HK1190819B - Ionization source apparatus and method for mass spectrometry - Google Patents

Description

Ionization source apparatus and method for mass spectrometry

This application is a divisional application filed on the filing date of 2007, 5 and 9, under the filing number 200780016909.4, entitled ionization source apparatus and method for mass spectrometry.

Technical Field

The present invention relates to the field of mass spectrometry, and more particularly to an apparatus and method that allows ionization of different chemical compounds using a unique ionization source, with greatly improved sensitivity compared to common Electrospray (ESI) and Atmospheric Pressure Chemical Ionization (APCI) techniques.

Background

Mass spectrometry is a widely spread technique for the analysis of various polar and non-polar compounds. In particular, liquid chromatography has been used to analyze compounds of various polarity degrees and various molecular weights. In fact, the characterization and quantification of these compounds is of great interest, and new methodologies are continually being developed for their analysis. In recent years, various techniques have been developed for analyzing various molecules by mass spectrometry. For example, the analysis of addictive drugs is one of the fields that liquid chromatography-mass spectrometry has recently made a great contribution. (Cristoni S, Bernardi LR, Gerthoux P, Gonella E, Mocarreip. Rapid Commun. Mass Spectrum.2004; 18: 1847; Marquet P, Lachatre G.J.Chromatogr.BBiomed. Sci appl.1999; 73: 93; Sato M, Hida M, Nagase H.Forensic Sci. int.2002; 128: 146). In particular, the technology enables the analysis of addictive drug compounds directly from urine samples without undergoing derivatization reactions. (Cristoni S, Bernardi LR, Gerthoux P, Gonella E, Mocarlli P. Rapid Commun. MassSpectrum.2004; 18: 1847). In fact, when these compounds are analyzed using gas chromatography-mass spectrometry (GC-MS), the reaction is indispensable, thereby increasing the cost of the analysis. Another area of interest is the analysis of macromolecules such as proteins, peptides and oligonucleotides. (Kim SY, Chudapongse N, Lee SM, Levin MC, Oh JT, Park HJ, Ho IK. brain Res.mol. brain Res.2005; 133: 58; Cristoni S, Bernardi LR. Mass. Spectrum. Rev.2003; 22: 369; Cristoni S, Bernardi LR, Biuno I, Tubaro M, Guiduglmess F. Rapid Commun. Mass. Spectrum.2003; 17: 1973; Willems AV, Deforme DL, LambertWE, Van Peteghem CH, Van Bocxlater JF. J. Mass. A.2004; 93. once these molecules have passed through the ionization source, the charged molecules are analyzed using a mass spectrometer (ion trap (IT), time of flight (TOF) spectrometer (TOF, TOF Q-25Q-bar, FTQ-25, four-level FTQ-bar, et al.

The ionization source is a key component in mass spectrometers. It converts neutral molecules into ions that can be analyzed by mass spectrometry. It must be emphasized that in order to ionize the analyte, various ionization sources need to be used, since various physicochemical ionization effects need to be used according to the physicochemical characteristics of the compound to be ionized. Indeed, the most used ionization sources are Electrospray (ESI), Atmospheric Pressure Chemical Ionization (APCI) and matrix-assisted laser desorption ionization (MALDI) techniques, which are highly efficient for generating ions in the gas phase for further analysis by mass spectrometry (Cristoni S, Bernardi LR. MassSpectrum. Rev. 2003; 22: 369). ESI and APCI operate on liquid phase samples, while MALDI is used to analyze solid phase samples.

In the case of ESI, a strong electric field is used to evaporate and ionize the analyte. In this case, multiply charged ions (giving more than one signal per molecule) of medium/high molecular weight compounds (e.g. proteins and oligonucleotides) are generated. The mass spectra thus obtained are difficult to analyze and can be analyzed using specific software algorithms (Pearcy JO, Lee TD.J.Am.Soc. Mass. Spectrum.2001; 12: 599; Wehofsky M, Hoffmann R.J. Mass. Spectrum.2002; 37: 223). Low molecular weight compounds typically produce a mass spectrum that is easier to analyze due to the formation of singly charged ions (only one signal per molecule). Therefore, such ionization sources are mainly used for analyzing medium-polar and high-polar compounds having low, medium, and high molecular weights.

In the case of APCI, the sample is first vaporized at high temperature (250-. This ionization pathway can be used to analyze low molecular weight (molecular weight <600Da) and medium low polarity (e.g. steroids, etc.) compounds.

In the case of MALDI, low charge state molecules (typically singly or doubly charged ions) are generated. In this case, the analyte co-crystallizes with a matrix compound which can absorb ultraviolet light with a wavelength of 337 nm. The co-crystallized sample is then placed in a vacuum zone (10)^-8Torr) and irradiated with an ultraviolet laser having a wavelength of 337 nm. A microexplosion phenomenon, called "ablation", occurs at the crystal surface, allowing the analyte and the matrix to be vaporized. In addition, the analyte is ionized by various reactions that typically occur between the analyte and the matrix. This approach is commonly used to analyze high molecular weight compounds with various polarities.

