathway, amino acids, and neurological disorder-related metabolites. The mass spectrometry was equipped with electrospray ionization in both positive and negative modes with scheduled multiple reactions monitoring. The validation of the method involved a comprehensive assessment of linearity, accuracy, precision, and matrix effect. The linear range was from 1.0 to 2000 ng mL-1 with a high correlation coefficient (r > 0.99). The LOD ranged from 0.1 to 10 ng mL-1, and the LOQ ranged from 0.1 to 25 ng mL-1. The overall recovery ranged from 81.3% to 117.8%, with RSD < 15.1%. Subsequently, an analysis of metabolites was conducted in dSH-SY5Y neuroblastoma cells with 6-hydroxydopamine, a commonly used neurotoxin in neurodegenerative diseases. The results demonstrate that neurotoxin-induced mitochondrial damage significantly altered related analytes, corroborating previous estimates and validating the feasibility and reliability of the bioanalytical platform.

Insights into the Chemical Exposome during Pregnancy: A Non-Targeted Analysis of Preterm and Term Births.

Human-made chemicals are ubiquitous, leading to chronic exposure to complex mixtures of potentially harmful substances. We investigated chemical exposures in pregnant women in New York City by applying a non-targeted analysis (NTA) workflow to 95 paired prenatal urine and serum samples (35 pairs of preterm birth) collected as part of the New York University Children's Health and Environment Study. We analyzed all samples using liquid chromatography coupled with Orbitrap high-resolution mass spectrometry in both positive and negative electrospray ionization modes, employing full scan and data-dependent MS/MS fragmentation scans. We detected a total of 1524 chemical features for annotation, with 12 chemicals confirmed by authentic standards. Two confirmed chemicals dodecyltrimethylammonium and N,N-dimethyldecylamine N-oxide appear to not have been previously reported in human blood samples. We observed a statistically significant differential enrichment between urine and serum samples, as well as between preterm and term birth (p < 0.0001) in serum samples. When comparing between preterm and term births, an exogenous contaminant, 1,4-cyclohexanedicarboxylic acid (tentative), showed a statistical significance difference (p = 0.003) with more abundance in preterm birth in serum. An example of chemical associations (12 associations in total) observed was between surfactants (tertiary amines) and endogenous metabolites (fatty acid amides).

A fragmentation study of disaccharide flavonoid C-glycosides using triple quadrupole mass spectrometry and its application for identification of flavonoid C-glycosides in Odontosoria chinensis.

Flavonoid C-glycosides have a wide range of pharmacological activities. However, there are few mass spectrometric research on C,O-disaccharide flavonoid C-glycosides and di-C,O-saccharide flavonoid C-glycosides. Their low-energy collision-induced dissociation (ESI-CID-MS/MS) fragmentation pattern and differences have not been reported, and the fragment ion library is incomplete. Therefore, it was essential to elucidate the fragmentation patterns of disaccharide flavonoid C-glycosides, which is described in this study. Four disaccharide flavonoid C-glycosides such as vitexin-4″-O-glucoside were analyzed by ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometer (UPLC-MS/MS) using electrospray ionization (ESI) in both positive and negative ion modes. Each ion and its proposed fragmentation pathways of the four disaccharide flavonoid C-glycosides were analyzed comprehensively. Finally, ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) and the established fragmentation patterns have been used to identify disaccharide flavonoid C-glycosides in Odontosoria Chinensis. The fragmentation pathways of C,O-disaccharide flavonoid C-glycosides and di-C,O-saccharide flavonoid C-glycosides are similar. They both have the mass spectrometric characteristics of O-glycoside and C-glycoside. Product ions after mixed pathways of neutral fragments such as saccharide ring fragment, O-glycosides, H2O, and CH2O elimination appeared in both types of flavonoid C-glycosides, but their relative abundances are significantly different. According to the established fragmentation patterns, di-C,O-saccharide flavonoid glycosides were also found in Odontosoria chinensis. The fragment ions at m/z 431, 413, 341, 311, 293, and 282 in negative ion mode and m/z 293, 282, 577, 559, 541, 523, 529, and 499 in positive ion mode can serves as the main characteristics for identifying C,O-disaccharide flavonoid C-glycosides and di-C,O-saccharide flavonoid C-glycosides.

