Quality analysis methods

Abstract

The present invention provides a mass spectrometry method comprising: a preparation step (step 1) for adding prescribed alkali metal ions 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 adduct ions obtained by adding the prescribed alkali metal to the target compound that has been generated in the ionization step or of product ions derived from the adduct ions.

Description

[Technical Field]

[0001] The present invention relates to a method for mass spectrometry of a target compound contained in a liquid sample. [Background technology]

[0002] Liquid chromatograph mass spectrometers are used to analyze target compounds contained in liquid samples. In liquid chromatograph mass spectrometers, various compounds contained in the liquid sample are separated using a liquid chromatograph column and then introduced into a mass spectrometer. The mass spectrometer ionizes the compounds introduced from the liquid chromatograph, separates the ions according to their mass-to-charge ratio, and measures the intensity of the ions for each mass-to-charge ratio.

[0003] Multiple reaction monitoring (MRM) measurements are used to quantify target compounds. In MRM, precursor ions with a mass-to-charge ratio characteristic of the target compound are selected from the ions generated from a liquid sample. Furthermore, product ions derived from these precursor ions with a specific mass-to-charge ratio are selected and their intensities are measured. MRM measurements narrow down the ions to those with a specific mass-to-charge ratio characteristic of the target compound through two steps: precursor ion selection and product ion selection. Therefore, even if the liquid sample contains contaminants that generate 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 by these contaminants. Furthermore, due to its high sensitivity, MRM measurements are particularly useful for quantifying trace amounts of target compounds in samples.

[0004] Electrospray ionization (ESI) is a well-known ionization method for ionizing liquid samples. When a compound is ionized using ESI (positive mode), many protonated ions are typically generated, in which hydrogen ions (protons) are added to the compound. Therefore, protonated ions are often used as precursor ions in MRM measurements.

[0005] However, when compounds with complex molecular structures, such as organic polymer compounds, are ionized, various ions other than proton adduct ions may be generated. These ions include, for example, dehydrated ions, solvent adduct ions, ammonium adduct ions, sodium adduct ions, and potassium adduct ions. For example, ammonium adduct ions are generated when ammonium ions contained in the mobile phase used in the analysis are added to the compound, or when ammonium ions derived from volatile salts such as ammonium formate or ammonium acetate that were contained in the mobile phase or solvent used in previous analyses and remained inside the mass spectrometer are added to the compound.

[0006] Non-Patent Document 1 presents the results of MRM measurement using a liquid chromatograph mass spectrometer on a liquid sample containing trace amounts (1 ng / mL) of ciguatoxins, known to be fish poisons. In this MRM measurement, sodium adduct ions were used as precursor ions. The solution used as the mobile phase in Non-Patent Document 1 does not contain sodium. Furthermore, there have been multiple reports of sodium adduct ions and potassium adduct ions being generated even when the mobile phase or solvent used in the past did not contain sodium or potassium. Therefore, it is presumed that these ions originate from the glass container containing the liquid sample or mobile phase, the glass capillary that constitutes the ESI probe, and the like. [Prior art documents] [Non-patent literature]

[0007] [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 [Problem to be solved by the invention]

[0008] The amount of previously used mobile phase and solvent remaining in the mass spectrometer is uncertain, making it impossible to control the amount of ammonium ions adsorbed to the target compound. Furthermore, sodium and potassium ions originate from the mobile phase and glass containers containing the liquid sample, and the amount of these ions adsorbed to the target compound cannot be controlled. Therefore, MRM measurements using adduct ions formed by these ions adsorbed to the target compound as precursor ions have poor measurement reproducibility, making it difficult to accurately quantify the target compound. Furthermore, when multiple ions are generated from a target compound, selecting any one of them as the precursor ion results in a lower amount of precursor ion compared to when only one ion is generated, resulting in poor measurement sensitivity. As mentioned above, MRM measurements are used to quantify trace amounts of target compounds contained in samples, so poor measurement sensitivity can prevent the target compound from being detected.

[0009] While the example described here is MRM measurement, the same problems as described above also exist in selected ion monitoring (SIM) measurements, which measure ions with specific mass-to-charge ratios, and MS / MS scan measurements, which select precursor ions in the same way as MRM measurements.

