Mass spectrometry device and mass spectrometry method

Abstract

A mass spectrometry device includes a reaction chamber (132) into which a precursor ion is introduced; a radical generation section (54) that generates a known radical; a radical supply section (5) that causes the precursor ion to react with the radical to generate a fragment ion and an adduct ion; a measurement control section (63) that measures an ion group including the precursor ion, the fragment ion, and the adduct ion to obtain a mass spectrum; and a precise mass estimation section (64) that determines a peak of the adduct ion by searching within a prescribed mass range centered on a mass value obtained by adding a mass of an atom or molecule derived from the radical to a mass calculated from a peak of the precursor ion, and estimates a precise mass of the precursor ion by subtracting a precise mass of the atom or molecule from a precise mass of the adduct ion.

Description

Technical Field

[0001] This invention relates to a mass spectrometry analysis device and a mass spectrometry analysis method. Background Technology

[0002] To identify the polymer components in a sample or to determine its structure, mass spectrometry is widely used: ions with a specific mass-to-charge ratio are selected from the ions originating from the sample components as precursor ions, and the various fragment ions generated by dissociating the precursor ions are separated according to their mass-to-charge ratio and then detected.

[0003] Most polymers are organic compounds with hydrocarbon chains as their main skeleton. To understand the properties of polymers, it is effective to obtain information such as the presence or absence of unsaturated bonds and characteristic functional groups on carbon atoms. Therefore, a radical-assisted dissociation method has recently been proposed, which involves attaching free radicals to precursor ions derived from a sample component to cause the precursor ions to dissociate at the positions of unsaturated bonds or specific functional groups on carbon atoms. For example, Patent Documents 1 and 2 describe the selective dissociation of precursor ions at peptide bond positions by attaching hydrogen free radicals or the like to the precursor ions. Furthermore, Patent Documents 3 and 4 describe the selective dissociation of precursor ions at the positions of unsaturated bonds contained in the hydrocarbon chain by attaching oxygen free radicals or the like to the precursor ions.

[0004] When using radical-assisted dissociation to analyze unknown components in a sample, data-dependent analysis (DDA) is performed, for example, using a mass spectrometer equipped with a pre-filter, collision cell, and post-filter. In DDA, for example, a liquid sample is introduced into a liquid chromatograph, and the components contained in the sample are separated within the column and then introduced into the mass spectrometer. MS and MS / MS analyses are then performed continuously while each component is introduced into the mass spectrometer. In MS analysis, ions generated from the sample components are separated according to their mass-to-charge ratio using either the pre-filter or post-filter, and these ions are detected to obtain mass spectrometry (MS) data. Then, the mass peaks in the mass spectrum that meet a specified benchmark (typically the most intense mass peak) are identified, and the ion corresponding to that mass peak is determined as the precursor ion. In MS / MS analysis, a pre-filter is used to screen precursor ions from ions generated by the sample components, and the precursor ions are dissociated in a collision cell to generate fragment ions. These fragment ions are then separated according to their mass-to-charge ratio using a post-filter and detected to obtain the product ion spectrum (MS / MS spectrum) data.

[0005] After MS and MS / MS analyses, the peak climaxes of the precursor ions in the MS or MS / MS spectra are determined, and the mass-to-charge ratio of the precursor ions is calculated to, for example, three decimal places. Based on this precise mass-to-charge ratio, the composition of the compound is estimated. Additionally, for fragment ions in the MS / MS spectra, the composition is also estimated based on the precise mass-to-charge ratio of the mass peaks, thereby estimating the partial molecular structures of the ions generated by the compound with the estimated composition. Then, the partial molecular structures estimated from the mass peaks of multiple fragment ions are combined to estimate the overall molecular structure of the compound, thus identifying the compound.

[0006] Existing technical documents

[0007] Patent documents

[0008] Patent Document 1: International Publication No. 2015 / 133259

[0009] Patent Document 2: International Publication No. 2018 / 186286

[0010] Patent Document 3: International Publication No. 2019 / 155725

[0011] Patent Document 4: International Publication No. 2020 / 240908

[0012] Patent Document 5: Japanese Patent Application Publication No. 2020-177784

[0013] Non-patent literature

[0014] Non-patent document 1: Hidenori Takahashi, Yuji Shimabukuro, Daiki Asakawa, Akihito Korenaga, Masaki Yamada, Shinichi Iwamoto, Motoi Wada, Koichi Tanaka, "IdentifyingDouble Bond Positions in Phospholipids Using Liquid Chromatography-TripleQuadrupole Tandem Mass Spectrometry Based on Oxygen Attachment Dissociation", Mass Spectrometry,Volume 8,Issue 2,Pages S0080,2019 Summary of the Invention

[0015] The problem the invention aims to solve

[0016] The fragment ions generated by the radical-assisted dissociation method described in Patent Documents 1-4 are only a few percent of the precursor ions. Therefore, in order to measure these fragment ions with sufficient intensity, the detector gain needs to be set relatively high. However, if the gain is increased in this way, the measurement intensity of the precursor ions saturates. As a result, the mass peak of the precursor ions is distorted, making it impossible to accurately determine the peak position, and the error in the precise mass-to-charge ratio of the precursor ions increases. In the aforementioned DDA, the composition of the compound is estimated based on the precise mass-to-charge ratio of the precursor ions. Therefore, if the error in the precise mass-to-charge ratio of the precursor ions increases, there is a problem that the composition of the compound cannot be accurately estimated, making it difficult to identify the compound.

[0017] The problem to be solved by the present invention is to provide a technique that can accurately determine the mass-to-charge ratio of the precursor ions while measuring the fragment ions with sufficient intensity during mass spectrometry analysis of fragment ions generated by dissociating precursor ions.