Medium-chain fatty acid triglycerides improve feed intake and oxidative stress of finishing bulls by regulating ghrelin concentration and gastrointestinal tract microorganisms and rumen metabolites

BackgroundAs a feed additive, medium-chain fatty acids (MCFAs)/medium-chain fatty acid triglycerides (MCTs) have been used in ruminant production, but mostly added in the form of mixed esters. Studies have shown that MCTs may have a positive effect on feed intake or oxidative stress in animals, but it is unclear which MCT could play a role, and the mechanism has not been elucidated. In this study, the effects of individual MCT on growth performance, serum intake-related hormones, and oxidative stress indices in finishing bulls were investigated and further studied the effects of MCT supplementation on gastrointestinal tract bacteria and rumen fluid metabolomics.ResultsFour ruminally fistulated Yanbian cattle (bulls) were selected in 4 × 4 Latin square designs and allocated to four treatment groups: a control group (CON) fed a basal diet (total mixed ration, TMR), three groups fed a basal diet supplemented with 60 g/bull/day glycerol monocaprylin (GMC, C8), glycerol monodecanoate (GMD, C10), and glycerol monolaurate (GML, C12), respectively. Compared with the CON group, GMD tended to increase the dry matter intake (DMI) of finishing bulls (P = 0.069). Compared with the CON group, GMD significantly increased the concentration of ghrelin O-acyl transferase (GOAT), total ghrelin (TG), acylated ghrelin (AG), and orexins (P < 0.05) and significantly decreased the concentrations of hydrogen peroxide (H2O2), malondialdehyde, reactive oxygen species (ROS), and lipopolysaccharides (LPS) in the serum of finishing bulls (P < 0.05). Compared with the CON group, GMD and GML significantly increased the concentrations of total antioxidant capacity (T-AOC), catalase, glutathione peroxidase (GSH-PX), glutathione reductase (GR), and nitric oxide (NO) in the serum of finishing bulls (P < 0.05). Compared with the CON group, there were 5, 14, and 6 significantly different bacteria in the rumen digesta in the C8, C10, and C12 groups, respectively; there were 3, 10, and 5 significantly different bacteria in the rumen fluid in the C8, C10, and C12 groups, respectively; and only one differential bacteria (genus level) in the feces among the four treatment groups. Compared with the CON group, there were 3, 14, and 15 significantly differential metabolites identified under positive ionization mode in the C8, C10, and C12 groups, respectively, while under negative ionization mode were 3, 11 and 14, respectively. Correlation analysis showed that there was a significant correlation between DMI, GOAT, AG, GSH-PX, LPS, gastrointestinal tract bacteria, and rumen fluid metabolites.ConclusionsOur findings revealed that different types of MCTs have different application effects in ruminants. Among them, GMD may improve the feed intake of finishing bulls by stimulating the secretion of AG. GMD and GML may change gastrointestinal tract microorganisms and produce specific rumen metabolites to improve the oxidative stress of finishing bulls, and ghrelin may also be involved. This study enlightens the potential mechanisms by which MCT improves feed intake and oxidative stress in finishing bulls.5d9kULzJFQsQ3ajLfa--XDVideo

MS2Lipid: A Lipid Subclass Prediction Program Using Machine Learning and Curated Tandem Mass Spectral Data

Background: Untargeted lipidomics using collision-induced dissociation-based tandem mass spectrometry (CID-MS/MS) is essential for biological and clinical applications. However, annotation confidence still relies on manual curation by analytical chemists, despite the development of various software tools for automatic spectral processing based on rule-based fragment annotations. Methods: In this study, we present a novel machine learning model, MS2Lipid, for the prediction of known lipid subclasses from MS/MS queries, providing an orthogonal approach to existing lipidomics software programs in determining the lipid subclass of ion features. We designed a new descriptor, MCH (mode of carbon and hydrogen), to increase the specificity of lipid subclass prediction in nominal mass resolution MS data. Results: The model, trained with 6760 and 6862 manually curated MS/MS spectra for the positive and negative ion modes, respectively, classified queries into one or several of 97 lipid subclasses, achieving an accuracy of 97.4% in the test set. The program was further validated using various datasets from different instruments and curators, with the average accuracy exceeding 87.2%. Using an integrated approach with molecular spectral networking, we demonstrated the utility of MS2Lipid by annotating microbiota-derived esterified bile acids, whose abundance was significantly increased in fecal samples of obese patients in a human cohort study. This suggests that the machine learning model provides an independent criterion for lipid subclass classification, enhancing the annotation of lipid metabolites within known lipid classes. Conclusions: MS2Lipid is a highly accurate machine learning model that enhances lipid subclass annotation from MS/MS data and provides an independent criterion.

