A tutorial: Laserspray ionization and related laser-based ionization methods for use in mass spectrometry.

This Tutorial is to provide a summary of parameters useful for successful outcomes of laserspray ionization (LSI) and related methods that employ a laser to ablate a matrix:analyte sample to produce highly charged ions. In these methods the purpose of the laser is to transfer matrix-analyte clusters into the gas phase. Ions are hypothesized to be produced by a thermal process where emitted matrix:analyte gas-phase particles/clusters are charged and loss of matrix from the charged particles leads to release of the analyte ions into the gas phase. The thermal energy responsible for the charge-separation process is relatively low and not necessarily supplied by the laser; a heated inlet tube linking atmospheric pressure with the first vacuum stage of a mass spectrometer is sufficient. The inlet becomes the "ion source", and inter alia, pressure, temperature, and the matrix, which can be a solid, liquid, or combinations, become critical parameters. Injecting matrix:analyte into a heated inlet tube using laser ablation, a shockwave, or simply tapping, all produce the similar mass spectra. Applications are provided that showcase new opportunities in the field of mass spectrometry.

Matrix-assisted ionization mass spectrometry in targeted protein analysis - An initial evaluation.

RATIONALE: Matrix-assisted ionization (MAI) is a relatively new ionization technique for analysis by mass spectrometry (MS). The technique is simple and has been shown to be less influenced by matrix effects than e.g. electrospray ionization (ESI). These features are of interest in the targeted analysis of proteins from biological samples. METHODS: Targeted protein determination by MAI-MS was evaluated using a triple quadrupole mass analyzer equipped with a stripped nanoESI source in selected reaction monitoring (SRM) mode. The proteins were analyzed using the bottom-up approach with stable isotopic labeled peptides as internal standards (IS). The MAI matrix was 3-nitrobenzonitrile dissolved in acetonitrile. Aqueous sample and matrix solution were mixed in a 1:3 volume ratio. One microlitre of the dried matrix/analyte sample was introduced into the inlet of the mass spectrometer where ionization commences. RESULTS: SRM settings established for ESI-SRM-MS of the peptides here investigated were applicable in MAI-SRM-MS for all evaluated peptides except one that is poorly soluble in water. Addition of IS provided efficient correction at most levels (relative standard deviation (RSD) ≤28% (except lowest digest level), r2 ≥ 0.995). This was also true for the more complex biological matrices, diluted urine (1:1; RSD = 20% a synthetic peptide, NLLGLIEAK) and diluted digested serum (1:100; RSD = 7% digested cytochrome C). Biological matrix influenced the signal intensity unless sufficiently diluted. CONCLUSIONS: The results demonstrate that MAI-SRM-MS has promising potential in targeted protein determination by the bottom-up approach because of its simplicity, ease of use, and speed. However, more data is needed to confirm the results prior to application in a clinical setting.

Toward understanding the ionization mechanism of matrix-assisted ionization using mass spectrometry experiment and theory.

RATIONALE: Matrix-assisted ionization (MAI) mass spectrometry does not require voltages, a laser beam, or added heat to initiate ionization, but it is strongly dependent on the choice of matrix and the vacuum conditions. High charge state distributions of nonvolatile analyte ions produced by MAI suggest that the ionization mechanism may be similar to that of electrospray ionization (ESI), but different from matrix-assisted laser desorption/ionization (MALDI). While significant information is available for MAI using mass spectrometers operating at atmospheric and intermediate pressure, little is known about the mechanism at high vacuum. METHODS: Eleven MAI matrices were studied on a high-vacuum time-of-flight (TOF) mass spectrometer using a 266 nm pulsed laser beam under otherwise typical MALDI conditions. Detailed comparisons with the commonly used MALDI matrices and theoretical prediction were made for 3-nitrobenzonitrile (3-NBN), which is the only MAI matrix that works well in high vacuum when irradiated with a laser. RESULTS: Screening of MAI matrices with good absorption at 266 nm but with various degrees of volatility and laser energies suggests that volatility and absorption at the laser wavelength may be necessary, but not sufficient, criteria to explain the formation of multiply charged analyte ions. 3-NBN produces intact, highly charged ions of nonvolatile analytes in high-vacuum TOF with the use of a laser, demonstrating that ESI-like ions can be produced in high vacuum. Theoretical calculations and mass spectra suggest that thermally induced proton transfer, which is the major ionization mechanism in MALDI, is not important with the 3-NBN matrix at 266 nm laser wavelength. 3-NBN:analyte crystal morphology is, however, important in ion generation in high vacuum. CONCLUSIONS: The 3-NBN MAI matrix produces intact, highly charged ions of nonvolatile compounds in high-vacuum TOF mass spectrometers with the aid of ablation and/or heating by laser irradiation, and shows a different ionization mechanism from that of typical MALDI matrices.

