Gas chromatography/multiphoton ionization/time-of-flight mass spectrometry using a femtosecond laser

A laser can be used for the selective excitation and subsequent ionization of a molecule with an absorption band at the laser wavelength. This technique of multiphoton ionization (MPI), when combined with time-of-flight mass spectrometry (TOF-MS), permits the efficient detection of induced ions in mass analysis. This combination of MPI/TOF-MS can be coupled with gas chromatography (GC) to achieve even more enhanced selectivity. Thus, GC/MPI/TOF-MS can be employed for trace analysis of samples containing numerous chemical species. A variety of laser sources have been used for this purpose. Since molecules that are classified as environmental pollutants frequently contain chlorine and bromine atoms, the lifetime of the excited state can be decreased by energy transfer from the singlet to triplet levels by spin–orbit interaction. A high-power femtosecond laser with a pulse width shorter than the lifetime of the analyte molecule provides femtogram or even subfemtogram detection limits, which have not yet been achieved using the most sensitive high-resolution double-focus sector-type mass spectrometers. Numerous environmental pollutants such as dioxins in soils and pesticides in foods have been successfully quantified using GC/MPI/TOF-MS, and this technique has proven itself to be a useful and practical method for trace analysis.

Ionization of pesticides using a far-ultraviolet femtosecond laser in gas chromatography/time-of-flight mass spectrometry

The fourth harmonic emission (200 nm) of a femtosecond Ti:sapphire laser (35 fs) was generated and used in the multiphoton ionization of 49 pesticides in gas chromatography/time-of-flight mass spectrometry. The limit of detection was improved when the ionization source from the third harmonic emission (267 nm) was replaced with the fourth harmonic emission for several pesticide molecules that contained no conjugated double bonds since their absorption bands are located in the far-ultraviolet region. This analytical instrument was used in the analysis of a series of real samples including potatoes, carrots, and cabbage, and a signal suspected to arise from di-allate was observed for the potato sample.

Quantification of Saquinavir from Lysates of Peripheral Blood Mononuclear Cells Using Microarrays and Standard MALDI-TOF-MS

Drug monitoring is usually performed by liquid chromatography coupled with optical detection or electrospray ionization mass spectrometry. More recently, matrix-assisted laser desorption/ionization (MALDI) in combination with triple quadrupole or Fourier-transform (FT) mass analyzers has also been reported to allow accurate quantification. Here, we present a strategy that employs standard MALDI time-of-flight (TOF) mass spectrometry (MS) for the sensitive and accurate quantification of saquinavir from an extract of blood peripheral mononuclear cells. Unambiguous identification of saquinavir in the mass spectra was possible because of using internal mass calibration and by an overall low chemical noise in the low mass range. Exact mass determination of the constant background peaks of the cell extract, which were used for recalibration, was performed by an initial MALDI-FT-MS analysis. Fast and multiplexed sample analysis was enabled by microarray technology, which provided 10 replicates in the lower nL range for each sample in parallel lanes on a chip. In order to validate the method, we employed various statistical tests, such as confidence intervals for linear regressions, three quality control samples, and inverse confidence limits of the estimated concentration ratios.

Application of UPLC-QTOF-MS in MSE mode for the rapid and precise identification of alkaloids in goldenseal (Hydrastis canadensis)

Here, we describe a new application of ultra-performance liquid chromatography coupled with an electrospray ionization quadrupole time-of-flight mass spectrometry operating in MS^E mode (UPLC-QTOF-MS^E) for the sensitive, fast, and effective characterization of alkaloids in goldenseal (Hydrastis canadensis). This approach allowed identification of alkaloids using a cyclic low and high collision energy spectral acquisition mode providing simultaneous accurate precursor and fragment ion mass information. A total of 45 compounds were separated and 40 of them characterized including one new compound and 7 identified for the first time in goldenseal. The spectral data obtained using this method is comparable to those obtained by conventional LC-MSⁿ. However, the UPLC-QTOF-MS^E method offers high chromatographic resolution with structural characterization facilitated by accurate mass measurement in both MS and MS/MS modes in a single analytical run; this makes it suitable for the rapid analysis and screening of alkaloids in plant extracts.

