WO2018207903A1

WO2018207903A1 - Electrospray ionization device, mass analysis apparatus, electrospray ionization method, and mass analysis method

Info

Publication number: WO2018207903A1
Application number: PCT/JP2018/018263
Authority: WO
Inventors: 賢三平岡
Original assignee: 国立大学法人山梨大学
Priority date: 2017-05-12
Filing date: 2018-05-11
Publication date: 2018-11-15
Also published as: JP7109731B2; JPWO2018207903A1

Abstract

The present invention provides an electrospray ionization device comprising a probe member for contacting and thereby capturing a sample, and a voltage application member configured to apply a voltage to the probe member. The probe member is provided with a continuous supply mechanism or an intermittent supply mechanism for supplying a liquid solvent to the sample. Alternatively, the electrospray ionization device further comprises a solvent supply member provided with a continuous supply mechanism or an intermittent supply mechanism for supplying a liquid solvent to the sample.

Description

Electrospray ionization apparatus, mass spectrometer, electrospray ionization method, and mass spectrometry method

The present invention relates to an electrospray ionization apparatus, a mass spectrometry instrument, an electrospray ionization method, and a mass spectrometry method.

In recent years, mass spectrometry (MS) has been widely used in the fields of organic chemistry and biochemistry to ionize molecules in a sample to be analyzed and detect the mass of the ions. In mass spectrometry, molecules in a sample can detect ions with a single charge or ions with multiple charges, and also detect ions derived from the association or dissociation of molecules in a sample. A wealth of information can be obtained. Therefore, it is very useful as a means for identifying a known substance and determining the structure of an unknown substance, and research and development of more excellent mass spectrometry has been actively conducted.

For example, in Patent Document 1, a sample is captured at the tip of a metal probe, and a voltage is applied to the metal probe to generate an electrospray. A probe electrospray ionization method (PESI) and a mass spectrometer using PESI (PESI) PESI-MS) has been reported. According to Patent Document 1, it is disclosed that PESI can use biological tissue or the like without pretreatment as a target sample, and a metal probe or sample can ionize the sample under atmospheric pressure. .

Patent Document 2 reports PESI-MS that enables the analysis of all components contained in a sample by sequentially ionizing components that are easily ionized to components that are not easily ionized by spraying a solvent in high temperature vapor onto the probe. Has been.

Patent 4862167 Patent 5034092

In the PESI-MS of Patent Document 1, since the amount of the solvent contained in the sample was extremely small, electrospray could be generated only for a short time. For this reason, among components in the sample, only components that are easily ionized are detected, and components that are relatively difficult to ionize cannot be detected.

The PESI-MS in Patent Document 2 has the function of supplying a solvent by spraying the solvent as a high-temperature vapor on the sample, thereby enabling analysis of the entire components contained in the sample including components that are difficult to ionize. However, this PESI-MS requires a process of heating the solvent to vapor and a process of spraying the vapor, and the sample is ionized by supplying the solvent by a simple method in a milder environment. There was a need for an apparatus and method.

The present invention has been made in view of the above circumstances, and provides a conventional PESI-MS by supplying a solvent continuously or intermittently (continuously) in a mild environment and in a simple manner. The present invention provides an electrospray ionization apparatus, a mass spectrometry instrument, an electrospray ionization method, and a mass spectrometry method that can ionize components that are difficult to ionize and analyze.

According to the present invention, an electrospray ionization apparatus, comprising: a probe member that captures a sample by contact; and a voltage application member configured to apply a voltage to the probe member, The probe member has a liquid solvent continuous supply mechanism or liquid solvent intermittent supply mechanism to the sample, or the electrospray ionization apparatus has a liquid solvent continuous supply mechanism or liquid solvent intermittent supply mechanism to the sample. An electrospray ionization apparatus is further provided that further includes a solvent supply member.

FIG. 1 is a schematic view of an example of an electrospray ionization apparatus according to the present invention. FIG. 2 is a schematic view of an example of an electrospray ionization apparatus according to the present invention. FIG. 3 is a conceptual diagram showing how the repetitive movement of the probe member and the application of voltage are synchronized. FIG. 4 is a schematic view of an example of the first embodiment in the electrospray ionization apparatus of the present invention. FIG. 5 is a schematic view of an example having the manipulator of the first embodiment in the electrospray ionization apparatus of the present invention. FIG. 6 is a schematic view of an example of the second embodiment of the electrospray ionization apparatus of the present invention. FIG. 7 is a schematic view of an example of the third embodiment in the electrospray ionization apparatus of the present invention. FIG. 8 is a schematic diagram of an example of the mass spectrometer of the present invention. 9A to 9C are spectrum diagrams showing the results of mass spectrometry using the first embodiment of the electrospray ionization apparatus of the present invention. FIGS. 10A to 10B are spectrum diagrams showing the results of performing mass spectrometry using the second embodiment of the electrospray ionization apparatus of the present invention. FIGS. 11A to 11E are spectrum diagrams showing the results of performing mass spectrometry using the second embodiment of the electrospray ionization apparatus of the present invention. 12A to 12B are spectrum diagrams showing the results of mass spectrometry using the third embodiment of the electrospray ionization apparatus of the present invention.

Hereinafter, the present invention will be described in detail by exemplifying embodiments of the present invention. The present invention is not limited by these descriptions. Various features of the embodiments of the present invention described below can be combined with each other. In addition, the invention is independently established for each feature.

