JP4491299B2 - Mass spectrometer - Google Patents

Description

  The present invention relates to a separation / analysis system for a sample containing many kinds of components, and more particularly to a liquid chromatograph / mass spectrometer and an analysis method used for analyzing biologically relevant substances such as proteins, peptides, sugar chains, and metabolites.

  In recent years, proteome analysis, glycome analysis, and metabolome analysis that comprehensively analyze biological substances such as proteins, sugar chains, and metabolites contained in biological tissues and body fluids have been effective mainly in the fields of biotechnology, drug discovery, and food It is becoming recognized as a strategy. For these analyses, development of high-throughput analysis technology mainly using mass spectrometry is regarded as important. Such an analysis is characterized in that the sample contains a large number of unknown substances and their amounts (expression amount) may differ by as much as 10 digits (for example, (Non-patent Document 1)). ). That is, it is required to accurately identify or quantify a trace substance contained in a sample in which many contaminating components are mixed.

In the high-throughput analysis, a liquid chromatograph / tandem mass spectrometer (LC / MS ⁿ ) is often used. In the liquid chromatograph (LC), a liquid sample is separated, and the separated substance is converted into gaseous ions at the interface and introduced into a tandem mass spectrometer (MS ⁿ ) to determine the mass of ions and the mass of dissociated ions. From the viewpoint of high-sensitivity analysis, it is known that reduction of the liquid flow rate is suitable for high-efficiency ionization in the ionization of a liquid sample at the interface with the mass spectrometer. On the other hand, in a liquid chromatograph that performs multi-component analysis, a high-separation is required in order to handle a liquid sample containing many kinds of substances. For this reason, it is considered effective to use nano LC that achieves high separation at extremely low flow rates.

  In actual analysis, even if separation is performed with nano LC, which achieves high separation, many types of substances are often detected simultaneously by a mass spectrometer. In tandem mass spectrometry, data-dependent analysis in which tandem mass spectrometry is performed in order from a substance having a high ionic strength is often used. However, since the number of types of substances that can be subjected to tandem mass spectrometry with a mass spectrometer is limited to several types, substances that could not be tandem mass analyzed are left unanalyzed. In a typical data-dependent analysis, tandem mass spectrometry is performed on about four types of ions in descending order of peak intensity. On the other hand, it is also possible to create a list by inputting the masses of ions that do not need to be subjected to tandem mass spectrometry, and to exclude the ions described in the list from the objects of dandem mass spectrometry (for example, (Non-patent Document 2)). . In this case, the mass of the detected ion is first determined by performing a normal mass analysis, and the ions not included in the tandem mass analysis, that is, the ions listed are excluded from the tandem mass analysis targets. Then, tandem mass spectrometry is preferentially performed on other detected ions.

An analysis example by LC / MS using a separation column having a length of about 80 cm at a flow rate of 20 nanoL (liter) / min has been reported (for example, (Non-patent Document 3)). In this example, as many as about 400 peptides have been identified in a single LC / MS ² analysis. Furthermore, the analysis of the same sample is repeated, and the total number of identified peptides is gradually increased. This is because the order of the ionic strength is slightly changed for each analysis, and ions (substances) to be newly subjected to tandem mass spectrometry are generated. By repeating the LC / MS ² analysis as many as about 30 times, about 1400 kinds of peptides have been identified.

  Further, a technique for storing mass information of ions subjected to tandem mass spectrometry in a mass spectrometer as an internal database has been reported (for example, (Patent Document 1)). In this example, non-analytical ions such as contaminant components registered in the internal database are excluded from the analysis target by the high-frequency electric field generated by the mass spectrometer. Further, in order to preferentially perform tandem mass analysis on the analysis candidate substance registered in the internal database, a high-frequency electric field for eliminating other ions is generated.

  For the three-dimensional mass spectrum obtained with the liquid chromatograph mass spectrometer, set the time width before and after the retention time when the target substance appears, cut out the three-dimensional mass spectrum within the time width, and determine the intensity peak due to the target substance. It has been reported that the intensity peaks affected by other substances are distinguished from each other, and the intensity peaks affected by substances other than the target substance are excluded when the molecular weight is estimated (for example, (Patent Document 2)). ). This technique determines the exact mass of ions of the separation material after the analysis is complete.

In addition, a technology that performs sample analysis and internal standard compound analysis, finds a correction curve that corrects the retention time using the internal standard compound, calculates the calculated retention time of the quantitative compound from the correction curve, and assigns a specific peak Has been reported (for example, (Patent Document 3)).
Also, in the liquid chromatograph mass spectrometer (LC / MS), when the signal intensity at each time point during peak detection exceeds the intensity corresponding to the upper limit of the dynamic range of the mass spectrometer, detection from the mass spectrometer There is a report of a technique for controlling a mass spectrometer in order to avoid generation of a saturated portion per signal (for example, (Patent Document 4)).

JP 2004-71420 A

JP-A-8-334493 JP-A-11-344482 JP2002-181784 Molecular & Cellular Proteomics (2002) p. 845-867 (Molecular and Cellular Proteomics 2002, 845 to 867) “Product Support Bulletin”, [online], Thermofinigan, Inc. [Search May 28, 2004], Internet <URL: www.thermo.com/eThermo/CMA/PDFs/Articles/articlesFile_10918.PDF> Analytical Chemistry Vol. 76 (2004) p. 1134-1144 (Analytical Chemistry, Vol. 76, 2004, Paragraphs 1134 to 1144)

A sample extracted from a living body (crude sample) may contain a great variety of substances, and the amount (concentration) of each substance may vary by several orders of magnitude. In such a sample analysis, it is difficult to analyze a trace component contained in the sample.
In conventional data-dependent analysis, a substance with a high detection signal amount is preferentially subjected to tandem mass spectrometry. Therefore, there is a problem that even if the method described in (Non-Patent Document 3) is used, it is difficult to analyze a substance with a very small amount of detection signal that is not subject to data dependence analysis.

Furthermore, in the method described in (Non-patent Document 3), it is necessary to repeat the analysis many times, and there is a problem that much labor and time are required. When creating a list obtained by inputting masses of ions that do not need to be subjected to tandem mass spectrometry as described in (Non-patent Document 2), it takes a lot of labor to input, so it is not realistic.
An object of the present invention is to provide a mass spectrometer that quickly analyzes a very small amount of components in analysis of a sample containing many kinds of components such as a sample extracted from a living body.

