Liquid Chromatography Coupled To Mass Spectrometer Research Articles

P046 Crohn Disease (CD) untargeted fecal metabolome shows strong correlation with specific salivary associated microbial taxa, and with disease activity

Abstract Background The fecal metabolome is comprised from derivatives of the gut microbiome, the host, the environment, and the diet. Thus, it is a central output and effector that is linked with Crohn disease (CD). We aimed to characterize metabolomics profiles that are associated with CD and link metabolites with gut microbial diversity and composition. Methods Fecal metabolomics was performed using liquid chromatography coupled Mass Spectrometer (LC/MS). Microbiome was defined by 16Sseq. Multivariate analyses in MaAsLin2 identified differentially abundant metabolites and these were used for pathways enrichment analyses using Metacyc (PMID: 31586394). Hierarchical All-against-All association (HAllA; PMID: 35758795) was used to identify significant relationships between fecal metabolites and microbial amplicon sequence variants (ASVs). Results The cohort included 294 samples from 85 subjects (mean age 36, 51% males). 250 samples were from CD patients (n=59) and 44 samples were from controls (n=26). 545 metabolites were included in our predefined library and were subsequently analyzed. Multivariate analyses identified 86 increased and 18 decreased metabolites in CD, after controlling for age and gender (FDR<0.25). These included reduction in 9 long-chain fatty acids and increase of standard and post-translational modified amino-acids. Enriched pathways in CD included amino-acids biosynthesis and degradation, electron carrier biosynthesis, guanine and guanosine salvage, and indole-3-acetate inactivation (FDR<0.1). Within the CD comparison, 29 metabolites were higher in active and 97 in quiescent CD. Acetylated amino-acids, dopamine, glucuronolactone and decanoic acid were all reduced in active CD. These metabolites and others were also significantly correlated with fecal calprotectin, microbial alpha diversity (Faith’s PD) and CD dysbiosis index (PMID: 24629344) (FDR<0.25; Fig. 1). To correlate fecal metabolomics with the gut microbial dataset, we applied the HAllA analysis (n= 231) that indicated 7,184 significant associations (FDR<0.05; Fig. 2). An interesting positive correlation was noted between a group of bacteria enriched with saliva bacteria (s_parvula, g_Actinomyces, g_Streptococcus, etc.) and metabolites like fructose, stachyose, glucose, raffinose etc., that are involved in stachyose metabolism (cluster 16). Conclusion CD patients have distinct fecal metabolic signature which is also correlated with disease activity. Furthermore, we identified significant interactions between specific fecal metabolites and gut microbial ASVs enriched with saliva bacteria that were previously linked with CD. More research is needed to elucidate the factors governing this newly discovered link.

DATA MODELLING OF LC/MS-BASED METABOLOMIC PROFILING TO COMPARE BETWEEN HUMAN PLASMA AND URINE SAMPLES ASSOCIATED WITH BEETROOT JUICE

Data modelling- based untargeted metabolomic researches is one of the best approaches which can be used to compare among biological fluid samples to provide a comprehensive and reliable sight about the changes of metabolomic profiling. This study sought to compare between human urine and plasma to investigate metabolomic changes of a diet pre- and postintake beetroot juice that offer unique metabolomic fingerprint associated to the potential effects of beetroot juice. A current pilot study of metabolomic patterns used Liquid Chromatography coupled to Mass Spectrometer (LC-MS) to carry out an analysis for seventytwo plasma and urine samples, equally. Samples were collected from nine adult healthy subjects (4 samples per subject) at pre-, as baseline, and post-intake beetroot juice in three stages, after 2hrs, 4hrs, and 8hrs. On the basis of the validation of data modelling, robust separation was observed between urine samples pre and post-intake beetroot juice and was more fitting and significant than the separation between plasma samples. The results of pilot study indicate that metabolomics screening of urine samples may be the best tool and a potential approach to predict the metabolomic profiling than plasma samples to assess the metabolic effect of a diet pre- and post-intake beetroot juice. As a result of the effects of beetroot juice, the present results also uncover significantly changes in most important metabolites including amino acid, peptide, Co-factors and vitamins which may contribute to the consolidation of the using of plant metabolites and natural substances to synthesis the nanoparticles for the biomedical applications. Keywords: Metabolomic Profiling, Beetroot Juice (BJ), Biological Fluid, LC-MS, Principal Components Analysis (PCA), Orthogonal Partial Least Squares- Discriminant Analysis (OPLSDA)