Systematic Characterisation of the Fragmentation of Flavonoids Using High-Resolution Accurate Mass Electrospray Tandem Mass Spectrometry

Flavonoids are a class of polyphenolic secondary metabolites found in plants. Due to their ubiquity in our daily dietary intake and their major anti-oxidative, anti-inflammatory and anti-mutagenic activities, they have been a major focus of wide-ranging research for the past two decades. Mass spectrometry combined with liquid chromatography is one of the most popular techniques for the analysis of flavonoids. In this study, high-resolution accurate mass electrospray tandem mass spectrometry was used to study 30 flavonoids in both positive and negative ionisation modes. From the data obtained, common losses were summarised and compiled. Dominating neutral losses were tabulated. The radical loss of CH3· was observed in flavonoids containing methoxy groups and three key diagnostic product ions were identified. These were m/z 153 (indicative of two OH groups on ring A) m/z 167 (indicative of one OH and one methoxy group on ring A) and m/z 151 (a flavanol, with no ketone oxygen but two OH groups on ring A). These will be useful in structural elucidation of unknown flavonoids and flavonoid metabolites. Energy breakdown graphs were utilised to distinguish between three pairs of structural isomers, and to help rationalise proposed fragmentation pathways. Lastly, a competition of loss of CH3· and methane was reported for rhamnetin and isorhamnetin in the negative ion mode for the first time. Proposed fragmentation pathways were given to rationalise the differences in peak intensities for this competitive process.

Peroxydisulfate Activation by Biochar from Banana Peel Promoted with Copper Phosphide for Bisphenol S Degradation in Aqueous Media

In this work, the decomposition of bisphenol S (BPS) by biochar derived from banana peel (BPB) promoted by copper phosphide (Cu3P) was examined. Different materials with Cu3P loadings from 0.25 to 4.00 wt.% on biochar were synthesized, characterized using the Brunauer–Emmett–Teller (BET) method, X-ray diffraction (XRD) and a scanning electron microscope (SEM) equipped with an energy dispersive spectrometer (EDS), and evaluated. Nearly all of the synthesized materials exhibited low to moderate adsorption capacity, attributable to their limited surface area (&lt;3.1 m2/g). However, in the presence of sodium persulfate (SPS), the 2%Cu3P/ΒPB/SPS system was capable of removing 90% of 500 μg/L BPS in less than 10 min. The system’s performance was enhanced under inherent pH, and the reaction rate followed pseudo-first-order kinetics with respect to BPS and persulfate concentrations. Interestingly, the presence of 250 mg/L of sodium chloride had a negligible effect, while low to moderate inhibition was observed in the presence of bicarbonates and humic acid. In contrast, significant retardation was observed in experiments performed in real matrices, such as secondary effluent (WW) and bottled water (BW). According to scavenging experiments, both radical and non-radical mechanisms participated in the BPS degradation. Four transformation products were identified using the UHPLC/TOF-MS system in negative ionization mode, with two of them having higher molecular weights than BPS, while the other two TBPs involved the ring-opening reaction, and a BPS decomposition pathway was proposed.

The association between metabolite profiles and impaired bone microstructure in adult growth hormone deficient rats

BackgroundAdult growth hormone deficiency (AGHD) is associated with an increased risk of fractures and impaired bone microstructure. Understanding the metabolic changes accompanying bone deterioration in AGHD might provide insights into mechanisms behind molecular changes and develop new biomarkers or nutritional strategies for bone destruction. Our study aimed to investigate the association between altered metabolite patterns and impaired bone microstructure in adult rats with growth hormone deficiency.MethodsThirty seven-week-aged adult Lewis dwarf homozygous (dw/dw) rats (five females and five males), and adult Lewis dwarf heterozygous (dw/ +) rats (five females and five males) rats were compared. Micro-computed tomography (Micro-CT) was used to examine the bone's microstructure. Hematoxylin and eosin (H&E) staining were used to quantify the histological characteristics. Liquid chromatography-mass spectrometry untargeted serum metabolomic analysis was applied in the study. ELISA was used to measure serum bone turnover markers and IGF-1 levels.ResultsAdult dw/dw rats exhibited great reductions in trabecular volume bone density (Tb.vBMD), bone volume/total volume (BV/TV), and cortical thickness (Ct. Th) compared with adult dw/ + rats (all p values < 0.05), indicating significant impairment in bone microstructure. The serum metabolite profiles revealed substantial differences between the dw/dw rats and dw/ + rats. A total of 134 differential metabolites in positive ion mode and 49 differential metabolites in negative mode were identified. Five metabolites, including Lysophosphatidylcholine(LPC) 20:3, LPC22:6, LPC22:4, cortisol and histamine levels were upregulated in dw/dw rats. The steroid hormone biosynthesis and bile secretion pathways were the main perturbed metabolic pathways. There were significant associations between differential metabolites and the impaired bone microstructure parameters, indicating that the selected metabolites might serve as potential biomarkers for deteriorated bone microstructure in AGHD.ConclusionAdult dw/dw rats exhibit impaired bone microstructure and distinct serum metabolic profiles, and the altered metabolites were significantly associated with bone microstructure destruction. This provides a new insight into understanding the mechanism of bone deterioration in AGHD patients from a metabolic perspective.