[0010] 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. [Means for solving the problem]

[0011] In order to solve the above problems, the mass spectrometry method according to the present invention comprises: A preparation step of adding predetermined 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 have been added; The target compound produced in the ionization step is added with the predetermined alkali metal ion a measuring step of measuring the intensity of an adduct ion to which Includes. [Effects of the Invention]

[0012] In the mass spectrometry method according to the present invention, a preparation step is carried out prior to mass spectrometry to add predetermined alkali metal ions to a liquid sample. The predetermined alkali metal ions may be added by adding the ions themselves (e.g., by adding a liquid containing the ions) or by dissolving an alkali metal salt in the liquid sample. Furthermore, when a liquid chromatograph mass spectrometer is used as in the examples described below, the alkali metal ions may be added to the mobile phase.

[0013] When the ionization step is performed with a liquid sample containing a predetermined alkali metal ion, the predetermined alkali metal adduct ion is mainly generated, and the generation of other types of adduct ions is suppressed. Furthermore, since most alkali metal ions are difficult to dissociate, the generation of dehydrated ions and solvent adduct ions is also suppressed.

[0014] Mass spectrometry according to the present invention method In the ionization step, the target compound is ionized with the predetermined alkali metal. ionThe intensity of the adduct ion to which the alkali metal is added, or the ion derived from the adduct ion, is measured. Therefore, the amount of ions measured is larger than when measuring one of the multiple types of ions generated from the target compound due to ions generated from the liquid sample or sample container, and the target compound can be measured with high sensitivity. Furthermore, since the mechanism by which alkali metal adduct ions are generated is reproducible, the target compound can be accurately analyzed. [Brief explanation of the drawings]

[0015] [Figure 1] 1 is a diagram showing the configuration of the main parts of a liquid chromatograph mass spectrometer used to carry out an embodiment of a mass analysis method according to the present invention; [Figure 2] 3 is a flowchart illustrating a mass spectrometry method according to the present embodiment. [Figure 3] 1 shows a mass spectrum obtained by a conventional mass spectrometry method and a mass spectrum obtained by the mass spectrometry method of this embodiment for CTX3C, a type of ciguatoxin. DETAILED DESCRIPTION OF THE INVENTION

[0016] An embodiment of a mass spectrometry method according to the present invention will be described below with reference to the drawings.

[0017] 1 is a diagram showing the configuration of the main components of a liquid chromatograph mass spectrometer used to carry out the mass analysis method of this embodiment. The liquid chromatograph mass spectrometer of this embodiment includes a liquid chromatograph 1, a mass spectrometer 2, and a control and processing unit 4 that controls the operations of these components.

[0018] The liquid chromatograph 1 comprises a mobile phase container 10 in which a mobile phase is stored, a pump 11 that draws in the mobile phase and delivers it at a constant flow rate, an injector 12 that injects a liquid sample into the mobile phase, and a column 13 that separates compounds contained in the liquid sample. When multiple liquid samples are to be analyzed continuously, an autosampler (not shown) is further provided, and multiple liquid samples set in the autosampler are introduced sequentially from the injector 12.

[0019] The mass spectrometer 2 includes an ionization chamber 20 and a vacuum chamber. The vacuum chamber is evacuated by a vacuum pump (not shown). Inside the vacuum chamber, in order from the ionization chamber 20 side, there are a first intermediate vacuum chamber 21, a second intermediate vacuum chamber 22, and an analysis chamber 23, and the structure is a multi-stage differential pumping system in which the degree of vacuum increases in this order.

[0020] An electrospray ionization (ESI) probe 201 that imparts an electric charge to a sample solution and sprays it is installed in the ionization chamber 20. The ionization chamber 20 and a first intermediate vacuum chamber 21 at the rear are connected via a thin-diameter heated capillary 202.

[0021] An ion lens 211 composed of multiple ring electrodes is disposed in the first intermediate vacuum chamber 21. The ion lens 211 focuses the flight path of the 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 at the top.

[0022] An ion guide 221 composed of a plurality of rod electrodes is disposed in the second intermediate vacuum chamber 22. Like the ion lens 211, the ion guide 221 also converges the flight path of ions along the ion optical axis C. The second intermediate vacuum chamber 22 and the analysis chamber are separated by a partition wall having small holes formed therein.

[0023] 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. A collision-induced dissociation (CID) gas is introduced into the collision cell 232 from a gas source (not shown).

[0024] The mass spectrometer 2 can perform selected ion monitoring (SIM) measurements, MS / MS scan (product ion scan) measurements, multiple reaction monitoring (MRM) measurements, etc. In SIM measurements, the front-stage quadrupole mass filter 231 does not select ions (does not function as a mass filter), and the mass-to-charge ratio of ions passing through the rear-stage quadrupole mass filter 234 is fixed to detect ions.

