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ABSTRACT The formulation of multi‐nutrient blocks based on low‐cost and locally available browse feed resources can be a valid feeding strategy in Sub‐Saharan Africa, where inadequate feed supply, both in quality and quantity, is a major constraint. We evaluated the four different inclusion percentages (M‐0%, M‐25%, M‐35% and M‐45%) of Moringa stenopetala leaf powder to multi‐nutrient blocks on their change on blood metabolite of dairy cows under practical, ranging conditions. Multi‐nutrient blocks with four inclusion rates of M. stenopetala leaves were applied as complementary feed for free ranging dairy cows. The study was performed on 24 free ranging dairy cows reared around Arba Minch town in the Southern Ethiopian Rift Valley. Blood samples were collected from the jugular vein of dairy cows both before and after supplementation. Plasma glucose, beta hydroxy butyrate (BHB), urea, creatinine, triglycerides and nonesterified fatty acids (NEFA) concentration was quantified spectrophotometrically. Dried serum spots were subject to quantitative electrospray tandem mass spectrometry to estimate changes in nutrient metabolism based on selected carnitines. Based on these measurements, the milk yield and body condition score were increased during the period of multi‐nutrient block supplementation. During the supplementation period, the cows got higher plasma glucose, triglyceride and urea concentrations and lower concentrations of BHB, NEFA and creatinine. From the metabolite profiles, a more efficient nutrient use could be concluded. Although no clear dose−response relationship was observed, the highest inclusion of the M. stenopetala leaves in the multi‐nutrient blocks gave the best performance. This outcome supports the idea of implementing M. stenopetala based multi‐nutrient blocks on tropical smallholder farms that are not easily accessible to conventional extension services. [ABSTRACT FROM AUTHOR]

Rationale: Optimizing the structure of digital polymers is an efficient strategy to ensure their tandem mass spectrometry (MS/MS) readability. In block‐truncated poly(phosphodiester)s, homolysis of C–ON bonds in long chains permits the release of smaller blocks amenable to sequencing. Yet the dissociation behavior of diradical blocks was observed to strongly depend on their charge state. Methods: Polymers were ionized in negative mode electrospray and activated in‐source so that blocks released as primary fragments can be investigated using ion mobility spectrometry (IMS) or sequenced in the post‐IMS collision cell. Collision cross sections (CCS) were derived from arrival times using a calibration procedure developed for polyanions using the IMSCal software. A multistep protocol based on quantum methods and classical molecular dynamics was implemented for molecular modeling and calculation of theoretical CCS. Results: Unlike their triply charged homologues, dissociation of diradical blocks at the 2– charge state produces additional fragments, with +1 m/z shift for those holding the nitroxide α‐termination and −1 m/z for those containing the carbon‐centered radical ω‐end. These results suggest cyclization of these diradical species, followed by H• transfer on activated reopening of this cycle. This assumption was validated using IMS resolution of the cyclic/linear isomers and supported by molecular modeling. Conclusions: Combining IMS with molecular modeling provided new insights into how the charge state of digital blocks influences their dissociation. These results permit to define new guidelines to improve either ionization conditions or the structural design of these digital polymers for best MS/MS readability. [ABSTRACT FROM AUTHOR]

Rationale: Sphingomyelins (SMs) and resulting metabolic products serve important functional and cell signaling roles and can act as potential biomarkers and therapeutic targets in many pathological disorders. SMs each contain a sphingoid base, an amide‐linked fatty acyl chain, and a phosphocholine headgroup. Despite these simple building blocks, variations and modifications of both the sphingoid base and the fatty acyl chain result in a diverse array of structurally complicated SM compounds. Conventional tandem mass spectrometry (MS/MS) using the collision‐induced dissociation (CID) method only provides limited structural information, necessitating other tools to unravel the structural complexity of these lipids. Methods: We utilize electron‐induced dissociation (EID) and sequential CID/EID approaches to elucidate detailed structural features of SMs. Integrating the CID/EID method into an imaging MS workflow enables accurate identification of SMs directly from kidney tissue. Results: The application of EID enables identification of SMs at the molecular species level, identifying the sphingosine base and the amide‐linked fatty acyl chains. Furthermore, removal of the phosphocholine headgroup via CID followed by sequential EID in an MS3 analysis (CID/EID) enhances the structural information obtained. CID/EID provides diagnostic fragmentation patterns revealing the hydroxylation site and double bond position in both the sphingosine base and amide‐linked fatty acyl chains. Conclusions: Detailed structural information of SMs from synthetic standards and biological tissue samples is obtained using an alternative electron‐based dissociation method. Accurate characterization of SMs promises to better inform studies of tissue biochemistry, lipid metabolism, and molecular pathology. [ABSTRACT FROM AUTHOR]

This article provides a personal account of the author's career in mass spectrometry, both in the United States and Spain. The author discusses their training in the US and their work with Dr. John Oró at the University of Houston, as well as their experiences with mass spectrometry and gas chromatography. They also mention their involvement in NASA research programs and the social and historic events they witnessed during their time in the US. The article then focuses on the challenges faced by the author in establishing a biomedical mass spectrometry laboratory in Spain, including the lack of support and funding for research. Despite these difficulties, the author and their team managed to publish papers and graduate students. The text also discusses the author's efforts to promote the use of biomedical mass spectrometry in Spain, including organizing international symposiums and meetings. The author reflects on the advancements in mass spectrometry technology during this time period. They also discuss their involvement in the organization of international conferences and the development of biomedical mass spectrometry in Spain. The author served on scientific committees and played a key role in organizing the 15th International Mass Spectrometry Conference in Barcelona in 2000. They also discuss the challenges and successes of their research group, including the creation of a proteomics MS lab. The author reflects on their career in the field of mass spectrometry, particularly in the medical community in Spain. They discuss the challenges they faced in conducting research and the lack of available resources and facilities. [Extracted from the article]

Agglutination of pathogenic microorganisms on the body surface is a significant phenomenon for the prevention of infection. In the present study, we show that an extract of the skin mucus from Japanese flounder (Paralichthys olivaceus) has agglutination activity against the yeast Saccharomyces cerevisiae. We purified this yeast‐binding protein, which consists of an approximately 35‐kDa homodimer, using affinity chromatography with yeast as a ligand. Multiple internal amino acid sequences of the protein, as determined using liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry, mapped to flounder glyceraldehyde 3‐phosphate dehydrogenase (GAPDH). An anti‐GAPDH antibody inhibited the yeast agglutination activity in the skin mucus extract and stained agglutinated yeast, indicating that flounder GAPDH could agglutinate yeast. The current study suggests that GAPDH, a well‐known protein as the sixth enzyme in the glycolytic pathway, is a significant player in mucosal immunity in teleosts. [ABSTRACT FROM AUTHOR]

Rationale: This study used proteomics‐based data‐independent acquisition (DIA) technology with the aim of screening for differential expression proteins in type I gastric neuroendocrine neoplasm (g‐NEN). Methods: Differential expression proteins in type I g‐NEN and peritumoral tissues were screened using DIA with liquid chromatography/tandem mass spectrometry (DIA‐LC/MS/MS). The identified proteins were then functionally analysed using bioinformatics methods. We selected the three most highly expressed proteins, combined with patients' clinical data, for prognostic analysis. Results: Compared with peritumoral tissues, 224 proteins were up‐regulated, and 70 were down‐regulated. The most significantly enriched biological processes and pathways were vacuolar proton‐transporting V‐type ATPase complex assembly and metabolism‐related pathways. PCSK1, FBXO2, ACSL1, IRS2, and PTPRZ1 expression was markedly up‐regulated in type I g‐NENs. High IRS2 expression significantly correlated with a shorter time to recurrence. Conclusions: Our study provides a comprehensive proteomic signature based on DIA‐LC/MS/MS and highlights high IRS2 expression as a potential prognostic marker for type I gNENs. [ABSTRACT FROM AUTHOR]

