Liquid chromatograph data processing device

Abstract

Peak tracking is performed regardless of the mass spectrum pattern. [Solution] A data processing device (20) for a liquid chromatograph, comprising: a memory unit (21) in which first chromatogram data and first mass analysis data of a main detector under first analysis conditions and second chromatogram data and second mass analysis data of a main detector under second analysis conditions are stored; a chromatogram peak extraction unit (34) that extracts a first peak and a second peak from the first chromatogram data and the second chromatogram data; a mass-to-charge ratio acquisition unit (35) that acquires the mass-to-charge ratio of ions in the first peak and the mass-to-charge ratio of ions in the second peak; a molecular weight calculation unit (36) that calculates molecular weights from the mass-to-charge ratio of the first peak and the mass-to-charge ratio of the second peak, respectively; and a peak correspondence unit (37) that associates the first peak with the second peak based on the molecular weights of the first peak and the second peak.

Description

[Technical Field]

[0001] The present invention relates to a data processing device for a liquid chromatograph. [Background technology]

[0002] Liquid chromatographs are widely used for analyzing liquid samples. Many liquid chromatographs are equipped with an absorbance detector that detects the absorbance of compounds separated in a column by irradiating them with light of a specific wavelength.

[0003] When analyzing a liquid sample containing a target compound (target compound) in pharmaceutical manufacturing and development, analytical conditions (analysis methods) are considered to ensure sufficient separation of the target compound from other compounds (e.g., impurities) in a column. When considering analytical conditions, multiple analytical conditions are established, e.g., by varying the composition and mixing ratio of the solvents constituting the mobile phase, and the target compound, etc., contained in the liquid sample is detected using each of these analytical conditions. Because the retention time of each compound varies depending on the analytical conditions, it is necessary to identify the peaks in the chromatogram corresponding to each compound (peak tracking) for each of the multiple analytical conditions. Conventionally, liquid samples are measured using a liquid chromatograph equipped with an additional mass spectrometer as a detector, and peak tracking is performed based on the mass-to-charge ratio of the base peak (the peak with the greatest intensity in the mass spectrum) of the mass spectrum obtained at the position of each peak in the chromatogram (e.g., Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Shinichi Fujisaki, "Improvement of analytical method development efficiency using MS peak tracking", [online], January 2024, Shimadzu Corporation, [Retrieved August 20, 2024], Internet <URL:https: / / www.an.shimadzu.co.jp / sites / an.shimadzu.co.jp / files / pim / pim_document_file / an_jp / applications / application_note / 22542 / an_01-00688-jp.pdf> Summary of the Invention [Problem to be solved by the invention]

[0005] When compounds with medium or larger molecular weights, such as proteins, peptides, and nucleic acids, are analyzed by mass spectrometry, multiple types of multiply charged ions with different valences are detected for the same compound. The intensity of each mass peak of a multiply charged ion varies depending on the analytical conditions. As a result, even for the same compound, the spectral pattern of the mass spectrum varies depending on the analytical conditions, and the mass-to-charge ratio of the base peak differs, making peak tracking difficult.

[0006] The problem to be solved by the present invention is to provide a technique that enables peak tracking regardless of changes in the spectral pattern of a mass spectrum. [Means for solving the problem]

