JP2017026586A - Mass spectrometry, mass spectrometer and mass spectrometry program - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simply and accurately acquire MSspectral data of a compound to be analyzed in the MSanalysis of electing precursor ions on the basis of MSspectral data acquired by MSanalysis (n is an integer of 2 or more) of the compound to be analyzed to perform a MSanalysis.SOLUTION: A mass spectrometry comprises: (the step S1) determining MSanalysis conditions including a measurement mass range; (the step S2) determining MSanalysis conditions including the conditions of electing precursor ions of a MSanalysis from MSspectral data of a compound to be detected and the measurement mass range; (the step S3) preparing an analysis execution file for performing the MSanalysis of the compound to be detected without introducing a collision gas and introducing the collision gas into a collision cell to perform an analysis equal to or more than the MSof the compound to be analyzed; and (the Step S4) performing the MSanalysis of the compound to be analyzed by performing the analysis execution file to acquire MSspectrum data.SELECTED DRAWING: Figure 3

Description

The present invention relates to a mass spectrometer capable of performing MS ⁿ analysis (n is an integer of 2 or more). The present invention also relates to a mass spectrometry method and a mass spectrometry program performed using such a mass spectrometer.

MS ² analysis is a method for identifying an analyte compound contained in a sample. The MS ² analysis is, for example, a triple quadrupole type having a front stage mass separation unit that sorts precursor ions, a collision cell that cleaves the precursor ions to generate product ions, and a rear stage mass separation unit that sorts product ions. This can be done using a mass spectrometer.

In MS ² analysis, in general, first, an analysis target compound is introduced into a mass spectrometer, MS ¹ analysis (MS analysis) is performed, and MS spectrum data is acquired. Then, the analyst selects one or more ions having a high detection intensity based on the MS spectrum data. Then, the compound to be analyzed is again introduced into the mass spectrometer, MS ² analysis is performed using one of the selected one or more ions as a precursor ion, and MS ² spectrum data is acquired. After acquiring MS ² spectrum data for all of the selected ions, they are collated with mass spectrum data of a large number of reference compounds stored in advance in a database to identify analysis target compounds.

Among mass spectrometers capable of performing MS ² analysis, predetermined selection conditions (for example, ions having a detection intensity equal to or higher than a threshold value are selected) from MS spectrum data of an analysis target compound obtained by MS analysis. Some precursor ions can be automatically selected and MS ² analysis can be performed following the MS analysis. Such an analysis is referred to as data dependent analysis (DDA) (for example, Patent Document 1). The DDA, the number of times of introducing the compounds of interest to the mass spectrometer has to be performed one time, and also, analyst acquires conveniently MS ² spectral data without the need to elect a precursor ion to check himself MS spectral data There is an advantage that you can. In some cases, it is called auto MS instead of DDA (for example, Patent Documents 2 and 3).

Japanese Unexamined Patent Publication No. 2007-309661 JP 2008-298427 A Japanese Unexamined Patent Publication No. 2014-170013

In DDA, in order to perform MS analysis and MS ² analysis continuously, a collision gas for cleaving a precursor ion in MS ² analysis is introduced into the collision cell before starting the analysis. Therefore, ions also collide with the collision gas when passing through the collision cell during MS analysis, and some ions are cleaved. Hereinafter, problems caused by such ion cleavage will be described.

FIG. 1A shows an example of an MS spectrum in the case where ions generated from the analysis target compound are detected without being cleaved. On the other hand, FIG. 1B shows an example of an MS spectrum when a part of ions generated from the analysis target compound is detected by cleavage. In these MS spectra, when ions corresponding to mass peaks having an intensity exceeding a preset threshold value (that is, ions detected at an intensity exceeding the threshold value) are selected and subjected to MS ² analysis, the following results.