[0025] On the other hand, in MS / MS scan measurements and MRM measurements, both the front quadrupole mass filter 231 and the rear quadrupole mass filter 234 function as mass filters. The front quadrupole mass filter 231 allows only ions designated as precursor ions to pass through. Furthermore, CID gas is supplied to the collision cell 232, and precursor ions are accelerated and introduced into the collision cell 232, causing the precursor ions to collide with the CID gas and promote fragmentation of the precursor ions. In MS / MS scan measurements, the mass-to-charge ratio of ions passing through the rear quadrupole mass filter 234 is scanned, while in MRM measurements, the mass-to-charge ratio of ions passing through the rear quadrupole mass filter 234 is fixed, allowing only product ions with a specific mass-to-charge ratio to pass through.

[0026] The control and processing unit 4 has a memory unit 41. The memory unit 41 stores a compound database (compound DB) 411 containing information including measurement conditions and analysis methods for multiple known compounds. The measurement conditions stored in the compound DB 411 include, for example, information on the time (retention time) for the target compound to flow out of the column 13, SIM measurement conditions, MRM measurement conditions, MS / MS scan measurement conditions, etc. The SIM measurement conditions include information on the mass-to-charge ratio of the ions to be measured. The MRM measurement conditions include information on the mass-to-charge ratio of the precursor ions, the magnitude of the collision energy, and the mass-to-charge ratio of the product ions. The MS / MS scan measurement conditions include information on the mass-to-charge ratio of the precursor ions, the magnitude of the collision energy, and the mass-to-charge ratio range (mass scan range) of the product ions. Note that for the ions to be measured in SIM measurement and the precursor ions and product ions in MRM measurement, information on the mass-to-charge ratios that differ for each type of alkali metal ion, as described below, is stored. The analysis methods described in the compound DB411 include peak picking techniques, parameter values ​​used during peak picking, and calibration curve information.

[0027] The control and processing unit 4 has, as its functional blocks, a measurement condition setting unit 42, a measurement control unit 43, and an analysis processing unit 44. The actual entity of the control and processing unit 4 is a personal computer, and the above-mentioned units function by executing a program pre-installed in the computer on a processor. Furthermore, an input unit 5 and a display unit 6 are connected to the control and processing unit 4.

[0028] Hereinafter, a procedure for analyzing a target component contained in a liquid sample using the liquid chromatograph mass spectrometer of this embodiment will be described with reference to the flowchart of FIG.

[0029] Prior to measurement, the user adds alkali metal ions to the mobile phase (Step 1). The addition of alkali metal ions may be performed by adding a solution in which the alkali metal ions are dissolved to the mobile phase, or by adding an alkali metal salt to the mobile phase. When adding an alkali metal salt to the mobile phase, it is preferable to add a volatile salt of the alkali metal. Note that a volatile salt refers to a salt that does not precipitate as a solid when the liquid mobile phase is desolvated in the ion source of the mass spectrometer.

[0030] Specifically, for example, an aqueous lithium solution or a lithium salt (lithium hydroxide, lithium hydroxide monohydrate, lithium nitrate, lithium chloride, lithium bromide, etc.) is added to the mobile phase, or an aqueous sodium solution or a sodium salt (sodium hydroxide, sodium hydroxide monohydrate, sodium nitrate, sodium chloride, sodium bromide, etc.) is added to the mobile phase.

[0031] At this time, the pH of the mobile phase may change depending on the type of aqueous solution or salt added to the mobile phase. In many cases, the pH range of the mobile phase that can be used for each column 13 is determined for the liquid chromatograph 1. Therefore, if the pH of the mobile phase falls outside the usable range of the column 13 due to the addition of alkali metal ions, it is necessary to add more aqueous solution or salt to adjust the pH of the mobile phase. Adjuster For example, when sodium hydroxide is added, the pH of the mobile phase can be adjusted by mixing it with an acidic volatile salt (such as a salt of trifluoroacetic acid, formic acid, or acetic acid).

[0032] If a non-volatile salt is added to the mobile phase, the salt may precipitate during desolvation in the ionization chamber 20 of the mass spectrometer 2, potentially clogging the capillary 202. Furthermore, if the non-volatile salt in the mobile phase is taken into the vacuum chamber, it may clog small-diameter openings such as the skimmer 212. Furthermore, if salt precipitates on the surfaces of the ion lens 211, ion guides 221 and 233, front-stage quadrupole mass filter 231, or rear-stage quadrupole mass filter 234, it may disrupt the electric field formed by these elements.

