Patent Application
20230245875
The present invention relates to a mass spectrometer capable of an MS/MS analysis as well as a method for mass spectrometry in the same mass spectrometer.
MS/MS analysis, which is one of the techniques for mass spectrometry, is useful for identifying compounds having high molecular weights as well as analyzing their chemical structures. As for the mass spectrometer capable of an MS/MS analysis, a triple quadrupole mass spectrometer and a quadrupole time-of-flight mass spectrometer (which is hereinafter called a “Q-TOF mass spectrometer”) are commonly known. These types of mass spectrometers normally include a collision cell. Ions having a predetermined amount of energy (collision energy) are introduced into the collision cell and made to collide with a collision gas so as to induce collision induced dissociation (CID) and thereby dissociate the ions. A mass spectrometric analysis of the product ions resulting from the dissociation is performed to create a mass spectrum (product ion spectrum).
Since the bond energies of various binding sites in a compound vary from portion to portion, the ease of breaking the binding sites also varies from site to site. Therefore, in the previously described type of mass spectrometer, when the collision energy (which may be hereinafter abbreviated as “CE”) which an ion has when introduced into the collision cell is changed, the form of dissociation changes even when the ion is derived from the same compound, so that the resulting product ion spectrum has a different peak pattern.
In general, for the identification or structural analysis of a compound having a complex chemical structure, it is advantageous to find out the masses of various fragments originating from that compound. Accordingly, in a conventionally known analytical technique called a “collision energy spread method” (which is hereinafter called the “CES method”), a product-ion scan measurement is repeated for one target compound while sequentially changing the CE value to a plurality of levels, and the thereby obtained mass spectra are accumulated to create a mass spectrum in which various kinds of product ions are observed (see Patent Literature 1 or other related documents). The mass spectrum thus created by the accumulation (which is hereinafter called the “cumulative mass spectrum”) is a mass spectrum including a mixture of peaks originating from various product ions resulting from the dissociation under different CE values.
In the CES method, it is preferable to accumulate mass spectra obtained under various CE values. However, the number of CE values (the number of levels to which the CE value is changed; this number may be hereinafter called the “CE-value number”) used in the CES method is constrained by analysis conditions, such as the number of times of the accumulation of mass spectra as well as the measurement time. In the conventional mass spectrometer, the CE values in the CES method are determined by dividing the range of CE values (from the lower to the upper limit; this range may be hereinafter called the “CE-value range”) specified directly or indirectly as one of the analysis conditions, by the number of CE values determined from specific constraints, such as the number of times of the spectrum accumulation and the period of time allotted to the measurement by the CES method. For example, when the CE-value range is from 20 to 50 V and the CE-value number is 5, the CE values (V) in the CES method are set at five levels of 20, 27.5, 35, 42.5 and 50. When the CE-value number is 4 while the CE-value range is identical to the aforementioned one, the CE values (V) in the CES method are set at four levels of 20, 30, 40 and 50. Although CE values may be expressed in eV, it is often a common practice to represent a CE value by the voltage which gives the collision energy. Accordingly, the unit V is used throughout the present description.
Thus, even when the CE-value range is the same, the CE values significantly change with a change in the CE-value number, or analysis conditions, with the result that the pattern of the cumulative mass spectrum may significantly change. Cumulative mass spectra are useful for identifying compounds by pattern matching based on a database (or similar identification methods). However, if the pattern of the cumulative mass spectrum significantly changes with a change in the CE-value number, a problem may possibly occur in the aforementioned task of identifying compounds. Another problem is that it is difficult to compare cumulative mass spectra obtained under different conditions.
The present invention has been developed to solve the previously described problems. Its primary objective is to provide a method for mass spectrometry and a mass spectrometer which can avoid a significant change in the pattern of the cumulative mass spectrum with a change in CE-value number when obtaining cumulative mass spectra by the CES method.