All of the ionization approaches described above are not suitable for analyzing non-polar compounds such as benzene, toluene, etc. Thus, a new ionization source, called atmospheric pressure photoionization, was developed and used to analyze various compounds (Raffaelli A, Saba A. MassSpectrum Rev. 2003; 22; 318). As in the case of APCI, the liquid sample solution is vaporized at high temperature. The analyte is then irradiated with ultraviolet light (10eV krypton (Kr) light) and ionized by various physicochemical reactions, mainly charge and proton exchange and photoionization reactions.

A new ionization pathway, called "surface activated chemical ionization-SACI", has also recently been developed to improve the performance of commercial mass spectrometers in analyzing various compounds extracted from biological matrices (PCT No WO 2004/034011). The device is based on the introduction of a surface for the ionization of neutral molecules in an atmospheric pressure chamber. SACI (Cristoni S, Bernardi LR, Biuno I, Tubaro M, Guidougli F. RapidCommun. Mass. Spectrum.2003; 17:1973) has been obtained by upgrading Atmospheric Pressure Chemical Ionization (APCI) sources. In fact, it was observed that the introduction of an element with a discotic active surface into an APCI ionization chamber could produce a high sensitivity beyond expectations and make it possible to probe molecules with a wide range of molecular weights (Cristoni S, Bernardi LR, Biuno I, Tubaro M, Guiguguli F. Rapid Commun. Mass Spectrum.2003; 17: 1973; Cristoni S, Bernardi LR, Gerthoux P, Gonella E, Mocari. Rapid Commun. Mass. Spectrum.2004; 18: 1847; Crist S, Scanambr M, Bernardol I, Biuno I, Gerthoux P, Russo G, Chiumello G, Mora S. Rapid Commun. Mass Spectrum.2004; 18: 1392).

However, there is no ionization source that can gently ionize all compounds. This is mainly due to their different physicochemical properties, and therefore requires the use of different physicochemical effects to ionize the analyte.

Objects and description of the invention, and improvements over the prior art

The present invention relates to a method and apparatus called Universal Soft Ionization Source (USIS) (fig. 1) capable of ionizing all classes of compounds and increasing the instrument sensitivity with respect to the commonly used Atmospheric Pressure Ionization (API) technique. The core of the present invention is based on a surface on which various physicochemical stimuli are combined to amplify the ionization effect. This approach is very different from SACI (PCT No WO 2004/034011). In practice, SACI uses an ionization surface inserted into an Atmospheric Pressure Ionization (API) chamber and ionizes the sample simply by applying a low voltage (200V) to it. The main difference with the current USIS technology is that only moderately to highly polarized compounds can be ionized by SACI. Thus, although higher sensitivity can be achieved, the class of compounds that can be ionized is the same as ESI. It must be noted that the USIS technique yields a strongly improved sensitivity compared to the ESI and APCI techniques. The application of various physicochemical stimuli (UV light, tunneling, electrostatic potential, ultrasound and microwaves) on surfaces makes it possible to strongly ionize the analyte of interest and to reduce the ionization of solvent molecules which can lead to an increase in chemical noise and thus to a decrease in the S/N ratio. It has been observed that analytes are typically soft ionized by charge transfer or proton transfer reactions (the ions of the analyte do not fragment in the ionization source and reach the detector intact).

Another innovative aspect of the present invention is the possibility to be used in a wide range of experimental conditions. Typically the ESI and APCI ionization sources are operated using different flow rates of analyte solution into the ionization chamber. In particular, ESI is typically operated at ionization flows below 0.3mL/min, while APCI operates in the range of 0.5-2 mL/min. Due to the specific combination of physicochemical ionization effects, the USIS ionization source can operate over the entire flow range (0.010-2 mL/min). This makes it possible to analyse any compound with high instrument sensitivity and strongly increases the versatility of mass spectrometry instruments operating in liquid phase.

Brief description of the drawings

FIG. 1 shows a schematic view of a

One embodiment of a USIS ionization source according to the present invention is shown. The parts of the apparatus are: (1) a mass spectrometer inlet, (2) a USIS flange, (3) a cavity, (4) a surface, (5) a connector, (6) an assembly device, (7) a power connector, (8) a screw, (9) a screw, (10) a sample access hole, (11) an inlet assembly, (12) a nebulizer region, (13) a charging region, (14) a nebulizer gas line, (15) a nebulizer gas line, (16) a power connector, (17) a screw, (18) a screw, (19) an assembly, (20) a power connector, (21) a UV-VIS or IR laser or lamp, (22) a UV-VIS or IR laser or lamp, (23) a power connector for ultrasound applications, (24) a power connector for a lamp or laser, (25) a tube under vacuum or gas pressure, (26) a power supply, (27) a power supply, (28) a power supply, (29) power supply, (30) power connector, (31) power supply.

FIG. 2 (Tunnel effect)

An enlarged view of the ionization surface used in the USIS ionization approach.

FIG. 3

Proton transfer ionization reactions that can occur using USIS. In this case, the molecules are solvated by solvent molecules (clusters). The surface (4') is excited with various effects (ultrasound, UV light, electrostatic potential) to concentrate the energy of these physical effects on the surface. When the cluster containing the solvent hits the excited surface (4'), the solvent is detached from the analyte, generating positive or negative ions due to proton exchange or other kind of reaction. Various effects applied to the surface provide activation energy to strongly enhance the ionization activity. The ionization step is: A) spraying the clusters onto the surface using a nebulizer jet (2.5L/min or higher), B) collision of the clusters with the surface, and C) ionization of the analyte on the surface after the solvent has been detached by reaction with the excited surface.