[0007] The present invention, which has been made to solve the above problems, is a data processing device for a liquid chromatograph equipped with a column for separating compounds contained in a liquid sample, and a main detector other than a mass spectrometer and a mass spectrometer for detecting the compounds separated by the column, comprising: a memory unit in which first chromatogram data obtained by measuring a liquid sample containing a target compound with the main detector under first analytical conditions and first mass analysis data obtained by measuring the liquid sample with the mass spectrometer, and second chromatogram data obtained by measuring the liquid sample with the main detector under second analytical conditions and second mass analysis data obtained by measuring the liquid sample with the mass spectrometer are stored; a chromatogram peak extraction unit that extracts a first peak from the first chromatogram data and a second peak from the second chromatogram data; a mass-to-charge ratio acquiring unit that acquires mass-to-charge ratios of a plurality of ions detected at the position of the first peak from the first mass analysis data, and acquires mass-to-charge ratios of a plurality of ions detected at the position of the second peak from the second mass analysis data; a molecular weight calculation unit that calculates molecular weights of compounds corresponding to the plurality of ions from the mass-to-charge ratios of the plurality of ions detected at the position of the first peak, and calculates molecular weights of compounds corresponding to the plurality of ions from the mass-to-charge ratios of the plurality of ions detected at the position of the second peak; a peak association unit that associates the first peak with the second peak based on the molecular weight calculated for the first peak and the molecular weight calculated for the second peak; Equipped with. [Effects of the Invention]

[0008] In the data processing device for a liquid chromatograph according to the present invention, the molecular weights of compounds corresponding to a first peak extracted from first chromatogram data are calculated based on the mass-to-charge ratios of the ions detected by a mass spectrometer at the position of the first peak. The molecular weights of compounds corresponding to the ions are also calculated based on the mass-to-charge ratios of the ions detected by a mass spectrometer at the position of a second peak extracted from second chromatogram data. The first peak and the second peak are then associated with each other based on the molecular weights calculated for the first peak and the second peak. In the present invention, peak tracking is performed based on molecular weights rather than the spectral pattern of the mass spectrum, and therefore peak tracking can be performed regardless of changes in the spectral pattern of the mass spectrum. [Brief explanation of the drawings]

[0009] [Figure 1] 1 is a diagram showing the configuration of a main part of a liquid chromatograph system including a data processing device for a liquid chromatograph according to the present invention. [Figure 2] 1 is a flowchart showing a procedure for examining analytical conditions using the liquid chromatograph system of the present embodiment. [Figure 3] 10 is an example of a screen display of analysis results in the liquid chromatograph system of the present embodiment. [Figure 4] 2 is a chromatogram obtained by measuring a sample containing a nucleic acid compound under first analytical conditions. [Figure 5] A mass spectrum based on measurement data obtained at peak positions in a chromatogram obtained by measuring a sample containing a nucleic acid compound under first analytical conditions. [Figure 6] chromatogram obtained by measuring a sample containing a nucleic acid compound under second analytical conditions. [Figure 7] A mass spectrum based on measurement data obtained at peak positions in a chromatogram obtained by measuring a sample containing a nucleic acid compound under second analytical conditions. DETAILED DESCRIPTION OF THE INVENTION

[0010] An embodiment of a data processing device for liquid chromatography according to the present invention will be described below with reference to the drawings. Hereinafter, analysis of nucleic acids will be described as a specific example, but the compound to be analyzed is not limited to nucleic acids.

[0011] FIG. 1 is a diagram showing the configuration of the main parts of a liquid chromatograph system 1 including a data processing device for a liquid chromatograph according to this embodiment (hereinafter also simply referred to as a "data processing device").

[0012] The liquid chromatograph system 1 of this embodiment includes a liquid chromatograph 10 and an analysis and processing device 20. The analysis and processing device 20 corresponds to the data processing device of the liquid chromatograph in the present invention.

[0013] The liquid chromatograph 10 is an analytical device that combines a component separation unit 11, an absorbance detector 12 (corresponding to the main detector in the present invention), and a mass spectrometer 13. In the component separation unit 11, a liquid sample is introduced into a column, and various compounds contained in the liquid sample are separated by the column and allowed to flow out. In the absorbance detector 12, the absorbance is detected by irradiating the compounds flowing out from the column with light of a predetermined wavelength. In this embodiment, the absorbance detector 12 is a PDA detector.

