Negative Mode Research Articles

In vivo detection of the epimer metabolites of sweroside via ultra-performance liquid chromatography time-of-flight mass spectrometry combined with DNPH derivatization.

The metabolites of sweroside were first investigated in vivo with ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOF-MS) in combination with 2,4-dinitrophenylhydrazine derivatization. In addition, the mass detection sensitivity of the major metabolites, epinaucledal and naucledal, via UPLC-TOF-MS was significantly enhanced, and the epimer metabolites were distinctly discovered from plasma following gavage of sweroside in rats. The plasma concentration of epinaucledal and naucledal was quantified via UPLC-TOF-MS in negative mode using erythrocentaurin as the internal standard. The maximum mean plasma concentrations of naucledal and epinaucledal were 75.36 ± 20.10and 43.52 ± 15.60 ng/ml within 2h, respectively, following gavage of sweroside at 20 mg/kg. Moreover, the area under the concentration-time curve of naucledal was three times that of epinaucledal. The metabolic process of conversion of sweroside to epinaucledal and naucledal was deduced, and the pharmacological effects of epinaucledal and naucledal will clarify the clinical efficacy of sweroside.

Network pharmacology combined with experimental validation reveals the mechanism of action of erpixing granules on functional dyspepsia

Ethnopharmacological relevanceFunctional dyspepsia (FD) is a prevalent gastrointestinal disorder characterised by high incidence and recurrence rates, posing significant health risks. Erpixing Granules (EPX), approved by the National Food and Drug Administration in 2002, are known for their spleen and stomach invigorating properties, effectively treating FD. However, its mechanism of action remains unclear. Aim of the studyThis study aims to elucidate EPX's mechanism of treating FD through network pharmacology, and experimental validation using FD animal models. MethodsIn this study, the chemical composition of EPX in positive and negative ion modes was analyzed by UHPLC-Q-TOF MS. The mass spectral data were processed and analyzed using MS-DIAL software to automatically match compound fragment information and identify the known components with the compound database to obtain the active components of EPX. SwissTargetPrediction was used to obtain EPX targets, while FD-related targets were sourced from GeneCards, OMIM and DisGeNET databases. A protein-protein interaction (PPI) network was constructed using the STRING platform, and potential signalling pathways of EPX were determined through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Finally, an FD model was established in rates by administering a 0.1% iodoacetamide sucrose solution, followed by tail clamp stimulation to experimentally validate the network pharmacology findings. ResultsOur results revealed 139 effective ingredients in EPX, targeting 60 core FD-related genes. PPI network analysis identified EGFR, CTNNB1 and NFκB1 as core target genes. The KEGG pathway analysis indicated that EPX can modulate FD progression through the PI3K/AKT signalling pathway. Animal experiments demonstrated EPX's capacity to increase body mass, food intake and food utilisation efficiency in FD rats, alongside increased gastric juice secretion, pepsin activity, trypsin activity, cholesterol, bile acid and bilirubin activity. HE examination revealed that EPX improved the inflammatory infiltration of gastric mucosal cells in rats. Furthermore, EPX also promoted gastric emptying and intestinal propulsion in mice. These results suggest that EPX improves spleen and stomach function, enhances the protective effect on the spleen and stomach and promotes food digestion and absorption. Immunofluorescence studies revealed upregulated expression of PI3K, AKT and ANO1 proteins in gastric tissue following EPX administration, while Western blotting indicated increased expression of SCF and C-kit proteins. ConclusionSuggesting EPX's anti-FD effect may involve the regulation of the SCF/C-kit signalling pathway and activation of downstream PI3K/AKT signalling pathway, thereby promoting gastrointestinal motility and improving FD symptoms.

