WO2020152871A1

WO2020152871A1 - Substance identification method using chromatograph

Info

Publication number: WO2020152871A1
Application number: PCT/JP2019/002586
Authority: WO
Inventors: 真希山田; 茜村山
Original assignee: 株式会社島津製作所
Priority date: 2019-01-25
Filing date: 2019-01-25
Publication date: 2020-07-30
Also published as: JP7056767B2; JPWO2020152871A1

Abstract

The present invention is a substance identification method for identifying a substance included in a sample by referring, on the basis of chromatogram data obtained by using a chromatograph to separate the substance from the sample and analyzing said substance, to an identification table 34 in which the expected retention time of a plurality of types of substances under analysis which may be included in the sample and the expected retention time of standard substances are registered. The plurality of types of substances under analysis are grouped on the basis of trends in retention time variation when a sample including said plurality of types of substances under analysis is subjected to chromatographic analysis under a plurality of different conditions, each group being allocated at least one standard substance having a trend in retention time variation similar to a substance under analysis belonging to the group.

Description

Chromatographic substance identification method

The present invention relates to a substance identification method using a chromatograph.

When identifying substances contained in a sample using a chromatograph such as a liquid chromatograph or gas chromatograph, the substances contained in the sample are temporally separated, and then each substance is detected by a detector. Data representing a chromatogram (hereinafter referred to as chromatogram data) in which the time is plotted on the vertical axis and the signal intensity is plotted on the vertical axis, and peaks appearing in the chromatogram are detected from the chromatogram data. Then, the substance corresponding to the detected peak position (retention time) is identified with reference to a preset identification table.

In the identification table, the expected retention times of multiple known substances that are expected to be contained in the sample to be analyzed are registered. Then, if the detected peak position is within the allowable range of the expected retention time of a certain known substance registered in the identification table, it can be determined that the peak corresponds to the known substance. Here, the allowable range refers to a range from [expected retention time-allowable width] to [estimated retention time+allowable width]. The allowable width is a value set by the user, and it is recommended to set the allowable width within 0.08 minutes. The identification table is prepared for each type of sample to be analyzed. For example, when identifying metabolites contained in a biological sample, an identification table in which expected retention times of a plurality of types of metabolites are registered is used, When identifying residual pesticide components contained in food, an identification table in which expected retention times of a plurality of pesticide components are registered is used.

The elution time of each substance in chromatography depends on various factors such as column, detector type, age, frequency of use, and other conditions related to the chromatograph device, column temperature, mobile phase type, flow rate, and other analytical conditions. Known to fluctuate. Therefore, even when the same analysis conditions are set, if the conditions of the apparatus are different, the elution time of each substance changes, and the peak position corresponding to the substance, that is, the retention time, changes. Therefore, in the identification table, the retention time obtained when the known substance is measured under the analysis conditions suitable for the known substance using a standard chromatograph device is registered as the expected retention time. However, if the retention time fluctuates beyond the tolerance set by the user, peak identification will fail.

Therefore, conventionally, a standard substance having a known retention time is previously used by using an apparatus under the same conditions as a chromatographic analysis for identifying a substance contained in a sample, and by analyzing under the same conditions, the apparatus conditions and the Obtain the retention time of the standard substance under the analysis conditions, and predict the expected retention of the known substance registered in the identification table by the difference (retention time deviation amount) between this value (measured value of retention time) and the known retention time. Correction was performed to add or subtract time (Patent Document 1).

As the standard substance, a substance having a known retention time that is as close as possible to the expected retention time of the known substance, and the peak appearing in the chromatogram can be separated from the peak of the known substance and is physically and chemically stable is selected. .. It is virtually impossible to prepare standard substances for all known substances in terms of cost. Therefore, multiple types of known substances registered in the identification table are divided into groups so that those with close expected retention times belong to the same group, and the known substances contained in each group and the standard substances with close expected retention times are It was assigned as a standard substance common to the group. Then, the expected retention time of the known substance was corrected by using the retention time shift amount of the standard substance for each group.

JP 07-092152 JP

Chromatography is a method of separating multiple substances by utilizing the difference in separation characteristics such as the size and polarity of the substances. Therefore, when a substance contained in a certain sample is separated by chromatography, it is preferable to use an apparatus or analysis condition that is suitable for the separation characteristics of the substance contained in the sample. Do not necessarily have the same separation characteristics. Therefore, even for known substances belonging to the same group, the amount of deviation of the retention time may differ due to the difference in the device condition and the analysis condition.

