Mass spectrometry method

Abstract

A mass spectrometry method includes: a preparation step (step 1) of adding a predetermined alkali metal ion to a liquid sample; an ionization step (step 3) for ionizing a target compound contained in the liquid sample to which the alkali metal ions have been added; and a measurement step (step 7) for measuring the intensity of an adduct ion generated in the ionization step and obtained by adding the prescribed alkali metal to the target compound or a product ion derived from the adduct ion.

Description

Technical Field

[0001] The present invention relates to a method for performing mass spectrometric analysis on a target compound contained in a liquid sample. Background Art

[0002] In order to analyze the target compound contained in the liquid sample, a liquid chromatography-mass spectrometry device is used. In the liquid chromatography-mass spectrometry device, various compounds contained in the liquid sample are separated by the column of the liquid chromatograph and introduced into the mass spectrometry device. In the mass spectrometry device, the compound introduced from the liquid chromatograph is ionized, and these ions are separated according to the mass-to-charge ratio, and the intensity of the ions is measured for each mass-to-charge ratio.

[0003] When quantifying the target compound, a multiple reaction monitoring (MRM) measurement is performed. In the MRM measurement, ions with a mass-to-charge ratio characteristic of the target compound are screened from the ions generated from the liquid sample as precursor ions, and product ions with a specific mass-to-charge ratio are screened from the product ions derived from the precursor ions and their intensities are measured. In the MRM measurement, through the two stages of precursor ion screening and product ion screening, ions with a specific mass-to-charge ratio characteristic of the target compound are locked. Therefore, even in the case where the liquid sample contains an impurity for generating ions with the same mass-to-charge ratio as the precursor ion of the target compound, the target compound can be accurately quantified without being affected. In addition, due to its high sensitivity, the MRM measurement is particularly used to quantify trace amounts of target compounds contained in the sample.

[0004] As a representative ionization method for ionizing a liquid sample, the electrospray ionization (ESI) method is known. When a compound is ionized using the ESI method (positive mode), a large amount of proton-added ions are usually generated by the addition of hydrogen ions (protons) to the compound. Therefore, in MRM measurement, proton-added ions are often used as precursor ions.

[0005] However, when compounds having a complex molecular structure such as organic polymer compounds are ionized, various ions other than proton addition ions are sometimes generated. Such ions include, for example, dehydrated ions, solvent addition ions, ammonium addition ions, sodium addition ions, potassium addition ions, and the like. For example, ammonium addition ions are generated by the addition of ammonium ions contained in the mobile phase used in the analysis to the compound, or by the addition of ammonium ions of volatile salts such as ammonium formate or ammonium acetate, which are contained in the mobile phase or solvent used in the past analysis and remain inside the mass spectrometer, to the compound.

[0006] In non-patent document 1, the results obtained by MRM determination of a liquid sample containing a very small amount (1 ng / mL) of ciguatoxin, a toxin known as a fish toxin, are shown using a liquid chromatography-mass spectrometry device. In this MRM determination, sodium addition ions are used as precursor ions. Sodium is not contained in the solution used as a mobile phase in non-patent document 1. In addition, multiple cases where sodium addition ions or potassium addition ions are generated even when sodium or potassium is not contained in the mobile phase or solvent used in the past have been reported. Therefore, it is estimated that these ions originate from a glass container containing a liquid sample or a mobile phase, or a glass capillary constituting an ESI probe, etc.

[0007] Prior art literature

[0008] Non-patent literature

[0009] Non-patent document 1: Kentaro Yogi et al., "Detailed LC-MS / MS Analysis of Ciguatoxins Revealing Distinct Regional and Species Characteristics in Fish and Causative Alga from the Pacific", Anal. Chem., 2011, 83, 8886-8891 Summary of the invention

[0010] Problem that the invention aims to solve

[0011] The amount of mobile phase or solvent used in the past that remains in the mass spectrometry device is uncertain, so it is impossible to adjust the amount of ammonium ions added to the target compound from it. In addition, sodium ions and potassium ions originate from glass containers containing mobile phases or liquid samples, and the amount of these ions added to the target compound cannot be adjusted. Therefore, in the MRM measurement in which the adduct ions obtained by adding these ions to the target compound are used as precursor ions, there is a problem that the reproducibility of the measurement is poor and it is difficult to accurately quantify the target compound. In addition, when multiple ions are generated from the target compound, no matter which one is screened as the precursor ion, the amount of the precursor ion is small and the measurement sensitivity is poor compared to the case where only one ion is generated. As mentioned above, since the MRM measurement is used to quantify the trace amount of the target compound contained in the sample, there is a problem that the target compound cannot be detected if the measurement sensitivity is poor.

[0012] Here, the case of MRM measurement is described as an example, but the same problem as above also exists in selected ion monitoring (SIM) measurement for measuring ions of a specific mass-to-charge ratio and MS / MS scanning measurement for screening precursor ions as in MRM measurement.