In the example of FIG. 1A, three ions (Pa, Pb, Pc) are detected with an intensity equal to or higher than a threshold value, and MS ² analysis using these ions as precursor ions is performed following the MS analysis. On the other hand, in the example of FIG. 1B, the detection intensity of these three ions (Pa, Pb, Pc) decreases, and a plurality of new ions (Pd, Pe, Pf) that are not detected in FIG. Detected with an intensity greater than the threshold. This is because Pa, Pb, and Pc collide with the collision gas and cleave, and ions such as Pd, Pe, and Pf are newly generated. As a result, MS ² analysis using Pa, Pd, Pe, and Pf as precursor ions is performed.

Of the four precursor ions (Pa, Pd, Pe, Pf) analyzed by MS ^{2 in} the example of FIG. 1 (b), three ions (Pd, Pe, Pf) are ions generated by cleavage, and originally It is an ion to be detected in MS ² analysis. Therefore, there is no that apply also against the MS ² spectra data of these reference compounds MS ² spectra data acquired by MS ² analysis to precursor ions. In addition, MS ² spectrum data of precursor ions (Pb, Pc) to be analyzed by MS ² cannot be obtained. Therefore, unless the selection conditions for the precursor ions are changed and the MS ² scan measurement is performed again, the original MS ² spectrum data cannot be acquired, which takes time and effort.

  By the way, in DDA, ions that are not selected regardless of the detection intensity can be set in advance as excluded ions. Thus, it is conceivable to avoid the above problem by setting ions that are supposed to be generated by undesired cleavage during MS analysis as excluded ions. However, it is actually impossible to assume in advance all ions generated by cleavage, and it takes time to set a large number of excluded ions.

Here, MS ² analysis has been described as an example. However, in MS ³ or higher analysis using an ion trap time-of-flight mass spectrometer (IT-TOF), ions are selected from MS spectrum data as described above. And there is the same problem because there is a process of MS ² analysis.

The problem to be solved by the present invention is that MS ^n-1 spectrum data is obtained by MS ^n-1 analysis (n is an integer of 2 or more) of a compound to be analyzed, and a precursor ion is obtained based on the MS ^n-1 spectrum data. In MS ⁿ analysis that selects and executes MS ⁿ analysis, it is easy and accurate to acquire MS ⁿ spectral data of the analysis target compound.

The first aspect of the present invention made to solve the above-described problem is that a compound to be analyzed is analyzed by MS ⁿ analysis (n is 2 or more) using a mass spectrometer having a mass separation unit before and after the collision unit. Integer) method,
a) MS ¹ analysis conditions including the measured mass range in MS ¹ analysis are determined,
From MS ^n-1 spectrum data of b) compounds of interest and conditions of selecting the precursor ion of MS ⁿ analysis, the measurement mass range of MS ⁿ analysis, the MS ⁿ analysis conditions including determining,
c) Perform the MS ¹ analysis of the analytes on the basis of the MS ¹ analysis condition without introducing collision gas for cleaving the ions into the collision portion, said collision portion of the collision gas for cleaving an ionic An analysis execution file for executing analysis of MS ² or more of the analysis target compound based on the MS ⁿ analysis conditions is created,
d) the compounds of interest were analyzed MS ⁿ by performing the analysis execution file and acquires the MS ⁿ spectrum data.

Examples of the mass spectrometer include a triple quadrupole mass spectrometer (Triple-Q), a quadrupole time-of-flight mass spectrometer (Q-TOF), and an ion trap time-of-flight mass spectrometer (IT-TOF). It is. In the triple quadrupole mass spectrometer and the quadrupole time-of-flight mass spectrometer, the collision cell corresponds to the collision part, and in the ion trap time-of-flight mass spectrometer, the ion trap corresponds to the collision part.
The conditions for selecting the precursor ion for the MS ⁿ analysis include, for example, selecting ions whose detection intensity is equal to or higher than a threshold, selecting a predetermined number of ions in descending order of detection intensity, or setting by the user in advance. If one or more preferential ions are detected, selection can be used regardless of the detected intensity of the ions. It is also possible to use as the above condition that any one or a plurality of excluded ions set in advance by the user is not selected.