One mode of the method for mass spectrometry according to the present invention developed for solving the previously described problem is a method for mass spectrometry using a mass spectrometer capable of an MS/MS analysis and provided with a measurement section which includes a collision cell configured to dissociate an ion and a mass separator configured to perform a mass spectrometric analysis of product ions resulting from dissociation, the method for mass spectrometry including the step of executing a collision energy spread method in which collision energy is changed into a plurality of levels and a mass spectrum obtained at each level of the collision energy is accumulated to obtain a cumulative mass spectrum, and the method for mass spectrometry including:
a CES-method-condition determination step for determining a plurality of collision-energy values to be used in the collision energy spread method, according to given conditions including the range of collision-energy values and the number of collision-energy values, in such a manner that n+1 collision-energy values to be used when the number is n+1 (where n is an integer equal to or greater than three) are determined so that the n+1 collision-energy values include n collision-energy values used when the number is n and one additional collision-energy value different from any one of the n collision-energy values; and
an analysis execution step for sequentially setting the collision energy at the n+1 collision-energy values determined in the CES-method-condition determination step and controlling the measurement section so as to execute an MS/MS analysis under each collision-energy value and thereby obtain a mass spectrum for each collision-energy value.
One mode of the mass spectrometer according to the present invention developed for solving the previously described problem is a mass spectrometer capable of an MS/MS analysis and provided with a measurement section which includes a collision cell configured to dissociate an ion and a mass separator configured to perform a mass spectrometric analysis of product ions resulting from dissociation, the mass spectrometer including:
a CES-method-condition determiner configured to determine a plurality of collision-energy values to be used in the collision energy spread method, according to given conditions including the range of collision-energy values and the number of collision-energy values, in such a manner that n+1 collision-energy values to be used when the number is n+1 (where n is an integer equal to or greater than three) are determined so that the n+1 collision-energy values include n collision-energy values used when the number is n and one additional collision-energy value different from any one of the n collision-energy values;
an analysis controller configured to sequentially change the collision energy to the n+1 collision-energy values determined in the CES-method-condition determiner and control the measurement section so as to execute an MS/MS analysis under each collision-energy value; and
a data processor configured to obtain a cumulative mass spectrum by accumulating mass spectra each of which is obtained under each of the different collision-energy values under the control of the analysis controller.
In the previously described modes of the method for mass spectrometry and the mass spectrometer according to the present invention, even when the number of CE values specified within the CE-value range for performing the CES method is changed, the spectrum pattern of the cumulative mass spectrum undergoes only a small change since there is a considerable number of common CE values. Therefore, for example, the identification of compounds by pattern matching using a database of cumulative mass spectra (or similar methods) can be performed with a high level of accuracy. Additionally, the task of comparing cumulative mass spectra obtained for different samples will be easier.
A Q-TOF mass spectrometer as one embodiment of the mass spectrometer according to the present invention is hereinafter described with reference to the attached drawings.
As shown in
The ionization chamber 101 is provided with an electrospray ionization (ESI) probe 11. The ESI probe 11 is supplied with an eluate, for example, from the exit end of a column in a liquid chromatograph (LC, which is not shown) located in the previous stage. The ionization chamber 101 communicates with the first intermediate vacuum chamber 102 through a thin desolvation tube 12. The first intermediate vacuum chamber 102 communicates with the second intermediate vacuum chamber 103 through an orifice formed at the apex of a skimmer 14. The first and second intermediate vacuum chambers 102 and 103 contain multipole ion guides 13 and 15, respectively.
The first high vacuum chamber 104 contains a quadrupole mass filter 16 and a collision cell 17 which contains a multipole ion guide 18. An array of ring electrodes arranged from the first high vacuum chamber 104 to the second high vacuum chamber 105 across the partition between these chambers forms an ion guide 19. The second high vacuum chamber 105 contains an orthogonal acceleration time-of-flight mass separator and an ion detector 24. The mass separator includes an orthogonal accelerator 20, a flight tube 22 forming a flight space inside, and a reflectron 23.
The control-and-processing unit 3 controls the measurement unit 1 to carry out an analysis as well as performs data processing based on the detection signals obtained with the ion detector 24. The control-and-processing unit 3 includes an analysis controller 30, analysis condition setter 31, CES-method-condition determiner 32 and data processor 33 as its functional blocks. The CES-method-condition determiner 32 includes a CE-value number determiner 320 and CE value determiner 321 as sub-functional blocks.