[0013] The problem to be solved by the present invention is to provide a mass spectrometry method capable of quantifying a target compound contained in a liquid sample with high sensitivity and accuracy.

[0014] Solutions for solving problems

[0015] The mass spectrometry analysis method involved in the present invention, which is completed to solve the above-mentioned problems, includes: a preparation step of adding specified alkali metal ions to a liquid sample; an ionization step of ionizing a target compound contained in the liquid sample to which the alkali metal ions are added; and a measurement step of measuring the intensity of an adduct ion generated in the ionization step and obtained by adding the specified alkali metal to the target compound or an ion derived from the adduct ion.

[0016] Effects of the Invention

[0017] In the mass spectrometry method of the present invention, a preparation step is performed before mass spectrometry analysis, and a predetermined alkali metal ion is added to the liquid sample in advance. The addition of the predetermined alkali metal ion can be performed by adding the ion itself (for example, adding a liquid containing the ion) or by dissolving an alkali metal salt in the liquid sample. In addition, when a liquid chromatography-mass spectrometry device is used as in the embodiments described later, the alkali metal ion can also be added to the mobile phase.

[0018] When the ionization step is performed in a state where a predetermined alkali metal ion is supplied to a liquid sample, the predetermined alkali metal addition ion obtained by adding the alkali metal ion is mainly generated, and the generation of other types of addition ions is suppressed. In addition, since most alkali metal ions are ions that are difficult to dissociate, the generation of dehydrated ions and solvent addition ions is also suppressed.

[0019] In the mass spectrometer of the present invention, the intensity of the adduct ion generated in the ionization step and obtained by adding the predetermined alkali metal to the target compound or the ion derived from the adduct ion is measured. Therefore, compared with the case of measuring one ion among a plurality of ions generated from the target compound due to ions generated from a liquid sample or a sample container, the amount of the measured ion is large, and the target compound can be measured with high sensitivity. In addition, the mechanism for generating the alkali metal adduct ion has reproducibility, so the target compound can be accurately analyzed. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a diagram showing the main structure of a liquid chromatography-mass spectrometry apparatus for executing one embodiment of the mass spectrometry method according to the present invention.

[0021] Figure 2 This is a flow chart related to the mass spectrometry method of this embodiment.

[0022] Figure 3 The mass spectrum of CTX3C, which is a type of ciguatoxin, obtained by a conventional mass spectrometry method and the mass spectrum obtained by the mass spectrometry method of the present embodiment are shown. DETAILED DESCRIPTION

[0023] Hereinafter, one embodiment of the mass spectrometry method according to the present invention will be described with reference to the accompanying drawings.

[0024] Figure 1 1 is a diagram showing the main structure of a liquid chromatography-mass spectrometry apparatus for carrying out the mass spectrometry method of the present embodiment. The liquid chromatography-mass spectrometry apparatus of the present embodiment includes a liquid chromatograph 1, a mass spectrometer 2, and a control / processing unit 4 for controlling the operations thereof.

[0025] The liquid chromatograph 1 includes a mobile phase container 10 storing a mobile phase, a pump 11 for sucking the mobile phase and delivering the mobile phase at a constant flow rate, a syringe 12 for injecting a liquid sample into the mobile phase, and a column 13 for separating compounds contained in the liquid sample. In the case of continuously analyzing a plurality of liquid samples, an automatic sampler (not shown) is further provided, and a plurality of liquid samples provided in the automatic sampler are sequentially introduced from the syringe 12.

[0026] The mass spectrometer 2 includes an ionization chamber 20 and a vacuum chamber. The vacuum chamber is evacuated by a vacuum pump (not shown). The vacuum chamber includes a first intermediate vacuum chamber 21, a second intermediate vacuum chamber 22, and an analysis chamber 23 in order from the ionization chamber 20 side, and has a multi-stage differential exhaust system structure in which the vacuum degree increases in this order.

[0027] An electrospray ionization (ESI) probe 201 for applying electric charge to a sample solution and then spraying it is provided in the ionization chamber 20. The ionization chamber 20 and a subsequent first intermediate vacuum chamber 21 are connected via a heating capillary 202 having a small diameter.

[0028] An ion lens 211 composed of a plurality of annular electrodes is disposed in the first intermediate vacuum chamber 21. The ion lens 211 converges the flight path of ions along the ion optical axis C. The first intermediate vacuum chamber 21 and the second intermediate vacuum chamber 22 are separated by a skimmer 212 having a small hole on the top.

[0029] An ion guide 221 composed of a plurality of rod electrodes is disposed in the second intermediate vacuum chamber 22. The ion guide 221 also converges the flight path of ions along the ion optical axis C, similarly to the ion lens 211. The second intermediate vacuum chamber 22 is separated from the analysis chamber by a partition wall having a small hole.

