JP7282324B2 - Ionization device, mass spectrometer and method for analyzing fluid sample - Google Patents

Description

The present invention relates to an ionization device that ionizes a fluid sample, a mass spectrometer that performs mass analysis of the fluid sample ionized by the ionization device, and a fluid sample analysis method.

In a mass spectrometer, components in a sample to be measured are ionized. Electrospray ionization (ESI) is known as a method for ionizing components in a sample. Also, as one of the ionization methods using the ESI method, a probe electrospray ionization (PESI) method is attracting attention.

As disclosed in Patent Document 1 below, a PESI ion source that performs ionization by the PESI method includes a conductive probe whose tip diameter is about several hundred nanometers and a high voltage that applies a high voltage to the probe. and a voltage generator. At the time of measurement, at least one of the probe and the sample is moved so that a minute amount of the sample adheres to the surface of the tip of the probe. Subsequently, the probe is separated from the sample, the high voltage generator is driven, and a high voltage is applied to the probe. As a result, a strong electric field acts on the sample adhering to the tip of the probe, causing an electrospray phenomenon, and component molecules in the sample are ionized while being detached.

In general, ionization using the electrospray phenomenon has higher ionization efficiency than other ionization methods, such as ionization methods using laser light irradiation. Therefore, the PESI ion source can efficiently ionize a very small amount of molecules in the sample. By using the PESI method, for example, a very small amount of biological tissue collected from a subject can be ionized as it is without any pretreatment including dissolution and dispersion.

By using a PESI ion source with such a high ionization efficiency, for example, as described in Non-Patent Document 1 below, a probe can be directly inserted into a biological tissue to generate a TCA (TriCarboxylic Acid) circuit component. and changes in drug metabolism can be monitored.

A microdialysis method is known as a method for observing changes in neurotransmitters in the brain of experimental animals under free movement. Extracellular fluid can be collected by perfusing perfusate through a guide cannula implanted in the brain of an experimental animal.

High Performance Liquid Chromatography (HPLC) and mass spectrometry are used as methods for measuring components of extracellular fluid. These methods for measuring extracellular fluid require pretreatment of the sample before measurement, so off-line measurement without real-time capability is performed.

JP 2014-44110 A

Construction of in vivo real-time monitoring method by PESI/MS/MS (Shimadzu Review VOL.74 No.1/2 September 2017)

The technique disclosed in Patent Document 1 makes it possible to efficiently ionize a sample and perform measurement. By using the PESI method, it is possible to perform measurement without pretreatment on a biological sample, and as shown in Non-Patent Document 1 above, it is possible to measure changes in biological components in a living state. Monitoring is possible. However, in order to measure a fluid sample (extracellular fluid) collected by brain microdialysis by the PESI method, it is necessary to temporarily store the collected fluid sample by some means and then supply it to the PESI ion source. Therefore, the process of measuring a fluid sample collected by the brain microdialysis method by the PESI method is offline. In order to measure changes in components of fluid samples collected by the brain microdialysis method, such processing must be repeated, and changes can only be monitored on the order of several minutes (at intervals of several minutes). Therefore, it is difficult to capture real-time changes in brain neurotransmitters collected by brain microdialysis.

Moreover, in order to increase the ionization efficiency by the PESI method, it is necessary to keep the concentration of the solvent (mixture of water and alcohols) constant. Therefore, in order to monitor a fluid sample collected by the brain microdialysis method for a long time by the PESI method, it is difficult to keep the concentration of the solvent constant.

An object of the present invention is to provide an ionization apparatus, a mass spectrometer, and a method of analyzing a fluid sample that are capable of real-time analysis of fluctuations in components of a fluid sample collected from a living body.

(1) An ionization device according to a first aspect of the invention is an ionization device for ionizing a component of a fluid sample, comprising a probe, a voltage applying section for applying a voltage to the probe, and a probe drive for moving the probe. a solvent storage section for storing a solvent; and a sample storage section connected to a sample supply path for receiving a fluid sample supplied from the outside of the ionization device. The solvent storage part has first and second openings facing each other, and the first opening is closed by the first film body to store the solvent, and the outside of the container part, A second membrane disposed between the first membrane and the sample containing portion is included. The probe driving section causes the probe to enter the container through the second opening in a state in which the solvent is accommodated in the container, and the probe penetrates the first and second membranes to reach the sample accommodation section. move to pick up a fluid sample supplied to the .