The present inventor has conducted intensive studies and found that the electrospray ionization apparatus used in conventional PESI-MS is continuously (continuously or intermittently) in a mild environment and in a simple manner with the solvent in a liquid state. In other words, the present invention has completed the present invention.

In the electrospray ionization apparatus of the present disclosure, the probe member holds a sample containing a liquid solvent at the tip, and when a voltage is applied, an electrochemical reaction occurring at the tip causes the same sign as the applied voltage. Excess charge is supplied to the droplet and the droplet is charged. Then, the evaporation of the solvent and the increase in surface charge density proceed, the repulsive force between the charges exceeds the surface tension of the liquid, and finally a coulomb explosion occurs due to the repulsion of the electrostatic force, resulting in a fine charged liquid containing sample molecules Drops are released as a spray (electrospray). Through such a process, it is considered that the sample molecules are ionized by being supplied with electric charge in the charged droplets.

Hereinafter, various embodiments of the present invention will be exemplified. The following embodiments can be combined with each other.
Preferably, the probe member includes a probe, the solvent supply member includes a solvent storage unit and an operation unit, and the operation unit brings the probe into contact with a liquid solvent stored in the solvent storage unit. The solvent is supplied to the sample.
Preferably, the time for which the probe contacts the liquid solvent is 10 milliseconds or more and 100 milliseconds or less.
Preferably, the operating part includes a repetitive moving means for repetitively moving the probe member in the longitudinal direction of the probe member.
Preferably, the operating part includes a manipulator.
Preferably, the probe member includes a probe and a solvent loading tip, and the probe protrudes from a lower end of the solvent loading tip, and is configured to store a liquid solvent inside the solvent loading tip. Has been.
Preferably, the probe protrudes from the lower end of the solvent loading tip in a range of 0 mm to 0.2 mm.
Preferably, the probe member includes a probe and an insulator covering the probe, the insulator holds a liquid solvent on the surface, and the solvent is removed when a part of the sample is ionized. It is configured to supply.
Preferably, the insulator includes borosilicate or epoxy resin.
Preferably, it further includes repetitive moving means for repetitively moving the probe member in the longitudinal direction of the probe member.
Preferably, it further includes a manipulator.

According to another aspect of the present invention, any one of the above electrospray ionization apparatuses, and an analysis apparatus that separates ions ionized by the electrospray ionization apparatus,
There is provided a mass spectrometer comprising: an ion detector that detects ions separated by the analyzer.

According to another aspect of the present invention, a capturing step in which a probe member captures a sample, an ionization step in which a voltage is applied to the probe member to ionize the sample, and the sample is continuously or intermittently applied. And a solvent supply step of supplying a liquid solvent.

Hereinafter, various embodiments according to this aspect will be exemplified. The following embodiments can be combined with each other.
Preferably, the probe member includes a probe, and in the solvent supply step, the probe is brought into contact with a liquid solvent to supply a liquid solvent to the sample.
Preferably, the time for which the probe contacts the liquid solvent is 10 milliseconds or more and 100 milliseconds or less.
Preferably, in the solvent supply step, the probe member is repeatedly moved in the longitudinal direction of the probe member.
Preferably, in the capturing step, the probe member collects the sample from all directions.
Preferably, the probe member includes a probe and a solvent loading tip, the probe protrudes from a lower end of the solvent loading tip, and has a solvent filling step of filling the solvent loading tip with a liquid solvent. In the solvent supply step, the probe member is brought into contact with the sample, and the liquid solvent is supplied from the inside of the solvent loading chip.
Preferably, the probe member includes a probe and an insulator covering the probe, and in the capturing step, the probe member contacts the sample at a depth of greater than 0 mm and 2 mm or less. In the ionization step, a part of the sample is ionized, and in the solvent supply step, a liquid solvent held on the surface of the insulator is supplied to the sample.
Preferably, in the capturing step, the probe member collects the sample from all directions.

According to another aspect of the present invention, any one of the electrospray ionization methods described above, an analysis step of separating ions ionized by the ionization method, and ions that detect ions separated in the analysis step A mass spectrometric method having a detection step.

1. Electrospray ionization apparatus A schematic view of an example of an electrospray ionization apparatus according to the present invention is shown in FIG. The electrospray ionization apparatus 1 includes a probe member 10 that captures a sample by contact, and a voltage application member 20 configured to apply a voltage to the probe member 10, and the probe member 10. Is configured to continuously supply the solvent to the sample, or further includes a solvent supply member configured to continuously supply the solvent to the sample.

In other words, the electrospray ionization apparatus 1 includes a probe member 10 that captures a sample by contact, and a voltage application member 20 that is configured to apply a voltage to the probe member 10. The needle member 10 has a liquid solvent continuous supply mechanism or a liquid solvent intermittent supply mechanism to the sample, or the electrospray ionization apparatus has a solvent continuous supply mechanism or a liquid solvent intermittent supply mechanism to the sample. It further has a member.

The electrospray ionization apparatus 1 only needs to have an amount of sample that is attached to the tip of the probe member 10, and can ionize even a very small amount of sample such as 1 pL (picoliter) or 3 pL. Even if the sample targeted by the electrospray ionization apparatus of the present invention is a solid sample such as a powder, the sample itself is in a liquid state, or a liquid sample such as a dispersion in which the sample is dispersed in a solvent or a solution in which the sample is dissolved It may be.