Biliary atresia and cholestasis plasma non-targeted metabolomics unravels perturbed metabolic pathways and unveils a diagnostic model for biliary atresia

The clinical diagnosis of biliary atresia (BA) poses challenges, particularly in distinguishing it from cholestasis (CS). Moreover, the prognosis for BA is unfavorable and there is a dearth of effective non-invasive diagnostic models for detection. Therefore, the aim of this study is to elucidate the metabolic disparities among children with BA, CS, and normal controls (NC) without any hepatic abnormalities through comprehensive metabolomics analysis. Additionally, our objective is to develop an advanced diagnostic model that enables identification of BA. The plasma samples from 90 children with BA, 48 children with CS, and 47 NC without any liver abnormalities children were subjected to metabolomics analysis, revealing significant differences in metabolite profiles among the 3 groups, particularly between BA and CS. A total of 238 differential metabolites were identified in the positive mode, while 89 differential metabolites were detected in the negative mode. Enrichment analysis revealed 10 distinct metabolic pathways that differed, such as lysine degradation, bile acid biosynthesis. A total of 18 biomarkers were identified through biomarker analysis, and in combination with the exploration of 3 additional biomarkers (LysoPC(18:2(9Z,12Z)), PC (22:5(7Z,10Z,13Z,16Z,19Z)/14:0), and Biliverdin-IX-α), a diagnostic model for BA was constructed using logistic regression analysis. The resulting ROC area under the curve was determined to be 0.968. This study presents an innovative and pioneering approach that utilizes metabolomics analysis to develop a diagnostic model for BA, thereby reducing the need for unnecessary invasive examinations and contributing to advancements in diagnosis and prognosis for patients with BA.

The cervicovaginal metabolome in women with favorable induction cervix and those unfavorable for induction when delivering at term

BackgroundCervical ripening is crucial for induction. However, its influencing factors, mechanistic understanding, and effective risk stratification are still challenging. Recent research suggested that microorganisms and their metabolites in vaginal spaces correlate to preterm birth. However, it remains unclear whether the cervicovaginal metabolome is related to the natural physiological process of cervical maturation. ObjectiveWe aimed to analyze the cervicovaginal metabolome in women with favorable induction cervix and those unfavorable for induction when delivering at term. Study designCervicovaginal swabs were collected between 40 and 41 weeks gestation from the following 2 different groups of patients: Ripe group (n = 25) which was favorable for the induction cervix and Unripe group which was unfavorable for the induction cervix (n = 25). Samples were tested using untargeted metabolomics analysis and analyzed by a bioinformatics platform. The correlation analysis between the metabolome and the previously acquired microbiome was also performed. ResultsA total of 629 metabolites were identified in cervicovaginal fluid. The cervicovaginal metabolome was significantly different between the women with the ripe cervix and those with the unripe cervix, especially within each stratum of the same CST. Metabolites within the amino acid, carbohydrate, and dipeptide pathways may play a role in this distinction. Thirty-four metabolites were significantly upregulated, and the remaining fourteen were significantly downregulated in the Unripe group with an unripe cervix unfavorable for induction. Statistical modeling identified Arachidonic Acid and Nicotinate associated with the risk of cervical maturation disorder (AUC 0.87) in negative ion mode. A combination of Choline and d-Mannose identified a risk of cervical maturation disorder (AUC 0.80) in positive ion mode, improved by Lactobacillus relative abundance (AUC 0.89). ConclusionThese data suggested that the cervicovaginal space was metabolically active during pregnancy and significantly altered among the women with the mature and immature cervix. Combining the genera-level phylotypes and metabolites could build better cervix maturity prediction models. By using cervicovaginal fluid samples, we demonstrated the potential of multi-data type integration for developing composite models toward understanding the contribution of the vaginal environment to the remodeling of cervix during term pregnancy.

Association of litter size with the ruminal microbiome structure and metabolomic profile in goats