Compact Plasma Ionization for Ion Mobility Spectrometry Using a 4.3 MHz Miniature Tesla Coil.

In this study, a low-cost 4.3 MHz plasma ionization source for ion mobility spectrometry (IMS), utilizing a miniaturized Tesla coil, is presented. This compact design, combined with a 3D printed cyclic olefin copolymer (COC) housing, delivers a stable and directed plasma suitable for ionization in IMS applications. The 3D printed housing ensures chemical resistance and low off-gassing, which are crucial for maintaining sample integrity. The Tesla coil produces a consistent sine wave at 4.3 MHz, and when connected to stainless steel screw electrodes it generates a stable plasma capable of ionizing analytes such as limonene, MTBE, nicotine, 2-octanone, and propofol. Measurements were conducted in both positive and negative ion modes. The results demonstrate the Tesla coil's effectiveness as a low-cost and reliable ionization source for IMS, offering comparable performance to traditional Ni63 β-emitters. This advancement in plasma ionization technology could facilitate more accessible and flexible IMS systems for diverse analytical applications. The integration of 3D printing in the development of this ionization source underscores the potential for customized, low-cost analytical instrumentation, promoting innovation in laboratory environments and commercial applications.

Atmospheric Pressure Photoionization with Halogen Anion Attachment for Mass Spectrometric Analysis of Hydrocarbons and Hydrocarbon-Based Polymers.

Aliphatic hydrocarbons and hydrocarbon-based synthetic polymers are of interest in many fields, but their characterization by mass spectrometric methods is generally limited due to their poor ionizability. Recently, atmospheric pressure photoionization (APPI), combined with halogen anion attachment in negative-ion mode, has drawn attention as a potential method for ionizing various polymers without extensive fragmentation or other unwanted side reactions. In this work, the applicability of halogen anion attachment with APPI was studied using several synthetic polymers, including polyethylene, polypropylene, polyisoprene, and polystyrene, as well as simple n-alkanes of various chain lengths. For hydrocarbon-based polymers, the method produced clear distributions of intact polymer adduct ions when different halogen anions were used. It was found that increasing the halogen anion size decreased ionization efficiency, particularly in the absence of π-bonds in the polymer structure. Testing with simple n-alkanes showed that only molecules containing fifty or more carbon atoms formed detectable halogen adducts, possibly due to the low gas-phase stabilities of the lighter n-alkane adduct ions. In conclusion, halogen anion attachment with negative-ion APPI appears to be a highly promising method for polymer analysis, providing structural data and clean polymer mass spectra with minimal fragmentation, which can be useful for the identification of unknown samples.

Study on the Mechanism of Raspberry (Rubi fructus)in Treating Type 2 Diabetes Based on UPLC-Q-Exactive Orbitrap MS, Network Pharmacology, and Experimental Validation.

