Patent Application
20250069878
Words/expressions/meanings used in the disclosure of this invention which may in some circumstances be used interchangeably include: (1) Port: Passageway, inlet, dock, hole, entrance, entry, opening, access, doorway, ingress, admission (site), conduit, channel, shaft, tube, trough, sluice, as available options to describe the area between the interior (sub-atmospheric pressure region) and the exterior region of the vacuum chamber assembly, and have in common that they will vent the analyzer device if not configured for limiting the gas flow by use of valve, restriction such as inlet tube, skimmer, pinhole, valve, valve plate, or other options known to those practiced in the art. Additional vacuum pumps may be added but this is not necessary but may broaden even further ionization source/sampling devices/methods combinations. Multiple Ports referred to as a plurality or multiport be used interchangeably. These combinations are flexible but may be permanent fixtures. Also, an analyzer device can be configured around the vacuum chamber assembly and not just retrofitted to existing analyzer devices. (2) Ions: Gaseous analyte molecules with n charge, either positive of negative. (3) Charged particles: Sometimes referred to charged clusters or charged droplets or non-ions or charged agglomerations. The charged particle may be comprised of solvent or solid compound, referred to as a matrix and analyte. Loss of molecules comprising the charged particle may be referred to “other molecules” or a matrix and may result in bare singly or multiply charged ions of the gas phase analyte molecules. For the simplicity of this text, in the following a particle is typically referred to as comprising a matrix although it is generally understood that it may be any molecule surrounding the analyte. Under proper conditions charged particles are converted to gaseous ions. (4) Transfer: The ion transfer is increased when charged particles and possibly neutral particles are broken down to the gaseous ions. This improves the ion transmission and the sensitivity of an experiment. (5) Vacuum chamber assembly: We define conduit to mean the interior channel or passageway within the vacuum chamber assembly that, in use, is at sub-atmospheric pressure. Otherwise, we use terms such as “channel”, passageway, tube, inlet, hole to have the meaning of a conduit but to distinguish these conduits from the principle conduit of the disclosed invention. All of which have in common gas flow and being in fluid communication with the analyzer device and that the sum of gas flows cannot exceed the vacuum/pumping capacity value associated/established for the analyzer device for a single use Port of, e.g., solely an API source.
Mass spectrometry (MS) is an analytical measurement technology providing a wealth of information with accuracy and specificity, doing so even from highly complex samples, often not readily obtainable with any other analytical technology. This technology and developed methods have the inherent advantage of requiring a minute amount of sample which can be studied directly or after clean-up. Biological, inorganic, and synthetic materials are examples of samples from which the analyte compositions and amounts may be identified using MS. Related technologies and methods are ion mobility spectrometry (IMS) and charge detector mass spectrometry (CDMS) and certain spectroscopy instruments. Liquid and gas separation methods are frequently used hyphenated with a mass spectrometer. Mass spectrometers and IMS are often hyphenated to provide nearly instantaneous separations by ion mobility and mass measurement of gas phase ions by MS. Both methods have in common measurement of gaseous ions, thereby providing information on the chemical/molecular compositions or structural information. Using the most suitable sampling, ionization source, and method and analyzer devices, gaseous ions of small, and large molecules (typically referred to as “analyte”) are created and separated/detected (analyzed) from samples of interest and may constitute one or more or thousands of “components”, also referred to as molecules, compounds, or analytes making up the ‘chemical’ and ‘molecular’ composition of a material, substance, fabric, biopsy, film, tissue, smear, bacteria, fungi, virus, biological matrix (saliva, blood, urine, feces, tears, sputum, etc.), chemical or biological reactions.
Data obtained may be indicative of certain disease(s) and/or disease state(s), or an environmental or security threat, warfare agents (chemical, biological, nuclear, etc.). The results from various ionization sources and methods complement each other, or in certain cases are exclusively applicable. However, the ‘right’ choice may require an entirely different mass spectrometer system in order to have available the ‘right’, or necessary condition, therefore increasing the need of user expertise and money, and to some degree trial and error and therefore time and loss in productivity or prevention of immediate knowledge of a health, environmental, or security threat.
A key to MS analyses is converting compounds, salts, or elements into gaseous ions. Direct analysis, without use of expensive and time-consuming hyphenated separation methods is gaining popularity in MS analyses; however, biological fluids and other ‘dirty’ or ‘salty’ samples are problematic because of resulting suppression of analyte ionization, or carryover, and ionization source (sometimes referred to “ion source”) or instrument contamination. In electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), matrices (sometimes referred to as “matrixes”), solvents (ESI) or solids (MALDI), additives, and matrix combinations are frequently used. Certain forms of sample preparation may cause sample degradation prior to analyses and some ionization sources and methods are known to undesirably fragment the molecule, sometimes already in solution (e.g., acid labile functionalities) or its related molecular ion during the ionization process, and historically referred to as “harsh” or “hard” ionization. This is as especially prevalent with electron ionization (EI) of small molecule analyses. Atmospheric solids analysis probe (ASAP) and direct analysis in real time (DART) provide direct access from atmospheric pressure (AP) through one of the well-established atmospheric pressure ionization (API) source configurations for use in MS, with minimal sample preparation, to volatile and semi-volatile compounds from liquids and solids offering ionization ‘soft’ enough to prevent excessive fragmentation and straightforward means of mass analyses and data interpretation. Both methods have the intrinsic advantage of being capable of potentially analyzing gases but are not suitable for nonvolatile compounds. Other direct ionization methods such as vacuum matrix assisted ionization (vMAI) and desorption-ESI (DESI), as examples, are effective in ionizing large nonvolatile compounds. MS technologies are, in comparison to other technologies, high performing but expensive in their initial cost (e.g., laser, high-voltage supply, operator), heavy and bulky, use of consumables (e.g., high pressure gases, specialized hardware), and upkeep costs including instrument maintenance and operator expertise (e.g., training, safety).
An inherent necessity in MS is the separation achieved under vacuum in and by the mass analyzer or ion mobility analyzer (in the following also referred to in the general form of ‘analyzer device’) with detection of gaseous ions by various detector devices. The gaseous ions are formed in an ionization region, often referred to as an ionization or ion source. Creating gaseous ions and effectively transferring them to and through the analyzer to the detector are functions of the ionization region as well as the analyzer device. High ionization efficiency needs to be accompanied with high ion transmission to the detector). In MS, ions are separated according to their mass-to-charge (m/z) values which occurs in sub-atmospheric pressure (sub-AP), referred to a vacuum, conditions. With some ionization sources/methods, charged droplets or solid matrix particles are initially formed by the ionization process and must shed ‘desolvate’ the matrix molecules to release the bare ion for analysis. The bare ions may be in various charge states, either negatively or positively charged. The polarity of extraction lens and ion guides within the mass analyses determines if positive or negatively charged ions are detected.
Within the first vacuum stage region, various ion optics devices have been established such as the ion funnel, S-lens, stacked-ring ion guide, Einzel lens, tube lens, as well as ion guides such as quadrupole, hexapole, and octapole, some of which are also used as collision or reaction cells as known by the practitioner of the art. Also, devices to keep the instrument from getting too dirty to quickly such as jet disrupters, obstructions, off axis alignments are included as known by the practitioner of the art. This is also accomplished through the various source geometries (IonMax inlet tube, Z-spray skimmer cone, as examples). Extraction lenses or repulsive electric fields might be included to accelerate charged particles by exposure to voltages in a desired direction.
Prominent examples of ionization sources/methods are ESI, MALDI, matrix-assisted ionization (MAI), solvent-assisted ionization (SAI), DESI, atmospheric solids analysis probe (ASAP), direct analysis in real time (DART), electron ionization (EI), and numerous others. Ionization methods can be categorized into those where ionization occurs at or near AP (e.g., ESI, AP-MALDI, DESI, DART, ASAP); those where the sampling process is initiated at AP, but ionization occurs after the material enters sub-AP conditions as it is being transferred through the inlet and referred to as inlet ionization (e.g., MAI, SAI, laserspray ionization (LSI), and laser ablation (LA) SAI); or by vacuum ionization where the material being analyzed is inserted on a substrate (e.g., a probe or plate) from AP directly into vacuum (sub-AP) where ionization occurs (e.g., vacuum (v) MALDI, vMAI, vLSI, field desorption (FD), solid probe introduction chemical ionization (CI) and EI). Note, vMALDI is not a typical term in literature but used here for consistency of this text. Also, e.g., vMAI and MAI, may use the same matrix but the introduction to the system where ionization commences is different and does not mean a different ionization process.
Typically, mass spectrometers are dedicated to either API, or vacuum ionization, and within vacuum ionization, instruments are dedicated to either EI/CI for low-mass and volatile compounds, or vMALDI, a direct ionization method used primarily for high-mass and nonvolatile compounds, and less frequently, methods such as FD. Inlet ionization uses an inlet tube, frequently heated, in which ions are created within the pressure differential traversing from higher pressure to lower pressure and no matrix is required although matrix may be used for specific applications, as detailed below. Inlet ionization may be performed in some limited instances and on certain API inlets configured for API methods of, e.g., ESI, DESI, ASAP, and DART. In some cases, API mass spectrometers need to be retrofitted with an inlet tube for inlet ionization in order to achieve good ion abundance. A few mass spectrometer manufacturers have made it possible to physically switch between API sources/methods and MALDI (called intermediate pressure MALDI, herein vMALDI, to distinguish it from high vacuum MALDI time-of-flight (ToF) mass spectrometers) with certain upgraded mass spectrometers. Having this option is expensive and switching between methods time consuming as it requires venting and pumping down the instrument with each switchover. Bruker sells mass spectrometers which has API and vacuum (v) MALDI available without physical switching over or venting the instrument, but it is only available for specific Bruker mass spectrometers which are in the upper price range. The installation is permanent, limiting flexibility and maximum utility. The plurality of ionization sources/methods and approaches (sometimes referred to techniques) in MS, and IMS, and related instruments is because no ionization source, method, or sampling device works for all samples. This is made worse because only a limited number of ionization sources/methods, or more precisely categories (API, inlet ionization, and vacuum ionization) are operational on the same mass spectrometer. This limits analyses capabilities and the breadth and depth of any one analyses, ensures inflexibility, and unnecessary expenses. While there are a number of positive attributes relative to API and inlet ionization sources and methods for use with a mass spectrometer, the need for high pumping capacity is a disadvantage and addressed through ever better pumping systems or through vacuum ionization methods.
Efficient ion transfer or transmission is frequently achieved using ion optics, where electric fields are used to guide and focus gaseous ions and charged particles, but frequently in a mass dependent manner. Gas flow may also be used to carry ions, charged particles, and neutral molecules and neutral particles. The efficacy of gas flow in the transmission of ions and particles is related to the structure of the passageway, and in particular the inner diameter. Frequently, both methods are combined. MSTM's invention of the vacuum chamber assembly democratizes the issue, as detailed below.
Commercial mass spectrometers are often designed for ESI, and employ desolvation methods such as a heated inlet to release the bare ions from charged droplets. but also use means to prevent any particles from entering the mass analyzer rather than employ methods to desolvate these charged particles to gain analyte ion abundance. Improved desolvation of charged particles is a means for increasing sensitivity, while removing neutral particles keeps the ion optic elements cleaner for longer. Charged particles and droplets are crucial with the more recent inventions of inlet ionization and vacuum ionization, in addition to ESI. While some commercial sources can be used with these newer sources/methods, they may not perform optimally, so there is room for improvements and need for ion and (charged) particle transfer to maximize and optimize ion transmission into the analyzer device.
The present invention provides a simple, low-cost, compact, and relatively light weight vacuum chamber assembly device which can be configured with Ports providing a multifunctional assembly for, in principle, interfacing almost immediate access to and use of API, inlet ionization, and vacuum ionization sources and methods to any API mass spectrometer and ion mobility and related instruments. Additional functionalities may be incorporated to enhance ion/charged particle transfer, and therewith ionization or more precisely improve ionization efficiency through improvements in transfer/transmission, desolvation, fragmentation, if desired, and induce reactions with gases, and more. A Port may be used to seal the vacuum within the analyzer device, such as a mass spectrometer, from the pressure within the vacuum chamber conduit allowing Ports on the high-pressure side of the valve assembly to be open to AP without venting the analyzer device. A probe can be configured for this purpose allowing simple opening and closing of the passageway leading from the vacuum assembly into the analyzer device.
Common to API inlets, a variety of API sources/methods may be interfaced to a single Port or have a separate Port for a preferred ionization source, sampling device, and method. The present invention allows either option, and also allows nearly instant access to the vacuum ionization methods of vMALDI and vMAI with exceptional sensitivity and robustness to contamination using additional Ports on the same vacuum chamber assembly device, ensuring that the mass analyzer need not be vented circumventing complexity and pump down time. The invention constitutes an ion/(charged) particle transfer region in fluid communication with an analyzer device, and having multiple Ports configured to interface with either API, inlet ionization, or vacuum ionization sources/sampling devices allowing multiple methods to be used in rapid succession. The vacuum chamber assembly may be configured to receive the commercial ion sources available with the instrument being modified by inserting between the ion source and the analyzer device as an add-on giving additional functionality, and vacuum ionization capabilities. The Ports are in fluid communication with the vacuum chamber assembly conduit region and ready to receive and transmit at least one ion, as well as at least one particle or charged particle to the vacuum region where desolvation converts the charged particles to bare ions, which along with any remaining charged or neutral particles, are transmitted into the ion optics of the analyzer device. A Port may be used to aid desolvation of particles and charged particles by placing an obstruction in passageway leading to the mass analyzer from the conduit. The obstruction designed to transmit bare ions and very low mass charged particles while mostly deflecting neutral molecules, neutral particles, and remaining large particles out of the ion path leading to the detector. Ports are available for gaseous ion/(charged) particle transfer/transmission enhancements and may be operated to achieve the desired ionization for specific or more complete sample analysis obtaining chemical, nuclear, and/or biological molecular information. Thus, the disclosed invention allows the user to select the best ionization sources/sampling devices/methods (sometimes also referred to as “approaches”) rapidly and conveniently for the sample (material) at hand.