Comprehensive chiral GC-MS/MS and LC-MS/MS methods for identification and determination of N-acyl homoserine lactones

N-acyl homoserine lactones (N-HLs) are signaling molecules synthesized by gram-negative bacteria to communicate in a process called quorum sensing. Most reported methods for the analysis of N-HLs, which are chiral molecules, do not distinguish between enantiomers. Typical examples include biosensors, liquid chromatography with UV detection, gas chromatography coupled with a mass spectrometer (GC-MS) and liquid chromatography coupled with mass spectrometer (LC-MS). Recently, the production of both D,L-N-HLs have been reported in Vibrio fischeri and Burkholderia cepacia. Concentrations of the D-N-HLs were found at the limit of quantification for the employed method. Therefore, for further studies of the role of the D-N-HLs in bacterial physiology, more sensitive, reliable, and selective analytical methods are necessary. In this work, such comprehensive chiral analytical methods for the identification and determination of 18 N-HLs using solid phase extraction followed by GC-MS/MS and LC-MS/MS analyses were developed. Extraction recoveries for the more hydrophilic C4 N-HLs were <10% of all other N-HLs, thus offering a possible explanation as to their lack of detection in previous studies. The chiral separations of all 18 N-HLs derivatives were accomplished by the complementary GC-MS/MS and LC-MS/MS methods. The limit of detection for LC-MS/MS method was as low as 1 ppb. The limit of detection for the GC-MS/MS method was found to be one to three orders of magnitude higher than the LC-MS/MS method. Due to the high extraction recovery and a preconcentration factor of 100, concentrations as low as 10 ppt can be detected by LC-MS/MS in biological samples. The LC-MS/MS approach provided greater enantioselectivity for the larger, more hydrophobic N-HLs while GC-MS/MS provided better enantioselectivity for the smaller N-HLs.

The role of iron-doped g-C3N4 heterogeneous catalysts in Fenton-like process investigated by experiment and theoretical simulation

The Fenton-like process provides a promising method to produce reactive radicals for the degradation of refractory organic pollutants in environmental remediation. In this study, the iron-doped g-C3N4 (CNF) coupled with H2O2 was applied to enhance the degradation of carbamazepine (CBZ). And the role of CNF was elucidated by experiment and density functional theory (DFT) calculations. The largest CBZ removal efficiency of 99.02% was achieved in acid condition (pH = 3) with the addition of 0.5 g/L 14% CNF and 50 mM H2O2. Thirteen intermediates were identified based on high performance liquid chromatography coupled with mass spectrometer (LCMS) and DFT calculations. The degradation products of CBZ were mainly protein-like materials with a low humification degree. During the degradation process, H2O2 was preferentially adsorbed on the active Fe-O site of CNF by hydrogen bonding, hence the promoted electron transfer from H2O2 to Fe(III) contributed to the generation of Fe(II). Certain deformation of H2O2 was observed that the O-O bond length increased to about 1.51 Å. As a result, large amounts of radical species (·OH) were generated by H2O2 decomposition, which was responsible for attacking the CBZ molecules and proceeding the degradation reaction.

Development and validation of a liquid chromatography coupled to mass spectrometer (LC-MS) method for the simultaneous quantification of estrone-3-sulfate, progesterone, estrone and estradiol in serum of mares and American bisons

Steroid concentrations in serum are fluctuating during pregnancy of many mammal species. The current knowledge about endocrinology of gestation is mainly based on immunoassays. However, the lack of specificity of these assays hampers the reliability of the results. In the present work, we developed and validated a methodology associating liquid chromatography (LC) and mass spectrometry (MS) to simultaneously quantify, with high specificity and accuracy, estrone-3-sulfate (E3S), progesterone (PRO), estrone (E1) and estradiol (E2) in serum of two different mammal species.The sample preparation procedure is based on a simple protein precipitation and a derivatization with dansyl chloride. After the chromatographical separation, compounds were analyzed with a triple-quadrupole mass spectrometer operating in multiple reaction monitoring. Mare and American bison serum samples were analyzed with the validated method and results were compared with concentrations measured with commercial radioimmunoassay (RIA), enzyme linked immunosorbent assay (ELISA) and chemiluminescent microparticle immunoassay (CMIA).Following these criterions: relative standard deviation <15% and relative bias <15%, lower limits of quantification of 0.5 ng/mL (E3S), 0.1 ng/mL (PRO) and 2 pg/mL (E1 and E2) were achieved. Most of the comparison between immunoassays and LC-MS showed poor correlation and proportional differences.Our LC-MS method is able to simultaneously quantify several steroid hormones with high specificity, accuracy and sensitivity in serum of two different mammal species. Our method constitutes a useful and performant tool for veterinary clinicians and LC-MS should thus be used to update and refine the current knowledge about the endocrinology of pregnancy in mammals.