As mentioned above, the grouping of known substances registered in the identification table and the assignment of standard substances to each group are performed based on the expected retention time length, and the separation characteristics are not taken into consideration. Therefore, if the expected retention time of a known substance belonging to the same group is uniformly corrected using the retention time shift amount of the standard substance common to the group, identification may fail.

The problem to be solved by the present invention is to improve the identification accuracy of a substance using a chromatograph.

The substance identification method using the chromatograph according to the present invention made to solve the above problems,
Based on the chromatogram data obtained by separating a substance from a sample using a chromatograph and analyzing the substance, expected retention times and standard substances of a plurality of types of analysis target substances that may be contained in the sample Regarding a method for identifying a substance contained in the sample, with reference to an identification table in which the expected retention time of
The plurality of types of analysis target substances are grouped based on the tendency of fluctuations in retention time when a sample containing the plurality of types of analysis target substances is subjected to chromatographic analysis under a plurality of different conditions. At least one standard substance having a similar tendency of variation in retention time to a substance to be analyzed belonging to the group is assigned to the group.

In the substance identification method according to the present invention, in the identification table referred to when identifying the substance contained in the sample, a plurality of analysis target substances, the retention time of when observed chromatographic analysis under different conditions The substances included in the sample are grouped in consideration of the tendency of fluctuations, and each group is assigned a standard substance that has a similar tendency to fluctuations in retention time as the analysis target substance belonging to that group. Can be identified with high accuracy.

FIG. 1 is a schematic configuration diagram of an embodiment of an LC-MS for carrying out a substance identification method using a chromatograph according to the present invention. 5 is a flowchart showing a procedure for grouping analysis target substances registered in an identification table in the LC-MS of the present embodiment. The flowchart which shows the identification procedure of the substance contained in a sample. An example of the operation screen for the identification processing operation of the substance contained in the sample. An example of the operation screen in which the measured value of the holding time and the corrected holding time are displayed. The figure which shows the result of having grouped based on expected retention time in the identification table in which the lipid mediator derived from an arachidonic acid cascade and its related substance are registered as an analysis target substance (the 1). The figure which shows the result of having grouped based on expected retention time in the identification table in which the lipid mediator derived from an arachidonic acid cascade and its related substance are registered as a substance to be analyzed (Part 2). The figure which shows the result of having grouped based on expected retention time in the identification table in which the lipid mediator derived from an arachidonic acid cascade and its related substance are registered as an analysis target substance (the 3). The figure (1) which shows the result of having newly grouped based on the tendency of the change of holding time. The figure which shows the result of having newly grouped based on the tendency of the change of holding time (the 2). The figure (3) which shows the result of having newly grouped based on the tendency of the change of holding time.

Hereinafter, the substance identification method using the chromatograph according to the present invention will be described in detail with reference to the drawings. FIG. 1 is a schematic configuration diagram of an embodiment of an LC-MS used for carrying out the substance identification method according to the present invention.

The LC-MS includes a liquid chromatograph (LC) unit 1 having a standard substance addition unit 11, a mass spectrometry unit 2, a data processing unit 3, an analysis control unit 4, a central control unit 5, an input unit 6, and a display unit 7. Prepare

The data processing unit 3 is adapted to receive a signal from the mass spectrometry unit 2, and includes a data storage unit 30, a chromatogram creation unit 31, a peak detection unit 32, a peak identification unit 33, etc. as functional blocks.

The analysis control unit 4 has a function of controlling the operations of the LC unit 1 and the mass analysis unit 2 under the instruction of the central control unit 5. The central control unit 5 has a user interface through the input unit 6 and the display unit 7, and also has overall control of the entire system. The storage device included in the central control unit 5 stores a control program 8 for executing the characteristic control of the present invention, which will be described later, and the CPU or the like controls each unit through the analysis control unit 4 according to the program 8. By doing so, analysis and data processing necessary for identifying the substance contained in the sample are executed.

The central control unit 5 and the data processing unit 3 realize their respective functions by using, for example, a personal computer as a hardware resource and executing dedicated control/processing software preinstalled in the computer. It can be configured. In this case, the input unit 6 is a keyboard or a pointing device (mouse, etc.) attached to the computer, and the display unit 7 is a display monitor of the computer.