[0030] The analysis chamber 23 is provided with a front-stage quadrupole mass filter 231, a collision cell 232, a rear-stage quadrupole mass filter 234, and an ion detector 235. An ion guide 233 is provided inside the collision cell 232. In addition, a collision-induced dissociation (CID) gas is introduced into the collision cell 232 from a gas source (not shown).

[0031] The mass spectrometer 2 can perform selected ion monitoring (SIM) measurement, MS / MS scanning (product ion scanning) measurement, multiple reaction monitoring (MRM) measurement, etc. In SIM measurement, the front-stage quadrupole mass filter 231 does not filter ions (does not function as a mass filter), but the mass-to-charge ratio of ions passing through the rear-stage quadrupole mass filter 234 is fixed to detect ions.

[0032] On the other hand, in MS / MS scanning measurement and MRM measurement, both the front-stage quadrupole mass filter 231 and the rear-stage quadrupole mass filter 234 are made to function as mass filters. In the front-stage quadrupole mass filter 231, only ions set as precursor ions are allowed to pass. In addition, CID gas is supplied to the interior of the collision chamber 232 and the precursor ions are accelerated and introduced into the interior thereof, so that the precursor ions collide with the CID gas and cause the precursor ions to break. In the MS / MS scanning measurement, the mass-to-charge ratio of the ions passing through the rear-stage quadrupole mass filter 234 is scanned, and in the MRM measurement, the mass-to-charge ratio of the ions passing through the rear-stage quadrupole mass filter 234 is fixed and only product ions with a specific mass-to-charge ratio are allowed to pass.

[0033] The control / processing unit 4 has a storage unit 41. A compound database (compound DB) 411 is stored in the storage unit 41, and the compound database 411 records information including measurement conditions and analytical methods related to multiple known compounds. The measurement conditions recorded in the compound DB 411 include, for example, information on the time (retention time) when the target compound flows out of the column 13, SIM measurement conditions, MRM measurement conditions, MS / MS scanning measurement conditions, etc. The SIM measurement conditions include information on the mass-to-charge ratio of the measurement object ion. The MRM measurement conditions include information on the mass-to-charge ratio of the precursor ion, the magnitude of the collision energy, and the mass-to-charge ratio of the product ion. The MS / MS scanning measurement conditions include information on the mass-to-charge ratio of the precursor ion, the magnitude of the collision energy, and the mass-to-charge ratio range (mass scanning range) of the product ion. In addition, for the measurement object ion in the SIM measurement, and the precursor ion and product ion in the case of the MRM measurement, information on the mass-to-charge ratio different for each type of alkali metal ion described later is recorded. In addition, the analytical method recorded in the compound DB 411 includes the method of peak extraction and the values ​​of the parameters used in peak extraction, as well as information on the calibration curve.

[0034] The control / processing unit 4 includes a measurement condition setting unit 42, a measurement control unit 43, and an analysis processing unit 44 as functional blocks. The entity of the control / processing unit 4 is a personal computer, and the processor executes the program pre-installed in the computer to function as the above-mentioned units. In addition, the control / processing unit 4 is connected to the input unit 5 and the display unit 6.

[0035] Below, refer to Figure 2 The process of analyzing a target component contained in a liquid sample using the liquid chromatography-mass spectrometry apparatus of this embodiment is described with reference to the flowchart of FIG.

[0036] The user adds alkali metal ions to the mobile phase before the measurement (step 1). The addition of alkali metal ions can be carried out by adding a solution in which alkali metal ions are dissolved to the mobile phase, or by adding a salt of an alkali metal to the mobile phase. When an alkali metal salt is added to the mobile phase, it is preferred to add a volatile salt of the alkali metal. In addition, a volatile salt refers to a salt that does not deposit solid matter when the liquid mobile phase is desolvated in the ion source of a mass spectrometer.

[0037] Specifically, for example, a lithium aqueous solution or a lithium salt (lithium hydroxide, lithium hydroxide monohydrate, lithium nitrate, lithium chloride, lithium bromide, etc.) is added to the mobile phase. Alternatively, for example, a sodium aqueous solution or a sodium salt (sodium hydroxide, sodium hydroxide monohydrate, sodium nitrate, sodium chloride, sodium bromide, etc.) is added to the mobile phase.

[0038] At this time, the pH of the mobile phase is sometimes changed according to the type of aqueous solution or salt added to the mobile phase. In the liquid chromatograph 1, the range of the pH of the mobile phase that can be used is determined by each column 13 in most cases. Therefore, when the pH of the mobile phase is outside the use range of the column 13 by adding alkali metal ions, it is preferred to further add an aqueous solution or salt for adjusting the pH of the mobile phase as a pH modulator. For example, when sodium hydroxide is added, it can be mixed with an acidic volatile salt (salts of trifluoroacetic acid, formic acid, acetic acid, etc.) to adjust the pH of the mobile phase.