[0018] Solution for solving the problem

[0019] The mass spectrometry analysis method of the present invention, which addresses the above-mentioned problems, includes the following steps:

[0020] The precursor ions react with known types of free radicals to produce fragment ions and adduct ions;

[0021] The detector gain is set to a gain such that the fragment ions and the adduct ions do not produce a halo, but the precursor ions do produce a halo;

[0022] The detector is used to determine the ion cluster containing the precursor ion, the fragment ion, and the adduct ion to obtain a mass spectrum;

[0023] The peak corresponding to the precursor ion is determined based on the mass spectrometry.

[0024] The peak of the adduct ion is determined by searching within a specified mass range centered on the following mass value, which is obtained by adding the mass of the peak corresponding to the precursor ion to the mass of the adduct ion, assuming that the mass of the atoms or molecules originating from the free radical is added to the adduct ion.

[0025] The precise mass of the precursor ion is estimated by subtracting the precise mass of the atoms or molecules derived from the free radical, presumed to be attached to the adduct ion, from the precise mass determined based on the peak of the determined adduct ion; and

[0026] Peaks in the mass spectrum other than those of the identified precursor ions and the identified adduct ions are identified as candidate peaks for the fragment ions.

[0027] Furthermore, the mass spectrometry analysis apparatus of the present invention, which was completed to solve the above-mentioned problems, includes:

[0028] The reaction chamber is in which precursor ions are introduced;

[0029] The radical generation section generates known types of free radicals;

[0030] A free radical supply unit supplies free radicals generated by the free radical generation unit to the reaction chamber where precursor ions have been introduced, thereby generating fragment ions and adduct ions;

[0031] The measurement control unit measures an ion cluster comprising the precursor ion, the fragment ion, and the adduct ion to obtain a mass spectrometer; and

[0032] The precise mass estimation unit determines the peak corresponding to the precursor ion in the mass spectrum by searching within a predetermined mass range centered on a mass value that is obtained by adding the mass of atoms or molecules derived from the free radical that are assumed to be attached to the adduct ion, based on the mass of the peak corresponding to the precursor ion. The precise mass of the precursor ion is estimated by subtracting the precise mass of atoms or molecules derived from the free radical that are assumed to be attached to the adduct ion from the precise mass of the peak of the adduct ion.

[0033] The effects of the invention

[0034] In radical-assisted dissociation methods, only a few percent of the precursor ions react to generate adduct ions or fragment ions. Therefore, if the detector gain is increased to a sufficient intensity to measure fragment ions, the intensity of the precursor ion's mass peak saturates. On the other hand, the amount of adduct ions generated by the radical attaching to the precursor ion is the same as the amount of fragment ions generated by the dissociation of the precursor ion. In the mass spectrometry method and apparatus of the present invention, by setting the detector gain to measure fragment ions with sufficient intensity, adduct ions can also be measured with sufficient intensity, and the precise mass of the adduct ion can be accurately determined. Then, by subtracting the precise mass of the atoms or molecules derived from the radical assumed to be attached to the adduct ion from the precise mass determined from the adduct ion peak, the mass-to-charge ratio of the precursor ion can be accurately determined. Attached Figure Description

[0035] Figure 1 This is a structural diagram of the main parts of an embodiment of the mass spectrometry analysis device involved in the present invention.

[0036] Figure 2 This is a schematic diagram of the free radical generation section in the mass spectrometry analysis apparatus of this embodiment.

[0037] Figure 3 This is a schematic diagram of the free radical transport path in the mass spectrometry analysis device of this embodiment.

[0038] Figure 4 The product ion spectrum is determined by setting the applied voltage of the ion detector to the upper limit to allow phosphatidylcholine (PC18:2 / 18:0) to react with oxygen free radicals.

[0039] Figure 5 It shows Figure 4 The product ion spectrum is a graph showing the range near the protonated ion.

[0040] Figure 6 It is the difference between the precise mass-to-charge ratio of the adduct ion generated when the precursor ion reacts with various free radicals and the precise mass-to-charge ratio of the precursor ion. Detailed Implementation

[0041] Hereinafter, embodiments of the mass spectrometry analysis apparatus and mass spectrometry analysis method involved in the present invention will be described with reference to the accompanying drawings.

[0042] Figure 1 This is a structural diagram of the main parts of the liquid chromatography-mass spectrometry (LC-MS) coupling device 100 obtained by combining the mass spectrometry analysis device 1 and the liquid chromatograph 2 in this embodiment.

[0043] The liquid chromatograph 2 includes a mobile phase container 20 containing the mobile phase, a delivery pump 21 for delivering the mobile phase, a syringe 22, and a column 23. Additionally, the syringe 22 is connected to an autosampler 24, which introduces multiple liquid samples into the syringe in a predetermined sequence.

[0044] The mass spectrometry analysis apparatus 1 includes an ionization chamber 10 at approximately atmospheric pressure, a main body consisting of a vacuum chamber, and a control / processing unit 6. Inside the vacuum chamber, a first intermediate vacuum chamber 11, a second intermediate vacuum chamber 12, a third intermediate vacuum chamber 13, and an analysis chamber 14 are arranged sequentially from the ionization chamber 10 side, and the apparatus has a structure of a multi-stage differential exhaust system that increases the vacuum level in this order.

[0045] An electrospray ionization probe (ESI probe) 101 is provided in the ionization chamber 10 to spray a liquid sample by applying a charge. Sample components separated by column 23 of liquid chromatograph 2 are sequentially introduced into the ESI probe 101.

[0046] The ionization chamber 10 is connected to the first intermediate vacuum chamber 11 via a thin-diameter heating capillary 102. An ion lens 111 is disposed in the first intermediate vacuum chamber 11. The ion lens 111 is composed of multiple annular electrodes of different diameters and is used to focus ions near the central axis of the ion flight path, i.e., the ion optical axis C.

[0047] The first intermediate vacuum chamber 11 and the second intermediate vacuum chamber 12 are separated by a skimmer 112 with a small hole at the top. An ion guide 121 is disposed in the second intermediate vacuum chamber 12. The ion guide 121 is composed of a plurality of rod electrodes arranged in a manner surrounding the ion optical axis C, for focusing ions in the vicinity of the ion optical axis C.