Although not shown, the LC unit 1 separates a plurality of compounds contained in the sample, a pump for sucking the mobile phase and feeding it at a constant flow rate, an injector for injecting a fixed amount of the sample into the mobile phase. And a column. The standard substance addition unit 11 adds a predetermined amount of standard substance to the sample before being introduced into the injector. The standard substance is selected so that it is clear that it does not exist in the sample to be analyzed and the expected retention time is known. The standard substance addition unit 11 includes a container in which a plurality of types of standard substances are stored, and a plurality of types of standard substances are taken out of the container and added to the sample.

The mass spectrometric unit 2 is a quadrupole mass spectroscope equipped with an atmospheric pressure ion source such as an electrospray ionization (ESI) method. However, the mass spectrometric section 2 is not limited to this, and can be replaced with a mass spectrometric apparatus having another configuration such as a Q-TOF type mass spectroscope or an ion trap time-of-flight mass spectroscope.

Each substance in the sample separated by the column of the LC section 1 is introduced into the mass spectrometric section 2 with a different delay. Each substance in the sample introduced into the mass spectrometric section 2 is sequentially ionized by the atmospheric pressure ion source. The ions thus generated are introduced into the quadrupole mass filter, the ions having a specific mass-to-charge ratio that have passed through the quadrupole mass filter sequentially reach the ion detector, and a signal corresponding to the amount of the ions is generated. It is output to the data processing unit 3. The substance itself to be analyzed is known, regardless of whether it is actually contained in the sample. Therefore, the mass-to-charge ratio of the ions to be detected derived from the substance to be analyzed is also known, and the retention time of the substance is also known. Therefore, in the mass spectrometric unit 2, if the SIM (selective ion monitoring) measurement in which the mass-to-charge ratio to be detected is determined within a predetermined measurement time range near the retention time for each analysis target substance, Ions can be detected without leakage.

In the data processing unit 3, the data obtained by the ion detector is temporarily stored in the data storage unit 30 of the data processing unit 3. In the data processing unit 3, the chromatogram creating unit 31 creates a mass chromatogram based on the data stored in the data storage unit 30. The peak detector 32 detects a peak in a mass chromatogram. The peak identifying unit 33 identifies the substance corresponding to the peak from the position (holding time) of the detected peak. The substance is identified with reference to the identification table 34.

In the identification table 34, expected retention times of a large number of known analysis target substances that are expected to be contained in the sample are registered. For example, when identifying physiologically active substances and metabolites contained in biological samples such as serum and plasma, all of these physiologically active substances and their related substances, or metabolites and their related substances are the substances to be analyzed. .. When the detected peak position is within the permissible range of the expected retention time of one of the many analyte substances registered in the identification table 34, the peak identifying unit 33 determines that the substance corresponding to the peak is Presumed to be the substance to be analyzed.

In the present embodiment, an identification table 34 necessary for identifying a large number of known analysis target substances contained in a sample using LC-MS is prepared in advance for each type of analysis target substance. .. The identification table 34 is usually not created by the user who uses the device, but by the manufacturer who sells the device. The procedure for creating the identification table 34 will be described below with reference to FIGS. 2 and 3. FIG. 2 is a flowchart showing a procedure for grouping the substances to be analyzed contained in the identification table 34.

In the analysis for substance identification performed in the LC-MS of the present embodiment, a standard substance is introduced into the LC unit 1 in a state where a fixed amount of the standard substance is added to the sample to be analyzed. Therefore, a peak derived from the substance contained in the sample and a peak derived from the standard substance appear in the mass chromatogram created based on the data obtained by the ion detector of the mass spectrometric section 2. The peak detection unit 32 detects all the peaks appearing on the mass chromatogram, and the peak identification unit 33 obtains the positions of the detected peaks (that is, the measured values of the retention time), and refers to the identification table 34. , Specify the peak of the standard substance from all the peaks. Therefore, the expected retention time of the standard substance is registered in the identification table 34 in addition to the substance to be analyzed.

The peak identification unit 33 obtains a difference between the expected retention time of the standard substance registered in the identification table 34 and the measured value of the retention time of the standard substance, and uses this difference as a correction value to determine the expected retention time of the substance to be analyzed. to correct. In the identification table 34, a large number of substances to be analyzed are divided into a plurality of groups, and a common reference substance is assigned to each group. For the correction of the expected retention time of the substance to be analyzed belonging to the same group, the correction value obtained for the standard substance assigned to the group is used.