BACKGROUND: Peanut peptides have good chelating ability with metal ions. However, there are few studies on the chelation mechanism of peanut peptides with calcium and absorption properties of peptide–calcium complex. RESULTS: Peptides with high calcium chelating rate were isolated and purified from peanut protein hydrolysate (PPH), and the chelation rate of component F21 was higher (81.4 ± 0.8%). Six peptides were identified from component F21 by liquid chromatography–tandem mass spectrometry, and the frequency of acidic amino acids and arginine in the amino acid sequence was higher in all six peptides. Peanut peptide–calcium complex (PPH21‐Ca) was prepared by selecting component F21 (PPH21). Ultraviolet analysis indicated that the chelate reaction occurred between peanut peptide and calcium ions. Fourier transform infrared analysis showed that the chelating sites were carboxyl and amino groups on the amino acid residues of peptides. Scanning electron microscopy revealed that the surface of peanut peptide had a smooth block structure, but the surface of the complex had a granular morphology. Caco‐2 cell model tests revealed that the bioavailability of PPH21‐Ca was 58.4 ± 0.5%, which was significantly higher than that of inorganic calcium at 37.0 ± 0.4%. CONCLUSION: Peanut peptides can chelate calcium ions by carboxyl and amino groups, and the peptide–calcium complex had higher bioavailability. This study provides a theoretical basis for the development of new calcium supplement products that are absorbed easily. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Cyclodepsipeptides (CDPs) represent a huge family of chemically and structurally diverse molecules with a wide ability for molecular interactions. CDPs are cyclic peptide‐related natural products made up of both proteinogenic and nonproteinogenic amino acids linked by amide and ester bonds. The combined use of different analytical methods is required to accurately determine their integral structures including stereochemistry, thus allowing deeper insights into their often‐intriguing bioactivities and their possible usefulness. Our goal is to present the various methods developed to accurately characterize CDPs. Presently, Marfey's method and NMR (nuclear magnetic resonance) are still considered the best for characterizing CDP configuration. Nevertheless, electrospray‐high resolution tandem mass spectrometry (ESI‐HRMS/MS) is of great value for efficiently resolving CDP's composition and sequences. For instance, recent data shows that the fragmentation of cationized CDPs (e.g., [M + Li]+ and [M + Na]+) leads to selective cleavage of ester bonds and specific cationized product ions (b series) useful to get unprecedented sequence information. Thus, after a brief presentation of their structure, biological functions, and biosynthesis, we also provide a historic overview of these various analytical approaches as well as their advantages and limitations with a special emphasis on the emergence of methods based on HRMS/MS through recent fundamental works and applications. [ABSTRACT FROM AUTHOR]

Clinical expression of coronavirus disease 2019 (COVID‐19) infectionis widely variable including fatal cases and patients with mild symptoms and a rapid resolution. We studied saliva from 63 hospitalized COVID‐19 patients and from 30 healthy controls by integrating large‐scale proteomics, peptidomics and targeted metabolomics to assess the biochemical alterations following the infection and to obtain a set of putative biomarkers useful for noninvasive diagnosis. We used an untargeted approach by using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for proteomics and peptidomics analysis and targeted LC–multiple reaction monitoring/MS for the analysis of amino acids. The levels of 77 proteins were significantly different in COVID‐19 patients. Among these, seven proteins were found only in saliva from patients with COVID‐19, four were up‐regulated and three were down‐regulated at least five‐folds in saliva from COVID‐19 patients in comparison to controls. The analysis of proteins revealed a complex balance between pro‐inflammatory and anti‐inflammatory proteins and a reduced amount of several proteins with immune activity that possibly favours the spreading of the virus. Such reduction could be related to the enhanced activity of endopeptidases induced by the infection that in turn caused an altered balance of free peptides. In fact, on a total of 28 peptides, 22 (80%) were differently expressed in severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and control subjects. The multivariate analysis of such peptides permits to obtain a diagnostic algorithm that discriminate the two populations with a high diagnostic efficiency. Among amino acids, only threonine resulted significantly different between COVID‐19 patients and controls, while alanine levels were significantly different between COVID‐19 patients with different severity. In conclusion, the present study defined a set of molecules to be detected with a quick and easy method based on mass spectrometry tandem useful to reveal biochemical alterations involved in the pathogenesis of such a complex disease. Data are available via ProteomeXchange with identifier PXD045612. [ABSTRACT FROM AUTHOR]

Triphenylamine (TPA) and N‐heterotriangulene (N‐HTA) scaffolds with up to three oligophenyl extensions are investigated by electrospray ionization (tandem) mass spectrometry (ESI‐[MS/]MS). Due to their low oxidation potentials, all molecules readily form radical cations in the electrospray process. The energy‐resolved collision‐induced dissociation behaviour of the molecular ions is contrasted to that of the silver(I) adducts. Complexation with Ag(I) leads to the expected [1:1] and [2:1] complexes (MAg+ and M2Ag+); however, even [1:2] complexes (MAg22+) can be detected for molecules with two and three large π‐expansions to allow stabilization of two charges. The TPA scaffolds decompose only at high collision energies through the loss of peripheral tert‐butyl groups. A general mechanism for this is proposed commencing with a methyl loss and followed by the release of isobutene and butyl radical moieties. The N‐HTA‐based scaffolds are considerably less stable and molecular ions fragment at low collision energies. This is caused by the facile loss of methyl radicals from the dimethylmethylene‐bridged triangulene core. In contrast, complexation with Ag+ leads to a dramatic stabilization. Most interestingly, dissociation eventually proceeds via the loss of neutral AgCH3, which is indicative of strong bidentate, tweezer‐like bonding of Ag+ to the molecules. [ABSTRACT FROM AUTHOR]

Rationale: Spironolactone is a steroidal drug prescribed for a variety of medical conditions and is extensively metabolized quickly after administration. Measurement of spironolactone and its metabolites remains challenging using mass spectrometry (MS) due to in-source fragmentation and relatively poor ionization using electrospray ionization. Therefore, improved methods of measurements are needed, particularly in the case of small sample volumes. Methods: Girard's reagent P (GP) derivatization of spironolactone was employed to improve response and provide an MS-based solution to the measurement of spironolactone and its metabolites. We performed ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESIMS/MS) and ion mobility spectrometry (IMS)-high-resolution mass spectrometry (HRMS) to fully characterize the GP derivatization products. Analytes were studied in positive ionization mode, and MS/MS was performed using nonresonance and resonance excitation collision-induced dissociation. Results: We observed the successful GP derivatization of spironolactone and its metabolites using authentic chemical standards. A signal enhancement of 1-2 orders of magnitude was observed for GP-derivatized versions of spironolactone and its metabolites. Further, GP derivatization eliminated in-source fragmentation. Finally, we performed GP derivatization and ultra-high-performance liquid chromatography- high-resolution mass spectrometry (UHPLC-HRMS) in a small volume of murine serum (20 µL) from spironolactone-treated and control animals and observed multiple spironolactone metabolites only in the spironolactone-treated group. Conclusions: GP derivatization was proven to have advantageous mass spectral performance (e.g., limiting in-source fragmentation, enhancing signals, and eliminating isobaric analytes) for spironolactone and its metabolites. This work and the detailed characterization using ultra-high-performance liquid chromatography- high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) and IMS serve as the foundation for future developments in reaction optimization and/or quantitative assay development. [ABSTRACT FROM AUTHOR]

Rationale: Illegal addition of anti-infective drugs to cosmetics at low concentrations has been found. The illicit addition of anti-infective drugs encompasses a wide variety of medications. The current sample purification methods are inadequate to detect all these compounds. A sensitive, wide-coverage, and weak-matrix-effect measurement method needs to be established to address this issue. Methods: Samples were extracted using acetonitrile, diluted 25 times, and then analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect 111 anti-infective drugs. The method was validated and assessed for matrix effect before being applied to cosmetic products. Results: The calibration curves for the analytes exhibited a strong correlation coefficient (r > 0.995). The limit of detection ranged from 0.006 to 0.6 mg/kg. Matrix effects were significantly improved after a 25-fold dilution. The method was successfully applied to various cosmetics. Two of 82 samples tested contained lincomycin and miconazole, respectively. Conclusions: The developed method is quick and reliable to analyze anti-infective drugs in cosmetics, with potential for both qualitative and quantitative analyses. It is a valuable tool for cosmetic research and development, contributing to safer and more effective cosmetic products. [ABSTRACT FROM AUTHOR]

Nanoelectromechanical systems (NEMS)‐based mass spectrometry (MS) is an emerging technique that enables determination of the mass of individual adsorbed particles by driving nanomechanical devices at resonance and monitoring the real‐time changes in their resonance frequencies induced by each single molecule adsorption event. We incorporate NEMS into an Orbitrap mass spectrometer and report our progress towards leveraging the single‐molecule capabilities of the NEMS to enhance the dynamic range of conventional MS instrumentation and to offer new capabilities for performing deep proteomic analysis of clinically relevant samples. We use the hybrid instrument to deliver E. coli GroEL molecules (801 kDa) to the NEMS devices in their native, intact state. Custom ion optics are used to focus the beam down to 40 μm diameter with a maximum flux of 25 molecules/second. The mass spectrum obtained with NEMS‐MS shows good agreement with the known mass of GroEL. [ABSTRACT FROM AUTHOR]

