Chromatograph mass spectrometer

Abstract

An exact centroid spectrum with a mass number corrected is determined from a profile spectrum adjacent to a plurality of peaks. Regarding a profile spectrum determined by a mass spectrometer, overlapping with adjacent peaks occurs, and compounds having a plurality of peaks with different overlapping degrees is measured, a correction function is created from a relationship between an overlapping degrees with respect to the plurality of peaks and a shift of the mass number, and a centroid peak is corrected by the correction function when the profile spectrum is converted into the centroid spectrum.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings:

FIG. 1 is a structural diagram of a liquid chromatograph mass spectrometer using an HPLC;

FIG. 2 is a structural diagram of an ion trap mass analysis portion;

FIG. 3 is a graph illustrating a an example of an MS profile spectrum;

FIG. 4A illustrates an example of the MS profile spectrum in an APCI mode;

FIG. 4B illustrates an example of the MS profile spectrum in an ESI mode;

FIG. 5 illustrates the MS profile spectrum and the centroid spectrum;

FIGS. 6A and 6B are graphs each illustrating respective parameters for creating a correction function of correcting a shift of the centroid spectrum;

FIG. 7 illustrates s an example of a correlation obtained at a peak of a sample for creating a correction function;

FIG. 8 illustrates an example of a DDA condition setting screen;

FIG. 9 illustrates a flowchart of apparatus adjustment processing; and

FIG. 10 illustrates a flowchart of measurement processing.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

Hereinafter, operation of the present invention in a case of performing MS/MS measurement using DDA will be described with reference to the drawings. FIG. 1 illustrates an entire configuration of an IT-TOF, and FIG. 2 illustrates an exemplary configuration of an ion trap portion 11. A chemical compound eluted from a column 4 of an LC is guided to an MS portion 5 via a flow path switching valve 18. The MS portion 5 includes an atomizing chamber 7 in which an ion spray portion 6 is provided, and an ion analysis chamber 10 in which the ion trap portion 11, an ion flight electrode 12, and an ion detecting unit 14 are provided, and two ion introducing chambers 9 are provided between the atomizing chamber 7 and the analysis chamber 10. The atomizing chamber 7 and an ion introducing chamber 15 in one stage are communicated with each other through a desolvating tube 8. A detection signal of the ion detecting unit 14 of the MS portion 5 is input to a signal processing portion 15, and is processed by the signal processing portion 15 as described later to be given to a parameter input/data display portion 17 as chromatogram data. The control unit 16 controls the operation in each part of the MS portion 5.

The operation of the MS portion 5 is as follows. When the chemical compound eluted from the column 4 reaches the ion spray portion 6, the compound is sprayed in the atomizing chamber 7 as liquid droplets charged with a high voltage applied to the ion spray portion. The flown liquid droplets strike gas molecules in the atmosphere, further are crushed into fine liquid droplets and dried rapidly (desolvated). As a result, molecules are vaporized. The gas fine particles effect an ion evaporation reaction to be ionized. The fine liquid droplets containing the generated ions jump into the desolvating tube 8, and desolvation further proceeds while the fine liquid droplets pass through the desolvating tube 8. The ions are sent to the ion analysis chamber 10 through the two ion introducing chambers 9. The ions are once accumulated in the ion trap portion 11 provided in the ion analysis chamber 10, and thereafter, are discharged to the ion flight electrode portion 12. In the ion analysis chamber 10, a voltage applied to electrodes constituting the ion trap portion 11 is changed. As a result, the MS measurement, MS/MS measurement, MS/MS/MS measurement, and the like can be conducted. During the MS measurement, first, in order to accumulate the ions in the ion trap portion, an inlet end cap electrode 21 is supplied with a potential of negative several V and an outlet end cap electrode 23 is supplied with a potential of positive several V (in a case where the ions are positive). As a result, the ions are confined. At a time when the ions enter the ion trap, a high frequency potential is applied to a ring electrode 22, and the confined ions are collected in a center portion of the ion trap electrode with gas introduced from a cooling gas introducing portion 24 and a high-frequency potential applied to the ring electrode 22 (referred to as cooling). After that, the high-frequency potential of the ring electrode 22 is turned off, and a potential of tens of KV is applied to the inlet end gap electrode 21 and a potential of the ion flight electrode portion 12 provided in the latter stage is applied to the outlet end cap electrode 23. As a result, the ions are discharged from the ion trap portion 11.

In the ion flight electrode portion 12, the ions fly in a drift space in accordance with the conservative law of energy with a voltage applied to the ion flight electrode portion 12. In the course of flight, the ions are pushed back again to the ion flight electrode portion 12 by a reflectron electrode 13 provided on an opposite side of the ion trap portion 11, and reach the ion detecting unit 14. Regarding the time required for the ions to reach the ion detecting unit 14, the ions with a smaller (lighter) m/z value reach the ion detecting unit 14 faster. Therefore, the time required for the ions to be discharged from the ion trap portion 11 and reach the ion detecting unit 14 is measured, the time information is converted into mass number information in a signal processing portion 15a of an operation portion 15, and a current in accordance with the number of ions having reached is taken out in the ion detecting unit 14.