  As a means to solve the above problems, it has an internal database storing ion mass information and retention time, and identifies a sample separated by a liquid chromatograph, or identifies non-analyzed ions and eliminates them with a high-frequency electric field, Provided is a mass spectrometer that performs optimization of mass spectrometry or dandem mass spectrometry during analysis time (real time).

  A mass spectrometer according to the present invention includes a database for storing mass and retention time information of a plurality of substances, a chromatograph for separating a sample, an ion source for ionizing the separated sample, and a sample ionized by the ion source. Mass analysis unit for mass analysis, detection unit for detecting the analysis result of the mass analysis unit, retention time measurement unit for measuring the retention time of the sample from the detection result of the detection unit, and retention time measured by the retention time measurement unit And an information processing unit that performs a comparison process with the retention time information stored in the database, and a database control unit that corrects the retention time information based on the result of the comparison process of the information processing unit. Is characterized in that the mass spectrometry is controlled based on the result of the comparison process.

  The mass of the sample and the retention time information may be predetermined mass and retention time information of the analysis target substance, or may be predetermined mass and retention time information of the non-analysis target substance. The information processing unit may identify the separated sample based on the information on the mass of the ionized sample input from the mass analyzing unit and the corrected holding time information. The information processing unit further includes a high frequency power supply, and the information processing unit issues a high frequency application command to the high frequency power supply based on the mass information of the ionized sample input from the mass analyzing unit and the corrected holding time information. You may send it.

  According to the present invention, when a sample mixed with many kinds of substances, such as a sample extracted from a living body, is analyzed quickly, a substance with a small amount of detection signal that is not subject to data dependence analysis is reliably analyzed. be able to.

An example in which an ion trap is used in the mass spectrometer is shown in FIG. It is known that ion traps have an upper limit on the number of ions that can be trapped at one time due to space charge effects. However, in the very abundant ions A ⁺ and trace ions B ⁺ are analysis of a sample mixed, too low a number of trace ions B ⁺ are trapped, is as shown in (a), B ⁺ detection It can be difficult. However, as shown in (b), if a high-frequency electric field that eliminates a very large amount of ion A ⁺ is applied at the time of trapping or before the trap, it is possible to preferentially trap trace ions B ^+. It is. As a result, it is possible to concentrate a trace amount of ion B ⁺ and to perform a highly sensitive analysis. In this way, eliminating very large amounts of ions with a high-frequency electric field is effective for analyzing trace ions, even in mass spectrometers using linear ion traps, quadrupoles, or other multipole filters. It is. In general, if space charge effects can be involved, ion analysis is adversely affected.

Therefore, if the mass information of ions is stored as an internal database and non-analyzed ions are excluded by a high-frequency electric field and real-time optimization of tandem mass spectrometry is performed, the internal database can be created and updated automatically, and trace components can be automatically updated. Effective for high sensitivity analysis.
The method of eliminating ions having a very high abundance ratio with a high-frequency electric field as described above is extremely effective when the types of substances contained in the sample are limited. However, when the number of types of substances contained in the sample is enormous, there is a possibility that ions having a mass similar to that of ions not to be analyzed are contained in the sample. That is, it is often found in a crude sample that different materials are contained in the sample while having the same mass. Then, since these ions are excluded without being distinguished, there may be a problem that the analysis is hindered. For this reason, the accuracy of the mass of ions alone is insufficient to specify the substance.

  Therefore, not only ion mass information but also retention time information is included in the internal database and used for ion identification to improve ion identification accuracy. In this specification, the retention time is the time during which a sample is retained for analysis, and mainly refers to the time from the start of analysis by the liquid chromatograph mass spectrometer to the detection of ions. However, the time from when the separated sample passes through the liquid chromatograph until it is ionized and detected as ions is about milliseconds, and the width of one detection peak (band) derived from one separated sample is 10 Considering that it is about 2 seconds, the time from the start of analysis by the liquid chromatograph mass spectrometer to the ion detection and the time from the start to the detection of the separated sample at the end of the liquid chromatograph are substantially Can also be identified. Therefore, the retention time may be the retention time in the liquid chromatograph, that is, the time from the start of analysis until the detection of the separated sample at the end of the liquid chromatograph by UV detection or the like.

  When not only ion mass information but also retention time information is used for ion identification, it is necessary to pay attention to a shift in retention time information. In general, it is considered that when the column deteriorates, the holding time shifts. Furthermore, when the analysis conditions (gradient conditions and separation columns) are changed, the existing internal database may not be used. And restructuring the database under new analysis conditions requires a lot of effort and time from the user. In addition, analyzing a standard sample and correcting an existing internal database has a great burden on the user and it is difficult to guarantee the accuracy of the retention time information. This is because the shift in the retention time information may be different for each analysis. In addition, although the shift | offset | difference of holding time changes with the flow rates etc. of a liquid chromatograph, the shift | offset | difference of about 10 minutes may arise.

  The present invention provides a mass spectrometer that quickly analyzes a trace amount component in an analysis of a sample including many kinds of components such as a sample extracted from a living body, and further can correct an internal database according to the analysis. An object is to provide a mass spectrometer. Examples according to the present invention will be described below.

  FIG. 1 shows a system configuration diagram according to an embodiment of the present invention. This system includes a database for an analysis target substance (ion) or a database for a non-analysis target substance (ion), which stores information such as ion mass and retention time information. As a database used for reliably detecting ions to be analyzed, there is information on analysis candidate ions, a first database prepared in advance, information on ions that are not analysis candidates, and a second prepared in advance. The database is mainly used.

  A sample in which a plurality of substances are mixed is separated by a chromatograph, and the separated sample is introduced into an ion source. Next, the substance derived from the separated sample converted into gaseous ions by the ion source is introduced into the mass analysis unit, subjected to mass spectrometry or tandem mass analysis, and detected by the detection unit. The output of the detection unit is introduced into the holding time measuring unit, and the holding time is measured. Further, the output of the detection unit and the output of the retention time measurement unit are introduced into the information processing unit, and the ion mass information and the retention time information are integrated and compared with the database information.

