JP7056767B2 - Substance identification method using chromatograph - Google Patents

Description

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 a gas chromatograph, the horizontal axis is obtained by separating the substances contained in the sample in time and then detecting each substance with a detector. Data representing a chromatogram with time and signal intensity on the vertical axis (hereinafter referred to as chromatogram data) is acquired, and a peak appearing in the chromatogram is detected from the chromatogram data. Then, with reference to a preset identification table, the substance corresponding to the peak is identified from the position (retention time) of the detected peak.

In the identification table, the expected retention times of each of a plurality of 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 permissible range refers to a range from [expected holding time-permissible width] to [expected holding time + permissible width]. The allowable width is a value set by the user, and it is recommended to set the allowable width within 0.08 minutes. An identification table is prepared for each type of sample to be analyzed. For example, when identifying a metabolite contained in a biological sample, an identification table in which the expected retention times of a plurality of types of metabolites are registered is used. When identifying residual pesticide components contained in foods, an identification table in which the expected retention times of a plurality of types of pesticide components are registered is used.

The elution time of each substance in chromatography depends on various factors such as the column, the type of detector, the number of years of use, the conditions related to the chromatograph device such as the frequency of use, the temperature of the column, and the analysis conditions such as the type and flow rate of the mobile phase. It is 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 fluctuates, and the peak position corresponding to the substance, that is, the holding time fluctuates. 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 allowable range arbitrarily set by the user, peak identification fails.

Therefore, conventionally, a standard substance having a known retention time is analyzed in advance using an apparatus under the same conditions as the chromatograph analysis for identifying the substance contained in the sample, and the apparatus conditions and the apparatus conditions thereof are analyzed under the same conditions. The retention time of the standard substance under the analytical conditions was obtained, and the expected retention of the known substance registered in the identification table by the difference between this value (actual measurement value of retention time) and the known retention time (retention time deviation amount). Correction was performed to add or subtract time (Patent Document 1).

As the reference substance, a substance whose known retention time is as close as possible to the expected retention time of the known substance, whose peak appearing in the chromatogram is separable from the peak of the known substance, and which is physically and chemically stable is selected. .. It is practically impossible to prepare a standard substance for all known substances in terms of cost. Therefore, multiple types of known substances registered in the identification table are grouped so that those with similar expected retention times belong to the same group, and standard substances with similar expected retention times to the known substances contained in each group are classified. It was assigned as a common standard substance to the group. Then, for each group, the expected retention time of the known substance was corrected by using the retention time deviation amount of the standard substance.

Japanese Patent Application Laid-Open No. 07-092152

Chromatography is a method for separating a plurality of substances by utilizing differences in separation characteristics such as size and polarity of the substances. Therefore, when separating substances contained in a sample by chromatography, it is preferable to use equipment and analytical conditions suitable for the separation characteristics of the substances contained in the sample, but all substances contained in the sample. Separation characteristics are not always the same. Therefore, even known substances belonging to the same group may have different retention time deviations due to differences in equipment conditions and analysis conditions.

As mentioned above, the grouping of known substances registered in the identification table and the allocation of standard substances to each group are based on the length of expected retention time, 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 by using the retention time deviation 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, which was made to solve the above problems, is
Based on the chromatogram data obtained by separating the substance from the sample using a chromatograph and analyzing the substance, the expected retention time and standard substance of multiple types of substances to be analyzed that may be contained in the sample. Regarding the method of identifying the substance contained in the sample with reference to the identification table in which the expected retention time of the sample is registered.
The plurality of types of analysis target substances are grouped based on the tendency of the retention time to fluctuate when a sample containing the plurality of types of analysis target substances is subjected to chromatograph analysis under a plurality of different conditions. A group is assigned at least one standard substance that has a similar tendency of retention time variation to that of the substance to be analyzed that belongs to that group.