Development of a robotics platform for automated multi-ionization mass spectrometry.

RATIONALE: Successful coupling of a multi-ionization automated platform with commercially available mass spectrometers provides improved coverage of compounds in complex mixtures through implementation of new and traditional ionization methods. The versatility of the automated platform is demonstrated through coupling with mass spectrometers from two different vendors. Standards and complex biological samples were acquired using electrospray ionization (ESI), solvent-assisted ionization (SAI) and matrix-assisted ionization (MAI). METHODS: The MS™ prototype automated platform samples from 96- or 384-well plates as well as surfaces. The platform interfaces with Thermo Fisher Scientific mass spectrometers by replacement of the IonMax source, and on Waters mass spectrometers with additional minor source inlet modifications. The sample is transferred to the ionization region using a fused-silica or metal capillary which is cleaned between acquisitions using solvents. For ESI and SAI, typically 1 µL of sample solution is drawn into the capillary tube and for ESI slowly dispensed near the inlet of the mass spectrometer with voltage placed on the delivering syringe barrel to which the tubing is attached, while for SAI the sample delivery tubing inserts into the inlet without the need for high voltage. For MAI, typically, 0.2 µL of matrix solution is drawn into the syringe before drawing 0.1 µL of the sample solution and dispensing to dry before insertion into the inlet. RESULTS: A comparison study of a mixture of angiotensin I, verapamil, crystal violet, and atrazine representative of peptides, drugs, dyes, and herbicides using SAI, MAI, and ESI shows large differences in ionization efficiency of the various components. Solutions of a mixture of erythromycin and azithromycin in wells of a 384-microtiter well plate were mass analyzed at the rate of ca 1 min per sample using MAI and ESI. In addition, we report the analysis of bacterial extracts using automated MAI and ESI methods. Finally, the ability to perform surface analysis with the automated platform is also demonstrated by directly analyzing dyes separated on a thin-layer chromatography (TLC) plate and compounds extracted from the surface of a beef liver tissue section. CONCLUSIONS: The prototype multi-ionization automated platform offers solid matrix introduction used with MAI, as well as solution introduction using either ESI or SAI. The combination of ionization methods extends the types of compounds which are efficiently ionized and is especially valuable with complex mixtures as demonstrated for bacterial extracts. While coupling of the automated multi-ionization platform to Thermo and Waters mass spectrometers is demonstrated, it should be possible to interface it with most commercial mass spectrometers.

Comparison of gaseous ubiquitin ion structures obtained from a solid and solution matrix using ion mobility spectrometry/mass spectrometry.