  As shown in FIG. 15, in the analysis, the information processing unit detects a difference between the holding time information stored in the database and the actual holding time, and corrects the information stored in the database while performing the analysis. be able to. Here, the actual holding time is determined as the time from the start of analysis of the liquid chromatograph mass spectrometer to the time when the ion intensity becomes maximum by examining the time change (chromatogram) of the detected ion intensity. In addition, the deviation is detected every time ions are detected.

  For example, when ion 1 is detected by the mass analyzer, ion detection time (ion detection information) is transmitted to the retention time measurement unit, and ion mass information and retention time are processed from the mass analysis unit and the retention time measurement unit, respectively. Sent to the department. Next, the information processing unit searches the database for detected ion information (mainly requests for retention time information in the database), receives transmission of retention time information from the database, and calculates a shift in retention time. To do. If the holding time shift is larger than a certain value, it is determined that the shift has been detected, and the storage time correction information is requested to be recorded in the database. The database control unit updates the retention time correction information by recording it in the database.

And a control part requests | requires the holding time information after correction | amendment about the ion i (namely, ion detected by the detection part after the ion 1) from a database, and uses the holding time information after the said correction | amendment, and ion i And whether or not the ion i is an analysis target is determined. In addition, when performing the dandem mass analysis such as the data dependence analysis, the control unit determines the analysis order of the analysis target ions, and issues a dandem mass analysis command to the mass analysis unit based on the determination.
By the above flow, it becomes possible to identify ions not only by ion mass information but also by retention time information, and by improving the accuracy of ion identification, the accuracy of mass spectrometry can be increased.

  FIG. 2 shows a system configuration diagram according to another embodiment of the present invention. This system includes a high frequency power supply in addition to the system shown in FIG. 1, and based on ion information (such as mass and retention time) listed and stored in a database, a specific mass detected at a specific retention time ( In order to exclude non-analyzed ions having mass (or mass / charge number) in the mass spectrometer or other than analysis candidate ions having specific mass (or mass / charge number) detected at a specific retention time In order to exclude non-target ions by the mass spectrometer, a high frequency voltage is applied to the mass analyzer from a high frequency power source. The following are typical methods for removing ions by a high-frequency electric field generated for ions trapped three-dimensionally or two-dimensionally by a quadrupole electric field. When excluded ions are identified, a method of applying a high-frequency electric field that resonates with those ions is convenient. Due to the resonant high frequency electric field, the ions are heated and excluded (from the ion trajectory). When the mass range of excluded ions is specified, a method of scanning a resonance electric field, a method of applying a resonance electric field to ions of various mass / charge (m / z) at random, a quadrupole electric field Can be used to shift to an unstable region where only excluded ions cannot be trapped (low mass cut-off). Which method is used depends on m / z of ions to be analyzed and ions to be analyzed. With this high-frequency voltage, ions not to be analyzed are excluded by the mass analyzer, and other analysis candidate ions and the like are analyzed.

  As shown in FIG. 16, in the analysis, the information processing unit detects a deviation between the holding time information stored in the database and the actual holding time, and corrects the ion information stored in the database while performing the analysis. can do. Here, the actual holding time is determined as the time from the start of analysis of the liquid chromatograph mass spectrometer to the time when the ion intensity becomes maximum by examining the time change (chromatogram) of the detected ion intensity. In addition, the deviation is detected every time ions are detected.

  For example, when ions are detected by the mass analyzer, the ion detection time (ion detection information) is transmitted to the retention time measurement unit, and the ion mass information and the retention time are respectively transmitted from the mass analysis unit and the retention time measurement unit to the information processing unit. Sent. Next, the information processing unit searches the database for detected ion information (mainly requests for retention time information in the database), receives transmission of retention time information from the database, and calculates a shift in retention time. To do. If the holding time shift is larger than a certain value, it is determined that the shift has been detected, and the storage time correction information is requested to be recorded in the database. The database control unit updates the retention time correction information by recording it in the database. Then, the control unit having the correction information determines the high frequency application timing based on the correction information so that the high frequency voltage is applied to the mass analysis unit during the holding time when the ion detection is predicted, and the high frequency power supply command is sent to the high frequency power supply. Put out.

  When non-analysis ions other than analysis candidate ions registered in the updated database or non-analysis ions registered in the updated database are detected at a predetermined holding time, Non-analyzed ions are excluded from the mass spectrometer. Then, tandem mass spectrometry such as data dependence analysis is performed on the ions detected without being excluded. However, if the non-analysis target ions are completely eliminated by the high-frequency electric field applied, the non-analysis target ions cannot be detected at all, and correction of the database becomes difficult. Therefore, it is important to set the voltage and frequency of the high-frequency electric field for ion exclusion so as to leave a certain amount of ions. The purpose of ion exclusion is to prevent the space charge effect that occurs when the analysis unnecessary ions, that is, ions to be excluded, are overwhelmingly larger than other ions, as shown in FIG. Therefore, if ion exclusion is performed so that the intensity is the same as the intensity of ions detected at the same time, ions excluded can be detected and the database can be corrected. Typically, by removing 90% of ions to be excluded and leaving only 10%, the ions can be detected.

  According to the above flow, mass analysis is performed after ions that are not candidates for analysis that are not listed in the first database before primary mass spectrometry, or ions that are not subject to analysis that match the data in the second database are excluded by high-frequency voltage. It can be performed. Thus, analysis candidate ions can be reliably detected by mass spectrometry. In that case, it is possible to eliminate the non-analytical ions with high accuracy before performing the primary mass analysis while omitting the trouble of specifying the non-analyzing ions for each analysis by using a database. Analysis can be easily achieved.

  In addition, when the analysis conditions are changed, main parameters (eg, gradient conditions) can be input from the input unit, and information stored in the database can be corrected in advance. When a database applicable to the changed analysis condition is held, the database name and the analysis condition can be displayed on the display unit, and the database can be selected on the input unit. In addition, while performing analysis, the information processing unit can detect a deviation between the retention time information stored in the database and the actual retention time, and the ion information stored in the database can be corrected. In this way, the database can be corrected with a minimum of effort. Even if the analysis conditions are not changed, the retention time may slightly change due to deterioration of the column or the like. Even in this case, the change can be corrected in real time while the analysis is performed, so that the non-analyzed ions can be eliminated with high frequency voltage with high accuracy, which is optimal for mass spectrometry and tandem mass spectrometry. Is realized. As a result, analysis candidate ions can be subjected to tandem mass spectrometry with high sensitivity and high accuracy.

