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This study describes the first reported development of a rapid, generic gradient Ultra-High Performance Liquid Chromatography (UHPLC) methodology with targeted triple quadrupole MS/MS using electrospray positive ionisation to detect and unambiguously confirm the identity of 33 substituted 1, 2-diarylethamine (or diphenidine) derivatives in solid drug samples. The in-house synthesised library included a range of derivatives possessing either electron donating/withdrawing substituents, commonly included in combinatorial libraries, of varying size and lipophilicity on the phenyl ring. These test probes were used to investigate if their order of elution and that of their regioisomers were dependent on the position and type of the substituent on the phenyl ring. In addition, investigations into the retention mechanism of the diphenidines under reverse-phase UHPLC conditions were undertaken. Common adulterants found within seized bulk samples were assessed to prove that the methodology was specific, and the developed UHPLC-MS/MS (t G = 10 min) protocol was applied to confirm the identity of the psychoactive components within four seized bulk samples provided by law enforcement.

Analysis of oxylipins by liquid chromatography mass spectrometry (LC/MS) is challenging because of the small mass range occupied by this diverse lipid class, the presence of numerous structural isomers, and their low abundance in biological samples. Although highly sensitive LC/MS/MS methods are commonly used, further separation is achievable by using drift tube ion mobility coupled with high-resolution mass spectrometry (DTIM-MS). Herein, we present a combined analytical and computational method for the identification of oxylipins and fatty acids. We use a reversed-phase LC/DTIM-MS workflow able to profile and quantify (based on chromatographic peak area) the oxylipin and fatty acid content of biological samples while simultaneously acquiring full scan and product ion spectra. The information regarding accurate mass, collision-cross-section values in nitrogen (DTCCSN2), and retention times of the species found are compared to an internal library of lipid standards as well as the LIPID MAPS Structure Database by using specifically developed processing tools. Features detected within the DTCCSN2 and m/ z ranges of the analyzed standards are flagged as oxylipin-like species, which can be further characterized using drift-time alignment of product and precursor ions distinctive of DTIM-MS. This not only helps identification by reducing the number of annotations from LIPID MAPS but also guides discovery studies of potentially novel species. Testing the methodology on Salmonella enterica serovar Typhimurium-infected murine bone-marrow-derived macrophages and thrombin activated human platelets yields results in agreement with literature. This workflow has also annotated features as potentially novel oxylipins, confirming its ability in providing further insights into lipid analysis of biological samples.

Obesity is a complex disorder where the genome interacts with diet and environmental factors to ultimately influence body mass, composition, and shape. Numerous studies have investigated how bulk lipid metabolism of adipose tissue changes with obesity and, in particular, how the composition of triglycerides (TGs) changes with increased adipocyte expansion. However, reflecting the analytical challenge posed by examining non-TG lipids in extracts dominated by TGs, the glycerophospholipid composition of cell membranes has been seldom investigated. Phospholipids (PLs) contribute to a variety of cellular processes including maintaining organelle functionality, providing an optimized environment for membrane-associated proteins, and acting as pools for metabolites (e.g. choline for one-carbon metabolism and for methylation of DNA). We have conducted a comprehensive lipidomic study of white adipose tissue in mice which become obese either through genetic modification (ob/ob), diet (high fat diet), or a combination of the two, using both solid phase extraction and ion mobility to increase coverage of the lipidome. Composition changes in seven classes of lipids (free fatty acids, diglycerides, TGs, phosphatidylcholines, lyso-phosphatidylcholines, phosphatidylethanolamines, and phosphatidylserines) correlated with perturbations in one-carbon metabolism and transcriptional changes in adipose tissue. We demonstrate that changes in TGs that dominate the overall lipid composition of white adipose tissue are distinct from diet-induced alterations of PLs, the predominant components of the cell membranes. PLs correlate better with transcriptional and one-carbon metabolism changes within the cell, suggesting that the compositional changes that occur in cell membranes during adipocyte expansion have far-reaching functional consequences. Data are available at MetaboLights under the submission number: MTBLS1775.