In normal cases, the control-and-processing unit 3 is actually a personal computer, on which the aforementioned functional blocks can be embodied by executing, on the computer, dedicated control-and-processing software (computer program) installed on the same computer. This type of computer program can be offered to users in the form of a non-transitory computer-readable record medium holding the program, such as a CD-ROM, DVD-ROM, memory card, or USB memory (dongle). The program may also be offered to users in the form of data transferred through the Internet or similar communication networks. The program can also be preinstalled on a computer provided as a part of a system before a user purchases the system.
An operation for a product-ion scan measurement, which is one form of the MS/MS analysis carried out in the mass spectrometer according to the present embodiment, is hereinafter schematically described.
The ESI probe 11 sprays an introduced liquid sample (e.g., an eluate from an LC column) into the ionization chamber 101 while imparting imbalanced electric charges to the liquid. The charged droplets generated by the spraying process come in contact with the surrounding high-temperature gas and are thereby atomized. The compound contained in each droplet is turned into a gas ion through the vaporization of the solvent from the droplet. The generated ions are sent through the desolvation tube 12 into the first intermediate vacuum chamber 102 and subsequently transported through the ion guide 13, the orifice of the skimmer 14 and the ion guide 15, to be introduced into the quadrupole mass filter 16 in the first high vacuum chamber 104.
A predetermined voltage composed of an RF (radio frequency) voltage superposed on a DC voltage is applied to each of the plurality of rod electrodes forming the quadrupole mass filter 16. An ion having a specific mass-to-charge ratio (m/z) corresponding to that voltage is selected as a precursor ion. After passing through the quadrupole mass filter 16, the precursor ion enters the collision cell 17, having a collision energy corresponding to the DC potential difference between the quadrupole mass filter 16 and the entrance electrode of the collision cell 17. Meanwhile, a collision gas, such as argon gas, is introduced into this collision cell 17. The precursor ion collides with this collision gas and is fragmented into various kinds of product ions through the CID process. The generated product ions exit the collision cell 17 and are transported through the ion guide 19 into the orthogonal accelerator 20.
The orthogonal accelerator 20 simultaneously ejects all of the introduced ions in a substantially orthogonal direction (Z-axis direction) to their direction of introduction (X-axis direction). Each ejected ion flies in the flight space 21 at a speed corresponding to its m/z value. After being repelled by the reflective electric field created by the reflectron 23 as indicated by the alternating long-dashed and double short-dashed lines in
The data processor 33 in the control-and-processing unit 3 digitizes the detection signal and creates a mass spectrum by converting, into an m/z value, the time of flight of each ion as measured from the point in time of the ejection of the ions from the orthogonal accelerator 20. The display unit 5 shows the thus created mass spectrum on its screen.
As already noted, the form of dissociation within the collision cell 17 changes with a change in the collision energy used in the MS/MS analysis, which leads to a change in the kinds and intensities of the mass peaks in the mass spectrum. The CES method is an analytical technique utilizing this fact, in which at least one mass spectrum is obtained under each of the plurality of different CE values, and the obtained mass spectra are accumulated to obtain a cumulative mass spectrum in which various kinds of ions originating from the target component are reflected.
A characteristic control and processing for carrying out a CES method in the mass spectrometer according to the present embodiment is hereinafter described with reference to
In advance of an analysis, a user (operator) inputs parameter values of the analysis conditions required for the CES method by operating the input unit 4. The analysis condition setter 31 receives the input of the parameter value of each analysis condition. The analysis conditions include the total number of times of the accumulation of mass spectra as well as the central value and width of variation of the CE value. The measurement time allotted to the measurement by the CES method and the number of times of the accumulation per one CE value may also be included in the analysis conditions. Needless to say, one or more of those parameter values may be given as fixed values which normal users are not allowed to change.
