JPH08334493A - Liquid chromatograph mass spectroscope - Google Patents

Abstract

PURPOSE: To estimate molecular weights with good sensitivity and to precisely fix molecules by providing means for addition, estimation, judgment,, and processing, and judging the similarity between fluctuations in intensity in the time direction of a three-dimensional mass spectrum. CONSTITUTION: An addition means is used to set predetermined time widths in front of and behind a holding time during which a desired material appears, and three-dimensional mass spectra within the time widths are added together in time direction to produce a two-dimensional mass spectrum (S2). Next, an estimation means is used to extract (S3), from a number of intensity peaks in the two-dimensional mass spectrum, such a set of intensity peaks (peak set) as may be produced by polyvalent ions derived from a certain material. Then, using a judgment means and in consideration of the mass numbers of a plurality of intensity peaks, chromatograms of the time-relative intensity at each of the mass numbers are cut out (S4) from the three-dimensional mass spectra, and are compared (S5) to judge the similarity. Further, a processing means is used for estimating molecular weights from the peak set based on the result of judgment on the similarity and for fixing molecules (S7).

Description

Detailed Description of the Invention

[0001]

The present invention relates to a liquid chromatograph (L
The present invention relates to a liquid chromatograph / mass spectrometer that separates substances in a sample by C) and then analyzes the separated substances using mass spectrometry (MS), and particularly to a three-dimensional multiply charged ion analysis in the analyzer. Regarding improvement.

[0002]

2. Description of the Related Art A liquid chromatograph mass spectrometer is a liquid chromatograph (L) that separates substances contained in a liquid sample on a time axis by passing a liquid sample through a column.
C: Liquid Chromatography) and a mass spectrometer (MS) that ionizes the separated substance and separates it according to the mass number (mass / charge ratio: m / z) of the ion, and creates a mass spectrum according to the generated number. : Mass Spectrometer). FIG. 1 is a block diagram showing the basic configuration of the LC-MS analyzer. The LC unit 10 is an eluent tank 11,
It is composed of a pump 12, a sample injection unit 13, and a column 14, and the MS unit 20 includes a volatilization unit 21, an ionization unit 22, a mass separation unit 23, a detector 24, an amplifier 25, and a data processing unit 2.
6 and a recording unit 27.

The sample injection section 13 is supplied with the eluent sucked from the eluent tank 11 by the pump 12. The liquid sample injected into the sample injection unit 13 is guided into the column 14 by the flow of this eluent. The substances in the sample are separated as the eluent containing the sample passes through the column 14 filled with the separation material for fine particles, and the substances are retained in the column 14 for a retention time specific to each substance, and then the column 14 is removed. Is discharged. Each substance separated in the LC section 10 is separated into a volatilization section 21.
Is vaporized into a gas phase state and introduced into the ionization section 22 in a vapor state. In the ionization section 22, ions of the introduced substance are generated by, for example, electron impact. The charge of this ion is determined by the number of electrons that fly out of the molecule. That is, n-electrons (n-heavily-charged) ions are n-electrons protruding from the molecule, and this n is called valence.

In the mass separation unit 23, the ions generated in the ionization unit 22 are separated according to the mass number. As a method of separating the ions, various methods such as a method of focusing using a high-frequency quadrupole electric field and a method of separating ions having different mass numbers depending on the strength of the magnetic field are used. The detector 24 detects the number of generated ions separated for each mass number. The detection signal is amplified by the amplifier 25 and then input to the data processing unit 26. In the data processing unit 26,
A mass spectrum indicating the relationship between the number of generated ions, that is, the relative intensity and the mass number is created. Since this mass spectrum is measured at predetermined time intervals with respect to the sample supplied from the LC unit 10 to the MS unit 20, the final mass spectrum has a three-dimensional structure including the time axis, the mass number, and the relative intensity. It will be Then, the molecular weight of the molecule in the sample is estimated and the molecule is identified based on the three-dimensional mass spectrum, and the result is recorded by the recording unit 27.

In the analysis of multiply-charged ions, in particular, a plurality of multiply-charged ions having different valences are generated from one molecule in the ionization section 22, and the plurality of intensity peaks appearing on the mass spectrum by the multiply-charged ions. The molecular weight is estimated by analyzing.

