Segmented Flow Strategies for Integrating Liquid Chromatography-Mass Spectrometry with Nuclear Magnetic Resonance for Lipidomics.

Building an accurate lipid inventory relies on coordinated information from orthogonal analytical capabilities. Integrating the familiar workflow of liquid chromatography (LC), high-resolution mass spectrometry (HRMS), and tandem mass spectrometry (MS/MS) with proton nuclear magnetic resonance spectroscopy (1H NMR) would be ideal for building that inventory. For absolute lipid structural elucidation, LC-HRMS/MS can provide lower-level structural information with superior sensitivity, while 1H NMR can provide invaluable higher-order structural information for the disambiguation of isomers with absolute chemical specificity. Digitization of the LC eluent followed by splitting the microfractions into two flow paths in a defined ratio for HRMS/MS and NMR would be the ideal strategy to permit correlation of the MS and NMR data as a function of chromatographic retention time. Here, we report an active segmentation platform to transform analytical flow rate LC eluent into parallel microliter segmented flow queues for high confidence correlation of the MS, MS/MS, and NMR data. The practical details in implementing this strategy to achieve an integrated LC-MS-NMR platform are presented, including the development of an active segmentation technology using a four-port two-way valve to transform the LC eluent into parallel segmented flows for online MS analysis followed by offline segment-specific 1H NMR and optimization of the detector response toward segmented flow. To demonstrate the practicality of this novel platform, it was tested using lipid mixture samples.

Why tandem mass spectrometry for trace analysis: Concepts of tandem analytical techniques.

RATIONALE: The triple quadrupole mass spectrometer, typically in combination with a gas or liquid chromatograph (GC/MS/MS and LC/MS/MS), is perhaps the most iconic example today of a tandem analytical instrument. Here I present the concepts of tandem or hyphenated techniques for trace analysis (that is, the detection and/or quantitation of one or more analytes present in a mixture at low levels). METHODS: This tutorial presents the principles of tandem trace analytical techniques such as GC/MS/MS and LC/MS/MS, including the capabilities and requirements for such tandem techniques, the role of sensitivity and selectivity in tandem techniques, ways to assess the "informing power" of these techniques, and a comparison of tandem techniques with individual techniques at high resolution. These points are illustrated with several examples of trace analysis using tandem analytical techniques. RESULTS: Several characteristics of the triple quadrupole have made it the "laboratory workhorse" for trace analysis, including the remarkable efficiency of the low-energy collision-induced dissociation (CID) process in a radiofrequency (RF)-only multipole collision cell, the ease of computer control, and the capability for rapid scanning, rapid switching from mass to mass, and high transmission efficiency, enabling a wide variety of MS/MS scans. The efficiency of selected reaction monitoring means that triple quadrupoles dominate MS/MS for detection and quantitation of targeted compounds. CONCLUSIONS: This special issue addresses the intriguing question of how the triple quadrupole mass spectrometer progressed from "bleeding edge" to "the laboratory workhorse" over the last 40 years. This tutorial on the principles of tandem trace analytical techniques provides perspectives and insights into answering that question and should help educate the novice and stimulate the sophisticate.

Per- and Polyfluoroalkyl Substances (PFAS) in Street Sweepings.

One hundred and seventeen street sweeping samples were collected and analyzed for per- and polyfluoroalkyl substances (PFAS). Fifty-six samples were collected in one city (Gainesville, Florida) allowing for an in-depth city-wide characterization. Street sweepings from five other urban areas, (Orlando, n = 15; Key West, n = 15; Pensacola, n = 12; Tampa, n = 13; and Daytona Beach, n = 6) were analyzed to provide a city-to-city comparison of PFAS. Within our analytical workflow, 37 PFAS were quantified across all samples, while the maximum number of PFAS quantified at one site was 26. Of those PFAS quantified in Gainesville, 60% were perfluoroalkyl acids (PFAAs) and 33% were precursors to PFAA. Among the PFAAs, short-chain perfluoroalkyl carboxylic acids (PFCAs) were the dominant class representing 26% of the total PFAS by concentration. In the comparison across different urban cities, the dominant compound by concentration and frequency of detection varied; however, perfluorooctanoic acid (PFOA) and linear perfluorooctanesulfonic acid (PFOSlin) were the two PFAS that were detected the most frequently. This study documents the first-time detection of hexadecafluorosebacic acid and perfluoro-3,6,9-trioxaundecane-1,11-dioic acid within environmental samples.