Overcoming Selectivity and Sensitivity Issues of Direct Inject Electrospray Mass Spectrometry via DAPNe-NSI-MS

Direct inject electrospray mass spectrometry offers minimal sample preparation and a “shotgun” approach to analyzing samples. However, complex matrix effects often make direct inject an undesirable sample introduction technique, particularly for trace level analytes. Highlighted here is our solution to the pitfalls of direct inject mass spectrometry and other ambient ionization methods with a focus on trace explosives. Direct analyte-probed nanoextraction coupled to nanospray ionization mass spectrometry solves selectivity issues and reduces matrix effects while maintaining minimal sample preparation requirements. With appropriate solvent conditions, most explosive residues can be analyzed with this technique regardless of the nature of the substance (i.e., nitroaromatic, oxidizing salt, or peroxide).

Combination of UHPLC/Q-TOF-MS,NMR spectroscopy,and ECD calculation for screening and identification of reactive metabolites of gentiopicroside in humans

The metabolic investigation of natural products is a great challenge because of unpredictable metabolic pathways, little knowledge on metabolic effects, and lack of recommended analytical methodology. Herein, a combined strategy based on ultrahigh-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC/Q-TOF-MS), nuclear magnetic resonance (NMR) spectroscopy, and electronic circular dichroism (ECD) calculation was developed and employed for the human metabolism study of gentiopicroside (GPS), a naturally hepato-protective iridoid glycoside. The whole metabolic study consisted of three major procedures. First, an improved UHPLC/Q-TOF-MS method was used to separate and detect a total of 15 GPS metabolites that were obtained from urine samples (0 to 72 h) of 12 healthy male participants after a single 50-mg oral dose of GPS. Second, a developed “MS-NMR-MS” method was applied to accurately identify molecular structures of the observed metabolites. Finally, given that the associated stereochemistry may be a crucial factor of the metabolic activation, the absolute configuration of the reactive metabolites was revealed through chemical calculations. Based on the combined use, a pair of diastereoisomers (G05 and G06) were experimentally addressed as the bioreactive metabolites of GPS, and the stereochemical determination was completed. Whereas several novel metabolic transformations, occurring via oxidation, N-heterocyclization and glucuronidation after deglycosylation, were also observed. The results indicated that GPS has to undergo in vivo metabolism-based activation to generate reactive molecules capable of processing its hepato-protective activity.

Imaging of Noncovalent Complexes by MALDI-MS

Noncovalent interactions govern how molecules communicate. Mass spectrometry is an important and versatile tool for the analysis of noncovalent complexes (NCX). Electrospray mass spectrometry (ESI-MS) is the most widely used MS technique for the study of NCXs because of its softer ionization and easy compatibility with the solution phase of NCX mixtures. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has also been used to study NCXs. However, successful analysis depends upon several experimental factors, such as matrix selection, solution pH, and instrumental parameters. In this study, we employ MALDI imaging mass spectrometry to investigate the location and formation of NCXs, involving both peptides and proteins, in a MALDI sample spot.

Multidimensional nano-HPLC coupled with tandem mass spectrometry for analyzing biotinylated proteins

Multidimensional high-performance liquid chromatography (HPLC) is a key method in shotgun proteomics approaches for analyzing highly complex protein mixtures by complementary chromatographic separation principles. Here, we describe an integrated 3D-nano-HPLC/nano-electrospray ionization quadrupole time-of-flight mass spectrometry system that allows an enzymatic digestion of proteins followed by an enrichment and subsequent separation of the created peptide mixtures. The online 3D-nano-HPLC system is composed of a monolithic trypsin reactor in the first dimension, a monolithic affinity column with immobilized monomeric avidin in the second dimension, and a reversed phase C18 HPLC-Chip in the third dimension that is coupled to a nano-ESI-Q-TOF mass spectrometer. The 3D-LC/MS setup is exemplified for the identification of biotinylated proteins from a simple protein mixture. Additionally, we describe an online 2D-nano-HPLC/nano-ESI-LTQ-Orbitrap-MS/MS setup for the enrichment, separation, and identification of cross-linked, biotinylated species from chemical cross-linking of cytochrome c and a calmodulin/peptide complex using a novel trifunctional cross-linker with two amine-reactive groups and a biotin label.