FIG. 4

USIS ionization source

FIG. 5

Full scan mass spectra obtained from analysis of a 50ng/mL solution of MDE using a) APCI, b) ESI and c) USIS ionization sources, respectively. The sample was dissolved with water. The flow rate of the directly injected sample was 20. mu.L/min. The surface potential, electrospray tip voltage (13) and surface temperature were 50V, 0V and 110 ℃ respectively. The UV lamp and ultrasound were turned off. The gas flow rate of the sprayer is 2L/min.

FIG. 6

MS/MS mass chromatograms obtained by analysis of MDE contained in urine samples using a) APCI, b) ESI and c) USIS ionization sources, respectively. Urine samples were diluted 20-fold prior to analysis. The gradient was carried out with two phases: A) water + 0.05% formic acid and B) CH₃CN + 0.05% formic acid. In particular, 15% of phase B was kept for 2 minutes and then a linear gradient from 15% to 70% was performed over 8 minutes, reaching the initial state within 2 minutes. The acquisition time was 24 minutes to re-equilibrate the column. A thermoelectric C8150 x1mm column was used. The eluent flow rate was 100. mu.L/min. The surface potential, electrospray tip voltage (13) and surface temperature were 50V, 0V and 110 ℃ respectively. The UV lamp and ultrasound were turned off. The gas flow rate of the sprayer is 2L/min.

FIG. 7

The full scan mass spectra obtained for the 100ng/mL arginine standard solution were analyzed using a) APCI, b) ESI, and c) USIS ionization sources, respectively. The sample was dissolved with water. The flow rate of the directly injected sample was 20. mu.L/min. The surface potential, electrospray tip voltage (13) and surface temperature were 50V, 0V and 110 ℃ respectively. The UV lamp was turned off and the ultrasound was turned on. The gas flow rate of the sprayer is 2L/min.

FIG. 8

MS3 mass chromatograms obtained from arginine extracted from human plasma samples were analyzed using a) APCI, b) ESI, and d) USIS ionization source, respectively. The gradient was carried out with two phases: A) CH (CH)₃OH/CH₃CN 1: 1+ 0.1% formic acid + ammonium formate (20. mu. mol/L) and B) H₂O + 0.1% formic acid + ammonium formate (20. mu. mol/L). Arginine was extracted from plasma using a protein precipitation method based on phase a as protein precipitant. Analysis was performed using 4% B under isocratic conditions. The acquisition time was 6 minutes to re-equilibrate the column. A water SAX 100x 4.1mm column was used. The eluent flow rate was 1000. mu.L/min. The surface potential, electrospray tip voltage (13) and surface temperature were 50V, 0V and 110 ℃ respectively. The UV lamp was turned off and the ultrasound was turned on. The gas flow rate of the sprayer is 2L/min.

FIG. 9

Full scan MS direct injection analysis of 3. mu.g/mL of a standard solution of P2 peptide (PHGGGWGQPHGGGWGQ MW: 1570) obtained using a) APCI, b) ESI and c) USIS ionization source, respectively. The sample was dissolved with water. The flow rate of the directly injected sample was 20. mu.L/min. The surface potential, electrospray tip voltage (13) and surface temperature were 50V, 350V and 50 ℃. The UV lamp was turned off and the ultrasound was turned on. The gas flow rate of the sprayer is 2L/min.

FIG. 10 shows a schematic view of a

Analysis of 10 of oligonucleotides with molecular weight of 6138Da^-7Mass spectrum obtained for M solution. Triethylamine (tryethyylamine) was present at 1% in the solution. The following atmospheric pressure ionization sources were used: a) APCI, b) ESI and c) USIS. It can be seen that in case a), b) and c) no oligonucleotide ion signal was detected, whereas in case d) the signal was clearly detected. Count/second value of 10⁷The S/N ratio of the peak of the abundance was 150. Surface potential, electricityThe spray tip voltage (13) and surface temperature were 50V, 350V and 50 deg.C, respectively. The UV lamp was turned off and the ultrasound was turned on. Also shown is a deconvolution spectrum in which the molecular masses of the analyzed oligonucleotides obtained using USIS are shown (see spectrum c).

FIG. 11

Analysis of 10 of oligonucleotides with molecular weight of 6138Da^-7Mass spectrum obtained for M solution. The solution had 1% triethylamine and a concentration of 5 x 10^-6NaCl salt of M. The following atmospheric pressure ionization sources were used: a) APCI, b) ESI, and c) USIS ionization source. It can be seen that in this case too, the oligonucleotide multi-charge signal can only be detected using the USIS ionization pathway. Count/second value of 10⁶The S/N ratio of the abundance peak is 30. The surface potential, electrospray tip voltage (13) and surface temperature were 50V, 350V and 50 ℃. The UV lamp was turned off and the ultrasound was turned on. Also shown is a deconvolution spectrum in which the molecular masses of the analyzed oligonucleotides obtained using USIS are shown (cf. spectrum c)

FIG. 12

Full scan mass spectra obtained from analysis of a 50ng/mL standard estradiol solution using a) APCI, b) ESI and c) USIS ionization source, respectively. CH for sample₃OH is dissolved. The flow rate of the directly injected sample was 20. mu.L/min. The surface potential, electrospray tip voltage (13) and surface temperature were 50V, 0V and 110 ℃ respectively. The UV lamp was turned on and the ultrasound was turned off. The gas flow rate of the sprayer is 2L/min.