Supplemental Digital Content is available in the text., Background: Common chromosome 9p21 single nucleotide polymorphisms (SNPs) increase coronary heart disease risk, independent of traditional lipid risk factors. However, lipids comprise large numbers of structurally related molecules not measured in traditional risk measurements, and many have inflammatory bioactivities. Here, we applied lipidomic and genomic approaches to 3 model systems to characterize lipid metabolic changes in common Chr9p21 SNPs, which confer ≈30% elevated coronary heart disease risk associated with altered expression of ANRIL, a long ncRNA. Methods: Untargeted and targeted lipidomics was applied to plasma from NPHSII (Northwick Park Heart Study II) homozygotes for AA or GG in rs10757274, followed by correlation and network analysis. To identify candidate genes, transcriptomic data from shRNA downregulation of ANRIL in HEK-293 cells was mined. Transcriptional data from vascular smooth muscle cells differentiated from induced pluripotent stem cells of individuals with/without Chr9p21 risk, nonrisk alleles, and corresponding knockout isogenic lines were next examined. Last, an in-silico analysis of miRNAs was conducted to identify how ANRIL might control lysoPL (lysophosphospholipid)/lysoPA (lysophosphatidic acid) genes. Results: Elevated risk GG correlated with reduced lysoPLs, lysoPA, and ATX (autotaxin). Five other risk SNPs did not show this phenotype. LysoPL-lysoPA interconversion was uncoupled from ATX in GG plasma, suggesting metabolic dysregulation. Significantly altered expression of several lysoPL/lysoPA metabolizing enzymes was found in HEK cells lacking ANRIL. In the vascular smooth muscle cells data set, the presence of risk alleles associated with altered expression of several lysoPL/lysoPA enzymes. Deletion of the risk locus reversed the expression of several lysoPL/lysoPA genes to nonrisk haplotype levels. Genes that were altered across both cell data sets were DGKA, MBOAT2, PLPP1, and LPL. The in-silico analysis identified 4 ANRIL-regulated miRNAs that control lysoPL genes as miR-186-3p, miR-34a-3p, miR-122-5p, and miR-34a-5p. Conclusions: A Chr9p21 risk SNP associates with complex alterations in immune-bioactive phospholipids and their metabolism. Lipid metabolites and genomic pathways associated with coronary heart disease pathogenesis in Chr9p21 and ANRIL-associated disease are demonstrated.

Lipids are a large and highly diverse family of biomolecules, which play essential structural, storage and signalling roles in cells and tissues. Although traditional mass spectrometry (MS) approaches used in lipidomics are highly sensitive and selective, lipid analysis remains challenging due to the chemical diversity of lipid structures, multiple isobaric species and incomplete separation using many forms of chromatography. Ion mobility (IM) separates ions in the gas phase based on their physicochemical properties. Addition of IM to the traditional lipidomic workflow both enhances separation of complex lipid mixtures, beneficial for lipid identification, and improves isomer resolution. Herein, we discuss the recent developments in IM-MS for lipidomics.

Obesity is a complex disorder where the genome interacts with diet and environmental factors to ultimately influence body mass, composition and shape. Numerous studies have investigated how bulk lipid metabolism of adipose tissue changes with obesity, and in particular how the composition of triglycerides (TGs) changes with increased adipocyte expansion. However, reflecting the analytical challenge posed by examining non-TG lipids in extracts dominated by TGs, the glycerophospholipid (PL) composition of cell membranes has been seldom investigated. PLs contribute to a variety of cellular processes including maintaining organelle functionality, providing an optimised environment for membrane-associated proteins and as pools for metabolites (e.g. choline for one-carbon metabolism and for methylation of DNA). We have conducted a comprehensive lipidomic study of white adipose tissue in mice who become obese either through genetic modification (ob/ob), diet (high fat diet) or a combination of the two using both solid phase extraction and ion mobility to increase coverage of the lipidome. Composition changes in seven classes of lipid (free fatty acids, diglycerides, TGs, phosphatidylcholines, lyso-phosphatidylcholines, phosphatidylethanolamines, and phosphatidylserines) correlated with perturbations in one-carbon metabolism and transcriptional changes in adipose tissue. We demonstrate that changes in TGs that dominate the overall lipid composition of white adipose tissue are distinct from diet-induced alterations of PLs, the predominant components of the cell membranes. PLs correlate better with transcriptional and one-carbon metabolism changes within the cell, suggesting the compositional changes that occur in cell membranes during adipocyte expansion have far-reaching functional consequences.

BACKGROUND: Inflammatory bowel disease is a group of pathologies characterised by chronic inflammation of the intestine and an unclear aetiology. Its main manifestations are Crohn's disease and ulcerative colitis. Currently, biopsies are the most used diagnostic tests for these diseases and metabolomics could represent a less invasive approach to identify biomarkers of disease presence and progression. OBJECTIVES: The lipid and the polar metabolite profile of plasma samples of patients affected by inflammatory bowel disease have been compared with healthy individuals with the aim to find their metabolomic differences. Also, a selected sub-set of samples was analysed following solid phase extraction to further characterise differences between pathological samples. METHODS: A total of 200 plasma samples were analysed using drift tube ion mobility coupled with time of flight mass spectrometry and liquid chromatography for the lipid metabolite profile analysis, while liquid chromatography coupled with triple quadrupole mass spectrometry was used for the polar metabolite profile analysis. RESULTS: Variations in the lipid profile between inflammatory bowel disease and healthy individuals were highlighted. Phosphatidylcholines, lyso-phosphatidylcholines and fatty acids were significantly changed among pathological samples suggesting changes in phospholipase A2 and arachidonic acid metabolic pathways. Variations in the levels of cholesteryl esters and glycerophospholipids were also found. Furthermore, a decrease in amino acids levels suggests mucosal damage in inflammatory bowel disease. CONCLUSIONS: Given good statistical results and predictive power of the model produced in our study, metabolomics can be considered as a valid tool to investigate inflammatory bowel disease., This study was funded by Agilent Technologies, Regione Autonoma della Sardegna (L.R.7/2007, Grant Number F71J12001180002), and the Medical Research Council UK (Grant Number MR/P011705/1).