[0048] The third intermediate vacuum chamber 13 is equipped with a quadrupole mass filter 131, a collision cell 132, and an ion guide 134. The quadrupole mass filter 131 separates ions according to their mass-to-charge ratio. The collision cell 132 has a multi-polar ion guide 133 inside, and the ion guide 134 is used to transport ions released from the collision cell 132. The ion guide 134 is composed of multiple annular electrodes of the same diameter.

[0049] The collision pool 132 is connected to the collision gas supply unit 4. The collision gas supply unit 4 includes: a collision gas source 41, a gas inlet flow path 42 for introducing gas from the collision gas source 41 into the collision pool 132, and a valve 43 for opening and closing the gas inlet flow path 42. The collision gas is, for example, an inert gas such as nitrogen or argon.

[0050] Furthermore, the collision pool 132 is also connected to the free radical supply unit 5. The free radical supply unit 5 has the same structure as described in Patent Document 5 or Non-Patent Document 1. Figure 1 and Figure 2 As shown, the free radical supply unit 5 includes: a free radical source 54, which has a free radical generation chamber 51 formed inside; a vacuum pump (not shown) for venting the free radical generation chamber 51; a raw material gas supply source 52 for supplying gas (raw material gas) as a raw material for free radicals; and a high-frequency power supply unit 53. A valve 56 for adjusting the flow rate of the raw material gas is provided in the flow path from the raw material gas supply source 52 to the free radical generation chamber 51.

[0051] exist Figure 2 A cross-sectional view of the radical source 54 is shown. The radical source 54 has a tubular body 541 made of a dielectric material such as alumina (e.g., alumina, quartz, aluminum nitride), the internal space of which is called a radical generation chamber 51. The tubular body 541 is fixed by a plunger 545 in a state of being inserted into the interior of a hollow cylindrical magnet 544. A helical antenna 542 is wound around the outer periphery of the portion of the tubular body 541 located inside the magnet 544. Figure 2(dashed line).

[0052] In addition, a high-frequency power supply section 546 is provided in the free radical source 54. High-frequency power is supplied to the high-frequency power supply section 546 from the high-frequency power supply section 53. The free radical source 54 also has a flange 547 for fixing the front end portion of the free radical source 54. A hollow cylindrical magnet 548, which is the same diameter as the magnet 544, is housed inside the flange 547. A magnetic field is generated inside the tubular body 541 (free radical generation chamber 51) by the magnets 544 and 548, and the generation and maintenance of plasma are facilitated by the action of this magnetic field.

[0053] At the outlet end of the free radical source 54, a delivery pipe 58 is connected via a valve 582 for transporting the free radicals generated in the free radical generation chamber 51 to the collision pool 132. The delivery pipe 58 is an insulating pipe, such as a quartz glass tube or a borosilicate glass tube.

[0054] like Figure 3 As shown, a plurality of heads 581 are provided in the portion of the delivery pipe 58 that is arranged along the wall of the collision cell 132. Each head 581 is provided with an inclined, cone-shaped inlet, which introduces free radicals in a direction intersecting the central axis (ion optical axis C) of the ion flight direction. As a result, free radicals can be uniformly supplied into the interior of the collision cell 132.

[0055] The analysis chamber 14 includes: an ion transport electrode 141 for transporting ions incident from the third intermediate vacuum chamber 13; an orthogonal accelerating electrode 142, consisting of a set of ejector electrodes 1421 and an inlet electrode 1422 arranged facing each other across the incident optical axis (orthogonal accelerating region) of the ions; an accelerating electrode 143 for accelerating ions sent into the flight space using the orthogonal accelerating electrode 142; a reflecting electrode 144 for forming a refracting trajectory for the ions within the flight space; an ion detector 145; and a flight tube 146 for defining the outer edge of the flight space. The ion detector 145 is, for example, an electron multiplier tube or a microchannel plate.

[0056] The control / processing unit 6 has the function of controlling the operation of each part and saving and analyzing the data obtained by the ion detector 145. The control / processing unit 6 includes a storage unit 61. The storage unit 61 contains a compound database 611 and a free radical information database 612. In addition, the storage unit 61 also stores method documents describing the measurement conditions for performing the measurement described later, and information for converting the time of flight of ions into the mass-to-charge ratio of ions. The control / processing unit 6 also includes a measurement condition setting unit 62, a measurement control unit 63, a precise mass estimation unit 64, and a compound identification unit 65 as functional blocks. The entity of the control / processing unit 6 is a general personal computer connected to an input unit 7 and a display unit 8, and the above functional blocks are implemented by using a processor to execute a pre-installed mass spectrometry analysis program.

[0057] Next, as an example of the mass spectrometry analysis method according to the present invention, the analysis process using the liquid chromatography-mass spectrometry apparatus 100 of this embodiment will be described. In this example, data-dependent analysis (DDA) is performed to identify unknown components contained in the liquid sample. In this example, the retention time of the unknown component is known. DDA can be performed throughout the entire time of the liquid chromatography operation, even when the retention time of the unknown component is unknown. In DDA, during the introduction of each sample component into the mass spectrometry apparatus 1, an MS scan is first performed to determine the precursor ion, followed by an MS / MS scan to dissociate the precursor ion and obtain the product ion spectrum. Furthermore, before performing the following measurements, the gain of the ion detector 145 is set to a value that can measure fragment ions with sufficient intensity. The same sample can be measured with multiple gain settings, and the gain of the ion detector 145 can be set based on the results. Alternatively, even without preliminary measurements, it can be determined by theoretical values ​​or based on the analyst's experience. Furthermore, as in the measurement example described later, the detector gain can be set to maximum when the expected intensity of fragment ions and adduct ions is low. In this embodiment, the precursor ions are reacted with oxygen radicals during MS / MS scanning.