As described above, a three-dimensional mass spectrum can be obtained by liquid chromatograph mass spectrometry, but in reality, the S / N ratio of the mass spectrum obtained by mass spectrometry is not so good. Therefore, in the data processing unit 26, the three-dimensional mass spectrum is integrated in the time direction within the range of the time in which one substance separated in the LC unit 10 is introduced into the MS unit 20 to be two-dimensional. It is generally done. As a result, the desired intensity peak becomes steep, and the peak position can be easily identified.

[0007]

When the substances contained in the sample are completely separated in the time direction by the liquid chromatograph, the S / N ratio can be improved by executing the two-dimensional processing as described above. Since a two-dimensional mass spectrum is obtained,
It is possible to accurately estimate the molecular weight and identify the substance. However, if the above-described two-dimensional processing is performed on a substance that could not be completely separated by liquid chromatography, the mass spectra of two or more substances overlap, making it difficult to accurately detect the peak position. .

This will be described below with a specific example. In the mass spectrum of the substance of molecular weight X, the mass number pi at which the intensity peak appears is represented by the following formula (1). pi = (X + i) / i (1) Here, i is the valence of the ion. That is, if the valence of the ion is different, the charge is different, so the mass number (mass / mass /
The charge ratio) is also different. Therefore, in the multiply charged ion analysis, a plurality of intensity peaks (hereinafter referred to as “peak set”) having different peak positions are obtained from one molecule. The molecular weight is estimated from the position of this peak set.

Now, it is assumed that there are two substances that can obtain the following results when they are individually measured. [Substance A] ・ Separation result by LC: Has a peak of time width of TW1 around time T1 ・ Molecular weight: 6700 ・ Valence measured: 5-9 ・ Mass number at which intensity peak occurs: 1341, 1117.67, 958.1
4, 838.5, 745.44 [Substance B] -Separation result by LC: Peak of TW2 time width around time T2-Molecular weight: 1117-Measured valence: 1-2-Mass number at which intensity peak occurs : 1118, 559.5

FIG. 4 shows a liquid chromatographic separation result, that is, a chromatogram when the sample is a mixture of the substance A and the substance B, and time T1 = 10 and time T2 = 15.5. Further, the three-dimensional mass spectrum at this time is shown in FIG. 5 in the range of mass numbers 1113.3333 to 1120 and time 0 to 29. Further, FIG. 6 shows a two-dimensional mass spectrum obtained by integrating the three-dimensional mass spectrum of FIG. 5 in the time direction in the time range of 0 to 20 to make it two-dimensional. As shown in FIG. 4, two substances A
When B and B are not completely separated, the position of the hexavalent intensity peak of substance A and the position of the monovalent intensity peak of substance B appear very close on the time-mass plane. (See FIG. 4). Therefore, when the three-dimensional mass spectrum is integrated in the time direction, the two peaks are fused and recognized as one peak as shown in FIG. At this time, the mass number of the peak is 1117.8, which is 6 for substance A.
Although the molecular weight is different from the mass number of the valence, the erroneous value is used as one of the peak sets to estimate the molecular weight, resulting in an error in the estimated molecular weight.

Further, when the original substance is identified based on the peak set of multiply charged ions in the mass spectrum, it is erroneously recognized as one of the peak sets of other substances due to the shift of the peak positions as described above. If it coincides with the position that is caused, that is, the position due to the multiply charged ion of another molecule, it may even be identified as an incorrect molecule.

The present invention has been made to solve such a problem, and its object is to accurately estimate the molecular weight even when the separation of a plurality of substances by liquid chromatography is poor. Another object of the present invention is to provide a liquid chromatograph / mass spectrometer capable of performing well and accurately identifying a molecule.

[0013]

DISCLOSURE OF THE INVENTION The present invention, which has been made to solve the above-mentioned problems, provides a liquid chromatograph mass spectrometer for separating a plurality of substances contained in a sample by a liquid chromatograph and then analyzing them by a mass spectrometer. And
In an analyzer for identifying the substance and estimating the molecular weight by three-dimensional multiply charged ion analysis, a) In the three-dimensional mass spectrum which is the mass analysis result,
Accumulating means for obtaining a two-dimensional mass spectrum by integrating the three-dimensional mass spectrum in the time direction in the vicinity of the retention time at which the target substance appears, and b) generating by the target substance in the two-dimensional mass spectrum. Estimating means for obtaining a plurality of intensity peaks that are estimated to have been performed, and c) focusing on the mass number in which the intensity peaks occur for each of the plurality of intensity peaks, and changing the intensity in the time direction of the three-dimensional mass spectrum. And a processing means for performing the identification or molecular weight estimation of the target substance from the plurality of intensity peaks based on the result of the determination means.