Therefore, in order to properly classify the analysis target substances into groups and select the reference substances assigned to each group, LC-MS is performed under the same conditions for all the analysis target substances and the reference substances registered in the identification table 34. Is used to determine the peak position of each. This peak position is a reference value for the retention time of the substance to be analyzed and the standard substance, and this reference value is registered in the identification table 34 as the expected retention time (step 1). Here, a standard LC-MS device is used, and analysis is performed under the analysis conditions optimized for the substance to be analyzed and the standard substance. Then, a plurality of analysis target substances and a plurality of standard substances are arranged in order of the length of the expected retention time, and grouping is performed so that substances having similar expected retention times belong to the same group (step 2). At this time, the number of groups is set so that each group always contains one or more standard substances. The grouping in step 2 corresponds to the conventional grouping.

In addition, when the reference value of the retention time of the analysis target substance and the standard substance registered in the identification table is known, the known reference value can be used as the expected retention time of each analysis target substance and each standard substance. ..

Next, the peak positions of all the analytes and standard substances are determined using a device different from the LC-MS device used in step 1 (step 3). Hereinafter, this peak position will be referred to as a measured value of the holding time. The analysis conditions of the analysis performed in step 3 and the analysis performed in step 1 may be the same or different as long as the LC-MS device is different. In addition, the analysis performed in step 3 and the analysis performed in step 1 may be the same in the LC-MS apparatus and different in the analysis conditions.

Next, for each substance to be analyzed, the difference between the expected retention time and the measured value of the retention time (hereinafter referred to as "retention time deviation amount") is calculated (step 4). Then, among the multiple groups formed by the conventional method, the retention time deviation amounts of the analysis target substance and the standard substance belonging thereto are similar (that is, the tendency of the variation of the retention time is similar. ), and a plurality of groups having similar expected retention times are reorganized as the same group (step 5). In general, it is more accurate to correct the expected retention time when the substances to be analyzed that have similar expected retention times are included in the same group, so the groups with similar expected retention times should be integrated as much as possible. In addition, when there is one or more analysis target substances in which the retention time deviation amount greatly differs from the retention time deviation amounts of other analysis target substances in all the analysis target substances, the one or more Organize the substances to be analyzed in a separate group. Further, the analytes and standard substances having similar chemical structures may have similar retention time fluctuation tendencies, so the analyte substances having similar chemical structures are grouped together.

The number of groups created by the conventional method of grouping and the number of groups reorganized may be the same or different, but be sure to make sure that all groups created by regrouping Ensure that one or more standards are included. The finer the grouping, the higher the accuracy of identification. However, if the number of groups is large, it is necessary to prepare many kinds of standard substances and the cost increases. Therefore, when there are a large number of substances to be analyzed such as 100 or more, it is preferable that the substances to be analyzed registered in one identification table be divided into groups of about 5 to 30.

The steps 3 to 5 may be performed once, or may be repeated multiple times. The fluctuation of the retention time is due to the difference in the type of LC-MS device used to obtain the reference value and the actual measurement value of the retention time and the difference in the analysis conditions. And/or using multiple LC-MS devices of different types, the analysis is performed multiple times, and the retention time of the substance to be analyzed belonging to the same group is based on the retention time shift amount obtained in each analysis. The groups are grouped so that the trends of fluctuations are similar.

Next, a procedure for identifying a substance in a sample in the data processing unit 3 having an identification table in which the retention times of a large number of analysis target substances and standard substances grouped in the above-described procedure are registered. The operation of LC-MS will be described with reference to FIGS. 3 and 4. FIG. 3 shows a flowchart showing the operation of the LC-MS, and FIG. 4 shows a display screen of the display unit 7.

First, an analyst prepares a sample that has been subjected to a predetermined pretreatment operation in advance and a standard substance to be added to the sample, and sets these at a predetermined location of the LC-MS. The type of standard substance to be added is designated in advance according to the type of sample to be analyzed. Next, the analyst operates the input unit 6 to input and set the measurement conditions, and then gives an instruction to start the analysis (step 11).