The time during which the electrospray ionization apparatus 1 can generate electrospray is preferably 1 second or longer, more preferably 2 seconds or longer, 3 seconds or longer, and 5 seconds or longer.

The probe member 10 is not particularly limited as long as it has a function of capturing a sample and can apply a voltage. Typically, a conductive probe such as a metal probe or a conductive probe is typically used. Those covered with other materials.

The voltage application member 20 is not particularly limited as long as a voltage high enough to ionize the sample can be applied to the probe member 10, but the voltage that can be applied to the probe member 10 by the voltage application member 20 is 0.5 kV. (Kilovolt) or more is preferable, 1 kV or more is more preferable, 10 kV or less is preferable, and 8 kV or less is more preferable. 2 ± 0.5 kV is more preferable.

As a case where the probe member 10 is configured to continuously or intermittently supply the solvent to the sample, for example, the probe member described in the second embodiment or the third embodiment described later is used. May be used. The solvent supply member configured to continuously or intermittently supply the solvent to the sample may be the solvent supply member 30 described in the first embodiment described later.

The solvent used is not particularly limited as long as it can dissolve or disperse the sample, but is preferably a polar solvent from the viewpoint of efficiently supplying electric charge to the sample, and efficiently removes the charged sample. From the viewpoint, a volatile solvent is preferable. The solvent used may be a mixed solvent, typically a mixed solvent of water and a volatile polar solvent. Examples of the volatile polar solvent include alcohols such as methanol and ethanol, and acetonitrile. Preferably used.

The solvent used is preferably 0 ° C. or higher and 80 ° C. or lower, more preferably 70 ° C. or lower, 60 ° C. or lower, and 50 ° C. or lower, and even more preferably room temperature, from the viewpoint of preventing sample denaturation. Moreover, it is preferable that the solvent used is not supplied as a vapor | steam or a mist from a viewpoint which simplifies an apparatus.

The electrospray ionization method using the electrospray ionization apparatus 1 includes a capturing step in which the probe member 10 captures a sample, an ionization step in which a voltage is applied to the probe member 10 to ionize the sample, and the sample A solvent supply step of supplying the solvent continuously or intermittently.

The timing for applying a voltage to the probe member 10 is not particularly limited, and a voltage may be constantly applied, or a voltage may be applied only after capturing a sample or after supplying a solvent.

FIG. 2 shows an example of the electrospray ionization apparatus of the present invention. The voltage supply member 20 in FIG. 2 includes a power source 21 (for example, HV power supply), a switch 22 (for example, HV switch), and a digital delay pulse generator 23 (for example, digital delay pulse). generator etc.). In the present embodiment, the digital delay pulse generator 23 supplies a trigger signal to the repetitive moving means 33 to cause the repetitive movement of the probe member 10. Further, the power source 21 constantly applies a voltage, the digital delay pulse generator 23 supplies a trigger signal to the switch 22, and controls whether the voltage applied by the power source 21 is transmitted to the probe member 10. is doing. For example, in a period in which the tip of the probe member 10 is in the vicinity of the lower solstice point, the switch 22 is turned off during the period in which the tip of the probe member 10 is in the vicinity of the lower solstice. Can synchronize the repetitive movement of the probe member 10 and the application of voltage, such as by turning on the switch 22 and applying a voltage to the probe member 10 to ionize the sample.

FIG. 3 is a conceptual diagram showing how the repeated movement of the probe member 10 and the application of voltage are synchronized. When the repetitive moving means 33 receives the trigger signal, repetitive movement is performed in which the tip of the probe member 10 moves to the lower end point and returns to the upper end point. Further, the switch 22 is turned on with a delay of Δt time from the trigger signal, and a voltage is applied to the probe member 10 for a certain period of time. In this way, the repeated movement of the probe member 10 and the application of voltage are synchronized. By repeating the above steps, sample collection and ionization can be repeated, or solvent supply can be repeated.

Hereinafter, more specific first to third embodiments of the present invention will be described. The features of these embodiments can be combined with each other.

(1) 1st Embodiment An example of 1st Embodiment of the electrospray ionization apparatus 1 of this indication is shown in FIG. In the first embodiment, in addition to the configuration of the electrospray ionization apparatus 1 shown in FIG. 1, the probe member 10 includes the probe 11, the solvent supply member 30 includes the solvent storage unit 31 and the operation unit 32, and operates. The part 32 moves the probe 11 so that the probe 11 is brought into contact with the solvent stored in the solvent storage unit 31.

The electrospray ionization apparatus 1 according to the first embodiment is configured such that the sample repeatedly obtains a solvent by operating the probe 11 to which the sample is attached to the solvent storage unit 31 by operating the probe 32. is there. That is, an operation of dipping (dipping) the probe 11 to which the sample is attached to the solvent in the solvent storage unit 31 is executed at least once.

In the electrospray ionization apparatus 1 according to the first embodiment, the probe 11 to which a sample is attached is operated by the operating unit 32 and brought into contact with the solvent storage unit 31, so that the sample is repeatedly supplied to the sample, that is, the solvent is intermittently supplied. It is made to be done. In the present embodiment, an intermittent supply mechanism of the liquid solvent may be configured by the solvent supply member 30 including the solvent storage unit 31 and the operation unit 32.

