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.

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.

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.

Direct sub-atmospheric pressure ionization mass spectrometry: Evaporation/sublimation-driven ionization is amazing, fundamentally, and practically.

This paper covers direct sub-atmospheric pressure ionization mass spectrometry (MS). The discovery, applications, and mechanistic aspects of novel ionization processes for use in MS that are not based on the high-energy input from voltage, laser, and/or high temperature but on sublimation/evaporation within a region linking a higher to lower pressure and modulated by heat and collisions, are discussed, including how this new reality has guided a series of discoveries, instrument developments, and commercialization. A research focus, inter alia, is on how best to understand, improve, and use these novel ionization processes, which convert volatile and nonvolatile compounds from solids (sublimation) or liquids (evaporation) into gas-phase ions for analysis by MS providing reproducible, accurate, sensitive, and prompt results. Our perception on how these unprecedented versus traditional ionization processes/methods relate to each other, how they can be made to coexist on the same mass spectrometer, and an outlook on new and expanded applications (e.g., clinical, portable, fast, safe, and autonomous) is presented, and is based on ST's Opening lecture presentation at the Nordic Mass spectrometry Conference, Geilo, Norway, January 2023. Focus will be on matrix-assisted ionization (MAI) and solvent-assisted ionization (SAI) MS covering the period from 2010 to 2023; a potential paradigm shift in the making.

Instrumentation development, improvement, simplification, and miniaturization: The multifunctional plate source for use in mass spectrometry.

In remembrance of Prof. Dr Przybylski, we are presenting a vision towards his beloved mass spectrometry (MS) and its far-reaching promises outside of the academic laboratory. Sub-atmospheric pressure (AP) ionization MS is well positioned to make a step-change in direct ionization, a concept that allows sublimation/evaporation ionization and mass analyses of volatile and nonvolatile molecules from clean or dirty samples, directly, accurately, sensitively, and in a straightforward manner that has the potential to expand the field of MS into unchartered application areas. Contrary to ambient ionization MS, ionization commences in the sub-AP region of the mass spectrometer, important for practical and safety reasons, and offers inter alia, simplicity, speed, sensitivity, and robustness directly from real-world samples without cleanup. The plate source concept, presented here, provides an easy to use, rapid, and direct sample introduction from AP into the sub-AP of a mass spectrometer. Utilizing sub-AP ionization MS based on the plate source concept, small to large molecules from various environments that would be deemed too dirty for some direct MS methods are demonstrated. The new source concept can be expanded to include multiple ionization methods using the same plate source "front end" without the need to vent the mass spectrometer between the different methods, thus allowing ionization of more compounds on the same mass spectrometer for which any one ionization method may be insufficient. Examples such as fentanyl, gamma-hydroxybutyric acid, clozapine, 1-propionyllysergic acid, hydrocodone angiotensin I and II, myoglobin, and carbonic anhydrase are included.

A Combination MAI and MALDI Vacuum Source Operational from Atmospheric Pressure for Fast, Robust, and Sensitive Analyses.

Previously, vacuum matrix-assisted ionization (vMAI) was employed with matrix/analyte sample introduction into the vacuum of a mass spectrometer on a probe sample introduction device. Low attomole detection was achieved, while no carryover was observed even for concentrated samples. Here, we report a new vacuum ionization source designed to duplicate the sensitivity and robustness of probe device while providing fast multisample introduction to vacuum and rapid sequential ionization. Exposure of a sample to the vacuum of the mass spectrometer provides spontaneous ionization of volatile as well as nonvolatile analytes without the need for external energy input. However, the novel source design described herein, in addition to vMAI, can employ a laser to obtain vacuum matrix-assisted laser desorption/ionization (vMALDI). In particular, ionization by vMAI or vMALDI is achieved by using the appropriate matrix. Switching between ionization modes is accomplished in a few seconds. We present results demonstrating the utility of the two ionization methods in combination to improve the molecular analyses of sample composition. In both ionization modes, multiple samples can be sequentially and rapidly acquired to increase throughput in MS. With the prototype source, samples were acquired in as little as 1 s per sample. Exchanging multisample plates can be accomplished in as little as 2 s, suggesting low-cost high-throughput automation when properly developed.

Sublimation Driven Ionization for Use in Mass Spectrometry: Mechanistic Implications.