In the mass spectrometry method according to the present invention, since MS ¹ analysis (MS analysis) is performed without using a collision gas, ions are not cleaved during MS analysis and undesired ions are not generated. Therefore, a precursor ion is correctly selected from MS spectrum data acquired by MS analysis, MS ² analysis is executed, and MS ² spectrum data can be acquired. Further, it is possible to acquire the MS ³ or more spectral data by performing an analysis of the MS ³ after based on the correct MS ² spectra data.
Also, the mass spectrometry method according to the present invention, the analyst determines the MS ¹ analysis conditions and MS ⁿ analysis conditions, only performs an analysis execution file, a conveniently analyzed compound was analyzed MS ⁿ MS ⁿ spectra Data can be acquired.

The mass-to-charge ratio of product ions generated by cleavage of monovalent precursor ions is smaller than the mass-to-charge ratio of precursor ions, but the mass-to-charge ratio of product ions generated by cleavage of divalent precursor ions is the mass-to-charge ratio of precursor ions. May be greater than the ratio. Therefore, normally, the measurement mass range of MS analysis and the measurement mass range of MS ⁿ analysis are the same.

Therefore, in the mass spectrometry method according to the present invention, the MS ⁿ analysis condition is determined before setting the MS ¹ analysis condition, and the measurement mass range included in the MS ⁿ analysis condition is directly measured in the MS ¹ analysis. It can be configured to determine the mass range. This eliminates the need for the analyst to determine the MS ¹ analysis conditions, and allows the MS ⁿ analysis of the analysis target compound to be performed more easily.

A second aspect of the present invention made to solve the above problems is a mass spectrometer having a mass separation unit on the front and rear sides of the collision unit,
a) MS ¹ analysis condition setting unit for setting MS ¹ analysis conditions including a measurement mass range in MS ¹ analysis based on user input;
b) Conditions for selecting precursor ions for MS ⁿ analysis from MS ^{n -1} spectra obtained by MS ^n-1 analysis (n is an integer of 2 or more) based on user input, and measured mass in MS ⁿ analysis and MS ⁿ analysis condition setting unit for setting a MS ⁿ analysis conditions including the range and,
c) Perform the MS ¹ analysis of the analytes on the basis of the MS ¹ analysis condition without introducing collision gas for cleaving the ions into the collision portion, said collision portion of the collision gas for cleaving an ionic An analysis execution file creation unit that creates an analysis execution file that is introduced into the MS ⁿ analysis condition and performs analysis of MS ² or more of the analysis target compound based on the MS ⁿ analysis conditions;
d) An analysis execution unit that performs MS ⁿ analysis on the analysis target compound to acquire MS ⁿ spectrum data by executing the analysis execution file based on a predetermined operation by a user.

The third aspect of the present invention, which has been made to solve the above-mentioned problems, is a program for MS ⁿ analysis of a compound to be analyzed using a mass spectrometer having a mass separation unit before and after the collision unit. The computer is caused to function as each part of the mass spectrometer of the second aspect.

By using the mass spectrometric method, the mass spectroscope, or the mass spectrometric program according to the present invention, MS ⁿ spectrum data can be obtained by MS ⁿ analysis of the compound to be analyzed simply and accurately.

Figure ions MS ¹ the time of analysis to explain the problem in the case of cleavage. The principal part block diagram of the chromatograph mass spectrometer which is one Example of the mass spectrometer which concerns on this invention. The flowchart regarding the mass spectrometry method in a present Example.

  Hereinafter, an embodiment of a mass spectrometry method, a mass spectrometer, and a mass spectrometry program according to the present invention will be described with reference to the drawings. FIG. 2 is a configuration diagram of a main part of the chromatographic mass spectrometer of the present embodiment, and FIG. 3 is a flowchart relating to the mass spectrometry method of the present embodiment.

  The chromatograph mass spectrometer of the present embodiment is a liquid chromatograph mass spectrometer composed of a liquid chromatograph section 1, a mass analyzer section 2, and a control section 4 that controls these operations.