[0039] If a non-volatile salt is added to the mobile phase, the salt may be deposited and clog the capillary 202 during desolvation in the ionization chamber 20 of the mass spectrometer 2. In addition, if the non-volatile salt in the mobile phase is introduced into the vacuum chamber, it may clog the small-diameter opening of the separator 212 and the like. Furthermore, if the salt is deposited on the surface of the ion lens 211, the ion guides 221, 233, the front-stage quadrupole mass filter 231, or the rear-stage quadrupole mass filter 234, it may also cause disturbance in the electric field formed by them.

[0040] In addition, at least more (high concentration) alkali metal ions than the assumed amount (concentration) of the target compound contained in the liquid sample are added to the mobile phase in advance. It is preferred to add a sufficient amount (excess) relative to the assumed amount of the target compound to the mobile phase. In addition, a sufficient amount (excess) of alkali metal ions relative to the doping compound contained in the liquid sample and the doping ions that can flow out from each part of the liquid chromatography-mass spectrometry device are added to the mobile phase. The doping ions mentioned here particularly refer to compounds that affect the ion species generated from the target compound. As such doping ions, for example, ammonium ions contained in the mobile phase of the liquid chromatograph used in past analyses, sodium ions or potassium ions flowing out from a glass bottle containing a liquid sample or a mobile phase, etc. can be cited.

[0041] When the user performs a predetermined input operation for instructing the measurement of the liquid sample, the measurement condition setting unit 42 displays a screen for the user to input the measurement conditions on the display unit 6. The screen includes, for example, a screen for the user to select a target compound and a type of mass spectrometry (SIM measurement, MRM measurement, or MS / MS scanning measurement) from a list of a plurality of known compounds. The screen also includes, for example, a screen for the user to select the type of alkali metal ions added to the liquid sample by the user in the above-mentioned step 1 from a list of a plurality of alkali metal ions.

[0042] When the user selects a target compound, a type of mass spectrometry, and an alkali metal ion, the measurement condition setting unit 42 reads out the measurement conditions of the compound selected by the user with reference to the compound DB 411 stored in the storage unit 41. As described above, in the compound DB 411, for each compound, the mass-to-charge ratio values ​​of the measurement target ions (in the case of SIM measurement) and the mass-to-charge ratio (or mass-to-charge ratio range) values ​​of the precursor ions and the product ions (in the case of MRM measurement or MS / MS scanning measurement) that are different for each type of alkali metal ions are stored. Therefore, the measurement condition setting unit 42 reads out the information of the mass-to-charge ratio of the measurement target ions or the precursor ions and the product ions corresponding to the combination of the target compound and the alkali metal ion selected by the user, and creates a method file that records the measurement conditions containing them and a batch file for executing the method file.

[0043] After the batch file is created, when the user sets the liquid sample in the syringe 12 (or the automatic sampler) and instructs to start the measurement, the measurement control unit 43 supplies the mobile phase contained in the mobile phase container 10 to the column 13 using the pump 11. In addition, in the mass spectrometer 2, the first intermediate vacuum chamber 21, the second intermediate vacuum chamber 22, and the analysis chamber 23 in the vacuum chamber are exhausted to a predetermined pressure by a vacuum pump (not shown). Then, the measurement control unit 43 operates each part of the liquid chromatograph according to the measurement conditions recorded in the method file and starts the measurement. The operation in the case of performing MRM measurement or MS / MS scanning measurement is described below.

[0044] First, a liquid sample is introduced from a syringe 12 into the liquid chromatograph 1 (step 2). The liquid sample introduced from the syringe 12 is introduced into the column 13 along with the flow of a mobile phase to which alkali metal ions are added. In the column 13, various compounds contained in the liquid sample are separated from each other.

[0045] The compounds separated in the column 13 are sequentially introduced into the ESI probe 201 and ionized (step 3). The ions generated in this way are converged by the ion lens 211 and the ion guide 221 so as to fly along the ion optical axis C, and then enter the analysis chamber 23.

[0046] In the front-stage quadrupole mass filter 231, only ions having a mass-to-charge ratio set as precursor ions of the target compound among the ions incident into the analysis chamber 23 are screened (step 4). The ions screened as precursor ions are given collision energy, thereby being accelerated and incident into the collision chamber 232. In the collision chamber 232, the precursor ions collide with molecules of the collision gas introduced into the collision chamber 232 and break, thereby generating product ions (step 5).

[0047] The product ions generated in the collision cell 232 are incident on the post-stage quadrupole mass filter 234. When MRM measurement is selected, in the post-stage quadrupole mass filter 234, only ions with a mass-to-charge ratio set as the product ions of the target compound are screened out (step 6). When MS / MS scanning measurement is selected, in the post-stage quadrupole mass filter 234, mass scanning is repeatedly performed within the mass-to-charge ratio range set as the measurement condition to screen the product ions (step 6). The product ions screened out by the post-stage quadrupole mass filter 234 are incident on the ion detector 235, and their intensity is measured (step 7). The output data from the ion detector 235 is sent to the control / processing unit 4 in sequence and stored in the storage unit 41.