More detailed information relative to the background of the invention is provided in the following with emphasis on ‘sources'/‘sampling devices'/‘methods’, although ionization sources, sampling devices, and methods are not part of this invention, but are important for describing the significance of the multitude of Ports vacuum chamber assembly able to receive a multitude of ionization sources and therefore sampling devices and methods that, more particularly, comprise sample preparation, sample transfer, ionization, and transfer of ions and charged and neutral particles. The importance of a simple and nearly universal approach using a vacuum assembly with multiple Ports capable of docking API, inlet ionization, and/or vacuum ionization sources simultaneously or in short succession from each other will be made apparent.
Technically, the vacuum assembly uses a combination of a low gas flow and electrical elements (ion optics) to aid transfer of ions and charged particles into the analyzer device. The vacuum assembly is therefore ideally suited in implementing or using, essentially seamlessly, a specific function and/or enhancing specific functions as needed or desired. Other Ports (or docking systems) referred to as auxiliary device(s) may be interfaced to enhance or cause certain effects within the vacuum chamber assembly or at certain other Port(s). Heat and collisions may be used to enhance the ionization process such as in, e.g., ESI or SAL. This can be accomplished with an API inlet (Chait patent) or an inlet ionization inlet tube (McEwen patents), as examples, or additionally within the vacuum assembly conduit through one of the additional Ports (e.g., heated gas flow). Shutters or valves within the assembly or inserted through one or more auxiliary Port may be used to block gas flow in order to remove parts of the assembly for replacement or cleaning without having to vent the instrument as described above. Similarly, fluid communication restrictions (e.g., small, <1 mm inner diameter (ID)) inlet tubes may be used to exchange a Ports' ionization source/sampling device, or a valve assembly (e.g., ball valve) may be used to maintain the instrument vacuum when inserting a probe from AP to the vacuum within the vacuum chamber assembly. Relative positions of the individual Ports within the assembly and relative to each other also play a role (e.g., increase gas flow when and where needed or enhance use of a laser aligned in reflection geometry for producing and transferring ions and charged particles).
Sampling devices can be categorized broadly and in a variety of ways but have in common that they are associated with certain ionization sources for conversion of materials (samples) to at least one gaseous ion or charged particle. One reason for a sampling device is to provide a cleaning or separation step prior to sample introduction and ionization as in chromatography (1-dimensional (1D) or 2-D), including liquid (LC), gas (GC), gel permeation (GPC), and paper chromatography, plates (thin layer (TLC)), gels (1D- and 2D- (sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)), slab gels, Western Blot, as examples. These none-MS separation approaches typically serve to increase the specificity and sensitivity of the experiment, but at additional expense and time; some may be readily hyphenated with MS such as LC and GC. Another reason for sampling devices, complementary to the first reason, is for a sampling device to avoid or reduce sample treatment by using direct mass analyses which decreases the need for highly trained personnel and the cost, especially if interfaced with computer approaches for data interpretation such as machine learning and artificial intelligence. MS is superbly accurate and with direct ionization approaches also very fast. To further improve the specificity for, e.g., identifying a drug or protein, intentional fragmentation (MS/MS) can be included with minimal additional time. Examples of sampling devices for direct analyses include, but are not limited to, ASAP, DART, MAI, SAI, and more specifically surface SAI without and with the use of a laser. Sampling devices also serve flexible and distant operations relative to the ionization source. Sampling devices (in certain cases also referred to as remote sampling or sampling from a distance) may be used with surface SAI, laser ablation SAI, and including transformative sampling-transfer-ionization technology to read the chemical composition from surfaces from a distance (e.g., “The Surface Reader”). Recent names described in literature (a number of papers are included into the reference list), with sometimes rather limited information provided, include and are not limited to REIMS ranging from “iKnife” to “LA-REIMS”, “zero volt paper spray”, “droplet assisted ionization inlet” (“DAII”) (or “DAI”), “vibrating sharp-edge spray ionization” (VSSI)”, a “MasSpec Pen”, “Spidermass”/“water assisted laser desorption/ionization” (“WALDI”), as examples. Yet another reason for sampling devices and their operations is to use robotics where the human is replaced for convenience or safety.
A brief synopsis of ionization methods include API methods such as “ambient ionization” (e.g., DESI), AP chemical ionization (APCI), AP photoionization (APPI), ESI and associated methods (e.g., nanoESI), nano-DESI, DART, electrospray-assisted laser desorption ionization (ELDI), laser ablation ESI (LAESI), laser desorption/ionization (LDI), liquid extraction surface analyses (LESA) including other liquid extraction and liquid junction approaches, MALDI, ESI (MALDESI), paper spray and variations thereof, surface acoustic wave nebulization (SAWN), and others known to the practitioner of the art. Alternatively, inlet ionization sources and methods are in many respects even simpler and more direct than ambient ionization. The category of inlet ionization (Trimpin papers) includes LSI, (previously described as laserspray ionization inlet (LSII)), MAI (previously described as matrix-assisted ionization inlet (MAII)), SAI (previously described as matrix assisted ionization inlet (SAII)), laser ablation (LA) MAI, LA-SAI, voltage SAI (VSAI) (also referred to as ESI inlet, ESII), DAI (Johnston paper), zero volt paper spray, VSSI and variations thereof, as examples. Yet another alternative category, vacuum ionization sources/methods have in common that the step leading to the analyte ionization occurs under sub-AP and are therefore generally more sensitive than API methods. However, the mechanism to introduce the sample to vacuum to perform vacuum ionization is typically slow with the exception of some newer inventions (Pophristic pending patent). Vacuum ionization methods include chemical ionization (CI), EI, fast atom bombardment (FAB), sub-AP ESI (Smith patents), LDI, vMAI, vMALDI, vLSI, FD, secondary ionization MS (SIMS), and others well-known to the practitioner of the art.
ESI-based methods form multiply charged ions and MALDI methods predominantly singly charged ions; APCI based ions are also singly charged. Advantages of singly charged ions include the relative ease in data interpretation. Advantages of multiply charged ions, even of large proteins and protein complexes, are the ability to mass measure high mass compounds on mass range limited mass spectrometers because mass spectrometers measure m/z rather than mass which can be mathematically calculated, as long as the z of the detected ion is sufficiently clearly delineated. For example, a compound of molecular weight (MW) 10,000 with 10 charges (10,010 assuming protons are the charge carrier) will appear at m/z 1001 whereas with 1 charge, at m/z 10,001. Alternatively, if the z of an ion at m/z 1001 is 1 and ion received its charge through protonation (proton charge charrier), the MW of the molecule is about 1000 whereas if the charge is 10 the MW is about 10,000. These mathematical examples demonstrate the important aspect of the charge z in identifying the MW. Other more common charge carriers are sodium, potassium, and ammonium cations. Chloride adductions or deprotonation (abstraction of a proton) can lead to negative ions. Electron attachment or removal are yet other means of ionization of the molecule, although this frequently leads to a higher degree of fragmentation of the molecular ions, unlike other ionization means. Because of multiply charging, API high performance mass spectrometers, intrinsically limited in mass range, are readily applicable to high mass compounds with well-known advantages such as mass accuracy, mass resolution and precision, as well as other advanced measurement technologies such as ion activation for structural details and specificity (e.g., collision induced dissociation (CID), electron capture dissociation (ECD), electron detachment dissociation (EDD), electron transfer dissociation (ETD), in source decay (ISD), negative ETD (NETD), surface induced dissociation (SID), sustained off-resonance irradiation collision-induced dissociation (SORI-CID) and others known to the practitioners of the art) for complementary (or only) MS/MS or MSn fragmentation capabilities of the respective ions formed. Low chemical background allows ready detection of small and large molecules, some difficult or impossible by MALDI because of the typical large chemical background in the low mass range associated with the use of a less volatile solid matrix. Significant signal width in the higher mass range is associated with metal cation and matrix adductions as well as the highly restrictive mass range on API mass spectrometers because of the singly charged ions that are formed by AP-MALDI.
As samples become increasingly complex (e.g., tissue, bacteria/fungus smears, films of natural or synthetic origin), high performance mass spectrometers, become more critical, as does advanced data analyses for accurate and fast analyses. Programs are used to assign the charge state, readily extractable from the isotopic distribution of the mass spectral data. Deconvolution programs are typically used to provide the mass (MW) representation of the data resulting from multiply charged ions. Statistical methods such as principal component analysis (PCA), machine learning, and artificial intelligence, and others known to the practitioners of the art, are recently being utilized more heavily in order to quickly evaluate (interpret) the sample composition without the need for specific interpretations of a single analyte (as in, e.g., conventional MS/MS) but the sample composition as a whole, frequently supported through an extensive database using match/mismatch (positive/negative, go/no-go) decisions which enables interpretation of complex sample compositions on the fly (nearly instantaneously) and independent of the degree of the training of the end user. Open Mass Fingerprinting Framework (OMFF) used for classification studies is a software platform for processing, storage, and used to query fingerprint reference data obtained from mass analyses. Other software may include amongst other utilities IMS and/or chromatography data to enhance (e.g., improve and/or speed up) the data interpretation as well known by the practitioner of the art.
APCI is a method for ionizing molecules in the gas phase at AP, typically by protonation in positive ion and deprotonation in negative ion, or by cation or anion adduction. Ions are formed using an electric discharge. ASAP (McEwen patent), is a technique in which a sample is introduced to an APCI vented enclosure and vaporized by a hot gas stream for ionization by APCI. A safety enclosure is used as fumes associated with vaporized materials may be harmful. Only small molecules that can be vaporized are amenable to either APCI or ASAP. DART (Cody patents) is a similar method producing nearly identical results.
ESI produces through application of high voltages (typically 2-5 kV) high and low-mass ions regardless of volatility through production of charged liquid droplets when a solution flowing through a capillary tube experiences a high voltage gradient upon exiting the tube at AP. Various solvent (“matrices), solvent combination and additives such as acids and bases may be used to improve the results. In ESI, effective means for desolvation of the liquid droplets are provided (counter gas flow, jet disrupter are examples, Smith patents) to prevent the solvent from freezing. Desolvation of the charged gas phase droplets results in bare gas phase ions with little or no fragmentations of the analyte. ESI is frequently hyphenated with LC to deal with suppression effects and improve utility of the approach. nanoESI uses nanoflow liquid flow employing nano-emitters improving sensitivity at the expense of expertise, cost, and time.
SAI (McEwen patents) uses solvent (liquid matrices) similar to ESI, but rather than form charged droplets at AP, the sample in solution is introduced into a tube that links AP and the first vacuum stage of the mass spectrometer and no voltage is required. With, e.g., methanol as solvent good sensitivity can be achieved at room temperature, but with less volatile solvents such as water, ion abundance and sensitivity is increased by heating the inlet tube. Both positive and negative ions are formed similar to ESI, but at inlet tube temperature exceeding 400 degree Celsius, singly charged ions typically dominate, but without significant fragmentation. In related DAI, droplets are introduced and significant heat employed (UP TO 900 degree Celsius) (Johnson paper).
vMALDI is a popular ionization method for MS, most commonly performed on high vacuum ToF mass spectrometers, where, typically, the co-crystallized matrix:analyte samples on a MALDI plate is introduced mechanically (ca. 2 minutes) to the vacuum of the ion source region. A laser is used to ablate the sample, consisting of analyte associated with a small molecule compound called a matrix (which may consist of binary matrix combinations and additives). The sample preparation may also be important, e.g., “dried droplet” and “thin layer”. The matrix is present in large excess in order to produce gas phase ions of the analyte(s). Typically, the laser beam traverses from the laser housed at AP through a window that seals the vacuum of the mass spectrometer from AP and using lenses and mirrors strike the sample on the sample plate in, typically, reflection geometry laser alignment and in a relatively focused spot, typically <100 microns diameter. Higher spatial resolution is achievable through a number of different ways and of them is, e.g., using the laser alignment in transmission geometry; this arrangement has the intrinsic advantage of one shot one mass spectrum, hence, the ability to be fast while employing an affordable laser with relatively low repetition rates. MALDI has also been demonstrated on API mass spectrometers, but is generally accepted to be less sensitive than vMALDI which is typically performed on Tof mass spectrometers (e.g., Bruker, Shimadzu, and previously Thermo, Waters/Micromass, and Sciex) and less common from intermediate pressure associated with quadrupole-Tof (QTof) mass spectrometers (Waters/Micromass and previously Thermo). A notable disadvantage is the chemical background associated with MALDI. Another stark disadvantage is that singly charged ions are predominantly formed in MALDI which limits MALDI on API mass spectrometers (e.g., Fourier Transform (FT) instruments such as Orbitraps, as well as QTof's) because of the inherent mass range limitations of API mass spectrometers. Yet, another limitation of MALDI (as in MALDI-ToF) is that with increasing mass (MW) the signal width becomes broad (because of a combination of cation and matrix adduction(s) as well as potentially fragmentation) making the interpretation of masses above 100 kDa differing by ca. 10 kDa a challenge to achieve. While MALDI-Tof may be routinely employed and for complex samples of moderate sizes, once a satisfactory sample preparation and acquisition method has been established, limitations include chemical background (typical operation is with a mass range above m/z 800 limiting low mass applications such as drugs), undesired fragmentation (limiting applications to fragile compounds such as posttranslational modifications (PTM's), and chemical modifications), matrix and variable metal cation adductions, and “hot spot” phenomenon (limiting reproducibility and quantification). Ionization sources operational from intermediate pressure may help with AP issues relative to sensitivity of the measurements but the mass range limitations remain. Although the introduction of the sample plate into the (high) vacuum region of the mass spectrometer complicates and slows down the overall speed of analyses, fast analyses are achieved with MALDI-Tof-MS by using expensive high repetition lasers.