  In the system shown in FIG. 1 and FIG. A configuration in which the display unit and the input unit are omitted may be employed. In this case, since the system configuration is simplified, the price can be reduced. When routine analysis is performed with the analysis conditions kept constant, even with such a system configuration, the retention time stored in the database while performing the analysis according to the same procedure as shown in FIGS. A deviation between the information and the actual holding time can be detected by the information processing unit, and the ion information stored in the database can be corrected. However, if the analysis conditions are changed, it is necessary to create a new database.

  The present invention relates to a liquid chromatograph / mass spectrometer. Hereinafter, separation in a liquid chromatograph will be described. In a liquid chromatograph, a sample substance is once adsorbed on a separation column. A specific substance is desorbed and released into the mobile phase solution according to the composition of the mobile phase solution introduced into the separation column. Therefore, by sweeping the composition of the mobile phase solution introduced into the separation column, the sample substance adsorbed on the separation column is sequentially released to the mobile phase. This is the principle of separating substances in a liquid chromatograph.

  In general, the main factors that determine the separation conditions are the sweep method of the composition and mixing ratio of the solution (liquid A, liquid B, etc.) contained in the mobile phase solution, and the separation column. Usually, two types of solutions having significantly different solvent ratios are prepared, and the composition of the mobile phase solution can be swept (changed) with time by changing the mixing ratio with time. Typical two types of solutions include 5% acetonitrile / 95% water / 0.1% formic acid (solution A) and 95% acetonitrile / 5% water / 0.1% formic acid (solution B). For example, when a program for sweeping the mixing ratio of liquid A and liquid B from 100% / 0% to 0% / 100% in about one hour is used, the acetonitrile ratio (solvent ratio) is 5% to 95%. Corresponds to being swept in time.

FIG. 4 schematically shows the relationship between the substance separated by liquid chromatography and the solvent ratio R. Here, the ratio of the solvent is not the mixing ratio of the A liquid and the B liquid described above, but the ratio of the solvent (acetonitrile in the above example) to water in the mobile phase solution prepared by mixing the A liquid and the B liquid. It is. In the example of FIG. 4, the solvent ratio R is constant during the time t ₀ after the liquid chromatograph starts to operate. This corresponds to the fact that a certain time is required for the mobile phase liquid to reach the detector installed downstream of the separation column. Specifically, for example, when t = 0 is the liquid mixing start time of the liquid chromatograph, the time t ₀ is the pipe from the mixer (mixing unit) to the detector in the liquid chromatograph pump and the internal volume of the separation column. Corresponds to the amount (time) divided by the liquid flow rate. In a liquid chromatograph / mass spectrometer, this detector corresponds to a mass spectrometer. In FIG. 4, after time t ₀ , the ratio R increases in proportion to time t. In this case, a straight line having an inclination k shown by a solid line in FIG. 4 can be expressed as follows.

_{R = R 0 + k (t} - t 0) ( Equation 1).

Here, k represents the rate of change of the ratio R with time, and R ₀ is the solvent ratio at the start (solvent ratio of the liquid A). The detection of the separated substance is shown as a data point (●) shown on the straight line with the slope k, and the solvent ratio R can be given as a function of the time (holding time t) detected by the detector. Since the retention time is substantially the same as described above, the time from the start of analysis by the liquid chromatograph mass spectrometer to the ion detection, and the end of the liquid chromatograph from the start of analysis. The time until the detection of the separated sample can be taken as either. In addition, the sweep method as shown in (Formula 1) is called a linear gradient. The sweep method can be programmed by a computer controlling the liquid chromatograph, and more complicated sweep methods can be performed as shown in FIG. In this example, the slope k is set low only in the central portion where the separated substances are expected to concentrate. This can prevent a large number of separated substances from being detected simultaneously.

  In the region where the separated substance is not detected so much, the entire analysis time can be optimized by increasing the slope k. Whatever the gradient conditions (sweep method), the sweep method can be approximated to a straight line (linear) in a short time range. Correction, and thus the accuracy of liquid chromatograph mass spectrometry can be improved. In the embodiment shown in FIG. 1 and FIG. 2 and other embodiments, the correction of the holding time is performed by the above method, but other methods can be used as long as the deviation of the holding time can be corrected. It may be used. In addition, the correction of the holding time is calculated by the information processing unit and transmitted to the database control unit as deviation detection information, and the database control unit corrects the database.

  The internal database in the present invention is assumed to be used for LC / MS analysis as described above. Therefore, as shown in FIG. 17, not only the retention time information of the detection substance (that is, R or t which is information regarding the retention time) but also mass information is included. Therefore, even if the data points (●) in FIGS. 4 and 5 are very dense, it is possible to specify a substance based on retention time information and mass information.

Hereinafter, a data correction method will be described. In the example of FIG. 4, the actually detected retention time t ′ tends to be slightly delayed compared to the retention time information t of the detection substance stored in the database. In such a case, based on the difference (retention time difference) between the information on the substance detected at the beginning of the analysis and the information stored in the database, the information on the substance that is expected to be detected (retention time) It can be corrected. As a basic correction method, by applying fitting to information on a substance that has already been detected using t ₀ or k in (Equation 1) as a variable parameter, the detection of the substance that is expected to be detected after the fitting is performed. A method for correcting the information (holding time) can be mentioned. Therefore, in the example of FIG. 4, the corrected data is fitted with a straight line (dotted line) having a slope lower than the solid line with the slope k, but there is no problem even if fitting with a higher-order function. In this case, although the number of parameters increases, there is a feature that it is easy to cope with a sudden change in deviation.