RATIONALE: Examining surface protein conformations, and especially achieving this with spatial resolution, is an important goal. The recently discovered ionization processes offer spatial-resolution measurements similar to matrix-assisted laser desorption/ionization (MALDI) and produce charge states similar to electrospray ionization (ESI) extending higher-mass protein applications directly from surfaces on high-performance mass spectrometers. Studying a well-interrogated protein by ion mobility spectrometry-mass spectrometry (IMS-MS) to access effects on structures using a solid vs. solvent matrix may provide insights. METHODS: Ubiquitin was studied by IMS-MS using new ionization processes with commercial and homebuilt ion sources and instruments (Waters SYNAPT G2(S)) and homebuilt 2 m drift-tube instrument; MS™ sources). Mass-to-charge and drift-time (td )-measurements are compared for ubiquitin ions obtained by inlet and vacuum ionization using laserspray ionization (LSI), matrix- (MAI) and solvent-assisted ionization (SAI), respectively, and compared with those from ESI under conditions that are most comparable. RESULTS: Using the same solution conditions with SYNAPT G2(S) instruments, td -distributions of various ubiquitin charge states from MAI, LSI, and SAI are similar to those from ESI using a variety of solvents, matrices, extraction voltages, a laser, and temperature only, showing subtle differences in more compact features within the elongated distribution of structures. However, on a homebuilt drift-tube instrument, within the elongated distribution of structures, both similar and different td -distributions are observed for ubiquitin ions obtained by MAI and ESI. MAI-generated ions are frequently narrower in their td -distributions. CONCLUSIONS: Direct comparisons between ESI and the new ionization methods operational directly from surfaces suggest that the protein in its solution structure prior to exposure to the ionization event is either captured (frozen out) at the time of crystallization, or that the protein in the solid matrix is associated with sufficient solvent to maintain the solution structure, or, alternatively, that the observed structures are those related to what occurs in the gas phase with ESI- or MAI-generated ions and not with the solution structures.

An overview of biological applications and fundamentals of new inlet and vacuum ionization technologies.

RATIONALE: The developments of new ionization technologies based on processes previously unknown to mass spectrometry (MS) have gained significant momentum. Herein we address the importance of understanding these unique ionization processes, demonstrate the new capabilities currently unmet by other methods, and outline their considerable analytical potential. METHODS: The inlet and vacuum ionization methods of solvent-assisted ionization (SAI), matrix-assisted ionization (MAI), and laserspray ionization can be used with commercial and dedicated ion sources producing ions from atmospheric or vacuum conditions for analyses of a variety of materials including drugs, lipids, and proteins introduced from well plates, pipet tips and plate surfaces with and without a laser using solid or solvent matrices. Mass spectrometers from various vendors are employed. RESULTS: Results are presented highlighting strengths relative to ionization methods of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization. We demonstrate the utility of multi-ionization platforms encompassing MAI, SAI, and ESI and enabling detection of what otherwise is missed, especially when directly analyzing mixtures. Unmatched robustness is achieved with dedicated vacuum MAI sources with mechanical introduction of the sample to the sub-atmospheric pressure (vacuum MAI). Simplicity and use of a wide array of matrices are attained using a conduit (inlet ionization), preferably heated, with sample introduction from atmospheric pressure. Tissue, whole blood, urine (including mouse, chicken, and human origin), bacteria strains and chemical on-probe reactions are analyzed directly and, especially in the case of vacuum ionization, without concern of carryover or instrument contamination. CONCLUSIONS: Examples are provided highlighting the exceptional analytical capabilities associated with the novel ionization processes in MS that reduce operational complexity while increasing speed and robustness, achieving mass spectra with low background for improved sensitivity, suggesting the potential of this simple ionization technology to drive MS into areas currently underserved, such as clinical and medical applications.

Expression and In Vivo Characterization of the Antimicrobial Peptide Oncocin and Variants Binding to Ribosomes.

Peptides have important biomedical applications, but poor correlation between in vitro and in vivo activities can limit their development for clinical use. The ability to generate peptides and monitor their expression with new mass spectrometric methods and biological activities in vivo would be an advantage for the discovery and improvement of peptide-based drugs. In this study, a plasmid-based system was used to express the ribosome-targeting peptide oncocin (19 amino acids, VDKPPYLPRPRPPRRIYNR) and to determine its direct antibacterial effects on Escherichia coli. Previous biochemical and structure studies showed that oncocin targets the bacterial ribosome. The oncocin peptide generated in vivo strongly inhibits bacterial growth. In vivo dimethyl sulfate footprinting of oncocin on the rRNA gives results that are consistent with those of in vitro studies but reveals additional binding interactions with E. coli ribosomes. Furthermore, expression of truncated or mutated peptides reveals which amino acids are important for antimicrobial activity. Overall, the in vivo peptide expression system can be used to investigate biological activities and interactions of peptides with their targets within the cellular environment and to separate contributions of the sequence to cellular transport. This strategy has future applications for improving the effectiveness of existing peptides and developing new peptide-based drugs.