Mycoplasma synoviae (MS) infection is a serious threat to poultry industry in China. Tilmicosin is a semisynthetic macrolide antibiotic used only in animals and has shown potential efficacy against MS, but there were no reported articles concerning the pharmacokinetics/pharmacodynamics (PK/PD) interactions of tilmicosin against MS in vitro and vivo. This study aimed to assess the antibacterial activity of tilmicosin against MS in vitro and in vivo using PK/PD model to provide maximal efficacy. The minimum inhibitory concentration (MIC) and killing rates of different drug concentrations were measured using the microdilution method in vitro. Then, tilmicosin was administered orally to the MS‐infected chickens at doses of 7.5 and 60 mg/kg, and the PK parameters of tilmicosin in joint dialysates were determined using high‐pressure liquid chromatography/tandem mass spectrometry (HPLC‐MS/MS) combined with the microdialysis technique. The antibacterial effect (△E) was calculated when the infected chickens were administered a single oral dose of tilmicosin at 4, 7.5, 15, 30, and 60 mg/kg b.w. The PK and PD data were fitted using the Sigmoid Emax model to evaluate the PK/PD interactions of tilmicosin against MS. The bactericidal activity of tilmicosin against MS was concentration dependent. Furthermore, the PK/PD index of AUC0–72h/MIC exhibited the most optimal fitting results (R2 = .98). The MS load decreased by 1, 2, and 3 Log10 CFU/mL, then AUC/MIC was determined as 13.99, 20.53, and 28.23 h, respectively, and the bactericidal effect can be achieved when the dose of MS‐infected chickens is at 31.64 mg/kg b.w. The findings of this study hold significant implications for optimizing the treatment regimen for MS infection. [ABSTRACT FROM AUTHOR]

ABSTRACT Canagliflozin (CFZ) is a sodium‐glucose cotransporter‐2 inhibitor that has shown promising results as a drug for the treatment of insulin dysregulation in horses. Even though CFZ is used clinically, no pharmacokinetic data has previously been published. In this study, the pharmacokinetics of CFZ after administration of a single oral dose of 1.8 mg/kg in eight healthy Icelandic horses was examined. Additionally, the effect of treatment on glucose and insulin levels in response to a graded glucose infusion was investigated. Plasma samples for CFZ quantification were taken at 0, 0.33, 0.66, 1, 1.33, 1.66, 2, 2.33, 2.66, 3, 3.5, 4, 5, 6, 8, 12, 24, 32, and 48 h post administration. CFZ was quantified using UHPLC coupled to tandem quadrupole mass spectrometry (UHPLC‐MS/MS). A non‐compartmental analysis revealed key pharmacokinetic parameters, including a median Tmax of 7 h, a Cmax of 2350 ng/mL, and a t1/2Z of 28.5 h. CFZ treatment reduced glucose (AUCGLU, p = 0.001) and insulin (AUCINS, p = 0.04) response to a graded glucose infusion administered 5 h after treatment. This indicates a rapid onset of action following a single dose in healthy Icelandic horses. No obvious adverse effects related to the treatment were observed. [ABSTRACT FROM AUTHOR]

A detailed approach into electronic effects of ligands on gold‐alkyne interactions has been carried out in the gas phase using tandem mass spectrometry. A semi‐empirical method coupled to DFT calculations was used to quantify ligands effects on Au−CC bond strength. To this end, the critical energies of 16 [L−Au‐hex‐3‐yne]+ complexes with different L ligands were determined through collision induced dissociation. It was observed that electron‐rich ligands destabilize the gold‐alkyne bond, whereas phosphites and carbenes strengthened it. The influence of alkyne electronic effects on the gold‐alkyne bond has also been studied by dissociation in tandem mass spectrometry on four different alkynes. In an original approach, two successive ion‐molecule reactions within the mass spectrometer were used to measure the relative affinity of gold to an alkyne by exchange reactions. The result is that the more electron‐rich the alkyne, the stronger the gold‐alkyne bond. [ABSTRACT FROM AUTHOR]

Pidotimod is a chiral drug that possesses two chiral centers, resulting in three isomeric impurities (analytes, A). This study employs electrospray ionization ion trap mass spectrometry (ESI‐MS) through collision‐induced dissociation (CID) to investigate the chiral recognition of pidotimod and its three isomers to eliminate chromatographic separation. Three approaches were explored: (1) Protonated molecules in CID exhibited discriminative potential for diastereomers, with the ability to distinguish between S,S and R,R configurations, albeit with an Rchiral value of ~1.8. However, differentiation between R,S and S,R configurations was not achievable. (2) Alkali adductions (lithium and sodium) only discerned diastereomers. The Rchiral values of the diastereomers obtained from alkali adduct ions were significantly lower than those obtained from protonated ions. (3) Therefore, a third approach was used to address the challenge of distinguishing between R,S and S,R configurations, including the introduction of chiral references (ref) and transition metals (MII) to form metal‐bound complexes [MII(A)(ref)‐H]+. Additionally, we synthesized a novel ligand, 4‐(N‐tert‐butoxycarbonyl [Boc]‐L‐prolinamido)phenol (denoted as ligand A), by modifying N‐t‐Boc‐L‐Pro with 2‐aminophenol, which, in combination with CuII and NiII, enabled simultaneous differentiation of all four isomers. CuII complexes exhibited significant chiral selectivity between R,S and S,R configurations. Density functional theory calculations were performed to further elucidate the stereodynamic behavior and stoichiometry of these ions in the gas phase. These calculations revealed the interaction energy and coordination sites of the precursor ions in the gas phase, correlating well with MS/MS experiment results. Additionally, the logarithm of the CuII complexes' characteristic fragment ion abundance ratio demonstrated a strong linear relationship with enantiomeric excess (ee). This study presents a novel strategy for chiral drug quality control that eliminates chromatographic separation. [ABSTRACT FROM AUTHOR]

This study aims to elucidate the detailed metabolic implications of varying monacolin K levels and sterilization methods on Monascus‐fermented rice products (MFRPs), acclaimed for their health benefits and monacolin K content. Advanced metabolite profiling of various MFRP variants was conducted using ultrahigh‐performance liquid chromatography coupled with tandem time‐of‐flight mass spectrometry (UHPLC‐Q‐TOF MS). Statistical analysis encompassed t‐tests, ANOVA, and multivariate techniques including PCA, PLS‐DA, and OPLS‐DA. Notable variations in metabolites were observed across MFRPs with differing monacolin K levels, particularly in variants such as MR1‐S, MR1.5‐S, MR2‐S, and MR3‐S. Among the 524 identified metabolites, significant shifts were noted in organic acids, derivatives, lipids, nucleosides, and organic oxygen compounds. The study also uncovered distinct metabolic changes resulting from different sterilization methods and the use of highland barley as a fermentation substitute for rice. Pathway analysis shed light on affected metabolic pathways, including those involved in longevity regulation, cGMP‐PKG signaling, and the biosynthesis of unsaturated fatty acids. The research provides critical insights into the complex metabolic networks of MFRPs, underscoring the impact of fermentation substrates and conditions on monacolin K levels and their health implications. This study not only guides the nutritional optimization of MFRPs but also emphasizes the strategic importance of substrate choice and sterilization techniques in enhancing the nutritional and medicinal value of these functional foods. [ABSTRACT FROM AUTHOR]

Recently, our group has shown that fentanyl and many of its analogues form prototropic isomers ("protomers") during electrospray ionization. These different protomers can be resolved using ion mobility spectrometry and annotated using mobility‐aligned tandem mass spectrometry fragmentation. However, their formation and the extent to which experimental variables contribute to their relative ratio remain poorly understood. In the present study, we systematically investigated the effects of mixtures of common chromatographic solvents (water, methanol, and acetonitrile) and pH on the ratio of previously observed protomers for 23 fentanyl analogues. Interestingly, these ratios (N‐piperidine protonation vs. secondary amine/O = protonation) decreased significantly for many analogues (e.g., despropionyl ortho‐, meta‐, and para‐methyl fentanyl), increased significantly for others (e.g., cis‐isofentanyl), and remained relatively constant for the others as solvent conditions changed from 100% organic solvent (methanol or acetonitrile) to 100% water. Interestingly, pH also had significant effects on this ratio, causing the change in ratio to switch in many cases. Lastly, increasing conditions to pH ≥ 4.0 also prompted the appearance of new mobility peaks for ortho‐ and para‐methyl acetyl fentanyl, where all previous studies had only showed one single distribution. Because these ratios have promise to be used qualitatively for identification of these (and emerging) fentanyl analogues, understanding how various conditions (i.e., mobile phase selection and/or chromatographic gradient) affect their ratios is critically important to the development of advanced ion mobility and mass spectrometry methodologies to identify fentanyl analogues. [ABSTRACT FROM AUTHOR]