[0014] The mass spectrometer 13 sequentially ionizes compounds passing through the absorbance detector 12, and the generated ions are separated and detected according to their mass-to-charge ratios in a mass separator. The mass spectrometer 13 of this embodiment is equipped with an atmospheric pressure ion source such as ESI or APCI and a quadrupole mass separator. The mass spectrometer 13 can perform mass analysis (MS scan measurement, SIM measurement) of ions generated from compounds contained in a sample. These ion sources and mass separators are merely examples, and other types may also be used. For example, a mass separator equipped with a time-of-flight mass separator (ToF), a triple quadrupole mass separator, or a quadrupole-time-of-flight mass separator (Q-ToF) may also be used. Using a mass separator equipped with ToF allows for obtaining accurate mass information (e.g., values ​​to five decimal places) of various ions with high precision (e.g., mass accuracy of several ppm or less). Furthermore, using a mass separator equipped with a triple quadrupole mass separator or Q-ToF allows for MS / MS scan measurement, MRM measurement, and the like.

[0015] The analysis and processing device 20 includes a memory unit 21. The analysis and processing device 20 also includes, as functional blocks, a compound information input receiving unit 31, an analysis condition setting unit 32, a measurement data acquisition unit 33, a chromatogram peak extraction unit 34, a mass-to-charge ratio acquisition unit 35, a molecular weight calculation unit 36, a peak matching unit 37, an identification unit 38, and an analysis result display processing unit 39. The analysis and processing device 20 is actually, for example, a general personal computer, and these functional blocks are realized by executing a dedicated analysis and processing device program pre-installed on the processor. The analysis and processing device 20 is also connected to an input unit 41 including a mouse, keyboard, etc., and a display unit 42 such as a liquid crystal display.

[0016] Next, an example of examining analytical conditions (analysis methods) using the liquid chromatograph system 1 of this embodiment will be described with reference to the flowchart in Fig. 2. Such examination of analytical conditions is carried out to determine optimal analytical conditions when a target compound contained in a sample to be analyzed is separated using a liquid chromatograph column.

[0017] For example, nucleic acid drugs are produced by chemical synthesis, but the synthesis process generates many impurities related to incomplete nucleotide elongation and incomplete removal of protecting groups. Consequently, the resulting product contains a vast number of impurity-containing oligonucleotides. To ensure the safety of nucleic acid drugs, the individual concentrations of these impurities must be below specified limits. To confirm this, each compound must be properly separated. In liquid chromatography, reversed-phase ion-pair chromatography (RP-IP) is a separation mode commonly used to separate charged substances. In RP-IP, the separation pattern of each compound changes depending on the concentration of ion-pair reagents added and the organic solvent composition used in the mobile phase. However, the behavior of the change in separation pattern varies depending on the oligonucleotide chain length, base composition, and the presence or absence of modified bonds. Therefore, analytical conditions must be considered for each target sequence to find the optimal separation conditions.

[0018] When the user issues an instruction to start analysis by performing a predetermined input operation, the compound information input receiving unit 31 displays a screen on the display unit 42 that prompts the user to input the molecular weight of the target compound (Step 1).

[0019] When the user inputs the molecular weight of the target compound, the analytical condition setting unit 32 displays on the display unit 42 a screen for setting analytical conditions for separating and detecting the target compound contained in the sample using a liquid chromatograph. These analytical conditions include, for example, the type of mobile phase (various organic solvents and aqueous solvents) used in the liquid chromatograph, their mixing ratio, flow rate, column oven temperature, etc. They also include the wavelength of light irradiated onto the sample liquid in the absorbance detector 12 and analytical conditions for the mass spectrometer (e.g., selection of a mass-to-charge ratio range for MS scan measurement). In developing analytical conditions, multiple separation conditions, each with different types and mixing ratios of mobile phases, are often set, while the same analytical conditions are set for the wavelength of light irradiated onto the sample liquid in the absorbance detector 12 and the analytical method for the mass spectrometer. In this embodiment, ten analytical conditions with different mobile phase mixing ratios are set. Of course, this number is merely an example and can be changed as desired.