In this ionization device, a fluid sample is supplied to the sample storage section through the sample supply path. Under the control of the probe driver, the probe enters the container containing the solvent, penetrates the first and second membranes, and collects the fluid sample supplied to the sample container. By applying a voltage to the probe under the control of the voltage applying section, the fluid sample collected by the probe is ionized. According to this configuration, the fluid sample supplied from the outside of the ionization device can be ionized in real time without undergoing pretreatment. The ionization of the fluid sample can be performed continuously while the fluid sample is supplied.

(2) The fluid sample supplied from the sample supply channel may contain a substance recovered from the living body by the microdialysis method. According to this configuration, the substance recovered from the living body by the microdialysis method can be ionized in real time. Fluctuations in components in the extracellular fluid can be monitored in real time with high resolution (for example, on the order of several seconds) while the substance is being supplied from the living body by the microdialysis method.

(3) The fluid sample supplied from the sample supply channel may contain an internal standard substance. With this configuration, it is possible to improve the quantitativeness of the fluid sample.

(4) The solvent storage unit may be connected to a solvent supply channel for receiving solvent supply from the outside of the ionization device. According to this configuration, the concentration of the solvent in the solvent container can be kept constant. As a result, ionization can be performed while the concentration of the solvent is kept constant even while the fluid sample is continuously supplied to the sample storage section.

(5) A mass spectrometer according to a second aspect of the present invention comprises an ionization device according to any one of (1) to (4) above, and a mass spectrometer that analyzes components of a fluid sample based on ions generated by the ionization device. And prepare. According to this configuration, it is possible to perform mass spectrometry on the fluid sample supplied to the sample storage section in real time.

(6) A method for analyzing a fluid sample according to a third aspect of the invention is a method for analyzing a fluid sample using the ionization device according to (1) above, wherein the solvent is contained in the solvent container and the sample is supplied through the sample supply path. preparing a state in which the fluid sample is continuously supplied to the sample containing portion by using the probe driving portion to move the probe through the solvent contained in the container; collecting a fluid sample by penetrating the probe through the first and second membranes to reach the fluid sample supplied to the sample storage unit; After the needle is separated from the fluid sample, the probe is again passed through the solvent contained in the container section, and moved to a predetermined position, a voltage is applied to the probe by the voltage applying section to move the probe. The steps include ionizing a fluid sample attached to the tip and mass spectrometrically analyzing the ionized fluid sample. According to this method, it is possible to ionize the fluid sample supplied to the sample storage section in real time while continuously supplying the fluid sample to the sample storage section. This makes it possible to perform real-time mass spectrometry on the fluid sample supplied to the sample container.

According to the present invention, fluctuations in the components of fluid samples collected from living organisms can be analyzed in real time.

1 is an overall view of a mass spectrometer according to an embodiment of the invention; FIG. 1 is a diagram showing the configuration of an ionization apparatus according to an embodiment of the present invention to which a microdialysis method is applied; FIG. It is a side cross-sectional view showing a solvent storage unit provided in the ionization device. It is a perspective view which shows the solvent storage part with which an ionization apparatus is provided. 3 is a perspective view showing a sample storage section and a solvent storage section provided in the ionization device; FIG. It is a figure which shows the modification of this invention.

DETAILED DESCRIPTION OF THE INVENTION An ionization apparatus according to an embodiment of the present invention, a configuration of a mass spectrometer equipped with the ionization apparatus, and a method for analyzing a fluid sample will now be described in detail with reference to the drawings.

(1) Configuration of Mass Spectrometer FIG. 1 is an overall view showing the configuration of a mass spectrometer 1 according to an embodiment of the present invention. The mass spectrometer 1 according to the embodiment is a PESI ionization mass spectrometer using probe electrospray ionization (PESI). The mass spectrometer 1 comprises an ionization chamber 4, a first intermediate vacuum chamber 6, a second intermediate vacuum chamber 7, an analysis chamber 8 and a computer 9, as shown in the figure.