  In the liquid chromatograph mass spectrometer of the present embodiment, the liquid chromatograph unit 1 includes a mobile phase container 10 in which a mobile phase is stored, a pump 11 that sucks the mobile phase and delivers it at a constant flow rate, and a mobile phase. And a column 13 for separating various compounds contained in the sample solution in the time direction.

  The mass analysis unit 2 includes first and second vacuums whose degree of vacuum is increased stepwise between the ionization chamber 20 at approximately atmospheric pressure and the high-vacuum analysis chamber 23 evacuated by a vacuum pump (not shown). 2 A multi-stage differential exhaust system having intermediate vacuum chambers 21 and 22 is provided. The ionization chamber 20 is provided with an electrospray ionization probe (ESI probe) 201 for spraying while applying a charge to the sample solution. The ionization chamber 20 and the first intermediate vacuum chamber 21 in the subsequent stage communicate with each other through a small heating capillary 202.

  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, and ions are focused in the first intermediate vacuum chamber 21 and the second intermediate vacuum chamber 22, respectively. However, ion guides 211 and 221 for transporting to the subsequent stage are installed. The analysis chamber 23 sandwiches a collision cell 232 in which a multipole ion guide (q2) 233 is installed, and the former quadrupole mass filter (Q1) 231 that separates ions according to the mass-to-charge ratio, A quadrupole mass filter (Q3) 234 and an ion detector 235 are installed to separate the two according to the mass-to-charge ratio.

  A collision gas such as argon and nitrogen can be supplied into the collision cell 232 continuously or intermittently. The power supply unit 24 applies predetermined voltages to the ESI probe 201, the ion guides 211, 221, 233, the quadrupole mass filters 231, 234, and the like. Each of the quadrupole mass filters 231 and 234 has a pre-rod electrode for correcting the disturbance of the electric field at the inlet end in front of the main rod electrode. The pre-rod electrode is different from the main rod electrode. A voltage can be applied.

The control unit 4 includes a storage unit 41 and functionally includes an MS ¹ analysis condition setting unit 42, an MS ⁿ analysis condition setting unit 43, an analysis execution file creation unit 44, and an analysis execution unit 45 as functional blocks. Yes. In addition, the function of controlling the operation of each part such as the pump 11 and the injector 12 of the liquid chromatograph part 1, the power supply part 24 and the collision gas supply part (not shown) of the mass analysis part 2 in accordance with the operation of each part described above. Have.

  The entity of the control unit 4 is a personal computer, and can function as each of the above units by executing a mass analysis program installed in advance in the computer. An input unit 6 and a display unit 7 are connected to the control unit 4.

Hereinafter, the mass spectrometry method in the present embodiment will be described with reference to the flowchart of FIG. In this example, the analysis target compound is separated from the sample by the chromatograph unit 1, and MS ² analysis is performed by the mass separation unit 2. In the present embodiment, MS ² analysis will be described, but the configuration of the present invention can be applied to MS ⁿ analysis in general.

The MS ¹ analysis condition setting unit 42 displays an MS ¹ analysis condition input screen on the display unit 7 and prompts the analyst to input each condition. When the analyst inputs MS ¹ analysis conditions including the measurement mass range and scan speed, they are set as MS ¹ analysis conditions and stored in the storage unit 41 (step S1). Note that the scan speed included in the MS ¹ analysis condition and the MS ⁿ analysis condition described later in this example is an analysis condition when a triple quadrupole mass analysis unit is used for the mass analysis unit 2, and is a quadrupole flight. When using other types of mass spectrometers, such as a time-type mass spectrometer and an ion trap time-of-flight mass spectrometer, analysis conditions are set according to each.

Next, the MS ⁿ analysis condition setting unit 43 displays an MS ² analysis condition input screen on the screen of the display unit 7 and prompts the analyst to input each condition. MS ⁿ analysis conditions (= MS ² analysis conditions) include precursor mass of MS ⁿ analysis (= MS ² analysis) from MS ^{n -1} spectrum data (= MS ¹ spectrum data) in addition to the measurement mass range and scan speed. The conditions for selection are included.