The probe 11 preferably has a tip radius of curvature of, for example, 10 μm (micrometer) or less, 5 μm or less, 1 μm or less, or 0.7 μm or less from the viewpoint of capturing a fine sample. Further, from the viewpoint of easy availability and manufacturing cost, the curvature radius of the tip is preferably 0.1 μm or more, 0.3 μm or more, or 0.5 μm or more, for example. The diameter of the body of the probe 11 is not particularly limited, and is, for example, 5.0 mm or less, 1.0 mm or less, 0.5 mm or less, and 0.1 mm or more, 0.3 mm or more. In this way, when the diameter of the body and the tip of the probe 11 is very small, the invasiveness to the sample is very low, so that it can be applied to the medical field such as biopsy and sampling with an endoscope. Is preferred.

The probe 11 is preferably made of metal from the viewpoint of conductivity and high hardness. Examples of the metal probe include the metals such as stainless steel, titanium, aluminum, iron, silver, gold, and steel. It is preferable that the alloy is included. In addition, when the probe contains titanium, the probe surface is rich in irregularities, so that separation of each component occurs remarkably.

The solvent storage unit 31 is not particularly limited, but is typically a dish-shaped or cup-shaped container, and is preferably made of a material that does not elute into a solvent such as glass.

The time for the operating unit 32 to contact the probe 11 with the solvent stored in the solvent storage unit 31 is, for example, 10 milliseconds or more and 100 milliseconds or less, but is 80 milliseconds or less from the viewpoint of preventing sample elution. It is preferable that it is 50 milliseconds or less. The time for contacting the probe with the solvent stored in the solvent storage unit is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, It suffices to be within the range of any two values among 85, 90, 95, and 100 milliseconds. The depth at which the operating unit 32 brings the probe 11 into contact with the solvent stored in the solvent storage unit 31 is preferably set so that all the sample captured by the probe 11 is in contact with the solvent.

The operating unit 32 may include a repetitive moving means 33 that repetitively moves the probe 11 in the longitudinal direction of the probe 11.

In addition to the dipping operation described above, the operating unit 32 may perform a capturing operation for attaching or capturing the sample to the probe 11. As in the example of the dipping operation described above, the sample may be captured by the probe 11 by the vertical movement of the probe 11 (that is, the operation of moving the probe 11 up and down in the longitudinal direction of the probe 11).

The operating unit 32 may include a manipulator 34. FIG. 5 shows an example of the first embodiment in which the operating unit 32 includes a manipulator 34. When the operating unit 32 includes the manipulator 33, since the sample can be captured from all directions, the sample having a large volume can be captured and ionized. It is suitable for 3D (three-dimensional) imaging mass spectrometry equipment capable of performing mass spectrometry.

For example, when measuring the three-dimensional component distribution of a biological sample, the sample is captured and measured at a plurality of measurement points of the three-dimensional sample. At this time, it is possible to easily perform the component analysis of the plurality of points by using the operating unit 32 including a manipulator. The operation control, measurement, and data collection of the manipulator can be controlled by using a computer or the like, and can be automated.

In the present disclosure, a manipulator refers to a device that has a movement function similar to that of a human arm or hand and is intended to replace human manual work. For example, one or more movable parts that can be bent and rotated are included. The magic hand etc. which have are mention | raise | lifted.

The electrospray ionization method using the electrospray ionization apparatus 1 according to the first embodiment includes the above-described capturing step, solvent supply step, and ionization step, the probe member 10 includes a probe 11, and the solvent supply step. Thus, the probe 11 is brought into contact with a liquid solvent to supply a liquid solvent to the sample.

Here, the capturing step is a step of attaching or capturing the sample to the probe 11 using the operating unit 32, and the solvent supplying step is that the operating unit 32 contacts the solvent stored in the solvent storage unit 31 by the operating unit 32. The ionization step is a step of applying a voltage (high voltage) to the probe 11 that has captured the sample to ionize the sample.
Note that the ionization step is desirably performed in the vicinity of an ion inlet (not shown) of the analyzer 3 described later.

The ionization step and the solvent supply step are preferably performed a plurality of times in order to achieve the object of the present invention to ionize components that are difficult to ionize, for example, 3 times or more, 5 times or more, 10 times or more. The order in which the ionization step and the solvent supply step are performed may be reversed.

When the operation part 32 includes the repetitive movement means 33, the probe 11 can repetitively move in the longitudinal direction of the probe 11, and when the operation part 32 includes the manipulator 34, the probe 11 can move relative to the sample. Can be captured from all directions. By the operation of the operating unit 32, the sample can be captured and the solvent can be supplied.

In the first embodiment, since the solvent is supplied by contact between the tip of the probe 11 and the solvent, a liquid sample having a high concentration (which cannot be handled by ordinary electrospray), a dry sample, or a solid sample may be used. It can be ionized. Furthermore, after capturing a liquid sample, the sample can be dried and transported once, and a solvent can be supplied and ionized at a remote location.

(2) Second Embodiment FIG. 6 shows an example of the second embodiment of the electrospray ionization apparatus 1 of the present disclosure. In the second embodiment, in addition to the configuration of the electrospray ionization apparatus 1 shown in FIG. 1, the probe member 10 includes a probe 11 and a solvent loading chip 13 (for example, a gel loading chip). The solvent loading chip 13 protrudes from the lower end in a range of 0 mm to 0.2 mm, and is configured to store the solvent inside the solvent loading chip 13.