[0058] When the user instructs the start of analysis through the prescribed input operation, the measurement condition setting unit 62 displays a screen on the display unit 8 for inputting measurement conditions. These conditions include information on the mass scan range in the DDA, the selection conditions for precursor ions, the type of free radical, and collision-induced dissociation (CID). For example, the mass scan range or scan rate for MS and MS / MS measurements is input. For the selection conditions for precursor ions, the ion with the highest intensity measured in the MS measurement data is input. For the type of free radical, the type of free radical that reacts with the precursor ion or the type of feed gas used to generate the free radical is input. Regarding CID, the user selects whether a collision gas is introduced in the MS / MS measurement, the type of collision gas, and the magnitude of the collision energy. When the user sets the desired measurement conditions including these, the measurement condition setting unit 62 creates a batch file containing these conditions. When this batch file is executed, the measurement control unit 63 continuously performs MS and MS / MS measurements in accordance with the retention time of the unknown component, as follows.

[0059] When the user instructs to begin the measurement after the batch file is generated, the interior of the free radical generation chamber 51 is first evacuated to a predetermined vacuum level using a vacuum pump, and a raw material gas (oxygen in this embodiment) is introduced into the free radical generation chamber 51 from the raw material gas supply source 52. Next, a high-frequency voltage is supplied to the helical antenna 542 from the high-frequency power supply unit 53, thereby generating plasma in the free radical generation chamber 51. Oxygen free radicals are then generated from the oxygen supplied to the free radical generation chamber 51. However, at this point, the valve 582 is closed, and oxygen free radicals are not introduced into the collision cell 132.

[0060] Next, the liquid sample, pre-placed by the user in the autosampler 24, is introduced into the syringe 22. The liquid sample is then introduced into the column 23 along with the flow of the mobile phase delivered from the mobile phase container 20 by the delivery pump 21. After the components contained in the liquid sample are separated by time in the column 23, they are sequentially introduced into the electrospray ionization probe 101 for ionization.

[0061] Ions generated from the sample are introduced into the first intermediate vacuum chamber 11 through the heated capillary 102 due to the pressure difference between the ionization chamber 10 and the first intermediate vacuum chamber 11. In the first intermediate vacuum chamber 11, the ions are focused near the ion optical axis C by the ion lens 111.

[0062] The ions focused in the first intermediate vacuum chamber 11 then enter the second intermediate vacuum chamber 12, are refocused by the ion guide 121 to the vicinity of the ion optical axis C, and then enter the third intermediate vacuum chamber 13.

[0063] MS scanning is performed after the start of the measurement and once the time (retention time) during which the unknown component elutes from column 23 is reached. In the MS scanning, all ions are passed directly through the third intermediate vacuum chamber 13 without activating the quadrupole mass filter 131 or the collision cell 132. Ions passing through the collision cell 132 are focused near the ion optical axis C by the ion guide 134 and then enter the analysis chamber 14.

[0064] Ions entering the analysis chamber 14 are transported to the orthogonal accelerating electrode 142 via the ion transport electrode 141. A voltage is applied to the orthogonal accelerating electrode 142 at a predetermined period, causing the ion's flight direction to deflect in a direction approximately orthogonal to its previous flight direction. The ions, now deflected, are accelerated by the accelerating electrode 143 and ejected into the flight space. The ions ejected into the flight space travel along a predetermined flight path defined by the reflecting electrode 144 and the flight tube 146, with a time corresponding to the mass-to-charge ratio of each ion, and are incident on the ion detector 145. The ion detector 145 outputs a signal corresponding to the incident ion quantity each time an ion is incident on it. The output signals from the ion detector 145 are sequentially stored in the storage unit 61. The storage unit 61 stores measurement data based on the ion's flight time and detection intensity.

[0065] When the MS scan measurement is completed, the measurement control unit 63 reads the measurement data and the information used to convert the ion flight time into the ion mass-to-charge ratio stored in the storage unit 61, and converts them into mass spectrometry data with the ion mass-to-charge ratio and ion detection intensity as axes.

[0066] The measurement control unit 63 then identifies the peak with the highest intensity in the mass spectrometry data and obtains its mass-to-charge ratio. In the electrospray ionization probe 101 used in this embodiment, the protonated ions generated by the addition of protons to the sample molecules typically produce the most. Therefore, in this embodiment, the protonated ions are identified as the precursor ions corresponding to the peak with the highest intensity. Then, the mass-to-charge ratio of the precursor ions is determined using an estimated value (e.g., a value with one or two decimal places of precision). Furthermore, it is possible to calculate the value to three or more decimal places, but due to intensity saturation, even if an accurate mass-to-charge ratio is obtained, the value usually contains errors.

[0067] After determining the mass-to-charge ratio of the precursor ions, the measurement control unit 63 then performs an MS / MS scan. First, valve 582 is opened to introduce oxygen free radicals into the collision cell 132.

[0068] In MS / MS scanning measurements, the ions generated by the sample are also focused near the ion optical axis C during the passage through the first intermediate vacuum chamber 11 and the second intermediate vacuum chamber 12, and then enter the third intermediate vacuum chamber 13, just as in MS scanning measurements.

[0069] In the third intermediate vacuum chamber 13, ions with a mass-to-charge ratio determined based on the MS scan results described above are selected as precursor ions using a quadrupole mass filter 131 and introduced into the collision cell 132. Oxygen radicals are introduced into the collision cell 132 as described above. These oxygen radicals attach to the precursor ions, thereby generating fragment ions from the dissociated precursor ions and adduct ions that remain undissociated and retain the attached oxygen radicals. The fragment ions and adduct ions generated in the collision cell 132 (hereinafter, they are also collectively referred to as "product ions") are focused near the ion optical axis C by the ion guide 134 and then enter the analysis chamber 14.

[0070] In the analysis chamber 14, similar to MS scanning, product ions travel along a predetermined flight path defined by the reflective electrode 144 and the flight tube 146, with a time corresponding to the mass-to-charge ratio of each ion, and are incident on the ion detector 145. The ion detector 145 outputs a signal corresponding to the incident amount of ions each time an ion is incident on it. The output signals from the ion detector 145 are sequentially stored in the storage unit 61. The storage unit 61 stores measurement data with the ion flight time and ion detection intensity as axes.