Background: Maize stalk rot (MSR) caused by Fusarium graminearum is the primary factor contributing to the reduction in maize yield and quality. However, this soil‐borne disease presents a significant challenge for sustainable control through field management and chemical agents. The screening of novel biocontrol agents can aid in developing innovative and successful strategies for MSR control. Results: A total of 407 strains of bacteria were isolated from the rhizosphere soil of a resistant maize inbred line. One strain exhibited significant antagonistic activity in plate and pot experiments, and was identified as Burkholderia ambifaria H8. The strain could significantly inhibit the mycelial growth and spore germination of F. graminearum, induce resistance to stalk rot, and promote plant growth. The volatile compounds produced by strain H8 and its secondary metabolites in the sterile fermentation broth exhibited antagonistic activity. The primary volatile compound produced by strain H8 was identified as dimethyl disulfide (DMDS) using gas chromatography tandem mass spectrometry. Through in vitro antagonistic activity assays and microscopic observation, it was confirmed that DMDS was capable of inhibiting mycelial growth and disrupting the mycelial structure of F. graminearum, suggesting it may be the major active compound for strain H8. The transcriptome data of F. graminearum further indicated that strain H8 and its volatile compounds could alter pathogenic fungi metabolism, influence the related metabolic pathways, and potentially induce cell apoptosis within F. graminearum. Conclusion: Our results showed that B. ambifaria H8 was capable of producing the volatile substance dimethyl disulfide, which influenced the synthesis and permeability of cell membranes in pathogens. Thus, B. ambifaria H8 was found to be a promising biological control agent against MSR. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Rationale: Perillae Fructus (PF) is a common traditional Chinese medicine (TCM) for the treatment of asthma. It has not been effectively characterized by rosmarinic acid (RosA), which is currently designed as the sole quality indicator in the Chinese Pharmacopoeia. Methods: This study introduced a database‐aided ultrahigh‐performance liquid chromatography equipped with quadrupole‐Exactive‐Orbitrap mass spectrometry (UHPLC/Q‐Exactive‐Orbitrap MS/MS) technology to putatively identify the compounds in PF, followed by literature research, quantum chemical calculation, and molecular docking to screen potential quality markers (Q‐markers) of PF. Results: A total of 27 compounds were putatively identified, 16 of which had not been previously found from PF. In particular, matrine, scopolamine, and RosA showed relatively high levels of content, stability, and drug‐likeness. They exhibited interactions with the asthma‐related target and demonstrated the TCM properties of PF. Conclusions: The database‐aided UHPLC/Q‐Exactive‐Orbitrap MS/MS can identify at least 27 compounds in PF. Of these, 16 compounds are unexpected, and three compounds (matrine, scopolamine, and RosA) should be considered anticounterfeiting pharmacopoeia Q‐markers of PF. [ABSTRACT FROM AUTHOR]

Mitapivat is a novel, first‐in‐class orally active pyruvate kinase activator approved by the US Food and Drug Administration in 2022 for the treatment of hemolytic anemia. There is no literature available regarding the identification of degradation impurities of mitapivat. The present study deals with the degradation behavior of mitapivat under various stress conditions such as hydrolytic, photolytic, thermal, and oxidative stress. The multivariate analysis found that the independent variables, that is, molarity, temperature, and time, are interacting with each other to affect the degradation of mitapivat. A specific, accurate, and precise high‐performance liquid chromatographic (HPLC) method was developed to separate mitapivat from its degradation products. The separation was achieved on the C‐18 column (250 mm × 4.6 mm × 5 µm) using the combination of 0.1% formic acid buffer and acetonitrile in gradient elution profile. The method was validated as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use Q2(R2) guideline. LC‐electrospray ionization‐Quadrupole‐time of flight was employed to identify degradation products. A total of seven novel degradation products of mitapivat were identified based on tandem mass spectrometry and accurate mass measurement. In‐silico toxicity of mitapivat and its degradation products was qualitatively evaluated by the DEREK toxicity prediction tool. [ABSTRACT FROM AUTHOR]

Introduction Methods Results Discussion Phosphatidylethanol (PEth) is a long‐term marker of alcohol consumption used frequently in clinical scenarios such as liver transplant evaluation. Recent cases have demonstrated that packed red blood cell (pRBC) transfusion creates the potential for artificial elevation or decrease of observed PEth concentrations in recipients. Very little is known about the prevalence or stability of PEth in pRBCs.Apheresis and whole‐blood (WB) donations were tested for PEth using liquid chromatography – tandem mass spectrometry with limit of quantitation 10 ng/mL. Units were stored under routine blood bank conditions to evaluate the stability of PEth and the impact of irradiation.Over 40% of apheresis and WB donors had PEth ≥10 ng/mL (maximum observed 587 ng/mL). As WB units were processed into component pRBCs, PEth concentrations increased and were higher than donor WB levels (EDTA sample) prior to collection (maximum observed 711 ng/mL). Storage for up to 5 weeks post donation resulted in mean 17.3% decrease in PEth‐positive units; in contrast to a prior report, we observed no PEth formation in units with negative (<10 ng/mL) baseline concentrations. Irradiation of pRBCs did not substantially affect PEth concentrations in either PEth‐positive or PEth‐negative units.PEth concentrations in healthy blood donors may potentially confound alcohol use or abstinence assessment in pRBC recipients. Transfusion medicine services and clinical practices such as transplantation and behavioral medicine should recognize this phenomenon and collaborate on testing protocols to appropriately interpret PEth in pRBC recipients. [ABSTRACT FROM AUTHOR]

BACKGROUND RESULTS CONCLUSION To extract anthocyanins with high efficiency, a hypothesis for high‐speed shear homogenization extraction (HSHE) method was established through a combination of solvent and ultrasonic‐assisted extractions. The efficacy of this hypothesis was demonstrated by performing qualitative and quantitative analyses of 16 anthocyanins extracted from five northern vegetables, and five berry fruits using ultra‐high‐performance Q‐Exactive Orbitrap tandem mass spectrometry. Single‐factor experiments were conducted by varying ethanol concentration, temperature, pH and extraction cycles to determine the optimal conditions for this method.Optimal extraction conditions (ethanol 70–80%, 40–50 °C, pH 3–4, performed twice) were determined using an HSHE (5 min, 10 000 rpm, 25 °C) assisted shaker (60 min) and ultrasonication (40 kHz, 160 W cm−2, 30 min, 25 °C) procedure. Compared to the traditional non‐HSHE method, the total anthocyanin content obtained through HSHE extraction showed a significant increase, ranging from 1.0 to 3.9 times higher, with purple cabbage exhibiting the most pronounced enhancement in content. More types of anthocyanins were detected in blueberry (9), black bean (7) and raspberry (5), of which malvidin was the major anthocyanin (0.426 g kg−1) in blueberry, having an amount five times than previously obtained.The established HSHE method has been proven to be a superior technique for anthocyanin extraction, with higher extraction efficiency and concentrations. This technique also provides a new avenue for extracting bioactive compounds from diverse food sources, with potential applications in improving the functional properties of food products. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

In this study, a rapid and sensitive ultra‐high performance liquid chromatography‐tandem mass spectrometry method was established for the simultaneous determination of four Vitexin‐4"‐O‐glucoside (VOG), Vitexin‐2"‐O‐rhamnoside (VOR), vitexin (VIT), and hyperoside (HP) in plasma, which are representative flavonoids constituents of hawthorn leaves. The four components were separated by CORTECS UPLC C18 column (100 × 3.0 mm, 1.6 µm), using acetonitrile‐water containing 0.02% formic acid and 2 mmol/L ammonium formate as the mobile phase, the gradient elution flow rate was 0.30 mL/min. The analyte was quantified using an electrospray ionization triple quadrupole mass spectrometer. The analytes and baicalin internal standard were performed at m/z 593.2/413.2 for VOG, m/z 577.2/413.1 for VOR, m/z 431.1/310.9 for VIT, m/z 463.2/300 for HP and m/z 445.1/269 for internal standard. Among the four flavonoids, the correlation coefficient of the standard curve of each component is between 0.9939 and 0.9987. After oral administration of hawthorn leaf extract, VOG and VOR showed similar pharmacokinetic characteristics; VIT was eliminated in the body more slowly than the other three flavonoids and distributed more in the tissues; HP can be quickly absorbed in the blood. The developed analytical method has high sensitivity, precision, and accuracy with no interference from plasma components and has been successfully used in pharmacokinetic studies. [ABSTRACT FROM AUTHOR]

Amphetamines (AMPHs) and their derivatives are potent central nervous system stimulants, in 2020 an astonishing 34 million regular users were reported worldwide. Consequently, there has been a critical need for the development of methods capable of detecting these compounds in alternative biological matrices. The review article discusses the analysis of amphetamines and their derivatives in alternative biological matrices, such as hair and oral fluid (OF), using liquid chromatography‐mass spectrometry. Compiles data from 28 articles published between 2012 and 2022, focusing on 13 articles on OFs and 15 on hair analysis. The article highlights the advantages of using these alternative matrices, including less invasive collection methods, greater sample stability, and simplified extraction processes. Additionally, prevalent analytical practices such as the use of C18 columns and the incorporation of formic acid and methanol into mobile phases are discussed. Furthermore, it demonstrates that the most used method for collecting OF is the Quantisal device, as well as solid phase extraction is also the most used for the same matrix. The review serves as a guide for researchers interested in analytical methods for identifying AMPHs, offering insights into advances, challenges, and the most used in the area. [ABSTRACT FROM AUTHOR]