FIG. 13

Full scan mass spectra obtained from analysis of a 50ng/mL standard estradiol solution using a) APCI, b) ESI and c) USIS ionization source, respectively. CH for sample₃And dissolving CN. The flow rate of the directly injected sample was 20. mu.L/min. The surface potential, electrospray tip voltage (13) and surface temperature were 50V, 0V and 110 ℃ respectively. The UV lamp was turned on and the ultrasound was turned off. The gas flow rate of the sprayer is 2L/min.

Description of a preferred embodiment and examples of application of the invention

A schematic diagram of the USIS ionization source is shown in fig. 1. The USIS ionization source produces ions that are analyzed by the mass spectrometer under a wide range of experimental conditions (e.g., polar and non-polar solvents, various flow rates, etc.).

A mass spectrometer comprises an ionization source, an analyzer or filter for separating ions by mass to charge ratio, a detector for counting the ions, and a data processing system. Since the structure of the mass spectrometer is conventional, it will not be described in detail. The ionization source device of the present invention comprises an inlet assembly (11) in fluid communication with an ionization chamber (3).

The ionization chamber (3) contains an exit orifice (1), typically less than 1mm in diameter, for communication between the ionization chamber and the analyzer or filter. Typically, the axis of the inlet assembly (11) is at an angle of about 90 ° to the axis passing through the aperture, but different relative positions may be devised. A tray (4) is placed inside the ionization chamber (3). The disc (4) has at least one active surface (4') facing the inner opening of the inlet assembly (11). Preferably, the disc (4) is perpendicular or at an angle of 45 ° with respect to the axis of the nebulizer (12) (fig. 2 and 3). Different physical ionization effects (such as UV radiation, ultrasound and electrostatic potential) can be concentrated at the surface to strongly increase the ionization efficiency. In addition, the selectivity of the pathway is increased. Indeed, the combination of different physical ionization effects on the surface allows selective ionization of the analyte of interest.

The disc (4) may have different geometries and shapes (as in fig. 2 and 3), such as square, rectangular, hexagonal, etc., without thereby departing from the scope of the invention. It has been found that when the active surface (4') is increased, the sensitivity of the assay is increased. Thus, the surface of the disc (4) is preferably distributed over 1cm²To 4cm²And generally its upper limit is dictated by the physical dimensions of the ionization chamber (3). The area of the active surface (4 ') can still be increased in a number of ways, keeping the dimensions of the disc (4) constant, for example by creating wrinkles in the surface (4'). In special cases, for example when high molecular weight molecules have to be analyzed, high electric field amplitudes are required. Under these circumstances, mentionProviding an active surface (4') with a plurality of point-like corrugations may be advantageous for increasing the amplitude of the electric field therein. It was also observed that the sensitivity increases strongly when a strong turbulence is generated by placing the surface (4') perpendicular to the axis of the atomizer (12) and applying a strong gas flow (typically 10L/min or more of nitrogen) through the atomization zone (12). Various geometries and angles relative to the inlet assembly (11) may be used to increase turbulence effects. The preferred configuration is that the surface (4') is positioned at an angle of perpendicular or 45 ° to the axis of the nebulizer region (12) and the surface is close to the inlet aperture (1) of the mass spectrometer to create a multiple collision phenomenon of solvent analyte clusters that results in ionization of the analyte and directing the gas stream and analyte ions into the inlet aperture (1). The flow rate of the analyte solution through the inlet system (11) may be between 0.0001 and 10000. mu.L/min, with a preferred flow rate of 100. mu.L/min.

The active surface (4') can be made of various materials, which can be conductive or non-conductive in nature. Preferred materials may be metals such as iron, steel, copper, gold or platinum, silica or suicide materials such as glass or quartz, polymeric materials such as PTFE (teflon) and the like. When the active surface (4 ') consists of an electrically non-conductive material, the body of the disc (4) will be made of an electrically conductive material, such as metal, and at least one of its faces will be coated with the electrically non-conductive material in the form of a layer or film to produce the active surface (4'). For example, a stainless steel disk (4) may be coated with a PTFE membrane. In fact, it is important that the active surface (4 ') is subjected to charge polarization even if the active surface (4') is made of a non-conductive material. This can be achieved by applying a potential difference from a power supply (26) to the disk, thereby inducing polarization also on the active surface (4') by induction. On the other hand, if the surface (4') is electrically conductive, the disc (4) need not be coated. In this case, the ionization source of the invention achieves good performance even without the application of a potential difference, i.e. the surface (4') is kept at ground potential and allowed to drift (float). However, this result can also be obtained if a latent charge polarization is applied to the conductive surface (4').