New mass spectrometry concepts for characterization of synthetic polymers and additives.

RATIONALE: New ionization processes have been developed for biological mass spectrometry (MS) in which the matrix lifts the nonvolatile analyte into the gas phase as ions without any additional energy input. We rationalized that additional fundamental knowledge is needed to assess analytical utility for the field of synthetic polymers and additives. METHODS: Different mass spectrometers (Thermo Orbitrap (Q-)Exactive (Focus); Waters SYNAPT G2(S)) were employed. The formation of multiply charged polymer ions upon exposure of the matrix/analyte(/salt) sample to sub-atmospheric pressure directly from the solid state and surfaces facilitates the use of advanced mass spectrometers for detection of polymeric materials including consumer products (e.g., gum). RESULTS: Astonishingly, using nothing more than a small molecule matrix compound (e.g., 2-methyl-2-nitropropane-1,3-diol or 3-nitrobenzonitrile) and a salt (e.g., mono- or divalent cation(s)), such samples upon exposure to sub-atmospheric pressure transfer nonvolatile polymers and nonvolatile salts into the gas phase as multiply charged ions. These successes contradict the conventional understanding of ionization in MS, because can nonvolatile polymers be lifted in the gas phase as ions not only by as little as a volatile matrix but also by the salt required for ionizing the analyte through noncovalent metal cation adduction(s). Prototype vacuum matrix-assisted ionization (vMAI) and automated sources using a contactless approach are demonstrated for direct analyses of synthetic polymers and plasticizers, minimizing the risk of contamination using direct sample introduction into the mass spectrometer vacuum. CONCLUSIONS: Direct ionization methods from surfaces without the need of high voltage, a laser, or even applied heat are demonstrated for characterization of detailed materials using (ultra)high-resolution and accurate mass measurements enabled by the multiply charged ions extending the mass range of high-performance mass spectrometers and use of a split probe sample introduction device. Our vision is that, with further development of fundamentals and dedicated sources, both spatial- and temporal-resolution measurements are within reach if sensitivity is addressed for decreasing sample-size measurements.

1000-Fold Preconcentration of Per- and Polyfluorinated Alkyl Substances within 10 Minutes via Electrochemical Aerosol Formation.

We present a simple and efficient method for preconcentrating per- and polyfluorinated alkyl substances (PFAS) in water. Our method was inspired by the sea-spray aerosol enrichment in nature. Gas bubbles in the ocean serve to scavenge surface active material, carrying it to the air-ocean interface, where the bubbles burst and form a sea-spray aerosol. These aerosol particles are enriched in surface-active organic compounds such as free fatty acids and anionic surfactants. In our method, we in situ generate H2 microbubbles by electrochemical water reduction using a porous Ni foam electrode. These H2 bubbles pick up PFAS as they rise through the water column that contains low concentration PFAS. When these bubbles reach the water surface, they burst and produce aerosol droplets that are enriched in PFAS. Using this method, we demonstrated ∼1000-fold preconcentration for ten common PFAS in the concentration range from 1 pM to 1 nM (or ∼0.5 ng/L to 500 ng/L) in 10 min. We also developed a diffusion-limited adsorption model that is in quantitative agreement with the experimental data. In addition, we demonstrated using this method to preconcentrate PFAS in tap water, indicating its potential use for quantitative analysis of PFAS in real-world water samples.

Novel ionization processes for use in mass spectrometry: 'Squeezing' nonvolatile analyte ions from crystals and droplets.