Before an actual measurement operation is started, an apparatus is adjusted. For adjusting the apparatus, a standard sample filling a standard sample liquid tank 20 is used. The standard sample is a combination of a sample for calibrating a mass number (sample in which overlapping with an adjacent peak does not occur in a profile spectrum. For example, sodium acetate trifluoride) and a sample for obtaining a correction function (sample in which overlapping with an adjacent peak occurs in a profile spectrum, and a plurality of profile spectra having different degrees of overlapping can be measured. For example, myoglobin).

The operation of adjusting a mass spectrometer will be described with reference to a flowchart shown in FIG. 9. After the flow path switching valve 18 is switched so as to feed a standard sample from the standard sample liquid tank 20 to the MS portion 5, a standard sample feed pump 19 is operated. As a result, the standard sample in the standard sample liquid tank 20 is introduced to the MS portion 5 (S101). In this state, the control values of each electrode of the ion introducing chamber 9 and the ion trap portion 11, and the reflectron electrode 13 are optimized so that the detection sensitivity becomes maximum in the MS portion 5 (S102). After that, the subsequent processes S103 to S105 are repeated by the number of peaks of a mass number calibration sample (mass number calibration processing).

An MS profile spectrum in the vicinity of the calibration mass number of the mass number calibration sample is measured (S103).

The determined MS profile is converted into a centroid spectrum in a conversion processing portion 15b (S104).

A peak corresponding to a calibration mass number is searched for from a list of peaks in the determined centroid spectrum, and the flight time of the peak is stored in a storage unit 26 (S105).

Due to the mass number calibration processing, Table 1 showing a relationship between the calibration mass number of the mass number calibration sample and the flight time is created, and a relationship between the known mass number and the measured flight time can be obtained.

TABLE 1
Mass number Flight time
158.96458   22952.15891
566.88900   43262.79403
838.83862   52606.90998
1246.76305  64115.19855

Based on Table 1, a relational expression between a flight time and a mass number is created (S106).

Flight time (t)=g(Square root of mass number (m/z))  (1)

In measurement, the flight time of the centroid peak is converted into a mass number, using an inverse function expression (2) of Expression (1).

Square root of mass number (m/z)=g′(flight time (t))  (2)

Further, processes S107 to S110 are repeated by the number of peaks of the mass number correction sample contained in the standard sample (mass number correction processing).

An MS profile spectrum in the vicinity of the correction mass number of the mass number correction sample is conducted (S107).

The determined MS profile spectrum is subjected to the centroid conversion of a peak of a sample for creating a correction function, and the flight time of the centroid peak is converted into a mass number by Expression (2) (S108).

The degree of overlapping with respect to adjacent peaks is determined (S109).

Overlapping degree of peak=Overlapping intensity/peak intensity  (3)

The difference between the mass number of the determined centroid peak and the true mass number of the peak is determined (S110).

Shift from true value=Mass number of centroid peak−True mass number of peak  (4)

The mass number correction processing is conducted by the number of peaks of the sample for creating a correction function. As a result, the figure as shown in FIG. 7 is obtained. This shows that the relation between the overlapping degree and the shift from a true value is substantially a quadric function. Herein, if the overlapping degree is 0, there is no shift from the true value of a centroid peak, and the value of the shift becomes 0.

A correlation function (Expression 5) between the overlapping degree of peaks and the shift from a true value is created. MS measurement processing is conducted using a sample with a known mass number in which peaks of a profile spectrum overlap each other as shown in FIG. 6B. As a result, the difference between the true mass number and the mass number of a centroid spectrum, and the overlapping degree at that time (ratio between the intensity of a portion to be a valley in a profile spectrum and the intensity of a peak top) are determined. Regarding a plurality of peaks having different overlapping degrees, the data thereof are measured, and a correction function (5) is created using a plurality of pieces of information [overlapping degree and shift of a mass number) (S111).

Shift of mass number=f(Overlapping degree)  (5)

As the overlapping degree of a target peak, the overlapping degree in a rising portion of a peak and the overlapping degree in a falling portion of the peak are determined simultaneously, and finally, Expression (6) for correcting the centroid peak position of each peak is created, and stored in the storage unit 26.

Centroid peak position=Centroid peak position as in conventional example+f(Overlapping degree in a rising portion)−f(Overlapping degree in a falling portion)  (6)

Herein, the overlapping degree in a rising portion is corrected in a +(plus) direction, and the overlapping degree in a falling portion is corrected in a −(minus) direction. Therefore, even in a case of expressing a correction function by a third or more order function, the correction function does not take a negative value.

Thus, the adjustment processing of the apparatus is completed. Next, an actual measurement operation is conducted. The actual measurement operation will be described with reference to the flowchart shown in FIG. 10. For an actual measurement, measurement completion conditions, a measurement mass range in an MS spectrum measurement (hereinafter, referred to as “MS measurement conditions”), precursor ion selection conditions for measuring an MS/MS spectrum, and measurement conditions of the MS/MS spectrum measurement mass range (referred to as “DDA conditions”) are created by the parameter input/data display portion 17. The created MS measurement condition and DDA condition are stored in the storage unit.