Sublimation has been known at least since the middle ages. This process is frequently taught in schools through the use of phase diagrams. Astonishingly, such a well-known process appears to still harbor secrets. Under conditions in which compound sublimation occurs, gas-phase ions are frequently detected using mass spectrometry. This was exploited in matrix-assisted ionization in vacuum (vMAI) by adding analyte to subliming compounds used as matrices. Good vMAI matrices were those that ionize the added analyte with high sensitivity, but even matrices that fail this test often produce ions of likely matrix impurities suggesting that they may be good matrices for some compound types. We also show that binary matrices may be manipulated to provide desired properties such as fast analyses and improved sensitivity. These results imply that sublimation in some cases is more complicated than just molecules leaving a surface and that understanding the physical force responsible, and how the nonvolatile compound becomes charged, could lead to improved ionization efficiency for mass spectrometry. Here we provide insights into this process and an explanation of why this unexpected phenomenon has not previously been reported.

Spontaneous Charge Separation and Sublimation Processes are Ubiquitous in Nature and in Ionization Processes in Mass Spectrometry.

Ionization processes have been discovered by which small and large as well as volatile and nonvolatile compounds are converted to gas-phase ions when associated with a matrix and exposed to sub-atmospheric pressure. Here, we discuss experiments further defining these simple and unexpected processes. Charge separation is found to be a common process for small molecule chemicals, solids and liquids, passed through an inlet tube from a higher to a lower pressure region, with and without heat applied. This charge separation process produces positively- and negatively-charged particles with widely different efficiencies depending on the compound and its physical state. Circumstantial evidence is presented suggesting that in the new ionization process, charged particles carry analyte into the gas phase, and desolvation of these particles produce the bare ions similar to electrospray ionization, except that solid particles appear likely to be involved. This mechanistic proposition is in agreement with previous theoretical work related to ion emission from ice. Graphical Abstract ᅟ.

High Sensitivity Analysis of Nanoliter Volumes of Volatile and Nonvolatile Compounds using Matrix Assisted Ionization (MAI) Mass Spectrometry.

First results are reported using a simple, fast, and reproducible matrix-assisted ionization (MAI) sample introduction method that provides substantial improvements relative to previously published MAI methods. The sensitivity of the new MAI methods, which requires no laser, high voltage, or nebulizing gas, is comparable to those reported for MALDI-TOF and n-ESI. High resolution full acquisition mass spectra having low chemical background are acquired from low nanoliters of solution using only a few femtomoles of analyte. The limit-of-detection for angiotensin II is less than 50 amol on an Orbitrap Exactive mass spectrometer. Analysis of peptides, including a bovine serum albumin digest, and drugs, including drugs in urine without a purification step, are reported using a 1 µL zero dead volume syringe in which only the analyte solution wetting the walls of the syringe needle is used in the analysis.

Analysis of SAW properties in ZnO/AlxGa1-xN/c-Al2O3 structures.

Piezoelectric thin films on high acoustic velocity nonpiezoelectric substrates, such as ZnO, AlN, or GaN deposited on diamond or sapphire substrates, are attractive for high frequency and low-loss surface acoustic wave devices. In this work, ZnO films are deposited on AlxGa1-xN/c-Al2O3 (0 < or = chi < or = 1) substrates using the radio frequency (RF) sputtering technique. In comparison with a single AlxGa1-xN layer deposited on c-Al2O3 with the same total film thickness, a ZnO/AlxGa1-xN/c-Al2O3 multilayer structure provides several advantages, including higher order wave modes with higher velocity and larger electromechanical coupling coefficient (K2). The surface acoustic wave (SAW) velocities and coupling coefficients of the ZnO/AlxGa1-xN/c-Al2O3 structure are tailored as a function of the Al mole percentage in AlxGa1-xN films, and as a function of the ZnO (h1) to AlxGa1-xN (h2) thickness ratio. It is found that a wide thickness-frequency product (hf) region in which coupling is close to its maximum value, K(2)max, can be obtained. The K(2)max of the second order wave mode (h1 = h2) is estimated to be 4.3% for ZnO/GaN/c-Al2O3, and 3.8% for ZnO/AlN/c-Al2O3. The bandwidth of second and third order wave modes, in which the coupling coefficient is within +/- 0.3% of K(2)max, is calculated to be 820 hf for ZnO/GaN/c-Al2O3, and 3620 hf for ZnO/AlN/c-Al2O3. Thus, the hf region in which the coupling coefficient is close to the maximum value broadens with increasing Al content, while K(2)max decreases slightly. When the thickness ratio of AlN to ZnO increases, the K(2)max and hf bandwidth of the second and third higher wave modes increases. The SAW test devices are fabricated and tested. The theoretical and experimental results of velocity dispersion in the ZnO/AlxGa1-xN/c-Al2O3 structures are found to be well matched.