The ionization chamber 4 accommodates an ionization device 3 that ionizes the components in the sample under atmospheric pressure. The analysis chamber 8 performs mass separation and detection of ions in a high vacuum atmosphere. Between the ionization chamber 4 and the analysis chamber 8, a first intermediate vacuum chamber 6 and a second intermediate vacuum chamber 7 forming a multistage differential pumping system are provided. The degree of vacuum in the mass spectrometer 1 is increased stepwise by the first intermediate vacuum chamber 6 and the second intermediate vacuum chamber 7 . Although not shown in FIG. 1, generally, the first intermediate vacuum chamber 6 is evacuated by a rotary pump, and the second intermediate vacuum chamber 7 and analysis chamber 8 are evacuated by a turbomolecular pump in addition to the rotary pump. be done.

The ionization device 3 has a probe holder 21 and a metal probe 22 . The probe 22 is held by a probe holder 21 in the upper space of the ionization chamber 4, which is substantially atmospheric pressure. The probe holder 21 is movable in the vertical direction (the Z-axis direction in FIG. 1) by a probe drive unit 101 including a motor, a deceleration mechanism, an actuator, and the like. As a result, the probe 22 is vertically movable in the ionization chamber 4 . A high voltage of about several kV at the maximum is applied to the probe 22 from the high voltage generator 102 . In this embodiment, the metal probe 22 is used, but the probe 22 is not limited to metal, and other conductive materials can be used.

The ionization device 3 includes a solvent storage section 3A and a sample storage section 3B. The solvent containing portion 3A and the sample containing portion 3B are arranged below the probe 22 . A door (not shown) is provided in the ionization chamber 4, and the user can install and adjust the solvent storage section 3A and the sample storage section 3B with the door open. The configurations of the solvent storage section 3A and the sample storage section 3B will be described later in detail.

The ionization chamber 4 and the first intermediate vacuum chamber 6 are connected through a desolvation pipe 51 which is a thin capillary. The gas in the ionization chamber 4 is drawn into the first intermediate vacuum chamber 6 through the desolvation tube 51 due to the pressure difference between the openings at both ends of the desolvation tube 51 .

The first intermediate vacuum chamber 6 is provided with an ion guide 61 called a Q array in which four virtual rod electrodes are arranged around the ion optical axis C. As shown in FIG. A virtual rod electrode is composed of a plurality of disk-shaped electrode plates arranged along the ion optical axis C. As shown in FIG. The first intermediate vacuum chamber 6 and the second intermediate vacuum chamber 7 are connected through a small diameter orifice formed at the top of the skimmer 52 . An octapole type ion guide 71 in which eight rod electrodes are arranged around the ion optical axis C is installed in the second intermediate vacuum chamber 7 .

In the analysis chamber 8, a front-stage quadrupole mass filter 81 and a rear-stage quadrupole mass filter 84 are arranged in front of and behind the collision cell 82 with the collision cell 82 interposed therebetween. Both the front-stage quadrupole mass filter 81 and the rear-stage quadrupole mass filter 84 have a configuration in which four rod electrodes are arranged around the ion optical axis C. FIG.

A quadrupole or more multipole ion guide 83 is arranged inside the collision cell 82 . An ion detector 85 that outputs a signal corresponding to the amount of ions that have arrived is arranged downstream of the post-stage quadrupole mass filter 84 . Although illustration of each signal line is omitted, a predetermined voltage is applied to each part including the desolvation pipe 51 from the voltage generator 103 .

The mass spectrometer 1 has a computer 9 . The computer 9 has a control section 100 , a data processing section 105 , an input section 106 and a display section 107 . The control unit 100 controls the probe driving unit 101, the high voltage generating unit 102, the voltage generating unit 103, etc. in order to perform mass spectrometry on the fluid sample supplied to the sample containing unit 3B. A signal detected by the ion detector 85 is input to the data processing section 105 . After the detection signal is digitally converted in the data processing unit 105, processing for creating a mass spectrum, a chromatogram, or the like is executed. The probe driving unit 101, the high voltage generating unit 102, the voltage generating unit 103, and the like are arranged anywhere in the apparatus housing housing the ionization chamber 4 and the analysis chamber 8. FIG.