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

The schematic block diagram of one Embodiment of LC-MS which carries out the substance identification method using the chromatograph which concerns on this invention. The flowchart which shows the procedure of grouping of the analysis target substance registered in the identification table in LC-MS of this embodiment. The flowchart which shows the identification procedure of the substance contained in a sample. An example of an operation screen for an operation to identify a substance contained in a sample. An example of an operation screen in which the actual measurement value of the holding time and the corrected holding time are displayed. The figure which shows the result of grouping based on the expected retention time in the identification table in which the lipid mediator derived from the arachidonic acid cascade and the related substance are registered as the substance to be analyzed (No. 1). FIG. 2 is a diagram showing the results of grouping based on the expected retention time 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 (No. 2). FIG. 3 is a diagram showing the results of grouping based on the expected retention time 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 (No. 3). The figure which shows the result of having performed the grouping newly based on the tendency of the fluctuation of the holding time (the 1). The figure which shows the result of having performed the grouping newly based on the tendency of the fluctuation of the holding time (the 2). The figure which shows the result of having performed the grouping newly based on the tendency of the fluctuation of the holding time (the 3).

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 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. Be prepared.

The data processing unit 3 receives 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, and the like as functional blocks.

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

The central control unit 5 and the data processing unit 3 realize their respective functions by, for example, using a personal computer as a hardware resource and executing dedicated control / processing software pre-installed in the computer on the computer. It can be configured. In this case, the input unit 6 is a keyboard or pointing device (mouse or the like) 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 pump that sucks a mobile phase and feeds it at a constant flow rate, an injector that injects a constant amount of a sample into the mobile phase, and a plurality of compounds contained in the sample. It has a column and. The standard substance addition unit 11 adds a predetermined amount of the standard substance to the sample before it is introduced into the injector. As the standard substance, a substance that is clearly not present in the sample to be analyzed and has a known expected retention time is selected. 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 from the container and added to the sample.

The mass spectrometer 2 is a quadrupole mass spectrometer equipped with an atmospheric pressure ion source such as an electrospray ionization (ESI) method. However, the mass spectrometer 2 is not limited to this, and can be replaced with a mass spectrometer having other configurations such as a Q-TOF type mass spectrometer and an ion trap time-of-flight mass spectrometer.

Each substance in the sample separated by the column of the LC unit 1 is introduced into the mass spectrometry unit 2 with a delay of a different time. Each substance in the sample introduced into the mass spectrometer 2 is sequentially ionized by an atmospheric pressure ion source. The ions thus generated are introduced into the quadrupole mass filter, and the ions having a specific mass-to-charge ratio that have passed through the quadrupole mass filter reach the ion detector in sequence, and a signal corresponding to the amount of the ions is generated. It is output to the data processing unit 3. Regardless of whether or not it is actually contained in the sample, the substance itself to be analyzed is known. Therefore, the mass-to-charge ratio of the ion to be detected derived from the substance to be analyzed is also known, and the retention time of the substance is also known. Therefore, if the mass spectrometric unit 2 performs 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 holding time for each substance to be analyzed, it is derived from the substance to be analyzed. 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 detection unit 32 detects the peak in the mass chromatogram. The peak identification unit 33 identifies the substance corresponding to the peak from the position (retention time) of the detected peak. Identification of the substance is performed with reference to the identification table 34.

In the identification table 34, the expected retention times of a large number of known substances to be analyzed that are expected to be contained in the sample are registered. For example, when identifying a bioactive substance or a metabolite contained in a biological sample such as serum or plasma, all the bioactive substances and their related substances, or the metabolite and its related substances are the substances to be analyzed. .. When the detected peak position is within the allowable range of the expected retention time of one of the many substances to be analyzed registered in the identification table 34, the peak identification unit 33 determines that the substance corresponding to the peak is in the allowable range. It is presumed to be the substance to be analyzed.

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

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

The peak identification unit 33 obtains the difference between the estimated 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 standard substance is assigned to each group. For the correction of the expected retention time of the substances to be analyzed belonging to the same group, the correction value obtained for the standard substance assigned to the group is used.

Therefore, all the analysis target substances and reference substances registered in the identification table 34 are LC-MS under the same conditions so that the grouping of the analysis target substances and the selection of the standard substances assigned to each group are appropriately performed. The analysis using is performed, and each peak position is obtained. 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 apparatus is used, and the analysis target substance and the standard substance are analyzed under optimized analysis conditions. Then, a plurality of substances to be analyzed 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 grouping by the conventional method.