A rapid and robust method for amino acid quantification using a simple N-hydroxysuccinimide ester derivatization and liquid chromatography-ion mobility-mass spectrometry.

The vast majority of mass spectrometry (MS)-based metabolomics studies employ reversed-phase liquid chromatography (RPLC) to separate analytes prior to MS detection. Highly polar metabolites, such as amino acids (AAs), are poorly retained by RPLC, making quantitation of these key species challenging across the broad concentration ranges typically observed in biological specimens, such as cell extracts. To improve the detection and quantitation of AAs in microglial cell extracts, the implementation of a 4-dimethylaminobenzoylamido acetic acid N-hydroxysuccinimide ester (DBAA-NHS) derivatization agent was explored for its ability to improve both analyte retention and detection limits in RPLC-MS. In addition to the introduction of the DBAA-NHS labeling reagent, a uniformly (U) 13C-labeled yeast extract was also introduced during the sample preparation workflow as an internal standard (IS) to eliminate artifacts and to enable targeted quantitation of AAs, as well as untargeted amine submetabolome profiling. To improve method sensitivity and selectivity, multiplexed drift-tube ion mobility (IM) was integrated into the LC-MS workflow, facilitating the separation of isomeric metabolites, and improving the structural identification of unknown metabolites. Implementation of the U-13C-labeled yeast extract during the multiplexed LC-IM-MS analysis enabled the quantitation of 19 of the 20 common AAs, supporting a linear dynamic range spanning up to three orders of magnitude in concentration for microglial cell extracts, in addition to reducing the required cell count for reliable quantitation from 10 to 5 million cells per sample.

Tissue-specific analysis of lipid species in Drosophila during overnutrition by UHPLC-MS/MS and MALDI-MSI.

Diets high in calories can be used to model metabolic diseases, including obesity and its associated comorbidities, in animals. Drosophila melanogaster fed high-sugar diets (HSDs) exhibit complications of human obesity including hyperglycemia, hyperlipidemia, insulin resistance, cardiomyopathy, increased susceptibility to infection, and reduced longevity. We hypothesize that lipid storage in the high-sugar-fed fly's fat body (FB) reaches a maximum capacity, resulting in the accumulation of toxic lipids in other tissues or lipotoxicity. We took two approaches to characterize tissue-specific lipotoxicity. Ultra-HPLC-MS/MS and MALDI-MS imaging enabled spatial and temporal localization of lipid species in the FB, heart, and hemolymph. Substituent chain length was diet dependent, with fewer odd chain esterified FAs on HSDs in all sample types. By contrast, dietary effects on double bond content differed among organs, consistent with a model where some substituent pools are shared and others are spatially restricted. Both di- and triglycerides increased on HSDs in all sample types, similar to observations in obese humans. Interestingly, there were dramatic effects of sugar feeding on lipid ethers, which have not been previously associated with lipotoxicity. Taken together, we have identified candidate endocrine mechanisms and molecular targets that may be involved in metabolic disease and lipotoxicity.

Ultrahigh-Performance Liquid Chromatography-High-Resolution Mass Spectrometry Metabolomics and Lipidomics Study of Stool from Transgenic Parkinson's Disease Mice Following Immunotherapy.

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta of the brain, as well as the degeneration of motor and nonmotor circuitries. The cause of neuronal death is currently unknown, although chronic neuroinflammation, aggregated α-synuclein, mitochondrial dysfunction, and oxidative stress have all been implicated. Gliosis has been shown to exacerbate neuroinflammation via secretion of proinflammatory cytokines, and there is a subsequent infiltration of T lymphocytes (T-cells), into the brain of PD patients. Using liquid chromatography-high-resolution mass spectrometry (LC-HRMS), we have observed metabolomic changes in stool samples, thought to be associated with the potential disease-modifying effect of immunotherapy administered to transgenic Parkinsonian (A53T) mice. Significant elevations (p < 0.05) in metabolites associated with immune response (taurine, histamine, and its methylated product, 3-methylhistamine) are identified as being higher in the mice undergoing immunotherapy. Furthermore, a reduction in triacylglycerol (TG) and diacylglycerol (DG) expressions in stool following immunotherapy suggests a regulation of lipid breakdown or biosynthesis with the vaccine. These "omics" markers (among others reported in this article) along with weight gain and increased life expectancy suggest that immunotherapy is positively modifying the disease state.