The CE-value number determiner 320 determines the number of levels of the CE value to be set in the CES method (which is the number of CE values that are different from each other; this number is hereinafter called the “CE-value number”) based on the total number of times of the accumulation of mass spectra, measurement time, number of times of the accumulation per one CE value and other related conditions. For example, if the total number of times of the accumulation of mass spectra under the constraint of the measurement time is 10, and the number of times of the accumulation per one CE value is 2, then the CE-value number is 5. The CE-value number determiner 320 also determines the range over which the CE value is to be varied (this range is hereinafter called the “CE-value range”) from the central value and width of variation of the CE value. As shown in
After the CE-value number and the CE-value range have been determined, the CE value determiner 321 determines a plurality of CE values as follows: Suppose that the CE-value is currently m. Initially, which of the following equations (1) and (2) the value of m satisfies is determined:
m=2p+1 (1)
2p+1<m<2p+1+1 (2)
where p is a natural number whose value is determined from equation (1) or (2). If equation (1) holds, gain G is calculated by the following equation (3):
G=width of CE-value range/(m−1) (3)
The m CE values are given by the following equation (4) using gain G:
[CE]=lowest voltage value in the CE-value range+G×(q−1) (4)
where q represents all natural numbers equal to or less than m.
On the other hand, when equation (2) holds, excess number H and two gains G1 and G2 are determined by the following equations (5)-(7):
H=m−(2p+1) (5)
G1=width of CE-value range/2p (6)
G2=width of CE-value range/2p+1 (7)
The m CE values are given by the following equations (8) and (9) using excess number H and gain G1 and G2:
For the range of q≤H×2:
[CE]=lowest voltage value in the CE-value range+G2×(q−1) (8)
For the range of q>H×2:
[CE]=lowest voltage value in the CE-value range+G1×(q−1−H) (9)
As one example, consider the case where the CE-value range is from 20 to 50 V (the width of the CE-value range is 30 V) and m=5, as shown in
In this case, since m=5=22+1, equation (1) holds, and G=30[V]/4=7.5 from equation (3). Consequently, the five CE values are 20, 27.5, 35. 42.5 and 50 from equation (4).
As another example, consider the case where the CE-value range is from 20 to 50 V and m=7, as shown in
In this case, since 22+1<m=7<23+1, equation (2) holds. From equations (5)-(7), H=7−(22+1)=2, G1=30[V]/4=7.5, and G2=30[V]/8=3.75. The CE values for the range of q≤2×2=4 are [CE]=20, 23.75, 27.5 and 31.25 from equation (8), while the CE values for the range of q>4 are [CE]=42.5 and 50 from equation (9). Combining the two results gives the seven CE values of 20, 23.75, 27.5, 31.25, 35, 42.5 and 50.
The present method for determining CE values is also characterized by the way in which a new CE value is added when the CE-value number is to be increased by one: This new CE value is equal to the middle value of a pair of existing adjacent CE values, where a pair of smaller CE values is preferentially selected as the two values between which the middle value should be inserted. Technical meanings of these features will be described later.
After the CE values in the CES method have been determined in the previously described manner, the information is sent to the analysis controller 30. The analysis controller 30 operates the measurement unit 1 so that an MS/MS analysis is carried out under each of the specified CE values. Specifically, a voltage generator (not shown) in the measurement unit 1 is controlled so that the precursor ion has a specified amount of collision energy when it is introduced into the collision cell 17.
The data processor 33 receives detection signals obtained by MS/MS analyses under different CE values and accumulates mass spectra corresponding to those MS/MS analyses to obtain a cumulative mass spectrum. This cumulative mass spectrum shows mass peaks corresponding to the product ions generated by the CID process under the different CE values. The data processor 33 displays this cumulative mass spectrum on the display unit 5. It also calculates the degree of matching between the pattern of the cumulative mass spectrum obtained by the actual measurement and that of each of the cumulative mass spectra which, for example, are recorded in a database of cumulative mass spectra prepared beforehand. Based on this degree of matching, the data processor 33 identifies the compound.
In the mass spectrometer according to the present embodiment, the CE values are determined by the previously described characteristic procedure. This provides the following advantages, or technical meanings.