[0071] When the measurement is completed, the measurement control unit 63 reads the measurement data and the information used to convert the flight time of the ions into the mass-to-charge ratio of the ions stored in the storage unit 61, and converts them into mass spectrometry (product ion spectrum) data with the mass-to-charge ratio of the ions and the detection intensity of the ions as axes.

[0072] Next, the precise mass estimation unit 64 determines the mass peak of the adduct ion obtained by the addition of oxygen atoms to the precursor ion in the product ion spectrum. In the radical-addition dissociation method as described in this analytical example, only a few percent of the precursor ions react to generate product ions. Therefore, the mass peak with the highest intensity in the product ion spectrum becomes the mass peak of the precursor ion. Then, a peak within a specified mass range centered at a mass 16 Da greater than that mass peak is searched, and this peak is identified as the peak of the adduct ion, where 16 Da corresponds to the estimated mass of the oxygen atom. This specified mass range can be appropriately determined based on the degree of saturation of the peak corresponding to the precursor ion. The specified mass range can be expanded to account for the possibility that the larger the mass range of precursor ion detection intensity saturation, the greater the error in the estimated mass-to-charge ratio of the precursor ion. As a specific value, it can be set to ±1 Da or ±0.5 Da.

[0073] The precise mass estimation unit 64 then calculates the precise mass (mass-to-charge ratio) of the adduct ion's mass peak to four decimal places, and subtracts the precise mass of the oxygen atom (15.9949 Da) from this precise mass. This value is the precise mass-to-charge ratio of the precursor ion. Generally, determining one adduct peak is sufficient, but multiple adduct peaks can be determined in this case. For example, when oxygen radicals are used as in the above embodiment, multiple adduct peaks appear with intensities corresponding to the presence ratios of oxygen isotopes (15.9949 Da, 16.9991 Da, 17.9991 Da). Therefore, these multiple adduct peak groups can also be determined.

[0074] When the precise mass-to-charge ratio of the precursor ion is calculated, the compound identification unit 65 estimates the composition of the unknown component based on this precise mass-to-charge ratio. Furthermore, the mass peaks obtained by removing the mass peaks of the precursor ion and the adduct ion from the product ion spectrum are identified as fragment ion mass peak candidates. Then, the composition of the unknown component is determined based on the precise mass-to-charge ratio of the fragment ion mass peak candidates, and a partial molecular structure of the unknown component is estimated based on this composition. The partial molecular structures estimated from multiple fragment ions are combined and compared with the molecular structures of known compounds stored in the compound database 611 to identify the unknown component. The process of estimating partial molecular structures from fragment ions to identify compounds can be performed in the same manner as described in Patent Documents 1-4, and therefore, its description is omitted here.

[0075] In the mass spectrometry analysis method and liquid chromatography-mass spectrometry coupled apparatus 100 of this embodiment, as described above, precursor ions derived from the sample components react with oxygen free radicals to generate product ions containing adduct ions with additional oxygen atoms.

[0076] In the radical-assisted dissociation method, only a few percent of the precursor ions react to generate fragment ions. If the gain of the ion detector 145 is increased to a sufficient intensity to measure the fragment ions, the intensity of the mass peak of the precursor ions will saturate. On the other hand, the amount of adduct ions generated by the radical attaching to the precursor ions is the same as the amount of fragment ions generated by the dissociation of the precursor ions. Therefore, by setting the gain of the ion detector 145 to measure the fragment ions with sufficient intensity, it is possible to measure the adduct ions with the same sufficient intensity and to accurately determine the mass-to-charge ratio of the adduct ions. Thus, the mass-to-charge ratio of the precursor ions can be accurately determined based on the mass-to-charge ratio of the adduct ions and the mass of the atoms or molecules attached to the adduct ions. In particular, for electron multiplier tubes or microchannel plates, which are widely used as ion detectors in mass spectrometry analysis devices, the dynamic range of the detector gain is limited to a narrow range, and the measurement intensity is prone to saturation. Therefore, the mass spectrometry analysis method and mass spectrometry analysis device 1 of the above embodiments can be preferably used.

[0077] Furthermore, in DDA, the gain of the ion detector 145 is the same during MS scanning and MS / MS scanning. Therefore, the intensity of the precursor ion is saturated in the mass spectrum obtained by MS scanning. However, since the mass-to-charge ratio required for screening precursor ions in MS / MS scanning is 1 to 2 decimal places, there will be no particular problem even if the intensity of the precursor ion mass peak is saturated in the mass spectrum.

[0078] Next, refer to Figure 4 and Figure 5 To illustrate the actual measurement examples. Figure 4 This is the product ion spectrum determined by reacting phosphatidylcholine (PC18:2 / 18:0) with oxygen free radicals. In this measurement, a microchannel plate (MCP) was used as the ion detector. Based on prior experience in measuring the reaction of phospholipids with various free radicals, the inventors know that the intensity of fragment ions and adduct ions generated when phospholipids react with oxygen free radicals is low. Therefore, in this measurement, the applied voltage was set to an upper limit of 2.5 kV (i.e., the detector gain was set to maximum).

[0079] Figure 5 It shows Figure 4 The protonated ion (precursor ion) in the product ion spectrum. [M+H] + The range is near the exact mass-to-charge ratio of 786.600731. In the product ion spectrum, it can be seen that the intensity of the mass peak of the protonated ion (a single isotope ion where all hydrogen atoms are 1H) is saturated, such as... Figure 4The shape of the mass peak is distorted as shown. Furthermore, the mass-to-charge ratio at the peak tip is 786.6364. This value deviates significantly from the true value (786.600731 Da), with a mass accuracy of approximately 50 ppm. In addition, the original mass accuracy of the mass spectrometry apparatus used in this determination is 3 ppm.