Nitazoxanide (NTZ) is an effective antiparasitic drug with potent antiviral and antimicrobial activity. This randomized, open‐label, 2‐sequence, 2‐period crossover trial was designed to evaluate the bioequivalence (BE) of the NTZ dry suspension in healthy subjects and investigated the effect of food intake on the pharmacokinetic (PK) properties of tizoxanide (an active metabolite of NTZ, TIZ). Sixty healthy Chinese subjects were enrolled and received a single dose of 500 mg/25 mL of preparations on days 1 and 4 under overnight fasting or fed conditions, respectively. The plasma concentration of TIZ was determined using high‐performance liquid chromatography/tandem mass spectrometry. PK parameters were calculated using WinNonlin 8.2 and BE was evaluated using SAS 9.4. The 90% confidence intervals for the geometric mean ratio (test/reference) of maximum concentration (Cmax), the area under the curve from time 0 to the time of the last quantifiable concentration (AUC0‐t), and the area under the curve from time 0 to extrapolation to infinity (AUC0‐∞) were all within the equivalent interval of 80%‐125%, compliant with BE requirements. In comparison with fasting, on taking the reference and test preparations of the NTZ dry suspension after a meal, the AUC0‐t increased by 48.9% and 47.3%, respectively, the AUC0‐∞ increased by 48.4% and 48.3%, respectively, and the post‐meal Tmax was prolonged by 1.8‐2 hours. Our results demonstrate that the test and reference preparations were bioequivalent. High‐fat meals significantly improve the degree of drug absorption and delay the rate of drug absorption. [ABSTRACT FROM AUTHOR]

In this study, we performed a quantitative analysis of 12 compounds derived from Piper sarmentosum extract (PSE) and guava leaf extract (GE). In addition, we investigated the effects of mixed extract (ME) of PSE and GE (1:1) on piglets' gut microbiome and metabolome. A total of 200 piglets (Duroc × Landrace × Large Yorkshire, 21‐day‐old) were randomly assigned into two groups with five replicates of 20 piglets/pen having the same initial body weight. Piglets were fed a basal diet supplemented with ME at 0 (T0) or 200 mg/kg (T1) for 3 weeks. The quantitation results by ultraperformance liquid chromatography linked to triple‐quadrupole tandem mass spectrometry showed that vitexin 2‐O‐rhamnoside and pellitorine were the greatest abundant among six compounds detected in the PSE. In addition, quercetin, isoquercitrin and avicularin were found to be the richest of all detected compounds in the GE. Findings on experimental animals indicated that three differential metabolites, comprising L‐alanine, sarcosine and dihydrofolic acid, in T1 compared with T0 groups, have exactly opposite levels trends in serum and faeces. Moreover, two metabolic pathways (i.e., urea cycle and glutamate metabolism) differed significantly in the serum and faeces of piglets between T0 and T1 (p < 0.05). At the same time, T1 had significantly higher relative abundances of Agathobacter and Alloprevotella than T0 at genus level (p < 0.05). Correlation analysis revealed that the genus Agathobacter correlated positively with carbamoyl phosphate (p < 0.01) and oxoglutaric acid (p < 0.05), and negatively with succinic acid (p < 0.01) and ornithine (p < 0.05). These four differential metabolites were also involved in the urea cycle and/or glutamate metabolism pathways. The results here indicated that the tested plant extract mixture represents a worthy feed additive with obvious antioxidative properties. [ABSTRACT FROM AUTHOR]

Neurocysticercosis (NCC), a major cause of global acquired epilepsy, results from Taenia solium larval brain infection. T. solium adult worms release large numbers of infective eggs into the environment contributing to high levels of exposure in endemic areas. This study identifies T. solium proteins in the sera of individuals with and without NCC using mass spectrometry to examine exposure in endemic regions. Forty‐seven patients (18–51 years), 24 parenchymal NCC (pNCC), 8 epilepsy of unknown aetiology, 7 glioma, 8 brain tuberculoma, and 7 healthy volunteers were studied. Trypsin digested sera were subject to liquid chromatography–tandem mass spectrometry and spectra of 375–1700 m/z matched against T. solium WormBase ParaSite database with MaxQuant software to identify T. solium proteins. Three hundred and nineteen T. solium proteins were identified in 87.5% of pNCC and 56.6% of non‐NCC subjects. Three hundred and four proteins were exclusive to pNCC sera, seven to non‐NCC sera and eight in both. Ten percent, exhibiting immune‐modulatory properties, originated from the oncosphere and cyst vesicular fluid. In conclusion, in endemic regions, T. solium proteins are detected in sera of individuals with and without pNCC. The immunomodulatory nature of these proteins may influence susceptibility and course of infection. [ABSTRACT FROM AUTHOR]

Jiawei Huoxiang Zhengqi Pill (JHZP) is a commonly used Chinese patent medicine for the clinical treatment of headache, dizziness, chest tightness as well as abdominal distension, and pain caused by wind‐cold flu. In this study, a comprehensive strategy combining ultra‐high performance liquid chromatography with diode array detector (UHPLC‐DAD) fingerprinting and multi‐component quantitative analysis was established and validated for quality evaluation of JHZP. A total of 49 characteristic common peaks were selected in a chromatographic fingerprinting study to assess the similarity of 15 batches of JHZP. Furthermore, 109 compounds were identified or preliminarily identified from JHZP by coupling with an advanced hybrid linear ion trap‐Orbitrap mass spectrometer. For quantification, the optimized ultra‐performance liquid chromatography with tandem mass spectrometry (UPLC‐MS/MS) method was employed for the simultaneous determination of 13 target compounds within 12 min. The sensitivity, precision, reproducibility, and accuracy of the method were satisfactory. This validated UPLC‐MS/MS method was successfully applied to analyzing 15 batches of JHZP. The proposed comprehensive strategy combining UHPLC‐DAD fingerprinting and multi‐component UPLC‐MS/MS analysis proved to be highly efficient, accurate, and reliable for the quality evaluation of JHZP, which can be considered as a reference for the overall quality evaluation of other Chinese herbal formulations. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a neurodegenerative disorder associated with behavioral and cognitive impairments. Unfortunately, the drugs the Food and Drug Administration currently approved for AD have shown low effectiveness in delaying the progression of the disease. The focus has shifted to non‐pharmacological interventions (NPIs) because of the challenges associated with pharmacological treatments for AD. One such intervention is environmental enrichment (EE), which has been reported to restore cognitive decline associated with AD effectively. However, the therapeutic mechanisms by which EE improves symptoms associated with AD remain unclear. Therefore, this study aimed to reveal the mechanisms underlying the alleviating effects of EE on AD symptoms using histological, proteomic, and neurotransmitter‐related analyses. Wild‐type (WT) and 5XFAD mice were maintained in standard housing or EE conditions for 4 weeks. First, we confirmed the mitigating effects of EE on cognitive impairment in an AD animal model. Then, histological analysis revealed that EE reduced Aβ accumulation, neuroinflammation, neuronal death, and synaptic loss in the AD brain. Moreover, proteomic analysis by liquid chromatography–tandem mass spectrometry showed that EE enhanced synapse‐ and neurotransmitter‐related networks and upregulated synapse‐ and neurotransmitter‐related proteins in the AD brain. Furthermore, neurotransmitter‐related analyses showed an increase in acetylcholine and serotonin concentrations as well as a decrease in polyamine concentration in the frontal cortex and hippocampus of 5XFAD mice raised under EE conditions. Our findings demonstrate that EE restores cognitive impairment by alleviating AD pathology and regulating synapse‐related proteins and neurotransmitters. Our study provided neurological evidence for the application of NPIs in treating AD. [ABSTRACT FROM AUTHOR]

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 spectrometry method 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 6 ng/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. [ABSTRACT FROM AUTHOR]

A sensitive and reliable LC–MS/MS method was developed and validated for the quantification of oxycodone and metabolites in human plasma. The method has a runtime of 6 min and a sensitivity of 0.1 μg/L for all analytes. Sample preparation consisted of protein precipitation. Separation was performed on a Kinetix biphenyl column (2.1 × 100 mm, 1.7 μm), using ammonium formate 5 mm in 0.1% aqueous formic acid and methanol LC–MS grade 100% in gradient elution at a flow rate of 0.4 ml/min. Detection was performed in multiple reaction monitoring mode using positive electrospray ionization. The method was linear over the calibration range of 0.1–25.0 μg/L for oxycodone, noroxycodone and noroxymorphone and 0.1–5.0 μg/L for oxymorphone. The method demonstrated good performance in terms of intra‐ and interday accuracy (86.5–110.3%) and precision (CV 1.7–9.3%). The criteria for the matrix effect were met (CV < 15%) except for noroxymorphone, for which an additional method was applied to compensate for the matrix effect. Whole blood samples were stable for 4 h at room temperature. Plasma samples were stable for 24 h at room temperature and 3 months at −20°C. Furthermore, the method was successfully applied in a pharmacokinetic drug interaction study of oxycodone and enzalutamide in patients with prostate cancer. [ABSTRACT FROM AUTHOR]