Matrices may be at room temperature. Matrices in MS are widely used and may be in pure or mixed forms and may include additives and salts depending on the need for the analyte to gain a type of charge. The “matrix” is particularly important for ionizing nonvolatile compounds such as used in ESI, MALDI, their variations (e.g., MALDESI, (nano)DESI, LAESI), as well as SAI, MAI, LSI, and their variations (e.g., LA-SAI), and more recently in other methods such as in sonic spray as example. Relatively low volumes can be handled, e.g., <1 μl sample solution and <1 μl matrix solution mixed together and <1 μl of the mixed solution dried on the sample plate (‘dried droplet method’). Instruments are available which can automate the matrix:analyte sample preparation. In APCI methods such as ASAP and DART, no matrix is required (McEwen patent, Cody patents).
Common matrices used in MALDI are optimized for stability under vacuum and “sufficient” absorption at the laser wavelength employed. Typical examples of “MALDI matrices” include alpha-cyano-4-hydroxycinnamic acid (CHCA), anthranilic acid, 6-aza-2-thiothymine, caffeic acid, 2,5-dihydroxyacetophenone (2,5-DHAP), 2,6-dihydroxyacetophenone (2,6-DHAP), 2,5-dihydroxybenzoic acid (2,5-DHB), 2,6-dihydroxybenzoic acid (2,6-DHB), dithranol, 3-hydroxypicolinic acid, nicotinic acid, 4-nitroaniline, salicylamide, sinapinic acid (SA), 2,4,6-trihydroxyacetophenone, and tetracyanoquinodimethane (TCNQ), among numerous others compounds that are used to assist in analyte ionization by a MALDI process; combination of matrices (or so-called binary matrices) have been used, e.g., “super-DHB”). ESI and SAI use various solvents, sometimes with additives such as ammonium tartrate for matrices.
Another laser-based source/method is LSI (Trimpin patents) which appears softer than MALDI and produces ESI-like multiply charged ions directly from surfaces, in some cases even using a matrix that may be used for MALDI (e.g., 2,5-DHAP, 2,5-DHB, CHCA, SA), producing significantly less chemical background than, e.g., MALDI and is less prone to metal cation attachments contrary to both ESI- and MALDI-based methods. LSI from AP typically requires a heated inlet tube, and with some matrices substantially heated (≥450 degree Celsius, e.g., CHCA, SA). Many compounds act as LSI matrices, but only a few at low inlet temperatures and in the absence of an “inlet tube”, “channel” or “conduit”, as is the case with vacuum LSI (vLSI), e.g., 2-nitrophloroglucinol (2-NPG) matrix. LSI is operational from AP, intermediate pressure to high vacuum relative to the source chamber pressure.
Still another newer ionization source/method is vMAI (Trimpin patents), and like LSI and MALDI uses a solid matrix, but unlike LSI and MALDI does not require a laser; a laser can be used and one typical effects is faster removal of the matrix:analyte sample from the surface from where the sampling occurs. Adding heat is not necessary, depending on the matrix used. Certain volatile matrices sublime/evaporate spontaneously, to degrees specific to a specific matrix or matrix combination, and, importantly, converts compounds contained in the sample to “bare” gas phase ions, with reasonable certainty, through charged matrix particles. vMAI also produces multiply charged ions, but similar to LSI, conditions imparting energy (e.g., voltage, higher laser energy) into the ionized species reduces the charge states observed. A number of specific matrices for vMAI have been discovered, including but not limited to 3-nitrobenzonitrile (3-NBN or NBN), 2H-chromen-2-one (or also known as coumarin), 1,2-dicyanobenzene, 2-methyl-2-nitropropan-1,3-diol, 2-bromo-2-nitro-1,3-propanediol (also known as bronopol), isopropyl N-phenylcarbamate (or also known as propham), and methyl-2-methyl-3-nitrobenzoate, among others. Matrix combinations (e.g., different MAI matrices, MAI/MALDI matrices (e.g., 3-NBN/CHCA although no laser is required to be used), and MAI matrices with compounds that are not “matrices”) and matrix(es)/additive combinations including solvent (as in SAI, as example) may be utilized to enhance the effectiveness of analyte ionization providing more specificity or breadth and depth analyzing samples. Matrices for use with (v)MAI have in common high volatility, more so than (v)LSI and contrary to MALDI matrices. Some of these matrices are deemed safe and even eatable (coumarin, bronopol, ice). Depending on the matrix used and the inlet conditions employed (v)MAI is applicable from below ambient temperature to high heat applications (>positive 450 degree Celsius and higher). vMAI has been shown to ionize proteins as large as bovine serum albumin (66 kDa) using an intermediate pressure MALDI source of a QTof without need of laser ablation. It is expected that higher masses will be readily applicable under the appropriate conditions, as is the case with MALDI, LSI, SAI, and ESI. Examples of biomedical and clinical relevance include, as representative examples, drugs of abuse, specific metabolites, gangliosides, cardiolipins (e.g., associated with Barth Syndrome), carbohydrate-based vaccine candidates, Ebola related proteins, membrane proteins such as bacteriorhodopsin, various toxins, bacteria, fungi, and microbe colonies, among other biological samples using, e.g., vMAI. Yet others include synthetic materials such as monolayers, synthetic polymers, dendrimers, molecular ions of salts including but not limited to lanthanides and actinides such as uranium salts.
Combined with IMS and the formation of multiply charged ions, the direct ionization approach is especially useful for highly complex samples and for cases where LC applications are difficult or nearly impossible to employ (e.g., in the field or for imaging applications). Mass spectra from a wide variety of compounds and materials (including, as examples, urine, tissue, whole blood, and samples with high concentrations of salt(s) or detergent(s)) produce analyte ions with good sensitivity, even with the inlet at ambient temperature using certain MAI or SAI matrices. In some cases, introducing MAI/SAI/LSI matrix:analyte samples to the inlet of an AP mass spectrometer in some cases may have some drawbacks, principle among these are carryover (prolonged observation of ions from introduction of a sample), and common with other direct ionization methods (e.g., ESI, AP-MALDI). inlet contamination over time when using highly contaminated or high concentration samples. Carryover limits control and the speed with which samples can be sequentially analyzed without ions from prior samples being observed. The robustness to inlet contamination is expected to be also over time problematic with sample introduction from AP but much less so when introduced from vacuum. More specifically, 3-NBN ionizes under vacuum conditions drugs, peptides, and proteins, monolayers of inorganic complexes in the positive detection mode whereas uranyl salts ionize using a matrix (Wellons paper) in the presence of nitric acid in the negative ion mode. The matrix 1,2-dicyanobenzene ionizes preferably lipids (e.g., gangliosides, cardiolipins), potential synthetic magnetic resonance imaging agents while bronopol matrix readily ionizes carbohydrate-based vaccine candidates. SAI ionizes compound classes that are less effectively ionized by, e.g., (v)MAI, such as the general class of carbohydrates and other less basic compounds. Unlike other matrices used in MS (e.g., CHCA in MALDI, acetonitrile or methanol in ESI), some of the SAI (e.g., pure water or ethanol) and MAI matrices are safe and even edible (coumarin) or used in consumer products such as bronopol making even in vivo applications and temporal resolution measurements possible such as from living species. While the vMAI source/method is highly sensitive, only a minute amount of nonvolatile material is removed during the ionization process. Thus, carryover between subsequent samples as well as instrument contamination for dirty and high concentration samples is essentially eliminated. While probe introduction (Trimpin papers) has a number of positive attributes, it is not at the level of high throughput or easily automated. The vacuum vMAI plate source (Pophristic pending patent), however, offers an expansive repertoire of additional measurement capabilities (high throughput, automation, robotics, unmanned, field-operation, are examples); although a laser is not necessary, a laser can be used (laser ablation (v)MAI) or switching matrices to perform vMALDI and/or vLSI.
Various improvements in ionization have been implemented for the various sampling devices, sources, and methods operational from AP. These include use of gases at AP along with heat, voltages, collisions, as well as other means to reduce/remove the matrix from the charged particle to increase bare analyte ions. Ionization under vacuum conditions allows large apertures that do not restrict ion/(charged) particle transfer/transmission, and rim losses (Willoughby patent) do not occur but requires a mechanical means for introducing the sample (materials) into the vacuum in fluid communication with the analyzer region. Thus, improved sensitivity is achieved with vacuum ionization source/methods as has been demonstrated with MALDI-Tof mass spectrometers relative to AP-MALDI and other API methods; the most typical use of MALDI is with Tof mass spectrometers operating at high vacuum. A method termed Super-AP MS has been implemented for increasing the sensitivity in ESI (Hiraoka paper). Other means are the use of collisions (e.g., jet disrupters, gases, voltages, heat) for increasing sensitivity and applicability in traditional ESI methods (e.g., Smith papers). Collisions within the inlet tube, or prior to, and after the tube region have been employed for inlet and vacuum ionization (e.g., Trimpin patents, McEwen patents, Trimpin papers).
Many of the deficiencies of API approaches are cured by introducing the liquid or solid sample directly into the vacuum of the mass spectrometer near the inlet to the analyzer device (e.g., Trimpin patents, McEwen patents, Trimpin papers). The vMAI sample may be introduced directly to sub-AP using a probe such that the sample experiences a limited gas flow when the volume into which the sample is inserted is evacuated. The matrix sublimes from the probe surface leaving any nonvolatile compounds (including, as one example, salts contained in samples) which are not ionized remaining on the probe to be removed from the instrument when the analysis is complete. The result from inserting the matrix:analyte sample on a probe tip directly into the sub-AP close to the ion transfer optics is a steady emission of gaseous analyte ions over a period of time. The time of ionization is determined by the rate of sublimation of the matrix and the amount of matrix placed on the probe. Conditions, which accelerate sublimation increase the ion abundance and shorten the overall time ions are produced and may include conductive and radiative heat. Conditions that slow the sublimation (e.g., cooling, lower pressure/less gas flow), cause ion formation to occur with less momentary ion abundance, but over a longer period of time (beneficial for, e.g., MS/MS). All matrices of this spontaneous ionization method sublime in vacuum near room temperature. In other inlet and vacuum ionization methods evaporation has to play a role; whether sublimation or evaporation, the process described is to remove (desolvate) matrix, solid, or liquid, from charged and neutral particles to transport the analyte into the gas phase as bare ions for analysis by MS or IMS or others. It has been shown that vMAI is applicable on a broad range of instruments from (ultra) high performance to portable and from small to large molecules, and directly from their natural environment.
By moving, or rastering, the sample through, e.g., a laser beam, a solvent stream or gas stream in a coherent manner and collecting mass spectra denoted by sample (plate) position, an image of the surface (or 2-D array) can be reconstructed for any m/z ratio mass analyzed and detected by the analyzer and detector devices and displayed in, e.g., the mass spectra (typically thousands per surface, e.g., mouse brain tissue section. This type of highly systematic measurement is referred to as imaging MS or MS imaging.
A sampling line of variable diameter, length and material, can be used to implement remote analyses from a distance, overcoming physical limitations and safety aspects (Trimpin patents, McEwen patents, Trimpin papers). A matrix, solid or liquid, may be added to the surface to be analyzed and the transfer line, if desired. A laser or other means may be used to remove material (sample) into the gas phase such as the inlet ionization source or into a transfer line first. Mass spectra are collected from the surface of the sample by typically summing several laser shots to produce more representative chemical information contained in the obtained mass spectra with acceptable signal-to-noise (S/N). In more recent years “imaging by MS” and “surface analyses” have been adapted with a variety of different ionization methods including but not limited to DESI, MALDESI, LAESI, LSI, SAI, and vMAI from API, inlet or vacuum ionization, respectively. Various lasers (Trimpin patents, McEwen patents, Trimpin papers) can be used (deep UV to far IR, ultra-fast and low repetition lasers, high and low power lasers, etc.). As understood by the practitioner of the art, in the newer inventions of inlet ionization and vacuum ionization methods, the laser is not directly involved in the sample (analyte) ionization but assists in dislodging the sample content into the gas phase for transport through a variable line toward the analyzer device where ionization occurs on the fly by conditions described for inlet ionization. As is also understood by the practitioner of the art, charged particles are converted to bare gaseous analyte ions under sub-AP conditions. Conditions for transfer of ions (e.g., created by ESI) versus those created by (e.g., SAI) are expected to optimize under different source and method conditions, even though they may use the sane matrix (solvent).
Some form of sample cleanup is generally utilized with MS methods, especially with ESI and APCI-based ionization sources and methods, which increase time, cost of analysis, and risk to the operator if the samples are too dangerous to be handled (e.g., toxic, contagious, or deadly such as bacterial, viral, fungi, or radioactive compounds, biological and/or chemical warfare agents contained in certain materials; several of these analytical challenges have been accomplished in a straightforward and safe manner (note ionization in both inlet ionization and vacuum ionization occurs under vacuum) using the newer ionization inventions (Trimpin patents, McEwen patents, Trimpin papers). Liquid chromatography (LC) may be used for separating complex samples prior to MS analyses. RapidFire from Agilent includes a form of quick LC separation but suffers from robustness of the setup and experiments requiring significant user expertise in running, operating and maintaining the instrument and methods. The ionization is based on ESI and achievable speed is at ca. 6 seconds per sample.
Liquid extraction methods have had a long history (Das/Bhatia paper; Brodbelt/Eberlin paper) and have been more recently combined with MS in a more direct fashion such as DESI, LESA, and MasSpec Pen, e.g., Eberlin papers). In common is that a solvent is used to extract the analyte(s) from a surface that are than ionized either by applying a voltage as in ESI or a pressure differential and preferably heat as in inlet ionization (specifically SAI or VSAI).