  In addition, it is considered that the accuracy of correction is increased by fitting the data with a short time difference with a higher weight. Retention time corrections are performed sequentially during the analysis, that is, whenever a peak is detected, more specifically, whenever a peak corresponding to a data point (substance) registered in the internal database is detected. It is desirable to implement. ) By performing this correction, it is possible to perform high-precision correction of the database. Based on the corrected prediction information (◯), ion identification, ion-selective mass analysis, specifically, predetermined ions by applying high-frequency voltage It becomes possible to perform tandem mass spectrometry such as data-dependent analysis on the detected ions that are excluded from the mass analysis unit (or ions other than the predetermined ions). Further, as shown in FIG. 17, the information stored in the database includes not only the retention time information and the mass information but also errors thereof. As the mass information error, a value based on mass accuracy and resolution of the mass spectrometer and ion intensity is mainly input.

  On the other hand, since the error in the retention time information depends on the chromatograph, the piping method, the separation column, and the like, the actual analysis data shift is detected and input. A typical error is considered to be about 1% of the retention time, but data that deviates more than the set error can no longer be handled. It is. Some substances have a large retention time error. For this reason, if a substance having a small retention time error is known, it is effective to correct the retention time by preferentially using the data of the substance.

  Furthermore, it is possible to correct the mass information as well as the holding time information. That is, by storing mass information with extremely high accuracy such as theoretical values as known ion information stored in the database in advance, the mass information can be corrected by comparison with actual detection data. At this time, the actual detected data mass is corrected to highly accurate mass information based on a theoretical value or the like. Mass spectrometers with relatively high mass accuracy, such as time-of-flight mass spectrometers and Fourier transform ion cyclotron resonance mass spectrometers, are based on detected ion information, and the time dependence of subtle deviations in mass number of mass spectrometers Is found, the mass number can be corrected. This correction work may be performed during the analysis (performed in real time) or after the analysis is completed.

FIG. 6 shows a system configuration example. In this system, a high-frequency power source that applies a high-frequency voltage for eliminating ions that are not analysis candidates is used. However, when applied to the system described in FIGS. 1 and 15, the high-frequency power source may be omitted. . When changing the length of the separation column or changing the gradient conditions of the liquid chromatograph, enter the R ₀ and k values in (Equation 1) as initial values from an input unit such as a personal computer. It can be corrected in advance. When a plurality of databases can be used, the database to be used can be selected by a display unit such as a personal computer or an input unit. Thereafter, the analysis is started, and the holding time information of the database can be corrected while performing the analysis. This makes it possible to further fine-correct the database content that was corrected first in accordance with the actual analysis, thereby improving the accuracy of the database information. As a result, the database once created can be used effectively even under different separation conditions. Further, the storage of the corrected database is also executed by input from the input unit.

  FIG. 17 shows a screen display example of database contents. A database has a name, and a plurality of databases can be selected. The database includes separation conditions and ion information. The separation conditions need to be updated if the gradient conditions of the liquid chromatograph are changed. At that time, the retention time t of the ion information is actually updated after the entire analysis is completed according to the change of the separation condition. Furthermore, the value of t ′ is shown as the predicted retention time for the ions that are predicted to be detected after the detected ions are detected based on the retention time of the detected ions, and according to the prediction It is to be corrected. It is desirable to perform this correction sequentially each time ions included in the database are detected. When the updated database is saved, the update date is changed and saved. (When creating a database for the first time, save the date and time of creation).

  Although t ′ is a predicted value, t is an actual measurement value, and t ′ is basically updated with correction every time an ion included in the database is detected. Updated when analysis is complete. If all the values of t have been updated, it is expected that t ′ and t are almost the same, and it is desirable to save the database in this state. However, when only a part of the ions registered in the database is detected in the analysis, the one updated to the value of t and the one not updated are mixed. In that case, it is desirable to save the database by replacing the value of t ′ at the stage when the analysis is completed with t. Thereby, also about the ion which was not detected, prediction value t 'is preserve | saved as t, and the precision regarding the retention time information as the whole database can be improved.

  Prior to the analysis, the input unit may select or load a database to be used and input analysis parameters. It is effective to automatically read the separation condition information from the gradient program of the liquid chromatograph. Actually, the relationship between the mixing ratio of the liquid A and the time t is input by a PC or the like, but based on the input information, it is converted into the relationship between the solvent ratio R and the time t in the input unit or the information processing unit. Can be matched. Next, the analysis is started by instructing the start of the analysis on the screen. Then, the information of the substance stored in the first or second database is sequentially compared with the detected ion information, and if a deviation is found in the retention time information, the retention time of the substance expected to be detected in the database Correct the information. Each time a peak derived from an analysis candidate ion whose information is registered in the first database is detected, the retention time is corrected.

FIG. 18 shows an example of the analysis flow in the embodiment shown in FIG. Here, N is the set number of times that tandem analysis can be performed in one sequence, and the retention time information is corrected using a database that includes retention time information and information on analysis target ions or information on non-analysis targets. Analyze as you go.
In order to make it difficult to detect unnecessary ions for analysis, as shown in FIG. 16, the information processing unit transmits an instruction for controlling the high-frequency voltage applied to the mass spectrometer (MS) to the high-frequency voltage power source. Prior to secondary mass analysis, non-analytical ions not listed in the first database or non-analytical ions that match the data in the second database are excluded to obtain a primary mass spectrometry spectrum.

  As a result, if there are ions of the candidate substances to be analyzed listed in the first database, the non-analyzed ions are excluded when acquiring the primary mass spectrometry data, and the ions are mainly listed in the first database. A primary mass spectrometry spectrum consisting of the spectrum of ions of the candidate substance to be analyzed can be obtained. Furthermore, tandem mass spectrometry can be performed as necessary. Information on detected ions is sequentially searched in the first database stored in the database storage means. Since the number of types of ions to be subjected to secondary mass analysis is determined in advance, an instruction to preferentially perform secondary mass analysis (tandem mass spectrometry) is performed on ions that match the data in the first database. To the power supply involved in CID. In addition, when ions other than the analysis target are known, a second database for recording data of substances other than the analysis target candidate is created in the same manner as the first database described above, and is applied during the primary mass analysis. It can be used to control a high frequency voltage.