The Yunshang black goat is a renowned mutton specialist breed mainly originating from China that has excellent breeding ability with varying litter sizes. Litter size is an important factor in the economics of goat farming. However, ruminal microbiome structure might be directly or indirectly regulated by pregnancy-associated factors, including litter sizes. Therefore, the current experiment aimed to evaluate the association of different litter sizes (low versus high) with ruminal microbiome structure by 16S rRNA gene sequencing and metabolomic profiling of Yunshang black does. A total of twenty does of the Yunshang Black breed, approximately aged between 3 and 4 years, were grouped (n = 10 goats/group) into low (D-l) and high (D-h) litter groups according to their litter size (the lower group has ≤ 2 kids/litter and the high group has ≧ 3 kids/litter, respectively). All goats were sacrificed, and collected ruminal fluid samples were subjected to 16S rRNA sequencing and LC–MS/MC Analysis for ruminal microbiome and metabolomic profiling respectively. According to PCoA analysis, the ruminal microbiota was not significantly changed by the litter sizes among the groups. The Firmicutes and Bacteroidetes were the most dominant phyla, with an abundance of 55.34% and 39.62%, respectively. However, Ruminococcaceae_UCG-009, Sediminispirochaeta, and Paraprevotella were significantly increased in the D-h group, whereas Ruminococcaceae_UCG-010 and Howardella were found to be significantly decreased in the D-l group. The metabolic profiling analysis revealed that litter size impacts metabolites as 29 and 50 metabolites in positive and negative ionic modes respectively had significant differences in their regulation. From them, 16 and 24 metabolites of the D-h group were significantly down-regulated in the positive ionic mode, while 26 metabolites were up-regulated in the negative ionic mode for the same group. The most vibrant identified metabolites, including methyl linoleate, acetylursolic acid, O-desmethyl venlafaxine glucuronide, melanostatin, and arginyl-hydroxyproline, are involved in multiple biochemical processes relevant to rumen roles. The identified differential metabolites were significantly enriched in 12 different pathways including protein digestion and absorption, glycerophospholipid metabolism, regulation of lipolysis in adipocytes, and the mTOR signaling pathway. Spearman’s correlation coefficient analysis indicated that metabolites and microbial communities were tightly correlated and had significant differences between the D-l and D-h groups. Based on the results, the present study provides novel insights into the regulation mechanisms of the rumen microbiota and metabolomic profiles leading to different fertility in goats, which can give breeders some enlightenments to further improve the fertility of Yunshang Black goats.

A comparative metabolomics analysis of Açaí (Euterpe oleracea Mart.) fruit, food powder, and botanical dietary supplement extracts.

Euterpe oleracea Mart. (açaí) is a botanical of interest to many who seek functional foods that provide antioxidant and anti-inflammatory properties. Cancer patients are increasingly taking botanical dietary supplements containing açaí to complement their conventional therapeutics, which may lead to serious adverse events. Before testing our açaí extracts in vitro for botanical-drug interactions, the goal is to chemically characterize our extracts for compounds whose biological activity in açaí is unknown. The objective of this work was to develop a chemical fingerprinting method for untargeted characterization of açaí samples from a variety of sources, including food products and botanical dietary supplement capsules, made with multiple extraction solvents. An optimized LC-MS method was generated for in-depth untargeted fingerprinting of chemical constituents in açaí extracts. Statistical analysis models were used to describe relationships between the açaí extracts based on molecular features found in both positive and negative mode ESI. In an attempt to elucidate the differences in metabolites among açaí extracts from different cultivars, we identified or tentatively identified 173 metabolites from the 16 extracts made from 6 different sources. Of these compounds, there are 138 reported in açaí for the first time. Statistical models showed similar yet distinct differences between the extracts tested based on the polarity of compounds present and the origin of the source material. A high-resolution mass spectrometry method was generated that allowed us to greatly characterize 16 complex extracts made from different sources of açaí with different extraction solvent polarities.

Investigating the Chemical Profile of Underexplored Parts of Dipteryx alata (Baru) Using the PS-MS Technique.

The baru (Dipteryx alata Vog.), a fruit native to the Cerrado biome, is well-known for its almonds, which are extensively exploited and exported. Unfortunately, the remaining parts of this fruit are often discarded. This study investigates the fixed chemical constituents of the baru, including the bark, pulp, endocarp, and almonds, using the PS-MS technique in positive and negative ionization modes. Notably, this research presents the first chemical profile of baru almonds in both their raw and roasted states. The analysis identified 57 compounds reported for the first time in a baru and 24 common compounds. The majority of these compounds are classified as flavonoids. In both ionization modes, the peel exhibited a higher proportion of phenolic compounds, although the chemical compounds varied among the peel, pulp, almond, and endocarp. These findings highlight the perspective of bioeconomy and biotechnology. By staggering baru fruit production alongside extractivists, we can optimize the utilization of all parts of the fruit. Furthermore, given the knowledge of the biological properties of flavonoids and the baru composition, we recommend additional studies to analyze their potential in preventing chronic non-communicable diseases.