Structural Characterization of Polymers by MALDI Spiral-TOF Mass Spectrometry Combined with Kendrick Mass Defect Analysis

High-resolution mass spectrometry (HRMS) continues to play an important role in the compositional characterization of larger organic molecules. In the field of polymer characterization, however, the application of HRMS has made only slow progress because of lower compatibility between matrix-assisted laser desorption/ionization (MALDI) and ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICRMS). In this study, a newly developed type of MALDI high-resolution time-of-flight mass spectrometry (TOFMS) with a spiral ion trajectory (MALDI spiral-TOFMS) was applied to the structural and compositional characterization of polymers. To create a graphical distribution of polymer components on a two-dimensional plot converted from complex mass spectra, we adopted a slightly modified Kendrick mass defect (KMD) analysis based on accurate masses determined using spiral-TOFMS. By setting the Kendrick mass scale based on the mass of the repeating units of a given polymer, components with common repeat units lined up in the horizontal direction on the KMD plot, whereas those components with different structures were shifted vertically. This combination of MALDI spiral-TOFMS measurement and KMD analysis enabled the successful discrimination of the polymer components in a blend of poly(alkylene oxide)s, the compositional analysis of poly(ethylene oxide)/poly(propylene oxide) block copolymers, and profiling of the end-group distribution of poly(ε-caprolactone)s synthesized under different conditions.

Screening of Complicated Matrixes with Paper Assisted Ultrasonic Spray Ionization Mass Spectrometry

To analyze compounds in complicated matrixes using mass spectrometry, we describe a novel ambient ionization approach, termed paper assisted ultrasonic spray ionization (PAUSI). The ionization process is based on the ultrasonic vibration of the piezoelectric ceramic disk, on which the samples are placed. Porous materials are utilized to generate fine initial droplet, which could alleviate matrix effect during ionization process for complicated matrix. PAUSI was evaluated as an attractive tool to screen analytes from complicated matrixes, such as (1) bovine serum with NaCl 150 g/L, (2) viscous samples, and (3) biological fluid, without any sample preparation. Moreover, it provides great advantage in simplifying the mass spectrometry analysis process, and the ionization device is inexpensive and easy to operate.

Rapid screening of active ingredients in drugs by mass spectrometry with low-temperature plasma probe

A high-throughput method for rapid screening of active ingredients in drugs has been developed with mass spectrometry coupled to a low-temperature plasma (LTP) probe ion source. Without sample preparation or pretreatment, the active ingredients of 11 types of commercial pharmaceuticals, including hormones, antipyretic analgesics, cardiovascular, digestant, neuro-psychotherapeutic, diuretic, antithyroid, sulfa anti-inflammatory, antiparastic, sedative-hypnotics, and antibacterial, were directly desorbed/ionized and detected by a linear ion trap mass spectrometry (MS). The structures of these ingredients were elucidated by tandem MS. The analysis of 18 methyltestosterone tablets could be accomplished within 1.9 min, which allows fast detection with a speed of approximate 600 samples within 1 h. This work demonstrated that LTP probe ion source combined with MS is a high-throughput method for screening of pharmaceuticals and potentially applied to on-line quality control in pharmaceutical industry.

Laser desorption VUV postionization MS imaging of a cocultured biofilm

Laser desorption postionization mass spectrometry (LDPI-MS) imaging is demonstrated with a 10.5 eV photon energy source for analysis and imaging of small endogenous molecules within intact biofilms. Biofilm consortia comprised of a synthetic Escherichia coli K12 coculture engineered for syntrophic metabolite exchange are grown on membranes and then used to test LDPI-MS analysis and imaging. Both E. coli strains displayed many similar peaks in LDPI-MS up to m/z 650, although some observed differences in peak intensities were consistent with the appearance of byproducts preferentially expressed by one strain. The relatively low mass resolution and accuracy of this specific LDPI-MS instrument prevented definitive assignment of species to peaks, but strategies are discussed to overcome this shortcoming. The results are also discussed in terms of desorption and ionization issues related to the use of 10.5 eV single-photon ionization, with control experiments providing additional mechanistic information. Finally, 10.5 eV LDPI-MS was able to collect ion images from intact, electrically insulating biofilms at ～100 μm spatial resolution. Spatial resolution of ～20 μm was possible, although a relatively long acquisition time resulted from the 10 Hz repetition rate of the single-photon ionization source.