[0080] When estimating molecular structure using MS / MS scanning, the composition is estimated based on the precise mass of the precursor ion. Furthermore, the mass peaks obtained by removing the mass peaks of the precursor ion and adduct ion from the product ion spectrum are identified as candidate mass peaks for fragment ions. Then, based on the estimated composition and the mass-to-charge ratio of the identified candidate mass peaks for each fragment ion, the candidate mass peaks for each fragment ion are assigned. Therefore, if like... Figure 5 As illustrated in the example, if the measurement of intensity saturation leads to a large error in the mass-to-charge ratio of the precursor ions, then accurate structural analysis cannot be performed. Generally, a mass accuracy of 10 ppm or higher is required to accurately estimate the composition based on the mass-to-charge ratio of the precursor ions.

[0081] like Figure 5 As shown, due to the high applied voltage of the detector, weak fragment ions were observed. As described above, the intensity of the mass peak of the precursor ion was saturated, while the signal intensity of the adduct ion with the added oxygen radical was low; therefore, the mass peak did not saturate, and its mass-to-charge ratio was 802.6026. Since the precise mass of the oxygen radical is 15.9949 Da, the precursor ion [M+H]... + The mass value was obtained as 802.6026 - 15.9949 = 786.6077. This value is consistent with the true value (786.600731 Da) with an accuracy of more than 10 ppm.

[0082] The above embodiment is an example, and modifications can be made appropriately according to the spirit of the present invention. The above embodiment uses a liquid chromatography-mass spectrometry (LC-MS) apparatus, but a gas chromatography-mass spectrometry (GC-MS) apparatus can be used, or a mass spectrometry analysis apparatus can be used without a chromatograph. In the above embodiment, an orthogonal accelerated time-of-flight (COF) mass filter was used as the downstream mass filter, but multiple circulation type, magnetic field fan type, and other types of mass filters can also be used.

[0083] Furthermore, a precise mass is simply a mass with the precision to which the composition of a compound as an unknown sample can be estimated based on its precise mass-to-charge ratio using precursor ions. In the above embodiments, the precise mass was calculated with a precision of four decimal places, but the required precision varies depending on the characteristics of the compound to be measured (such as the number of compounds with similar structures), and is not limited to the precision described in the above embodiments.

[0084] In the above embodiments, a protonated ion, which is a positive ion, is used as the precursor ion and reacts with an oxygen free radical. However, the combination of the polarity of the precursor ion and the type of free radical can be appropriately changed. When using a negative precursor ion, for example, sodium adducts, ammonia adducts, potassium adducts, and chloride adducts can be used as precursor ions.

[0085] Besides oxygen radicals, other free radicals that react with precursor ions include hydrogen radicals, hydroxyl radicals, nitrogen radicals, methyl radicals, chlorine radicals, fluorine radicals, phosphate radicals, and silicon radicals. Furthermore, suitable feedstock gases for generating such free radicals include hydrogen gas (which generates hydrogen radicals), oxygen or ozone gas (which generates oxygen radicals), water vapor (which generates hydrogen, oxygen, and hydroxyl radicals), nitrogen gas (which generates nitrogen radicals), methyl gas (which generates methyl radicals), chlorine gas (which generates chlorine radicals), fluorine gas (which generates fluorine radicals), phosphoric acid vapor (which generates phosphate radicals), and silane gas (which generates silicon radicals). Figure 6 The diagram shows the adduct ions obtained using various free radicals (X) (the common adduct ion [M+H(+X)]). + (Central column) The adduct ion formed after the hydrogen atom is removed: [M+H(+XH)] + (Left column) and adduct ions with added hydrogen atoms [M+H(+X+H)] + The difference between the precise mass-to-charge ratio of the precursor ion and the precise mass-to-charge ratio of the precursor ion.

[0086] In the above embodiments, the precursor ions passing through the collision cell 132 react with the free radicals. However, an ion trap can also be used instead of the collision cell 132 to capture the precursor ions in the ion trap so that the precursor ions react with the free radicals.

[0087] Depending on the sample composition, during radical-induced dissociation, due to collisions with the background gas, H2O, CO2, polar groups, etc., may detach (neutral loss), resulting in the precursor ion itself sometimes being undetectable. In this case, the precise mass of the precursor ion can be calculated based on the mass value of the radical-added ion relative to the neutral loss peak.

[0088] In the above embodiments, a measurement example without CID was described. However, it is also possible to introduce collision gas into the collision cell 132 while supplying free radicals, and generate fragment ions through CID. By using CID simultaneously, more types of fragment ions can be generated for analysis. However, if all precursor ions are broken due to CID, the accurate mass-to-charge ratio of the precursor ions cannot be determined by the method described in the above measurement example. In such cases, the measurement control unit 63 only needs to be configured to sequentially (separately) perform MS / MS scan measurements of the reaction with free radicals and MS / MS scan measurements of CID after MS scan measurements (the measurement condition setting unit 62 only needs to create a batch file for performing measurements in this order).

[0089] Furthermore, while the above embodiments described the cases of performing MS scanning and MS / MS scanning, the MS method that generates fragment ions by capturing precursor ions in an ion trap and performing multiple dissociations is also described. n This invention can also be applied in scanning measurements.

[0090] [Way]

[0091] Those skilled in the art will understand that the above-described exemplary embodiments are specific examples of the following approaches.

[0092] (First item)

[0093] One approach involves a mass spectrometry analysis method that includes the following steps:

[0094] The precursor ions react with known types of free radicals to produce fragment ions and adduct ions;

[0095] The detector gain is set to a gain such that the fragment ions and the adduct ions do not produce a halo, but the precursor ions do produce a halo;

[0096] The detector is used to determine the ion cluster containing the precursor ion, the fragment ion, and the adduct ion to obtain a mass spectrum;

[0097] The peak corresponding to the precursor ion is determined based on the mass spectrometry.