Selective Synthesis of Deuterated cis- and trans-Isohumulones and trans-Isohumulinones

Deuterated isohumulones can be prepared directly from humulones by an acyloin ring contraction under either magnesium catalyzed basic conditions or photochemical induced reaction in deuterated solvents. Reaction of humulones with biphasic methylene chloride/aqueous NaOD and MgSO4 in D2O gave stereoselective formation of cis-3d- isohumulones (cis-trans ratio of 82:18) as the magnesium salts in 71-83% yield. Greater than 95% incorporation of three deuterons was observed in the C5 position of the pentenone ring and the methylene position of the C4 acyl group. Photochemical isomerization with a 400 nm blue LED source provided stereospecific formation of deuterated trans-isohumulones in 36-82% yield with greater than 95% incorporation of deuterium in the C5 ring position. Oxidation of humulones with cumene hydroperoxide in basic D2O gave isohumulinones with partial 55-73% incorporation of deuterium due to keto-enol isomerization of the methylene substituent of the C4 acyl group. Structural identity of the deuterated products was determined by a combination of negative mode electrospray mass spectrometry (MS-ESI-) and 2D heteronuclear proton-carbon HMQC nmr analysis.

A simple and sensitive LC-MS/MS method for the determination of loganic acid in rat plasma and its pharmacokinetic analysis.

Loganic acid is an iridoid compound extracted from Gentianaceae plant Gentiana macrophylla Pall. It can effectively inhibit inflammation and tumor migration and has antioxidant activity. In this paper, we establish a simple, fast, sensitive and validated LC-MS method with the purpose of quantification of loganic acid in rat plasma with gliclazide as an internal standard (IS). Methanol was used to precipitate the protein in the plasma sample, and a C18 column (2.1 × 50 mm, 1.7μm) was used for the separation of the target compound. Meanwhile, 0.1% formic acid water-methanol was employed as the mobile phase. Multiple reaction monitoring detection mode was adopted in detection with m/z 375.1 > 213.2 for loganic acid and m/z 322.1 > 169.9 for the IS, respectively, in negative ion scan mode. The linear range of calibration curve was 5.77-11,540.00 ng/ml, and the lower limit of detedtion was 2.89 ng/ml. The inter-day and intra-day precision and accuracy were <15% for lower limit of quantitation, low, middle and high quality control samples. This method was successfully used for the pharmacokinetic study of loganic acid in rat plasma at a dose range of 50-150 mg/kg for oral administration and 2mg/kg for intravenous administration. The pharmacokinetic results showed that the oral bioavailability of loganic acid was low (2.71-5.58%).

A Rapid, Sensitive, and High-throughput Method for the Simultaneous Determination of Antihypertensive Drug Combinations in Dog Plasma by UHPLC-MS/MS: The Assessment of Predicable Bioequivalence of In-vitro Dissolution Condition.

Essential hypertension is a common clinical disease and a risk factor for cardiovascular and cerebrovascular diseases. Olmesartan medoxomil, amlodipine, and hydrochlorothiazide are commonly used antihypertensive drugs. The aim of this study was to establish a robust UPLC-MS/MS method for the simultaneous determination of olmesartan medoxomil, amlodipine, and hydrochlorothiazide in dog plasma. At the same time, the release in vivo and in vitro studies were conducted, and a preliminary in vitro-in vivo correlation (IVIVC) evaluation was performed. The bioequivalence experiment was conducted with a double-crossed design. Three major components were extracted and analyzed by UHPLC-MS/MS. With the MRM scan, olmesartan and amlodipine were quantified by fragment conversion (m/z 447.10→190.10) and (m/z 408.95→294.00) under positive ESI mode, while hydrochlorothiazide was quantified with fragment conversion (m/z 295.90→268.90) under negative ESI mode. The in vitro release studies were performed using a USP paddle, and the dissolution medium was chosen from pH 6.0 to pH 6.8 according to the BCS classification of compounds. The IVIVC was calculated using the Wagner-Nelson equation. The linear ranges of olmesartan, amlodipine, and hydrochlorothiazide in the plasma were 5.0-2500, 0.1-50, and 3.0-1500 ng/mL, respectively. All accuracies were within 3.8% of the target values, and the findings revealed that intra-day and inter-day accuracy was less than 12.1%. Moreover, the recoveries exceeded 88.3%, the matrix effect tests were positive, and the stability tests were positive. With the establishment of correlation, the distinguishable dissolution condition (pH 6.8) was selected as the predictable condition. The established method was suitable for the preclinical pharmacokinetic study of tripartite drugs with strong specificity and high sensitivity. Through the evaluation of IVIVC, the connection between in vivo and in vitro drug testing was initially established.</p>.