The plate (4) is connected to a handle means (6) by means of a connecting means (5), the handle means (6) allowing the plate (4) to move in all directions. The handle tool (6) is movable into the ionization chamber and can be rotated. The connecting means (5) can be made of different conductive materials and can have various geometries, shapes and sizes. Preferably, it is shaped and sized to facilitate the orientation of the disc (4) in an inclined position. The disc (4) is electrically connected to an electric power supply means (26) to apply a potential difference to the active surface (4'). The disc (4) typically has a thickness of between 0.05 and 100mm, preferably between 0.1 and 3 mm.

Various physical stimuli may be applied to the surface (4'). The laser (21) may illuminate the surface (4 ') to enhance ionization of analytes striking the surface (4') or deposited on the surface. The laser operates in the ultraviolet-visible (UV-VIS) or Infrared (IR) spectral region using various wavelengths (typically between 0,200 and 10.6 μm), with the preferred wavelength being 337nm for the UV-VIS band and 10.6 μm for the IR band. The lamp, UV laser, is connected to a commercially available external power supply (27). A molecule that absorbs UV-VIS or IR wavelengths is added to the sample solution to further increase the ionization efficiency. For example, sinapinic acid (synapinic acid) or caffeic acid may be used for this purpose. These molecules are actually excited by laser irradiation. These excited species react with the sample molecules and cause the formation of analyte ions. UV-VIS or IR lamps (22) may also be used to illuminate the surface (4) and the liquid sample reaching the surface (4) through the inlet device (11). The surface (4) or (4') may, upon interaction with photons, result in the formation of electrons or other ions, which may ionize the analyte molecules. The laser and lamp light may be placed inside or outside the ionization chamber and may illuminate the solvent and the surface (4) or (4') or only the surface through a nearby conduit (25) (see enlarged view of fig. 2) that avoids direct interaction of the solvent and analyte with the light. When connected to a pump, the tubing may be under vacuum; and when the vacuum pump is turned off, the pipe is at atmospheric pressure. When the device is operated under vacuum, it is possible to use the tunnel effect to ionize the analyte to reduce chemical noise. In this case the surface must be thin (0.05-0.1 mm, preferably 0.05mm) to allow electrons generated in the channel to pass through the surface and interact with the analyte, eventually causing it to ionize. In fact, the direct interaction of laser or UV light with the nebulizer gas and solvent can lead to the formation of a large number of charged solvent species, resulting in a strong increase in chemical noise. The tubing connecting the laser and lamp light to the thin surface can be maintained at various pressures (vacuum, atmospheric pressure) and filled with different gases (e.g., air, nitrogen). In addition, the temperature of the surface (4) can be varied by means of a commercially available power supply (31) connected to a resistor inserted in the surface (4'). The surface is cooled by means of a commercially available power supply which is also connected to a peltier device placed on the surface (4'), making it possible to cool the surface. The temperature of the surface (4) may be between-100 ℃ and +700 ℃, preferably between 25 ℃ and 100 ℃. An electric connector (16) or (23) makes it possible to apply to the ionization chamber (3) an ultrasonic excitation effect through a surface (4) or (4 ') subjected to an ultrasonic ionization effect generated by an electric power supply (26) connected to the connector (16) or (23) connected to the surface (4 ') by means of an electrically conductive material (copper, steel, gold) and to a piezoelectric device connected to the surface (4 ') and generating an ultrasonic sound at a frequency of 40-200kHz, preferably between 185 and 190kHz, more preferably at 186 kHz. Referring now to the inlet assembly (11), a liquid sample containing an analyte is introduced into the chamber through the sample inlet hole (10). The inlet assembly (11) comprises an internal duct opening out through the access opening (10), said internal duct leading to an atomization zone (12). The nebulization region is in liquid communication with at least one, typically two, gas lines (14), (15) (the gas typically being nitrogen) which intersect the main flow of the sample at different angles to perform both the function of nebulizing and bringing the analyte solution to the ionization chamber (3). An electrical power connector (23) may be used to apply an electrical potential difference between the region (13) and the inlet (1) of the mass spectrometer. The potential difference may be set to between-10000V and 10000V, preferably between-1000V and 1000V, but 0-500V is generally employed. This potential difference can be used to a) generate analyte ions in solution, and b) evaporate the solvent and analyte by the electrospray effect, thereby allowing the generation of analyte gas phase ions. The power connector (7) makes it possible to set the temperature of the atomizer area (12) and of the surface (4 ') by means of a commercially available power supply (31) connected to a thermal resistor or peltier device which is inserted in the atomizer area (12) and in the surface (4'). The temperature may be between-100 ℃ and +700 ℃. The preferred temperature is in the range of 100-200 deg.C, with a more preferred temperature of 200 deg.C. The inner conduit of the inlet assembly (11) ends inside the ionization chamber (3) in a position such that the analyte solvent droplets hit the active surface (4') of the disc (4), where the neutral molecules of the analyte are ionized. A variety of chemical reactions may occur on a surface without being limited to any particular theory: such as proton transfer reactions, reactions with thermal electrons, reactions with reactive molecules located on the surface, gas phase ionic molecular reactions, molecular excitation by electrostatic induction or photochemical effects. For example, one possible ionization mechanism is shown in FIG. 3. In this case, the analyzed molecules are dissolved in the solvent molecules (clusters). When the clusters collide with the ionization surface, the solvent is detached from the analyte, resulting in the generation of negative or positive ions of the analyte. Alternatively, it is also possible that the dipolar solvent is attracted to the active surface (4') by means of polarization of the charges induced thereon, thereby allowing the deprotonation or protonation source to form ions. As mentioned above, the disc (4) may be allowed to drift and a potential difference may be applied. Such a potential difference, the absolute value of which is preferably in the range of 0 to 15000V (in practice, it may be between 0V and 1000V, depending on the kind of polarization desired on the active surface (4'), preferably between 0 and 500V, more preferably between 0 and 200V.