[0020] Once the user sets multiple analytical conditions (Step 2), the measurement data acquisition unit 33 performs measurement of the liquid sample using each of the multiple analytical conditions (Step 3). In this measurement, compounds contained in the liquid sample are separated in the component separation unit 11 of the liquid chromatograph 10, and the separated compounds are measured by the absorbance detector 12 and the mass spectrometer 13, respectively. The measurement data obtained by the absorbance detector 12 and the mass spectrometer 13 are stored in the memory unit 21. The absorbance detector 12 obtains measurement data representing the change in the intensity of light transmitted through the sample liquid over time. By converting the light intensity in this measurement data to absorbance, chromatogram data representing the change in absorbance over time is obtained. In addition, the mass spectrometer 13 obtains measurement data, for example, by repeatedly performing MS analysis within a predetermined mass-to-charge ratio range during measurement. This results in three-dimensional data representing the change in the measured ion intensity along two axes: the time axis and the mass-to-charge ratio axis. From this measurement data, a total ion current chromatogram (TICC) that shows the change over time in the sum of the intensities of all ions, an extracted ion chromatogram (EIC, also called a mass chromatogram) that shows the change over time in the intensities of ions with a specific mass-to-charge ratio, and mass spectrum (MS spectrum) data for a specific time (time range) can be obtained.

[0021] When the measurement data is obtained, the chromatogram peak extraction unit 34 reads out the measurement data of the absorbance detector 12 from the storage unit 21, creates a chromatogram, and extracts peaks present on the chromatogram (step 4).

[0022] When a peak is extracted from the chromatogram of the absorbance detector 12, the mass-to-charge ratio acquisition unit 35 reads out the measurement data obtained by the mass spectrometer 13 in the same measurement from the storage unit 21. Then, a mass spectrum (a mass spectrum obtained by MS scan measurement) is created from the measurement data obtained by the mass spectrometer 13 at the position (retention time) of the peak extracted from the chromatogram of the absorbance detector 12, and the mass-to-charge ratio values ​​of the multiple mass peaks present in the mass spectrum are obtained (Step 5). When a nucleic acid, which is the target compound in this embodiment, is ionized using an atmospheric pressure ion source, multiple types of multivalent ions with different valences are often generated, and the mass-to-charge ratio values ​​of each of these multiple types of multivalent ions are obtained.

[0023] When the mass-to-charge ratios of the multiple mass peaks on the mass spectrum at the peak position of the chromatogram are obtained, the molecular weight calculation unit 36 ​​calculates the molecular weight of the compound that generated those multiply charged ions based on the values ​​of the multiple mass-to-charge ratios obtained. This process is called multiply charged ion analysis or multiply charged ion deconvolution. For example, a negative multiply charged ion from which multiple protons have been released is [M+nH] n- In addition, a positive multiply charged ion with multiple protons is expressed as [M+nH] n+ The molecular weight calculation unit 36 ​​calculates the mass-to-charge ratio of the multiply charged ion obtained by the mass-to-charge ratio acquisition unit 35 as [M+nH] in accordance with the analysis conditions (polarity of the ions generated in the ion source). n- or [M+nH] n+ The molecular weight M of the compound that generated the multiply charged ions is calculated by applying [M+nH] n- or [M+nH] n+ It is sufficient to calculate the molecular weight based on a plurality of mass-to-charge ratios that fit the above formula, and it is not necessary to use all of the mass-to-charge ratios acquired by the mass-to-charge ratio acquisition unit 35. The mass spectrum may contain noise peaks resulting from noise. The mass-to-charge ratio of such noise peaks is expressed as [M+nH] n- or [M+nH] n+Therefore, the molecular weight calculation unit 36 ​​calculates the molecular weight of the compound by excluding the mass-to-charge ratio values ​​of such mass peaks (noise peaks, etc.).

