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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 ( 1 H 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 1 H 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 1 H 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.

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.

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.

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.

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.

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.

The utility of adding ion mobility (IM) to liquid chromatography/mass spectrometry (LC/MS) for quantitation of 25-hydroxyvitamin D 3 (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.

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.

Liquid-microjunction surface sampling (LMJ-SS) is an ambient ionization technique based on the continuous flow of solvent using an in situ microextraction device in which solvent moves through the probe, drawing in the analytes in preparation for ionization using an electrospray ionization source. However, unlike traditional mass spectrometry (MS) techniques, it operates under ambient pressure and requires no sample preparation, thereby making it ideal for rapid sampling of thicker tissue sections for electrophysiological and other neuroscientific research studies. Studies interrogating neural synapses, or a specific neural circuit, typically employ thick, ex vivo tissue sections maintained under near-physiological conditions to preserve tissue viability and maintain the neural networks. Deep brain stimulation (DBS) is a surgical procedure used to treat the neurological symptoms that are associated with certain neurodegenerative and neuropsychiatric diseases. Parkinson's disease (PD) is a neurological disorder which is commonly treated with DBS therapy. PD is characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta portion of the brain. Here, we demonstrate that the LMJ-SS methodology can provide a platform for ex vivo analysis of the brain during electrical stimulation, such as DBS. We employ LMJ-SS in the ex vivo analysis of mouse brain tissue for monitoring dopamine during electrical stimulation of the striatum region. The mouse brain tissue was sectioned fresh post sacrifice and maintained in artificial cerebrospinal fluid to create near-physiological conditions before direct sampling using LMJ-SS. A selection of metabolites, including time-sensitive metabolites involved in energy regulation in the brain, were identified using standards, and the mass spectral database mzCloud was used to assess the feasibility of the methodology. Thereafter, the intensity of m/z 154 corresponding to protonated dopamine was monitored before and after electrical stimulation of the striatum region, showing an increase in signal directly following a stimulation event. Dopamine is the key neurotransmitter implicated in PD, and although electrochemical detectors have shown such increases in dopamine post-DBS, this is the first study to do so using MS methodologies.

As advances in analytical separation techniques, mass spectrometry instrumentation, and data processing platforms continue to spur growth in the lipidomics field, more structurally unique lipid species are detected and annotated. The lipidomics community is in need of benchmark reference values to assess the validity of various lipidomics workflows in providing accurate quantitative measurements across the diverse lipidome. LipidQC addresses the harmonization challenge in lipid quantitation by providing a semiautomated process, independent of analytical platform, for visual comparison of experimental results of National Institute of Standards and Technology Standard Reference Material (SRM) 1950, "Metabolites in Frozen Human Plasma", against benchmark consensus mean concentrations derived from the NIST Lipidomics Interlaboratory Comparison Exercise.

Preparation of tissue for matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) generally involves embedding the tissue followed by freezing and cryosectioning, usually between 5 and 25 μm thick, depending on the tissue type and the analyte(s) of interest. The brain is approximately 60% fat; it therefore lacks rigidity and poses structural preservation challenges during sample preparation. Histological sample preparation procedures are generally transferable to MALDI-MSI; however, there are various limitations. Optimal cutting temperature compound (OCT) is commonly used to embed and mount fixed tissue onto the chuck inside the cryostat during cryosectioning. However, OCT contains potential interferences that are detrimental to MALDI-MSI, while fixation is undesirable for the analysis of some analytes either due to extraction or chemical modification (i.e., polar metabolites). Therefore, a method for both fixed and fresh tissue compatible with MALDI-MSI and histology is desirable to increase the breadth of analyte(s), maintain the topographies of the brain, and provide rigidity to the fragile tissue while eliminating background interference. The method we introduce uses precast gelatin-based molds in which a whole mouse brain is embedded, flash frozen, and cryosectioned in preparation for mass spectrometry imaging (MSI).