[0098] The peak of the adduct ion is determined by searching within a specified mass range centered on the following mass value, which is obtained by adding the mass of the peak corresponding to the precursor ion to the mass of the adduct ion, assuming that the mass of the atoms or molecules originating from the free radical is added to the adduct ion.

[0099] The precise mass of the precursor ion is estimated by subtracting the precise mass of the atoms or molecules derived from the free radical, presumed to be attached to the adduct ion, from the precise mass determined based on the peak of the determined adduct ion; and

[0100] Peaks in the mass spectrum other than those of the identified precursor ions and the identified adduct ions are identified as candidate peaks for the fragment ions.

[0101] (Second item)

[0102] One method involves a mass spectrometry analysis device that includes:

[0103] The reaction chamber is in which precursor ions are introduced;

[0104] The radical generation section generates known types of free radicals;

[0105] A free radical supply unit supplies free radicals generated by the free radical generation unit to the reaction chamber where precursor ions have been introduced, thereby generating fragment ions and adduct ions;

[0106] The measurement control unit measures an ion cluster comprising the precursor ion, the fragment ion, and the adduct ion to obtain a mass spectrometer; and

[0107] The precise mass estimation unit determines the peak corresponding to the precursor ion in the mass spectrum by searching within a predetermined mass range centered on a mass value that is obtained by adding the mass of atoms or molecules derived from the free radical that are assumed to be attached to the adduct ion, based on the mass of the peak corresponding to the precursor ion. The precise mass of the precursor ion is estimated by subtracting the precise mass of atoms or molecules derived from the free radical that are assumed to be attached to the adduct ion from the precise mass of the peak of the adduct ion.

[0108] In the mass spectrometry method of the first item and the mass spectrometry apparatus of the second item, precursor ions derived from the sample component react with a known type of free radical to generate adduct ions obtained by attaching atoms or molecules corresponding to the type of free radical to the precursor ions, and fragment ions obtained by breaking the precursor ions. Then, a mass spectrum is obtained by measuring the ion cluster containing the precursor ions, fragment ions, and adduct ions. At this time, the detector gain is preset to a gain such that fragment ions and adduct ions do not produce halos while the precursor ions do. The same sample can be measured using multiple gain settings, and the detector gain can be set based on the results. Alternatively, even without preliminary measurements, it can be determined by theoretical values ​​or based on the analyst's experience. Furthermore, the detector gain can be set to maximum when the intensity of fragment ions and adduct ions is expected to be low. In short, as long as the measured result is set to a gain that allows for the measurement of fragment ions and adduct ions with sufficient intensity without saturation, it is acceptable.

[0109] Next, the peaks corresponding to the precursor ions are determined based on the mass spectrum. Generally, the peak with the largest height or area in the mass spectrum corresponds to the precursor ion. Therefore, it is sufficient to determine the largest peak in the mass spectrum as the peak corresponding to the precursor ion. Then, the mass of the precursor ion is calculated based on this peak. As mentioned above, since the detector gain is set to cause a halo effect on the precursor ion, the precursor ion peak is saturated. The mass calculated based on the saturated peak contains errors, but the mass calculated at this time point only needs to be an integer estimate and correct; for example, a small error is acceptable for values ​​after the decimal point. Next, the peak of the adduct ion is determined by searching within a specified mass range centered on the following mass value: the mass value is the estimated value plus the mass of the atoms or molecules derived from the free radical assumed to be attached to the adduct ion. The specified mass range can be appropriately determined based on the degree of saturation of the peak corresponding to the precursor ion; for example, it can be set to ±1 Da or ±0.5 Da. Next, the precise mass of the precursor ion is estimated by subtracting the precise mass of the atoms or molecules assumed to be attached to the adduct ion from the precise mass calculated based on the peak of the determined adduct ion. Finally, peaks in the mass spectrum other than those of the precursor ion and adduct ion are identified as candidates for fragment ion peaks.

[0110] In the radical-assisted dissociation method, only a few percent of the precursor ions react to generate adduct ions or fragment ions. Therefore, if the detector gain is increased to a sufficient intensity to measure fragment ions, the intensity of the precursor ion mass peak will saturate. On the other hand, the amount of adduct ions generated by the radical attaching to the precursor ion is the same as the amount of fragment ions generated by the dissociation of the precursor ion. Therefore, by setting the detector gain to measure fragment ions with sufficient intensity, it is possible to measure adduct ions with equally sufficient intensity and accurately determine the mass ratio of the adduct ions. Then, by subtracting the accurate mass of the atoms or molecules derived from the radical that are assumed to be attached to the adduct ion from the accurate mass determined from the adduct ion peak, the mass-to-charge ratio of the precursor ion can be accurately determined.

[0111] (Third item)

[0112] According to the mass spectrometry analysis apparatus described in the second item, wherein...

[0113] The free radical is any one of hydrogen free radical, oxygen free radical, hydroxyl free radical, nitrogen free radical, methyl free radical, chlorine free radical, fluorine free radical, phosphate free radical, and silicon free radical.

[0114] Similar to the mass spectrometry apparatus described in the third item, in the mass spectrometry apparatus described in the second item, the free radical that reacts with the precursor ion can be any of the following: hydrogen free radical, oxygen free radical, hydroxyl free radical, nitrogen free radical, methyl free radical, chlorine free radical, fluorine free radical, phosphate free radical, and silicon free radical. These free radicals are readily generated, and the atoms or molecules attached to the precursor ion can be predicted; therefore, the accurate mass-to-charge ratio of the precursor ion can be obtained through simple measurement.

[0115] (Item 4)

[0116] According to the mass spectrometry analysis apparatus described in the second or third item, wherein...

[0117] It also features electron multiplier tubes or microchannel plates as ion detectors for detecting the ion clusters.

[0118] Electron multiplier tubes or microchannel plates used in the mass spectrometry analysis apparatus described in the fourth item are widely used as ion detectors, but since the dynamic range of the detector gain is limited to a narrow range, the mass spectrometry analysis apparatus described in the second or third item is particularly preferred.