In the electrospray ionization apparatus 1 of the second embodiment, the probe 11 contacts the sample and captures the sample, and at the same time, the solvent stored in the solvent loading chip 13 is supplied to the sample by capillary action. Further, when a voltage is applied to the sample captured by the probe 11 and the sample or solvent is sprayed and reduced, the solvent in the solvent loading chip 13 is further supplied.

In this embodiment, since the probe 11 and the solvent loading chip 13 which are the probe members 10 have the above-described functions, the solvent can be continuously or intermittently supplied to the sample by a simple operation of simply touching the sample. it can. Therefore, it is possible to easily analyze a dried solid sample or a sample having a high concentration.

That is, continuous or intermittent solvent supply enables continuous electrospray generation even for samples that have been difficult to analyze. In the present embodiment, the probe member 10 including the probe 11 and the solvent loading chip 13 may constitute a liquid solvent continuous supply mechanism or a liquid solvent intermittent supply mechanism.

In the present embodiment, the probe 11 similar to that in the first embodiment can be used. The solvent loading chip 13 is preferably made of a hydrophobic material from the viewpoint of preventing the sample from adhering to the solvent loading chip 13 when the sample is hydrophilic. Examples of the solvent loading chip 13 include epT. I. P. S etc. are mentioned. Thereby, what is called cross contamination when many samples are continuously measured can be suppressed.

The electrospray ionization apparatus 1 may further include repetitive moving means for repetitively moving the probe member 10 in the longitudinal direction of the probe member 10. The sample can be attached to or captured by the probe 11 by the movement. Moreover, the electrospray ionization apparatus 1 may be provided with a manipulator, so that the sample can be captured from all directions as in the first embodiment.

The electrospray ionization method using the electrospray ionization apparatus 1 according to the second embodiment includes the above-described capturing step, ionization step, and solvent supply step, and the probe member 10 includes the probe 11 and the solvent loading chip 13. The probe 11 protrudes from the lower end of the solvent loading tip 13 in the range of 0 mm to 0.2 mm, and has a solvent filling step of filling the solvent loading tip 13 with the solvent. The needle member 10 is brought into contact with the sample, and the solvent is supplied from the inside of the solvent loading chip 13.

Here, if the protrusion of the probe 11 is smaller than 0 mm from the lower end of the solvent loading tip 13 (so as to be accommodated in the solvent loading tip 13), the spray generation point fluctuates. When the protrusion of the probe 11 is made larger than 0.2 mm from the lower end of the solvent loading tip 13, the tip of the sample is not wetted by the solvent from the solvent loading tip 13. By causing the probe 11 to protrude from the lower end of the solvent loading tip 13 within a range of 0 mm or more and 0.2 mm or less, stable spraying can be generated.

In the stage of capturing the sample (for example, the capturing step described above), the probe 11 protrudes from the lower end of the solvent loading tip 13 to a desired length (for example, 4 to 5 mm), and the tip of the probe member 11 is used as the sample. Puncture at a penetration depth of about 1 mm to capture the sample and perform electrospray (for example, the ionization step) so that the probe 11 is within the range of 0 mm to 0.2 mm from the lower end of the solvent loading chip 13. It may be. Such a mechanism may be incorporated in the probe member 10 or the operating unit 32. If comprised in this way, a hydrophobic sample etc. can also be capture | acquired easily, for example.

The amount of the solvent filled inside the solvent loading chip 13 is not particularly limited, but is, for example, 5 μL or more, 10 μL or more, 15 μL or more, and is 45 μL or less, 30 μL or less, 20 μL or less.

The electrospray ionization apparatus 1 of this embodiment stores a solvent in the solvent loading chip 13 in advance before measurement. As described above, this structure is a very simple structure, and a sufficient amount of solvent for electrospray can be supplied to the sample on the probe member 10.

(3) Third Embodiment FIG. 7 shows an example of the third embodiment of the electrospray ionization apparatus 1 of the present disclosure. In the third embodiment, in addition to the configuration of the electrospray ionization apparatus 1 shown in FIG. 1, the probe member 10 includes a probe 11 and an insulator 15 covering the probe 11, and the insulator 15 is on the surface. It is further characterized in that it is configured to supply a solvent when a solvent is retained and a part of the sample is ionized.

The electrospray ionization apparatus 1 of the third embodiment holds the liquid sample on the surface of the insulator 15 by bringing the probe member 10 into contact with the liquid sample. Thereafter, when a voltage is applied to the probe member 10 to generate electrospray at its tip, a solvent is supplied from the liquid sample held on the surface of the insulator 15 toward the tip of the insulator 15.

In the present embodiment, a liquid solvent continuous supply mechanism or a liquid solvent intermittent supply mechanism can be configured by the insulator 15 covering the probe 11.

In this embodiment, by covering the probe 11 with the insulator 15, electrospray can be generated over a long period of time, for example, 5 seconds or more. Further, since the spraying time lasts for a long time, it is suitable for a tandem mass spectrometer equipped with two mass spectrometers or a mass spectrometer equipped with three or more mass spectrometers.

The thickness of the probe member 10 covered with the insulator 15 is not particularly limited, but is, for example, 1 μm or more and 1000 μm or less, 5 μm or more and 500 μm or less.