If the reference values for the retention times of the analysis target substance and the standard substance registered in the identification table are known, the known reference values can be used as the expected retention time of each analysis target substance and each standard substance. ..

Next, the peak position is obtained for all the analysis target substances and the standard substances by using an apparatus different from the LC-MS apparatus used in step 1 (step 3). Hereinafter, this peak position will be referred to as an actually measured value of the holding time. The analysis conditions performed in step 3 and the analysis performed in step 1 may be the same or different as long as the LC-MS apparatus is different. Further, the analysis performed in step 3 and the analysis performed in step 1 may have the same LC-MS apparatus and different analysis conditions.

Subsequently, for each substance to be analyzed, the difference between the expected retention time and the measured value of the retention time (hereinafter, this is referred to as “retention time deviation amount”) is obtained (step 4). The retention time deviations of the substances to be analyzed and the standard substances belonging to the multiple groups formed by grouping by the conventional method are similar (that is, the tendency of the retention time fluctuation is similar). ) And reorganize a plurality of groups having similar expected retention times into the same group (step 5). In general, the accuracy of correction of the expected retention time is higher when the substances to be analyzed with close expected retention times are included in the same group. Therefore, the groups with close expected retention times should be integrated as much as possible. In addition, when there is one or more analysis target substances whose retention time deviation amount is significantly different from the retention time deviation amount of other analysis target substances among all the analysis target substances, the one or a plurality of the analysis target substances. Organize the substances to be analyzed into different groups. Furthermore, since the substances to be analyzed and the reference substances having similar chemical structures may have similar trends in the fluctuation of the retention time, the substances to be analyzed having similar chemical structures should be in the same group.

The number of groups created by conventional grouping and the number of groups created by reorganization may be the same or different, but be sure to include all groups created by regrouping. It should contain one or more standard substances. The finer the grouping, the higher the accuracy of identification, but the larger the number of groups, the more types of standard substances need to be prepared, which has the disadvantage of increasing costs. Therefore, when there are a large number of substances to be analyzed, for example, 100 or more, it is preferable that the substances to be analyzed registered in one identification table are divided into groups of about 5 to 30.

The processes of steps 3 to 5 may be performed once, but may be repeated a plurality of times. Since the fluctuation of the holding time is due to the difference in the type of LC-MS device and the analysis conditions used to obtain the reference value and the measured value of the holding time, under a plurality of different analysis conditions. And / or, the analysis was performed multiple times using a plurality of different types of LC-MS devices, and the retention time of the substances to be analyzed belonging to the same group based on the amount of difference in the retention time obtained in each analysis. Group them so that their fluctuation trends are similar.

Next, the procedure for identifying the substance in the sample in the data processing unit 3 provided with the identification table in which the retention times of a large number of substances to be analyzed and the reference substance grouped by the above 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 LC-MS, and FIG. 4 shows a display screen of the display unit 7.

First, the 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 in a predetermined place of LC-MS. The type of standard substance to be added is specified 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 instructs to start the analysis execution (step 11).

When the start of 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 spectrometry unit 2 according to the control program 8, so that the LC / MS analysis is started (step). 12). That is, the standard substance is added to the sample by the standard substance addition unit 11, and the sample for analysis is prepared. This analytical sample is injected into the mobile phase before it is introduced into the LC unit 1, whereby the substances contained in the analytical sample are temporally separated in the process of the mobile phase passing through the column of the LC unit 1. .. The substances separated in time by the column of the LC unit 1 are introduced into the mass spectrometry unit 2 with a delay of different times, and the ionic strength data reflecting the amount of ions derived from each substance is stored in the data storage unit 30. To.

When the analysis is completed, the chromatogram creating unit 31 reads out the ionic strength data from the data storage unit 30 in the data processing unit 3 and creates mass chromatogram data based on the data. The peak detection unit 32 detects peaks in the mass chromatogram created by the mass chromatogram data, and collects the position data of each peak, that is, the measured value data of the holding time (step 13). The collected measured 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 instruct the execution of the peak position identification process, the central control unit 5 changes to the display unit 7 and the display unit 7 according to the control program 8. Execution of the identification process is started by controlling the data processing unit 3 (step 14). As a result, the identification table 34 according to the type of sample set by the data processing unit 3 is read out, 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 the operation screen 40, a standard substance selection unit 41 for selecting a plurality of types of standard substances registered in the identification table 34, an actually measured holding time display unit 42, a corrected holding time display unit 43, and three types of operations are displayed. Buttons (clear button 44, execute button 45, copy button 46) are displayed.