[0033] Furthermore, alkali metal ions are added to the mobile phase in a concentration greater than the expected amount (concentration) of the target compound contained in the liquid sample. Preferably, an amount (excess amount) sufficiently greater than the expected amount of the target compound is added to the mobile phase. Furthermore, an amount (excess amount) of alkali metal ions sufficiently greater than the expected amount of the target compound is added to the mobile phase to cover impurity compounds contained in the liquid sample and impurity ions that may leak from various parts of the liquid chromatograph mass spectrometer. The impurity ions referred to here are compounds that affect the ion species generated from the target compound. Examples of such impurity ions include ammonium ions contained in the mobile phase of a liquid chromatograph used in a previous analysis, and sodium and potassium ions leaking from glass bottles containing the liquid sample and mobile phase.

[0034] When the user performs a predetermined input operation to instruct measurement of the liquid sample, the measurement condition setting unit 42 displays a screen on the display unit 6 that prompts the user to input measurement conditions. This screen includes, for example, a screen that prompts the user to select a target compound from a list of multiple known compounds and a type of mass spectrometry (SIM measurement, MRM measurement, or MS / MS scan measurement). This screen also includes, for example, a screen that prompts the user to select, from a list of multiple alkali metal ions, the type of alkali metal ion that the user added to the liquid sample in step 1 above.

[0035] When the user selects a target compound, a type of mass spectrometry, and an alkali metal ion, the measurement condition setting unit 42 refers to the compound DB 411 stored in the storage unit 41 and reads out the measurement conditions for the compound selected by the user. As described above, the compound DB 411 stores, for each compound, the mass-to-charge ratio values ​​of the target ions that differ for each type of alkali metal ion (in the case of SIM measurement) and the mass-to-charge ratios (or mass-to-charge ratio ranges) of the precursor ions and product ions (in the case of MRM measurement or MS / MS scan measurement). Therefore, the measurement condition setting unit 42 reads out information on the target ions or the mass-to-charge ratios of the precursor ions and product ions that correspond to the combination of the target compound and alkali metal ion selected by the user, and creates a method file describing the measurement conditions including these, and a batch file for executing the method file.

[0036] After the batch file has been created, when the user places a liquid sample in the injector 12 (or autosampler) and issues a command to start 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 evacuated to predetermined pressures using a vacuum pump (not shown). The measurement control unit 43 then operates each component of the liquid chromatograph according to the measurement conditions described in the method file to start measurement. The following describes the operations when performing MRM measurement or MS / MS scan measurement.

[0037] First, a liquid sample is introduced into the liquid chromatograph 1 through the injector 12 (step 2). The liquid sample introduced through the injector 12 is carried along with the flow of the mobile phase to which alkali metal ions have been added and introduced into the column 13. In the column 13, various compounds contained in the liquid sample are separated from each other.

[0038] The compounds separated in the column 13 are sequentially introduced into the ESI probe 201 and ionized (step 3). The ions thus generated 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.

[0039] In the front-stage quadrupole mass filter 231, only ions having a mass-to-charge ratio set as precursor ions of the target compound are selected from among the ions entering the analysis chamber 23 (step 4). Collision energy is imparted to the ions selected as precursor ions, which are then accelerated and enter the collision cell 232. In the collision cell 232, the precursor ions collide with molecules of a collision gas introduced into the collision cell 232, causing fragmentation and generating product ions (step 5).

[0040] The product ions generated in the collision cell 232 are incident on the post-quadrupole mass filter 234. When MRM measurement is selected, the post-quadrupole mass filter 234 selects only ions having a mass-to-charge ratio set as product ions of the target compound from among the product ions (step 6). When MS / MS scan measurement is selected, the post-quadrupole mass filter 234 repeatedly performs mass scanning within the mass-to-charge ratio range set as the measurement conditions to select product ions (step 6). The product ions selected by the post-quadrupole mass filter 234 are incident on the ion detector 235, where their intensity is measured (step 7). The output data from the ion detector 235 is sequentially transmitted to the control and processing unit 4 and stored in the memory unit 41.

[0041] After the measurement is completed, the analysis processor 44 creates a chromatogram using the output data from the ion detector 235. The analysis processor 44 creates an extracted ion current chromatogram if an MRM measurement is performed, and creates a total ion current chromatogram if an MS / MS scan measurement is performed. Then, using the peak picking technique and parameters read from the compound DB 411 as the analysis method for the target compound, the analysis processor 44 extracts peaks from the chromatogram and calculates their areas. Furthermore, the analysis processor 44 calculates the quantitative value of the target compound by comparing the peak area with a calibration curve (step 8). The quantitative value calculated by the analysis processor 44 is displayed on the screen of the display unit 6 together with the chromatogram. Furthermore, in the case of an MS / MS scan measurement, a product ion spectrum is created and compared with the product ion spectrum of the compound recorded in the compound DB 411 to confirm (qualify) that the measured compound is the target compound.