The ionization chamber (3) may also be microwave excited through the USIS flange (2) to apply microwaves to the ionization chamber (3). The microwaves are applied by an internal power supply (28) which is connected to the faraday box by a connector (20). The microwave frequency may be between 915 and 2450MHz, preferably between 2000 and 2450MHz, more preferably 2450 MHz. Microwaves are mainly used to evaporate water.

To summarize, the essential feature of the present invention is the exposure of one ionizing active surface (4') to a combination of different physical effects (at least two) in order to ionize a wide range of organic analytes (polar and non-polar). In addition, the present approach allows for improved sensitivity and selectivity in the analysis of one target compound.

It should be understood that the above description is intended to illustrate the principles of the present invention and not to limit any further modifications that may occur to those skilled in the art upon publication of this patent application. FIG. 4 illustrates a typical internal view of one exemplary embodiment of a USIS ionization chamber.

The following examples further illustrate the invention.

Example 1: analysis of MDE addictive drugs in diluted urine samples

USIS ionization source was used to analyze 3, 4-Methylenedioxyethylamphetamine (MDE) addictive drugs. An increase in sensitivity was observed compared to commonly used techniques (ESI and APCI). FIGS. 5a, b and c show full scan direct injection spectra obtained from analysis of a standard solution of 50ng/mL MDA using APCI, ESI and USIS ionization sources, respectively. The sample was dissolved with water. The flow rate of the directly injected sample was 20. mu.L/min. The surface potential, electrospray tip voltage (13) and surface temperature were 50V, 0V and 110 ℃ respectively. The UV lamp and ultrasound were turned off. The gas flow rate of the sprayer is 2L/min. It can be seen that in the APCI spectroscopic case, no MDE ion signal was detected, and in the ESI case, there was high chemical noise. Using USIS technology, [ M + H ] at M/z 208 is clearly detected]⁺MDE signal, thereby obtaining a full scan spectrum. A good signal-to-noise ratio (S/N:100) was achieved using USIS.

FIGS. 6a, b and c show the liquid chromatography-Tandem mass spectrometry (LC-MS/MS) results of MDE obtained using a) APCI, b) ESI and c) USIS ionization sources, respectively. Urine samples were diluted 20-fold prior to analysis. The gradient was carried out with two phases: A) water + 0.05% formic acid and B) CH₃CN + 0.05% formic acid. In particular, 15% of phase B is kept for 2 minutes and then a linear gradient from 15% to 70% of phase B is performed in 8 minutes, reaching the initial state within 2 minutes. The acquisition time was 24 minutes to re-equilibrate the column. Make itWith thermolec control C₈150X1mm column. The eluent flow rate was 100. mu.L/min. The surface potential, electrospray tip voltage (13) and surface temperature were 50V, 0V and 110 ℃ respectively. The UV lamp and ultrasound were turned off. The gas flow rate of the sprayer is 2L/min. It can be seen that the only technology that can detect MDE is USIS (S/N: 120). The high sensitivity and selectivity obtained using the MS/MS approach allows for clear identification of MDEs.

Example 2: analysis of arginine plasma samples

The plasma samples were analyzed for arginine using a USIS ionization source. In this case, an increase in sensitivity is also observed with respect to the commonly used techniques (ESI and APCI). FIGS. 7a, b and c show full scan direct injection spectra obtained by analysis of a 100ng/mL arginine standard solution with a) APCI, b) ESI and c) USIS ionization sources, respectively. The sample was dissolved with water. The flow rate of the directly injected sample was 20. mu.L/min. The surface potential, electrospray tip voltage (13) and surface temperature were 50V, 0V and 110 ℃ respectively. The UV lamp was turned off and the ultrasound was turned on. The gas flow rate of the sprayer is 2L/min. In the APCI spectrum (fig. 7a), no arginine ion signal was detected. The fact that in the ESI case (fig. 7b) there is high chemical noise in the spectrum makes the ion signal of arginine practically undetectable in the spectrum obtained in full scan mode. Using the USIS technique to obtain a full scan spectrum, [ M + H ] at M/z 175 is clearly detected]⁺The MDE signal. In particular, good signal-to-noise ratios (S/N:70) were obtained using USIS.