Also, the probe member 10 may be a glass capillary using an insulator 15 filled with an electrically conductive liquid, for example, an ionic liquid, or an inner surface plated. When such a probe member is used, the electric field is stronger than when a metal probe is inserted, which is preferable in that the ability to ionize the sample is enhanced.

The insulator 15 is not particularly limited, but from the viewpoint of easy attachment of a solvent, availability, and ease of processing, borosilicate and epoxy resin are preferable, and there is little contamination by a sample, and from the viewpoint that it can be used repeatedly. More preferred are borosilicates.

The electrospray ionization method according to the third embodiment of the present disclosure includes the above-described capturing step, solvent supply step, and ionization step, and the probe member 10 includes a probe 11 and an insulator 15 that covers the probe, In the capturing step, the probe member 10 contacts the sample at a depth greater than 0 and 2 mm or less to capture the sample. In the ionization step, a part of the sample is ionized and the solvent supply step The solvent held on the surface of the insulator 15 is supplied to the sample.

The depth at which the probe member 10 is brought into contact with the sample may be greater than 0 and 2 mm or less when minimal invasiveness is to be considered for the sample to be measured, such as a biological examination. Preferably it is 1 mm or less, More preferably, it is 0.5 mm or less. Even if the depth at which the probe member 10 is brought into contact with the sample is within these ranges, this method can perform sufficient measurement.

The third embodiment shows that electrospraying is possible even when the probe 11 is covered with an insulator 15 such as borosilicate as shown in Example 4 described later. Furthermore, the performance is comparable to the performance of other embodiments and conventional PESI.

2. Mass Spectrometer The mass spectrometer 2 of the present disclosure includes an electrospray ionizer 1, an analyzer 3, and an ion detector 5. As the electrospray ionization apparatus 1 provided in the mass spectrometer 2 of the present disclosure, the same ones as described above can be used.

The analyzer 3 provided in the mass spectrometer 2 of the present disclosure is not particularly limited, and includes a magnetic field deflection type, a quadrupole type, an ion trap type, an orbitrap type, a time-of-flight type, a Fourier transform ion cyclotron resonance type, a tandem type, and the like. General equipment can be used. Since the mass spectrometer 2 of the present disclosure uses the electrospray ionization apparatus 1 having a function of continuously supplying a solvent to the target sample, the electrospray can be continuously caused. Therefore, the mass spectrometer 2 of the present disclosure has a slow sweep rate like a quadrupole mass spectrometer, which is difficult to apply in the conventional PESI-MS in which the electrospray time of the present disclosure is less than 1 second. A mass spectrometer can be combined, and a tandem type mass spectrometer having two analyzers can be used.

The ion detection device 5 included in the mass spectrometer of the present disclosure is detected by, for example, a format in which ions selected by the analysis device 3 are sensitized with an electron multiplier or a microchannel plate, an orbitrap format, or a Faraday cup. And the form of counting.

The mass spectrometry method of the present disclosure includes the above-described electrospray ionization method, an analysis step of separating ions ionized by the ionization method, and an ion detection step of detecting ions separated in the analysis step. It is what you have.

An example of the mass spectrometer of the present invention is shown in FIG. The voltage supply member 20 in FIG. 8 has the same configuration as in FIG. 2, and the switch 22 is turned off during the period in which the tip of the probe 11 is in contact with the solvent near the bottom point, and the tip of the probe 11 is After the ascent, the repetitive movement of the probe member 10 and the on / off of the switch 22 can be synchronized, such as ionizing the sample by turning on the switch 22 during a period in which the tip of the probe 11 is in the vicinity of the highest point it can. Thereby, since the solvent can be repeatedly supplied to the sample, a component that is relatively less ionized in the sample can be ionized.

In addition to the above, if the following points are taken into consideration, the electrospray ionization apparatus is more comfortable.
For example, in the electrospray ionization apparatus 1, when voltage is applied to generate electrospray, sample components are first sprayed for several seconds, and then electrospray of the solvent itself is generated. Electrospraying continues until the solvent is completely removed, but if the voltage is turned off when the sample jumps, solvent consumption can be saved. When the solvent is electrosprayed to a certain extent so that the sample captured at the tip is completely blown out, the sample can be washed and carry over can be prevented.

Also, when electrospray is generated by the electrospray ionization apparatus 1, the solvent itself is likely to volatilize when the ambient temperature is high, so that the electrospray is likely to become unstable and the solvent consumption rate increases. Therefore, it is better that the ambient temperature is room temperature.

In the case where sample collection and ionization are repeatedly performed or solvent supply is repeatedly performed, a combination with a robot or the like can be performed in the above-described embodiment. In this way, the operability of the 3D imaging mass spectrometer can be further improved.

In the second embodiment, if the head portion (in the vertical direction) of the solvent loading chip 13 is too large (50 mm or more), when the sample is brought into contact with the sample, the solvent flows out in one direction and the sample capturing efficiency decreases. A thickness of about 30 to 35 mm is appropriate. In this case, it is possible to wet the solid sample even if the probe is vertical, oblique, or up-side down. The tip of the solvent loading tip 13 remains thin, and the amount of solvent can be increased regardless of the head by making the upper part thicker). When the head is about 20 mm (or less), the liquid is sucked into the chip, and a signal derived from the solvent may not be generated even when electrospraying is performed. That is, the cleaning effect of the sample trapped at the tip by the subsequent continuous solvent electrospray is diminished. Therefore, the head is preferably 20 mm or more and 50 mm or less, and more preferably 30 to 35 mm.