Ammonium nitrate (AN) and urea nitrate (UN) are commonly used materials in improvised explosive devices (IEDs). Detection by mass spectrometry (MS) and/or ion mobility spectrometry (IMS) is traditionally difficult. The major challenges of detecting these species arise from their ionic nature and their low mass (for MS detection) and size (for IMS detection). Although AN and UN both produce characteristic higher mass (and size) cluster ions when ionized by electrospray ionization (ESI), detection of AN/UN using cluster ions poses difficulty at trace levels because their formation is concentration-dependent. The addition of modifiers to the ESI process is demonstrated here to overcome some of these challenges for the detection of AN and UN using MS and/or IMS.

Isotopic ratio outlier analysis (IROA) is a (13)C metabolomics profiling method that eliminates sample to sample variance, discriminates against noise and artifacts, and improves identification of compounds, previously done with accurate mass liquid chromatography/mass spectrometry (LC/MS). This is the first report using IROA technology in combination with accurate mass gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS), here used to examine the S. cerevisiae metabolome. S. cerevisiae was grown in YNB media, containing randomized 95% (13)C, or 5%(13)C glucose as the single carbon source, in order that the isotopomer pattern of all metabolites would mirror the labeled glucose. When these IROA experiments are combined, the abundance of the heavy isotopologues in the 5%(13)C extracts, or light isotopologues in the 95%(13)C extracts, follows the binomial distribution, showing mirrored peak pairs for the molecular ion. The mass difference between the (12)C monoisotopic and the (13)C monoisotopic equals the number of carbons in the molecules. The IROA-GC/MS protocol developed, using both chemical and electron ionization, extends the information acquired from the isotopic peak patterns for formulas generation. The process that can be formulated as an algorithm, in which the number of carbons, as well as the number of methoximations and silylations are used as search constraints. In electron impact (EI/IROA) spectra, the artifactual peaks are identified and easily removed, which has the potential to generate "clean" EI libraries. The combination of chemical ionization (CI) IROA and EI/IROA affords a metabolite identification procedure that enables the identification of coeluting metabolites, and allowed us to characterize 126 metabolites in the current study.

We demonstrate the global metabolic analysis of Caenorhabditis elegans stress responses using a mass-spectrometry-based technique called isotopic ratio outlier analysis (IROA). In an IROA protocol, control and experimental samples are isotopically labeled with 95 and 5% (13)C, and the two sample populations are mixed together for uniform extraction, sample preparation, and LC-MS analysis. This labeling strategy provides several advantages over conventional approaches: (1) compounds arising from biosynthesis are easily distinguished from artifacts, (2) errors from sample extraction and preparation are minimized because the control and experiment are combined into a single sample, (3) measurement of both the molecular weight and the exact number of carbon atoms in each molecule provides extremely accurate molecular formulas, and (4) relative concentrations of all metabolites are easily determined. A heat-shock perturbation was conducted on C. elegans to demonstrate this approach. We identified many compounds that significantly changed upon heat shock, including several from the purine metabolism pathway. The metabolomic response information by IROA may be interpreted in the context of a wealth of genetic and proteomic information available for C. elegans . Furthermore, the IROA protocol can be applied to any organism that can be isotopically labeled, making it a powerful new tool in a global metabolomics pipeline.

Phospholipid oxidation has been implicated in the pathogenesis and progression of numerous age-related and neurodegenerative diseases. Despite these implications, this broad class of biomolecules remains poorly characterized. In this work, the fragmentation patterns of [M + H](+) and [M + Na](+) ions of intact phosphatidylcholine oxidation products (OxPCs) were characterized by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI MS(n), n = 2, 3, and 4). MS(2) of both the [M + H](+) and [M + Na](+) ions of short-chain OxPCs yielded product ions related to the PC headgroup and the fatty acid substituents. MS(3) of the [M + Na - N(CH3)3](+) ions yielded fragmentation indicative of the OxPC modification; specifically, a product ion corresponding to the neutral loss of CO2 (NL of 44) was observed for OxPCs containing a terminal carboxylic acid rather than an aldehyde. Furthermore, MS(4) of the [M + Na - HPO4(CH2)2N(CH3)3](+) ions resulted in fragmentation pathways dependent on the sn-2 fatty acid chain length and type of functional group(s). Specifically, CHO-containing OxPCs with palmitic acid esterified to the sn-1 position of the glycerol backbone yielded a NL of 254, 2 u less than the nominal mass of palmitic acid, whereas the analogous terminal COOH-containing OxPCs demonstrated a NL of 256. Finally, the presence of a γ-ketone relative to the terminal carboxyl group resulted in C-C bond cleavages along the sn-2 substituent, providing diagnostic product ions for keto-containing OxPCs. This work illustrates the enhanced selectivity afforded by MS(n) on the linear ion trap and develops a method for the identification of individual products of PC oxidation.