At least some of the functions of the control unit 100 and the data processing unit 105 are realized by using the CPU, memory, etc. of the computer 9 as hardware resources, and by operating the control software installed in the computer 9 on the computer 9. be able to. A personal computer may be used as the computer 9 . Alternatively, the computer 9 may be incorporated in an apparatus housing housing the ionization chamber 4 and the analysis chamber 8 .

(2) Configuration of Solvent Storage Unit and Sample Storage Unit FIG. 2 is a diagram showing the configuration of an ionization device 3 according to an embodiment of the present invention to which the microdialysis method is applied. As shown in the figure, the ionization device 3 includes a solvent containing portion 3A and a sample containing portion 3B.

The solvent stored in the solvent tank 311 is continuously supplied to the solvent storage section 3A. In the present embodiment, solvent tank 311 stores solvent 200 in which heparin (0.5 mg/mL), which is an anticoagulant for preventing blood coagulation, is added to 100 μL of 50% ethanol aqueous solution. ing. As the solvent 200, water, alcohols, or mixtures thereof are generally used. The components of the solvent can be appropriately selected depending on the fluid sample to be supplied. Depending on the fluid sample, the anticoagulant heparin may not be added.

Solvent tank 311 is connected to solvent storage section 3A via tube 313 and connection pipe 314 . A liquid transfer pump 312 is provided on the tube 313 . The solvent 200 stored in the solvent tank 311 is supplied to the solvent container 3A through the tube 313 and the connecting pipe 314 by the pumping action of the liquid-sending pump 312 . The tube 313 and connecting pipe 314 correspond to the "solvent supply channel" in the present invention. Although the configuration of the liquid transfer pump 312 is not particularly limited, for example, a peristaltic pump (registered trademark) can be used.

FIG. 3 is a side cross-sectional view showing the solvent container 3A. FIG. 4 is a perspective view showing the solvent container 3A. The solvent storage part 3A includes a cylindrical member 31, an upper film body 32, a lower film body 33 and spacers . The cylindrical member 31 is a cylindrical body made of synthetic resin and having upper and lower openings. An upper membrane 32 made of polyvinylidene chloride is attached to the lower portion of the cylindrical member 31 so as to block the opening of the lower portion of the cylindrical member 31 . A container for containing the solvent 200 is formed by the cylindrical member 31 and the upper film body 32 .

Below the upper film 32, a lower film 33 made of polyvinylidene chloride is arranged substantially parallel to the upper film 32 with a spacer 34 interposed therebetween. The upper film body 32 corresponds to the "first film body" in the present invention, and the lower film body 33 corresponds to the "second film body" in the present invention. Further, the container capable of containing the solvent 200, which is composed of the cylindrical member 31 and the upper film body 32, corresponds to the "container section" in the present invention.

In the present embodiment, the cylindrical member 31 having the entire top and bottom openings is used as a constituent element of the container portion, but the present invention is not limited to this. A member having upper and lower portions that are partially open may be used as the member constituting the container portion, and one of the openings may be closed with the first membrane. It is sufficient that the cylindrical member 31, together with the upper film body 32, constitutes a container capable of accommodating the solvent 200, and the shape and material thereof are not particularly limited. The lower opening of the cylindrical member 31 corresponds to the "first opening" in the present invention, and the upper opening of the cylindrical member 31 corresponds to the "second opening" in the present invention.

The distance between the upper film 32 and the lower film 33 corresponds to the thickness d of the spacer 34 . The spacer 34 has, for example, a cylindrical shape concentric with the cylindrical member 31, and an opening through which liquid can pass is provided at an appropriate position on the wall surface thereof. Both the upper film 32 and the lower film 33 have liquid impermeability, and preferably have appropriate elasticity.

In the present embodiment, polyvinylidene chloride is used as the upper film 32 and the lower film 33, but the material is not limited to this. A composite resin material or the like can be used. In this embodiment, the cylindrical member 31 has an inner diameter of 9.4 mm and a height of 5.0 mm. The film thickness of the upper film body 32 and the lower film body 33 is 11 μm. A distance d between the upper film body 32 and the lower film body 33 is 1.0 mm.