Removing sample content from, e.g., tissue and other 2- or 3-dimensional surfaces (e.g., fruits, meats, and other consumer products), other than by liquid extraction methods, also has a long history and have been in more recent years also coupled with mass spectrometers and related instruments. In common is that a force is used to remove the analyte(s) from a surface (volatilize the sample into the gas phase) that are than ionized (sometimes also referred to post-ionized) either by applying a voltage as in ESI or a pressure differential and collisions as in inlet ionization. A common limitation is that the analyses are physically limited to one sampling device, source and certain methods, and some analytes ionize (in some cases significantly) better with another source and method or are exclusively applicable by one or another sampling device, ionization source, or method. Inlet ionization (e.g., LSI, MAI, SAI, LA-MAI, LA-SAI, surface SAI based on liquid extraction or LA methods, LC, to name a few) and more recently DAI, as one of a number of examples of “new” ionization processes offering a variety of methods and devices to expand the utility of analytical, medical, security, biological, chemical, environmental, forensics, nuclear (and others) measurements.
Multi-ionization source capabilities (patents, papers) were initially introduced as a combination of ESI and APCI, which had previously required separate ion sources. Sources are still exchanged or modified to switch from ESI, to nanoESI, APCI, ASAP, DESI, nanoDESI, DART, AP-MALDI, photoionization and glow discharge ionization. among others that are commonly available from mass spectrometer vendors or homebuilt employing API mass spectrometers. For some of these “individual” ionization sources and methods, patent protection has been successfully sought. One example is that the interchangeable ionization sources offer the intrinsic advantage of tailoring the “right” ionization method (e.g., small molecule by APCI versus large molecules by ESI) to be used on the same mass spectrometer introducing less initial purchasing cost, footprint, and upkeep of more than one mass spectrometer, among other advantages such as a more comprehensive chemical analyses. AP-MALDI (TransMIT, Germany; MassTech, USA), paper spray (Thermo), DESI (Prosolia, now Micromass/Waters), LAESI, ELDI, ASAP (M&M), DART (IonSense, now Bruker), UniSpray (Micromass/Waters), iKnife (Medimass KFT, now Micromass/Waters), MasSpec Pen, SpiderMass, Unispray, WALDI, Echo (Sciex) are typical examples for sources (sampling devices, sometimes also referred to as add-on's) and methods which are frequently ‘standalone’ but are commonly, but not always, interchangeable on the same mass spectrometer, but may include venting the mass spectrometer (breaking vacuum). AP-MALDI, DART, ASAP, Unispray, iKnife, and MasSpec Pen have not been shown to be effective for larger molecules, either because of general ionization limitations or because of the mass range restrictions of API mass spectrometers for singly charged ions. DART and ASAP are applicable to volatile and semivolatile compounds producing singly charged ions. The exchange of sources, or when cleaning an API source inlet, can require minutes or hours (e.g., overnight baking), depending on the mass spectrometer and inlet geometry, adding to cost and downtime. Intermediate pressure MALDI sources are available (Waters) which can be replaced against an API source (e.g., ESI/APCI) but the vacuum needs to be broken. Breaking the vacuum (venting) the instrument is a significant issue in terms of time, expertise, and component use/wear and tear, as examples. This may be necessary for interchanging sources/sampling devices/methods or cleaning a source, such as, e.g., vMALDI, or exchanging with other ionization sources such as ESI. Intermediate pressure MALDI sources, however, under certain conditions may be used with LSI-MS on the same vacuum source of commercially available mass spectrometers (Trimpin patents and papers). The exchange of sources, sampling devices, methods without breaking the vacuum of the mass spectrometer is from a practical standpoint notably more desirable than having to deal with the aftermath of having to vent the instrument.
The Bruker dual ionization source capabilities include an API source and vacuum MALDI which are in a permanent location, expensive, and only on a select series of a high-performance mass spectrometers. A related patent is included (Linden patent), but to our knowledge was never commercialized. Select series of Waters mass spectrometers offer the exchange of API and intermediate pressure MALDI sources; patent coverage is not obvious, instead a paper is included (Trimpin papers). In even more limited instances, Thermo offered a vacuum MALDI or API ionization configuration on select mass spectrometers; for the exchange of the ionization sources, the mass spectrometer was required to be shipped back to the manufacturer. These examples give a principle feel of the general utility but also the significant technical difficulties of exchanging vacuum ionization source.
No ionization source, sampling device, or method, ‘can do it all’, meaning ionize every possible molecular composition. All ionization sources, sampling devices, and methods thereof have unique capabilities as well as downsides towards their respective applicability to small and large, hydrophobic and hydrophilic, and volatile and nonvolatile components within a mixture. Additionally, “soft” and “hard” ionization leads to desired and undesired fragmentation, different background ions, and mixed ionization of, e.g., protonation, deprotanation and cation, anion adduction, respectively, thus complicating interpretation of the results. Interchangeable ionization sources, sampling devices, and methods, where ionization is initiated to address shortcomings of any one approach, have been introduced in the past to, in part, alleviate the problems (limitations) and maximize the utility of a specific API or intermediate pressure ionization mass spectrometer and maximize the utility to some degree of the more expensive component, the analyzer device itself. This, however, has traditionally been accomplished in a manner which is both time-intensive, limited, expertise demanding, and impractical, especially when the instrument needs to be vented to switch between atmospheric and intermediate pressure ionization sources, sampling devices, and methods. Likely, the most common exchanges of ionization sources and methods are related to API sources (numerous patents and papers included to reference list) in which the ion entrance remains the same, but the source housing with the respective ion generation means is associated with the source housing such as high voltages, desolvation gas, etc., leading to capabilities of ESI, APCI (ASAP, DART), DESI, MALDESI, LAESI, nanoESI, as examples, for which the switchover does not require breaking the vacuum of the mass spectrometer (switchovers are in the order of minutes for the majority of these examples). In these cases, the ionization occurs at AP and gaseous ions, and to some degree charged particles (droplets) enter the sub-AP of the mass spectrometer.
Desolvation gases, voltages, and ionization source geometries help guide and focus the gaseous ions toward the mass spectrometer and help remove the (charged) particles (droplets) by either desolvation or ‘removal’, e.g., onto surfaces and if volatile into the rough pump. This helps keep the mass spectrometer cleaner for longer. Such API source/device/methods exchanges may include ESI, DESI, APCI including ASAP and DART (e.g., Prosolia, now Waters; M&M Mass Spec, and Advion Interchim; IonSense, now Bruker). A number of patents on this basic idea have been listed (more specifically, e.g., U.S. Pat. Nos. 3,886,365A, 4,037,108A, EP0423454A2, US20040079881A1, GB2394830A, GB2406705A) indicating the importance of such principle capabilities in exchange of an ionization source.
More recently, Waters received patent protection for a multi SAI inlet (Brown, U.S. Pat. No. 9,761,428B2) in which one inlet is used for calibration purposes. It also assumes that one inlet can be used with ESI/APCI. Data was not presented in this patent so it cannot be determined if the device was ever constructed or tested. Similarly, Waters received patent protection for a vacuum ionization configuration (Brown, U.S. Ser. No. 10/319,575B2). Again, no data was presented in this patent so accessing any form of analytical validity is not possible from the patent. MSTM recently introduced a plate-based vacuum source (Pophristic pending patent) that allows straightforward execution of vMAI and vMALDI methods through the same ‘Port’ and plate by simply without (e.g., vMAI) and with the use of an appropriate laser (e.g., vMALDI,). ESI capabilities were also retrofitted onto the plate-based vacuum ionization source, as a convenient way for, e.g., instrument calibration and other basic applications. This plate-based vacuum source is a specific type of multi-ionization capabilities, which, uses a single. This plate-based vacuum ionization source invention for directly introducing samples (e.g., vMAI) or gaseous ions (e.g., ESI) to vacuum constitutes a significant advantage over previous ionization sources. These examples give a feel of the general utility and opportunities of the new ionization processes, which proceed through stages of charged particles at sub-AP versus traditional ionization processes which are largely associated with bare gaseous ions under vacuum conditions. For AP methods such as ESI, the charged droplets were desolvated into bare ions before reaching the vacuum ion transfer region, or discarded early in the process of transfer/transmission, and in vacuum MALDI, only bare ions needed to be considered. The new ionization processes of SAI, MAI, vMAI, and LSI, produce charged matrix:analyte particles which, for optimum sensitivity must be transmitted, along with bare ion, well into the analyzer device, because, due to the volatility of the matrix, desolvation of the charged particles, and production of the desired bare ions continues throughout the lifetime of these charged particles. It is also possible, but not proven, that neutral gas phase matrix:analyte particles may produce charged particles similar to what happens at the matrix:analyte surface to initiate ionization. Thus, optimum transmission of ions in a multi-ionization device which includes SAI, MAI, vMAI, LSI or vLSI needs to take this into consideration. Importantly, even the likely most sophisticated of these newer inlet and vacuum ionization sources, the plate-based ionization source, also does not address to the sometimes rather contradictive needs associated with ion/(charged) particle transfer/transmission of API, inlet ionization, and vacuum ionization sources/methods, and while being more flexible than other ionization sources and methods, ion transfer/transmission under vacuum is not fully yet optimized. The multiPort vacuum chamber assembly of the invention presented herein provides Ports capable of aiding optimization of transmission of ions and charged particles when needed. Thus, this is an enabling technology for an exceptionally wide range of MS ionization methods and in a compact, relatively light weight, and low-cost add-on device.
Low-cost to manufacture and operate are especially important for measurements such as those desired or required during physician visits, airport screening, mobile response clinics, or health crisis such as emergency and operating rooms, as well as epidemics and pandemics, and monitoring cancer progression, fungal, bacterial or viral infection(s), the substance use disorder (SUD) epidemics including but not limited to opioid use disorder (OUD) (e.g., fentanyl) and stimulant use disorder (StUD) (e.g., cocaine) as well as prescription drugs (e.g., ketamine), safe and timely drug developments, homeland security, including energy and serosurveilance (e.g., uranium, bacterial, or viral detection/monitoring), as wide-ranging examples, because of the present invention's unique abilities to ionize small and large molecules for rapid and an unmatched breath of the measurement technology and its enabling sampling devices and, e.g., surface analyses (e.g., SAI, LA-SAI) methods. Similarly, simple sampling strategies including (filter) paper, meshes, needles, smears, and swabs, as well as other means known to practitioners may be directly applicable but may need enrichment (extraction and concentration) or even necessitate cell cultures and growing efforts (for some of the more difficult bacteria, as just one example). Filter paper has the intrinsic advantage of being able to add a brief separation/cleanup step, and even if it is as simple as exposing the opposite side to the vacuum to where the sample was applied. Other biological materials can be monitored rapidly and accurately for type and stages of cancers (blood, tissue including biopsies from organs), especially effective using a machine learning approach for fast and reliable interpretation including chemometric analysis. Surfaces may be wiped off or sampled using a stream of solvent, for which all that is required is to guide the solid or liquid into the sub-AP for ionization on the fly by (vMAI or SAI, as examples or with added complexity (e.g., use of a laser, MALDI, LA-SAI, LA-MAI, LSI, as examples). This is particularly true because of the robustness of the multiPort vacuum chamber assembly enabling the permanent or flexible use of ionization sources, sampling devices, methods, as well as auxiliaries afforded by the apparatus of this invention providing complementary ionization and chemical coverage of a material (sample) analyzed through traditional and orthogonal ionization methods that are well-known by the practitioner of the art. The outstanding sensitivity and chemical molecular information of small to large molecules achievable using the apparatus and methods of this invention are disclosed in more details in the following.
Not all Ports of multiPort vacuum chamber assembly need to be used. The device can be customized for specific use and the remaining Ports sealed for future use. Care must be exercised to prevent carryover by eliminating dead volumes (volumes without or with minimal gas flow), especially with MAI and vMAI. Use of filter paper with vMAI greatly reduces carryover while providing numerous other advantages. This invention supports an unprecedented number of sampling devices, unlike anything before developed in this scientific area. While the multiPort may sound complex, it is exceptional simple to operate, especially considering most users will employ a single Port for most analyses, with additional Ports only being employed when necessary to gather additional information to make or confirm an analysis. If physical exchange of sources/methods (approaches) is desired it may be accomplished without venting the vacuum of the instrument by blocking the vacuum chamber's fluid communication with the vacuum of the analyzer device. As mentioned above, it can be retrofitted on existing mass spectrometers or built as fit for purpose, and when the purpose changes, the ionization methods may be exchanged. No existing technology offer the ease or comprehensiveness of ionization sources, methods, and sampling devices, and especially at such a low cost to build.
The invention disclosed herein is a vacuum chamber assembly device specifically related to a gaseous ion/charged particle transfer region for use with an ‘analyzer device’, which may be a mass spectrometer, ion mobility spectrometer, and combinations thereof, and where the term ‘charged particles’ encompass gas phase droplets, particles, or clusters of matrix molecules, solution or solid, as well as one or more analyte and one or more charges of one sign (positive or negative) exceeding that of the opposite sign. The term ‘ion(s)’ encompasses bare analyte molecule(s) in the gas phase having one or more charges of one sign (positive or negative) than the opposite sign. Analyte(s) are part of a sample and can be in the form of solid and/or liquid condensed phase, or in some instances in the gas phase. The vacuum chamber assembly device is preferably attached (interfaced) to the analyzer device at the first vacuum stage region of the analyzer device. The ion/(charged) particle transfer region is defined as a vacuum conduit which may have multiple Ports (multiPort) (or docks)) which link the interior of the conduit to the exterior of the vacuum chamber assembly; the exterior residing at or near atmospheric pressure (AP) with the exception of the portion docked to the analyzer device and residing in the sub-AP region of the analyzer device. The pressure within the vacuum chamber conduit is generally slightly higher than the pressure in the analyzer region of the analyzer device so that the flow of gas is in the direction from AP through the vacuum chamber conduit to the analyzer device and is important for transmission of ions and particles.