[0014]

In the three-dimensional multiply charged ion analysis in the liquid chromatograph mass spectrometer according to the present invention, first, the three-dimensional mass spectrum is obtained as the analysis result of the sample, and then the retention time at which the target substance appears by the integrating means. A predetermined time width is set before and after, and a two-dimensional mass spectrum is created by integrating the three-dimensional mass spectrum within the time width in the time direction. Then, the estimation means extracts a set of intensity peaks (peak set) that can be generated by multiply charged ions of a certain substance from the many intensity peaks in this two-dimensional mass spectrum.

Subsequently, in order to check the reliability of the positions of the plurality of intensity peaks constituting this peak set, the judging means pays attention to the mass numbers of the plurality of intensity peaks and pays attention to the respective mass numbers from the three-dimensional mass spectrum. The time-relative intensity chromatograms at are cut out and compared to determine the similarity. That is, by this, in the peak set, the intensity peak only by the target substance and the intensity peak affected by the other substance are distinguished. Furthermore,
The processing means estimates the molecular weight from the peak set and identifies the molecule based on the determination result of the similarity. At this time,
It is possible to exclude from the peak set those that are judged to have low peak position reliability due to poor chromatogram similarity, and the reliability of each intensity peak can be determined according to the chromatogram similarity. The molecular weight may be calculated after weighting.

【The invention's effect】

As a result, according to the present invention, even when the separation of each substance in the liquid chromatograph is not sufficient, the reliability of the peak set in the mass spectrum is improved, so that the molecular weight can be obtained with high accuracy. The substance can be identified accurately.

[0017]

Embodiments of the present invention will be described below with reference to the drawings. FIG. 2 is a flow chart showing a processing procedure of three-dimensional multiply charged ion analysis in the analyzer according to the present invention, and FIG. 3 is a diagram for explaining a method of judging the reliability of the peak position.

In the liquid chromatograph mass spectrometer according to the present invention, the operation of the data processing unit 26 for executing the data processing of multiply charged ion analysis is greatly different from the conventional one. According to the flow chart of FIG.
The process of the three-dimensional multiply charged ion analysis according to the present invention when the substance and the substance B are not completely separated will be sequentially described.

First, like the conventional three-dimensional multiply charged ion analysis, a three-dimensional mass spectrum is prepared (step S).
1). The mass spectrum at this time is as shown in FIG.
Then, from this three-dimensional mass number spectrum, a two-dimensional mass number spectrum around the time when the target substance appears is obtained (step S2). That is, when the target substance is the substance A, the relative intensities are integrated in the time direction from time t = 3 to time t = 20. As a result, a two-dimensional mass spectrum as shown in FIG. 6 is obtained. Of course, FIG. 6 shows a mass spectrum only near the peak of the hexavalent ion of the substance A.
Intensity peaks due to 5 to 9 valence ions excluding valence also appear at each position (mass number).

Next, a plurality of intensity peaks appearing in the two-dimensional mass spectrum are examined to obtain a peak set (step S3). That is, the combinations of positions and valences of all the intensity peaks appearing in the two-dimensional mass spectrum are sequentially calculated, and the peak set estimated to have the highest possibility of being generated by multiply charged ions of one substance. Is extracted. In this embodiment, the peak positions are 1341, 1117.8,
It is judged that the five intensity peaks of 958.14, 838.5, and 745.44 are highly likely to be a peak set by a certain substance.

Subsequently, in order to examine the reliability of each peak position of the peak set obtained as described above, first, the time dependence of the relative intensity at the mass number at which each intensity peak occurs is examined. That is, when each intensity peak constituting the peak set is generated by a certain substance, it is considered that the chromatograms in the time direction in the mass number of the intensity peak have the same variation. Therefore, first, from the three-dimensional mass spectrum, a chromatogram showing the relationship between relative intensity and time at the mass number at which each intensity peak occurs is cut out (step S
4). For example, the chromatogram shown by the solid line in FIG.
The relationship between the relative intensity at the mass number 1117.8 and the time is cut out from the three-dimensional mass spectrum of FIG. 5, and the chromatogram indicated by the broken line is the relationship between the relative intensity at the mass number 1341 and the time. .