[0024] Once the above processing is completed for the measurement data obtained under all analytical conditions, the peak association unit 37 estimates that peaks on the chromatogram with the same obtained molecular weight value are peaks of the same compound and performs a process of associating these peaks. Note that the peak association unit 37 treats the molecular weight values ​​as being the same if the difference in molecular weight value is within a preset tolerance range. For example, molecular weights with a difference of 0.5% or less are treated as being the same. In this case, for example, for a target compound with a molecular weight of 716, molecular weights with a difference of 4 Da or less are treated as being the same. The tolerance range may be specified by a ratio relative to the molecular weight of the target compound, or by an absolute value.

[0025] When the above processing by the peak association unit 37 is completed, the identification unit 38 compares the molecular weight of the target compound received by the compound information input receiving unit 31 with the molecular weight of each peak set associated by the peak association unit 37, and identifies the peak set corresponding to the target compound based on whether the difference is within a predetermined range (for example, within ±1 Da).

[0026] When the processing by the identification unit 38 is completed, the analysis result display processing unit 39 displays a chromatogram based on the measurement data from the absorbance detector 12 obtained under each of the multiple analysis conditions. For each peak in the chromatogram, information about the target compound is displayed in association with the molecular weight value calculated by the molecular weight calculation unit 36. Furthermore, peaks in different chromatograms that have the same molecular weight value calculated by the molecular weight calculation unit 36 ​​are displayed in association with each other (step 7).

[0027] FIG. 3 shows an example of a display screen by the analysis result display processor 39. In this example, multiple chromatograms based on measurement data from the absorbance detector 12 are displayed vertically, and the molecular weight values ​​calculated for the peaks by the molecular weight calculation unit 36 ​​are displayed near the peak tops of the chromatograms. Furthermore, by displaying lines connecting peaks with the same molecular weight values, the correspondence between peaks presumed to originate from the same compound is clearly displayed. While lines connecting peaks are used here, various display formats are possible, such as displaying peaks with the same molecular weight values ​​in the same color. Furthermore, when the user selects one of the peaks on the screen shown in FIG. 3, a mass spectrum based on the measurement data from the mass spectrometer 13 obtained at the position (retention time) of that peak is displayed (not shown in FIG. 3). While only chromatograms obtained under three analytical conditions are displayed on the screen in FIG. 3, chromatograms obtained under other analytical conditions can be displayed by operating the scroll bar 51 on the screen.

[0028] The user checks the chromatograms obtained under each analytical condition displayed on the screen, and determines the analytical conditions that can sufficiently separate the target compound from other compounds (impurities, etc.) as the analytical conditions for that target compound. The determined analytical conditions are associated with information about the target compound and stored in memory unit 21. Furthermore, in the case of the development of nucleic acid drugs, for example, it is possible to confirm that the liquid sample does not contain any impurities at concentrations exceeding the standard value (i.e., that the nucleic acid drug is safe) based on the fact that, in a chromatogram in which the target compound and other compounds are sufficiently separated, the peak areas or peak top heights of compounds other than the target compound are both below predetermined standard values.

[0029] As shown in Figure 3, when the analytical conditions in the liquid chromatograph 10 are different, the retention time of the same compound changes. Furthermore, depending on the analytical conditions, multiple compounds may elute simultaneously (co-elute). Therefore, it is necessary to perform peak tracking, which identifies which peaks on the chromatograms obtained under multiple analytical conditions are derived from the same compound.

[0030] Conventionally, when performing peak tracking, peaks on multiple chromatograms are associated by identifying the peak with the greatest intensity (base peak) in the mass spectrum obtained at the position of each peak on the chromatogram.

[0031] In the case of low molecular weight compounds, most of the ions generated in the ion source of the mass spectrometer 13 are singly charged ions, and the peaks of these singly charged ions become base peaks. Therefore, by matching base peaks with the same mass-to-charge ratio, it is possible to match peaks in the chromatogram with each other.