The aim of this study is to analyze the chemical composition of raspberry using liquid chromatography-mass spectrometry (LC-MS) technology, predict the potential effects of raspberry in treating type 2 diabetes through network pharmacology, and conduct preliminary validation through invitro experiments. A Waters CORTECS C18 column (3.0 mm × 100 mm, 2.7 μm) was used; mobile phase A consisted of 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in acetonitrile. Gradient elution was performed with full-scan mode in both positive and negative ion modes, covering a mass range of m/z 100-1500. The chemical components of raspberry were analyzed and identified based on secondary spectra from databases and relevant literature. The disease targets related to type 2 diabetes were searched, and protein-protein interaction network analysis as well as gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted on the intersecting targets of the active components of raspberry and the disease. HepG2 cells were used for experimental validation, with high glucose-induced insulin resistance models established. The CCK-8 method was employed to assess the effects of raspberry on cell proliferation, while Western blotting was used to measure the expression of proteins related to the AGE/RAGE signaling pathway. A total of 47 components were identified, including 10 organic acids, 15 flavonoids, 12 phenols, 2 alkaloids, 4 terpenoids, 1 miscellaneous compound, 1 stilbene, 1 steroid and its derivatives, and 1 diterpenoid. Through database screening, seven active components were identified: kaempferol, epicatechin, ellagic acid, crocetin, stigmasterol, fisetin, and isorhamnetin. KEGG and GO results indicated that the therapeutic effects of raspberry on type 2 diabetes may be related to the advanced glycation end product (AGE)- receptor for advanced glycation end product (RAGE) signaling pathway. Establishment of an insulin resistance model in HepG2 cells demonstrated that, compared to the control group, the raspberry treatment group upregulated p53 protein expression while downregulating the expression of RAGE, Akt1, and Caspase-3 proteins. This study preliminarily elucidates that the therapeutic effects of raspberry in treating type 2 diabetes may be mediated through the inhibition of the AGE-RAGE signaling pathway, providing important references for the study of the pharmacological basis and clinical application of raspberry.

Development and validation of an LC‒MS/MS method for the determination of cyclocreatine phosphate and its related endogenous biomolecules in rat heart tissues

The cardioprotective drug cyclocreatine phosphate has been awarded Food and Drug Administration-orphan drug designation for the prevention of ischemic injury to enhance cardiac graft recovery and survival in heart transplantation. Cyclocreatine phosphate is the water-soluble derivative of cyclocreatine. Estimating the levels of Cyclocreatine phosphate, Adenosine triphosphate, Creatine Phosphate, Creatine and Cyclocreatine helps us in understanding the energy state as well as evaluating the heart cells’ function. The quantification of endogenous compounds imposes a challenging task for analysts because of the absence of a true blank matrix, whose use is required according to international guidelines. Recently, the International Council for Harmonization issued a new guideline that contains guidance on the validation of methods used to quantify endogenous components, such as the background subtraction approach that was employed in our current study. Specifically, we developed and validated a sensitive, reliable and accurate liquid chromatography-tandem mass spectrometry assay to determine simultaneously the levels of mentioned endogenous compounds in rat heart tissue. Tissue samples were prepared by protein precipitation extraction using water: methanol (1:1). Using Ultra Performance Liquid Chromatography, Chromatographic separation was achieved with ZORBAX Eclipse Plus C18 4.6 × 100 mm,3.5 μm column and conditions as following: ammonium acetate (pH 8.5): acetonitrile, 70:30 mobile phase, 0.7 mL/min flow rate and 25 °C temperature. Electrospray ionization mass detector with Multiple reaction monitoring mode was then employed, using both positive and negative modes, Analysis was carried out using 5.00–2000.00 ng/mL linear concentration range within 2 min for each analyte. According to Food and Drug Administration guidelines for bioanalytical methods, validation was carried out. We investigated the matrix effect, recovery efficiency and process efficiency for the analyte in neat solvent, postextraction matrix and tissue. The results stated mean percentage recoveries higher than 99%, accuracy 93.32–111.99%, and Relative Standard Deviation (RSD) below 15% within the concentration range of our study which indicated that target analytes’ stability in their real matrix is sufficient under the employed experimental conditions.

Simultaneous determination of 17 perfluoroalkyl substances in beef by EMR-Lipid dispersive solid-phase extraction and ultra performance liquid chromatography-tandem mass spectrometry