Typically, the vacuum chamber assembly may incorporate a minimum of three ports, but more typically four to eight Ports, but it should be understood that in some instances, more Ports may be desirable. The Ports may be at various angles extending from the exterior to the interior of the vacuum chamber. The Ports are in fluid communication with the sub-AP vacuum of the analyzer enabling transfer of ions and charged particles from AP or sub-AP into the first vacuum stage of an analyzer. The multiPorts are used for interfacing a variety of vacuum ionization, inlet ionization, and/or atmospheric pressure ionization (API) sources/sampling devices/methods used with MS or IMS, and combinations, as well as other auxiliary functionality to improve ion fragmentation, ion and charged particle generation and transfer into an analyzer devise; note, certain instruments contain more than one analyzer device, thus such instruments incorporating a mass or mobility analyzer are covered by this disclosure.
The multitude of Ports vacuum chamber assembly invention disclosed below can be used to assess, for example, biological, clinical, biomedical, forensic, environmental, agriculture related compounds, food and food products, authentication of samples, diet supplements, plant, spice, consumer products, drugs and drugs of abuse including but not limited to opioid use disorder (OUD) (e.g., fentanyl and its derivatives), stimulant use disorder (StUD) (e.g., cocaine and its derivatives), and prescription drugs (e.g., ketamines), pharmaceuticals, cannabis and cannabinoids, hallucinogens, and/or oil/petroleum samples, inorganic salts and complexes, molecular salts, nuclear compounds (e.g., uranyl nitrate), synthetic polymers, dendrimers, amongst others, as well as for identifying microbes as occurs in microbial infections, bacterial, fungal, and viral pathogens, cancers, and biomarkers for a variety of diseases ranging from small to large molecules. Tissue imaging, monolayer analyses, and single cell analyses are other examples in which the invention may provide detailed information from a sample of interest that relate to a specific disease and disease boundaries through in vitro and in vivo measurements. Depending on the application needs, this may be with or without a laser, or other energy inputs, with or without calibration or standards injections for calibration or quantitation purposes for improved measurement outcomes translating to faster answers, more precisely and accurately, for the end user (e.g., patient, health insurance). It should be understood that these uses are meant to be examples and not inclusive uses of the innovative, one of its kind ion/charged particle transmission vacuum chamber assembly based on the plentiful examples of traditional ionization processes and methods and complemented with inlet ionization and vacuum ionization processes and novel approaches, e.g., surface SAI and vMAI for gel separated proteins, and having access to inlet ionization and traditional ionization approaches, e.g., ESI, vMALDI, and ASAP, for chemical information obtained rapidly or even at the same time for small to large molecule analyses using best/most appropriate approaches at little additional time, efforts, expense, or expertise.
Practically up to 6 Ports are comfortably fitted on a reasonably sized vacuum chamber and the combinations of possibilities and solutions to numerous analytical challenges solvable using a single mass spectrometer or ion mobility spectrometer or combination instrument are unmatched. The advantages of this vacuum chamber assembly consisting of a multitude of Ports used with a variety of sources, sampling devices, and methods, as well as auxiliary functions for the analyses of a broad range of analytes fulfill the requirements of methods that include “biology”, “chemistry”, “human health”, “in the field”, “fit-to-purpose”, “point-of-need”, “point-of-care”, “bedside”, “doctors office”, “cancer”, “microbiome science” “integrative omics”, “food safety”, “forensics”, “remote”, “portable”, “analyses from a distance”, “environmental”, “environmental molecular sciences”, “earth system science”, “ecosystem science” “materials science”, “homeland security”, “law enforcement”, “chemical warfare”, “biological warfare”, “nuclear proliferation”, “scientific discovery”, etc. made possible by using the most fitting approach(es) for speed of analysis, cost effectiveness, simplicity of operation, and robustness to carryover and contamination and also at point-of-interest/need (in the field) measurements. The exceptional flexibility makes this multiPort assembly radically different from current mass spectrometer ionization sources, sampling devices, methods and applications. Further the invention is readily adaptable to how a sample is presented to the analyzer device, e.g., glass plate, filter paper, membrane, mesh, a tip (e.g., Qtip, toothpick), metal plate, thin layer chromatography (TLC) plates (1-dimensional (1D) or 2-D), gels (1D- and 2D-(sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)), swabs, and parts of leaf, hair, fruit, vegetable, or meat, as well as plastic and polymers including films, sections biological tissue, and gases. LC and other liquid clean up steps are applicable online (e.g., (nano)ESI, (nano)SAI, APCI), or offline (e.g., MAI, MALDI).
Quantification is rapidly achieved through the standard addition methods, internal standards method, and through relative quantification. Easy, rapid, and accurate detection on-demand and at high speed and sensitivity represent significant improvements in many analyses, and especially healthcare outcomes and costs. Forensics are readily executed by, e.g., rubbing one of a filter paper over a surface potentially exposed to harmful chemical(s).
The Ports provide an easily accessible interface from AP to the vacuum of the analyzer device, and provide for sequential or simultaneous transfer of bare ions and charged particles from multiple ionization sources configured for gas phase ion generation at or near AP, in the transfer region between AP and the sub-AP of the vacuum conduit, or directly in the sub-AP of the conduit within the vacuum chamber assembly. Ions and charged particles are transferred through the vacuum chamber conduit into the ion optics of the analyzer device by the flow of gas and/or, alternatively, with the aid of voltages applied to lens elements through one or more Ports. The invention relates to a device and methods for converting mass spectrometers designed for AP ionization (API) to being capable of rapid, seamless switching between multiple API, inlet ionization, and vacuum ionization methods without need of instrument venting, and including insertion of certain functionality (auxiliary functions) for collision induced fragmentation if desired, improved vacuum ionization, and improved ion/charged particle transfer, among others, at a reasonable cost while increasing breadth and depths of analyses capabilities.
Ionization may be initiated with and without a laser, without and with high voltages, and with or without a matrix added, with or without heating or cooling, from the general categories of (1) API, (2) inlet ionization, and (3) vacuum ionization, while the commercial abilities of existing mass spectrometers are not sacrificed. Additionally, we disclose auxiliary Ports which serve the flexibility and specificity of adding additional function or improving certain existing functions associated with the ionization sources, sampling devices and methods (1) to (3). The invention is not limited to retrofitting to existing instruments but also to design, construction of innovative new instrumentation in the general field of mass spectrometry, IMS, and combination instruments. A Port may have both ionization and auxiliary functions.
The multiPort vacuum chamber assembly may have walls which separate the environment within the vacuum chamber from the environment exterior to the vacuum chamber assembly. The vacuum chamber assembly may be as simple as the geometry of a tube (referred to herein also as a conduit), although other configurations, such as, but not limited to, an oval, rectangle, or spherical may be used. Considering a conduit, the two ends may be in fluid communication with each other and with the sub-AP (vacuum) of the analyzer device. The conduit may have Ports branching off and in fluid communication with the sub-AP within the conduit, and the sub-AP of the analyzer device. Ports may reside at the end of the conduit opposite the end that is docked to the analyzer device, or directly across the conduit from one another or in other positions along the length of the conduit and at 90 degrees to the conduit or at other angles such as, e.g., 60 (or 120) degrees.
After installation of the vacuum chamber assembly on an analyzer device, the gas flow is optimized for the desired configuration. In most configurations there are only two Ports that transmit air from AP to the sub-AP of the vacuum chamber assembly and one is used in conjunction with the vacuum ionization Port and is set for a small gas flow by the inner diameter of the tubing used which is typically <0.4 mm. The other Port transmitting gas (e.g., air, nitrogen, or others) and used for AP and inlet ionization has the gas flow optimized by selecting an inlet tube with length and inner diameter that produces the minimum gas flow and pressure within the conduit for optimum results. Other inlet tubes can be fitted on other Ports, but are typically blocked except for the one in use. As an example, there may be inlet tubes in two Ports, one for ESI/APCI and one for SAL. A cap is typically placed over one to block gas flow while the other is operational. Switching caps allows the other to be operational with optimal gas flow. If it is desired to have both operate simultaneously, it is necessary to compromise relative to optimum sensitivity in order to maintain operational pressure by using smaller inner diameter or longer tubes. An important feature is the ability to remove and replace inlet tubes without venting the by having a probe substantially block the flow of gas out of the conduit or operate a shutter device which can be opened or closed by manipulation from AP. Inlets with low gas flow may be used in some cases to enhance ionization in another Port such as a Port used for vacuum ionization.
The multiPort vacuum chamber assembly provides simplicity for production at relatively low cost, size and weight. The vacuum chamber provides flexibility for a cost-effective means of achieving maximum utility of an instrument both in terms of breadth and depth of analysis. It no longer requires selecting the instrument based on the ionization source/sampling devices because most currently known ionization methods can be retrofitted easily and cost effectively. By limiting the number of Ports which are deployed, the invention may provide enhanced portability of the instrument for fit for purpose, point of need, and fieldable instruments.
The multiPort vacuum chamber assembly is designed to be interfaced with API mass spectrometers and related instrumentation from various manufacturers and provide a cost-effective means of having rapid or simultaneous access to API, inlet ionization, and vacuum ionization sources/sampling devices/methods. Thus, a single mass spectrometer or ion mobility spectrometer, for the first time, provides, through the multiPort vacuum chamber assembly device invention, the capability to access at least one of the categories of (1) API such as electrospray ionization (ESI), AP chemical ionization (APCI), desorption ESI (DESI) direct analysis in real time (DART), atmospheric solids probe (ASAP), AP matrix-assisted laser desorption/ionization (MALDI), and others known to the practitioner of the art; (2) inlet ionization such as matrix-assisted ionization (MAI), solvent-assisted ionization (SAI), voltage SAI (VSAI), and sub-variants (laser ablation (LA) MAI and LA-SAI, droplet assisted ionization (DAI), laserspray ionization (LSI), as examples), and others known to the practitioner of the art; (3) vacuum ionization such as vMALDI, vacuum laser desorption/ionization (vLDI), vacuum laserspray ionization (vLSI), vacuum MAI (vMAI), and others known to the practitioner of the art. The practitioner in the art will know the many combinations that the limited number of sources/sampling devices/methods noted above offers for a multiPort vacuum chamber assembly ion/charged particle transfer chamber in which one Port interfaces the vacuum chamber conduit to the analyzer device and its related ion optics. The use of the other Ports may be simultaneous or sequentially accessed in the order of seconds for installed approaches selected from the categories of API, inlet ionization, vacuum ionization, and auxiliary functions and approaches which frequently may be simply and seamlessly assessed through a software mouse click. In these cases, the interchange between approaches does not require changing physical components, although this is an option to maximize the completeness of the functionalities of the vacuum chamber assembly as disclosed herein.
A Port may be a tube having walls encasing a channel or passageway with an inner diameter between >0.1 mm and <30 mm, and preferably between >0.3 mm and <7 mm. The Port may have a variety of lengths and shapes and may be open and transmitting air from AP or near AP into the interior of the vacuum chamber assembly, or sealed so that air does not pass through the Port. If the Port is open to AP or near AP, the opening must be sufficiently small so that the analyzer device maintains an operational pressure. Typically, the opening will be between 0.3 mm and 1.0 mm, but, as would be understood by someone practiced in the art, may under some circumstances be smaller or larger such as <0.1 mm and >1.5 mm. The opening may be a channel through a tube and defined by its inner diameter. Some Ports may be both sealed and open, as for example inserting a probe device into the vacuum chamber through the Port will use a means to seal the probe using, e.g., ‘O’ ring or ferrule seals, and have an opening on the sub-atmospheric pressure (sub-AP) side of the seal to provide a gas flow to aid sublimation, ion/charged particle transfer, or gas phase reactions, so long as the operating pressure of the analyzer device is maintained. A Port having a first end and a second end such that the first end is fitted to the vacuum chamber assembly and open to the interior of the conduit with a vacuum tight flange or fitting, and the second end in fluid contact with the first end except when a valve device is placed between the two ends and in a closed position. Ports may have different inner diameters, and in some instances it is preferential for one end of a Port to be larger than the other end depending on the application needs. The gas flow through all Ports simultaneously is important and must achieve a pressure within the vacuum chamber conduit that produces acceptable results. The desired pressure varies with the analyzer device, the conduit's internal diameter, ionization sources/sampling devices/methods being used and the analyze device's pumping capacity. Typically, pressure ranges are between 10 mbar and e−2 mbar, or a pressure which allow optimum operation of the analyzer device. Because the multiPort vacuum chamber assembly can be retrofitted with existing mass spectrometers, different gas flows and positions may be selected to provide best conditions for achieving maximum ions as well as charged particle transfer (or transmission) to achieve desired results.
An important aspect of this invention is that the different Ports can be configured to support and enhance each other. This synergy is unique and achieved through the implementation of three or more Ports in which one Port provides communication between the multiPort vacuum chamber assembly and the sub-AP of the analyzer device. In one configuration, the multiPort vacuum chamber interfaces on one end or side with the analyzer device, and on another end or side with the API source of the instrument that is being converted for multiPort use. In other words, the multiPort vacuum chamber inserts between the analyzer device and, e.g., the commercial API source, so that both are in fluid communication with each other and facilitating the transfer of the at least one analyte ion and/or the at least one charged particle from the ion source(s) to the vacuum region of the analyzer device. The second end may have a Port interfaced preferably with the commercial ion source that is supplied with the instrument, an API/inlet ionization inlet, or a plate source inlet. Between the first end and second end may reside a number of Ports enabling devices where the totality of these devices must maintain the pressure within the multiPort vacuum chamber within acceptable ranges for operation of the analyzer device. At least one of the Ports enable vacuum ionization methods. Adding an additional Port in line-of-sight with the surface onto which a matrix:analyte sample is applied, allows reflection geometry MALDI to be achieved by passing a laser beam through the added Port to impact the sample on the surface of the probe or plate device supported by the vacuum ionization Port.