The spectrum pattern of a mass spectrum obtained by an MS/MS analysis depends on the CE value. Therefore, even when the CE-value number is different, a similar spectrum pattern of the cumulative mass spectrum will be obtained if the percentage of the common CE values is high. Therefore, for example, when the compound identification using a database of cumulative mass spectra is performed in the previously described manner, the determination on the identification based on the pattern matching can be performed with no significant problem even when the CE-value number used for the acquisition of the cumulative mass spectra recorded in the database is not identical to the CE-value number used for the acquisition of the cumulative mass spectrum by the actual measurement.
There is also the advantage that it is not always necessary to equalize the CE-value numbers when determining the identity, structural similarity or other aspects of a compound by comparing cumulative mass spectra separately obtained for a plurality of samples. This increases the flexibility in the setting of the analysis conditions.
On the other hand, the impact of the same amount of change in CE value (e.g., 1 V) on the form of the dissociation by the CID process changes depending on whether the CE value is relatively small or large. In general, a smaller CE value leads to a more noticeable change in the form of the dissociation of an ion for the same amount of change in CE value. Therefore, if the neighboring CE values are always set at regular intervals as shown in
By comparison, as is evident from
In the previous description of the present embodiment, when CE values are determined in the CE value determiner 321, mathematical calculations and conditional judgments using computational expressions are performed to derive CE values from the set conditions. It is also possible to prepare and save a lookup table based on the numerical values obtained by those calculations. In that case, the lookup table can be configured to have the set conditions (e.g., the CE-value number and the CE-value range) as the input and the resulting CE values as the output. Needless to say, the previously described procedure is a mere example of determining CE values by mathematical calculations and conditional judgments using computational expressions; any procedure which can yield similar results may be used.
Although the mass spectrometer according to the previous embodiment is a Q-TOF mass spectrometer, it is evident that the present invention can be applied to other types of tandem mass spectrometers capable of an MS/MS analysis, such as a triple quadrupole mass spectrometer.
The previously described embodiment and its modifications are also mere examples of the present invention, and any change, modification or addition appropriately made within the gist of the present invention will evidently fall within the scope of claims of the present application.
[Various Modes]
It is evident for a person skilled in the art that the previously described illustrative embodiment is a specific example of the following modes of the present invention.
(Clause 1) One mode of the method for mass spectrometry according to the present invention is a method for mass spectrometry using a mass spectrometer capable of an MS/MS analysis and provided with a measurement section which includes a collision cell configured to dissociate an ion and a mass separator configured to perform a mass spectrometric analysis of product ions resulting from dissociation, the method for mass spectrometry including the step of executing a collision energy spread method in which collision energy is changed into a plurality of levels and a mass spectrum obtained at each level of the collision energy is accumulated to obtain a cumulative mass spectrum, and the method for mass spectrometry including:
a CES-method-condition determination step for determining a plurality of collision-energy values to be used in the collision energy spread method, according to given conditions including the range of collision-energy values and the number of collision-energy values, in such a manner that n+1 collision-energy values to be used when the number is n+1 (where n is an integer equal to or greater than three) are determined so that the n+1 collision-energy values include n collision-energy values used when the number is n and one additional collision-energy value different from any one of the n collision-energy values; and
an analysis execution step for sequentially setting the collision energy at the n+1 collision-energy values determined in the CES-method-condition determination step and controlling the measurement section so as to execute an MS/MS analysis under each collision-energy value and thereby obtain a mass spectrum for each collision-energy value.
(Clause 5) One mode of the mass spectrometer according to the present invention is a mass spectrometer capable of an MS/MS analysis and provided with a measurement section which includes a collision cell configured to dissociate an ion and a mass separator configured to perform a mass spectrometric analysis of product ions resulting from dissociation, the mass spectrometer including:
a CES-method-condition determiner configured to determine a plurality of collision-energy values to be used in a collision energy spread method, according to given conditions including the range of collision-energy values and the number of collision-energy values, in such a manner that n+1 collision-energy values to be used when the number is n+1 (where n is an integer equal to or greater than three) are determined so that the n+1 collision-energy values include n collision-energy values used when the number is n and one additional collision-energy value different from any one of the n collision-energy values;
an analysis controller configured to sequentially change the collision energy to the n+1 collision-energy values determined in the CES-method-condition determiner and control the measurement section so as to execute an MS/MS analysis under each collision-energy value; and
a data processor configured to obtain a cumulative mass spectrum by accumulating mass spectra each of which is obtained under each of the different collision-energy values under the control of the analysis controller.