The ability to accurately analyze perfluoroalkyl substance (PFAS) levels in beef is imperative in order to effectively assess food-safety risks and ensure consumer safety because PFASs are harmful and prevalent in beef. In this study, we developed a rapid and accurate method for the simultaneously determination of the 17 PFASs in beef using dispersive solid-phase extraction (d-SPE) and ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and optimized the mobile phase system, extraction solvent, and d-SPE materials. Samples were finally extracted using 0.1% (v/v) formic acid in acetonitrile, cleaned using d-SPE with PSA, C18, GCB, and EMR-Lipid, separated using an Acquity Premier BEH C18 column (100 mm×2.1 mm, 1.7 μm) with 0.5 mmol/L ammonium fluoride aqueous solution and methanol as the mobile phases at a flow rate of 0.3 mL/min. Analytes were detected in negative ion switching mode (ESI-) with multiple reaction monitoring (MRM) scanning, and quantitatively analyzed using the internal standard method. The 17 PFASs exhibited linearity in the 0.2-20.0 μg/L range under the optimal experimental conditions, with correlation coefficients of 0.9915-0.9999. The method delivered limits of detection (LODs) of 0.003-0.007 μg/kg and limits of quantification (LOQs) of 0.01-0.02 μg/kg. The 17 PFASs exhibited recoveries of 71.1%-127.4% with RSDs of 0.6%-14.4% when spiked at three levels (0.05, 0.5, and 1.8 μg/kg). We optimized the mobile phase system, which revealed that, compared with 2.0, 5.0, and 10.0 mmol/L ammonium formate or ammonium acetate in aqueous methanol, 0.5 mmol/L ammonium fluoride in aqueous methanol exhibited higher sensitivities for all the 17 PFASs, with PFASs bearing long-chain carboxylic acids (C10-C18) showing 1-2 fold increases in sensitivity. PFASs do not dissociate in acidic environments, favoring their entry into the organic phase. Therefore, we investigated the effect of extractant acidity, which revealed that the 17 PFASs were better extracted using 0.1% (v/v) formic acid in acetonitrile. The beef matrix has a complex composition; consequently, d-SPE adsorbents were required to purify samples and reduce matrix effects. The purification effects of four adsorbents (PSA, C18, GCB, and EMR-Lipid) toward the 17 PFASs and the amount of EMR-Lipid used were investigated, which revealed that 100 mg PSA+80 mg C18+40 mg GCB+150 mg EMR-Lipid exhibited superior matrix-purification behavior. We also investigated the effects of various injection solutions and types of syringe filter, with pure methanol selected for reconstitution and high-speed supernatant centrifugation applied prior to injection. The developed method is simple, rapid, sensitive, and reproducible, and can be used to simultaneously, rapidly, and accurately determine various perfluoroalkyl compounds in beef.

Targeted ultra-high performance liquid chromatography-tandem mass spectrometry assay for the quantification of medium-chain phosphatidylcholines in platelets of coronary artery disease patients

In a recent untargeted clinical lipidomics study of platelets of coronary artery disease (CAD) patients, medium-chain phosphatidylcholines (MCPCs) with C8 and C10 fatty acyl residues were found significantly upregulated in the patient group with acute coronary syndrome (ACS) as compared to chronic coronary syndrome (CCS) and healthy controls. To support this finding, this work presents the development and optimization of a targeted UHPLC-QTrap-MS/MS method with multiple reaction monitoring acquisition for the quantitative analysis of MCPCs (PC 10:0/8:0, PC 16:0/8:0, PC 10:0/20:4 and PC 10:0/10:0) in platelets for biomarker validation. A systematic optimization of chromatographic and mass spectrometric parameters was performed. A charged surface hybrid CSH C18 (1.7 µm, 130 Å) column and fine-tuned gradient elution with 2-propanol/acetonitrile and ammonium acetate as additive to the mobile phase was employed in the final method in ESI negative mode. Four selected PC standards (PC 6:0/6:0, PC 8:0/8:0, PC 10:0/10:0 and PC 12:0/12:0), which cover well the carbon and retention rime range of the target analytes, were used for the optimization process and calibration. Quantification was based on matrix-matched calibration with these four selected commercially available MCPC standards as surrogate calibrants and PC 6:0/6:0 (d22) as internal standard. Furthermore, an organic solvent and fatty acyl carbon number-corrected response factor approach gave also accuracies within acceptance limits of bioanalytical validation guidelines and has more generic applicability. Compared to the previous untargeted RPLC-ESI-QTOF-MS/MS method, the optimized targeted UHPLC-QTrap-MS/MS assay showed increased sensitivity and selectivity for the detection of medium-chain PCs in platelet samples of CAD (LOQs in the range of 0.5-5 nmol/L). The method performance parameters indicated its suitability for a future biomarker validation study of MCPCs in platelets.

Rapid and Effective Determination of Ethyl Glucuronide in Hair by Micro Extraction by Packed Sorbent (MEPS) and LC-MS/MS.