The wall surface of the cylindrical member 31 is provided with an opening into which the connecting pipe 314 can be inserted. A solvent 200 sent from a solvent tank 311 is supplied through a connecting pipe 314 to a container portion composed of a cylindrical member 31 and an upper membrane body 32 . Further, the wall surface of the cylindrical member 31 is provided with another opening into which the connection pipe 315 can be inserted. As shown in FIG. 2, connecting pipe 315 is connected to tube 316 . Tube 316 is connected to discharge tank 317 . Solvent 200 in the container part is discharged to discharge tank 317 through connecting pipe 315 and tube 316 .

In this embodiment, as shown in FIG. 3, the position where the connection pipe 315 is connected is higher than the position where the connection pipe 314 is connected. As a result, the liquid level of the solvent 200 supplied through the connection pipe 314 rises, and when it reaches the position of the connection pipe 315 , the solvent 200 is discharged to the discharge tank 317 .

In this manner, the present embodiment has a configuration for supplying the solvent to the solvent storage section 3A. Thereby, the concentration of the solvent in the solvent containing portion 3A can be kept constant. Even while the fluid sample is continuously supplied to the sample storage section 3B, the ionization device 3 can ionize the biological sample while keeping the concentration of the solvent constant.

The configuration of the sample container 3B will be described with reference to FIGS. 2 and 5. FIG. In the present embodiment, the sample storage section 3B is continuously supplied with a fluid sample from a fluid sample recovery device using the microdialysis method. As shown in FIG. 5, the sample storage section 3B includes a sample storage table 35 for storing a fluid sample and a sample supply pipe 325 embedded in the sample storage table 35 . A fluid sample is continuously supplied to the sample supply pipe 325 from the outside of the ionization device 3 . The lower film body 33 of the solvent storage part 3A is attached to the top of the sample storage table 35 . As a result, the solvent storage section 3A is placed on the upper portion of the sample storage table 35. As shown in FIG.

As shown in FIG. 2, a sampling hole 351 extending vertically is provided near the center of the sample holding table 35 . The upper end of the sampling hole 351 is open to the upper surface of the sample holding table 35 . A lower end of the sampling hole 351 is connected to the sample supply pipe 325 . The sample supply pipe 325 has an opening at a position corresponding to the lower end of the sampling hole 351 , and this opening is connected to the lower end of the sampling hole 351 . The tip of the probe 22 that has descended is inserted into the collection hole 351 . This allows the tip of the probe 22 to collect a fluid sample flowing through the sample supply pipe 325 .

Next, the configuration of the fluid sample recovery device using the microdialysis method connected to the sample storage section 3B will be described. As shown in FIG. 2, the fluid sample collection device comprises a perfusate tank 321 , a fluid pump 322 , a tube 323 , a microdialysis probe 328 and a tube 324 .

The perfusate 201 is stored in the perfusate tank 321 . As the perfusate 201, for example, cerebrospinal fluid, physiological saline, Ringer's solution, or the like is used. A tube 323 is connected to the perfusate tank 321 . The other end of tube 323 is connected to microdialysis probe 328 . The tube 323 is provided with a liquid feed pump 322 at a position between the perfusate tank 321 and the microdialysis probe 328 . The perfusate 201 stored in the perfusate tank 321 is sent to the microdialysis probe 328 through the tube 323 by the pumping action of the liquid-sending pump 322 . Although the configuration of the liquid-sending pump 322 is not particularly limited, for example, a peristaltic pump (registered trademark) can be used.

A dialysis membrane attached to the tip of the microdialysis probe 328 is left in the mouse M brain tissue. As a result, the extracellular fluid of the mouse M brain tissue can be collected by the dialysis membrane. The extracellular fluid collected by the dialysis membrane is sent as a fluid sample together with the perfusate 201 to the sample storage section 3B via the tube 324 . A sample supply pipe 325 is connected to the end of the tube 324 .

The sample supply pipe 325 is embedded inside the sample storage table 35 . In this embodiment, as shown in FIG. 5, the sample supply pipe 325 extends linearly inside the sample storage table 35, but the shape of the sample supply pipe 325 is not particularly limited. As described above, the sampling hole 351 extending vertically and connected to the sample supply pipe 325 is provided in the vicinity of the center of the sample holding table 35 . As a result, the tip of the descending probe 22 collects the fluid sample (extracellular fluid) flowing through the sample supply pipe 325 . The tube 324 and sample supply pipe 325 correspond to the "sample supply path" in the present invention.