  Precursor ion selection conditions include, for example, selecting ions whose ion detection intensity is equal to or higher than a predetermined threshold th, selecting a predetermined number of ions in descending order of ion detection intensity, and ions designated by an analyst. If (preferential ion) is detected, it can be selected regardless of the detected intensity. Moreover, even if the ion (exclusion ion) designated by the analyst is detected by intensity, the condition that the ion is not selected can be used. In this embodiment, ions detected with an intensity equal to or higher than the threshold th input by the analyst are selected.

When the analyst completes the input of the above conditions, the MS ⁿ analysis condition setting unit 43 sets them as MS ² analysis conditions and stores them in the storage unit 41 (step S2).

When the MS ¹ analysis condition and the MS ² analysis condition are set, the analysis execution file creation unit 44 controls each part of the apparatus based on them so that the analysis target compound is MS ² analyzed in the following steps S41 to S48. An analysis execution file describing the command to be executed is created (step S3).

Specifically, the internal space of the mass analyzer 2 (including the collision cell 232) is maintained in a vacuum state, and a sample is introduced from the injector 12 of the liquid chromatograph unit 1 (step S41) to hold the analysis target compound. MS ¹ analysis is executed according to the time (step S42), and MS ¹ spectrum data is acquired (step S43). Based on the MS ¹ spectrum data, ions detected with an intensity equal to or higher than the threshold th are selected and determined as precursor ions for MS ² analysis (step S44).

Subsequently, a collision gas is introduced into the collision cell 232 (step S45), a sample is again introduced from the injector 12 of the liquid chromatograph unit 1 (step S46), and the precursor previously selected in accordance with the retention time of the analysis target compound. MS ² analysis using each of the ions is performed (step S47), and MS ² spectrum data is acquired (step S48).

  When the creation of the analysis execution file is completed, the file is stored in the storage unit 41 and an analysis start button is displayed on the display unit 7.

When the analyst presses the analysis start button, the analysis execution unit 45 reads the analysis execution file from the storage unit 41 and executes it (step S4). Thereby, the above-described steps S41 to S48 are sequentially executed, and the MS ² spectrum data of the sample to be analyzed is acquired. Analysis execution unit 45 displays on the display unit 7 to create the MS ² spectra from the acquired MS ² spectral data (step S5).

As described above, when the mass spectrometry method, the chromatograph mass spectrometer, or the mass spectrometry program of the present embodiment is used, the analyst inputs the MS ¹ analysis conditions and the MS ² analysis conditions and presses the analysis start button. only performs a simple analysis sample can acquire the MS ² analysis and MS ² spectra.

Further, in this embodiment, the collision gas is not introduced into the collision cell 232, and the MS ¹ analysis is performed while maintaining the internal space of the mass analysis unit 2 in a vacuum state. Therefore, the MS ¹ analysis is executed as in the conventional DDA. There is no fear of occasional undesired ion cleavage, and a precursor ion for MS ² analysis can be correctly selected.

The above-described embodiment is an example, and can be appropriately changed in accordance with the gist of the present invention.
In the above example, the analyst inputs the MS ¹ analysis condition and the MS ² analysis condition, respectively. However, the analyst inputs only the MS ² analysis condition, and a part thereof (in the above example, the measurement mass range and the mass scanning speed). Etc.) and the MS ⁿ analysis condition setting unit automatically sets the MS ¹ analysis conditions.

Although the liquid chromatograph mass spectrometer is used in the above-described embodiments, the analysis target compound can be similarly analyzed in the gas chromatograph mass spectrometer. Also, if available pure analyte compounds, without using chromatographic can be MS ⁿ analysis by introducing analytes directly to the ionization section of the mass spectrometer.