High-coverage plasma lipidomics reveals novel sex-specific lipidomic fingerprints of age and BMI: Evidence from two large population cohort studies.

Obesity and related metabolic diseases show clear sex-related differences. The growing burden of these diseases calls for better understanding of the age- and sex-related metabolic consequences. High-throughput lipidomic analyses of population-based cohorts offer an opportunity to identify disease-risk–associated biomarkers and to improve our understanding of lipid metabolism and biology at a population level. Here, we comprehensively examined the relationship between lipid classes/subclasses and molecular species with age, sex, and body mass index (BMI). Furthermore, we evaluated sex specificity in the association of the plasma lipidome with age and BMI. Some 747 targeted lipid measures, representing 706 molecular lipid species across 36 classes/subclasses, were measured using a high-performance liquid chromatography coupled mass spectrometer on a total of 10,339 participants from the Australian Diabetes, Obesity and Lifestyle Study (AusDiab), with 563 lipid species being validated externally on 4,207 participants of the Busselton Health Study (BHS). Heat maps were constructed to visualise the relative differences in lipidomic profile between men and women. Multivariable linear regression analyses, including sex-interaction terms, were performed to assess the associations of lipid species with cardiometabolic phenotypes. Associations with age and sex were found for 472 (66.9%) and 583 (82.6%) lipid species, respectively. We further demonstrated that age-associated lipidomic fingerprints differed by sex. Specific classes of ether-phospholipids and lysophospholipids (calculated as the sum composition of the species within the class) were inversely associated with age in men only. In analyses with women alone, higher triacylglycerol and lower lysoalkylphosphatidylcholine species were observed among postmenopausal women compared with premenopausal women. We also identified sex-specific associations of lipid species with obesity. Lysophospholipids were negatively associated with BMI in both sexes (with a larger effect size in men), whilst acylcarnitine species showed opposing associations based on sex (positive association in women and negative association in men). Finally, by utilising specific lipid ratios as a proxy for enzymatic activity, we identified stearoyl CoA desaturase (SCD-1), fatty acid desaturase 3 (FADS3), and plasmanylethanolamine Δ1-desaturase activities, as well as the sphingolipid metabolic pathway, as constituent perturbations of cardiometabolic phenotypes. Our analyses elucidate the effect of age and sex on lipid metabolism by offering a comprehensive view of the lipidomic profiles associated with common cardiometabolic risk factors. These findings have implications for age- and sex-dependent lipid metabolism in health and disease and suggest the need for sex stratification during lipid biomarker discovery, establishing biological reference intervals for assessment of disease risk.

Effect of Gamma Radiation on Growth and Mycotoxin Production of Alternaria alternata

The ubiquitous genus Alternaria contains many species that are able to invade cereals, oleaginous plants and other crops. Alternaria alternata is considered one of the most important species and can produce several mycotoxins under favorable conditions of temperature and humidity, including the economically important toxins: alternariol (AOH) and alternariol monomethyl ether (AME). The aim of this study was to evaluate the radio-sensitivity of Alternaria alternata spores through different gamma radiation doses. A. alternata growth and the production of AOH and AME were then analyzed. After fungal irradiation with 2 kGy, 5 kGy and 7 kGy, the spores were suspended with sterile distilled water followed by inoculation on wheat grains. The count of the colony-forming units per gram (CFU/g) was performed using Dichloran Rose Bengal Chloramphenicol (DRBC) and Dichloran Chloramphenicol Agar Malt Extract (DCMA); AOH and AME were analyzed using Liquid Chromatography coupled with Mass Spectrometer (LC-MS). Results showed that fungal growth and toxin production increased with the increase of radiation dosage. The implications of these findings in relation to the resistance of A. alternata spores to gamma irradiation are discussed.