Generating analyte-specific distribution maps of compounds in a tissue sample by matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging (MSI) has become a useful tool in numerous areas across the biological sciences. Direct analysis of the tissue sample provides MS images of an analyte's distribution with minimal sample pretreatment. The technique, however, suffers from the inability to account for tissue-specific variations in ion signal. The variation in the makeup of different tissue types can result in significant differences in analyte extraction, cocrystallization, and ionization across a sample. In this study, a deuterated internal standard was used to account for these signal variations. Initial experiments were performed using pure standards and optimal cutting temperature compound (OCT) to generate known areas of ion suppression. By monitoring the analyte-to-internal-standard ratio, differences in ion signal were taken into account, resulting in images that better represented the analyte concentration. These experiments were then replicated using multiple tissue types in which the analyte's MS signal was monitored. In certain tissues, including liver and kidney, the analyte signal was attenuated by up to 90%; however, when the analyte-to-internal-standard ratio was monitored, these differences were taken into account. These experiments further exemplify the need for an internal standard in the MSI workflow.

Mass spectrometric imaging (MSI) is an analytical technique used to determine the distribution of individual analytes within a given sample. A wide array of analytes and samples can be investigated by MSI, including drug distribution in rats, lipid analysis from brain tissue, protein differentiation in tumors, and plant metabolite distributions. Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique capable of desorbing and ionizing a large range of compounds, and it is the most common ionization source used in MSI. MALDI mass spectrometry (MS) is generally considered to be a qualitative analytical technique because of significant ion-signal variability. Consequently, MSI is also thought to be a qualitative technique because of the quantitative limitations of MALDI coupled with the homogeneity of tissue sections inherent in an MSI experiment. Thus, conclusions based on MS images are often limited by the inability to correlate ion signal increases with actual concentration increases. Here, we report a quantitative MSI method for the analysis of cocaine (COC) from brain tissue using a deuterated internal standard (COC-d(3)) combined with wide-isolation MS/MS for analysis of the tissue extracts with scan-by-scan COC-to-COC-d(3) normalization. This resulted in significant improvements in signal reproducibility and calibration curve linearity. Quantitative results from the MSI experiments were compared with quantitative results from liquid chromatography (LC)-MS/MS results from brain tissue extracts. Two different quantitative MSI techniques (standard addition and external calibration) produced quantitative results comparable to LC-MS/MS data. Tissue extracts were also analyzed by MALDI wide-isolation MS/MS, and quantitative results were nearly identical to those from LC-MS/MS. These results clearly demonstrate the necessity for an internal standard for quantitative MSI experiments.

Although acute myocardial infarction (MI) is consistently among the top causes of death in the United States, the spatial distribution of lipids and metabolites following MI remains to be elucidated. This work presents the investigation of an in vivo rat model of MI using mass spectrometric imaging (MSI) and multivariate data analysis. MSI was conducted on cardiac tissue following a 24-h left anterior descending coronary artery ligation to analyze multiple compound classes. First, the spatial distribution of a small metabolite, creatine, was used to identify areas of infarcted myocardium. Second, multivariate data analysis and tandem mass spectrometry were used to identify phospholipid (PL) markers of MI. A number of lysophospholipids demonstrated increased ion signal in areas of infarction. In contrast, select intact PLs demonstrated decreased ion signal in the area of infarction. The complementary nature of these two lipid classes suggests increased activity of phospholipase A(2), an enzyme that has been implicated in coronary heart disease and inflammation.