Ethyl glucuronide (EtG) in hair is a reliable biomarker of alcohol consumption habits. Due to its small concentration incorporated into hair, analytical methods sensitive enough to reliably quantify EtG in this matrix are required. Sample preparation is critical in hair analysis, especially for EtG, for which extraction efficiency and matrix effect can strongly influence the results; furthermore, miniaturized methods are sought, to reduce solvent use and times of sample preparation. A micro extraction by packed sorbent (MEPS) procedure coupled to a high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for quantitation of EtG in human hair samples. Fifty milligrams of hair samples were cut into snippets and extracted in water. The cleanup of the extract was carried out by using a MEPS syringe packed with anion exchange sorbent (SAX); all parameters for conditioning, washing, loading and eluting steps were optimized and the eluted aqueous volume was directly injected in the LC-MS/MS system operating in the negative ionization mode. The method was fully validated assessing LOD, LOQ, calibration curve, repeatability, accuracy, matrix effect and carryover. The method was subsequently applied to QCs and authentic hair samples. The developed MEPS method is quick and effective, with low solvent purchase and discard costs, allowing the differentiation between social drinkers and chronic excessive alcohol consumers, according to the cut-offs established by the Society of Hair Testing (SoHT).

UPLC/HR-ESI-MS/MS and GC/MS profiling of Eriobotrya japonica L. fruit in correlation to its antioxidant, anti-inflammatory, and anti-arthritic effects.

Eriobotrya japonica Lindl. (Loquat) fruit is a subtropical edible fruit originally from China. It grows well in Egypt, but it is not widely known. In the current study, the fruit was extracted with 80% ethanol to get the total ethanol extract (TEE). A part of which was fractionated by dichloromethane to yield polar and nonpolar fractions (PF and NPF). The antioxidant and anti-inflammatory activities of the TEE were in vitro evaluated. The complete Freund's adjuvant (CFA) arthritis model was used to explore the in vivo biological assessment of the anti-arthritic properties in vivo of the TEE, PF, and NPF of the fruit. Additionally, the inspected limbs detached from all animals were subjected to histological inspection. Moreover, GC/MS analysis of the unsaponifiable (USF) and saponifiable (SF) fractions of the NPF was performed. Furthermore, 64 metabolites from various chemical classes were identified using UHPLC/HR-MS/MS analysis of the TEE of the fruit in both positive and negative ionization modes. The positive ionization mode of loquat fruit allowed for the first time the detection of two kinds of lyso-glycerophospholipids (Lyso-GPLs): lyso-glycerophosphoethanolamines (Lyso-PtdEtn) and lyso-glycerophosphocholines (Lyso-PtdCho). The fruit extracts exhibited a notable in vivo anti-arthritic activity by decreasing paw thickness in the treated rats and adjusting the inflammatory mediators. The TEE showed the highest anti-arthritic activity, followed by the PF that showed an observed activity, while the NPF exhibited the lowest activity. Histopathological findings showed a marked improvement in the arthritic condition of the excised limbs. Thus, E. japonica fruit may be considered as a promising natural antioxidant and anti-arthritic agent.

Toxicokinetics and Tissue Distribution of the Hepatotoxic Triterpenoid Saponin Pterocephin A in Rats Using the Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) Method.

Pterocephin A is a natural triterpenoid saponin isolated from Pterocephalus hookeri, a traditional Tibetan medicine with slight toxicity, which can induce liver injury in rats. This study aimed to establish a sensitive and reliable UPLC-MS/MS method for exploring the toxicokinetics and tissue distribution of pterocephin A following single intravenous and intragastric administration. Pterocephin A and prosapogenin 1C (internal standard, IS) were extracted using a simple protein precipitation technique with methanol as the precipitant for plasma samples and methanol/acetonitrile = 1:1 (v/v) for tissue samples. UPLC separation was achieved by gradient elution with 0.3 mL/min and a mobile phase consisting of 5 mM ammonium formate (A) and acetonitrile (B) (0-2 min 30% B; 2-4 min: 30-80% B; 4-5 min: 80-98% B; 5-6.5 min: 98% B; 6.5-7 min: 98-30% B; and 7-8 min: 30% B, v/v) with a column temperature of 35 °C. MS spectrometry adopted negative ion scanning mode, primary MS spectrometry adopted full scan monitoring mode, and secondary MS spectrometry adopted targeted MS2 scan monitoring mode. The assay exhibited a linear dynamic range of 0.02-15 μg/mL for pterocephin A in biological samples, with the low limit of quantification set at 0.02 μg/mL. Non-compartmental toxicokinetic parameters indicated that pterocephin A was well absorbed into the systemic circulation and had a long residual time after intravenous (10 mg/kg) and intragastric (60 mg/kg) administration, as it could still be detected after 72 h. Tissue distribution analysis revealed detectable levels of pterocephin A in various tissues, and a high concentration was maintained in the liver after intravenous (10 mg/kg) administration, with the highest concentration being 610.95 ± 25.73 ng/mL and a specific distribution pattern of liver > lung > kidney > intestine > spleen > testes > heart > stomach. The toxicokinetic process and tissue distribution characteristics of pterocephin A were expounded in this study, which can provide relevant data support for further research and clinical application of pterocephin A with its slight toxicity.