[0048] After the measurement is completed, the analysis processing unit 44 uses the output data from the ion detector 235 to create a chromatogram. In the case of MRM measurement, the analysis processing unit 44 creates an extracted ion current chromatogram, and in the case of MS / MS scanning measurement, the analysis processing unit 44 creates a total ion current chromatogram. Then, using the peak extraction method and parameters read from the compound DB 411 as the analysis method for the target compound, the peaks are extracted from the chromatogram and their areas are calculated. Furthermore, the analysis processing unit 44 compares the peak area with the calibration curve to calculate the quantitative value of the target compound (step 8). The quantitative value calculated by the analysis processing unit 44 is compared with the chromatogram. Figure 1 The result is displayed on the screen of the display unit 6. In the case of MS / MS scanning measurement, a product ion spectrum can be prepared and compared with the product ion spectrum of the compound included in the compound DB 411 to confirm (qualitatively identify) that the measured compound is the target compound.

[0049] The mass spectrometry method of this embodiment is characterized in that alkali metal ions are added before measuring the liquid sample. If a target compound having a complex molecular structure such as an organic polymer compound is ionized, various ions other than proton addition ions may be generated. Such ions include, for example, dehydrated ions, solvent addition ions, ammonium addition ions, sodium addition ions, potassium addition ions, and the like. For example, when the target compound is a sugar (monosaccharide, disaccharide, oligosaccharide), a pesticide (oxamyl, pentoxazone, emamectin and its metabolites, etc.), an animal medicine (cefuroxime, etc.), a fungal toxin (diacetoxyscirpenol, HT-2, T-2, neosolaniol, etc.), a shellfish toxin, a fish toxin (ciguatoxin, palytoxin, brevetoxin, etc.), or a functional compound (terpenoids, ginkgolide, bilobalide, etc.), such a variety of ions are easily generated.

[0050] The above-mentioned adduct ions are, for example, believed to be formed by the attachment of ammonium ions of the mobile phase used in the past, sodium ions, potassium ions, etc., which flow out from the glass container containing the mobile phase or liquid sample, or the glass capillary constituting the ESI probe 201, etc., to the molecules of the target compound. In the MRM measurement using the adduct ions obtained by adding these ions to the target compound as precursor ions, the reproducibility of the measurement is poor, and it is difficult to accurately quantify the target compound. In addition, when multiple ions are generated from the target compound, no matter which one is screened as the precursor ion, the amount of the precursor ion is small compared with the case where only one ion is generated, and the measurement sensitivity is poor. As described above, the MRM measurement is used to quantify the trace amount of the target compound contained in the sample, so if the measurement sensitivity is poor, the target compound may not be detected sometimes. In addition, in the case of MS / MS scanning measurement, the intensity of multiple product ions generated from such a small amount of precursor ions is measured to produce a product ion spectrum, so sometimes a part of the product ions cannot be detected and correct spectrum matching cannot be performed.

[0051] In contrast, in the mass spectrometry method of the present embodiment, alkali metal ions X are generated by adding alkali metal ions to the mobile phase. + [M+X] obtained by adding to the target compound molecule M contained in the liquid sample +As described above, in this embodiment, an excess amount of alkali metal ions relative to the target compound and the dopant compound is added to the mobile phase to suppress the generation of unintended addition ions such as dehydrated ions, solvent addition ions, ammonium addition ions, sodium addition ions, and potassium addition ions, so that the ion species generated from the target compound are concentrated on the alkali metal addition ions [M+X]. + Thus, by substantially generating only one alkali metal addition ion from the target compound and using the alkali metal addition ion as a precursor ion, the sensitivity and reproducibility in MRM measurement or MS / MS scanning measurement can be improved.

[0052] In addition, among alkali metal addition ions, lithium addition ions and sodium addition ions are difficult to break once they are generated. In particular, sodium addition ions are hardly broken. Therefore, in the case of adding lithium ions and sodium ions as alkali metal ions to the mobile phase for MRM determination, lithium addition ions or sodium addition ions are preferably used as precursor ions, and lithium addition ions or sodium addition ions are preferably used as product ions. For example, in the case of ions containing ions having the same mass-to-charge ratio as the mass-to-charge ratio of lithium addition ions or sodium addition ions derived from doping compounds, only the ions derived from doping compounds can be broken to accurately analyze only the target compound. In the case of generating lithium addition ions, not only the lithium addition ions as precursor ions and product ions are measured by MRM as described above, but also the lithium addition ions are used as precursor ions and the product ions generated by the rupture of the precursor ions are subjected to MS / MS scanning to obtain product ion spectra, thereby also being able to produce sounds for the identification (qualitative) of compounds.

[0053] Example

[0054] A measurement example in which one type of alkali metal addition ion is generated from a target compound that easily generates a plurality of types of ions by the mass spectrometry method of the above-described embodiment will be described.