When the start of the analysis execution is instructed, the analysis control unit 4 sends a predetermined control signal or the like to the LC unit 1 and the mass analysis unit 2 according to the control program 8 to start the LC/MS analysis (step 12). That is, the standard substance is added to the sample by the standard substance adding unit 11 to prepare an analytical sample. This analytical sample is injected into the mobile phase before being introduced into the LC section 1, and as a result, the substances contained in the analytical sample are temporally separated while the mobile phase passes through the column of the LC section 1. .. The substances temporally separated by the columns of the LC unit 1 are introduced into the mass spectrometric unit 2 with different delays, and the ion intensity data reflecting the amount of ions derived from each substance is stored in the data storage unit 30. It

When the analysis is completed, the chromatogram creation unit 31 in the data processing unit 3 reads the ion intensity data from the data storage unit 30 and creates mass chromatogram data based on the data. The peak detection unit 32 detects peaks in the mass chromatogram created from the mass chromatogram data, and collects position data of each peak, that is, measured value data of retention time (step 13). The collected actual measurement value data is temporarily stored in the data storage unit 30.

Next, when the analyst operates the input unit 6 to set the type of the sample to be analyzed and to instruct the execution of the peak position identification process, the central control unit 5 causes the display unit 7 and the display unit 7 to follow the control program 8. The identification process is started by controlling the data processing unit 3 (step 14). As a result, the identification table 34 corresponding to the type of sample set by the data processing unit 3 is read, and the screen for the identification processing operation is displayed on the display screen of the display unit 7. FIG. 4 is an example of the operation screen 40 displayed on the screen of the display unit 7. On this operation screen 40, a standard substance selection unit 41 having a plurality of types of standard substances registered in the identification table 34 as selection items, a measured retention time display unit 42, a corrected retention time display unit 43, and three types of operations. Buttons (clear button 44, execute button 45, copy button 46) are displayed.

In the standard substance selection unit 41, the standard substances belonging to the group of analysis target substances registered in the identification table 34 are collectively displayed. In the example shown in FIG. 4, the substances to be analyzed registered in the identification table 34 are divided into eight groups, and the first to third groups of the eight groups each have a plurality of reference substances. It can be seen that each of the 4th to 8th groups is assigned one standard substance.

On the operation screen 40, when the analyst selects the standard substance used for the identification process from the standard substance selection unit 41 and selects the copy button 46, the central control unit 5 causes the data storage unit 30 to measure the retention time data. Is read out and the measured value of the holding time is displayed on the measured holding time display section 42 of the display screen. Further, in the data processing unit 3, the peak identification unit 33 refers to the identification table 34 based on the measured value data of the retention time and obtains the measured value of the retention time of the selected standard substance (step 15). Here, the standard substances used in the identification process are basically all the standard substances added to the sample in step 11, but at least one standard substance belonging to each group may be selected. ..

Subsequently, when the analyst selects the execute button 45 on the operation screen 40, the data processing unit 3 calculates the difference between the measured retention time of the standard substance and the expected retention time of the standard substance registered in the identification table 34. Then, this is set as a correction value (step 15). In the data processing unit 3, the peak identifying unit 33 corrects the expected retention time of the substance to be analyzed registered in the identification table 34 using the correction value of the standard substance belonging to the same group as the substance to be analyzed (step 16). ). The corrected holding time is displayed on the corrected holding time display section 43.

FIG. 5 shows an example of the operation screen 40 in a state where the measured retention time display unit 42 displays the measured retention time value and the corrected retention time display unit 43 displays the corrected retention time. On this operation screen 40, all standard substances registered in the identification table 34 are selected.

The analyst selects the copy button 46 after checking the correction holding time displayed on the correction holding time display section 43 and confirming that there is no problem. Then, an identification table in which the correction holding time displayed on the correction holding time display unit 43 is registered is newly created. Hereinafter, the identification table in which the corrected retention time is registered is referred to as “identification table 34C”.

Further, the peak identification unit 33 reads the measured value data of the retention time stored in the data storage unit 30 and refers to the identification table 34C to identify the analysis target substance corresponding to each peak position. Finally, the identification result of the substance contained in the sample is displayed on the screen of the display unit 7.

According to the substance identification method of the present embodiment described above, in the identification tables 34 and 34C, the tendency of variation in retention time, which is observed when a plurality of substances to be analyzed are subjected to chromatographic analysis under different conditions, is considered. In addition to being divided into groups, each group is assigned with a standard substance that has a similar tendency of variation in retention time to the substance to be analyzed belonging to that group. Then, since the retention time of the substance to be analyzed is corrected for each group, it is possible to offset the amount of variation in the retention time of the substance contained in the sample due to the difference in the device used for LC/MS analysis and the analysis conditions. Therefore, the substance contained in the sample can be identified with high accuracy.

