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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.

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 ...

Introducing water or methanol containing a low concentration of volatile or nonvolatile analyte into an inlet tube cooled with dry ice linking atmospheric pressure and the first vacuum stage of a mass spectrometer produces gas-phase ions even of small proteins that can be detected by mass spectrometry. Collision-induced dissociation experiments conducted in the first vacuum region of the mass spectrometer suggest analyte ions being protected by a solvent cage. The charges may be produced by processes similar to those proposed for charge separation under freezing conditions in thunderclouds. By this process, the surface of an ice pellet is charged positive and the interior negative so that removal of surface results in charge separation. A reversal of surface charge is expected for a heated droplet surface, and this is observed by heating rather than cooling the inlet tube. These observations are consistent with charged supercooled droplets or ice particles as intermediates in the production of analyte ions under freezing conditions.

Matrix assisted ionization of nonvolatile compounds is shown not to be limited to vacuum conditions and does not require a laser. Simply placing a solution of analyte dissolved with a suitable matrix such as 3-nitrobenzonitrile (3-NBN) or 2,5-dihydroxyacetophenone on a melting point tube and gently heating the dried sample near the ion entrance aperture of a mass spectrometer using a flow of gas produces abundant ions of peptides, small proteins, drugs, and polar lipids. Fundamental studies point to matrix-mediated ionization occurring prior to the entrance aperture of the mass spectrometer. The method is analytically useful, producing peptide mass fingerprints of bovine serum albumin tryptic digest consuming sub-picomoles of sample. Application of 100 fmol of angiotensin I in 3-NBN matrix produces the doubly and triply protonated molecular ions as the most abundant peaks in the mass spectrum. No carryover is observed for samples containing up to 100 pmol of this peptide. A commercial atmospheric samples analysis probe provides a simple method for sample introduction to an atmospheric pressure ion source for analysis of volatile and nonvolatile compounds without using the corona discharge but using sample preparation similar to matrix-assisted laser desorption/ionization.

Solvent Assisted Ionization Inlet (SAII) produces ions in a heated inlet to a mass spectrometer from aqueous and aqueous/organic solutions with high sensitivity. However, the use of acid modifiers, which typically aids electrospray ionization, generally results in ion suppression in SAII. Here we demonstrate that the use of carbonation and other super-saturated gases as solution modifiers increases analyte ion abundance and reduces metal cation adduction in SAII. Carbonation is also found to enhance electrospray ionization. The mechanistic and practical utility of carbonation in mass spectrometry is addressed.

Combining electrospray ionization (ESI) and solvent assisted inlet ionization (SAII) provides higher ion abundances over a wide range of concentrations for peptides and proteins than either ESI or SAII. In this method, a voltage is applied to a union connector linking tubing from a solvent delivery device and the fused silica capillary, used with SAII, inserted into a heated inlet tube of an Orbitrap Exactive mass spectrometer (MS). The union can be metal or polymeric and the voltage can be applied directly or contactless. Solution flow rates from less than a 1 μL min(-1) to over 100 μL min(-1) can be accommodated. It appears that the voltage is only necessary to provide charge separation in solution, and the hot MS inlet tube and the high velocity of gas through the tube linking atmospheric pressure and vacuum provides droplet formation. As little as 100 V produces an increase in ion abundance for certain compounds using this method relative to no voltage. Interestingly, the total ion current observed with SAII and this electrosprayed inlet ionization (ESII) method are very similar for weak acid solutions, but with voltage on, the ion abundance for peptides and proteins increase as much as 100-fold relative to other compounds in the solution being analyzed. Thus, switching between SAII (voltage off) and ESII (voltage on) provides a more complete picture of the solution contents than either method alone.