In the standard substance selection unit 41, the standard substances belonging to each group of the 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 standard substances. Is assigned, and it can be seen that one standard substance is assigned to each of the 4th to 8th groups.

On the operation screen 40, when the analyst selects a standard substance to be used for the identification process from the standard substance selection unit 41 and selects and operates the copy button 46, the central control unit 5 displays the actual measurement value data of the holding time from the data storage unit 30. Is read out, and the measured value of the holding time is displayed on the measured holding time display unit 42 of the display screen. Further, in the data processing unit 3, the peak identification unit 33 obtains the actual measurement value of the retention time of the selected standard substance with reference to the identification table 34 based on the actual measurement value data of the retention time (step 15). Here, the standard substances used for 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 and operates the execution 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. This is used as the correction value (step 15). In the data processing unit 3, the peak identification unit 33 corrects the expected retention time of the analysis target substance registered in the identification table 34 by using the correction value of the standard substance belonging to the same group as the analysis target substance (step 16). ). The corrected holding time is displayed on the corrected holding time display unit 43.

FIG. 5 shows an example of the operation screen 40 in which the measured holding time value is displayed on the measured holding time display unit 42 and the corrected holding time is displayed on the corrected holding time display unit 43. In this operation screen 40, all the standard substances registered in the identification table 34 are selected.

The analyst selects and operates the copy button 46 after confirming that there is no problem by looking at the correction holding time displayed on the correction holding time display unit 43. 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 correction holding time is registered is referred to as "identification table 34C".

Further, the peak identification unit 33 reads out the measured value data of the holding time stored in the data storage unit 30, and refers to the identification table 34C to specify 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 the retention time fluctuation observed when a plurality of analysis target substances are subjected to chromatograph analysis under different conditions is taken into consideration. In addition to being divided into groups, each group is assigned a standard substance that has a similar tendency of retention time to the substance to be analyzed that belongs to that group. Then, in order to correct the retention time of the substance to be analyzed for each group, it is possible to offset the amount of fluctuation in the retention time of the substance contained in the sample due to the difference in the apparatus used for LC / MS analysis and the analysis conditions. Therefore, the substance contained in the sample can be identified with high accuracy.

Further, in the substance identification method of the present embodiment, the groups having similar tendencies of fluctuations in retention time and having similar expected retention times were reorganized into the same group. By grouping groups with similar expected retention times into one group in this way, the expected retention time can be corrected with high accuracy. In addition, when there are many compounds with similar trends in retention time, the number of groups can be reduced, so the types of standard substances added to the sample for the substance identification process contained in the sample should be reduced. Can be done.

[Example]
The results of grouping the substances to be analyzed 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-mentioned grouping procedure are shown in FIGS. 6A to 6C and FIGS. 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 whose compound name is shown in bold and whose compound name is underlined is the standard substance, and the other compounds are the objects to be analyzed. .. The standard substances are all deuteriums, 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 a conventional method based on the expected retention time, and one standard substance was assigned to each group. Hereinafter, the group divided by the conventional method is referred to as a "conventional group". The expected retention time of the substance to be analyzed and the standard substance was determined by performing chromatographic analysis under analytical conditions suitable for the lipid mediator using a standard device of LC-MS.

Next, the retention times of 196 substances to be analyzed and 18 standard substances were measured using a device different from the LC-MS device used for the analysis for dividing into groups, and all the analysis targets were measured. 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. FIGS. 6A to 6C show the expected holding time of 196 substances to be analyzed and 18 standard substances, the conventional group number, the measured value of the holding time, and the difference between the predicted holding time and the measured value.