Constituents of alexander’s celery (Smyrnium olusatrum) extracts and their antioxidant, enzyme inhibitory and anticancer effects based on in vitro, in silico and network pharmacology methods

This research delves into the medicinal significance of Smyrnium olusatrum by investigating ethyl acetate, ethanol, ethanol/water, and infusion extracts. The chemical composition analysed through LC-MS-qTOF metabolomic analysis, determine total phenolic and flavonoid contents, evaluate antioxidant potential using six in vitro tests (DPPH, ABTS, FRAP, CUPRAC, phosphomolybdenum assay (PBD), and metal chelating assay (MCA)), assess enzyme inhibition activity against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), tyrosinase, α-amylase, and α-glucosidase, and explore their anticancer effects on HEp-2 cells. Additionally, qPCR analysis is conducted on HEp-2 larynx cancer cells to examine the impact of S. olusatrum on self-renewal and apoptosis pathways, along with the expression levels of key genes associated with these pathways. The findings indicated that the infusion extraction method demonstrated the highest levels, with a recorded TPC of 35.02 mg GAE/g and a TFC of 15.08 mg RE/g. All four extracts of S. olusatrum exhibited a total of 328 entities. The most significant metabolites, primarily comprising polyphenolics, flavonoids, non-structural carbohydrates, and amino acids in negative ionization mode, as well as sesquiterpene lactone and amino acids in positive ionization, were identified. Infusion exhibited the highest antioxidant effect among the extracts, with peak values ranged from 55.55 mg TE/g (DPPH) to 100.07 mg TE/g (ABTS). The extracts exhibited variable enzyme activities. Notably, ethyl acetate also demonstrated superior α-Amylase inhibition (0.69 mmol ACAE/g), while the ethanol extract exhibited higher α-Glucosidase inhibition (1.70 mmol ACAE/g). The HEp-2 cells demonstrated the quickest attainment of half maximal inhibitory concentration values with ethanol and ethanol/water extracts, yielding IC50 values of 250 µg/mL (24 h) and 125 µg/mL (24 h), respectively, among the applied extracts. The qPCR results revealed that S. olusatrum inhibited all self-renewal pathways and activated the apoptotic pathway. The ethanol and ethanol/water extracts of S. olusatrum significantly suppressed WNT1, APC, LEF1, and TCF7 genes. Furthermore, the downregulation of NOTCH1, SHH, and SMO gene expressions in all extracts suggests the activation of the Type 1 non-canonical hedgehog pathway in laryngeal cancer. The findings not only underscore the therapeutic potential of these extracts but also open the way for further exploration of their applications in combating oxidative stress, enzyme-related disorders, and potential anti-cancer effects through modulation of crucial cellular pathways.

Safety and human health: The landscape of an effective UPLC-MS/MS method for the identification and quantification of N-Nitroso Hydrochlorothiazide impurity in Hydrochlorothiazide

N-nitrosamines (NSAs) and Nitrosamine Drug Substance Related Impurities (NDSRIs) are class of compounds that contains the nitroso functional group. These compounds are carcinogens and mutagens for both humans and animals. As a result, all global regulatory agenesis are requiring risk assessments of NSAs in each pharmaceutical product. The present research aims to provide a single pre-packaged mass spectrometry method parameter set, which includes Ultra performance liquid chromatography and a triple quadrupole mass spectrometer (UPLC-MS-MS) configuration technique for qualifying and quantifying NNHCT in HCT. The analysis was performed on a YMC ODS-A liquid chromatography column with an oven temperature of 35 °C. In this method, mobile phase was prepared by mixing 10 mM ammonium formate buffer and acetonitrile was mixed in the ratio of 60:40 (v/v) with an isocratic program and a flow rate of 0.8 mL/min. The mass detector was used along with electrospray ionization (ESI) Negative mode with multiple reaction monitoring (MRM). The method has been validated in accordance with the current regulatory guidelines and shown the ability to detect NNHCT with a LOQ of 0.01 ppm and LOD of 0.005 ppm.