Constant-Momentum Acceleration Time-of-Flight Mass Spectrometry with Energy Focusing

Fundamental aspects of constant-momentum acceleration time-of-flight mass spectrometry (CMA-TOFMS) are explored as a means to improve mass resolution. By accelerating all ions to the same momentum rather than to the same energy, the effects of the initial ion spatial and energy distributions upon the total ion flight time are decoupled. This decoupling permits the initial spatial distribution of ions in the acceleration region to be optimized independently, and energy focus, including ion turn-around-time error, to be accomplished with a linear-field reflectron. Constant-momentum acceleration also linearly disperses ions across time according to mass-to-charge (m/z) ratio, instead of the quadratic relationship between flight time and m/z found in conventional TOFMS. Here, CMA-TOFMS is shown to achieve simultaneous spatial and energy focusing over a selected portion of the mass spectrum. An orthogonal-acceleration time-of-flight system outfitted with a reduced-pressure DC glow discharge (GD) ionization source is used to demonstrate CMA-TOFMS with atomic ions. The influence of experimental parameters such as the amplitude and width of the time-dependent CMA pulse on mass resolution is investigated, and a useful CMA-TOFMS focusing window of 2 to 18 Da is found for GD-CMA-TOFMS.

A simple method to identify ether lipids in spermatozoa samples by MALDI-TOF mass spectrometry

Plasmalogens (alkenylacyl glycerophospholipids) are important lipid constituents of many tissues and cells (e.g., selected spermatozoa). Since the molecular weights of plasmalogens overlap with that of diacyl- or alkyl acyl lipids, sophisticated mass spectrometry (MS; including MS/MS) analysis is normally used for the unequivocal identification of plasmalogens. We will show here that a simple matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (without MS/MS capability) in combination with acidic hydrolysis and subsequent derivatization with 2,4-dinitrophenylhydrazine (DNPH) and/or digestion with phospholipase A₂ (PLA₂) is sufficient to determine the contributions of ether lipids in spermatozoa extracts. As neither diacyl nor alkylacyl lipids are sensitive to acids and do not react with DNPH, the comparison of the mass spectra before and after treatment with acids and/or DNPH addition readily provides unequivocal information about the plasmalogen content. Additionally, the released aldehydes are readily converted into the 2,4-dinitrophenylhydrazones and can be easily identified in the corresponding negative ion mass spectra. Finally, PLA₂ digestion is very useful in confirming the presence of plasmalogens. The suggested method was validated by analyzing roe deer, bovine, boar, and domestic cat spermatozoa extracts and comparing the results with isolated phospholipids.

Data-Independent Microbial Metabolomics with Ambient Ionization Mass Spectrometry

Atmospheric ionization methods are ideally suited for prolonged MS/MS analysis. Data-independent MS/MS is a complementary technique for analysis of biological samples as compared to data-dependent analysis. Here, we pair data-independent MS/MS with the ambient ionization method nanospray desorption electrospray ionization (nanoDESI) for untargeted analysis of bacterial metabolites. Proof-of-principle data and analysis are illustrated by sampling Bacillus subtilis and Pseudomonas aeruginosa directly from Petri dishes. We found that this technique enables facile comparisons between strains via MS and MS/MS plots which can be translated to chemically informative molecular maps through MS/MS networking. The development of novel techniques to characterize microbial metabolites allows rapid and efficient analysis of metabolic exchange factors. This is motivated by our desire to develop novel techniques to explore the role of interspecies interactions in the environment, health, and disease. This is a contribution to honor Professor Catherine C. Fenselau in receiving the prestigious ASMS Award for a Distinguished Contribution in Mass Spectrometry for her pioneering work on microbial mass spectrometry.