[0032] In contrast, when analyzing compounds with moderate or higher molecular weights (e.g., molecular weights of 2,000 or more), such as nucleic acids, proteins, and peptides described in the above embodiments, or when using an ion source that easily generates multiply charged ions even for low molecular weight compounds, the amount of multiple multiply charged ions generated varies depending on the analytical conditions. Furthermore, the valence of the generated ions may differ (the mass-to-charge ratios of the generated ions may differ). Therefore, even for the same compound, the mass-to-charge ratios of the base peaks may differ, making it difficult to associate chromatographic peaks derived from the same compound using conventional techniques. Furthermore, when using base peaks, if the measurement data contains significant noise, the noise peak may become the base peak. Because noise peaks typically appear suddenly and have variable mass-to-charge ratios, conventional techniques have also had the problem of making it impossible to associate chromatographic peaks when a noise peak becomes the base peak.

[0033] In contrast, in this embodiment, the molecular weights of the compounds that generated multiple types of multicharged ions are estimated from the mass-to-charge ratios of those ions, and chromatogram peaks with the same molecular weight are matched. That is, in this embodiment, information on the molecular weight of the compound is used instead of the spectral pattern of the mass spectrum (information on the intensity of the mass peak). By performing this processing, chromatogram peaks originating from the same compound can be properly matched even when the mass-to-charge ratio of the base peak varies depending on the analytical conditions or when different types of multicharged ions are generated. Furthermore, even if the measurement data contains significant noise, the molecular weight can be calculated regardless of this noise.

[0034] 4 to 7 show examples of measurements of actual samples. FIG. 4 is a chromatogram based on measurement data from a PDA detector in a measurement using a mobile phase in which acetonitrile (ACN) and methanol (MeOH) were mixed in a ratio of 40% to 60% as an organic solvent. FIG. 5 is a mass spectrum based on measurement data from a mass spectrometer 13 at the peak position of FLP (full length product of 20-mer nucleic acid) in FIG. 4. FIG. 6 is a chromatogram based on measurement data from a PDA detector in a measurement using a mobile phase in which acetonitrile (ACN) and methanol (MeOH) were mixed in a ratio of 60% to 40% as an organic solvent. FIG. 7 is a mass spectrum based on measurement data from a mass spectrometer 13 at the peak position of FLP (full length product of 20-mer nucleic acid) in FIG. 6.

[0035] In the chromatogram of Figure 4, the position (retention time) of the FLP peak is 21.077 min, and in the chromatogram of Figure 5, the position (retention time) of the FLP peak is 12.720 min, indicating that even for the same compound, the time it takes to exit the column changes depending on the analytical conditions.

[0036] In addition, in the mass spectrum of Figure 5, the mass peak at m / z = 795.38 is the base peak, but in the mass spectrum of Figure 7, the mass peak at m / z = 732.56 is the base peak, indicating that the mass-to-charge ratios of the base peaks are different even for the same compound.

[0037] In the case described above, conventional processing based on the base peak would result in a difference between the mass-to-charge ratio of 795.38 of the base peak in the mass spectrum corresponding to the peak at a retention time of 2.077 min in the chromatogram of Figure 4 and the mass-to-charge ratio of 732.56 of the base peak in the mass spectrum corresponding to the peak at a retention time of 12.720 min in the chromatogram of Figure 6, making it difficult to identify that the two are derived from the same compound.

[0038] In contrast, in this embodiment, the molecular weight value calculated based on the mass-to-charge ratios of the mass peaks of multiple multiply charged ions with different valences in the mass spectrum of FIG. 5 and the molecular weight value calculated based on the mass-to-charge ratios of the mass peaks of multiple multiply charged ions with different valences in the mass spectrum of FIG. 7 are the same (7167 in both cases), making it possible to appropriately identify that both are derived from the same compound. Note that in the examples shown in FIGS. 5 and 7, the molecular weights marked with circles are derived from multiply charged ions. Furthermore, the mass peak with a mass-to-charge ratio of 732.53 in FIG. 5 and the mass peak with a mass-to-charge ratio of 732.56 in FIG. 7 are presumed to be peaks derived from isotopes, etc.