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, the holding time deviation (difference between the measured holding time and the expected holding time) of LTC4 (No.84) and 11-trans-LTC4 (No.85) is 0.038 minutes and 0.036 minutes. The holding time lags of LTD4-d5 (No.86) and LTD4 (No.87) are relatively large at 0.122 minutes and 0.117 minutes. Under these circumstances, LTC4 (No.84), 11-trans-LTC4 (No.85), and LTD4 (No.8) in the same group 8 based on the retention time shift of the standard material LTD4-d5 (No.86). When the holding time of 87) is corrected, there is an inconvenience that the corrected holding time of LTC4 (No.84) and 11-trans-LTC4 (No.85) greatly deviates from the measured holding time. The reason for such a difference in the retention time shift amount is not clearly clarified, but it may be derived from the compound structure.

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

[New Group 1]: Integrate conventional groups 1 to 3
[New Group 2]: Integrate conventional groups 3-6
[New Group 3]: Integrate conventional groups 10 to 16 into new group 3. However, 2,3-dinor-TXB1 (No.12) and 2,3-dinor-TXB2 (No.14) of conventional group 1 are Since the structure is similar to TXB3 (No.26) of the conventional group 3, it was organized into the new group 2. In addition, 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, so they are added to the new group 6 together with Azelaoyl-PAF. I organized it.

[New Group 4]: The above-mentioned 9 analysis objects (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)) , Analysis target substances excluding two analysis target substances (11-trans-LTD4 (No.93), LTD4 (No.87)) whose difference between the predicted retention time and the actual measurement retention time is significantly different from other analysis target substances. Consists of

[New Group 5]: Consists of LTD4 (No.87), 11-trans-LTD4 (No.93), and standard substance LTD4-d5 (No.86), which were excluded from the above-mentioned nine analysis objects.
[New Group 6]: Among the substances to be analyzed belonging to the conventional groups 15 and 16, one standard substance PAF-d4 (No) having a similar difference between the predicted holding time and the measured holding time and having a similar chemical structure. .185))) and two analysis objects (PAF (No.186), Azelaoyl-PAF (No.198))
[New Group 7]: Of the substances to be analyzed that belong to the conventional groups 16 and 17, three substances to be analyzed (AEA (No. 203), 15-KEDE (AEA (No. 203)), which have similar chemical structures and close expected retention times. No.208), OEA (No.210)) and one standard substance (OEA-d4 (No.209))
[New Group 8]: Of the substances to be analyzed that belong to the conventional groups 17 and 18, three substances to be analyzed (EPA (No.211) and DHA (No.), which have similar chemical structures and close expected retention times. 212), AA (No.214)) and one standard substance (AA-d8 (No.213))

The results of the reorganization into eight new groups by the above-mentioned work are shown in FIGS. 7A-7C. By reorganizing the group, the amount of holding time shift can be reduced. Further, although there are groups to which a plurality of standard substances are assigned among the eight new groups shown in FIGS. 7A to 7C, at least one standard substance may be assigned to each of the new groups. .. Therefore, as a result of the number of groups in the new group being smaller than that in the conventional group, the types of standard substances can be reduced. In general, many standard substances are expensive, so a small number of standard substances leads to a reduction in the cost burden related to substance identification.

If only the expected retention time is to be corrected, it is sufficient if one standard substance is assigned to each group, but in the group to which multiple standard substances are assigned, the substances contained in the analysis sample are included. In addition to the accuracy of identification, the accuracy of quantification can also be improved. When quantitatively analyzing a substance contained in a sample for analysis, 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 area and peak intensity of the standard substance having more similar retention time and ionization efficiency characteristics to each analysis target substance can be used, the accuracy of quantification of each analysis target substance can be improved.

Further, FIGS. 7A to 7C show the results of correcting the expected retention time of the substances to be analyzed belonging to the new group based on the retention time deviation amount of the standard substance assigned to the group, and the measured values and corrections of the retention time. It shows the difference from the expected retention time later. Looking at FIGS. 7A to 7C, it can be seen that in the new group, the variation in the difference between the measured retention time of the substances to be analyzed belonging to the same group and the corrected expected retention time is small.

[Aspect]
It will be understood by those skilled in the art that the above-mentioned exemplary embodiments are specific examples of the following embodiments.