Other examples of Ports configured to support and enhance each other (auxiliary Ports), include a means for introducing a relatively low flow of gas (continuous or pulsed) for enhancing, e.g., the rate of ionization by vMAI and aiding transfer of ions, and charged matrix:analyte particles toward, and subsequently into, the ion optics of the analyzer device. A simple example is API or more particularly the very common “ESI”, which can serve as an “ionization source” or as an auxiliary function for another Port (e.g., gas flow and temperature control). Some of the various functionalities of the Ports are: a laser beam for vacuum MALDI passing through the Port using optical focusing or fiber optics; a shutter that allows a neighboring Port, ionization source or auxiliary device, to be exchanged or cleaned without venting the instrument, and addition of gases such as reaction gas(es) to cause reactions to occur with the ions or charged particles formed from another Port and includes deuterium gas to enable H/D exchange. Other uses of Ports include but are not limited to a laser beam for “MALDI-2”, a collision surface (cold or heated) for ionization, desolvation, or fragmentation purposes, a camera, or a manometer, as examples. A Port can be included to sufficiently block the flow of gas through the conduit from the higher pressure region to the lower pressure analyzer device. This can be achieved by using a probe device shaped to insert through an ‘O’ ring or ferrule, and insert into the conduit sufficiently tightly to provide the desired seal. The relative sizes of the vacuum chamber channel and that of this Port closely match each other.
With the exception of the “open” Port connecting the vacuum chamber conduit to the sub-AP of the analyzer device, all other Ports are configured to separate the sub-AP within the multiPort vacuum chamber from the higher pressure, typically AP, on the exterior of the vacuum chamber walls. These Ports may be fitted with valves, such as a ball valve to allow insertion of, e.g., a probe device from the higher-pressure region to be in fluid communication with the sub-AP of the multiPort vacuum chamber conduit in such a manner as to maintain the operating pressure (vacuum) at the analyzer device. The probe may be a device to expose analyte in a sample to sub-AP of the analyzer device, it may be a means to add a low gas flow in a specific region within the vacuum chamber, it may be a means to introduce a laser beam to a specific area of the vacuum chamber, among other uses. The Ports may also be fitted with a tube having a capillary inlet therethrough, or a skimmer cone with an opening therethrough to allow, e.g., gas flow, without the use of a valve, where the operating pressure of the analyzer device is maintained due to the small inner diameter of the tube or skimmer opening. Selection of one of the group of API, inlet ionization, vacuum ionization and auxiliary function maximizes utility through breadths depth, and simplicity, among other outstanding attributes. Excess Ports may be provided and capped for future use.
Two Ports may be used as API inlets, one for ESI and another for ASAP as examples; two Ports may be configured as inlet ionization inlets, such as SAI and MAI; and likewise two Ports can be dedicated to vacuum ionization inlets such as vMALDI and vMAI; or as is obvious to the practitioner of the art any other configuration or any other ionization source may be selected from the categories of API, inlet ionization and vacuum ionization not inclusively listed above. When so configured, it may be desirable that Ports not in use be sealed to prevent gas flow from AP into the vacuum chamber. Alternatively, as previously noted, one Port may serve multiple functions. In any case, the multiPort vacuum chamber assembly device may be configured so that changing ionization sources/methods and associated sampling devices, or other uses of the various possible Ports does not require venting the instrument and in most cases may be accomplished in minutes, if necessary. Alternatively, as disclosed below, additional Ports provide additional capabilities available for immediate use as well as to enhance the effectiveness of other ionization sources/sampling devices/methods on the multiPort vacuum chamber assembly. Auxiliary Ports are supporting functions other than that of a “traditional source”. As disclosed below, a ‘source’ docked on a certain Port can serve also as an auxiliary device to enhance another source docked on another Port, as, e.g., the gas flow through the inlet tube for API or Inlet ionization methods can alternatively be applied to enhance ionization in vMAI, and gaseous ion/charged particle transfer for vMAI and vMALDI.
With the multiPort vacuum chamber assembly, gaseous ion/charged particle transfer from AP to the ion optics of the analyzer device can be as efficient as the ion transfer with the commercial ion source. Thus, API methods are as sensitive, easy to operate, and have features found on the commercial “API-only” sources. This also is the case for inlet ionization, as well as vacuum ionization sources/sampling devices/methods. The reason is that with the multiPort vacuum chamber assembly, ion and charge particle transfer from the multiPort vacuum chamber to the ion optics of the analyzer device can be made as efficient as the ion transfer with the commercial ionization source and its associated ion optics by using ion extraction, focusing lens, ion guides, and/or gas flow known to practitioners of the art.
The multiPort vacuum chamber assembly may be configured to interface with different high- and low-performance commercial instruments designed for API by insertion between the commercial API source and the analyzer device and serves therefore as a high-performance extension of an ion/charged particle transfer region. Likewise, inlet ionization and vacuum ionization sources/sampling devices/methods may be applied to various instruments designed for API operation. This extended, ion/charged particle transfer region with its multiple Ports can be retrofitted on existing and on dedicated analyzer devices, and may have exchangeable or permanent features and functionalities selected by the user. For ionization methods that produce charged particles, the vacuum chamber assembly provides for additional desolvation of the charged particles and increased sensitivity. This is typically done by adjusting gas flow, use of a laser, and/or providing collisional surfaces through one of the Ports.
The vacuum chamber may contain ion optic elements to produce electric fields for extracting and transferring ions and charged particles from the respective Ports into the ion optics of the analyzer device. Gas flow (e.g., preferably air for ease and cost in use and alternatively nitrogen, argon, helium, or reaction gases, or combinations to sustain certain impact or reactions on the gaseous sample content) directs, in particularly, charged particles from the point(s) of origination into the ion optics of the analyzer device; whereas with ions, voltages are well known to be used to define and manipulate the ion paths. Through a Port, controlled gas flow may also be introduced and used to aid in removal of matrix (liquid or solid) from charged particles to aid release of the bare analyte ions. A Port may also be used to aid loss of matrix from charged particles by providing a means for a laser beam to impinge the charged particles thereby providing the energy necessary to aid removal of the matrix. A Port may also be used to enhance ionization through increased rate of sublimation of the matrix by providing energy to the matrix:analyte sample through gas/solid collisions. Heaters may be employed with Ports to provide optimum performance for the respective ion sources/sampling devices/methods used with Port(s) either globally or with specific Ports.
While dozens of ionization sources/sampling devices/methods and respective combinations may be used with this invention, we discus below at least one example from each general category of API (e.g., ESI), inlet ionization (e.g., SAI, MAI), and vacuum ionization (e.g., vMAI, vMALDI) and how auxiliary Ports may be used to enhance performance of a respective source/sampling devices/method, or how Ports may be used to enhance other Ports functions. This invention is not limited to the few specific examples provided in this text but the included examples instead give a general sense of what can be done, even with prominent and well-established sources/sampling devices/methods such as ESI and vacuum MALDI, in addition to inlet ionization and vacuum ionization sources/sampling devices/methods.
Other Port functionalities (auxiliaries) may be envisioned, including a Port for setup of remote sampling using a long tube made of PEEK, fused silica, or metal, as well as fixed (permanent) Ports which are not subject to exchange. The multiPort vacuum chamber assembly of this invention does not imply the invention of an ionization source or method. In fact, some of the ionization methods used in commercial ion sources can be readily converted to operate with patented ionization processes such as ASAP, DESI, DART, MAI, SAI, and VSAI. This is also true with the multiPort vacuum chamber assembly of this invention, and will require a license if these ionization methods are selected to be used. In this regard, the invention provides docking of API including so-called ambient ionization sources and methods, inlet ionization, and/or vacuum ionization sources/sampling devices/methods to the various Ports of the multiPort vacuum chamber and conduit therein to allow ready access to the principle categories of known ionization approaches and their nearly instantaneous, sequential or simultaneous use on a variety of analyzer devices by providing controlled temperature and gas flow as well as voltages and collisions favorably transferring ions and charged particles alike. This is possible because the disclosed invention facilitates and enhances the ion/charged particle transfer, without and with laser ablation/desorption. It is expected that this invention will also improve ionization approaches that may be invented in the future and may be docked to one of the Ports as desired.
An example is provided of how the multiPort vacuum chamber assembly may be used with mass spectrometry (MS), ion mobility spectrometry (IMS), and advanced capabilities such as MS/MS. Other uses and designs will be apparent to those practiced in the art. The multiPort vacuum chamber may be designed to fasten to the flange on the analyzer device that holds the commercial ion source provided for that analyzer device using an ‘O’ ring, gasket, or other means known to those practiced in the art to provide a vacuum-tight seal. In other words, the flange device may be made to fasten to a specific manufacturer's mass spectrometer or ion mobility spectrometer by replacing the instrument's commercial ionization source while the instrument is vented. This operation is only required the first time this invention is physically to be installed on an existing MS, IMS, or combination instrument. The flange device provides a vacuum seal with the instrument in a similar manner as the commercial API source, thus allowing the instrument to be pumped to the operating pressure. The commercial API source has electrical inputs which may be used if the commercial ion source is to be used with the vacuum chamber assembly in order to operate the ion source in the same manner as designed by the instrument manufacturer. In this case, the second end of the vacuum chamber assembly will be connected to a flange operationally identical to the flange the first end of the vacuum chamber assembly is attached to, i.e., the analyzer device flange that the commercial ion source connects to before modification. The disadvantage of using the commercial API source is that there is a physical limitation to the number of assessable Ports on the vacuum chamber assembly without extending its length. Also, voltages supplied with the instrument to operate the commercial source will not be readily available for functions which may be required to operate the various functions interfaced with the vacuum chamber assembly through its Ports (e.g., heaters, high voltages). The following sections describe the 4 categories of Port configurations and usages and are not limited to these examples.
One Port of the multiPort vacuum chamber assembly may have a capillary tube inserted through the Port linking the higher-pressure region outside the vacuum chamber walls with the sub-AP region inside the walls. The capillary tube may have lengths sufficient to extend from the conduit walls inside the vacuum chamber to exterior to the vacuum chamber and ranging from <1 mm to >1 m and where the longer length tubes are typically used for remote sampling. The tubes have a channel therethrough may have inner diameter between 0.1 to 1.5 mm allowing a restricted gas to flow from the higher-pressure region exterior to the vacuum chamber assembly to the lower pressure region inside the conduit. The capillary tube may be made of metal, fused silica, PEEK, or other materials known to those practiced in the art, and may be heated. This tube may be used to transfer gas phase ions, neutrals and charged particles from at or near AP to the sub-AP of the multiPort vacuum chamber where they are transferred into the analyzer device with electrical extraction and/or focusing lens operational by application of voltages, and/or gas flow (heated or not heated). The gas is typically air but other gases known to the practitioners are also applicable (e.g., nitrogen, helium, argon gas) may be used. Alternatively, a skimmer cone device or a plate with an opening slightly smaller than the inner diameter of the capillary tube may replace the capillary tube. The inner diameter (ID) of the capillary tube or skimmer opening or related restrictions must be sufficiently small to at least maintain and preferably be configured to easily control the operating pressure of the analyzer device. The gas flow acts as a carrier to aid transfer of ions and charged particles produced in the higher-pressure region, typically AP, into the ion optics of the analyzer device. Importantly, this gas flow may also be used to aid transfer of ions and charged particles produced directly in the low-pressure region to the ion optics of the analyzer device. Alternatively, the gas flow may be shut off as desired by, for example, capping the inlet.
Multiple Ports may be used for various probe devices and may be aligned such that one Port may be used to enhance ionization, transfer of gas-phase ions and charged particles to the analyzer device, and/or provide energy through collisions and heat transfer to aid removal of matrix from charged matrix:analyte particles. Probes may also be used for physical reasons such as a valve to block the conduit in a manner to seal the high pressure of the conduit from the analyzer device so that maintenance of the vacuum chamber assembly or changing of Ports may occur without venting the instrument. Alternatively, Ports may be used to provide a movable collision surface for, e.g., aiding removal of matrix from charged gaseous matrix:analyte particles or to cause intentional fragmentation. Voltages and additional heat can be applied to the surface. Any Port of the multiPort vacuum chamber, except the Port interfaced with the analyzer device vacuum, may be used for multiple purposes.
A Port may have a ball valve attached and configured to allow a probe device to pass through the ball valve when open so that a part of the probe device experiences the sub-AP of the multiPort vacuum chamber. The end of the probe may be inserted to a desired position within the channel (passageway) of the Port through which the probe shaft passes carrying the analyte or matrix:analyte to the optimum position for ionization to occur. The Port in this configuration may be a first tube having a channel open to the sub-AP of the conduit of the multiPort vacuum chamber on the first end and connected to the ball valve on the second end so that when the ball valve is in the closed position, without probe, no gas flows through the tube. On the opposite side of the ball valve is a second channel within a tube which is connected to the ball valve at the first end which is in fluid communication with the second end that is connected to a connector (fitting) such as a Swagelok fitting through a ferrule. The second end of the connector has a ferrule or ‘O’ ring which the probe rod passes through to make an airtight seal. When the ball valve is in the open position, the first tube and second tube with fitting are in fluid communication with one another and with the interior of the conduit of the multiPort vacuum chamber and thus with the analyzer device. In order to maintain operation pressure of the analyzer device, the second end of the channel passing through the first tube, the ball, and connector (fitting) must be sealed when the ball valve is in the open position to prevent venting the instrument which is common with the use of probe devices with vacuum ionization methods. The connector residing in the higher-pressure region, typically AP, provides a vacuum seal with the probe device passing through it. The connector may use an ‘O’ ring or ferrule to allow the probe to slide while preventing gas flow between the probe and seal. With the ball valve closed, the probe device may be inserted through the fitting so as to displace most of the air within the second tube connected to the ball valve on the high-pressure side. In this configuration, the ball valve may be opened and the probe may be inserted through the ball valve to a desired position with minimal gas load on the vacuum system. No additional pumping is necessary to maintain the analyzer device operational. The inner diameter of the first and second tubes and the ball valve may be 1/16″, ⅛″, 3/16″, ¼″, 5/16″, ⅜″, ¼″, as well as from <1 mm to >10 mm and other sizes which can interface with the multiPort vacuum chamber.