[0042] The mass spectrometry method of this embodiment is characterized by the addition of alkali metal ions to a liquid sample before measurement. When target compounds with complex molecular structures, such as organic polymer compounds, are ionized, various ions other than proton-adduct ions may be generated. These ions include adduct ions such as dehydrated ions, solvent-adduct ions, ammonium-adduct ions, sodium-adduct ions, and potassium-adduct ions. For example, when the target compounds are sugars (monosaccharides, disaccharides, and oligosaccharides), pesticides (e.g., oxamyl, pentoxazone, emamectin, and its metabolites), veterinary drugs (e.g., cefuroxime), mycotoxins (e.g., diacetoxyscirpenol, HT-2, T-2, and neosolaniol), shellfish poisons, fish poisons (e.g., ciguatoxin, palytoxin, and brevetoxin), and functional compounds (e.g., terpenoids, ginkgolides, and bilobalide), these various ions are likely to be generated.

[0043] These adduct ions are thought to be ammonium ions derived from the mobile phase used previously, sodium ions, or potassium ions escaping from the glass containers containing the mobile phase or liquid sample, or the glass capillaries that make up the ESI probe 201, which have attached to molecules of the target compound. MRM measurements using these ions as precursor ions resulting from the adduct ions of the target compound have poor measurement reproducibility, making it difficult to accurately quantify the target compound. Furthermore, when multiple ions are generated from a target compound, selecting any one of them as the precursor ion results in a smaller amount of precursor ion than when only one ion is generated, resulting in poor measurement sensitivity. As mentioned above, MRM measurements are used to quantify trace amounts of target compounds in samples, so poor measurement sensitivity can prevent the target compound from being detected. Furthermore, when performing MS / MS scan measurements, the product ion spectrum is created by measuring the intensities of multiple product ions generated from such a small amount of precursor ion, which can result in some product ions being overlooked, preventing accurate spectral matching.

[0044] In contrast, in the mass spectrometry method of this embodiment, alkali metal ions are added to the mobile phase, and the alkali metal ions X are ionized into the molecules M of the target compound contained in the liquid sample. + [M+X] + As described above, in this embodiment, an excess amount of alkali metal ions relative to the target compound and impurity compounds is added to the mobile phase to suppress the generation of unintended dehydrated ions and adduct ions such as solvent adduct ions, ammonium adduct ions, sodium adduct ions, and potassium adduct ions, and the ion species generated from the target compound are alkali metal adduct ions [M+X]. + In this way, by generating essentially only one type of alkali metal adduct ion from the target compound and using it as a precursor ion, it is possible to improve the sensitivity and reproducibility of MRM measurements and MS / MS scan measurements.

[0045] Among alkali metal adduct ions, lithium adduct ions and sodium adduct ions are difficult to fragment once generated. Sodium adduct ions, in particular, hardly fragment. Therefore, when performing MRM measurement by adding lithium ions or sodium ions as alkali metal ions to the mobile phase, it is recommended to use lithium adduct ions or sodium adduct ions as precursor ions and also use lithium adduct ions or sodium adduct ions as product ions. For example, when ions derived from impurity compounds having the same mass-to-charge ratio as the lithium adduct ions or sodium adduct ions are included, it is possible to fragment only the ions derived from the impurity compounds and accurately analyze only the target compound. When lithium adduct ions are generated, not only can the lithium adduct ions be subjected to MRM measurement as precursor ions and product ions as described above, but also the lithium adduct ions can be used as precursor ions and MS / MS scan measurement can be performed to obtain product ion spectra, thereby identifying (qualifying) the compound. thing It is also possible. [Example]

[0046] A measurement example will be described in which one type of alkali metal adduct ion is generated from a target compound that is prone to generate many types of ions using the mass spectrometry method of the above embodiment.