Together with my group and collaborators, I have been fortunate to have had a key role in the discovery of new ionization processes that we developed into new flexible, sensitive, rapid, reliable, and robust ionization technologies and methods for use in mass spectrometry (MS). Our current research is focused on how best to understand, improve, and use these novel ionization processes which convert volatile and nonvolatile compounds from solids or liquids into gas-phase ions for analysis by MS using e.g. mass-selected fragmentation and ion mobility spectrometry to provide reproducible, accurate, and improved mass and drift time resolution. In my view, the apex was the discovery of vacuum matrix-assisted ionization (vMAI) in 2012 on an intermediate pressure matrix-assisted laser desorption/ionization (MALDI) source without the use of a laser, high voltages, or any other added energy. Only exposure of the matrix:analyte to the sub-atmospheric pressure of the mass spectrometer was necessary to initiate ionization. These findings were initially rejected by three different scientific journals, with comments related to 'how can this work?', 'where do the charges come from?', and 'it is not analytically useful'. Meanwhile, we and others have demonstrated analytical utility without a complete understanding of the mechanism. In reality, MALDI and electrospray ionization are widely used in science and their mechanisms are still controversially discussed despite use and optimization of now 30 years. This Perspective covers the applications and mechanistic aspects of the novel ionization processes for use in MS that guided us in instrument developments, and provides our perspective on how they relate to traditional ionization processes.

Vacuum Matrix-Assisted Ionization Source Offering Simplicity, Sensitivity, and Exceptional Robustness in Mass Spectrometry.

Vacuum matrix-assisted ionization (vMAI) uses select matrix compounds which when exposed to the vacuum of a mass spectrometer produce gas-phase ions from associated volatile or nonvolatile analyte without external energy input. Here, a vMAI source was constructed to replace the commercial inlet of a Thermo Orbitrap mass spectrometer. This allowed for rapid introduction of the matrix/analyte sample by a probe, contrary to vacuum matrix-assisted laser desorption/ionization (MALDI) sources. The matrix/analyte sample is inserted into a region of the "S-lens" entrance, where the spontaneously formed ions can be effectively transferred to the mass analyzer. This specifically designed ion source requires no laser, high voltage, heat, or nebulizing gases. A low voltage is used to transmit the ions through the commercial "S-lens" assembly and airflow can be used to modulate the ionization event. A few picograms of the drug erythromycin, assisted by the 3-nitrobenzonitrile vMAI matrix, is sufficient to produce mass spectra for over 1 min with the MH+ ion as the base peak in each mass spectrum. There is minimal carryover when loading high concentration samples and complex mixtures, contrary to direct infusion electrospray ionization, providing the probe is thoroughly cleaned between each new sample acquisition. Analyses of biological fluids, bacterial extracts, tissue, and high concentration samples have so far shown no indication of inlet or instrument contamination with these samples. The typical ultrahigh resolution and mass accuracy of the mass spectrometer are achieved, and a path forward to potential high throughput acquisitions demonstrated. It is expected that robustness can be introduced to any mass spectrometer through implementation of such a simple source.

Unprecedented Ionization Processes in Mass Spectrometry Provide Missing Link between ESI and MALDI.

In the field of mass spectrometry, producing intact, highly-charged protein ions from surfaces is a conundrum with significant potential payoff in application areas ranging from biomedical to clinical research. Here, we report on the ability to form intact, highly-charged protein ions on high vacuum time-of-flight mass spectrometers in the linear and reflectron modes achievable using experimental conditions that allow effective matrix removal from both the sample surfaces and from the charged clusters formed by the laser ablation event. The charge states are the highest reported on high vacuum mass spectrometers, yet they remain at only around a third of the highest charge obtained using laser ablation with a suitable matrix at atmospheric pressure. Other than physical instrument modifications, the key to forming abundant and stable highly-charged ions appears to be the volatility of the matrix used. Cumulative results suggest mechanistic links between the ionization process reported here and traditional ionization methods of electrospray ionization and matrix-assisted laser desorption/ionization.

An LC/MS Method Providing Improved Sensitivity: Electrospray Ionization Inlet.