A tube 326 is connected to the end opposite to the end to which the tube 324 is connected to the sample supply pipe 325 embedded in the sample storage table 35 . Tube 326 is connected to discharge tank 327 . The perfusate 201 and the fluid sample (extracellular fluid) that have passed through the sample storage section 3B are discharged to the discharge tank 327 via the tube 326 .

(3) Analysis Processing Procedures and Operations Next, procedures and operations for real-time analysis of neurotransmitters in the brain of a living mouse M using the mass spectrometer 1 according to the present embodiment will be described. .

As shown in FIG. 2, the user implants a microdialysis probe 328 in the brain tissue of a freely moving mouse M. As shown in FIG. The perfusate 201 is stored in the perfusate tank 321, and the liquid feed pump 322, tube 323, tube 324, sample supply pipe 325, tube 326 and discharge tank 327 are placed at predetermined positions. The solvent 200 is stored in the solvent tank 311, and the liquid feed pump 312, tube 313, connection pipe 314, connection pipe 315, tube 316 and discharge tank 317 are arranged at predetermined positions.

In the present embodiment, the solvent tank 311, the perfusate tank 321, the liquid feed pumps 312 and 322, the tube 323, the microdialysis probe 328, the mouse M, the discharge tank 317 and the discharge tank 327 are included in the ionization chamber 4. Placed outside. Therefore, tubes 313 , 316 , 324 and 326 extend outside through tube insertion holes provided in ionization chamber 4 and are connected to the components described above outside ionization chamber 4 . Of course, this configuration is an example. If a space can be secured in the ionization chamber 4 , the devices including the device for performing the microdialysis method may be arranged in the ionization chamber 4 .

After the preparation of each part and apparatus is completed, the user operates the input unit 106 to instruct the start of analysis. Upon receiving the analysis start instruction, the control unit 100 sends a control signal to each unit to execute the analysis. When the analysis starts, the probe driver 101 drives the probe 22 under the control of the controller 100 . The probe drive unit 101 causes the probe 22 to enter the sampling hole 351 of the sample accommodation table 35 and lower it until the tip thereof is positioned inside the sample supply pipe 325 . Subsequently, the probe driver 101 raises the probe 22 to a predetermined position.

When the probe 22 descends, the pointed tip of the probe 22 passes through the solvent 200 contained in the solvent containing portion 3A, breaks through the upper film body 32 and the lower film body 33, respectively, and enters the sample supply pipe 325. reaches the fluid sample (extracellular fluid) within. Therefore, the solvent 200 adheres to the tip surface of the probe 22 before it reaches the fluid sample (extracellular fluid), and the probe 22 becomes wet with the solvent 200 . Then, when the tip of the probe 22 enters the sample supply pipe 325 , the fluid sample (extracellular fluid) adheres to the tip of the probe 22 .

When the probe 22 is pulled up, the tip of the probe 22 passes through the solvent 200 again, so the solvent 200 also adheres to the fluid sample (extracellular fluid) adhering to the tip of the probe 22 . As a result, the fluid sample (extracellular fluid) adhering to the tip of the probe 22 is wrapped in the solvent 200, so that the ionization efficiency can be improved.

After the probe 22 is pulled up to a predetermined position, the high voltage generator 102 applies a predetermined high voltage to the probe 22 . The polarity of the high voltage applied to the probe 22 depends on the polarity of ions to be measured. The high voltage generation section 102 corresponds to the "voltage application section" in the present invention. When a high voltage is applied to the tip of the probe 22, a large electric field acts on the fluid sample (extracellular fluid) adhering to the tip of the probe 22, and the components in the sample are biased due to Coulomb repulsion and the like. while desorbing. That is, the components in the fluid sample are ionized and then electrosprayed. Ions generated by electrospray are sucked into the desolvation pipe 51 along with the gas flow generated by the pressure difference as described above, and sent to the first intermediate vacuum chamber 6 .