FIG. 8 illustrates a screen of setting MS measurement conditions and DDA conditions. The m/z range of the MS measurement mass number is set to be 100.0000-1000.0000, and a tolerance value is set to be 0.050 regarding the determined m/z value. The conditions are as follows: an event execution trigger performs an MS/MS measurement when ions matched with the DDA conditions are found by the MS measurement in either mode of a total ion chromatogram (TIC) and a base peak chromatogram (BPC) during a period from a time when the signal intensity exceeds 10000 after the peak commencement of a chromatogram to a time when the signal intensity becomes less than 9000 before the peak completion, i.e., in a time band during which a component is separated in a time direction in the liquid chromatograph portion and eluted in a concentration to some degree. While the conditions are not satisfied, the MS/MS measurement is not conducted. The selection of a precursor ion is an item for performing an MS measurement and setting the n/z range of a precursor ion for performing an MS/MS measurement. A charge number filter appropriately sets which valence of ions are calculated in accordance with the kind of ionization and an object to be measured. In the monoisotopic item, it is determined whether or not only a monoisotopic mass is only targeted. The MSn conditions are used for setting the conditions for selecting only ions with a particular mass number and cleaving the selected ions.

Measurement processing is started from a time when the mixture of the compounds is introduced from the injection portion 3 (S201). Measurement execution means first performs the first MS spectrum measurement in accordance with the MS measurement conditions (S202). Then, for the MS/MS spectrum measurement, the determined MS profile spectrum is converted into a centroid spectrum (S203), and the overlapping degree is determined in rising and falling portions of a peak (S204). Then, using the correction function (Expression (6)) determined by the previous adjustment processing, the position correction processing of the centroid peak is performed in the correction processing portion 15c, and the determined results are set to be the mass number of a target peak (S205).

When the centroid conversion processing is completed over the entire region of the MS profile spectrum determined by the first MS measurement, a determination processing portion 15d determines whether or not the event trigger conditions of the DDA conditions in which the centroid spectrum determined by the conversion processing is set are satisfied with reference to the conditions stored in the storage unit 26. As the result of the determination, when the conditions are not satisfied, the measurement of an MS spectrum (S202) to the position correction processing (S205) of the centroid peak are repeated without performing the MS/MS measurement.

In a case where the event trigger conditions are satisfied, in order to search for the peak specified under the DDA conditions, charge number determination processing of the determined centroid peak is performed (S206). Although there are various methods for the charge number determination processing, the processing by any method may be conducted. The peaks on the centroid data are specified successively as standard peaks for identifying isotopes in a decreasing order of intensity, and emerging patterns of peaks arranged before and after the standard peak are compared with an emerging pattern of an isotope cluster predicted in a case where each charge number is assumed to perform processing of detecting an isotope cluster (invention of JP 2005-141845). As a result, charge number determination processing can be performed at a high speed.

A precursor ion matched with the precursor ion selection conditions is searched for with a centroid spectrum subjected to charge number determination processing (S207). In a case where the precursor ion matched with the conditions is found, an MS/MS spectrum measurement is performed. In a case where a precursor ion matched with the conditions is not found, the MS spectrum measurement is conducted again (S202) without conducting the MS/MS measurement. Such measurement processing is repeated until the measurement completion conditions are matched.

Due to the measurement operation, the LC/MS/MS measurement can be performed regarding a intended precursor ion, and a corrected true value can be determined regarding the mass number of the determined centroid spectrum.

Thus, the present invention has been described by way of an example of the liquid chromatograph mass spectrometer. However, the present invention is also applicable to the correction of a centroid peak position in the processing in which another separation apparatus is connected to a mass spectrometer. The above-mentioned example is merely an example of the present invention, and it is apparent that modifications or alterations are included in the present invention in the scope of the spirit of the present invention.

Claims

1. A chromatograph mass spectrometer, comprising: analysis execution means for obtaining a profile spectrum in a mass range based on a setting condition by one mass scanning;conversion means for converting the profile spectrum into a centroid spectrum;precursor ion selection means for setting an ion of a peak of the centroid spectrum matched with the setting condition to be a precursor ion;

2. A chromatograph mass analysis method, comprising: executing analysis of obtaining a profile spectrum in a mass range based on a setting condition by one mass scanning;converting the profile spectrum into a centroid spectrum;selecting an ion of a peak of the centroid spectrum matched with the setting condition as a precursor ion; andperforming mass scanning with the analysis execution means regarding the precursor ion,wherein a known calibrate sample in which overlapping between a compound with a known mass number and an adjacent peak occurs and which has a plurality of peaks having different overlapping degrees,a correction function is created from a relationship between an overlapping degree with respect to the plurality of peaks and a shift of a mass number, andthe centroid peak is corrected with the correction function when the profile spectrum is converted into the centroid spectrum.