High-field asymmetric waveform ion mobility spectrometry (FAIMS) separates ions by utilizing the mobility differences of ions at high and low fields. The shape of the waveform is one of the essential features affecting the resolution, transmission, and separation of FAIMS. Due to practical circuitry advantages, sinusoidal asymmetric waveforms are typically used in FAIMS, whereas theoretical studies indicate that square asymmetric waveforms improve ion separation, resolution, and sensitivity. Results from FAIMS using square and sinusoidal waveforms are presented, and effects of the waveforms on ion separation are discussed. A FAIMS system interfaced with a quadrupole ion trap mass spectrometer was used in this study. FAIMS spectra were generated by scanning the compensation voltage (CV) while operating the mass spectrometer in total ion mode. The identification of ions was accomplished through mass spectra acquired at fixed values of ions' CVs. Square waveform evaluation was done by acquiring data at three frequencies and six duty cycles of the square waveform generator. The performance of FAIMS using square and sinusoidal waveforms at 250, 333, and 500 kHz frequencies was compared, and trends were identified. For all frequencies, the best response of FAIMS was achieved at the lower amplitudes and under the lower duty cycles of the square waveform generator. The separation of FAIMS was better at the higher frequencies. These results demonstrate the potential to incorporate square-wave FAIMS into the design of a miniature device for detection of explosives in the field. SIMION version 8.0, the ion trajectory modeling program, was utilized to optimize the performance of the miniature FAIMS cell and to validate experimental results.

Matrix-assisted laser desorption/ionization (MALDI) based mass spectrometric imaging (MSI) is increasingly being used as an analytical tool to evaluate the molecular makeup of tissue samples. From the direct analysis of a tissue section, the physical integrity of sample is preserved; thus, spatial information of a compound's distribution may be determined. One limitation of the technique, however, has been the inability to determine the absolute concentration from a tissue sample. Here we report the development of a quantitative MSI technique in which the distribution of acetyl-L-carnitine (AC) in a piglet brain sample is quantified with MALDI MSI. An isotopically labeled internal standard was applied uniformly beneath the tissue section, and wide-isolation tandem mass spectrometry was performed. Normalizing the analyte ion signal by the internal standard ion signal resulted in significant improvements in MS images, signal reproducibility, and calibration curve linearity. From the improved MS images, the concentration of AC was determined and plotted producing a concentration-scaled image of the distribution of AC in the piglet brain section.

Imaging applied toward lignocellulosic materials requires high molecular specificity to map specific compounds within intact tissue. Although secondary ionization mass spectrometry (SIMS) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) with a single stage of MS have been used to image lignocellulosic biomass, the complexity of the plant tissue requires tandem MS, which limits the interpretation of simple MS. MALDI linear ion trap (LIT) tandem MS offers the high molecular specificity needed for lignocellulosic analyses. MALDI-LIT MS analyses of cellulose and xylan (hemicellulose) standards were performed to determine mass-to-charge ratios and fragmentation pathways for identification of these compounds in intact tissue. The MALDI-LIT-MS images of young Populus wood stem showed even distribution of both cellulose and hemicellulose ions; in contrast, the tandem MS images of cellulose and hemicellulose generated by plotting characteristic fragment ions resulted in drastically different images. This demonstrates that isobaric ions are present during MALDI-LIT-MS analyses of wood tissue and tandem MS is necessary to distinguish between isobaric species for selective imaging of carbohydrates in biomass.