(Clause 1) The substance identification method using the chromatograph according to one aspect is
Based on the chromatogram data obtained by separating the substance from the sample using a chromatograph and analyzing the substance, the expected retention time and standard substance of multiple types of substances to be analyzed that may be contained in the sample. It is a method of identifying a substance contained in the sample by referring to an identification table in which the expected retention time of the sample is registered.
The plurality of types of analysis target substances are grouped based on the tendency of the retention time to fluctuate when a sample containing the plurality of types of analysis target substances is subjected to chromatograph analysis under a plurality of different conditions. 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.

In the substance identification method according to the first item, the sample may be one containing one kind of substance or one containing a plurality of kinds of substances. In the case of a sample containing one type of substance, the substance is separated from the sample by a chromatograph, and in the case of a sample containing multiple types of substances, a plurality of types of substances are separated from the sample by a chromatograph, and the said. Multiple types of substances are separated in time. The chromatograph is a gas chromatograph or a liquid chromatograph. Further, the tendency of the retention time fluctuation refers to, for example, the direction in which the retention time fluctuates (that is, whether the retention time becomes longer or shorter) or the amount of fluctuation due to changes in the conditions for performing chromatographic analysis. .. The tendency of the retention time of each analysis target substance to fluctuate can be inferred from the chemical and physical properties of the analysis target substance, but there are multiple samples containing the analysis target substance registered in the identification table. It may be determined from the result of actual chromatograph analysis under different conditions.

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

(Item 2) In the substance identification method using the chromatograph described in item 1,
Multiple standards should be assigned to each group.

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

(Section 3) In the substance identification method using the chromatograph according to the first or second paragraph,
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 to be contained in the analytical sample. The process of collecting chromatogram data of substances and
A step of obtaining an actually measured value of the retention time of the standard substance contained in the analysis sample from the chromatogram data and the identification table, and
For each standard substance, a step of determining the difference between the expected retention time registered in the identification table and the measured value of the retention time as a retention time correction value, and
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.
It is preferable to provide.

According to the substance identification method using the chromatograph described in Section 3, the retention time of the substance to be analyzed registered in the identification table is measured for each group using the measured retention time of the standard substance belonging to the group. Therefore, it is possible to offset the amount of fluctuation in the retention time of the substance contained in the sample due to the difference in the conditions of the chromatograph analysis.

(Clause 4) In the substance identification method using the chromatograph according to any one of paragraphs 1 to 3.
It is preferable that the chromatogram data is obtained as a result of measuring the substance separated by using a chromatograph with a mass spectrometer.

According to the substance identification method using the chromatograph according to the fourth item, 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 are obtained. Since the mass-to-charge ratios of the two substances are different, it is possible to sufficiently distinguish a plurality of types of substances contained in the sample by the mass-to-charge ratio of the ions derived from each.

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

Claims (4)

Based on the chromatogram data obtained by separating the substance from the sample using a chromatograph and analyzing the substance, the expected retention time and standard substance of multiple types of substances to be analyzed that may be contained in the sample. In the substance identification method for identifying the substance contained in the sample with reference to the identification table in which the expected retention time of the sample is registered.
The plurality of types of analysis target substances are grouped based on the tendency of the retention time to fluctuate when a sample containing the plurality of types of analysis target substances is subjected to chromatograph analysis under a plurality of different conditions. A substance identification method using a chromatograph, to which at least one standard substance having a similar tendency of retention time fluctuation to that of the substance to be analyzed belonging to the group is assigned to. In the substance identification method using the chromatograph according to claim 1,
A substance identification method using a chromatograph, in which each group is assigned multiple standard substances having similar trends in retention time to the substances to be analyzed belonging to that group. In the substance identification method 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 to be contained in the analytical sample. The process of collecting chromatogram data of substances and
A step of obtaining an actually measured value of the retention time of the standard substance contained in the analysis sample from the chromatogram data and the identification table, and
For each standard substance, a step of determining the difference between the expected retention time registered in the identification table and the measured value of the retention time as a retention time correction value, and
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. In the substance identification method using the chromatograph according to claim 1,
A substance identification method 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.

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