Chemical Fingerprinting of PM2.5 via Sequential Speciation Analysis Using Electrochemical Mass Spectrometry.

Chemical fingerprinting to characterize the occurrence state and abundance of organic and inorganic constituents within fine particulate matter (PM2.5) is useful in evaluating the associated health risks and tracing pollution sources. Herein, an analytical strategy for the rapid analysis of metal and organic constituents in PM2.5 was developed employing a combination of sequential chemical extraction coupled with mass spectrometry detection. H2O, CH3OH, EDTA-2Na, electrochemical oxidation, and electrochemical reduction were sequentially utilized to extract the chemical constituents in PM2.5 samples on a homemade device employing simultaneous online detection using two linear trap quadrupole mass spectrometers (LTQ-MS) with electrospray ionization (ESI) in positive and negative modes. After a single analytical procedure, dozens of metals (e.g., Pb, Cr, and Cu), organic compounds (e.g., amines, polycyclic aromatic hydrocarbons, and aliphatic acids), and negative ions (e.g., NO3-, NO2-, and Cl-) were comprehensively detected in the water-soluble, liposoluble, insoluble, oxidizable, and reducible fractions of PM2.5 samples, and their physical and chemical relationships were established.

Global transcription and metabolic profiles of five tissues in pepper fruits

Studying the regulatory mechanisms in different tissues of pepper is crucial for understanding organ formation, growth, and development. However, relevant studies are far from sufficient. In the current study, the stipe, calyx, pericarp, placenta, and seed of ripe pepper were sampled, and metabolites were determined by the untargeted metabolomics method. Transcriptome sequencing was performed by Illumina NovaSeq 6000, and then a high-throughput data set was built. The results showed that a total of 4879 annotated metabolites were detected in 15 samples of the five tissues under positive and negative ion mode. A total of 110.66 Gb of clean data was obtained by transcriptome sequencing, the clean data of each sample reached 6.21 Gb, and a total of 35 336 annotated expression genes were obtained. Furthermore, validate the accuracy of the data by combining principal component analysis and other methods. In summary, this study provides valuable information for the genetic improvement and breeding of peppers, and it holds potential application value, particularly in enhancing the quality and nutritional value of pepper fruits.

Development of a Novel and Rapid LC-MS/MS Method for Measuring Phosphatidylethanol in Whole Blood

Abstract Alcohol consumption can lead to serious health repercussions, including motor impairments and organ failure. Testing for short-term alcohol use is routinely conducted using blood ethanol tests in clinical laboratories. However, there is an emergent need to identify heavy alcohol users, especially crucial for patients undergoing organ transplant evaluations. With the advent of various biomarkers to detect heavy alcohol use, phosphatidyl ethanol (PEth), a group of abnormal phospholipids formed in cell membranes due to alcohol consumption, has gained prominence. PEth has been analyzed in whole blood with liquid chromatography-tandem mass spectrometry (LC-MS/MS) as two isomers: 18:1 (fatty acid:double bond) and 18:2 (fatty acid:double bond). We have developed an LC-MS/MS method that can detect these isomers in 4.5 minutes at concentrations of 10-500 ng/mL with excellent linearity and precision. In contrast to most LC-MS/MS methods where phospholipids are removed to minimize ion suppression, this method requires retaining phospholipids because the analyte, PEth, is a type of phospholipid. This was achieved through a ‘reverse extraction’ approach using phospholipid removal cartridges. Subsequently, the extracted samples were separated in an Agilent Protoshell 120 HPLC column on a Vanquish UHPLC instrument using 1 mM ammonium formate with 0.2% formic acid in water as Solution A and 50/50 isopropanol/acetonitrile as Solution B. The separation is performed starting at 80% Solution B (rest is Solution A) followed by a gradient elution where Solution B was increased to 95% over 2.3 minutes. Ions were analyzed using a ThermoFisher TSQ Endura MS/MS instrument in the negative mode. Additionally, commercially available whole blood was found to contain detectable levels of PEth in random lots, rendering them unsuitable for use as blank blood in the preparation of calibrators and quality controls. Instead, synthetic was used for this purpose. Ion suppression was observed, and various strategies were attempted to minimize it, including adjusting additives in the mobile phase, extending LC separation times, among others. More details on the impact of these adjustments and their effectiveness in improving the method’s performance will be presented.