As described above, since both the upper film 32 and the lower film 33 have appropriate elasticity, the hole through which the tip of the probe 22 penetrates shrinks to some extent when the probe 22 is pulled up. . However, since this hole is not completely closed, liquid such as a fluid sample leaking from the sample supply pipe 325 as the probe 22 advances and retreats leaks onto the lower film body 33 through this minute hole.

There is a moderate gap space between the upper film body 32 and the lower film body 33, and a large amount of liquid does not leak out to fill this space in a short period of time. Therefore, a liquid such as a fluid sample leaking into this space spreads on the lower membrane 33 and is discharged to the outside of the spacer 34 through an opening formed in the spacer 34 . As a result, it is possible to prevent a liquid such as a fluid sample or blood that seeps out from the sample supply pipe 325 from mixing with the solvent 200 in the container portion of the solvent storage portion 3A.

Blood mixed in the fluid sample may adhere to the probe 22 . In this embodiment, heparin, which is an anticoagulant, is added to solvent 200 . As a result, the probe 22 passes through the solvent 200, thereby preventing coagulation of blood. By preventing coagulation of the blood adhering to the probe 22, high ionization efficiency can be maintained.

The ions sent into the first intermediate vacuum chamber 6 are converged and transported by the high-frequency electric field formed by the ion guide 61, and sent to the second intermediate vacuum chamber 7 through the orifice provided at the top of the skimmer 52. Further, the ions are converged by the high-frequency electric field formed by the ion guide 71 and sent to the analysis chamber 8 .

A voltage obtained by superimposing a high-frequency voltage on a DC voltage is applied from the voltage generator 103 to the front-stage quadrupole mass filter 81 . Only ions having a mass-to-charge ratio m/z corresponding to the applied voltage pass through the longitudinal space of the front-stage quadrupole mass filter 81 and enter the collision cell 82 . Argon gas or the like is introduced as a collision gas into the collision cell 82, and ions entering the collision cell 82 are dissociated upon contact with these gases. Product ions generated by dissociation are converged by the high-frequency electric field formed by the ion guide 83 , exit the collision cell 82 , and are introduced into the post-stage quadrupole mass filter 84 .

A voltage obtained by superimposing a high-frequency voltage on a DC voltage is also applied from the voltage generator 103 to the post-stage quadrupole mass filter 84 . Only product ions having a mass-to-charge ratio m/z corresponding to the applied voltage pass through the longitudinal space of the rear quadrupole mass filter 84 and reach the ion detector 85 .

For example, the front-stage quadrupole mass filter 81 allows only ions having a specific mass-to-charge ratio to pass through, and the rear-stage quadrupole mass filter 84 performs mass scanning over a predetermined mass-to-charge ratio range. As a result, the data processing unit 105 can obtain a product ion spectrum reflecting various product ions obtained by dissociating ions having a specific mass-to-charge ratio derived from the sample. Of course, in addition to product ion scan measurement, precursor ion scan measurement, neutral loss scan measurement, and multiple reaction monitoring (MRM) measurement may be performed. Ordinary scan measurement or Selected Ion Monitoring (SIM) measurement may be performed without dissociating ions in the collision cell 82 .

As described above, by using the ionization device 3 according to the present embodiment, a fluid sample supplied from the outside of the ionization device 3 can be ionized in real time without undergoing pretreatment. The ionization of the fluid sample can be performed continuously while the fluid sample is supplied. By using the mass spectrometer 1 according to the present embodiment, it is possible to perform mass spectrometry on the fluid sample supplied to the sample storage section 3B in real time.

Further, it is possible to apply the microdialysis method to the ionization device 3 of the present embodiment. This enables real-time ionization of substances recovered from living organisms by the microdialysis method. Fluctuations in components in the extracellular fluid can be monitored in real time with high resolution (for example, on the order of several seconds) while the substance is being supplied from the living body by the microdialysis method. For example, it is possible to monitor changes in neurotransmitters such as serotonin and dopamine collected by brain microdialysis in real time.

(4) Modification FIG. 6 is a diagram showing a modification of the present invention. In a modified example, an internal standard substance is mixed with the fluid sample supplied to the sample container 3B. An addition section 329A for adding an internal standard substance is provided in the perfusate tank 321, for example, as shown in the figure. The addition unit 329A adds an appropriate amount of internal standard substance according to the amount of perfusate stored in the perfusate tank 321 .