Pharmaceutical Analysis by Solid-Substrate Electrospray Ionization Mass Spectrometry with Wooden Tips

Electrospray ionization (ESI) using wooden tips as solid substrates allows direct ionization of various samples and their simple and efficient analyses by mass spectrometry (MS). In this study, wooden-tip ESI-MS was used for pharmaceutical analysis. A wide variety of active components present in pharmaceuticals with forms of tablets, capsules, granules, dry suspensions, suspensions, drops, and oral liquids, etc., were all successfully ionized directly for mass spectrometric analysis. Trace degradation products were also sensitively detected using wooden-tip ESI-MS. This strategy was extended to construct chemical fingerprints of herbal products containing complex and unknown components, and the fingerprints provided valuable information for their quality assessment and origin tracing. Our experimental data demonstrated that wooden-tip ESI-MS is a useful tool for rapid pharmaceutical analysis, with high sensitivity and wide applicability, showing promising perspectives for quality assessment and control, authentication, and origin tracing of pharmaceuticals.

Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI) Imaging Source Coupled to a FT-ICR Mass Spectrometer

Mass spectrometry imaging (MSI) allows for the direct monitoring of the abundance and spatial distribution of chemical compounds over the surface of a tissue sample. This technology has opened the field of mass spectrometry to numerous innovative applications over the past 15 years. First used with SIMS and MALDI MS that operate under vacuum, interest has grown for mass spectrometry ionization sources that allow for effective imaging but where the analysis can be performed at ambient pressure with minimal or no sample preparation. We introduce here a versatile source for MALDESI imaging analysis coupled to a hybrid LTQ-FT-ICR mass spectrometer. The imaging source offers single shot or multi-shot capability per pixel with full control over the laser repetition rate and mass spectrometer scanning cycle. Scanning rates can be as fast as 1 pixel/second and a spatial resolution of 45 μm was achieved with oversampling.

Studying Reaction Intermediates Formed at Graphenic Surfaces

We report in-situ production and detection of intermediates at graphenic surfaces, especially during alcohol oxidation. Alcohol oxidation to acid occurs on graphene oxide-coated paper surface, driven by an electrical potential, in a paper spray mass spectrometry experiment. As paper spray ionization is a fast process and the time scale matches with the reaction time scale, we were able to detect the intermediate, acetal. This is the first observation of acetal formed in surface oxidation. The process is not limited to alcohols and the reaction has been extended to aldehydes, amines, phosphenes, sugars, etc., where reaction products were detected instantaneously. By combining surface reactions with ambient ionization and mass spectrometry, we show that new insights into chemical reactions become feasible. We suggest that several other chemical transformations may be studied this way. This work opens up a new pathway for different industrially and energetically important reactions using different metal catalysts and modified substrate.

Selective Detection of Carbohydrates and Their Peptide Conjugates by ESI-MS Using Synthetic Quaternary Ammonium Salt Derivatives of Phenylboronic Acids

We present new tags based on the derivatives of phenylboronic acid and apply them for the selective detection of sugars and peptide-sugar conjugates in mass spectrometry. We investigated the binding of phenylboronic acid and its quaternary ammonium salt (QAS) derivatives to carbohydrates and peptide-derived Amadori products by HR-MS and MS/MS experiments. The formation of complexes between sugar or sugar-peptide conjugates and synthetic tags was confirmed on the basis of the unique isotopic distribution resulting from the presence of boron atom. Moreover, incorporation of a quaternary ammonium salt dramatically improved the efficiency of ionization in mass spectrometry. It was found that the formation of a complex with phenylboronic acid stabilizes the sugar moiety in glycated peptides, resulting in simplification of the fragmentation pattern of peptide-derived Amadori products. The obtained results suggest that derivatization of phenylboronic acid as QAS is a promising method for sensitive ESI-MS detection of carbohydrates and their conjugates formed by non-enzymatic glycation or glycosylation.

Mass Recalibration of FT-ICR Mass Spectrometry Imaging Data Using the Average Frequency Shift of Ambient Ions

Achieving and maintaining high mass measurement accuracy (MMA) throughout a mass spectrometry imaging (MSI) experiment is vital to the identification of the observed ions. However, when using FTMS instruments, fluctuations in the total ion abundance at each pixel due to inherent biological variation in the tissue section can introduce space charge effects that systematically shift the observed mass. Herein we apply a recalibration based on the observed cyclotron frequency shift of ions found in the ambient laboratory environment, polydimethylcyclosiloxanes (PDMS). This calibration method is capable of achieving part per billion (ppb) mass accuracy with relatively high precision for an infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) MSI dataset. Comparisons with previously published mass calibration approaches are also presented.