  In addition, information on ions that have been analyzed with ions listed in the first database can be transferred to the second database. As a result, it is possible to avoid analyzing the same ion repeatedly, and it is possible to analyze more kinds of trace samples. The substance is identified based on the result of the secondary mass spectrometry and the information of its parent ion. An example of the analysis flow is shown in FIG. Here, the set number of tandem analyzes that can be performed in one sequence is N, and information on ions that are detected S times in the first database is temporarily deleted by updating the first database. Such automatic update of the database can be designated by the screen operation in FIG. 1 (in the input screen of the display unit / input unit) before starting the analysis. When analyzing a very small amount of analysis target sample, as shown in FIG. 8, it is advantageous to detect the analysis target sample by turning off automatic update (deletion) of the first database.

  FIG. 9 shows a flow of the embodiment having the second database. If the non-analytical ions that are expected to have a very high ion intensity are known, information on the non-analytical ions is created as the second database in the same manner as the first database described above, and during the first mass analysis It is convenient to use it for controlling the applied high frequency voltage. These flows can be set by operating the screen in FIG.

  FIG. 10 shows an apparatus configuration diagram based on an embodiment of the mass spectrometry system of the present invention. This configuration performs quadrupole ion trap-time-of-flight mass spectrometry. The mass spectrometer described above is a combination of the ion transport unit 5, the ion trap 6 and the flight analysis type mass spectrometer 7 in FIGS. 10 to 11, and a combination of the ion transport unit 5 and the ion trap 6 in FIG. The structure which concerns on mass spectrometry centering on each of these is pointed out. The trajectory of ions ejected from the ion trap is bent at a substantially right angle in the acceleration part of the time-of-flight mass spectrometer. This structure is characterized in that it reduces the energy spread of ions in the acceleration part of the time-of-flight mass spectrometer and easily improves the mass resolution. A sample separated by a chromatograph such as a liquid chromatograph is introduced into an ion source and converted into gaseous ions. The generated gaseous ions are introduced into the differential exhaust part 2 through the pores 1.

  Furthermore, gaseous ions are introduced into the high vacuum part 4 from the pores 3, pass through the ion transport part 5 composed of a multipole pole or the like, and are introduced into the ion trap 6. A high frequency voltage is supplied to the ion trap 6 from a high frequency power source, and gaseous ions are trapped in the center of the ion trap 6 by a quadrupole electric field. A high-frequency voltage can be applied to the multipole pole of the ion transport portion so that ions that are not to be analyzed are excluded by the ion transport portion 5. Alternatively, non-analyzed ions may be excluded by the ion trap 6 and a high frequency voltage for trapping the analyzed ions may be applied to the ion trap 6. By applying the high-frequency voltage in this way, it becomes possible to exclude non-analytical ions before the primary mass analysis. In particular, when applying a high frequency voltage to the ion transport section 5, it is possible to exclude non-analyzed ions before reaching the ion trap 6, so that the analyte ions can be reliably trapped regardless of the space charge effect. Can do.

  Although FIG. 10 shows a configuration using two high-frequency power supplies, a configuration using any one of them may be used, and in this case, exclusion of ions not to be analyzed can be executed with a simple configuration. When two high-frequency power supplies are used, it is possible to increase the efficiency of eliminating ions not to be analyzed. In the embodiments shown in FIGS. 10 to 12, the configuration relating to the high frequency power supply is the same, and the effects obtained thereby are also the same. The gaseous ions trapped for a certain period of time are transported to the right by an electric force and introduced into the ion acceleration unit 8 of the time-of-flight mass spectrometer 7. In the ion acceleration unit 8, a pulsed high voltage is applied to the introduced gaseous ions at a specific timing, and the gaseous ions are accelerated until reaching a specific kinetic energy. The accelerated gaseous ions are subjected to a change of trajectory by the reflector 9, energy converged, and detected by the detector 10. Since the length of the ion trajectory from the ion accelerator 8 to the detector 10 is constant, and the ion velocity is lower as the ion m / z (mass / charge number) is larger, the detector 10 has a lower m / z. Sequentially detected from ions. The output of the detector 10 is introduced into the information processing unit, and the ion m / z is determined based on the ion detection time.

  From the primary mass analysis result thus obtained, the priority order of ions to be subjected to secondary mass analysis (tandem mass spectrometry) is determined by the information processing unit. In addition, when a substance whose information is stored in the database is detected and there is a difference between the retention time information and the actual retention time, based on the detected ion information, the retention time of ions expected to be detected later Correct the information.

  Next, in order to apply to the ion trap 6 a high frequency voltage for isolating only ions to be subjected to secondary mass analysis from the ions introduced into the ion trap 6, a high frequency is applied from the information processing unit. The power supply is instructed. Further, an instruction for dissociating the isolated ions with CID or the like is issued from the information processing unit to the high frequency power source, and the dissociated fragment ions are generated in the ion trap 6. The fragment ions are transported to the right by an electric force and introduced into the ion acceleration unit 8 of the time-of-flight mass spectrometer 7. In the ion acceleration unit 8, a pulsed high voltage is applied to the introduced gaseous ions at a specific timing, and the gaseous ions are accelerated until reaching a specific kinetic energy. The accelerated gaseous ions undergo a trajectory change by the reflector 9 and are detected by the detector 10. The output of the detector 10 is introduced into the information processing unit, and the ion m / z is determined based on the ion detection time.

In this way, secondary mass spectrometry is realized. A certain number of prioritized secondary mass analysis target ions are sequentially subjected to secondary mass analysis according to the priorities.
The information processing unit is a computer such as a PC and can measure time. Therefore, the holding time measuring unit can be substantially matched with the information processing unit by a computer.

  As the ion trap 6, a linear ion trap including a quadrupole pole as shown in FIG. 11 may be used instead of the quadrupole ion trap. This embodiment has the same function as the quadrupole ion trap of FIG. 10, but is characterized in that the amount of ions that can be trapped at a time can be increased. To the linear ion trap, non-analysis ions are removed, and a high frequency voltage that can trap the analysis ions is applied.