The probe device may be used to carry a sample containing analyte placed on the end that is inserted into the channel of the tube connecting the ball valve with the vacuum chamber assembly conduit or through the tube and into the conduit within the vacuum chamber assembly. Alternatively, the probe may contain a capillary within such as PEEK or fused silica tubing to be used to provide a gas flow to enhance ionization through sublimation of the matrix with vMAI, or to aid gas phase ion and particle transfer using vMAI or vMALDI. The probe may also have other uses such as the transmission of a laser beam from the higher-pressure region into the multiPort vacuum chamber conduit. Gas flow to enhance vMAI ionization and ion transfer in vMAI or vMALDI may be supplied into the first tube into which the probe is inserted using a tube having a small inner and outer diameter connected to the first tube so as to provide fluid communication between AP and the vacuum within the analyzer device. The second end of the vacuum chamber conduit may also have a Port to allow a low flow of gas for use in API sources/methods, or to provide gas flow over a probe inserted into the conduit downstream (towards the conduit interface to the analyzer device). If the probe end carries a vMAI matrix:analyte sample into or near the vacuum chamber conduit, the gas flow will enhance sublimation of the matrix and thus ionization of the analyte, as well as aid transfer of charged matrix:analyte particles and bare ions through the multiPort vacuum chamber conduit and to the analyzer device, as well as aiding loss of matrix (desolvation) from any charged particles to produce bare gas phase ions. The gas flow (or additional laser beam or obstructions, as examples) can also be used to enhance gaseous ion/(charged) particle loss of matrix molecules in vMALDI or vLSI, as well as by heating the gas.
One Port of the multiPort vacuum chamber assembly may be configured to interface the sub-AP of the vacuum chamber assembly with a plate device. In this case this Port of the multiPort vacuum chamber assembly is configured to have a flat surface on the high-pressure side of the Port with a channel linking the flat surface with the low-pressure region interior of the vacuum chamber conduit. By placing a flat plate over the flat surface of the Port, the lower pressure in the multiPort vacuum chamber is separated from the higher-pressure region by the plate, and gases from the higher pressure region are blocked from entering the conduit of the Port by the seal formed between two flat surfaces. With this arrangement, the plate can act as an ion source as described in Pophristic (pending patent). Briefly, in one arrangement, the plate may be a valve plate to block the flow of gas from the higher pressure into the sub-AP of the lower pressure in the vacuum chamber conduit by the seal formed by the contacting flat surfaces. The valve plate may fit into a groove to guide the plate along a path which goes over the open channel of the Port which is in fluid communication with the interior conduit of the multiPort vacuum chamber assembly. The valve plate typically have no channel or hole passing through it. The plate over the open channel leading to the interior of the conduit may also be a flat spacer plate, the spacer plate having a single or multiple channels (holes) or an adaptable grove cut therethrough. When the spacer plate covers the conduit of the Port, it acts as a valve blocking gas flow through the conduit so long as a channel or grove in the spacer plate is not aligned with the conduit opening. However, if a channel or groove is aligned with the conduit opening, the spacer plate must be covered on the high-pressure side to prevent excess air flow into the conduit. In this case, the spacer plate is sandwiched between the flat surface of the Port and a sample holder with no or limited gas flow therethrough. Thus, when the sample plate is placed in contact with the spacer plate so as to cover the channels in the spacer plate, no, or minimal, gas flows enter into the vacuum chamber as long as the spacer plate, sample plate, and Port surface are in contact and have flat surfaces to prevent or limit gas flow from the higher to the lower pressure regions. The spacer plate and sample plate make up a sample plate assembly which may slide over the flat Port surface without loss of the operating vacuum of the analyzer while sequentially exposing the channels in the spacer plate to the sub-AP of the vacuum chamber through the open conduit in the Port. By having flat edges, two sample assembly plates, a sample assembly plate and a valve plate, or two valve plates may slide with edges abutted to displace one another without loss of the operating vacuum of the analyzer device. One or both flat surfaces in contact with one another may use a polymeric material such as Teflon tape with a smooth surface adhered to the metallic or polymeric flat surface to enhance sliding the valve plate or sample plate assembly over the Teflon surface and maintain a vacuum seal.
With the plate device, matrix:analyte samples can be placed on a sample plate so as to align with the channels through the spacer plate. The sample plate assembly with the samples within the spacer plate channels is then placed in the grove in the Port device and by sliding along the flat face of the grove may displace the valve plate or other sample plate assembly and sequentially expose each sample in each spacer plate channel to the channel of the Port which leads to the interior conduit of the multiPort vacuum chamber assembly. With the spacer plate arrangement, samples on the sample plate do not come in direct contact with flat surface of the Port.
Alternatively, a paper substrate such as filter paper may be used to receive the vMAI samples from solution and the paper substrate with dried samples placed between the spacer plate, so that the samples align with the channels in the spacer plate, and an air impermeable membrane or plate. This plate in this configuration may be made of glass, metal, or polymeric material. By using a paper substrate, the ion signal from the analyte is stable and more reproducible. This is especially valuable with blood spotted on paper to show drugs, lipids, and even small proteins providing a quick method for blood spot analyses, as well as other biological materials such as urine. For example, MS/MS and IMS can be used to increase the specificity, sensitivity or add an additional separation dimension to an MS dimension. The number of samples which can be analyzed from a single sample plate, especially using filter paper substrate is flexible, typically ranging from 1 to 12. Continuous supply of sample spots can be envisioned (conveyer belt-like). Exposure of a single sample to vacuum and ionization, acquire a mass spectrum, and release the sample substrate requires as little as 5 sec in the current format. Eight or twelve samples may be spaced apart identical to linear rows of a 96-well microtiter plate. In this case, loading and removing the sample substrate requires 3 to 4 sec and each of the samples can be acquired every 4 sec by, e.g., vMAI, LA-vMAI, and less than 1 sec with vMALDI depending on the matrices or matrix combinations used (and with less volatile combinations a laser). Further, paper used with paper chromatography or thin layer chromatography (TLC) may be used and compounds in samples separated as is commonly done in paper chromatography or TLC. For this kind of analyses, a spacer plate with a grove therethrough is used. The groove must be configured to maintain the instrument vacuum in an operational range. Using TLC or paper chromatography, dirty or salty samples can be cleaned up without much effort and time before acquisition using vMAI. TLC plates with, for example, silica surface can also be used with this approach. Glass or plastic backed TLC plates may be used without the need of an air impermeable plate added separately. Likewise, backings for filter paper, such as a polymeric coating, can replace the need of a separate backing plate such as a glass microscope slide. Such a backing can be used to make the entire sample plate and backing disposable. Paper sample plates also allow bar codes for keeping track of samples, however, other sample supports can be used for the same purpose (e.g., glass, membranes). An important aspect of paper-based sample plates, besides providing smaller and more reproducible crystallization, is that they also provide a controlled limited flow of air over the sample. It is also an effective means to sampling surfaces by wiping over an area of concern, e.g., to investigate for fentanyl, and directly on-site as well as for collection/transport/shipping purposes to a more sophisticated lab for detailed high performance analyses (prescreening for samples worthy of detailed investigations) using the vacuum chamber assembly interfaced with a high-end high-performance instrument.
Selecting the paper and backing (e.g., polymer film) allows control of gas flow and crystallization, factors that have important consequences for reproducibility and quantification. Magnetic strips having a sticky side for adhering filter paper have also successfully been used as backing of filter paper. In addition, plastic having a sticky side and spaced holes can adhere to the filter paper and act as a spacer plate. In this case the paper sample substrate is sandwiches between two plastic sheets, preferentially stiff and clear. Polystyrene is good for this purpose because it lacks ionizable background. Such an arrangement may provide a sampling device for fit for purpose analyzer devices (e.g. illicit drug detection). Realistically, certain filter papers and membranes are made for best applications (see, e.g., Whatman product line) but in general filter papers have a wide range of applications as a sample holder in conjunction with this invention. Even printer paper can be used; good choices are materials that have gas flow and little chemical background when ionization occurs; if there is “too much gas flow”, this can be limited by backing devices. In a few cases, it might be advisable to match the substrate used to hold the sample with a specific application, e.g., dilute nitric acid solvent may not be compatible with some filter papers. In any case, uranyl nitrate can be readily analyzed from glass plates using >2% nitric acid in acetone, water, or ethanol. Similarly, to obtain the highest sensitivity, reproducibility, and accuracy one can choose specific life science (e.g., protein) filter papers, although this is not necessary but an option for high end applications such as accurate mass detection/analyses of proteins by coupling gels (e.g., SDS-PAGE gels via, e.g., filter paper media) with MS of this invention. Any plate used to separate the sample from the flat face of the Port, is referred to herein as a spacer plate. The plate source is adaptable to. automation for, e.g., high throughput applications with e.g. barcodes as an option. In other words, there is significant flexibility to the user's specific needs.
With vMAI, the ionization event leading to gas phase analyte ions begins when the matrix:analyte sample, using a vMAI matrix, is exposed to sub-AP conditions. Alternatively, across the conduit of the multiPort vacuum chamber assembly there may be another Port through which a laser beam can pass and strike the sample applied using a plate source or probe source, in this case composed of a MALDI or LSI matrix and thus producing gas phase ions by reflection vacuum MALDI or LSI. In the MALDI mode the Port providing the laser beam can have an optical lens embedded in the Port to focus the beam on the matrix:analyte surface for ionization. Specific angles can be implements either through or relative to the Port. An aperture can be used without a lens element as long as the aperture is sufficiently small to not cause detriment to the vacuum, especially using fiber optics cable to transmit the laser beam. In this case a larger surface area is ablated using higher laser energy. In order to extend the area being sampled with both methods, a convenient means is to have the laser beam strike the sample surface off-center of the probe end allowing new surface to be accessed by simply rotating the probe, or with a plate, moving the plate. If the sample plate is made of material, such as a glass microscope slide which can transmit a laser beam, then transmission geometry laser ablation can be used in which frequently a single or a few laser shots are sufficient for good analyte ion abundance. In either case, the laser beam can be transmitted and focused using optical lens and mirrors where necessary, or using fiber optics. In either case, with sufficient laser power, ionization can also be accomplished without a matrix, as in laser desorption/ionization (LDI), or with a laserspray ionization (LSI) matrix. Any use of the plate source may be enhanced by gas flow or voltage. For example, simply placing a voltage, between about 20 V and 1000 V, and preferably between about 100 V and 600 V, on the sample plate or the spacer plate, if metal (positive for positive ions and negative for negative ions), increases the ion abundances observed. As examples, larger molecules, such as proteins, have increased ion abundances when voltage is placed on the substrate, or spacer plate, if metal, whereas with smaller molecules (e.g., drugs and molecular salts as examples, application of voltage can be used to cause in-source fragmentation, however the utility is somewhat limited to relative clean samples. For direct MS approaches, mass selected MS/MS is the better options to increase both the sensitivity and specificity of the analyses.
The multiPort vacuum chamber assembly may have means to provide focusing and/or ion extraction and/or repulsive fields to aid transfer of charged species into the ion optics of the analyzer device. Electrical feedthroughs for the ion optics may be permanently provided or by use of, e.g., a probe device, likewise, lens elements may be permanently mounted as part of the multiPort vacuum chamber assembly or in the case of providing repulsive or extraction fields, lens elements may be inserted by use of a probe device. Focusing lenses or ion guides as known to those practiced in the art may be used within the multiPort vacuum conduit, within the open Port interfacing the vacuum chamber conduit with the analyzer device, or just exterior to the vacuum chamber conduit in for example a Port channel,
Typically, the most accessible Port will be used for the most used ionization method, and the most accessible Port is usually the one at the second end of the conduit directly opposite the entrance to the analyzer device. However, in some cases a Port is dependent on another Port for functionality. As an example, reflectron geometry vMALDI requires that a Port from which the laser beam is introduced is best positioned across from the vMALDI Port. Another advantage of this invention is that whatever configuration is selected, it can be easily adapted to another configuration if needs change. In addition by incorporating extra blank Ports, new configurations can be explored in the future.
The advantages of having mass spectrometers, ion mobility spectrometers, or combinations of these incorporate the multiPort vacuum chamber assembly is that the user has at their disposal, for relatively low costs, a wider variety of ionization methods and means of optimizing the analyses than is available with either API, inlet ionization, or vacuum ionization methods alone. For instruments purchased for a specific purpose, the multiPort arrangement more readily allows repurposing the instrument by selecting the most appropriate ionization source/sampling device/methods (approaches) for the new purpose whether API, inlet ionization or vacuum ionization, or all of them together or select combinations. Finally, the multiPort vacuum chamber of the current invention, provides the capability of using newer ionization technologies not currently available on commercial mass spectrometers. These include vMAI, MAI, SAI, VSAI, and vLSI providing ionization capabilities with exceptional sensitivity and simplicity for many compound classes. Simple, reliable, quick, and safe (avoiding exposure) operation for the professionals working with bodily fluids, trying to contain the problem (e.g., spill, forensic evidence), or trying to determine if uranyl nitrate or related compounds are present representing nuclear enrichment requires high sensitivity, specificity, reliability and accuracy, and prompt turnaround, enabled by this invention for this and other extremely important applications for which current approaches are more limited or fail. These requirements are fulfilled by this invention.