Design and Implementation of a Dual-Probe Microsampling Apparatus for the Direct Analysis of Adherent Mammalian Cells by Ion Mobility-Mass Spectrometry.

Atmospheric pressure sampling mass spectrometric methods are ideal platforms for rapidly analyzing the metabolomes of biological specimens. Several liquid extraction-based techniques have been developed for increasing metabolome coverage in direct sampling workflows. Here, we report the construction of a dual-probe microsampling device (DPM), based on the design of the liquid microjunction surface sampling probe, for analyzing the metabolome of live microglial cells by drift-tube ion mobility spectrometry (IMS) quadrupole time-of-flight mass spectrometry. Utilizing two distinct solvent systems in parallel is demonstrated to extract a wide structural variety of metabolites and lipids, enabling a more comprehensive analysis of intracellular metabolism. Employing the DPM-IM-MS method to adherent cells yielded the detection of 73 unique lipids and 79 small molecule metabolites from each optimized solvent system probe, respectively. Integration of multiplexed ion mobility scans is also shown to increase extracted analyte signal intensities between 2- and 10-fold compared to traditional single-pulse IMS, enabling the detection of 38 low-intensity features not previously detected by single-pulse DPM-IM-MS. To examine the ability of the DPM system to differentiate between sample treatment groups, microglia were stimulated with the endotoxin lipopolysaccharide (LPS). Several metabolic alterations were detected between sample treatment groups by DPM-IM-MS, many of which were not previously detected with conventional single-probe liquid microjunction surface sampling.

Analysis of Tryptophan Metabolites in Serum Using Wide-Isolation Strategies for UHPLC-HRMS/MS.

Current targeted metabolomic workflows are limited by design and thus sacrifice crucial information from a profiling standpoint that could lead to a more fundamental understanding of the metabolic processes of interest. One drawback to performing targeted analysis on ion trapping instruments is the potential for increased variability in analysis when analytes and standards are isolated and trapped individually for fragmentation. In addition, this sequential isolation process increases the duty cycle of the mass spectrometer and reduces the number of points collected across a chromatographic peak. To address this, the use of a wide-isolation window (12 Da) to encompass the target analyte and the isotope standard within a single fragmentation window ensures that fragmentation is consistent when quantitation relies on the ratio of the target to the internal standard. Additionally, the preservation of a faster scan rate ensures that optimal representation of chromatographic peaks is preserved for the purposes of both quantitative and qualitative analyses that require peak integration for statistical analysis. The use of this flexible method is promising in the investigation of pathways that require multiple targets and are highly integrated within the system. Here, we demonstrate the application of this method in a fast ultra-high performance liquid chromatography (UHPLC) analysis to integrate wide-isolation quantitative strategies for high-resolution mass spectrometry (HRMS) combined with profiling qualitative metabolomics for the analysis of tryptophan degradation metabolites in mouse serum. Analysis of tryptophan-deficient states as compared to control in both germ-free or E. coli gut microbiota states was used to quantitate pathway-specific metabolites as well as obtain full profiling information. The quantitative and qualitative results revealed the preservation of the primary pathways of degradation in the kynurenine pathway to potentially produce primary products such as nicotinamide during stress-induced dietary states.

Environmental lipidomics: understanding the response of organisms and ecosystems to a changing world.

BACKGROUND: Understanding the interaction between organisms and the environment is important for predicting and mitigating the effects of global phenomena such as climate change, and the fate, transport, and health effects of anthropogenic pollutants. By understanding organism and ecosystem responses to environmental stressors at the molecular level, mechanisms of toxicity and adaptation can be determined. This information has important implications in human and environmental health, engineering biotechnologies, and understanding the interaction between anthropogenic induced changes and the biosphere. One class of molecules with unique promise for environmental science are lipids; lipids are highly abundant and ubiquitous across nearly all organisms, and lipid profiles often change drastically in response to external stimuli. These changes allow organisms to maintain essential biological functions, for example, membrane fluidity, as they adapt to a changing climate and chemical environment. Lipidomics can help scientists understand the historical and present biofeedback processes in climate change and the biogeochemical processes affecting nutrient cycles. Lipids can also be used to understand how ecosystems respond to historical environmental changes with lipid signatures dating back to hundreds of millions of years, which can help predict similar changes in the future. In addition, lipids are direct targets of environmental stressors, for example, lipids are easily prone to oxidative damage, which occurs during exposure to most toxins. AIM OF REVIEW: This is the first review to summarize the current efforts to comprehensively measure lipids to better understand the interaction between organisms and their environment. This review focuses on lipidomic applications in the arenas of environmental toxicology and exposure assessment, xenobiotic exposures and health (e.g., obesity), global climate change, and nutrient cycles. Moreover, this review summarizes the use of and the potential for lipidomics in engineering biotechnologies for the remediation of persistent compounds and biofuel production. KEY SCIENTIFIC CONCEPT: With the preservation of certain lipids across millions of years and our ever-increasing understanding of their diverse biological roles, lipidomic-based approaches provide a unique utility to increase our understanding of the contemporary and historical interactions between organisms, ecosystems, and anthropogenically-induced environmental changes.