Moreover, as another modified example, the adding section 329B may add the internal standard substance in the middle portion of the tube 324 . The adding section 329B supplies the internal standard substance to the tube 324 at an appropriate flow rate according to the flow rate of the perfusate. By adding the internal standard substance to the fluid sample in this way, the mass spectrometer 1 can improve the quantification of the biological sample based on the amount of the internal standard substance.

In the above embodiment, the microdialysis method is applied to the mass spectrometer 1 of the present invention. The method of supplying the fluid sample to the ionization device 3 and the mass spectrometer 1 of the present invention is not limited to the microdialysis method. Any method may be used as long as it can continuously supply a fluid sample to the ionization device 3 and the mass spectrometer 1 of the present invention.

In the above embodiments, fluid samples are collected in drain tank 327 . Although the fluid sample collected in the drain tank 327 may be discarded, it can also be used for another analysis.

The specific configuration of the present invention is not limited to the above-described embodiment, and various changes and modifications are possible without departing from the gist of the invention. For example, the mass spectrometer 1 of the above embodiment has a tandem quadrupole configuration, but the configuration of the mass separator and the like is not limited to this as described above.

3... Ionization device, 3A... Solvent storage part, 3B... Sample storage part, 4... Ionization chamber, 6... First intermediate vacuum chamber, 7... Second intermediate vacuum chamber, 8... Analysis chamber, 21... Probe holder, 22 ... probe, 31 ... cylindrical member, 32 ... upper membrane body, 33 ... lower membrane body, 34 ... spacer, 200 ... solvent, 201 ... perfusate, 311 ... solvent tank, 317 ... discharge tank, 321 ... perfusate tank, 327... discharge tank, 328... microdialysis probe, M... mouse

Claims (5)

An ionization device for ionizing components of a fluid sample, comprising:
a probe;
a voltage applying unit that applies a voltage to the probe;
a probe drive unit for moving the probe;
a solvent storage unit that stores a solvent;
a solvent tank for storing the solvent;
a solvent supply path connecting the solvent tank and the solvent container;
a liquid feed pump that supplies the solvent stored in the solvent tank to the solvent storage unit through the solvent supply channel;
a drain tank containing the solvent;
a discharge channel connecting the solvent storage unit and the discharge tank;
a sample supply pipe for receiving the fluid sample from the outside of the ionization device;
with
The solvent storage unit is
a container part having first and second openings facing each other and capable of containing the solvent by closing the first opening with a first membrane;
a second membrane disposed between the first membrane and the sample supply pipe outside the container;
The probe drive unit causes the probe to enter the container through the second opening while the solvent is contained in the container, and moves the probe through the first and second films. moving through the body to collect the fluid sample flowing through the sample supply pipe ;
By driving the liquid feed pump, the solvent is continuously supplied from the solvent tank to the solvent storage unit, and when the solvent in the solvent storage unit exceeds a predetermined amount, the solvent is supplied to the solvent storage unit. to the discharge tank, thereby maintaining a constant concentration of the solvent in the solvent container . 2. The ionization device according to claim 1, wherein said fluid sample flowing through said sample supply pipe contains a substance recovered from a living body by a microdialysis method. 3. The ionization device according to claim 1, wherein the fluid sample flowing through the sample supply pipe contains an internal standard substance. an ionization device according to any one of claims 1 to 3 ;
and a mass spectrometer that analyzes the components of the fluid sample based on the ions generated by the ionization device. A method for analyzing a fluid sample using the ionization device according to claim 1,
preparing a state in which the solvent is stored in the solvent storage unit and the fluid sample is continuously supplied to the sample supply pipe;
By moving the probe by the probe driving section, the probe is caused to pass through the solvent contained in the container section, and the probe penetrates the first and second membranes. obtaining the fluid sample by causing the fluid sample to reach the fluid sample flowing in the sample supply pipe;
The probe is separated from the fluid sample by moving the probe by the probe drive unit, and the probe is passed again through the solvent contained in the container unit until it reaches a predetermined position. After the movement, the voltage applying unit applies a voltage to the probe to ionize the fluid sample adhering to the tip of the probe;
and mass spectrometry of said ionized fluid sample.

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