Electrospray ionization inlet (ESII) combines positive aspects of electrospray ionization (ESI) and solvent-assisted ionization (SAI). Similar to SAI, the analyte solution is directly introduced into a heated inlet tube linking atmospheric pressure and the initial vacuum stage of the mass spectrometer. However, unlike SAI, in ESII a voltage is applied to the solution through a metal union linking two sections of fused silica tubing through which solution flows into the inlet. Here, we demonstrate liquid chromatography (LC) ESII/MS on two different mass spectrometers using a mixture of drugs, a peptide standard mixture, and protein digests. This LC-ESII/MS approach has little dead volume and thus provides excellent chromatographic resolution at mobile phase flow rates from 1 to 55 µL min-1. Significant improvement in ion abundance and less chemical background ions were observed relative to ESI for all drugs and peptides tested at flow rates from 15 to 55 µL min-1. At a low inlet tube temperature, ESII has an ionization selectivity similar to that of ESI but, at higher inlet temperatures, appears to have the attributes of both ESI and SAI.

Rapid high mass resolution mass spectrometry using matrix-assisted ionization.

Matrix-assisted ionization (MAI) is demonstrated to be a robust and sensitive analytical method capable of analyzing proteins such as cholera toxin B-subunit and pertussis toxin mutant from conditions containing relatively high amounts of inorganic salts, buffers, and preservatives without the need for prior sample clean-up or concentration. By circumventing some of the sample preparation steps, MAI simplifies and accelerates the analytical workflow for biological samples in complex media. The benefits of multiply charged ions characteristic of electrospray ionization (ESI) and the robustness of matrix-assisted laser desorption/ionization (MALDI) can be obtained from a single method, making it well suited for analysis of proteins and other biomolecules at ultra-high resolution as demonstrated on an Orbitrap Fusion where protein subunits were resolved for which MALDI-time-of-flight failed. MAI results are compared with those obtained with ESI, MALDI, and laserspray ionization methods and fundamental commonalities discussed.

A broad-based study on hyphenating new ionization technologies with MS/MS for PTMs and tissue characterization.

Matrix-assisted ionization (MAI) is a newly discovered method for converting compounds from the solid phase to gas-phase ions having charge states similar to electrospray ionization (ESI), but without the need for high-energy sources such as lasers or high voltage. Laserspray ionization (LSI) is a subset of MAI that uses a laser to provide high spatial resolution analyses, but the laser is not directly involved in the ionization process. These methods produce multiply-charged analyte ions that are useful for characterizing compounds directly from surfaces using advanced characterization technologies. Because the multiply-charged ions originate from charged matrix clusters, efficient desolvation of the matrix is a prerequisite. Here, we report on the utility of collision-induced dissociation (CID) and electron transfer dissociation (ETD) coupled to mass spectrometry using several MAI and LSI matrices for peptide and protein characterization employing mass spectrometers from two manufacturers. The information obtained is similar to that using ESI for most analyses and superior to matrix-assisted laser desorption/ionization (MALDI) as is shown for intact proteins and protein digests directly from mouse brain tissue sections. The ionization processes are soft so that posttranslational modification (e.g. phosphorylation) sites are readily determined. Instances where ETD or CID in conjunction with MAI failed are attributed to lack of desolvation of charged matrix:analyte particles.

Matrix-Assisted Ionization on a Portable Mass Spectrometer: Analysis Directly from Biological and Synthetic Materials.

Matrix-assisted ionization (MAI)-mass spectrometry (MS) eliminates the need for high voltage, a heat source, lasers, and compressed gases in the ionization process and uses minimal solvents in sample preparation, thus making MAI ideal for field-portable mass spectrometers. The broad applicability of MAI is demonstrated by simple, rapid, and robust positive and negative detection mode analyses of low and high mass compounds including some pesticides, dyes, drugs, lipids, and proteins (186 Da to 8.5 kDa) from various materials including urine, biological tissue sections, paper, and plant material on a low pumping capacity, single-quadrupole mass spectrometer. Different sample introduction methods are applicable, including the use of a pipet tip or glass melting point tube, allowing integration of sample preparation with sample introduction for increased analytical utility and ease of operation, even when sampling directly from surfaces.

New ionization processes and applications for use in mass spectrometry.