Rationale: Tramadol (T) is a strong painkiller drug that belongs to the opioid analgesic group. Several accidental intoxication cases after oral administration of T have been reported in the past decade. Tramadol, its derivatives, and metabolites present information‐limited mass spectra with one prominent peak representing the amine‐containing residue; therefore, their structural determination based on both electron impact mass spectrometry (EI‐MS) and ESI‐MS/MS spectra could be misleading. Methods: A novel analytical method for the structural elucidation of tramadol, its four homologs, and its two main phase I metabolites (N‐desmethyltramadol and O‐desmethyltramadol) was developed using chemical modification and liquid chromatography–high‐resolution tandem mass spectrometry (LC‐HR‐MS/MS) with Orbitrap technology. Results: After chemical derivatization, each of the investigated T series exhibited informative mass spectra that enabled better exposition of their structures. The developed method was successfully implemented to explicitly identify the structures of tramadol and its N‐desmethyltramadol metabolite in urine samples at low ng/mL levels. Conclusions: An efficient derivatization‐aided strategy was developed for rapidly elucidating the structure of tramadol‐like compounds. The method is intended to assist forensic chemists in better diagnosing T and its analogs and metabolites in clinical or forensic toxicology laboratories. [ABSTRACT FROM AUTHOR]

Rationale: Although ubiquitous in explosives and ammunition, few trace methods for detection of heavy metal‐containing primary explosives from forensic samples are currently in practice. Methods: Extracts of cotton swabs or direct sampling of items were cleaned up using solid‐phase extraction to remove heavy metal contaminants (i.e., lead) while retaining the organic styphnate component. The styphnate was chromatographically separated using hydrophilic interaction chromatography (HILIC) and detected via high‐resolution tandem mass spectrometry (MS/MS) using a sensitive, targeted approach in five minutes or less. Results: A mass spectrometric method for the detection of styphnate, including limit of detection (LOD), sample stability, and interferences was developed. We present a validated method for the extraction, separation, and detection of styphnate from lead(II) styphnate with an estimated LOD of 257 ppt (pg/mL). Conclusions: We detail an improved LOD relative to previous reports for trace detection of styphnate and, for the first time to our knowledge, the post‐blast analysis of styphnate. [ABSTRACT FROM AUTHOR]

Rationale: 4,7‐Dichloroquinoline (DCQ) represents a group of synthetic molecules inspired by natural products with important roles in biological and biomedical areas. This work aimed to characterize DCQ and its derivatives by high‐resolution electrospray ionization (ESI) mass spectrometry and tandem mass spectrometry (ESI‐MS/MS), supported by theoretical calculations. Biological assays were carried out with DCQ and its derivatives to determine LC50 values against Aedes aegypti larvae. Methods: Five DCQ derivatives were synthesized by using previously described protocols. ESI‐MS/MS analyses were carried out with a quadrupole/time‐of‐flight and ion‐trap instrument. The proposed gas‐phase protonation sites and fragmentation were supported by density functional theory calculations. The larvicidal tests were performed with the Ae. aegypti Rockefeller strain, and the LC50 values were determined by employing five test concentrations. Larval mortality was determined after treatment for 48 h. Results: DCQ bromides or aldehydes (C‐3 or C‐8 positions), as well as the trimethylsilyl derivative (C‐3 position), were prepared. Detailed ESI‐MS/MS data revealed heteroatom elimination through an exception to the even‐electron rule, to originate open‐shell species. Computational studies were used to define the protonation sites and fragmentation pathways. High activity of DCQ and its derivatives against Ae. aegypti larvae was demonstrated. Conclusion: Our results provided a well‐founded characterization of the fragmentation reactions of DCQ and its derivatives, which can be useful for complementary studies of the development of a larvicidal product against Ae. aegypti. [ABSTRACT FROM AUTHOR]

In this work, the antioxidant components in persimmon (Diospyros kaki) leaves were separated by offline two‐dimensional liquid chromatography‐electrochemical detection (LC×LC‐ECD) and identified by LC‐tandem mass spectrometry (LC‐MS/MS). A total of 33 antioxidants, mainly proanthocyanidins, and glycosides of kaempferol and quercetin, were identified. The antioxidant assays demonstrated that the fractions collected from the first‐dimension LC (1D‐LC) possessed considerable radical scavenging capabilities, with correlation coefficients of peak area versus radical scavenging capability of 1,1‐diphenyl‐2‐picrylhydrazyl and 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) being 0.9335 and 0.9116, respectively. The fingerprinting showed that 37 peaks were present in all samples. The major antioxidant components of persimmon leaves were the glycosides of kaempferol and quercetin. Finally, fourteen antioxidants were quantitatively assessed. Offline LC×LC provided high peak capacity and separation; ECD enabled specific screening and detection of antioxidant components; and MS/MS provided excellent identification capability. In this study, the combination of the three approaches was utilized to screen for antioxidant components in persimmon leaves, with satisfactory findings. In conclusion, this technique is an effective means for rapid analysis of antioxidant components and quality control of medicinal plants, achieving rapid separation of congeners and facilitating more accurate qualitative and quantitative analyses. [ABSTRACT FROM AUTHOR]

Identification of molecules in complex natural matrices relies on matching the fragmentation spectra of ions under investigation and the spectra acquired for the corresponding analytical standards. Currently, there are many databases of experimentally measured tandem mass spectrometry spectra (such as NIST, MzCloud, and Metlin), and considerable progress has been made in the development of software for predicting tandem mass spectrometry fragments in silico using combinatorial, machine learning, and quantum chemistry approaches (such as MetFrag, CFM‐ID, and QCxMS). However, the electrospray ionization molecules can be ionized at different sites (protonated or deprotonated), and the fragmentation spectra of such ions are different. Here, we are using the combination of the in‐ESI source hydrogen/deuterium exchange reaction and MSn fragmentation for the investigation of the fragmentation pathways for different protomers of organic molecules. It is shown that the distribution of the deuterium in the fragment ions reflects the presence of different protomers. For several molecules, the distribution of deuterium was traced up to the MS5 level of fragmentation revealing many unusual and unexpected effects. For example, we investigated the loss of HF from the ciprofloxacin and norfloxacin ions and observed that for ions protonated at –COOH group, the eliminating hydrogen always comes from –NH group. When ions are protonated at another site, the elimination of hydrogen with a probability of 30% occurs from the –NH group, and with a probability of 70%, it originates from other sites on the molecule. Such effects were not described previously. Quantum chemical simulation was used for the verification of the protonated structures and simulation of the corresponding fragmentation spectra. [ABSTRACT FROM AUTHOR]

Chloroformates are prevalent in the atmosphere due to their utilization as fuel additives and industrial solvents. These species may undergo interactions with atmospheric water resulting in protonated chloroformates. This study delves into the gas‐phase dissociation of these protonated species. Tandem mass spectrometry was employed to scrutinize the unimolecular dissociation of protonated methyl (1), ethyl (2), neopentyl (3), and phenyl chloroformate (4). Notably, 1 and 4 exhibited HCl loss, yielding CH3OCO+ and C6H5OCO+, respectively, with 1 additionally generating neutral methanol and ClCO+. 4 additionally loses CO and CO2. In contrast, 2 and 3 each only exhibit a single fragmentation channel, with 2 losing C2H4 to generate protonated chloroformic acid and 3 generating protonated 2‐methylbutene by losing neutral chloroformic acid. Density functional theory at the B3LYP/6‐311+G(d,p) level of theory was employed to explore minimum energy reaction pathways for each ion, and CBS‐QB3 single‐point energy calculations were added to provide reliable energetics. The Rice–Ramsperger–Kassel–Marcus (RRKM) calculations of the rate constants for selected competing processes were carried out to link theory and experiment. One common unimolecular process observed was the 1,3‐H shift of the proton from the carbonyl oxygen to the ester oxygen before dissociation. [ABSTRACT FROM AUTHOR]