Further, the relationship between the relative intensities of the mass numbers of the other intensity peaks in the same set of peaks and the time also shows a variation tendency similar to the chromatogram indicated by the broken line in FIG.
That is, since the intensity peak at the mass number 1117.8 is due to both the substance A and the substance B, its chromatogram naturally has a shape in which the chromatogram of the substance A and the chromatogram of the substance B are synthesized. on the other hand,
The chromatograms at the mass numbers of the other intensity peaks have a relatively monotonous shape due to the substance A alone, because there is no influence of the substance B. Therefore, by judging the similarity or difference of this chromatogram, it is possible to identify whether or not each intensity peak is due to the ion of a single substance (step S5).

As a concrete method of judging the similarity of chromatograms, for example, the variation amount (the slope of the chromatogram at each time) is calculated by differentiating each chromatogram at every predetermined time interval, and the result is calculated. Similarity can be obtained by comparing. Also, other methods are possible.

Then, the reliability of each intensity peak is judged from the judgment result of the similarity of the chromatogram (step S
6) The molecular weight is estimated in consideration of this reliability (step S7). Various methods can be used to determine the reliability of each intensity peak from the similarity of the chromatograms. For example, the similarity is judged based on whether it is similar or not, and the intensity peak determined not to be similar is determined to be the intensity peak affected by another substance other than the target substance. However, it should be excluded when estimating the molecular weight. In the above example, it is determined that there is no similarity only in the chromatograms at the hexavalent peak positions, and the molecular weight is estimated from the four peak positions at 5, 7, 8, and 9 valences.

It is also possible to judge the similarity in multiple stages, weight the reliability of each intensity peak according to the similarity, and estimate the molecular weight using this reliability. For example, in the above example, only the chromatogram at the hexavalent peak position has low similarity, so the reliability is set to 0.5, and the reliability at other peak positions is set to 1.0. Generally, the molecular weight X can be calculated from the equation (1) as follows: X = i · Pi−i (2), but in this case, the molecular weight of the substance A is calculated by the following equation (3) considering reliability. It is calculated. X = {Σ (i = 5 to 9) [i · Pi−i] · Ri} / Σ (i = 5 to 9) Ri (3) where Ri is the reliability of the peak position due to the i-valent ion. Then, Ri = 0 to 1 is set. According to this, the reliability of the peak position is reflected in the estimation of the molecular weight.

Further, when not only estimating the molecular weight but also identifying the substance from the peak set, by excluding those having low peak position reliability as described above, an incorrect peak set is constructed. Since it will not occur, the accuracy of substance identification will be increased.

[Brief description of drawings]

FIG. 1 is a block configuration diagram of a liquid chromatograph mass spectrometer.

FIG. 2 is a flowchart showing a processing procedure of three-dimensional multiply charged ion analysis in the liquid chromatograph mass spectrometer according to the present invention.

FIG. 3 is a diagram for explaining a method of determining reliability of a peak position.

FIG. 4 is a diagram illustrating a chromatogram when substances are not separated in a liquid chromatograph.

FIG. 5 is a diagram illustrating a three-dimensional mass spectrum when substances are not separated by liquid chromatography.

FIG. 6 is a diagram showing a two-dimensional mass spectrum when the three-dimensional mass spectrum of FIG. 5 is two-dimensionally processed.

[Explanation of symbols]

 10 ... LC section 13 ... Sample injection section 14 ... Column 20 ... MS section 21 ... Volatilization section 22 ... Ionization section 23 ... Mass separation section 24 ... Detector 26 ... Data processing section

Claims (1)

[Claims] 1. A liquid chromatograph mass spectrometer which separates a plurality of substances contained in a sample by a liquid chromatograph and then analyzes them by a mass spectrometer, wherein the substances are identified and molecular weight is estimated by three-dimensional polyvalent ion analysis. A) in a three-dimensional mass spectrum that is the result of mass spectrometry,
Accumulating means for obtaining a two-dimensional mass spectrum by integrating the three-dimensional mass spectrum in the time direction in the vicinity of the retention time at which the target substance appears, and b) generating by the target substance in the two-dimensional mass spectrum. Estimating means for obtaining a plurality of intensity peaks that are estimated to have been performed, and c) focusing on the mass number in which the intensity peaks occur for each of the plurality of intensity peaks, and changing the intensity in the time direction of the three-dimensional mass spectrum. And a processing means for performing the identification or molecular weight estimation of the target substance from the plurality of intensity peaks based on the result of the determination means. Graph mass spectrometer.