In addition, in the substance identification method of this embodiment, groups having similar retention time fluctuation tendencies and close expected retention times are reorganized as the same group. In this way, by grouping the groups having a close expected retention time into one group, the expected retention time can be corrected with high accuracy. When there are many compounds with similar retention time fluctuation tendencies, the number of groups can be reduced.Therefore, the number of types of standard substances added to the sample for identification processing of the substance contained in the sample should be reduced. You can

[Example]
In the identification table in which the lipid mediator derived from the arachidonic acid cascade and its related substances are registered as the substances to be analyzed according to the above-described grouping procedure, the results of grouping the substances to be analyzed are shown in FIGS. 6A to 6C and FIG. This will be described with reference to 7A to 7C. In this example, 196 analysis target substances and 18 standard substances are registered in the identification table. In FIGS. 6A to 6C and FIGS. 7A to 7C, the compound name is shown in bold type and the compound name is underlined is the standard substance, and the other compounds are the analytes. .. All standard substances are deuterium compounds, and the compound name ends with "-dx (x is 4, 5, 6, 8, 11)".

First, the 196 substances to be analyzed registered in the identification table were divided into 18 groups by the conventional method based on the expected retention time, and 1 standard substance was assigned to each group. Hereinafter, the groups divided by the conventional method will be referred to as “conventional groups”. The expected retention time of the substance to be analyzed and the standard substance is obtained by performing a chromatographic analysis under the analytical conditions suitable for the lipid mediator using a standard device of LC-MS.

Next, the retention times of 196 target substances and 18 standard substances were measured using a device different from the LC-MS device used for the analysis to divide into the conventional groups, and all the target substances were analyzed. The difference (retention time deviation amount) between the measured retention time obtained for the substance and the standard substance and the expected retention time was determined. 6A to 6C show the expected retention times, the conventional group numbers, the measured values of the retention times, and the difference between the expected retention times and the measured values of 196 analysis target substances and 18 standard substances.

Here, in FIG. 6B, LTC4 (No.84), 11-trans-LTC4 (No.85), LTD4-d5 (No.86) and LTD4 (No.87) are assigned to the same conventional group 8. However, while the amount of difference in retention time of LTC4 (No.84) and 11-trans-LTC4 (No.85) (difference between measured retention time and expected retention time) is 0.038 minutes and 0.036 minutes, LTD4-d5 (No.86) and LTD4 (No.87) have relatively large time lag during holding at 0.122 minutes and 0.117 minutes. Under these circumstances, based on the retention time deviation of the reference material LTD4-d5 (No.86), LTC4 (No.84), 11-trans-LTC4 (No.85), LTD4 (No. When the retention time of 87) is corrected, there is a disadvantage that the corrected retention time of LTC4 (No.84) and 11-trans-LTC4 (No.85) deviates greatly from the measured retention time. The reason why such a difference in retention time shift amount occurs is not clearly known, but it is possible that it may be derived from the compound structure.

Therefore, for all substances to be analyzed, the group was reorganized in consideration of the expected retention time, retention time deviation amount, and chemical structure similarity. As a result, 196 analytes and 18 standard substances were divided into the following new groups 1-8.

[New group 1]: Conventional groups 1 to 3 are integrated
[New group 2]: Conventional groups 3-6 are integrated
[New group 3]: Conventional groups 10 to 16 are integrated into new group 3. However,

conventional group

1, 2,3-dinor-TXB1 (No.12) and 2,3-dinor-TXB2 (No.14) are Since the structure is similar to the TXB3 (No.26) of the conventional group 3, it was formed into the new group 2. Moreover, since PAF-d4 (No.185) and PAF (No.186) of the conventional group 15 are similar in structure to Azelaoyl-PAF (No198) of the conventional group 16 described later, they are included in the new group 6 together with Azelaoyl-PAF. Organized.