  Also, as shown in FIG. 12, the mass spectrometer may be a quadrupole ion trap mass spectrometer (or a linear ion trap) only. A sample solution separated by a liquid separation unit such as a liquid chromatograph is introduced into an ion source and converted into gaseous ions. The generated gaseous ions are introduced into the differential exhaust part 2 through the pores 1. Furthermore, it passes through the ion transport part 5 installed in the high vacuum part 4 from the pore 3 and is introduced into the ion trap 6. A high-frequency voltage is supplied to the in-trap 6 from a high-frequency power source, and gaseous ions are trapped in the center of the ion trap 6. The ion trap 6 is applied with a high-frequency voltage that removes ions not to be analyzed and traps the ions to be analyzed. The gaseous ions trapped for a certain period of time are discharged from the ion trap 6 according to the m / z of the ions and continuously detected by the detector 10 by continuously changing the high-frequency voltage applied to the ion trap 6. . The output of the detector 10 is introduced into the information processing unit, and the ion m / z can be determined (primary mass spectrometry) based on the ion detection time. Similar to the example of FIG. 2, secondary mass spectrometry can also be performed.

  In addition, if a substance whose information is stored in the database is detected and the difference between the retention time information and the actual retention time exceeds a certain value, detection is expected based on the detected ion information. The retention time information of ions to be corrected is corrected. Compared to time-of-flight mass spectrometers, quadrupole ion traps (or linear ion traps) tend to have lower mass analysis range, mass resolution, and mass accuracy, but the instrument can be made smaller and more sensitive. Is possible.

In the embodiments shown in FIGS. 10, 11 and 12, by applying a high frequency voltage in response to an instruction from the information processing unit, non-analytical ions are removed before the primary mass analysis, and a trace component to be analyzed. Certainly, mass spectrometry is performed. In particular, when the linear trap shown in FIG. 11 is used, the linear trap has a capacity two orders of magnitude higher than that of the quadrupole ion trap shown in FIG. be able to. The technology according to the present invention described so far can be applied to all mass spectrometers as long as the mass spectrometer uses a high-frequency electric field. For example, a quadrupole mass spectrometer-time-of-flight mass spectrometer (q-TOF) Applicable to triple quadrupole mass spectrometers (Triple Q). In addition, when the mass spectrometer is a quadrupole ion trap, a linear ion trap, or a Fourier transform ion cyclotron resonance mass spectrometer (FTICRMS), high-order tandem mass spectrometry {MS ⁿ (n = 3, 4 ,)} Can also be performed. Further, it is effective for analysis of a crude sample even if it is excluded from the target of tandem mass spectrometry without applying non-analytical ions by applying a high frequency voltage.

  FIG. 13 shows an apparatus configuration diagram based on an embodiment of still another mass spectrometry system of the present invention. This example is a two-dimensional liquid chromatography / mass spectrometry system. A liquid sample is introduced into the first-dimensional liquid chromatograph column (LC column) from the injection valve, but in order to apply a gradient to the mobile phase solution, two types of mobile phase solutions prepared in two liquid reservoirs 1 are pumped. 1 is mixed so that the total flow rate is constant and introduced into the injection valve. The liquid sample separated by the LC column is sequentially introduced into the switching valve 13 and adsorbed by the trap column. When the adsorption on the trap column is performed for a certain time, the pump 1 stops. Next, two types of mobile phase solutions prepared in the other two liquid reservoirs 2 are introduced into the trap column after the liquid flow rate is adjusted by the two pumps 2.

  Then, the separated sample adsorbed on the trap column is eluted, introduced into the second-dimensional liquid chromatograph column (LC column), and further separated. The separated samples are sequentially introduced into a mass spectrometer and subjected to mass analysis. After completion of the separation, the pump 2 is stopped and the pump 1 is operated. The liquid sample separated by the first dimension LC column is adsorbed for a certain time by the trap column, separated by the second dimension LC column, and subjected to mass spectrometry. In this way, the liquid sample that is two-dimensionally LC-separated is sequentially subjected to mass spectrometry. If the sample type is small, LC / MS analysis can be performed by separating only one-dimensional LC, but if the sample contains a very large number of substances, two-dimensional LC can be used completely. The separation sample cannot be separated, and the separation sample introduced into the MS is often a mixture. As described above, the types of ions that can be subjected to tandem mass analysis on the same sample are limited, and in the case of a small amount of sample, it is particularly effective to preferentially perform tandem mass analysis on the analyte. In this case, it is desirable to correct the holding time for each analysis.

  When the analysis target substance is clarified in advance and the number of analysis samples is large, high throughput analysis can be performed by parallel processing of LC columns. In the embodiment shown in FIG. 14, LC / MS analysis is performed, and two LCs and ion sources are used in parallel. If the LC elution time of the analysis target substance can be predicted, the time for generating the gaseous ions derived from the analysis target substance can be shifted by shifting the start time of the LC analysis. That is, after analyzing the gaseous ions derived from the analysis target substance generated from the ion source 1, the gaseous ions derived from the analysis target substance generated from the ion source 2 are analyzed. At the coupling portion between the ion source and the mass spectrometer, a plurality of ion sources are switched over time. The ion source and the LC column may be integrated. As another method, there is a method in which the ion source is fixed to one unit and the separation liquid sample introduced into the ion source is switched by a valve, etc., but the distance from the LC end to the ion source becomes longer, and separation occurs at the time of ion generation. The degree may decrease. In such parallel processing of LC, a waiting time (about 1 hour) during which gaseous ions derived from the analysis target substance are not analyzed can be effectively used, which is advantageous for high-throughput analysis. In the present embodiment, the number of parallel processes is 2, but the number may be increased. In order to be able to analyze multiple types of substances, the correspondence between the ion source movement time and the target ions listed in the first database and the LC analysis start time must be specified in the input section in advance. Is convenient.

  19 (a) to 19 (c) show examples of typical changes in ionic strength over time (mass chromatogram) according to the present invention. In this example, many types of ions are always detected. FIG. 19A shows an example in which the holding time is not shifted. On the other hand, FIGS. 19B and 19C are examples in which the retention time of a certain peak is shifted from t to t ′.

  FIG. 20 (a) shows a mass spectrum obtained at the retention time t in FIG. 19 (a). FIG. 20B shows a mass spectrum obtained without correction of the retention time information in the state of FIG. 19B, that is, a mass spectrum obtained at the retention time t, and FIG. ) Is completely different. Therefore, the mass spectrum of the substance for which a peak should have been obtained at the retention time t has not been acquired, and it is difficult to perform tandem mass spectrometry utilizing internal database information. FIG. 20C shows a mass spectrum obtained when the retention time is corrected in the present invention in the state of FIG. 19B, that is, a mass spectrum obtained at the retention time t ′. Although the holding time is shifted from t to t ′, almost the same mass spectrum as that in FIG. 20A can be acquired, and tandem mass spectrometry utilizing the internal database becomes possible.