Software tool for internal standard based normalization of lipids, and effect of data-processing strategies on resulting values.

BACKGROUND: Lipidomics, the comprehensive measurement of lipids within a biological system or substrate, is an emerging field with significant potential for improving clinical diagnosis and our understanding of health and disease. While lipids diverse biological roles contribute to their clinical utility, the diversity of lipid structure and concentrations prove to make lipidomics analytically challenging. Without internal standards to match each lipid species, researchers often apply individual internal standards to a broad range of related lipids. To aid in standardizing and automating this relative quantitation process, we developed LipidMatch Normalizer (LMN) http://secim.ufl.edu/secim-tools/ which can be used in most open source lipidomics workflows. RESULTS: LMN uses a ranking system (1-3) to assign lipid standards to target analytes. A ranking of 1 signifies that both the lipid class and adduct of the internal standard and target analyte match, while a ranking of 3 signifies that neither the adduct or class match. If multiple internal standards are provided for a lipid class, standards with the closest retention time to the target analyte will be chosen. The user can also signify which lipid classes an internal standard represents, for example indicating that ether-linked phosphatidylcholine can be semi-quantified using phosphatidylcholine. LMN is designed to work with any lipid identification software and feature finding software, and in this study is used to quantify lipids in NIST SRM 1950 human plasma annotated using LipidMatch and MZmine. CONCLUSIONS: LMN can be integrated into an open source workflow which completes all data processing steps including feature finding, annotation, and quantification for LC-MS/MS studies. Using LMN we determined that in certain cases the use of peak height versus peak area, certain adducts, and negative versus positive polarity data can have major effects on the final concentration obtained.

Rapid Quantitation of 25-Hydroxyvitamin D2 and D3 in Human Serum Using Liquid Chromatography/Drift Tube Ion Mobility-Mass Spectrometry.

Ion mobility was integrated with liquid chromatography/high resolution mass spectrometry (LC/IM-HRMS) to quantify 25-hydroxyvitamin D (25OHD) in human serum. It has previously been shown that 25OHD adopts two gas-phase conformations which are resolved using ion mobility; in contrast, the inactive epimer, 3-epi-25-hydroxyvitamin D (epi25OHD), only adopts one. Interference from epi25OHD was eliminated by filtering the chromatogram to retain the drift time that corresponds to the unique gas-phase conformation of 25OHD. Although ion mobility separates the epimers, some chromatography is required to separate compounds which interfere with ionization or fall at the same nominal m/z. Standards were prepared in 4% albumin solutions and compared against commercial serum quality controls. Standards and quality controls were analyzed and validated using a 2 min LC/IM-MS method. 25-Hydroxyvitamin D3 and D2 were quantified over the range between 2 and 500 ng/mL with bias and precision within 15%. When epi25OHD was spiked into quality control samples, no significant bias was introduced, and analysis of 30 patient samples shows good agreement between this LC/IM-MS and traditional LC/MS/MS methods. This work shows that ion mobility can be incorporated with liquid chromatography and mass spectrometry for rapid quantitation of 25OHD in human serum.

Effective Liquid Chromatography-Trapped Ion Mobility Spectrometry-Mass Spectrometry Separation of Isomeric Lipid Species.