Mechanistic arguments relative to matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) address observations that predominately singly charged ions are detected. However, recently a matrix assisted laser ablation method, laserspray ionization (LSI), was introduced that can use the same sample preparation and laser as MALDI, but produce highly charged ions from proteins. In MALDI, ions are generated from neutral molecules by the photon energy provided to a matrix, while in LSI ions are produced inside a heated inlet tube linking atmospheric pressure and the first vacuum region of the mass spectrometer. Some LSI matrices also produce highly charged ions with MALDI ion sources operated at intermediate pressure or high vacuum. The operational similarity of LSI to MALDI, and the large difference in charge states observed by these methods, provides information of fundamental importance to proposed ionization mechanisms for LSI and MALDI. Here, we present data suggesting that the prompt and delayed ionization reported for vacuum MALDI are both fast processes relative to producing highly charged ions by LSI. The energy supplied to produce these charged clusters/droplets as well as their size and time available for desolvation are determining factors in the charge states of the ions observed. Further, charged droplets/clusters may be a common link for ionization of nonvolatile compounds by a variety of MS ionization methods, including MALDI and LSI.

Exposures to organophosphorus nerve agents (OPNA) remain a threat to both civilian and military populations. Verification of exposures typically involves determinations of urinary metabolites or adducted proteins in blood. Urinary alkyl methylphosphonic acid metabolites resulting from hydrolysis of OPNAs provide a convenient marker for OPNA exposure. In a military setting, urine is a relatively easy sample to obtain, and a rapid turnaround for analyses for the identification of metabolites is critical for field commanders. Timely information on use and identity of OPNAs facilitates decisions regarding employment of personal protective equipment and additional strategies to mitigate additional exposure(s). Herein, we report the development of a rapid mass spectrometric (MS) method to identify OPNA metabolites directly from urine with no sample preparation. Synthetic urine spiked with multiple OPNA metabolites was analyzed using an atmospheric solids analysis probe (ASAP) attached to a high resolution mass spectrometer. The alkyl methylphosphonic acid metabolites resulting from hydrolysis of sarin, cyclosarin, soman, and Russian VX were clearly detectable down to a level of 1.0 ng/ml. The ability to rapidly detect OPNA metabolites in unprepared urine allows for the design of a field-deployable device that could afford field personnel the ability to rapidly screen individuals for specific OPNA exposure. In addition, this provides proof-of-concept evidence that a fieldable ASAP-MS device could afford personnel the ability to rapidly detect OPNAs on skin, equipment, and other porous surfaces. Published 2012. This article is a US Government work and is in the public domain in the USA.

RATIONALE Steroids can be injected to behave as therapeutic agents to promote muscle growth and strength. Areas of concern include synthetic steroids in consumer meat and milk products and the presence of anabolic steroids in athletes. Here we demonstrate a new ionization method for high sensitivity steroid analysis using liquid chromatography/mass spectrometry (LC/MS). METHODS Solvent-assisted inlet ionization (SAII) mass spectrometry was coupled directly to an infusion pump or to a liquid chromatograph to determine the limits of detection and quantitation for selected steroids. LC/MS/MS data was acquired on a quadrupole time-of-flight (QTOF) mass spectrometer and high resolution-accurate mass LC/MS data was obtained on an Orbitrap mass spectrometer. RESULTS The SAII limit of detection for infusion into the Orbitrap using high mass resolution and accurate mass was shown, for the steroids studied, to be low ppqt and the limit of quantitation using LC/MS was low ppt. Low ppb levels were detected with high signal-to-noise from spiked urine using a simple Ziptip procedure without sample concentration. CONCLUSIONS LC/SAII-MS is more sensitive than electrospray ionization (ESI) at similar mobile phase flow rates for the analysis of steroids. Previous studies have shown LC/SAII-MS to have high sensitivity for analysis of peptides. The combined results suggests this easy to implement ionization method may advantageously replace ESI for a wide range of analyses. Copyright © 2012 John Wiley & Sons, Ltd.