The assignment of structure by tandem mass spectrometry (MS/MS) relies on the interpretation of the fragmentation behavior of gas‐phase ions. Mass spectra were acquired for a series of heterocyclic mimetics of acidic amino acids and a related series of nitrile amino acids. All amino acids were readily protonated or deprotonated by electrospray ionization (ESI), and distinctive fragmentation processes were observed when the ions were subjected to collision‐induced dissociation (CID). The deprotonated heterocycles showed bond cleavages of the 3‐hydroxyfurazan ring with formation of oxoisocyanate and the complementary deprotonated nitrile amino acid. Further fragmentation of the deprotonated nitrile amino acids was greatly dependent on the length of the alkyl nitrile side chain. Competing losses of CO2 versus HCN occurred from α‐cyanoglycinate (shortest chain), whereas water was lost from 2‐amino‐5‐cyanopentanoate (longest chain). Interestingly, loss of acrylonitrile by a McLafferty‐type fragmentation process was detected for 2‐amino‐4‐cyanobutanoate, and several competing processes were observed for β‐cyanoalanate. In one process, cyanide ion was formed either by consecutive losses of ammonia, carbon dioxide, and acetylene or by a one‐step decarboxylative elimination. In another, complementary ions were obtained from β‐cyanoalanate by loss of acetonitrile or HN=CHCO2H. Fragmentation of the protonated 3‐hydroxyfurazan and nitrile amino acids resulted in the cumulative loss (H2O + CO), a loss that is commonly observed for protonated aliphatic α‐amino acids. Overall, the distinct fragmentation behavior of the multifunctional 3‐hydroxyfurazan amino acids correlated with the charged site, whereas fragmentations of the deprotonated nitrile amino acids showed cooperative interactions between the nitrile and the carboxylate groups. [ABSTRACT FROM AUTHOR]

Turmeric and ginger are extensively employed as functional ingredients due to their high content of curcuminoids and gingerols, considered the key bioactive compounds found in these roots. In this study, we present an innovative and fast method for the assay of curcuminoids and gingerols in different foods containing the two spices, with the aim of monitoring the quality of products from a nutraceutical perspective. The proposed approach is based on paper spray tandem mass spectrometry coupled with the use of a labeled internal standard, which has permitted to achieve the best results in terms of specificity and accuracy. All the calculated analytical parameters were satisfactory; accuracy values are around 100% for all spiked samples and the precision data result lower than 15%. The protocol was applied to several real samples, and to demonstrate its robustness and reliability, the results were compared to those arising from the common liquid chromatographic method. [ABSTRACT FROM AUTHOR]

P0 proteins encoded by the pepper vein yellow virus (PeVYV) are pathogenic factors that cause hypersensitive response (HR). However, the host gene expression related to PeVYV P0‐induced HR has not been thoroughly studied. Transcriptomic technology was used to investigate the host pathways mediated by the PeVYV P0 protein to explore the molecular mechanisms underlying its function. We found 12,638 differentially expressed genes (DEGs); 6784 and 5854 genes were significantly upregulated and downregulated, respectively. Transcriptomic and reverse‐transcription quantitative polymerase chain reaction (RT‐qPCR) analyses revealed that salicylic acid (SA) and jasmonic acid (JA) synthesis‐related gene expression was upregulated, and ethylene synthesis‐related gene expression was downregulated. Ultrahigh performance liquid chromatography‐tandem mass spectrometry was used to quantify SA and JA concentrations in Nicotiana benthamiana, and the P0 protein induced SA and JA biosynthesis. We then hypothesized that the pathogenic activity of the P0 protein might be owing to proteins related to host hormones in the SA and JA pathways, modulating host resistance at different times. Viral gene silencing suppression technology was used in N. benthamiana to characterize candidate proteins, and downregulating NbHERC3 (Homologous to E6‐AP carboxy‐terminus domain and regulator of choromosome condensation‐1 dmain protein 3) accelerated cell necrosis in the host. The downregulation of NbCRR reduced cell death, while that of NbBax induced necrosis and curled heart leaves. Our findings indicate that NbHERC3, NbBax, and NbCRR are involved in P0 protein‐driven cell necrosis. [ABSTRACT FROM AUTHOR]

Context: Qi‐dan‐dihuang decoction (QDD) has been used to treat diabetic kidney disease (DKD), but the underlying mechanisms are poorly understood. Objective: This study reveals the mechanism by which QDD ameliorates DKD. Materials and Methods: The compounds in QDD were identified by high‐performance liquid chromatography and quadrupole‐time‐of‐flight tandem mass spectrometry (HPLC‐Q‐TOF‐MS). Key targets and signaling pathways were screened through bioinformatics. Nondiabetic Lepr db/m mice were used as control group, while Lepr db/db mice were divided into model group, dapagliflozin group, 1% QDD‐low (QDD‐L), and 2% QDD‐high (QDD‐H) group. After 12 weeks of administration, 24 h urinary protein, serum creatinine, and blood urea nitrogen levels were detected. Kidney tissues damage and fibrosis were evaluated by pathological staining. In addition, 30 mmol/L glucose‐treated HK‐2 and NRK‐52E cells to induce DKD model. Cell activity and migration capacity as well as protein expression levels were evaluated. Results: A total of 46 key target genes were identified. Functional enrichment analyses showed that key target genes were significantly enriched in the phosphatidylinositol 3‐kinase (PI3K)/protein kinase B (AKT) and mitogen‐activated protein kinase (MAPK) signaling pathways. In addition, in vivo and in vitro experiments confirmed that QDD ameliorated renal fibrosis in diabetic mice by resolving inflammation and inhibiting the epithelial‐mesenchymal transition (EMT) via the p38MAPK and AKT‐mammalian target of rapamycin (mTOR) pathways. Discussion and Conclusion: QDD inhibits EMT and the inflammatory response through the p38MAPK and AKT/mTOR signaling pathways, thereby playing a protective role in renal fibrosis in DKD. [ABSTRACT FROM AUTHOR]

Rationale: Acetaminophen (APAP) overdose is the leading cause of acute liver failure (ALF) in North America. To investigate the effect of drug‐induced liver injury (DILI) on circulating bile acid (BA) profiles, serum from ALF patients and healthy controls were analyzed using a semitargeted high‐resolution mass spectrometry approach to measure BAs in their unconjugated and amidated forms and their glucuronide and sulfate conjugates. Methods: Human serum samples from 20 healthy volunteers and 34 ALF patients were combined with deuterated BAs and extracted, prior to liquid chromatography high‐resolution tandem mass spectrometry analysis. A mix of 46 standards helped assign 26 BAs in human serum by accurate mass and retention time matching. Moreover, other isomers of unconjugated and amidated BAs, as well as glucuronide and sulfate conjugates, were assigned by accurate mass filtering. In vitro incubations of standard BAs provided increased information for certain peaks of interest. Results: A total of 275 BA metabolites, with confirmed or putative assignments, were measured in human serum samples. APAP overdose significantly influenced the levels of most BAs, promoting glycine conjugation, and, to a lesser extent, taurine conjugation. When patient outcome was considered, 11 BAs were altered significantly, including multiple sulfated species. Although many of the BAs measured did not have exact structures assigned, several putatively identified BAs of interest were further characterized using in vitro incubations. Conclusion: An optimized chromatographic separation tailored to BAs of ranging polarities was combined with accurate mass measurements to investigate the effect that DILI has on their complex profiles and metabolism to a much wider extent than previously possible. The analysis of complex BA profiles enabled in‐depth analysis of the BA metabolism perturbations in ALF, including certain metabolites related to patient outcomes. [ABSTRACT FROM AUTHOR]

Rationale: Ensuring the global safety and effectiveness of agrochemicals has become imperative. An in‐depth understanding of impurity profiles of products is crucial, especially for high‐demand agrochemicals, where impurities may be more toxic and persistent than original agrochemicals. This study focuses on the detection and identification of impurities in a commercial chlorantraniliprole (CAP), an anthranilic diamide class broad‐spectrum insecticide. Methods: Commercial CAP was collected from an agrochemical supplier in India and was analyzed using a high‐performance liquid chromatography‐photodiode array (HPLC‐PDA) (Agilent 1260; wavelength, 220 nm) with a Zorbax RP SB‐C18 (250 × 4.6 mm, 5 μm) column and liquid chromatography–mass spectrometry (LC–MS) (Agilent 6545 quadrupole time of flight (Q‐TOF)) techniques to identify the impurities. The impurities were isolated by preparative HPLC using a Zorbax‐DB C18 (250 × 9.4 mm, 5 μm) column. liquid chromatography– tandem mass spectrometry (LC–MS/MS) experiments (Q‐TOF) were performed on CAP and its impurities to obtain their structural data. Results: HPLC‐PDA analysis of CAP showed four major impurities (IM‐1 to IM‐4) ranging from 0.76% to 4.1%. The positive ion electrospray ionization (ESI) mass spectra of CAP and its impurities showed dominant [M + H]+ ions in addition to [M + Na]+, [M + K]+, and [2M + Na]+ ions. High‐resolution mass spectrometry (HRMS) data provided the elemental composition of the compounds, and isotopic distribution patterns revealed the number of Cl and/or Br atoms present in them. The structures of impurities were proposed based on the LC–MS/MS) data and further confirmed by nuclear magnetic resonance (NMR) data on isolated impurities/synthesis. Conclusion: The quality and impurities of CAP, a popular insecticide, must be assessed and described for its efficacy and safety. In this study, four impurities of CAP were detected using HPLC and successfully characterized using LC‐HRMS, LC–MS/MS, and NMR data. The method is useful for verifying the purity of CAP as well as helping in the identification of its possible impurities. [ABSTRACT FROM AUTHOR]