Lipids are a major class of molecules that play key roles in different biological processes. Understanding their biological roles and mechanisms remains analytically challenging due to their high isomeric content (e.g., varying acyl chain positions and/or double bond locations/geometries) in eukaryotic cells. In the present work, a combination of liquid chromatography (LC) followed by high resolution trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) was used to investigate common isomeric glycerophosphocholine (PC) and diacylglycerol (DG) lipid species from human plasma. The LC dimension was effective for the separation of isomeric lipid species presenting distinct double bond locations or geometries but was not able to differentiate lipid isomers with distinct acyl chain positions. High resolution TIMS-MS resulted in the identification of lipid isomers that differ in the double bond locations/geometries as well as in the position of the acyl chain with resolving power ( R) up to ∼410 ( R ∼ 320 needed on average). Extremely small structural differences exhibiting collision cross sections (CCS) of less than 1% (down to 0.2%) are sufficient for the discrimination of the isomeric lipid species using TIMS-MS. The same level of performance was maintained in the complex biological mixture for the biologically relevant PC 16:0/18:1 lipid isomers. These results suggest several advantages of using complementary LC-TIMS-MS separations for regular lipidomic analysis, with the main emphasis in the elucidation of isomer-specific lipid biological activities.

Measuring the Integrity of Gas-Phase Conformers of Sodiated 25-Hydroxyvitamin D3 by Drift Tube, Traveling Wave, Trapped, and High-Field Asymmetric Ion Mobility.

Quantitation of the serum concentration of 25-hydroxyvitamin D is a high-demand assay that suffers from long chromatography time to separate 25-hydroxyvitamin D from its inactive epimer; however, ion mobility spectrometry can distinguish the epimer pair in under 30 ms due to the presence of a unique extended or "open" gas-phase sodiated conformer, not shared with the epimer, reducing the need for chromatographic separation. Five ion mobility mass spectrometers utilizing commercially available IMS technologies, including drift tube, traveling wave, trapped, and high-field asymmetric ion mobility spectrometry, are evaluated for their ability to resolve the unique open conformer. Additionally, settings for each instrument are evaluated to understand their influence on ion heating, which can drive the open conformer into a compact or "closed" conformer shared with the epimer. The four low-field instruments successfully resolved the open conformer from the closed conformer at baseline or near-baseline resolution at typical operating parameters. High-field asymmetric ion mobility was unable to resolve a unique peak but detected two peaks for the epimer, in contrast to the low-field methods that detected one conformer. This study seeks to expand the instrument space by highlighting the potential of each platform for the separation of 25-hydroxyvitamin D epimers.

Lipidomics for wildlife disease etiology and biomarker discovery: a case study of pansteatitis outbreak in South Africa.

INTRODUCTION: Lipidomics is an emerging field with great promise for biomarker and mechanistic studies due to lipids diverse biological roles. Clinical research applying lipidomics is drastically increasing, with research methods and tools developed for clinical applications equally promising for wildlife studies. OBJECTIVES: Limited research to date has applied lipidomics, especially of the intact lipidome, to wildlife studies. Therefore, we examine the application of lipidomics for in situ studies on Mozambique tilapia (Oreochromis mossambicus) in Loskop Dam, South Africa. Wide-scale mortality events of aquatic life associated with an environmentally-derived inflammatory disease, pansteatitis, have occurred in this area. METHODS: The lipidome of adipose tissue (n = 31) and plasma (n = 51) from tilapia collected from Loskop Dam were characterized using state of the art liquid chromatography coupled to high-resolution tandem mass spectrometry. RESULTS: Lipid profiles reflected pansteatitis severity and were significantly different between diseased and healthy individuals. Over 13 classes of lipids associated with inflammation, cell death, and/or oxidative damage were upregulated in pansteatitis-affected adipose tissue, including ether-lipids, short-chained triglyceride oxidation products, sphingolipids, and acylcarnitines. Ceramides showed a 1000-fold increase in the most affected adipose tissues and were sensitive to disease severity. In plasma, triglycerides were found to be downregulated in pansteatitis-affected tilapia. CONCLUSION: Intact lipidomics provided useful mechanistic data and possible biomarkers of pansteatitis. Lipids pointed to upregulated inflammatory pathways, and ceramides serve as promising biomarker candidates for pansteatitis. As comprehensive coverage of the lipidome aids in the elucidation of possible disease mechanisms, application of lipidomics could be applied to the understanding of other environmentally-derived inflammatory conditions, such as those caused by obesogens.

Pioglitazone improves hepatic mitochondrial function in a mouse model of nonalcoholic steatohepatitis.