In the second embodiment, the inner diameter of the tip of the solvent loading tip 13 may be selected to be about 0.2 mm. A probe 11 having an outer diameter of 0.12 mm (for example, a needle can be used) may be inserted into this. In this case, the gap at the tip of the solvent loading chip 13 is (0.2−0.12) /2=0.04 mm. In principle, the performance of electrospray increases as the tip diameter decreases. If the viewpoints of availability and manufacturing cost are ignored, the inner diameter of the chip may be 0.1 mm, 0.05 mm, or 0.01 mm or less in addition to the above. In this case, the probe 11 to be inserted may be thinner than the tip inner diameter.

In the third embodiment, the insulator 15 may be fused quartz, fused quartz coated with polyimide, or the like.

In the third embodiment, when depth direction analysis is performed, the depth at which the probe member 10 is brought into contact with the sample may be up to several mm (for example, about 10 mm). For example, it is possible to analyze a sample such as beef that has a greatly different mass spectrum when sampled only from the surface and deeply penetrated.

Example 1
Electrospray ionization apparatus and analysis apparatus (manufactured by JEOL) including a probe (Seirin, 0.12 × 40, 15Y05E1), a voltage application member, and a solvent supply member having a solvent storage unit and repetitive movement means A time-type mass spectrometer AccuTOF) and a mass spectrometer equipped with an ion detector were prepared. First, the tip of the probe is punctured by about 0.5 mm into the salmon roe. After puncturing, prepare a 1: 1 liquid mixture solvent of water: methanol in the solvent reservoir, and place it on the mass spectrometer so that the tip of the probe where the salmon is captured and the solvent reservoir are at a distance of 10 mm. Arranged. The probe is infiltrated into the solvent for 50 milliseconds to wet the sample at the tip of the probe with the solvent. After moistening, the probe was moved upward, and a 2.1 kV voltage was applied to the probe for 4 seconds at the topmost point, and the mass spectrum of salmon rosa just after the voltage application was started was measured. The puncture depth of the probe holding the sample into the solvent is about 1 mm.
9A to 9C show the results of sequential measurement of salmon PESI-MS spectra by repeating the above-mentioned liquid solvent supply and ionization steps once every 10 seconds. 9A is a spectrum measured at the first time, FIG. 9B is a spectrum measured at the fifth time, and FIG. 9C is a spectrum measured at the ninth time.

According to FIG. 9A, a peak derived from phosphatidylcholine (PC), which is a lipid, is observed at a mass / charge ratio in the vicinity of m / z 800-900 when the first electrospray is generated in Example 1. Further, according to FIG. 9B, when the electrospray is generated for the fifth time, a peak derived from PC is hardly observed, and instead, a peak derived from triacylglyceride (TAG) is observed around 900 to 1000 m / z. Yes. According to FIG. 9C, a peak considered to be a neutral lipid group different from PC is observed in the vicinity of m / z 800-900. Thus, in Example 1, not only ionized PC, but also neutral lipid groups different from TAG and PC, which were difficult to ionize by conventional PESI-MS, were successfully ionized and detected. is doing. Note that lipid groups such as TAG are components that are not easily ionized as compared to lipids such as PC because they originally have no charge.

(Example 2)
Metal probe so that it penetrates about 0.1 mm from the tip of the solvent loading tip (polypropylene, epTIPS, GEorder, Eppendorf, inner diameter about 0.2 mm, outer diameter about 0.3 mm, total length 65 mm, 20 μL). The needle was placed, and the other end of the solvent loading tip and the metal probe were sealed with epoxy resin. Further, a hole was made in the side surface of the solvent loading chip, and about 15 μL of a mixed solvent of water: methanol 1: 1 was injected. Instant coffee grains were brought into slight contact with the tip of the probe member to dissolve and capture the coffee component at the tip. This was set in the same mass spectrometer as in Example 1, a 2.2 kV voltage was applied to the probe for 2 seconds to generate electrospray from the tip, and the PESI-MS spectrum during that time was measured. Shown in ~ 10B. FIG. 10A is a spectrum measured 0.5 seconds after voltage application, and FIG. 10B is a spectrum measured 2 seconds after voltage application.

According to FIG. 10A, in the spectrum measured 0.5 seconds after the voltage application, a peak in which K ⁺ is added to caffeine and oligosaccharide is observed, but in the spectrum measured 2 seconds after the voltage application, A lipid-derived peak is observed around m / z 870. In addition, the peak of m / z 195 is a protonated form of caffeine (molecular weight 194), and m /

z

381, 543, and 705 are considered to be K ⁺ adducts of oligosaccharides. Thus, in Example 2, not only caffeine and oligosaccharides that are easily ionized, but also lipid-derived peaks that were difficult to ionize by conventional PESI-MS have been successfully ionized and detected. .

(Example 3)
Using the mass spectrometer used in Example 2, the result of measuring ink handwriting written on plain paper with a four-color ballpoint pen from Zebra using a mixed solvent of water: methanol: acetonitrile 1: 1: 1 FIG. 11A to FIG. 11D show the results of measuring ink handwriting written on plain paper with a pilot company's black ballpoint pen, as shown in FIG. 11E. The ink of the ballpoint pen was used after writing letters on paper and leaving it at room temperature for about 10 minutes. The tip of a probe member similar to that of Example 2 is lightly touched with a handwriting written with a ballpoint pen, and a part of the handwriting (about 0.3 mm in diameter) is dissolved in a solvent and extracted and captured at the tip. It was used for.