A feature of the present invention is that MS ¹ analysis is performed without using a collision gas, MS ¹ spectrum data is acquired, and a precursor ion for MS ² analysis is selected from the MS ¹ spectrum data, and the selected precursor ion is used. The MS ² analysis was automatically performed following the MS ¹ analysis. That is, a precursor ion for MS ³ analysis is selected from the MS ² spectrum data, that is, MS ³ or higher analysis can be performed in the same manner as in the conventional DDA. Therefore, by using an ion trap time-of-flight mass analysis unit instead of the triple quadrupole type mass separation unit in the above example, analysis of MS ³ or more can be performed as in the above example.

DESCRIPTION OF SYMBOLS 1 ... Liquid chromatograph part 10 ... Mobile phase container 11 ... Pump 12 ... Injector 13 ... Column 2 ... Mass spectrometry part 20 ... Ionization chamber 201 ... ESI probe 202 ... Heating capillary 21 ... First intermediate vacuum chamber 211 ... Ion guide 212 ... Skimmer 22 ... second intermediate vacuum chamber 23 ... analysis chamber 231 ... front quadrupole mass filter 232 ... collision cell 233 ... multipole ion guide 234 ... back quadrupole mass filter 235 ... ion detector 24 ... power supply 4 ... control Unit 41 ... Storage unit 42 ... MS ¹ analysis condition setting unit 43 ... MS ⁿ analysis condition setting unit 44 ... Analysis execution file creation unit 45 ... Analysis execution unit 6 ... Input unit 7 ... Display unit

Claims (4)

A method for performing MS ⁿ analysis (n is an integer of 2 or more) of a compound to be analyzed using a mass spectrometer having a mass separation unit before and after a collision part,
a) MS ¹ analysis conditions including the measured mass range in MS ¹ analysis are determined,
From MS ^n-1 spectrum data of b) compounds of interest and conditions of selecting the precursor ion of MS ⁿ analysis, the measurement mass range of MS ⁿ analysis, the MS ⁿ analysis conditions including determining,
c) Perform the MS ¹ analysis of the analytes on the basis of the MS ¹ analysis condition without introducing collision gas for cleaving the ions into the collision portion, said collision portion of the collision gas for cleaving an ionic An analysis execution file for executing analysis of MS ² or more of the analysis target compound based on the MS ⁿ analysis conditions is created,
mass spectrometry method characterized by d) the compounds of interest by performing the analysis executable by analyzing MS ⁿ to obtain the MS ⁿ spectrum data. The MS ⁿ analysis condition is determined before setting the MS ¹ analysis condition, and the measurement mass range included in the MS ⁿ analysis condition is directly determined as the measurement mass range of the MS ¹ analysis. Item 2. The mass spectrometry method according to Item 1. A mass spectrometer having a mass separation unit at the front and back across the collision unit,
a) MS ¹ analysis condition setting unit for setting MS ¹ analysis conditions including a measurement mass range in MS ¹ analysis based on user input;
b) Based on user input, conditions for selecting precursor ions for MS ⁿ analysis from MS ^{n -1} spectrum obtained by MS ^n-1 analysis (n is an integer of 2 or more), and measurement in MS ⁿ analysis and MS ⁿ analysis condition setting unit for setting, and MS ⁿ analysis conditions including the mass range,
c) Perform the MS ¹ analysis of the analytes on the basis of the MS ¹ analysis condition without introducing collision gas for cleaving the ions into the collision portion, said collision portion of the collision gas for cleaving an ionic An analysis execution file creation unit that creates an analysis execution file that is introduced into the MS ⁿ analysis condition and performs analysis of MS ² or more of the analysis target compound based on the MS ⁿ analysis conditions;
d) Mass spectrometry comprising: an analysis execution unit that performs MS ⁿ analysis of the analysis target compound to acquire MS ⁿ spectrum data by executing the analysis execution file based on a predetermined operation by a user apparatus. A program for ^MSn analysis of a compound to be analyzed using a mass spectrometer having a mass separation unit on the front and rear sides of a collision unit, wherein the computer functions as each unit of the mass spectrometer according to claim 3. A mass spectrometry program characterized by

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