Pioglitazone is effective in improving insulin resistance and liver histology in patients with nonalcoholic steatohepatitis (NASH). Because dysfunctional mitochondrial metabolism is a central feature of NASH, we hypothesized that an important target of pioglitazone would be alleviating mitochondrial oxidative dysfunction. To this end, we studied hepatic mitochondrial metabolism in mice fed high-fructose high-transfat diet (TFD) supplemented with pioglitazone for 20 wk, using nuclear magnetic resonance-based 13C isotopomer analysis. Pioglitazone improved whole body and adipose insulin sensitivity in TFD-fed mice. Furthermore, pioglitazone reduced intrahepatic triglyceride content and fed plasma ketones and hepatic TCA cycle flux, anaplerosis, and pyruvate cycling in mice with NASH. This was associated with a marked reduction in most intrahepatic diacylglycerol classes and, to a lesser extent, some ceramide species (C22:1, C23:0). Considering the cross-talk between mitochondrial function and branched-chain amino acid (BCAA) metabolism, pioglitazone's impact on plasma BCAA profile was determined in a cohort of human subjects. Pioglitazone improved the plasma BCAA concentration profile in patients with NASH. This appeared to be related to an improvement in BCAA degradation in multiple tissues. These results provide evidence that pioglitazone-induced changes in NASH are related to improvements in hepatic mitochondrial oxidative dysfunction and changes in whole body BCAA metabolism.

Influence of Experimental Conditions on the Ratio of 25-Hydroxyvitamin D3 Conformers for Validating a Liquid Chromatography/Ion Mobility-Mass Spectrometry Method for Routine Quantitation.

The utility of adding ion mobility (IM) to liquid chromatography/mass spectrometry (LC/MS) for quantitation of 25-hydroxyvitamin D3 (25OHD3) is evaluated. Sodiated 25OHD3 ions adopt both closed and open conformations, whereas the stereoisomer 3-epi-25-hydroxyvitamin D (epi25OHD), when sodiated, adopts only a closed gas-phase conformation. The unique open conformation for sodiated 25OHD3 permits unambiguous quantitation. Nonetheless, the ratio of open versus closed gas-phase conformations for sodiated 25OHD3 can vary with instrumental conditions; conversion from the open to the closed conformer is attributed to radio frequency (rf) heating within the ion accumulation trap. Ion heating becomes significant when space charge in the trap forces ions to larger radii where the rf field is higher. To avoid biasing quantitation, an isotopically labeled internal standard must be used to account for changes in conformer ratio. Thirty-three serum extract samples were evaluated by LC/IM-MS and were found to not be biased by changes in ion conformer ratios, permitting reliable quantitation of 25OHD3 without interference from the epimer.

Mass Spectrometric Methodologies for Investigating the Metabolic Signatures of Parkinson's Disease: Current Progress and Future Perspectives.

Parkinson's disease (PD) is a neurodegenerative disorder resulting from the loss of dopaminergic neurons of the substantia nigra as well as degeneration of motor and nonmotor basal ganglia circuitries. Typically known for classical motor deficits (tremor, rigidity, bradykinesia), early stages of the disease are associated with a large nonmotor component (depression, anxiety, apathy, etc.). Currently, there are no definitive biomarkers of PD, and the measurement of dopamine metabolites does not allow for detection of prodromal PD nor does it aid in long-term monitoring of disease progression. Given that PD is increasingly recognized as complex and heterogeneous, involving several neurotransmitters and proteins, it is of importance that we advance interdisciplinary studies to further our knowledge of the molecular and cellular pathways that are affected in PD. This approach will possibly yield useful biomarkers for early diagnosis and may assist in the development of disease-modifying therapies. Here, we discuss preanalytical factors associated with metabolomics studies, summarize current mass spectrometric methodologies used to evaluate the metabolic signature of PD, and provide future perspectives of the rapidly developing field of MS in the context of PD.

NIST lipidomics workflow questionnaire: an assessment of community-wide methodologies and perspectives.