The salinilactones, volatile marine natural products secreted from Salinispora arenicola, feature a unique [3.1.0]‐lactone ring system and cytotoxic activities through a hitherto unknown mechanism. To find their molecular target, an activity‐based protein profiling with a salinilactone‐derived probe is applied that disclosed the protein disulfide‐isomerases (PDIs) as the dominant mammalian targets of salinilactones, and thioredoxin (TRX1) as secondary target. The inhibition of protein disulfide‐isomerase A1 (PDIA1) and TRX1 is confirmed by biochemical assays with recombinant proteins, showing that (1S,5R)‐salinilactone B is more potent than its (1R,5S)‐configured enantiomer. The salinilactones bound covalently to C53 and C397, the catalytically active cysteines of the isoform PDIA1 according to tandem mass spectrometry. Reactions with a model substrate demonstrated that the cyclopropyl group is opened by an attack of the thiol at C6. Fluorophore labeling experiments showed the cell permeability of a salinilactone‐BODIPY (dipyrrometheneboron difluoride) conjugate and its co‐localization with PDIs in the endoplasmic reticulum. The study is one of the first to pinpoint a molecular target for a volatile microbial natural product, and it demonstrates that salinilactones can achieve high selectivity despite their small size and intrinsic reactivity. [ABSTRACT FROM AUTHOR]

Intracellular proteome aggregation is a ubiquitous disease hallmark with its composition associated with pathogenicity. Herein, this work reports on a cell‐permeable photosensitizer (P8, Rose Bengal derivative) for selective photo induced proximity labeling and crosslinking of cellular aggregated proteome. Rose Bengal is identified out of common photosensitizer scaffolds for its unique intrinsic binding affinity to various protein aggregates driven by the hydrophobic effect. Further acetylation permeabilizes Rose Bengal to selectively image, label, and crosslink aggregated proteome in live stressed cells. A combination of photo‐chemical, tandem mass spectrometry, and protein biochemistry characterizations reveals the complexity in photosensitizing pathways (both Type I & II), modification sites and labeling mechanisms. The diverse labeling sites and reaction types result in highly effective enrichment and identification of aggregated proteome. Finally, aggregated proteomics and interaction analyses thereby reveal extensive entangling of proteostasis network components mediated by HSP70 chaperone (HSPA1B) and active participation of autophagy pathway in combating proteasome inhibition. Overall, this work exemplifies the first photo induced proximity labeling and crosslinking method (namely AggID) to profile intracellular aggregated proteome and analyze its interactions. [ABSTRACT FROM AUTHOR]

SUMMARY: Extracellular ATP (eATP) is a key signaling molecule that plays a pivotal role in plant growth and defense responses. The receptor P2K1 is responsible for perceiving eATP and initiating its signaling cascade. However, the signal transduction mechanisms downstream of P2K1 activation remain incompletely understood. We conducted a comprehensive analysis of the P2K1 interactome using co‐immunoprecipitation‐coupled tandem mass spectrometry, leading to the identification of 121 candidate proteins interacting with P2K1. In silico analysis narrowed down the candidates to 47 proteins, including Ca2+‐binding proteins, ion transport‐related proteins, and receptor kinases. To investigate their involvement in eATP signaling, we employed a screening strategy based on changes in gene expression in response to eATP in mutants of the identified interactors. This screening revealed several Ca2+‐dependent protein kinases (CPKs) that significantly affected the expression of eATP‐responsive genes, suggesting their potential roles in eATP signaling. Notably, CPK28 and CPK6 showed physical interactions with P2K1 both in yeast and plant systems. Calcium influx and gene expression studies demonstrated that CPK28 perturbed eATP‐induced Ca2+ mobilization and some early transcriptional responses. Overexpression of CPK28 resulted in an antagonistic physiological response to P2K1‐mediated eATP signaling during both plant growth and defense responses to the necrotrophic pathogen Botrytis cinerea. Our findings highlight CPK28, among other CPKs, as a modulator of P2K1‐mediated eATP signaling, providing valuable insights into the coordination of eATP signaling in plant growth and immunity. Significance Statement: In the last two decades, extracellular ATP has become a pivotal signaling element in animal physiology and plant biology. Recognized for its role in plant growth and defense, the purinergic signal of eATP is well‐established. Despite some identified interactors of the eATP receptor P2K1, a comprehensive understanding of purinergic signaling has been hindered by the lack of a P2K1 interactome. Our study successfully generated this interactomics dataset, shedding light on eATP‐mediated signaling and revealing a key player, CPK28, a Ca2+‐dependent protein kinase. [ABSTRACT FROM AUTHOR]

A pinanamine‐based analog, (1R,2R,3R,5S)‐N‐((3‐cyclopropylthiophen‐2‐yl)methyl)‐2,6,6‐trimethylbicyclo[3.1.1]heptan‐3‐amine (M090) was synthesized and demonstrated with anti‐influenza activity to overcome the multi‐drug resistance. Herein, a rapid and robust liquid chromatography‐tandem mass spectrometry method was developed and validated to quantify M090 in rat plasma. With simple protein precipitation by methanol, the separation of M090 was performed on a Waters BEH‐C18 column (2.1 × 100 mm, 1.7 μm) by gradient elution at 0.4 mL/min with mobile phases consisting of water containing 0.1 % formic acid (phase A) and methanol (phase B). M090 and clenbuterol (internal standard) were detected using multiple reaction monitoring in positive electrospray ionization mode with transitions of m/z 290.3→137.0 and m/z 277.0→203.0, respectively. The method was validated over a linear range of 1.0–2400 ng/mL with a regression coefficient of 0.9974 and no endogenous interference. The intra‐ and inter‐batch precisions were within 8.29% and accuracy ranged from 100.3% to 108.1% for M090. The validated method was utilized to evaluate the in vitro metabolic stability and in vivo pharmacokinetics of M090 in rats. [ABSTRACT FROM AUTHOR]

Regular monitoring of pesticide residues in potatoes (Solanum tuberosum L.) is important to ensure a low risk of pesticide toxicity to consumers. An efficient, modified quick easy cheap effective rugged safe‐dispersive‐solid‐phase extraction (QuEChERS‐ d‐SPE) was optimized and validated for quantification of pesticide residues (metalaxyl, chlorpyrifos, λ‐cyhalothrin, and cypermethrin) in popular Kenyan potato varieties "Shangi," "Tigoni," and "Dutch Robyjn," grown under controlled field conditions using gas chromatography with tandem mass spectrometry (GC‐MS/MS). An evaluation of the effect of five spraying frequencies (unsprayed, 5, 7, 10, and 14‐day intervals) of metalaxyl‐based fungicides on residue levels in harvested potatoes was simultaneously done. Metalaxyl residue levels in the varieties were determined for each spraying regime. Method validation showed good linearity (r2) of 0.988–0.999, detection limit (0.37–0.61 µg/L), quantification limit (1.20–1.84 µg/L) while recovery (71%–94%), and precision (relative standard deviation of 2.3%–24.1%) were used to determine the accuracy of the method. Pyrethroids (cypermethrin and λ‐cyhalothrin) and organophosphates (metalaxyl and chlorpyrifos) were detected in the potato samples. Residue levels of the compounds were not significantly different (p = 0.05) between varieties except for chlorpyrifos. Cypermethrin was the residue compound detected at the highest mean level with 17.4 µg/L, followed by metalaxyl, chlorpyrifos, and λ‐cyhalothrin, at 2.88, 1.05, and 0.15 µg/L, respectively. Residue levels at different spraying frequencies varied statistically between varieties (p = 0.05). Dutch Robyjn sprayed at seven days' intervals had the highest residue level of 9.49 ± 0.26 µg/L. QuEChERS‐d‐SPE with GC‐MS/MS has shown suitability for routine pesticide residue monitoring in potatoes. The potatoes grown under the controlled experimental conditions did not present any concern for human health, as the pesticide residues detected were below Codex maximum residue levels of 500 µg/L. Core Ideas: Potato field production in Kenya is characterized by intensive pesticide application.There is limited data available locally about the type of pesticide or their residue levels present in potatoes.Pyrethroids and organophosphates were detected and quantified in the potato samples.Cypermethrin was the highest residue compound detected, though below Codex MRLs while λ‐cyhalothrin was the lowest.Highest residue in cypermethrin in Dutch Robyjn, metalaxyl in Shangi, and chlorpyrifos and λ‐cyhalothrin in Tigoni. [ABSTRACT FROM AUTHOR]