Effect of membrane properties on the odor emanating from training aids for explosive-detecting canines.

Canines are widely used for real-time detection of explosives and have proven to be on par with instrumental methods. Canines are thought to rely largely upon detection of volatile chemical constituents of the explosives, though not necessarily the explosive itself. Hence, it is crucial to understand the odor available to them as generated by training aids. Previous studies have established that the Training Aid Delivery Device (TADD) developed by SciK9 is a reliable training aid that reduces cross-contamination and doubles as a storage device. A TADD comprises a standardized container, a synthetic membrane, a membrane holder, and a lid. In the work presented, activated charcoal strips were placed above and below the TADD membrane to determine the relative amounts of volatiles emitted by dynamite (i.e., ethylene glycol dinitrate (EGDN) and trinitroglycerin (NG)). The strips were eluted and the extracts tested using gas chromatography-mass spectrometry in negative ion chemical ionization mode. A series of t-tests at 95% confidence level were performed to determine any differences in vapor composition above and below the membranes. Nine synthetic membranes and six glass fiber membranes were tested in this study. It was expected that the relative concentration of volatiles would remain the same on both sides of the membrane; however, selective removal of nitroglycerin by some membranes was observed. Synthetic membranes with larger pore sizes showed no alteration in the vapor composition. Both synthetic and glass fiber membranes did not show a significant change in relative concentration of the other volatile compound in dynamite, i.e., EGDN. Out of all the membranes tested, three synthetic membranes and four glass fiber membranes showed selective alteration in odor availability of nitroglycerin in dynamite. For training purposes, membranes that do not alter the vapor composition should be used in the training aid.

DART-HRMS reveals metabolic changes of whey through microparticulation and fermentations

Despite native whey (hereafter termed WHEY) being a watery by-product rich in high-value nutritional components, it poses environmental concerns. Recently, the techniques of thermal-mechanical microparticulation to produce microparticulated whey (MPW) and a further process to ferment MPW (FMPW) were suggested, in order to recycle whey into a protein concentrated soft dairy cream that acquires also fat mimicking functionality. Aiming at monitoring the effects of this recycling process on the products’ metabolic profiles, samples (n = 8) of WHEY, MPW and two sub-types of FMPW were analysed by combining direct analysis in real time coupled with high resolution mass spectrometry (DART-HRMS). The most informative ions were used to build a partial least squared discriminant analysis (PLS-DA) model to assess the biochemical compounds that characterize and differentiate WHEY from MPW and FMPW. The DART-HRMS fingerprints acquired in negative mode accurately differentiated samples throughout the steps of microparticulation and fermentation. Post-fermentation, greater relative abundances of lactic acid, glucosamine and histidyl-aspartic acid were detected in both subtypes of FMPW, which were moderately differentiated by DART-HRMS. Moreover, while WHEY and MPW contained high relative abundances of hydroxyglutaric and malic acids, the fermented derivates (FMPW) were characterised by elevated levels of volatile compounds (tetradecanal, hexadecene and tetradecene) and fermentative end-products (diethyltartrate and histidyl-aspartic acid). DART-HRMS successfully captured a pool of informative biomarkers useful to understand the chemical and metabolomic changes occurring in microparticulation and fermentation processes used to recycle whey. This analytical technique is an operative supporting step needed for comprehensive assessment of the nutritional and organoleptic properties of the proposed whey-based soft cream dairy food.

Effects of Combined Low-Dose Spironolactone Plus Vitamin E versus Vitamin E Monotherapy on Lipidomic Profile in Non-Alcoholic Fatty Liver Disease: A Post Hoc Analysis of a Randomized Controlled Trial.