INTRODUCTION: Efforts to harmonize lipidomic methodologies have been limited within the community. Here, we aimed to capitalize on the recent National Institute of Standards and Technology lipidomics interlaboratory comparison exercise by implementing a questionnaire that assessed current methodologies, quantitation strategies, standard operating procedures (SOPs), and quality control activities employed by the lipidomics community. OBJECTIVES: Lipidomics is a rapidly developing field with diverse applications. At present, there are no community-vetted methods to assess measurement comparability or data quality. Thus, a major impetus of this questionnaire was to profile current efforts, highlight areas of need, and establish future objectives in an effort to harmonize lipidomics workflows. METHODS: The 54-question survey inquired about laboratory demographics, lipidomic methodologies and SOPs, analytical platforms, quantitation, reference materials, quality control procedures, and opinions regarding challenges existing within the community. RESULTS: A total of 125 laboratories participated in the questionnaire. A broad overview of results highlighted a wide methodological diversity within current lipidomic workflows. The impact of this diversity on lipid measurement and quantitation is currently unknown and needs to be explored further. While some laboratories do incorporate SOPs and quality control activities, these concepts have not been fully embraced by the community. The top five perceived challenges within the lipidomics community were a lack of standardization amongst methods/protocols, lack of lipid standards, software/data handling and quantification, and over-reporting/false positives. CONCLUSION: The questionnaire provided an overview of current lipidomics methodologies and further promoted the need for community-accepted guidelines and protocols. The questionnaire also served as a platform to help determine and prioritize metrological issues to be investigated.

Integration of paper spray ionization high-field asymmetric waveform ion mobility spectrometry for forensic applications.

RATIONALE: Paper spray ionization (PSI) is an attractive ambient ionization source for mass spectrometry (MS) since it allows the combination of surface sampling and ionization. The minimal sample preparation inherent in this approach greatly reduces the time needed for analysis. However, the ions generated from interfering compounds in the sample and the paper substrate may interfere with the analyte ions. Therefore, the integration of PSI with high-field asymmetric ion mobility spectrometry (FAIMS) is of significant interest since it should reduce the background ions entering the mass analyzer without complicating the analysis or increasing analysis time. Here we demonstrate the integration of PSI with FAIMS/MS and its potential for analysis of samples of forensic interest. METHODS: In this work, the parameters that can influence the integration, including sampling and ionization by paper spray, the FAIMS separation of analytes from each other and background interferences, and the length of time that a usable signal can be observed for explosives on paper, were evaluated with the integrated system. RESULTS: In the negative ion analysis of 2,4,6-trinitrotoluene (TNT), pentaerythritol tetranitrate (PETN), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), and 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), amounts as low as 1 ng on paper were readily observed. The successful positive ion separation of a set of illicit drugs including heroin, methamphetamine, and cocaine was also achieved. In addition, the positive ion analysis of the chemical warfare agent simulants dimethyl methylphosphonate (DMMP) and diisopropyl methylphosphonate (DIMP) was evaluated. CONCLUSIONS: The integration of PSI-FAIMS/MS was demonstrated for the analyses of explosives in negative ion mode and for illicit drugs and CW simulants in positive mode. Paper background ions that could interfere with these analyses were separated by FAIMS. The compensation voltage of an ion obtained by FAIMS provided an additional identification parameter to be combined with the mass spectrum for each analyte.

Cation-Dependent Conformations in 25-Hydroxyvitamin D3-Cation Adducts Measured by Ion Mobility-Mass Spectrometry and Theoretical Modeling.

Ion mobility-mass spectrometry is a useful tool in separation of biological isomers, including clinically relevant analytes such as 25-hydroxyvitamin D3 (25OHD3) and its epimer, 3-epi-25-hydroxyvitamin D3 (epi25OHD3). Previous research indicates that these epimers adopt different gas-phase sodiated monomer structures, either the "open" or "closed" conformer, which allow 25OHD3 to be readily resolved in mixtures. In the current work, alternative metal cation adducts are investigated for their relative effects on the ratio of "open" and "closed conformers. Alkali and alkaline earth metal adducts caused changes in the 25OHD3 conformer ratio, where the proportion of the "open" conformer generally increases with the size of the metal cation in a given group. As such, the ratio of the "open" conformer, which is unique to 25OHD3 and absent for its epimer, can be increased from approximately 1:1 for the sodiated monomer to greater than 8:1 for the barium adduct. Molecular modeling and energy calculations agree with the experimental results, indicating that the Gibbs free energy of conversion from the "closed" to the "open" conformation decreased with increasing cation size, correlating with the variation in ratio between the conformers. This work demonstrates the effect of cation adducts on gas-phase conformations of small, flexible molecules and offers an additional strategy for resolution of clinically relevant epimers.