In the method for mass spectrometry according to Clause 1 and the mass spectrometer according to Clause 5, even when the number of CE values for performing the CES method is changed, the spectrum pattern of the cumulative mass spectrum undergoes only a small change since there is a considerable number of common CE values. Therefore, for example, the identification of compounds by pattern matching using a database of cumulative mass spectra (or similar methods) can be performed with a high level of accuracy. Additionally, the task of comparing cumulative mass spectra obtained for different samples will be easier.
(Clause 2) In the method for mass spectrometry according to Clause 1, the CES-method-condition determination step may include determining the one additional collision-energy value different from any one of the n collision-energy values in such a manner that two collision-energy values neighboring each other with a large interval of value in the sequence of the collision-energy values are preferentially selected as two collision-energy values between which a new collision-energy value is to be added.
(Clause 6) In the mass spectrometer according to Clause 5, the CES-method-condition determiner may determine the one additional collision-energy value different from any one of the n collision-energy values in such a manner that two collision-energy values neighboring each other with a large interval of value in the sequence of the collision-energy values are preferentially selected as two collision-energy values between which a new collision-energy value is to be added.
In the method for mass spectrometry according to Clause 2 and the mass spectrometer according to Clause 6, when a few or more collision-energy values are to be determined within a predetermined range of collision-energy values, the situation in which the interval between two neighboring collision-energy values becomes extremely wide can be avoided. This enables the acquisition of a cumulative mass spectrum in which the information of product ions generated by dissociation is satisfactorily reflected even when the dissociation of those product ions is particularly promoted at around a specific collision-energy value.
(Clause 3) In the method for mass spectrometry according to Clause 2, the CES-method-condition determination step may include adding a middle value of the two collision-energy values when adding a new collision-energy value between the two collision-energy values.
(Clause 7) In the mass spectrometer according to Clause 6, the CES-method-condition determiner may add a middle value of the two collision-energy values when adding a new collision-energy value between the two collision-energy values.
In the method for mass spectrometry according to Clause 3 and the mass spectrometer according to Clause 7, when a few or more collision-energy values are to be determined within a predetermined range of collision-energy values, the situation in which the collision-energy values are distributed in an extremely biased form within the aforementioned range can be avoided. This enables the acquisition of a cumulative mass spectrum in which the information of various product ions originating from a target compound is reflected with a satisfactory level of uniformity.
(Clause 4) In the method for mass spectrometry according to Clause 2 or 3, the CES-method-condition determination step may include adding the new collision-energy value in such a manner that, when there are a plurality of pairs of collision-energy values whose intervals are equal, a pair of relatively small energy values is preferentially selected as the two collision-energy values between which the new collision-energy value is to be added.
(Clause 8) In the mass spectrometer according to Clause 5 or 6, the CES-method-condition determiner may add the new collision-energy value in such a manner that, when there are a plurality of pairs of collision-energy values whose intervals are equal, a pair of relatively small energy values is preferentially selected as the two collision-energy values between which the new collision-energy value is to be added.
In the method for mass spectrometry according to Clause 4 and the mass spectrometer according to Clause 8, since the collision-energy values are preferentially allotted to a range of relatively small collision-energy values, the intervals of the neighboring collision-energy values within a range of small collision-energy values are likely to be narrower than those within a range of large collision-energy values. In general, the change in the form of the dissociation with a change in collision energy becomes more sensitive when the change in collision energy occurs within a range of smaller collision-energy values. Therefore, in the method for mass spectrometry according to Clause 4 and the mass spectrometer according to Clause 8, it is more likely that the information of various product ions is exhaustively obtained with a high level of sensitivity, which is favorable for the identification and structural analysis of a compound using a cumulative mass spectrum.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2022-015466 | Feb 2022 | JP | national |