Background/Objectives: Lipid dysmetabolism seems to contribute to the development and progression of nonalcoholic fatty liver disease (NAFLD). Our aim was to compare serum lipidomic profile between patients with NAFLD having received monotherapy with vitamin E (400 IU/d) and those having received combination therapy with vitamin E (400 IU/d) and low-dose spironolactone (25 mg/d) for 52 weeks. Methods: This was a post hoc study of a randomized controlled trial (NCT01147523). Serum lipidomic analysis was performed in vitamin E monotherapy group (n = 15) and spironolactone plus vitamin E combination therapy group (n = 12). We employed an untargeted liquid chromatography-mass spectrometry lipid profiling approach in positive and negative ionization mode. Results: Univariate analysis revealed 36 lipid molecules statistically different between groups in positive mode and seven molecules in negative mode. Multivariate analysis in negative mode identified six lipid molecules that remained robustly different between groups. After adjustment for potential confounders, including gender, omega-3 supplementation, leptin concentration and homeostasis model assessment-insulin resistance (HOMA-IR), four lipid molecules remained significant between groups: FA 20:5, SM 34:2;O2, SM 42:3;O2 and CE 22:6, all being higher in the combination treatment group. Conclusions: The combination of spironolactone with vitamin E led to higher circulating levels of four lipid molecules than vitamin E monotherapy, after adjustment for potential confounders. Owing to very limited relevant data, we could not support that these changes in lipid molecules may be beneficial or not for the progression of NAFLD. Thus, mechanistic studies are warranted to clarify the potential clinical significance of these findings.

Mass spectrometric characterization of aminophospholipids containing N-(2-hydroxyethyl)glycine in kombu algae extracts.

1,2-Diacyl-sn-glycero-3-phospho-O-[N-(2-hydroxyethyl)glycines] (PHEGs) are a class of rare aminophospholipids found specifically in brown algae, including kombu seaweed. Despite their potential importance in algal physiology, a comprehensive mass spectrometry (MS) characterization, useful to understand their biological behaviour, is still lacking. To establish the structural regiochemical features of PHEGs, we employed hydrophilic interaction liquid chromatography (HILIC). Following separation, the isolated band of PHEGs was analyzed using MS techniques. This included multistage tandem MS experiments, performed in both positive and negative electrospray ionization modes at low and high resolution. By comparing MS/MS and MS3 spectra acquired in negative ion mode, the regiochemical rules for PHEG identification were established. The most abundant PHEG species in kombu seaweed, from both Laminaria ochroleuca (European Atlantic) and Laminaria longissima (Japan), was identified as PHEG 20:4/20:4. Less abundant species included PHEG 20:4/20:5 and hydroxylated forms of both PHEG 20:4/20:4 (i.e. 40:8;O) and 20:4/20:5 (40:9;O). The presence of a lyso PHEG 20:4 was consistently detected but at very low levels. This study employed MS analysis to elucidate the regiochemical patterns of PHEGs in kombu seaweed. We identified PHEG 20:4/20:4 as the dominant species, along with several less abundant variants, including hydroxylated forms. These findings provide valuable insights into the potential roles and metabolism of PHEGs in brown algae, paving the way for further investigation into their biological functions.

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

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

Reassuring Quantitative Analysis of Glyphosate and Aminomethylphosphonic Acid Levels in Breast Milk Using Liquid Chromatography Mass Spectrometry.

Purpose: The World Health Organization's International Agency on Research for Cancer has determined that glyphosate is "probably carcinogenic to humans." There is a great public interest to investigate whether glyphosate are detected in breast milk. Thus, the goal of this study was to assess the concentration of glyphosate and its main metabolite in breast milk. Materials and Methods: Liquid chromatography was performed at 25°C using a Luna NH2, 50 × 2 mm, 3⎛ m (Phenomenex) analytical column. Electrospray ionization mass spectrometry was collected using negative ionization mode. The calibration curve for glyphosate ranged from 10 to 250 ng/mL. The detection limit was 1 ng/mL. Results: Breast milk samples were collected from 74 women, which included vegans (n = 26), vegetarians (n = 22), and nonvegetarians (n = 26). One of the 74 milk samples contained a detectable concentration of glyphosate and an additional 7 were found to contain aminomethylphosphonic acid. Conclusions: In breast milk samples collected mainly from women residing in urban regions of the United States, glyphosate detection was rare. Consistently, breastfed infants have a low or minimal risk of being exposed to glyphosate through ingestion of mother's milk. It is possible that the presence/absence and/or level of concentration of milk glyphosate depend on a place of residency and time of breastfeeding vis-à-vis time of its agricultural application.

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

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

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

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