  FIG. 19 (c) shows a case where ions having high ion intensity (indicated by a thick line in FIG. 20 (a)) detected at the holding time t in FIG. 19 (a) are registered in the database as non-analysis ions. In this case, the exclusion of the ions by the high frequency electric field is executed at a timing based on the corrected holding time. Here, the non-analysis target ions are detected as weak ions such as a peak indicated by a broken line centered on the retention time t ′. This ion can be used for database correction, but is excluded from the target of tandem mass spectrometry. FIG. 20D is a mass spectrum acquired at the retention time t ′ in this case, and shows that other ions detected simultaneously are detected more strongly. The reason for this is that, as shown in FIG. 3, ions other than the analysis target having a very large amount of ions are excluded, so that the space charge effect is relaxed and the detection efficiency of other ions is improved. As described above, tandem mass spectrometry utilizing the internal database is possible in spite of the retention time being deviated from t to t ′, and the detection efficiency of ions other than non-analyzed ions is improved. ing.

The apparatus block diagram of the mass spectrometry system based on one Example of this invention. The apparatus block diagram of the mass spectrometry system based on one Example of this invention. The schematic diagram regarding the trap of the ion in an ion trap. An example of the time dependence of the solvent ratio. An example of the time dependence of the solvent ratio. The apparatus block diagram of the mass spectrometry system based on one Example of this invention. 2 is an example of an analysis flow of a chromatograph / mass spectrometry system according to an embodiment of the present invention. 2 is an example of an analysis flow of a chromatograph / mass spectrometry system according to an embodiment of the present invention. 2 is an example of an analysis flow of a chromatograph / mass spectrometry system according to an embodiment of the present invention. The apparatus block diagram of the mass spectrometry system based on one Example of this invention using the quadrupole ion trap time-of-flight mass spectrometer. The apparatus block diagram of the mass spectrometry system based on one Example of this invention using the quadrupole linear ion trap time-of-flight mass spectrometer. The apparatus block diagram of the mass spectrometry system based on one Example of this invention using a quadrupole ion trap mass spectrometer. The apparatus block diagram of the mass spectrometry system based on one Example of this invention. The apparatus block diagram of the mass spectrometry system based on one Example of this invention. The example of the processing flow regarding the retention time information based on one Example of this invention. The example of the processing flow regarding the retention time information based on one Example of this invention. The example of a screen display of the database content of this invention. The example of the processing flow regarding the retention time information based on one Example of this invention. 2 is an example of a typical change in ionic strength over time according to the present invention. An example of a typical mass spectrum according to the present invention.

Explanation of symbols

1: pore, 2: differential exhaust part, 3: pore, 4: high vacuum part, 5: ion transport part, 6: in-trap, 7: time-of-flight mass spectrometer, 8: ion accelerator part, 9 : Reflector, 10: Detector.

Claims (11)

A database for storing mass and retention time information of a plurality of substances;
A chromatograph for separating the sample;
An ion source for ionizing the separated sample;
A mass analyzer for performing mass analysis of a sample ionized by the ion source;
A detection unit for detecting an analysis result of the mass analysis unit;
A holding time measuring unit for measuring the holding time of the sample from the detection result of the detecting unit;
An information processing unit for performing a comparison process between the holding time measured by the holding time measuring unit and the holding time information stored in the database;
A database control unit that corrects the retention time information of a substance that is expected to be detected later based on the result of the comparison process in the analysis real time ;
The information processing unit controls the mass spectrometry unit based on the corrected retention time information .   The liquid chromatograph mass spectrometer according to claim 1, wherein the mass of the sample and the retention time information are predetermined mass and retention time information of the analysis target substance.   The liquid chromatograph mass spectrometer according to claim 1, wherein the mass of the sample and the retention time information are predetermined mass and retention time information of a non-analyzed substance.   The information processing unit identifies the separated sample based on information on the mass of the ionized sample input from the mass analysis unit and corrected retention time information. Item 2. The liquid chromatograph mass spectrometer according to Item 1.   The apparatus further includes a high frequency power source that applies a high frequency to the mass analysis unit, and the information processing unit is based on the information on the mass of the ionized sample input from the mass analysis unit and the corrected retention time information. The liquid chromatograph mass spectrometer according to claim 1, wherein a high frequency application command is transmitted to the high frequency power source. The holding time information includes a predicted holding time and a holding time measured by the holding time measuring unit, and the holding time predicted value is determined when the detection unit detects the plurality of substances. The liquid chromatograph mass spectrometer according to claim 1, wherein the liquid chromatograph mass spectrometer is corrected by the unit.   7. The liquid chromatograph according to claim 6, wherein the information processing unit updates a relational expression between a solvent ratio in the liquid chromatograph and a retention time of the sample, and calculates the predicted retention time value. Mass spectrometer.   The liquid chromatograph mass according to claim 6, comprising a plurality of the databases, a display unit for displaying the plurality of databases, and an input unit for selecting the database. Analysis equipment. The mass spectrometer, a liquid chromatograph mass spectrometer according to claim 1, wherein the performing data tandem mass spectrometry. Separating the sample with a chromatograph;
Ionizing the separated sample;
Performing mass spectrometry of the ionized sample;
Measuring the retention time of the sample, and detecting a deviation between the retention time and the retention time information of the separated sample stored in a database;
Correcting each of the retention time information of a plurality of substances stored in the database based on the result of the detection in real time analysis ;
And a step of identifying the separated sample using the corrected retention time information. Separating the sample with a chromatograph;
  Ionizing the separated sample;
  Performing mass spectrometry of the ionized sample;
  Measuring the retention time of the sample, and detecting a deviation between the retention time and retention time information stored in a database;
  Correcting the retention time information of a plurality of substances stored in the database using the detection result in the analysis real time,
  In the step of performing mass spectrometry, mass analysis is performed after ions of non-analyzed substances determined in advance are removed by high frequency application, and the high frequency is applied at a timing determined based on the corrected retention time information. A mass spectrometric method characterized by the above.

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