[0119] (Item 5)

[0120] In any of the mass spectrometry analyzers described in items two through four,

[0121] The measurement control unit also performs MS scanning measurements to obtain mass spectrometry data by separating and measuring the ions generated from the sample. Based on the mass peaks contained in the mass spectrometry data, the precursor ion is determined under predetermined conditions. Then, an MS / MS scanning measurement is performed to cause the precursor ion to react with the free radical.

[0122] By using the mass spectrometry analysis device in the fifth section, data-dependent analysis (DDA) can be performed on unknown sample components.

[0123] (Item 6)

[0124] In the mass spectrometry analysis apparatus described in item five

[0125] It also includes a collision gas supply unit that supplies collision gas to the reaction chamber.

[0126] The measurement control unit performs MS / MS scanning measurements to cause the precursor ions to react with the free radicals and to supply the collision gas to the reaction chamber.

[0127] By using the mass spectrometry apparatus described in item six, it is possible to analyze the sample composition using both fragment ions generated by the radical additional dissociation method and fragment ions generated by the CID method. Furthermore, MS / MS measurements using the radical additional dissociation method and MS / MS measurements using the CID method can be performed simultaneously or independently. However, in cases where a large number of precursor ions are broken down by the CID method, it is preferable to perform both sequentially (independently).

[0128] Explanation of reference numerals in the attached figures

[0129] 100: Liquid Chromatography-Mass Spectrometry System; 1: Mass Spectrometry Analysis System; 10: Ionization Chamber; 101: Electrospray Ionization (ESI) Probe; 11: First Intermediate Vacuum Chamber; 12: Second Intermediate Vacuum Chamber; 13: Third Intermediate Vacuum Chamber; 131: Quadrupole Mass Filter; 132: Collision Cell; 133: Multipolar Ion Guider; 14: Analysis Chamber; 142: Orthogonal Accelerating Electrode; 144: Reflection Electrode; 145: Ion Detector; 146: Flight Tube; 2: Liquid Chromatograph; 20: Mobile Phase Container; 21: Pump; 22: Syringe; 23: Column; 24: Autosampler; 4: Collision Gas Supply Unit; 4 1: Collision gas source; 42: Gas introduction flow path; 5: Free radical supply unit; 51: Free radical generation chamber; 52: Raw material gas supply source; 53: High-frequency power supply unit; 54: Free radical source; 541: Tubular body; 542: Helical antenna; 544, 548: Magnets; 546: High-frequency power connection unit; 58: Delivery pipe; 6: Control / processing unit; 61: Storage unit; 611: Compound database; 612: Free radical information database; 62: Measurement condition setting unit; 63: Measurement control unit; 64: Precise mass estimation unit; 65: Compound identification unit; 7: Input unit; 8: Display unit; C: Ion optical axis.

Claims

1. A mass spectrometry analysis method, comprising the following steps: The precursor ions react with known types of free radicals to produce fragment ions and adduct ions; The detector gain is set to a gain such that the measurement intensity of the fragment ions and the adduct ions is unsaturated, but the measurement intensity of the precursor ions is saturated. The detector is used to determine the ion cluster containing the precursor ion, the fragment ion, and the adduct ion to obtain a mass spectrum; The peak corresponding to the precursor ion is determined based on the mass spectrometry. The peak of the adduct ion is determined by searching within a specified mass range centered on the following mass value, which is obtained by adding the mass of the peak corresponding to the precursor ion to the mass of the adduct ion, assuming that the mass of the atoms or molecules originating from the free radical is added to the adduct ion. The precise mass of the precursor ion is estimated by subtracting the precise mass of the atoms or molecules derived from the free radical that are assumed to be attached to the adduct ion from the precise mass determined based on the peak of the adduct ion. as well as Peaks in the mass spectrum other than those of the identified precursor ions and the identified adduct ions are identified as candidate peaks for the fragment ions.

2. A mass spectrometry analysis device, comprising: The reaction chamber is in which precursor ions are introduced; The radical generation section generates known types of free radicals; A free radical supply unit supplies free radicals generated by the free radical generation unit to the reaction chamber where precursor ions have been introduced, thereby generating fragment ions and adduct ions; The detector's gain is set such that the measurement intensity of the fragment ions and the adduct ions is unsaturated while the measurement intensity of the precursor ions is saturated. The measurement control unit obtains a mass spectrum by measuring an ion cluster containing the precursor ion, the fragment ion, and the adduct ion using the detector; and The precise mass estimation unit determines the peak corresponding to the precursor ion in the mass spectrum by searching within a predetermined mass range centered on a mass value that is obtained by adding the mass of atoms or molecules derived from the free radical that are assumed to be attached to the adduct ion, based on the mass of the peak corresponding to the precursor ion. The precise mass of the precursor ion is estimated by subtracting the precise mass of atoms or molecules derived from the free radical that are assumed to be attached to the adduct ion from the precise mass of the peak of the adduct ion.

3. The mass spectrometry analysis apparatus according to claim 2, wherein, The free radical is any one of hydrogen free radical, oxygen free radical, hydroxyl free radical, nitrogen free radical, methyl free radical, chlorine free radical, fluorine free radical, phosphate free radical, and silicon free radical.

4. The mass spectrometry analysis apparatus according to claim 2, wherein, It also features electron multiplier tubes or microchannel plates as ion detectors for detecting the ion clusters.

5. The mass spectrometry analysis apparatus according to claim 2, wherein, The measurement control unit also performs MS scanning measurements to obtain mass spectrometry data by separating and measuring the ions generated from the sample. Based on the mass peaks contained in the mass spectrometry data, the precursor ion is determined under predetermined conditions. Then, an MS / MS scanning measurement is performed to cause the precursor ion to react with the free radical.

6. The mass spectrometry analysis apparatus according to claim 5, wherein, It also includes a collision gas supply unit that supplies collision gas to the reaction chamber. The measurement control unit performs MS / MS scanning measurements to cause the precursor ions to react with the free radicals and to supply the collision gas to the reaction chamber.

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