[0047] Figure 3 shows a multi-SIM spectrum (upper row: MS spectrum) obtained for CTX3C, a type of ciguatoxin, by a conventional mass spectrometry method (measurement using a mobile phase without added alkali metal ions), and a multi-SIM spectrum (MS spectrum) obtained by the mass spectrometry method of the above embodiment (lower row: measurement using a mobile phase with added alkali metal ions). Note that a multi-SIM spectrum is a graph in which the horizontal axis represents the mass-to-charge ratio and the vertical axis represents the intensity of each ion obtained by individually SIM-measuring ions of multiple different mass-to-charge ratios, as in a normal mass spectrum. Conventional mass spectrometry methods mainly focus on dehydrated ions [M+H-HO]. +, protonated ion [M+H] + Two types of ions were detected, and two types of dehydrated ions [M+H-2H2O] + , ammonium adduct ion [M+NH4] + , sodium adduct ion [M+Na] + , potassium adduct ion [M+K] + was detected, whereas the mass spectrometry method of this example substantially detected the lithium adduct ion [M+Li] + Only α was detected at high intensity.

[0048] Similarly, multi-SIM spectra were obtained for CTX1B, another type of ciguatoxin, using both the conventional mass spectrometry method and the mass spectrometry method of the above embodiment. For CTX1B, the conventional mass spectrometry method mainly detected ammonium adduct ions [M+NH4]. + was detected, and other two dehydrated ions [M+H-2H2O] + , dehydrated ion [M+H-H2O] + , protonated ion [M+H] + , sodium adduct ion [M+Na] + , potassium adduct ion [M+K] + was detected, whereas the mass spectrometry method of this example substantially detected the lithium adduct ion [M+Li] + Only α was detected at high intensity.

[0049] Furthermore, for 51-hydroxy-CTX3C, another type of ciguatoxin, multi-SIM spectra were obtained using both the conventional mass spectrometry method and the mass spectrometry method of the above embodiment. For 51-hydroxy-CTX3C, the conventional mass spectrometry method mainly produced bimolecular dehydrated ions [M+H-2H2O]. + , dehydrated ion [M+H-H2O] + , protonated ion [M+H] + was detected, and sodium adduct ions [M+Na] + , potassium adduct ion [M+K] +was detected, whereas the mass spectrometry method of this example substantially detected the lithium adduct ion [M+Li] + Only α was detected at high intensity.

[0050] Furthermore, for 52-epi-54-deoxy-CTX1B, another type of ciguatoxin, multi-SIM spectra were obtained using both the conventional mass spectrometry method and the mass spectrometry method of the above embodiment. For 52-epi-54-deoxy-CTX1B, the conventional mass spectrometry method also obtained a bimolecular dehydrated ion [M+H-2H2O]. + , dehydrated ion [M+H-H2O] + , protonated ion [M+H] + , ammonium adduct ion [M+NH4] + , sodium adduct ion [M+Na] + In contrast, the mass spectrometry method of this example detected a variety of ions, such as lithium adduct ions [M+Li]. + Only α was detected at high intensity.

[0051] Furthermore, for CTX4A, another type of ciguatoxin, multi-SIM spectra were obtained using both the conventional mass spectrometry method and the mass spectrometry method of the above embodiment. For CTX4A, the conventional mass spectrometry method mainly obtained dehydrated ions [M+H-HO]. + , protonated ion [M+H] + , ammonium adduct ion [M+NH4] + , sodium adduct ion [M+Na] + Various ions were detected, and furthermore, the bimolecular dehydrated ion [M+H-2H2O] + was detected, whereas the mass spectrometry method of this example substantially detected the lithium adduct ion [M+Li] + Only α was detected at high intensity.

[0052] Although the above examples were all obtained by adding lithium ions to the mobile phase, similar results were confirmed when sodium ions were added to the mobile phase. As shown in these examples, by using the mass spectrometry method of this embodiment, it is possible to generate substantially only one type of alkali metal adduct ion from a target compound (especially an organic polymer). Then, by performing MRM measurement or MS / MS scan measurement using this single type of alkali metal adduct ion as a precursor ion, it is possible to quantify and identify the target compound with high accuracy.

[0053] As described in Non-Patent Document 1, when separating the components in a liquid sample containing ciguatoxins by liquid chromatography, it is common to use a mobile phase containing a mixture of multiple solvents and perform gradient analysis in which the mixture ratio is changed over time.

[0054] In such gradient analysis, it is possible to add alkali metal ions to both organic and inorganic solvents. However, the inventors of the present invention have investigated ciguatoxins under various conditions and 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, whereas when alkali metal ions and an acidic solution are added to an aqueous mobile phase, the alkali metal ions are effectively generated. ion It was found that in some cases adduct ions with the addition of α- and β-blocking groups were not generated. The above measurement example was also the result of using a liquid chromatograph mass spectrometer to separate and measure target compounds (CTX3C, etc.) in a liquid sample by gradient analysis in which the mixture ratio of an organic solvent, to which the pH was adjusted by adding formic acid, an acidic volatile salt, and a mixture of formic acid and water was changed over time.