[0039] The above embodiment is merely an example and can be modified as appropriate in accordance with the spirit of the present invention.

[0040] In the above embodiment, measurement data was obtained by measuring a sample containing the target compound using liquid chromatograph 10, but measurement data that was previously obtained and stored in a memory unit may also be read and processed in the same manner as above.

[0041] Although the above embodiments and measurement examples have been described with respect to the analysis of nucleic acids, similar configurations can be employed for the analysis of target compounds other than nucleic acids. Furthermore, while the above measurement examples involve the acquisition and analysis of mass spectra of negative ions, mass spectra of positive ions may also be acquired and analyzed. Furthermore, while the above embodiments calculate molecular weights from the mass-to-charge ratios of mass peaks of multiple types of multiply charged ions with different valences that appear in an MS spectrum, other methods may also be used to calculate molecular weights. For example, for multiple types of adduct ions (ions with atoms or molecules attached), the masses of atoms or molecules expected to be attached may be registered in advance, and molecular weights may be calculated from the mass-to-charge ratios of mass peaks of multiple different types of adduct ions. Furthermore, while the above embodiments calculate molecular weights from the mass-to-charge ratios of mass peaks in an MS spectrum, molecular weights may also be calculated from the mass-to-charge ratios of mass peaks in an MS / MS spectrum (product ion spectrum). Alternatively, both the mass-to-charge ratios of mass peaks in an MS spectrum and the mass-to-charge ratios of mass peaks in an MS / MS spectrum may be used.

[0042] In the above embodiment, the measurement data was analyzed with the user inputting the molecular weight of the target compound, i.e., with the molecular weight information of the target compound already known, and the identification unit 38 identified the target compound corresponding to the peak set associated by the peak association unit 37. However, in the present invention, peaks can be associated with each other simply by obtaining the molecular weight value corresponding to each peak of the chromatogram. Therefore, inputting the molecular weight of the target compound is not essential, and a similar configuration can be adopted when analyzing an unknown compound (where the molecular weight of the target compound is unknown). In this case, the above processing by the identification unit 38 is not performed.

[0043] In the above embodiment, a liquid chromatograph 10 equipped with an absorbance detector 12 was used as the main detector, but the type of main detector is arbitrary. However, many inspection standards for pharmaceuticals and the like are stipulated for measurements using an absorbance detector, and an absorbance detector 12 such as a PDA detector has higher robustness and measurement reproducibility than a mass spectrometer. For this reason, it is preferable to use a liquid chromatograph 10 equipped with an absorbance detector 12 as the main detector, as in the above embodiment.

[0044] [Aspect] It will be apparent to those skilled in the art that the above-described exemplary embodiments are examples of the following aspects.

[0045] (Section 1) One aspect of the present invention is an apparatus for processing data acquired by a liquid chromatograph equipped with a column for separating compounds contained in a liquid sample, and a mass spectrometer and a main detector other than a mass spectrometer for detecting the compounds separated by the column, the apparatus comprising: a memory unit in which first chromatogram data obtained by measuring a liquid sample containing a target compound with the main detector under first analytical conditions and first mass analysis data obtained by measuring the liquid sample with the mass spectrometer, and second chromatogram data obtained by measuring the liquid sample with the main detector under second analytical conditions and second mass analysis data obtained by measuring the liquid sample with the mass spectrometer are stored; a chromatogram peak extraction unit that extracts a first peak from the first chromatogram data and a second peak from the second chromatogram data; a mass-to-charge ratio acquiring unit that acquires mass-to-charge ratios of a plurality of ions detected at the position of the first peak from the first mass analysis data, and acquires mass-to-charge ratios of a plurality of ions detected at the position of the second peak from the second mass analysis data; a molecular weight calculation unit that calculates molecular weights of compounds corresponding to the plurality of ions from the mass-to-charge ratios of the plurality of ions detected at the position of the first peak, and calculates molecular weights of compounds corresponding to the plurality of ions from the mass-to-charge ratios of the plurality of ions detected at the position of the second peak; a peak association unit that associates the first peak with the second peak based on the molecular weight calculated for the first peak and the molecular weight calculated for the second peak; Equipped with.