Analysis of large peptides can be used to discover or to monitor biomarkers for various diseases. For example, the levels of such peptides can determine the effectiveness of an experimental drug or the progress of a disease. Many mass spectrometric methods for monitoring these peptides use MALDI-ToF instruments because of their high molecular weights, although such instruments typically lack MS/MS or MS(n) capabilities. Here, the m/z range of a MALDI-LIT instrument was extended to m/z 5500 for the MS or MS(n) analysis of large peptides. Instrument performance was examined using amyloid beta 1-40 and 1-42 (avg. MW 4330.8 and 4515.0, respectively), large peptides that comprise the bulk of neuritic plaques and are potential biomarkers for Alzheimer's Disease. The amyloid beta 1-40 was detected in the full-scan mass spectrum with sufficient resolution to distinguish and match the expected isotopic pattern. The MS/MS product ion spectra of both peptides matched the expected fragmentation patterns; up to MS(4) experiments were performed to verify the identity of the peptides. These experiments clearly demonstrate the advantages of this approach, including MS(n) experiments for structural elucidation and simplified spectra due to singly charged parent ions, for large peptides.

A hybrid linear ion trap/Orbitrap mass spectrometer was used to perform tandem mass spectrometry (MS/MS) experiments and high-resolution mass analysis of lipids desorbed from nerve tissue. A dramatic improvement in mass spectral resolution and a decrease in background are observed in the spectra collected from the Orbitrap mass analyzer, which allows generation of more accurate mass spectrometric images of the distribution of lipids within nerve tissue. Employment of both mass analyzers provides a rapid and reliable means of compound identification based on MS/MS fragmentation and high-resolution mass spectrometry (HRMS) accurate mass.

Derivatization of steroids is typically required before analysis by gas chromatography/mass spectrometry (GC/MS); nevertheless, the derivatization process can often be time-consuming and irreproducible. Although several strategies have been employed to enhance this process, few have the potential of microwave-accelerated derivatization (MAD) to be more efficient than traditional thermal derivatization methods. MAD using a synthesis microwave system was evaluated and compared to traditional thermal derivatization methods in terms of yield, reproducibility, and overall analysis time. Parameters affecting MAD, including reaction temperature, time, and power, were systematically optimized for several silyl reagents (BSTFA with TMCS, MSTFA, and BSA) and other derivatization procedures (MOX reagent and MTBSTFA). MSTFA was found to derivatize best with the microwave, as demonstrated by the enhanced relative response factors (RRFs). BSTFA with TMCS, on the other hand, did not couple as well, but RRF values improved significantly upon addition of polar solvents. The rapid (1 min) derivatization reactions associated with MAD had comparable RRFs for all reagents with those obtained with thermal heating (>30 min). This study highlights the best methods for analyzing a comprehensive variety of steroids and also provides ideal strategies for MAD of steroids on an individual or class level.

The direct analysis of tissue from both the central and peripheral nervous systems of control rats and those administered the potential neurotoxin dichloroacetate (DCA) was investigated using an intermediate-pressure matrix-assisted laser desorption/ionization (IP-MALDI) source coupled to a linear ion trap (LIT) mass spectrometer. The matrix, 2,5-dihydroxybenzoic acid, was applied to the tissue using a novel automated inkjet printer system. The MSn capabilities of the LIT allowed identification of lipids desorbed directly from tissue. A marked decrease is observed in the intensity of lipid ions in spinal cord and sciatic nerve tissues from rats exposed to DCA. The results also demonstrate the rapid, sensitive, and semiquantitative capabilities of this method.

Careful matrix deposition on tissue samples for matrix-assisted laser desorption/ionization (MALDI) is critical for producing reproducible analyte ion signals. Traditional methods for matrix deposition are often considered an art rather than a science, with significant sample-to-sample variability. Here we report an automated method for matrix deposition, employing a desktop inkjet printer (<$200) with 5760 x 1440 dpi resolution and a six-channel piezoelectric head that delivers 3 pL/drop. The inkjet printer tray, designed to hold CDs and DVDs, was modified to hold microscope slides. Empty ink cartridges were filled with MALDI matrix solutions, including DHB in methanol/water (70:30) at concentrations up to 40 mg/mL. Various samples (including rat brain tissue sections and standards of small drug molecules) were prepared using three deposition methods (electrospray, airbrush, inkjet). A linear ion trap equipped with an intermediate-pressure MALDI source was used for analyses. Optical microscopic examination showed that matrix crystals were formed evenly across the sample. There was minimal background signal after storing the matrix in the cartridges over a 6-month period. Overall, the mass spectral images gathered from inkjet-printed tissue specimens were of better quality and more reproducible than from specimens prepared by the electrospray and airbrush methods.