[0055] Figure 3 It is a multi-SIM spectrum (upper section. MS spectrum) obtained by the conventional mass spectrometry method (measurement using a mobile phase without the addition of alkali metal ions) for CTX3C, a type of ciguatoxin, and a multi-SIM spectrum (MS spectrum) obtained by the mass spectrometry method of the above embodiment (lower section. Measurement using a mobile phase with the addition of alkali metal ions). In addition, the multi-SIM spectrum refers to a spectrum of the intensity of each ion obtained by independently performing SIM measurement on ions of multiple different mass-to-charge ratios by setting the horizontal axis to the mass-to-charge ratio and the vertical axis to the intensity, as in a conventional mass spectrum. In the conventional mass spectrometry method, the dehydrated ion [M+HH 2 O] + 、Proton addition ion [M+H] +In addition to these two ions, the bimolecular dehydrate ion [M+H-2H 2 O] + 、Ammonium addition ions [M+NH 4 ] + 、Sodium addition ion [M+Na] + 、Potassium addition ion [M+K] + In contrast, in the mass spectrometry method of this embodiment, only lithium addition ions [M+Li] + Detected with high intensity.

[0056] In addition, for CTX1B, another ciguatoxin, multi-SIM spectra were obtained by the conventional mass spectrometry method and the mass spectrometry method of the above embodiment, respectively. Regarding CTX1B, in the conventional mass spectrometry method, ammonium addition ions [M+NH 4 ] + In addition, the bimolecular dehydrated ions [M+H-2H 2 O] + 、Dehydrated ions [M+HH 2 O] + 、Proton addition ion [M+H] + 、Sodium addition ion [M+Na] + 、Potassium addition ion [M+K] + In contrast, in the mass spectrometry method of this embodiment, only the lithium addition ions [M+Li] + Detected with high intensity.

[0057] In addition, for 51-hydroxy-CTX3C, another type of ciguatoxin, multi-SIM spectra were obtained by the conventional mass spectrometry method and the mass spectrometry method of the above embodiment, respectively. Regarding 51-hydroxy-CTX3C, in the conventional mass spectrometry method, the bimolecular dehydrated ion [M+H-2H 2 O] + 、Dehydrated ions [M+HH 2 O] + 、Proton addition ion [M+H] + In addition, sodium addition ions [M+Na] were detected + 、Potassium addition ion [M+K] + In contrast, in the mass spectrometry method of this embodiment, only lithium addition ions [M+Li] + Detected with high intensity.

[0058] In addition, for 52-epi-54-deoxy-CTX1B, another ciguatoxin, multiple SIM spectra were obtained by the conventional mass spectrometry method and the mass spectrometry method of the above embodiment, respectively. In the conventional mass spectrometry method, multiple ions such as the bimolecular dehydrated ion [M+H-2H 2 O] + 、Dehydrated ions [M+HH 2 O] + 、Proton addition ion [M+H] + 、Ammonium addition ions [M+NH 4 ] + 、Sodium addition ion [M+Na] + In contrast, in the mass spectrometry method of this embodiment, only lithium addition ions [M+Li] + Detected with high intensity.

[0059] In addition, for CTX4A, another ciguatoxin, multi-SIM spectra were obtained by the conventional mass spectrometry method and the mass spectrometry method of the above embodiment, respectively. Regarding CTX4A, in the conventional mass spectrometry method, multiple ions such as dehydrated ions [M+HH 2 O] + 、Proton addition ion [M+H] + 、Ammonium addition ions [M+NH 4 ] + 、Sodium addition ion [M+Na] + , and the bimolecular dehydrated ions [M+H-2H 2 O] + In contrast, in the mass spectrometry method of this embodiment, only lithium addition ions [M+Li] + Detected with high intensity.

[0060] The above are all examples obtained by adding lithium ions to the mobile phase, and the same results are confirmed when sodium ions are added to the mobile phase. As shown in these examples, by using the mass spectrometry method of the present embodiment, only one alkali metal addition ion can be substantially generated from the target compound (especially organic polymer). Then, by performing MRM measurement or MS / MS scanning measurement using the one alkali metal addition ion as a precursor ion, the target compound can be quantified and identified with high precision.

[0061] As described in Non-Patent Document 1, when separating components in a liquid sample containing ciguatoxins by liquid chromatography, a mobile phase containing a mixture of a plurality of solvents is generally used to perform gradient analysis in which the mixing ratio thereof is changed over time.

[0062] In such a gradient analysis, alkali metal ions can be added to either an organic solvent or an inorganic solvent, but when the present inventors investigated ciguatoxins under various conditions, they found that when alkali metal ions are added to an organic solvent, adduct ions to which the alkali metal ions are added are effectively generated, while on the other hand, when alkali metal ions and an acidic solution are added to an aqueous mobile phase, adduct ions to which alkali metals are added are sometimes not generated. The above-mentioned measurement example is also a result obtained by separating and measuring the target compound (CTX3C, etc.) in a liquid sample by gradient analysis in which the mixing ratio between an organic solvent and a mixed solution of formic acid and water changes over time using a liquid chromatography-mass spectrometry apparatus, and the organic solvent is an organic solvent obtained by adding lithium ions by mixing lithium hydroxide into acetonitrile, and then adding formic acid as an acidic volatile salt to adjust the pH.