A new inlet ionization method requiring no voltage or laser, and using water, methanol, or water/organic solvent mixtures, is shown to produce mass spectra similar to those obtained with electrospray ionization (ESI) for small molecules, peptides, and proteins, at least as large as carbonic anhydrase, with sensitivity that surpasses ESI. With the use of wide mass range acquisitions at 100,000 mass resolution on an Orbitrap Exactive, detection limits below parts per trillion are obtained for small molecules such as arginine, ciprofloxacin, and acetaminophen. Low attomoles of bovine insulin consumed produced a multiply charged mass spectrum. Ions are generated, even using pure water as solvent, within the heated inlet tube linking atmospheric pressure with the first vacuum stage of the Orbitrap Exactive. The extremely high sensitivity observed at this early stage of solvent assisted inlet ionization (SAII) development suggests that inlet ionization may surpass nanoelectrospray in sensitivity but without the need for extremely low solvent flows.

Laserspray ionization (LSI) is a new approach to producing multiply charged ions from solids on surfaces by laser ablation of matrixes commonly used in matrix-assisted laser desorption/ionization (MALDI). We show that the only necessity of the laser for producing multiply charged ions is to deliver particles or droplets of the matrix/analyte mixture to an ionization zone which is simply a heated inlet to the vacuum of the mass spectrometer. Several other methods for delivering sample are demonstrated to produce nearly equivalent results. One example shows the use of an air gun replacing the laser and producing mass spectra of proteins by shooting pellets into a metal plate which has matrix/analyte applied to the opposite side and near the ion entrance inlet to the mass spectrometer. Multiply charged ions of proteins are produced in the absence of any electric field or laser and with only the need of a heated ion entrance capillary or skimmer. The commonality of the matrix with MALDI and the mild conditions necessary for formation of ions brings into question the mechanism of formation of multiply charged ions and the importance of matrix structure in this process.

Inlet ionization is a new approach for ionizing both small and large molecules in solids or liquid solvents with high sensitivity. The utility of solvent based inlet ionization mass spectrometry (MS) as a method for analysis of volatile and nonvolatile compounds eluting from a liquid chromatography (LC) column is demonstrated. This new LC/MS approach uses reverse phase solvent systems common to electrospray ionization MS. The first LC/MS analyses using this novel approach produced sharp chromatographic peaks and good quality full mass range mass spectra for over 25 peptides from injection of only 1 pmol of a tryptic digest of bovine serum albumin using an eluent flow rate of 55 μL min(-1). Similarly, full acquisition LC/MS/MS of the MH(+) ion of the drug clozapine, using the same solvent flow rate, produced a signal-to-noise ratio of 54 for the major fragment ion with injection of only 1 μL of a 2 ppb solution. LC/MS results were acquired on two different manufacturer's mass spectrometers using a Waters Corporation NanoAcquity liquid chromatograph.

Exposures to organophosphorus nerve agents (OPNA) remain a threat to both civilian and military populations. Verification of exposures typically involves determinations of urinary metabolites or adducted proteins in blood. Urinary alkyl methylphosphonic acid metabolites resulting from hydrolysis of OPNAs provide a convenient marker for OPNA exposure. In a military setting, urine is a relatively easy sample to obtain, and a rapid turnaround for analyses for the identification of metabolites is critical for field commanders. Timely information on use and identity of OPNAs facilitates decisions regarding employment of personal protective equipment and additional strategies to mitigate additional exposure(s). Herein, we report the development of a rapid mass spectrometric (MS) method to identify OPNA metabolites directly from urine with no sample preparation. Synthetic urine spiked with multiple OPNA metabolites was analyzed using an atmospheric solids analysis probe (ASAP) attached to a high resolution mass spectrometer. The alkyl methylphosphonic acid metabolites resulting from hydrolysis of sarin, cyclosarin, soman, and Russian VX were clearly detectable down to a level of 1.0 ng/ml. The ability to rapidly detect OPNA metabolites in unprepared urine allows for the design of a field-deployable device that could afford field personnel the ability to rapidly screen individuals for specific OPNA exposure. In addition, this provides proof-of-concept evidence that a fieldable ASAP-MS device could afford personnel the ability to rapidly detect OPNAs on skin, equipment, and other porous surfaces. Published 2012. This article is a US Government work and is in the public domain in the USA.