[New Group 4]: 9 analytes mentioned above (14,15-LTC4 (No.73), 14,15-LTE4 (No.79), LTC4 (No.84), 11-trans-LTC4 ( No.85), LTE4 (No.89), LTF4 (No.90), 11-trans-LTE4 (No.99), LTD4 (No.87) and 11-trans-LTD4 (No.93)) , Analytical substances excluding two analytical substances (11-trans-LTD4 (No.93), LTD4 (No.87)) whose difference between predicted retention time and measured retention time is significantly different from other analytical target substances Composed of

[New group 5]: Consists of LTD4 (No.87), 11-trans-LTD4 (No.93) excluded from the above 9 analytes, and reference material LTD4-d5 (No,86)
[New group 6]: Among the substances to be analyzed belonging to the

conventional groups

15 and 16, one standard substance, PAF-d4 (No, which has a similar difference in predicted retention time and measured retention time and similar chemical structure) .185)) and two analytes (PAF (No.186) and Azelaoyl-PAF (No.198)).
[New group 7]: Among the analytes belonging to the

conventional groups

16 and 17, three analytes (AEA (No.203), 15-KEDE ( No.208), OEA (No.210)) and one standard substance (OEA-d4 (No.209)) [New group 8]: Chemical substance among the substances to be analyzed belonging to the

conventional groups

17 and 18 Three analytes (EPA (No. 211), DHA (No. 212), AA (No. 214)) and one standard substance (AA-d8) with similar structures and close expected retention times (No.213))

The results of reorganization into eight new groups by the above work are shown in Figures 7A-7C. Reorganization of groups can reduce the amount of retention time shift. Further, among the eight new groups shown in FIGS. 7A to 7C, there is a group to which a plurality of reference substances are assigned, but at least one reference substance may be assigned to each of the new groups. .. Therefore, as a result of the new group having a smaller number of groups than the conventional group, the types of reference materials can be reduced. Generally, many standard substances are expensive, so that the number of types of standard substances is small, which leads to reduction of cost burden related to substance identification.

It should be noted that if only the expected retention time is corrected, it is sufficient to assign one standard substance to each group, but in a group to which multiple standard substances are assigned, the substances contained in the sample for analysis In addition to the identification accuracy of, the accuracy of quantification can be improved. This is because when quantitatively analyzing a substance contained in an analytical sample, the peak area and peak intensity of the chromatogram obtained for the substance and the peak area and peak intensity and ratio of the chromatogram obtained for the standard substance are used. However, since the peak areas and peak intensities of the standard substances having similar retention time and ionization efficiency characteristics to the respective target substances can be used, the quantification accuracy of the respective target substances can be increased.

Further, FIGS. 7A to 7C show the results obtained by correcting the expected retention time of the substance to be analyzed belonging to the new group based on the amount of deviation of the retention time of the standard substance assigned to the group, and the measured value of the retention time and the correction. The difference with the expected retention time after is shown. From FIGS. 7A to 7C, it can be seen that in the new group, the difference in the difference between the measured retention time of the analysis target substance belonging to the same group and the corrected expected retention time is small.

[Aspect]
Those skilled in the art will appreciate that the exemplary embodiments described above are specific examples of the following aspects.

(Item 1) A method for identifying a substance using the chromatograph according to one aspect,
Based on the chromatogram data obtained by separating a substance from a sample using a chromatograph and analyzing the substance, expected retention times and standard substances of a plurality of types of analysis target substances that may be contained in the sample A method for identifying a substance contained in the sample by referring to an identification table in which the expected retention time of
The plurality of types of analysis target substances are divided into groups based on the tendency of variation in retention time when a sample containing the plurality of types of analysis target substances is subjected to chromatographic analysis under a plurality of different conditions. Is assigned to at least one standard substance having a similar tendency of variation in retention time to the substance to be analyzed belonging to the group.

In the substance identification method described in paragraph 1, the sample may be one containing one type of substance or one containing multiple types of substances. In the case of a sample containing one type of substance, the substance is separated from the sample by the chromatograph, and in the case of a sample containing a plurality of types of substance, the plurality of types of substances are separated from the sample by the chromatograph, and Multiple types of substances are temporally separated. Moreover, a chromatograph is a gas chromatograph or a liquid chromatograph. Furthermore, the tendency of the retention time to change refers to, for example, the direction in which the retention time changes with changes in the conditions when performing chromatographic analysis (that is, whether the retention time becomes longer or shorter) and the amount of change. .. The tendency of fluctuations in retention time of each analyte can be inferred from the chemical and physical properties of the analyte, but multiple samples containing the analyte registered in the identification table can be used. It may be determined from the results of actual chromatographic analysis under different conditions.

According to the substance identification method of Item 1, in the identification table that is referred to when identifying a substance contained in a sample, only a plurality of substances to be analyzed can be analyzed when chromatographic analysis is performed under different conditions. The samples are grouped in consideration of the tendency of variation in retention time, and each group is assigned a standard substance that has a similar tendency of variation in retention time to the analyte substance belonging to that group. The substance contained in can be identified with high accuracy.