A summary of some of the advantages or the multiPort vacuum chamber assembly are: (1) The methods of this disclosure do not rely on an additional pumping stage in addition to pumping already available with API mass spectrometers, although someone trained in the art can readily retrofit such capabilities for certain configurations with expected higher pumping requirements; (2) The time required to expose a single sample to the sub-AP region of the mass spectrometer on a sample plate device is rapid, reducing the time from as much as minutes with some commercial systems which use vacuum locks, to as little as two to three seconds; (3) The disclosed inventions can be used to rapidly sequentially acquire mass spectra from different samples while manipulating from AP the substrate that holds the samples; (4) The device of the disclosed invention can be used with (retrofitted to) commonly available commercial mass spectrometers designed for API or with dedicated new mass spectrometer designs; (5) The disclosed systems and methods are robust in avoiding carryover and instrument contamination and the assembly is configured for easy access to cleaning without the necessity of venting the instrument; (6) The disclosed systems and methods are inherently simple and low cost; (7) The disclosed multitude of Ports assembly allows multi-mode vacuum ionization including matrix-based methods such as vMALDI, vMAI, and vLSI, as well as matrix-free methods such as LDI, as examples; (8) The multiPort assembly supports multi-mode ionization from AP including but not limited to APCI, APPI, ASAP, ESI, nanoESI, DESI, nanoDESI, MAI, MALDESI, MALDI, SAI and acoustic ionization methods as examples, without having to vent the instrument when switching between ionization-modes by as little as a software command; (9) In (v)MALDI and (v)LSI, laser ablation (LA), laser desorption/ionization (LDI) can be by transmission geometry where the laser beam ablates the sample from the backside of the sample (plate) for convenience, speed (even with, inexpensive low repetition lasers), and spatial resolution, or by reflection geometry laser alignment where ablation is frontside (on the side of gaseous ion/(charged) particle transmission) and potentially more readily assessable for in vivo applications and/or with weaker lasers (e.g., LA-SAI, LA-MAI).
Reflection geometry laser alignment is key for samples, conditions, and requirements where the laser cannot or should not have to penetrate through the sample support or the entire sample itself, e.g., metal, certain glass (wavelengths), thin layer chromatography plates, micro- and nanostructured surfaces, paper, filter paper, membranes, as a few examples other than in vivo applications such as skin cancer or other cancers such as breast, head, neck, prostate, uterus, or colon associated with surgeries in which high accuracy, precision, and nearly instantaneous feedback of results is of extreme advantage for improved patient outcome and mounting costs. In certain configurations (inlet ionization), remote sampling with and without the use of a laser (e.g., surface SAI, LA-SAI, LA-MAI) or other means of dislodging (e.g., by simple and safe extraction protocols) a sample into the sub-AP and ultimately into the inlet tube, preferably heated, which serves as a unique ion source as it ionizes “on the fly” molecules traversing this region from higher pressure to lower pressure. Adduction of metal cations for providing the charge to the compounds are induced with higher inlet tube temperatures using liquid matrices and with certain matrices (e.g., bronopol) the attachment of, e.g., sodium cation(s) is achieved also at low or no added heat (e.g., under vMAI conditions). 1,2-Dicyanobenzene matrix works well in ionizing molecules in the negative mode; associated voltages can well exceed >300 V. Selectable ionization sources, sampling devices, and methods are disclosed herein enabling the best choice(s) for deciphering the composition of a sample of interest or serving the many needs of multiple users on the same mass spectrometer in a time and cost-effective way as attractive benefits of this multiPort vacuum chamber assembly and its broad and in-depths applications at little additional cost time and expertise.
These and other features and advantages of the present systems will be more fully disclosed by the following detailed description of the preferred embodiments, which are to be considered together with the accompanying drawings wherein the numbers refer to like parts and features wherein:
This description of preferred embodiments is intended to be read in connection with the accompanying drawings, which are to be considered part of the entire written description. The drawings/figures are not necessarily to scale and certain features of the invention may be shown exaggerated in scale or in somewhat schematic form in the interest of clarity and conciseness. Terms concerning attachments, coupling, interface, and the like such as “connected”, “interfaced”, and “interconnected”, refer to a relationship wherein structures are secured or attached to one another either directly or indirectly through intervening structures, as well as both movable and rigid attachments or relationships, unless expressly described otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Unless otherwise stated, all percentages, parts, ratios, or the like are by weight. When an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this is understood as specifically disclosing all ranges from any pair of any upper range limit or preferred value and any lower range limit or preferred value regardless of whether those ranges are explicitly disclosed.
The vacuum chamber assembly conduit [104] may have an interior [101] which may have the shape of a cylinder with one end providing fluid communication with the vacuum of the analyzer device [150], and the other end having a first Port [110] which may hold an inlet tube [113]. The conduit also has Port [120] which may serve as a means to introduce sample on a substrate into the sub-atmospheric pressure (sub-AP) existing in the conduit and the first tube [127] of [120] where ionization of the sample occurs. The Ports containing channel [111] and [121] provide fluid communication between [101] and a region of AP or near AP [103] on the exterior of the vacuum chamber assembly [100]. The Ports are configured to maintain operational and optimum pressure in [101] and analyzer device [150] through the use of gas flow restrictions as in [111] and valves [123] of [120], and additional Ports that may be interfaced with [101] to provide conditions that are synergistic with other Ports (gas flow, voltage, vacuum seal, collision, laser, and related means). Connected to Port [120] may be a ball valve [123] which in the closed position prevents gas flow from the higher pressure region [103] into the conduit [101] and when open provides fluid communication between the higher pressure region [103] and [101], which would vent the instrument unless the gas flow is blocked by inserting the probe rod [141] into connector [143] having ferrule or ‘O’ ring [144] shown in
Tube [113] with a restricted channel [111] can be used to retain the vacuum of the analyzer device while being open to [103] and used to supply a reactive gas (e.g., a gaseous composition that allows the molecules to react within the channel causing chemical alterations or aid fragmentation), supply a flow of gas (e.g., air, nitrogen, helium, argon) to aid transfer of ions, charged particles and neutral particles in, e.g., Port [120], and also aid sublimation of vMAI matrices which hastens the ionization of analyte for, e.g., more rapid analyses, or to hasten loss of matrix molecules (“desolvation”, “declustering”) from charged particles withing [101]. Alternatively, gas (e.g., reactive gas) can be employed to cause certain reactions. Additionally, with the configuration shown, a voltage may be placed on tube [113], provided [113] is not grounded, to repel ions and charged particles produced in Port [120] when they move into the conduit [101] of vacuum chamber conduit [104].
Additionally, Port [120] can be used to provide a small (regulated) gas flow that directly impinges the matrix:analyte sample on the tip of rod [121] of probe [140]. This gas flow increases the analyte ion abundance observed with vMAI and other methods and importantly allows good analyte ion abundance when the probe tip is at a distance from conduit [104], which is especially useful for the plate vacuum source described in
Unlike in
Just as Port [120] may be used for vMALDI, vLSI, or vMAI, the other Ports in a multiPort system may likewise have dual functions supporting each other synergistically and elevating the entire system to be greater than the sum of the individual parts. For example, Port [110] interfaced with an AP inlet tube [115](or other gas carrying extensions), that traditionally are configured to perform API or inlet ionization, may be used to supply gas to and through conduit [104] of the multiPort vacuum chamber assembly device [100] to direct ions and charged particles (and neutral particles) from the probe surface associated with Port [120] into optional extraction lens [105] and/or ion guide/focusing lens [106] of the multiPort vacuum chamber [100]. The sum of all gas flows must be adjusted to remain within the operational range of the analyzer device [150] and preferable at an optimum pressure for transfer and detection of gas phase analyte ions. A low voltage may be applied to inlet tube [113] in Port [110] to separate gaseous ions of opposite charge and repel gaseous ions of a desired charge into the ion optics, and to provide energetic collisions for charged particle loss of matrix (desolvation) and for analyte fragmentation when desired. Collisions of gaseous ions and charged particles with physical obstacles may also be employed to either enhance declustering and desolvation of charged particles and neutral particles or cause fragmentation of gaseous analyte ions. Likewise, extraction lens [105] may use voltages to attract gaseous ions and charged particles of opposite sign into the optional ion guide/focusing lens [106] of [100] and into [151] of analyzer device [150]. The attractive force is able to do all the aspects noted for the repulsive force. The two forces may be used simultaneously. Depending on the polarity, positively charged ions or negatively charged ions are detected at the detector associated with the analyzer device. The gas flow also aids sublimation of the vMAI matrix, thus enhancing the vMAI process of forming gaseous analyte ions. The gas flow can be controlled, allowing tuning for the highest analyte ion abundances. vLSI and vMALDI employ less volatile matrices so that the sublimation of the matrix and declustering of charged and neutral particles are more limited. Port [130] with the laser beam from laser [160] may in addition to transmitting a laser beam for reflection geometry laser ablation, also be used for gas flow directly towards the matrix:sample surface. With the proper design, Port [130] or [120] may be used to insert a probe [140] with a flexible end to allow conduit [104] to be sealed to gas flow from, e.g., the inlet tube [113] with channel [111] of Port [110] into the analyzer device [150]. Thus, in the multiPort design of this invention, each Port can have multiple uses, at least one of ionization source, method, sampling and auxiliary function, all readily accessible without venting the instrument or having to physically change hardware. A further distinct advantage of the multiPort design is that it may be made to interface with almost any API source/method of commercial analyzer devices in such a way as to insert [100] between the instruments API source and the analyzer device [150]. Inlet ionization sources/methods are also readily configured on such instruments. Additionally, at least some sub-AP vacuum ionization source/method operating with an intermediate pressure ionization source instruments can also be equipped with the multiPort front end to access API, inlet ionization, as well as vacuum ionization sources and respective sampling devices and methods on the same mass and/or ion mobility spectrometer and related instruments. Two of the same ionization sources/sampling devices/methods can be used on two different Ports to, e.g., improve measurements (quantification, mass accuracy, specificity, speed of analyses, sensitivity, as examples) or to minimize cleaning (avoiding switching between very different compound classes, e.g., polymer, drugs, proteomics versus lipidomics samples). Having two inlet tubes connected to [100] allows each to be used simultaneously or sequentially with automatic capping when momentarily not in use to improve throughput or for hyphenation with liquid chromatography or surface analyses devices without or with a laser. Remote sampling including imaging as well as portable applications are other applications also without or with a laser with or without the addition of a matrix (solid or liquid, or combinations) are enabled or improved through this invention.
A Port in certain configurations may be a “dock” and more precisely it can be a connector system that retains the vacuum within conduit [104] that is preferably created by the existing vacuum pump(s) of the analyzer device [150]. While the ionization sources and functionality (methods, sampling and auxiliary devices) associated with a Port or Ports may be regarded as permanent fixture(s)/choices, each Port may be used for different purposes as needs change, allowing flexible interchange even between API, inlet ionization, and vacuum ionization sources. This invention accomplishes this challenge because of conditions that enhance both gaseous analyte ion and charged particle transfer simultaneously minimize discrimination effects in current instrumentation. A key aspect of this invention is the supporting effect of Port(s) relative to each other's performance.
In
Alternatively, MALDI matrices can be used in conjunction with laser ablation to produce gaseous analyte ions by the MALDI process. Depending on the matrix, vLSI or no matrix, vLDI, may be performed. Port [130] may be used for features discussed above for the probe sample introduction, including passage of a laser beam. By using a glass or quartz sample plate such as a microscope slide, a laser beam may pass through the plate to strike the sample in so called transmission geometry. Thus, MALDI may be accomplished in transmission geometry as compared to reflection geometry, described above. At least parts of this assembly configuration can be automated and be further enhanced by robotics, as example. Certain gas flows from the same Port or adjacent (auxiliary) Port(s) can be used to improve the analyte ion abundance, speed of analyses, cleanliness of an experiment (no carryover), t name a few of the advantages enabled by invention [100].
The plate source does not need to be physically close to the vacuum conduit [104] for gaseous analyte ions and charged particles to reach conduit [104], but if at a distance, a low repulsive voltage or gas flow may be required or preferred to aid efficient gas phase ion and charged particle transfer. Alternatively, an extraction lens, such as [105] may be necessary to aid transfer of gaseous ions and charged particles into the vacuum of conduit [104]. It should be understood that ion transfer elements may also be incorporated such as a tube lens known to those practiced in the art, but may be less effective in transferring charged particles due to mass discrimination. Because gaseous ions and charged particles from any of the vacuum ionization sources/methods may enter the vacuum conduit [104] orthogonal to the direction between Port [110] and analyzer devices [150], gas flow and/or repulsive voltage from the inlet tube [113] and/or extraction voltage on the extraction lens [105] may be used to turn the gaseous ions/charged particles in the direction of the lens elements [151] leading to the mass or ion mobility analyzer device or related device [150]. It should be understood that with the plate source as with the probe source, other synergistic Ports may be incorporated and strategically employed to, for example, aid in desolvation of charged particles and thus improve ionization efficiency and sensitivity. Heater [116] may be used to heat the gas to aid this process. Ports and sampling devices/materials can also be used to aid this process. Some of the Ports may be made to be interchangeable, and others to be machined for purpose. Nevertheless, the simplicity of the design used here reduces the machining costs. As an example, for some applications, it may be desired to have the probe or plate source in the position shown in
This application claims the benefit of the filing date of U.S. Ser. No. 63/456,564 which was filed on Apr. 3, 2023 and U.S. Ser. No. 63/470,989, which was filed on Jun. 5, 2023. The content of these applications are incorporated by reference herein in its entirety.
This invention was made with U.S. government support under NSF SBIR Phase I 1913787 and NIH SBIR Phase I (NIH 1 R43 AI172667-01A1) to MSTM, LLC. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63470989 | Jun 2023 | US | |
| 63456564 | Apr 2023 | US |