Mass spectrometry (MS) continues to improve at a rapid pace as most prominently witnessed for mass analyzers and fragmentation technology. Ionization methods have also seen resurgence with ambient ionization approaches gaining a foothold because they often provide a convenient and direct means of sample analysis. Nevertheless, a vast majority of biological analyses using MS apply electrospray ionization or matrix-assisted laser desorption/ionization, methods introduced in the 1980s, or variants thereof. To further advance applications by MS such as protein characterization, and, for example, addressing their location within specific cell types, the progress in mass analyzer and fragmentation technology needs to be matched with similar advances in ionization technology. It is imperative to seek ionization methods that more efficiently convert molecules, to gas-phase ions in a way that allows high transfer efficiency to the mass analyzer and subsequently the detector to achieve a more complete picture of sample composition. This review provides a snapshot of fundamental aspects of new ionization methods and potential biological and medical applications.

High-throughput characterization of small and large molecules using only a matrix and the vacuum of a mass spectrometer.

Matrix assisted ionization vacuum (MAIV) rapidly generates gas-phase analyte ions from subliming solid-phase matrix:analyte crystals for analysis by mass spectrometry (MS). Ionization from the solid-phase allows the use of a variety of surfaces for introducing matrix:analyte samples to the vacuum of a mass spectrometer, including common laboratory materials, such as disposable pipet tips, filter paper, tooth picks, and nylon mesh. MAIV is shown here to be capable of analyses as fast as 3 s per sample with achievable sensitivities in the low femtomole range. MAIV-MS coupled with ion mobility spectrometry (IMS)-MS and tandem mass spectrometry (MS/MS) is shown to be especially powerful for analysis and characterization of a wide range of molecules ranging from small molecules such as drugs and metabolites (∼300 Da) to intact proteins (25.6 kDa). Automated sample introduction is demonstrated on two different commercial mass spectrometers using a programmable XYZ stage. A MAIV high-throughput nontargeted MS(E) approach is also demonstrated utilizing IMS for rapid characterization of small molecules and peptides from standard solutions, as well as drug spiked human urine.

Reproducibility and quantification of illicit drugs using matrix-assisted ionization (MAI) mass spectrometry.

Matrix-assisted ionization (MAI) mass spectrometry (MS) is a simple and sensitive method for analysis of low- and high-mass compounds, requiring only that the analyte in a suitable matrix be exposed to the inlet aperture of an atmospheric pressure ionization mass spectrometer. Here, we evaluate the reproducibility of MAI and its potential for quantification using six drug standards. Factors influencing reproducibility include the matrix compound used, temperature, and the method of sample introduction. The relative standard deviation (RSD) using MAI for a mixture of morphine, codeine, oxymorphone, oxycodone, clozapine, and buspirone and their deuterated internal standards using the matrix 3-nitrobenzonitrile is less than 10% with either a Waters SYNAPT G2 or a Thermo LTQ Velos mass spectrometer. The RSD values obtained using MAI are comparable to those using ESI or MALDI on these instruments. The day-to-day reproducibility of MAI determined for five consecutive days with internal standards was better than 20% using manual sample introduction. The reproducibility improved to better than 5% using a mechanically assisted sample introduction method. Hydrocodone, present in a sample of undiluted infant urine, was quantified with MAI using the standard addition method.

Matrix assisted ionization vacuum (MAIV), a new ionization method for biological materials analysis using mass spectrometry.

The introduction of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) for the mass spectrometric analysis of peptides and proteins had a dramatic impact on biological science. We now report that a wide variety of compounds, including peptides, proteins, and protein complexes, are transported directly from a solid-state small molecule matrix to gas-phase ions when placed into the vacuum of a mass spectrometer without the use of high voltage, a laser, or added heat. This ionization process produces ions having charge states similar to ESI, making the method applicable for high performance mass spectrometers designed for atmospheric pressure ionization. We demonstrate highly sensitive ionization using intermediate pressure MALDI and modified ESI sources. This matrix and vacuum assisted soft ionization method is suitable for the direct surface analysis of biological materials, including tissue, via mass spectrometry.