FIGS. 8a, b and c show the use of a) APCI, b) ESI and c) USIS ionization sources, respectively, and splitting the [ M + H at M/z 175]⁺Liquid chromatography-multiple collision analysis (LC-MS3) results for arginine obtained for the ion and its product ion at m/z 158. Gradient operation was performed with two phases: A) CH (CH)₃OH/CH₃CN + 0.1% formic acid + ammonium formate (20. mu. mol/L) and B) H₂O + 0.1% formic acid + ammonium formate (20. mu. mol/L). Arginine was extracted from plasma using a protein precipitation method based on phase a as protein precipitant. Analysis was performed using 4% B under isocratic conditions. The acquisition time was 6 minutes to re-equilibrate the column. Using water SAX 100x4.1mm column. The eluent flow rate was 1000 mL/min. The surface potential, electrospray tip voltage (13) and surface temperature were 50V, 0V and 110 ℃ respectively. The UV lamp was turned off and the ultrasound was turned on. The gas flow rate of the sprayer is 2L/min. Also in this case, the highest signal-to-noise ratio (S/N:100) is obtained using USIS. Thus MS³The high sensitivity and selectivity of the pathway makes it possible to clearly detect and identify arginine in the chromatograms obtained using USIS (fig. 8 c).

Example 3: analysis of peptides

Peptide P2 (PHGGGWGQPHGGGWGQ; partial sequence of PrPr protein) was analyzed using a) APCI, b) ESI, and c) USIS (FIG. 9a, b, and c). The peptide concentration was 3. mu.g/mL. The sample was dissolved with water. The flow rate of the directly injected sample was 20. mu.L/min. The surface potential, electrospray tip voltage (13) and surface temperature were 50V, 350V and 50 ℃. The UV lamp was turned off and the ultrasound was turned on. The gas flow rate of the sprayer is 2L/min. No signal was detected using APCI (fig. 9 a). In the case of ESI, [ M + H ]]⁺And [ M +2H]⁺The signals are all detected. Obtaining S/N ratio 80 at the peak of the abundance, and counting value per second is 2 x 10⁸. The USIS technique gives the best S/N ratio (S/N:180) at the peak of the abundance and gives a count of 1 x 10 per second⁷It is clearly shown that this ionization technique produces lower chemical noise.

Example 4: analysis of aqueous oligonucleotide solutions

FIGS. 10a, b and c show the spectra obtained by direct injection of a solution of oligonucleotide of molecular weight 6138 Da. The spectra were obtained using a) APCI, b) ESI and c) USIS ionization techniques, respectively. The concentration of the oligonucleotide solution was 10^-7And M. Triethylamine was added at 1% to the sample to avoid signal suppression effects due to formation of oligonucleotide cation complexes. It can be seen that no oligonucleotide mass ion signal was detected at this concentration level using APCI and ESI (fig. 10a and b). The situation changed dramatically when USIS ionization technique was used (fig. 10 c). In this case, in fact, the oligonucleotide multiply charged ions are clearly detected. The count value per second is 10⁷The S/N ratio of the peak of the abundance was 150. Oligo (A)The charge distribution of the nucleotide ions ranges from-10 to-4. The UV lamp was turned off and the ultrasound was turned on. It is emphasized that the chemical noise is rather low using the USIS ionization pathway (noise counts per second: 5 x 10)⁵)。

Example 5: analysis of aqueous oligonucleotide solutions containing inorganic salts, e.g. NaCl

FIGS. 11a, b and c show the spectra obtained by analysis of an oligonucleotide of molecular weight 6138Da using a) APCI, b) ESI and c) USIS ionization source, respectively. Concentration of 5 x 10^-6M NaCl was added to the sample solution to evaluate its performance in the presence of salt from a sensitivity point of view. The concentration of the oligonucleotide solution was 10^-7And M. 1% triethylamine was added to the sample solution to avoid signal suppression effects due to formation of oligonucleotide cation complexes. It can be seen that in this case too no oligonucleotide mass ion signal was detected using the APCI and ESI effects (fig. 11a and b). In the case of USIS (fig. 11c), the oligonucleotide multiply charged ion signal was clearly detected. The count value per second is 10⁶The S/N ratio of the peak of the abundance was 30. The charge distribution of the oligonucleotide ions ranges from-10 to-4. It is emphasized that the chemical noise is rather low using the USIS ionization pathway (noise counts per second: 5 x 10)⁴)。

Example 6: analysis of undetectable low polarity compounds (e.g., steroids, etc.) by direct injection using ESI and APCI at low concentration levels

Estradiol was analyzed using a) APCI, b) ESI and c) USIS. Using CH₃OH and CH₃CN as solvent, direct injection spectra were obtained (FIGS. 12a, b and c show the use of CH₃OH as solvent, while FIGS. 13a, b and c show the use of CH₃Spectrum obtained with CN as solvent). The concentration of estradiol was 50. mu.g/mL. The sample was dissolved with water. The flow rate of the directly injected sample was 20. mu.L/min. The surface potential, electrospray tip voltage (13) and surface temperature were 50V, 350V and 50 ℃. The UV lamp was turned on and the ultrasound was turned off. The gas flow rate of the sprayer is 2L/min. It can be seen that at this concentration level no signal was obtained using ESI and APCI (FIGS. 12a and b; FIG. 13)a and b), whereas using USIS, [ M ] is clearly detected.]⁺And [ M-H]⁺Ions. Using CH₃OH as solvent, [ M ].]⁺S/N ratio of 100, using CH₃The S/N ratio of CN as solvent was 102 (FIGS. 12c and 13 c). It must be emphasized that at higher estradiol concentration levels (1000. mu.g/mL) and CH is used₃OH as a solvent, the ESI soft ionization source typically produces [ M + H ] of the analyte]⁺But using CH₃CN as solvent, this signal is difficult to observe. In the case of USIS, two solvents (CH) are used for the analyte ions₃OH and CH₃CN) can be observed. This clearly demonstrates the potential of USIS.