Background High on treatment platelet reactivity (HTPR) is common in patients receiving clopidogrel following an acute coronary syndrome (ACS); it's also associated with increased morbidity and mortality. More potent and predictable antiplatelet drugs have addressed this issue at the expense of increased bleeding. Identification of HTPR and the targeted use of more potent antiplatelet drugs has, so far, broadly failed. We investigate this approach in terms of the timing of platelet function testing and how this can impact on the ability of these bedside tests to predict HTPR around the time of coronary intervention. Methods High risk ACS patients treated with 5 days of clopidogrel had platelet function assessed using the multiple electrode aggregometry system (MEA) pre, post and 24 h following percutaneous coronary intervention (PCI). Simultaneous detailed analysis of platelet status was undertaken with quantification of platelet bound and soluble p-selectin and mass spectrometry quantification of the eicosanoid 12-HETE. Results As assessed by MEA 40.5% of patients had HTPR pre-PCI; mean aggregation units (AU) in response to ADP were 499.1 ± 46.3 pre-PCI, 407.6 ± 37.7 post-PCI and 269.1 ± 24.6 AU 24 h post-PCI (pre to post PCI p > 0.05, pre to 24 h post-PCI p = 0.0002). This highly significant drop in platelet reactivity was contrasted with on-going expression of platelet bound p-selectin, increased soluble p-selectin and rising 12-HETE concentrations. Conclusions This study outlines significant changes in ex-vivo platelet aggregation that occur within 24 h of PCI in high risk NSTEMI patients using bedside PFT. Whilst there were no changes in antiplatelet therapy during the study period its clear that timing is crucial when assessing high on treatment residual platelet activity.

INTRODUCTION: Data processing is one of the biggest problems in metabolomics, given the high number of samples analyzed and the need of multiple software packages for each step of the processing workflow. OBJECTIVES: Merge in the same platform the steps required for metabolomics data processing. METHODS: KniMet is a workflow for the processing of mass spectrometry-metabolomics data based on the KNIME Analytics platform. RESULTS: The approach includes key steps to follow in metabolomics data processing: feature filtering, missing value imputation, normalization, batch correction and annotation. CONCLUSION: KniMet provides the user with a local, modular and customizable workflow for the processing of both GC-MS and LC-MS open profiling data.

Activated platelets generate an eicosanoid proposed to be 8-hydroxy-9,10-dioxolane A3 (DXA3). Herein, we demonstrate that significant amounts of DXA3 are rapidly attached to phosphatidylethanolamine (PE) forming four esterified eicosanoids, 16:0p, 18:0p, 18:1p and 18:0a/DXA3-PEs that can activate neutrophil integrin expression. These lipids comprise the majority of DXA3 generated by platelets, are formed in ng amounts (24.3±6.1 ng/2×108) and remain membrane bound. Pharmacological studies revealed DXA3-PE formation involves cyclooxygenase-1 (COX), protease-activated receptors (PAR) 1 and 4, cytosolic phospholipase A2 (cPLA2), phospholipase C and intracellular calcium. They are generated primarily via esterification of newly formed DXA3, but can also be formed in vitro via co-oxidation of PE during COX-1 co-oxidation of arachidonate. All four DXA3-PEs were detected in human clots. Purified platelet DXA3-PE activated neutrophil Mac-1 expression, independently of its hydrolysis to the free eicosanoid. This study demonstrates the structures and cellular synthetic pathway for a family of leukocyte-activating platelet phospholipids generated on acute activation, adding to the growing evidence that enzymatic PE oxidation is a physiological event in innate immune cells., Graphical abstract fx1, Highlights • Four enzymatically oxidized phospholipids are described, termed DXA3-PE in platelets. • DXA3-PE forms by COX-1 oxidation of arachidonate, then esterification into PE. • DXA3-PE formation requires calcium, phospholipase C and phospholipase A2. • DXA3-PEs purified from platelets activates neutrophil Mac-1 expression.