Haloacetic acids (HAAs) are environmentally and medically important chemicals. No analytical method is currently available to analyze EPA-regulated HAAs in biological samples at environmentally relevant low concentrations. Clinical studies of this class of chemicals are also limited by the lack of analytical techniques of high sensitivity and precision. We now report a new analytical method using gas chromatography/ion trap mass spectrometry for quantifying nine HAAs present inplasma, urine, and water at picogram per milliliter levels. The derivatization reactions of HAAs with pentafluorobenzyl bromide were optimized and detection with an electron capture negative ion chemical ionization mode was employed to enhance the sensitivity. Selected ion monitoring and selected reaction monitoring methods were utilized for quantitation. The detection limits of HAAs in plasma, urine, and water were 25-1000 pg/mL. Accuracies varied from 86.6 to 118.1% (intraday) and 81.7 to 119.6% (interday). Precisions (CV) varied from 0.9 to 19.9% (intraday) and 0.8 to 19.8% (interday), and linearities (r2) varied from 0.9732 to 0.9998 (intraday) and 0.9422 to 0.9987 (interday), respectively. Methyl tertbutyl ether and diethyl ether provided the highest extraction recoveries for the HAAs (74.9-107.2%). The method was applied successfully to a kinetic investigation of low levels of HAAs in humans consuming chlorinated drinking water.

A novel and practical technique for performing both parent and neutral loss (P&NL) monitoring experiments on a quadrupole ion trap mass spectrometer is presented. This technique is capable of performing scans analogous to the parent and neutral loss scans routinely applied on tandem-in-space instruments and allows for the screening of a sample to detect analytes of a specific compound class on a chromatographic time-scale. Acylcarnitines were chosen as the model compound class to demonstrate the analytical utility of P&NL monitoring because of their amenability to electrospray ionization (ESI), their unique and informative MS/MS fragmentation pattern, and their importance in biological functions. The [M + H]+ ions of all acylcarnitines dissociate to produce neutral losses of 59 and 161 amu and common product ions at m/z 60, 85, and 144. Both the neutral loss monitoring of 59 amu and the parent ion monitoring of m/z 85 are shown to be capable of identifying acylcarnitine [M + H]+ ions in a synthetic mixture and spiked pig plasma. The neutral loss monitoring of 59 amu is successful in detecting acylcarnitines in an unspiked pig plasma sample.

In the quadrupole ion trap, it has been noted that factors other than an ion's mass and charge may affect its measured m/z, resulting in compound-dependent, or "chemical", mass shifts. We propose that ions can exhibit a chemical mass shift because they are "fragile" and may fragment during the application of resonance ejection during mass analysis; these effects were studied using ions that include protonated, deprotonated, and adduct ions of explosives, acylcarnitines, and macrolide antibiotics. Fragile ions affect mass resolution by causing broader peaks than nonfragile ions, especially at slower scan speeds, as the result of the application of resonance ejection. Fragile ions may also be fragmented by the application of the isolation waveform during selection of the parent ion for tandem mass spectrometry experiments, making it impossible to achieve unit isolation of a fragile ion. To obtain adequate isolation intensity, the isolation waveform notch width must be increased and the time period of isolation must be decreased. Fragile ions also require lower optimum collision energy to achieve efficient collision-induced dissociation. We have developed criteria for the determination of the degree of ion fragility based upon experimental results.