[0063] Ciguatoxins are organic macromolecular compounds and therefore have a high affinity for the organic solvent contained in the mobile phase in the above-mentioned gradient analysis. Therefore, it is considered that the addition of alkali metal ions to the organic solvent efficiently generates adduct ions to which the alkali metal ions are added.

[0064] As described above, as compounds that generate various ions other than proton addition ions, for example, sugars (monosaccharides, disaccharides, oligosaccharides), pesticides (oxamyl, amyloxazolone, emamectin and its metabolites, etc.), animal medicines (cefuroxime, etc.), mycotoxins (serpentin, HT-2, T-2, new solanine fusarium, etc.), shellfish toxins, fish toxins (ciguatoxin, anemone toxin, brevetoxin, etc.), functional compounds (terpenoids, ginkgolide, bilobalide, etc.), most of which are organic polymer compounds like ciguatoxins. Therefore, when the target compound is an organic polymer compound, it is possible to efficiently generate the addition ion to which the alkali metal ion is added by adding a predetermined alkali metal ion to an organic solvent having a high affinity with the organic polymer compound. In addition, when the target compound is a low molecular weight compound or an inorganic compound such as a monosaccharide or a disaccharide, it is also possible to efficiently generate the addition ion to which the alkali metal ion is added by adding an alkali metal ion to an inorganic solvent.

[0065] The above-described embodiments and examples are merely examples and can be modified as appropriate in accordance with the spirit of the present invention.

[0066] In the above-mentioned embodiments and examples, a liquid chromatography-mass spectrometry device is used, and thus alkali metal ions are added to the target compound in the liquid sample by adding alkali metal ions to the mobile phase, but when only a mass spectrometer is used, it is sufficient to add alkali metal ions to the liquid sample itself. In addition, the structure of the mass spectrometer described in the above-mentioned embodiment (triple quadrupole mass spectrometer) is only an example, and mass spectrometers of various structures that can perform MRM measurement and MS / MS scanning measurement can be used.

[0067] [Way]

[0068] It is obvious to those skilled in the art that the above-mentioned exemplary embodiments are specific examples of the following aspects.

[0069] (First Item)

[0070] A mass spectrometry method according to one embodiment of the present invention includes:

[0071] A preparation step of adding a specified alkali metal ion to a liquid sample;

[0072] an ionization step of ionizing a target compound contained in the liquid sample to which the alkali metal ions are added; and

[0073] The step of measuring is to measure the intensity of the addition ion generated in the ionization step and obtained by adding the predetermined alkali metal to the target compound or the ion derived from the addition ion.

[0074] In the mass spectrometry method involved in the first item, a preparation step is performed before mass spectrometry analysis, and a predetermined alkali metal ion is added to the liquid sample in advance. The addition of the predetermined alkali metal ion can be performed by adding the ion itself (for example, adding a liquid containing the ion) or by dissolving an alkali metal salt in the liquid sample. When the ionization step is performed in a state where the predetermined alkali metal ion is supplied to the liquid sample in this way, the predetermined alkali metal addition ion to which the alkali metal ion is added is mainly generated, thereby suppressing the generation of other types of addition ions. In addition, since most alkali metal ions are ions that are difficult to dissociate, the generation of dehydrated ions and solvent addition ions is also suppressed.

[0075] In the mass spectrometer according to the first item, the intensity of the adduct ion obtained by adding the predetermined alkali metal to the target compound or the ion derived from the adduct ion among the ions generated in the ionization step is measured. Therefore, compared with the case where a plurality of ions are generated from the target compound due to ions generated from the liquid sample or the sample container, the amount of the measured ions is large, and the target compound can be measured with high sensitivity. In addition, the mechanism for generating the alkali metal adduct ions has sufficient reproducibility, so the target compound can be accurately quantified.

[0076] (Second Item)

[0077] The mass spectrometry method involved in the second item is the mass spectrometry method involved in the first item,

[0078] In the preparation step, a mobile phase containing an organic solvent to which the predetermined alkali metal ions are added is prepared, and the liquid sample is introduced into a column of a liquid chromatograph along with the flow of the mobile phase.

[0079] In the ionization step, the liquid sample flowing out of the column is ionized.

[0080] Compounds that generate various ions other than proton addition ions are mostly high molecular weight organic compounds and have a high affinity for organic solvents. In the mass spectrometry method involved in the second item, a liquid sample is introduced into a column of a liquid chromatograph by a mobile phase obtained by adding a predetermined alkali metal to an organic solvent, and the liquid sample flowing out of the column is ionized, so that the predetermined alkali metal is easily added to the target compound, and the alkali metal addition ions are efficiently generated.