As can be seen from FIGS. 11A to 11D, completely different spectra are obtained from the respective colors of the four-color ballpoint pens of Zebra. Furthermore, comparing FIG. 11A and FIG. 11E, different spectra are obtained for the same black ink, even for Zebra and Pilot. From this, the mass spectrometer of the present invention is expected to be applied to fields that require precise analysis such as handwriting appraisal.

Example 4
The tip of a glass capillary (manufactured by Prime Tech, PT Micropipettes for PMM, “Ultra-thin”, PINUS06-20FT) was heated and sealed, and a metal probe was inserted therein. After the insertion, an epoxy resin was bonded to the base of the glass capillary, and the probe and the glass capillary were fixed.
Otherwise, the results of mass spectrometry of yogurt (Meiji Co., Ltd .: Probio Yogurt R-1) using the same equipment as in Example 1 are shown in FIGS. 12A to 12B. FIG. 12A shows the spectrum after 1 second of voltage application, and FIG. 12B shows the spectrum after 5 seconds of voltage application.

As can be seen from FIGS. 12A to 12B, the peaks of lactose Na ⁺ and Ca ²⁺ adducts are observed immediately after voltage application. Furthermore, many components are newly observed around m / z 700 after 5 seconds. This component is expected to be added to protect lactic acid bacteria from stomach acid and transport them to the intestine in a protected state. Thus, in Example 4, application to the field of food safety and quality control can be expected.

1: Electrospray ionizer, 2: Mass spectrometer, 3: Analyzer, 5: Ion detector, 10: Probe member, 11: Probe, 13: Solvent loading chip, 15: Insulator, 20: Voltage application Member: 21: power source, 22: switch, 23: digital delay pulse generator, 30: solvent supply member, 31: soot solvent storage unit, 32: operating unit, 33: repetitive moving means, 34: manipulator

Claims

An electrospray ionization device,
A probe member that captures the sample by contact;
A voltage application member configured to apply a voltage to the probe member;
The probe member includes a continuous supply mechanism of liquid solvent or an intermittent supply mechanism of liquid solvent to the sample, or the electrospray ionization apparatus includes a continuous supply mechanism of liquid solvent or an intermittent supply mechanism of liquid solvent to the sample. A solvent supply member comprising:
Electrospray ionizer.
The probe member includes a probe and a solvent loading tip;
The probe protrudes from the lower end of the solvent loading tip;
Configured to store a liquid solvent inside the solvent loading chip,
The electrospray ionization apparatus according to claim 1.
The electrospray ionization apparatus according to claim 2, wherein the probe protrudes from a lower end of the solvent loading tip in a range of 0 mm to 0.2 mm.
The probe member includes a probe;
The solvent supply member includes a solvent storage unit and an operation unit,
The operating unit is configured to supply the solvent to the sample by bringing the probe into contact with a liquid solvent stored in the solvent storage unit.
The electrospray ionization apparatus according to claim 1.
The probe member includes a probe and an insulator covering the probe;
The insulator holds a liquid solvent on the surface, and is configured to supply the solvent when a part of the sample is ionized.
The electrospray ionization apparatus according to claim 1.
6. The electrospray ionization apparatus according to claim 1, further comprising repetitive movement means for repetitively moving the probe member in a longitudinal direction of the probe member.
The electrospray ionization apparatus according to any one of claims 1 to 6, further comprising a manipulator.
The electrospray ionization apparatus according to any one of claims 1 to 7,
An analyzer for separating ions ionized by the electrospray ionizer;
An ion detector for detecting ions separated by the analyzer;
A mass spectrometer.
A capturing step in which the probe member captures the sample;
An ionization step of ionizing the sample by applying a voltage to the probe member;
A solvent supply step of supplying a liquid solvent continuously or intermittently to the sample;
An electrospray ionization method comprising:
The probe member includes a probe and a solvent loading tip;
The probe protrudes from the lower end of the solvent loading tip;
Having a solvent filling step of filling the solvent loading chip with a liquid solvent;
The electrospray ionization method according to claim 9, wherein, in the solvent supply step, the probe member is brought into contact with the sample, and the liquid solvent is supplied from the inside of the solvent loading chip.
The probe member includes a probe;
Supplying the liquid solvent to the sample by bringing the probe into contact with the liquid solvent in the solvent supplying step;
The method of electrospray ionization according to claim 9.
The probe member includes a probe and an insulator covering the probe;
In the capturing step, the probe member contacts the sample at a depth greater than 0 mm and 2 mm or less to capture the sample,
A part of the sample is ionized in the ionization step, and in the solvent supply step, a liquid solvent held on the surface of the insulator is supplied to the sample.
The method of electrospray ionization according to claim 9.
The electrospray ionization method according to any one of claims 9 to 13, wherein the probe member is repeatedly moved in a longitudinal direction of the probe member in the solvent supply step.
The method of electrospray ionization according to any one of claims 9 to 13, wherein, in the capturing step, the probe member can collect the sample from all directions.
The method of electrospray ionization according to any one of claims 9 to 14,
An analysis step of separating ions ionized by the ionization method;
An ion detection step of detecting ions separated in the analysis step;
A mass spectrometric method.