Volatile compounds emanated from human skin were studied by gas chromatography/mass spectrometry (GC/MS). The purpose of this study was to identify compounds that may be human-produced kairomones which are used for host location by the mosquito, Aedes aegypti (L.). The procedure used to collect volatiles was chosen because of prior knowledge that attractive substances can be transferred from skin to glass by handling. Laboratory bioassays have shown that the residuum on the glass remains attractive to mosquitoes until the compounds of importance evaporate. The sampling and analytical procedures modeled the above-cited process as closely as possible except that the evaporation of compounds from the glass surface was accomplished by thermal desorption from glass beads in a heated GC injection port. This made possible the solventless injection of volatiles onto the column. The compounds were cryofocused on the head of the column with liquid nitrogen prior to GC separation. A single stage of mass spectrometry on a triple quadrupole instrument was used for mass analysis. A combination of electron ionization and pulsed positive ion/negative ion chemical ionization modes on two different GC columns (one polar, one relatively nonpolar) was used to identify most of the 346 compound peaks detected by this technique.

Handled glass has the ability to collect and concentrate nonaqueous human skin emanations while minimizing the collection of aqueous perspiration. Compounds originating from the skin and collected on glass have previously been found to attract the Aedes aegypti species of mosquito. Therefore, glass beads were used as the medium to collect skin emanations from humans for subsequent chemical analysis. This process consisted of a 5-15-min collection of sample on glass beads, followed by loading the beads into a gas chromatograph (GC) injector insert for subsequent desorption of the collected compounds onto the GC column. After cryofocusing by liquid nitrogen at the head of the column, the thermally desorbed compounds were analyzed by GC/MS. Microscale purge and trap introduction was also used to provide complementary information. In this case, the beads are held in a round-bottom flask, purged with nitrogen, and heated as the concentrator collects the headspace above the beads. The chromatograms produced by both of these sample introduction methods demonstrate good resolution of a complex sample. Cryofocusing volatiles from handled glass allowed identification of lactic acid, aliphatic fatty acids, and other polar to nonpolar compounds of moderate volatility while purge and trap allowed detection of nonpolar to moderately polar compounds of high volatility.

A promising instrumental technique has been investigated to rapidly screen complex environmental samples for chemical contaminants having the propensity to covalently bond to biomacromolecules such as DNA. Radical molecular ions of pyridine, a model compound for nucleophilic bases of DNA, were mass-selected and allowed to react with electrophilic environmental contaminants in the collision cell of a triple quadrupole mass spectrometer. Analytes were introduced into the collision cell via a gas chromatographic column. Reactive chemicals are then characterized by scanning Q3 to identify associative reaction products. A good qualitative correlation was observed for the gas-phase reactivity of a series of electrophilic reagents with both their alkylating reactivity in solution (4-(4-nitrobenzyl)pyridine) and AMES test mutagenicity which had been previously published. Femtomole limits of detection for specific associative reaction products were demonstrated. Gas-phase reactions of ions of environmental contaminants (introduced into the source) with neutral pyridine (in the collision cell) were also investigated. Reactions of the radical molecular ion of the allyl reagents with neutral pyridine were similar to results from the mass-selected reaction of the pyridine radical molecular ion with neutral allylic reagents.

The electrochemical oxidation pathway of uric acid was determined by on-line electrochemistry/thermospray/tandem mass spectrometry. Intermediates and products formed as a result of electrooxidation were monitored as the electrode potential was varied. Several reaction intermediates have been identified and characterized by tandem mass spectrometry. The tandem mass spectrometric results provide convincing evidence that the primary intermediate produced during the electrooxidation of uric acid has a quinonoid diimine structure. The results indicate that once formed via electrooxidation, the primary intermediate can follow three distinct reaction pathways to produce the identified final products. The final electrochemical oxidation products observed in these studies were urea, CO2, alloxan, alloxan monohydrate, allantoin, 5-hydroxyhydantoin-5-carboxamide, and parabanic acid. The solution reactions that follow the initial electron transfer at the electrode are affected by the vaporizer tip temperature of the thermospray probe. In particular, it was found that at different tip temperatures either hydrolysis or ammonolysis reactions of the initial electrochemical oxidation products can occur. Most importantly, the results show that the on-line combination of electrochemistry with thermospray/tandem mass spectrometry provides otherwise difficult to obtain information about redox and associated chemical reactions of biological molecules such as the structure of reaction intermediates and products, as well as providing insight into reaction pathways.