[0081] (Third Item)

[0082] The mass spectrometry method involved in the third item is the mass spectrometry method involved in the first item or the second item,

[0083] In the preparation step, a pH adjuster for adjusting the pH is also added to the mobile phase.

[0084] If alkali metal ions are added to the mobile phase by adding an alkali metal salt or the like, the pH of the mobile phase may change. In a liquid chromatograph, the range of the pH of the mobile phase that can be used is sometimes determined for each column. In the mass spectrometry method involved in the third item, by adjusting the pH of the mobile phase using a pH adjusting agent, a mobile phase having a pH suitable for the column and to which alkali metal ions are added can be used to measure the target compound contained in the liquid sample.

[0085] (4th item)

[0086] The mass spectrometry method involved in the fourth item is the mass spectrometry method involved in the first item,

[0087] In the preparation step, a predetermined volatile salt of an alkali metal is added to the liquid sample.

[0088] If a non-volatile salt is added to the mobile phase, the salt will be deposited when desolvation is performed to ionize the liquid sample, and if the salt is introduced into the mass spectrometry unit, it may clog the small-diameter opening of the separator or the like. In addition, if the salt is deposited on the surface of the ion guide or the mass filter, it may also cause disturbance of the electric field formed by them. In the mass spectrometry method involved in the fourth item, since a volatile salt of an alkali metal is added, such a problem does not occur.

[0089] (Fifth Item)

[0090] The mass spectrometry method according to item 5 is the mass spectrometry method according to any one of items 1 to 4,

[0091] The predetermined alkali metal ion is a lithium ion or a sodium ion.

[0092] When alkali metal ions, especially lithium ions or sodium ions, are added to the molecules of the target compound to form adduct ions, it is difficult to break them. Therefore, in the mass spectrometry method involved in the fifth item, the generation of other types of ions can be further suppressed.

[0093] (Sixth Item)

[0094] The mass spectrometry method according to item 6 is the mass spectrometry method according to any one of items 1 to 5,

[0095] In the measuring step, among the ions generated in the ionizing step, adduct ions obtained by adding the specified alkali metal to the target compound are selected as precursor ions, the precursor ions are subjected to a fragmentation treatment, and then the intensity of ions not fragmented in the fragmentation treatment is measured.

[0096] The adduct ions obtained by adding the alkali metal ions to the molecules of the target compound are difficult to fragment. In the mass spectrometry analysis method of the sixth item, the intensity of the ions remaining without fragmentation after the adduct ions are subjected to a fragmentation treatment using the adduct ions as precursor ions is measured. Thus, even in the presence of dopant ions having the same mass-to-charge ratio as that of the adduct ions of the target compound, only the dopant ions can be fragmented, thereby measuring only the ions derived from the target compound.

[0097] Description of Reference Numerals

[0098] 1: Liquid chromatograph; 10: Mobile phase container; 11: Pump; 12: Syringe; 13: Column; 2: Mass spectrometer; 20: Ionization chamber; 201: ESI probe; 21: First intermediate vacuum chamber; 211: Ion lens; 22: Second intermediate vacuum chamber; 221: Ion guide; 23: Analysis chamber; 231: Front quadrupole mass filter; 232: Collision chamber; 233: Ion guide; 234: Post quadrupole mass filter; 35: Ion detector; 4: Control / processing unit; 41: Storage unit; 411: Compound DB; 42: Measurement condition setting unit; 43: Measurement control unit; 44: Analysis processing unit; 5: Input unit; 6: Display unit; C: Ion optical axis.

Claims

1. A mass spectrometry analysis method, include: A preparation step of adding a specified alkali metal ion to a liquid sample; an ionization step of ionizing a target compound contained in the liquid sample to which the alkali metal ions are added; as well as The step of measuring is to measure the intensity of the addition ion generated in the ionization step and obtained by adding the predetermined alkali metal to the target compound or the ion derived from the addition ion.

2. The mass spectrometry method according to claim 1, in, In the preparation step, a mobile phase containing an organic solvent to which the predetermined alkali metal ions are added is prepared, and the liquid sample is introduced into a column of a liquid chromatograph along with the flow of the mobile phase. In the ionization step, the liquid sample flowing out of the column is ionized.

3. The mass spectrometry method according to claim 2, in, In the preparation step, a pH adjuster for adjusting the pH is also added to the mobile phase.

4. The mass spectrometry method according to claim 1, in, In the preparation step, a predetermined volatile salt of an alkali metal is added to the liquid sample.

5. The mass spectrometry method according to claim 1, in, The predetermined alkali metal ion is a lithium ion or a sodium ion.

6. The mass spectrometry method according to claim 1, in, In the measuring step, among the ions generated in the ionizing step, adduct ions obtained by adding the specified alkali metal to the target compound are selected as precursor ions, the precursor ions are subjected to a fragmentation treatment, and then the intensity of ions not fragmented in the fragmentation treatment is measured.

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