[0055] Ciguatoxins are organic polymer compounds, and therefore have a high affinity for the organic solvent contained in the mobile phase in the gradient analysis described above. Therefore, it is thought that adding alkali metal ions to the organic solvent efficiently generated adduct ions to which the alkali metal ions were added.

[0056] As described above, compounds from which various ions other than proton-adduct ions are generated include, for example, sugars (monosaccharides, disaccharides, oligosaccharides), pesticides (oxamyl, pentoxazone, emamectin and its metabolites, etc.), veterinary drugs (cefuroxime, etc.), mycotoxins (diacetoxyscirpenol, HT-2, T-2, neosolaniol, etc.), shellfish poisons, fish poisons (ciguatoxins, palytoxins, brevetoxins, etc.), and functional compounds (terpenoids, ginkgolides, bilobalide, etc.), many of which are organic polymer compounds, similar to ciguatoxins. Therefore, when the target compound is an organic polymer compound, adding a specific alkali metal ion to an organic solvent that has a high affinity for the organic polymer compound can efficiently generate an adduct ion to which the alkali metal ion is added. Note that when the target compound is a low-molecular-weight compound such as a monosaccharide or disaccharide, or an inorganic compound, adding an alkali metal ion to an inorganic solvent can also efficiently generate an adduct ion to which the alkali metal ion is added.

[0057] The above-described embodiments and examples are merely examples and can be modified as appropriate in accordance with the spirit of the present invention.

[0058] In the above embodiments and examples, a liquid chromatograph mass spectrometer was used, and alkali metal ions were added to the mobile phase to attach alkali metal ions to the target compound in the liquid sample. However, when using only a mass spectrometer, alkali metal ions can be added to the liquid sample itself. Furthermore, the configuration of the mass spectrometer described in the above embodiments (triple quadrupole mass spectrometer) is merely an example, and mass spectrometers of various configurations capable of performing MRM measurements and MS / MS scan measurements can be used.

[0059] [Aspect] It will be apparent to those skilled in the art that the above-described exemplary embodiments are examples of the following aspects.

[0060] (Section 1) A mass spectrometry method according to one aspect of the present invention includes the steps of: A preparation step of adding predetermined 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 have been added; The target compound produced in the ionization step is added with the predetermined alkali metal ion a measuring step of measuring the intensity of an adduct ion to which Includes.

[0061] In the mass spectrometry method according to paragraph 1, a preparation step is carried out prior to mass spectrometry to add predetermined alkali metal ions to a liquid sample. The addition of the predetermined alkali metal ions may be carried out by adding the ions themselves (e.g., by adding a liquid containing the ions) or by dissolving an alkali metal salt in the liquid sample. When the ionization step is carried out with the predetermined alkali metal ions supplied to the liquid sample in this manner, the predetermined alkali metal adduct ions, which are the additions of the alkali metal ions, are mainly produced, while the production of other types of adduct ions is suppressed. Furthermore, since many alkali metal ions are difficult to dissociate, the production of dehydrated ions and solvent adduct ions is also suppressed.

[0062] Mass spectrometry according to paragraph 1 method In the ionization step, the target compound is ionized with the predetermined alkali metal ion The intensity of the adduct ion to which the alkali metal is added or the ion derived from the adduct ion is measured. Therefore, the amount of ions measured is larger than when multiple types of ions are generated from the target compound due to ions generated from the liquid sample or sample container, and the target compound can be measured with high sensitivity. Furthermore, the mechanism by which the alkali metal adduct ion is generated has sufficient reproducibility, so the target compound can be accurately quantified.

[0063] (Section 2) The mass spectrometry method according to paragraph 2 is the mass spectrometry method according to paragraph 1, In the preparation step, a mobile phase containing an organic solvent to which the predetermined alkali metal ions have been added is prepared, and the liquid sample is introduced into a column of a liquid chromatograph in a flow of the mobile phase; In the ionization step, the liquid sample flowing out of the column is ionized.

[0064] Many of the compounds that produce various ions other than protonated ions are polymeric organic compounds, which have a high affinity with organic solvents. ion The liquid sample is introduced into a column of a liquid chromatograph using a mobile phase containing the compound, and the liquid sample flowing out of the column is ionized. ion is easily added, and alkali metal adduct ions are efficiently produced.