(Item 2) In the method for identifying a substance using the chromatograph according to Item 1,
It is recommended that multiple standards are assigned to each group.

If it is only necessary to identify the substance contained in the sample for analysis, it is sufficient to assign one standard substance to each group, but when quantitatively analyzing the substance, the chromatogram obtained for each substance is used. The ratio of the peak area value of the gram to the peak area value of the chromatogram determined for the standard is used. At this time, if a plurality of standard substances are used, the accuracy of quantitative analysis using the ratio can be improved.

(Claim 3) In the substance identification method using the chromatograph according to the

paragraph

1 or 2,
An analytical sample is prepared by adding the standard substance to the sample, and the substance contained in the analytical sample is separated and analyzed by using a chromatograph so that the analytical sample is contained in the analytical sample. Collecting the chromatogram data of the substance,
From the chromatogram data and the identification table, a step of obtaining an actual measurement value of the retention time of the standard substance contained in the analysis sample,
For each standard substance, a step of determining a difference between an expected retention time registered in the identification table and an actual measurement value of the retention time as a retention time correction value,
A step of correcting the expected retention time of each analysis target substance registered in the identification table based on the retention time correction value of the standard substance assigned to the same group as the analysis target substance,
Is preferably provided.

According to the substance identification method using the chromatograph described in the third paragraph, the retention time of the analysis target substance registered in the identification table is used for each group by using the measured value of the retention time of the standard substance belonging to the group. Since the correction is performed by the correction, the amount of the retention time of the substance contained in the sample that varies due to the difference in the chromatographic analysis conditions can be offset.

(Item 4) In the method for identifying a substance using the chromatograph according to any one of Items 1 to 3,
The chromatogram data is preferably obtained as a result of measuring a substance separated by using a chromatograph with a mass spectrometer.

According to the substance identification method using the chromatograph described in the fourth paragraph, even if the substances contained in the sample are not sufficiently separated by the column of the chromatograph, the ions obtained by ionizing the substances Since the mass-to-charge ratios of are different, a plurality of types of substances contained in the sample can be sufficiently distinguished by the mass-to-charge ratio of the ions originating from each.

DESCRIPTION OF SYMBOLS 1... LC part 11... Standard substance addition part 2... Mass analysis part 3... Data processing part 30... Data storage part 31... Chromatogram creation part 32... Peak detection part 33... Peak identification part 34... Identification table 4... Analysis control part 5... Central control unit 6... Input unit 7... Display unit 8... Control program

Claims

Based on the chromatogram data obtained by separating a substance from a sample using a chromatograph and analyzing the substance, expected retention times and standard substances of a plurality of types of analysis target substances that may be contained in the sample In the substance identification method for identifying the substance contained in the sample, referring to the identification table in which the expected retention time of
The plurality of types of analysis target substances are divided into groups based on the tendency of variation in retention time when a sample containing the plurality of types of analysis target substances is subjected to chromatographic analysis under a plurality of different conditions. A method for identifying a substance using a chromatograph, wherein at least one standard substance having a similar tendency of retention time fluctuation to the substance to be analyzed belonging to the group is assigned to.
A method for identifying a substance using the chromatograph according to claim 1,
A method for identifying substances using a chromatograph, in which a plurality of standard substances with similar retention time fluctuation tendencies to the analytes belonging to that group are assigned to each group.
A method for identifying a substance using the chromatograph according to claim 1,
An analytical sample is prepared by adding the standard substance to the sample, and the substance contained in the analytical sample is separated and analyzed by using a chromatograph so that the analytical sample is contained in the analytical sample. Collecting the chromatogram data of the substance,
From the chromatogram data and the identification table, a step of obtaining an actual measurement value of the retention time of the standard substance contained in the analysis sample,
For each standard substance, a step of determining a difference between an expected retention time registered in the identification table and an actual measurement value of the retention time as a retention time correction value,
A step of correcting the expected retention time of each analysis target substance registered in the identification table based on the retention time correction value of the standard substance assigned to the same group as the analysis target substance,
A method for identifying a substance using a chromatograph, comprising:
A method for identifying a substance using the chromatograph according to claim 1,
A method for identifying a substance using a chromatograph, wherein the chromatogram data is obtained as a result of measuring a substance separated by using a chromatograph with a mass spectrometer.