Claims (30)

1. An ionization source apparatus for ionizing an analyte in a liquid phase, comprising:

an inlet assembly in fluid communication with an ionization chamber, the ionization chamber having an exit orifice for communication between the ionization chamber and an analyzer or filter of a mass spectrometer; and

a disk or surface having at least one active surface within the ionization chamber,

wherein the analyte solvent droplets are arranged to impinge on the active surface of the disc or surface, where the neutral molecules of the analyte ionize.

2. The ionization source apparatus of claim 1 further comprising a nebulizer.

3. The ionization source apparatus according to claim 1 or 2, wherein the analyte molecules are arranged to dissolve in solvent molecules to form clusters, and wherein negative or positive ions of the analyte are generated when the clusters collide with the active surface.

4. The ionization source apparatus according to claim 1 or 2, further comprising: a power supply connected to the active surface through a conductive material to electrically charge or polarize the active surface.

5. The ionization source apparatus according to claim 1 or 2, further comprising: a power supply connected to the piezoelectric device for generating ultrasound in the region of the active surface.

6. The ionization source apparatus according to claim 1 or 2, further comprising: a UV-VIS or IR laser or lamp connected to an external power supply for irradiating light to the active surface.

7. The ionization source apparatus according to claim 1 or 2, further comprising: an external power supply connected to the faraday box through a connector for applying microwaves to the ionization chamber.

8. The ionization source apparatus according to claim 1 or 2, further comprising: a closed conduit connected to the active surface and to a pump for generating different pressures.

9. The ionization source apparatus according to claim 1 or 2, further comprising: a power supply for applying an electrical potential to a resistor inserted within the active surface to heat the active surface.

10. The ionization source apparatus according to claim 1 or 2, further comprising: a power supply connected to a Peltier device located on the active surface for cooling the active surface.

11. The ionization source apparatus of claim 1 or 2, wherein molecules of the analyte are ionized on the active surface and concentrated into the mass spectrometer analyzer inlet.

12. The ionization source apparatus of claim 1 or 2 wherein said disk or surface is coated with a non-conductive material to form said at least one active surface.

13. The ionization source apparatus of claim 12 wherein said non-conductive material comprises a silica or silicide derivative selected from glass or quartz, or a polymeric material selected from plastic.

14. The ionization source apparatus of claim 13 wherein said polymeric material is selected from the group consisting of PTFE, polyvinyl chloride (PVC), polyethylene glycol (PET).

15. The ionization source apparatus of claim 1 or 2, wherein the disk or surface is tilted at an angle relative to the axis of the inlet assembly, and wherein the angle of the disk or surface is changed using computer or manually controlled electronics connected to an external power supply.

16. The ionization source apparatus according to claim 1 or 2, wherein the inlet assembly comprises an access opening for feeding an analyte solution, and an internal conduit in liquid communication with the access opening, the internal conduit comprising an atomization region and a charging region and terminating inside the ionization chamber.

17. A mass spectrometer comprising an ionization source apparatus according to any one of the preceding claims.

18. The mass spectrometer of claim 17, further comprising:

means for separating or desalting molecules contained in the sample;

at least one analyser or filter for separating ions according to their mass-to-charge ratio;

a detector for counting the number of ions; and

and a data processing system for calculating and plotting the mass spectrum of the analyte.

19. The mass spectrometer of claim 18, wherein the means for separating or desalting molecules contained in the sample is a liquid chromatograph.

20. A method of ionizing an analyte in a liquid phase, the method comprising:

providing an ionization source apparatus comprising an inlet assembly in fluid communication with an ionization chamber having an exit orifice for communication between the ionization chamber and an analyzer or filter of a mass spectrometer;

providing a disk or surface within the ionization chamber, the disk or surface having at least one active surface; and

causing the analyte solvent droplets to impinge on the active surface of the disc or surface where the neutral molecules of the analyte ionize.

21. The method of claim 20, further comprising:

dissolving an analyte in a solvent; and

injecting the analyte solution into the ionization source device.

22. A method according to claim 21 wherein the analyte is dissolved in a solvent selected from H₂O, alcohol, acetonitrile, chloroform, tetrahydrofuran in a dipolar solvent.

23. A method according to claim 21 or 22, further comprising causing the analyte solution to be nebulised.

24. The method of claim 23, further comprising impinging the atomized analyte solution onto the active surface.

25. A method according to any of claims 20 to 22, further comprising causing the ionised analyte to be collected by an analyser or filter of the mass spectrometer.

26. The method of any of claims 20-22, further comprising maintaining the temperature of the active surface between-100 ℃ and 700 ℃.

27. The method of any of claims 20-22, further comprising maintaining the temperature of the active surface between 100 ℃ and 200 ℃.

28. A method according to any of claims 20-22, further comprising applying a potential difference of between 0 and 15000V to the active surface.

29. A method according to any of claims 20-22, further comprising applying a potential difference of between 0 and 1000V to the active surface.

30. A method according to any of claims 20-22, further comprising applying a potential difference of between 0 and 200V to the active surface.