[0065] (Section 3) The mass spectrometry method according to paragraph 3 is the mass spectrometry method according to paragraph 1 or 2, In the preparation step, a pH adjuster for adjusting the pH is further added to the mobile phase.

[0066] Adding alkali metal ions to a mobile phase, such as by adding an alkali metal salt, can change the pH of the mobile phase. In liquid chromatography, the pH range of the mobile phase that can be used for each column may be determined. In the mass spectrometry method according to paragraph 3, by adjusting the pH of the mobile phase using a pH adjuster, a target compound contained in a liquid sample can be measured using a mobile phase that has a pH suitable for the column and to which alkali metal ions have been added.

[0067] (Section 4) The mass spectrometry method according to paragraph 4 is the mass spectrometry method according to paragraph 1, In the preparation step, a volatile salt of a predetermined alkali metal is added to the liquid sample.

[0068] If a non-volatile salt is added to the mobile phase, the salt will precipitate when the liquid sample is desolvated to ionize it. If this salt is introduced into the mass analyzer, it may clog small openings such as skimmers. Furthermore, if salt precipitates on the surface of an ion guide or mass filter, it may disrupt the electric field formed by these elements. The mass analysis method according to paragraph 4 does not encounter such problems because a volatile alkali metal salt is added.

[0069] (Section 5) A mass spectrometry method according to paragraph 5 is a mass spectrometry method according to any one of paragraphs 1 to 4, The predetermined alkali metal ion is a lithium ion or a sodium ion.

[0070] Among alkali metal ions, lithium ions and sodium ions in particular are difficult to cleave once they are added to the molecules of the target compound to generate adduct ions, so the mass spectrometry method according to paragraph 5 can further suppress the generation of other types of ions.

[0071] (Section 6) A mass spectrometry method according to paragraph 6 is a mass spectrometry method according to any one of paragraphs 1 to 5, In the measuring step, the target compound is ionized by the predetermined alkali metal ions generated in the ionization step. ion The adduct ions to which the ions are added are selected as precursor ions, and after the precursor ions are subjected to a dissociation treatment, the intensity of the ions that are not dissociated by the dissociation treatment is measured.

[0072] Adduct ions formed when alkali metal ions are attached to molecules of a target compound are difficult to fragment. In the mass spectrometry method described in Section 6, these are used as precursor ions, and the intensity of the ions that remain unfragmented after the precursor ions are subjected to a dissociation process is measured. This allows for fragmentation of only the impurity ions, even when there are contaminant ions with the same mass-to-charge ratio as the adduct ions of the target compound, and allows for the measurement of only the ions derived from the target compound. [Explanation of symbols]

[0073] 1. Liquid chromatograph 10...Mobile phase container 11...Pump 12...Injector 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 room 231...Pre-quadrupole mass filter 232...Collision cell 233...Ion Guide 234...Post-quadrupole mass filter 235...Ion detector 4...Control and processing section 41...Storage section 411…Compound DB 42...Measurement condition setting section 43...Measurement control section 44...Analysis processing unit 5...Input section 6…Display section C...Ion optical axis

Claims

1. an alkali metal ion addition step of adding a predetermined alkali metal ion to an organic solvent; a mobile phase preparation step of preparing a mobile phase containing the organic solvent; a chromatographic step of introducing a liquid sample into a column of a liquid chromatograph in the flow of the mobile phase; an ionization step of ionizing a target compound contained in the liquid sample to which the alkali metal ions have been added and which flows out of the column; a measuring step of measuring the intensity of an adduct ion produced in the ionization step in which the predetermined alkali metal ion is added to the target compound, or an ion derived from the adduct ion; A mass spectrometry method comprising:

2. The mass spectrometry method according to claim 1 , wherein the mobile phase preparation step further comprises adding a pH adjuster to the mobile phase to adjust its pH.

3. The mass spectrometry method of claim 1, wherein the addition of the specified alkali metal ion to the organic solvent is by adding a volatile salt of the specified alkali metal.

4. 2. The mass spectrometry method according to claim 1, wherein the predetermined alkali metal ion is a lithium ion or a sodium ion.

5. 2. The mass spectrometry method according to claim 1, wherein in the measuring step, adduct ions in which the predetermined alkali metal ions are added to the target compound are selected as precursor ions from among the ions generated in the ionizing step, and the precursor ions are subjected to a dissociation process, and then the intensities of ions that are not dissociated by the dissociation process are measured.

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