[0046] In the data processing device for a liquid chromatograph according to paragraph 1, the molecular weight of a compound corresponding to a first peak extracted from first chromatogram data is calculated based on the mass-to-charge ratio of the plurality of ions detected by a mass spectrometer at the position of the first peak. The molecular weight of a compound corresponding to the ions is also calculated based on the mass-to-charge ratio of the plurality of ions detected by a mass spectrometer at the position of a second peak extracted from second chromatogram data. The first peak and the second peak are then associated based on the molecular weights calculated for the first peak and the second peak. In this invention, peak tracking is performed based on molecular weights rather than the spectral pattern of the mass spectrum, so peak tracking can be performed regardless of changes in the spectral pattern of the mass spectrum.

[0047] (Section 2) The data processing device for a liquid chromatograph according to paragraph 2 is the data processing device for a liquid chromatograph according to paragraph 1, The target compound includes any one of a protein, a peptide, and a nucleic acid.

[0048] (Section 3) The data processing device for a liquid chromatograph according to paragraph 3 is a data processing device for a liquid chromatograph according to paragraph 1 or 2, The molecular weight calculation unit calculates the molecular weight of a compound corresponding to the plurality of ions by performing multicharged ion analysis of the mass-to-charge ratios of the plurality of ions detected at the position of the first peak, and calculates the molecular weight of a compound corresponding to the plurality of ions by performing multicharged ion analysis of the mass-to-charge ratios of the plurality of ions detected at the position of the second peak.

[0049] When compounds with medium or larger molecular weights, such as proteins, peptides, and nucleic acids, are ionized, multiply charged ions are likely to be generated. Furthermore, the type and amount of multiply charged ions generated vary depending on the analytical conditions. Therefore, as described in Section 2, when the target compound includes any of proteins, peptides, and nucleic acids, the liquid chromatograph data processing device described in Section 1 can be suitably used. In such cases, multiply charged ion analysis can be suitably used when calculating molecular weights from the mass-to-charge ratios of ions, as described in Section 3.

[0050] (Section 4) The data processing device for a liquid chromatograph according to paragraph 4 is the data processing device for a liquid chromatograph according to any one of paragraphs 1 to 3, further comprising: a compound information input receiving unit that receives an input of the molecular weight of the target compound; an identifying unit that identifies a pair of the first peak and the second peak corresponding to the target compound by comparing the molecular weights calculated by the molecular weight calculating unit for the first peak and the second peak associated by the peak associating unit with the input molecular weight; and Equipped with.

[0051] In the data processing device for a liquid chromatograph according to paragraph 4, the peak set (the pair of the first peak and the second peak associated by the peak association unit) corresponding to the target compound whose molecular weight has been input by the user can be easily identified.

[0052] (Section 5) The data processing device for a liquid chromatograph according to paragraph 5 is a data processing device for a liquid chromatograph according to any one of paragraphs 1 to 4, The peak associating unit associates the first peak with the second peak when a difference between the molecular weight calculated for the first peak and the molecular weight calculated for the second peak is smaller than a predetermined value.

[0053] The peak association unit in the liquid chromatographic data processing device described in paragraph 1 can associate the first peak with the second peak when the difference between the molecular weight calculated for the first peak and the molecular weight calculated for the second peak is smaller than a predetermined value, as described in paragraph 5, for example. This allows the first peak to be associated with the second peak even when there is some error due to the mass accuracy of the mass spectrometer, etc. This predetermined value can be determined appropriately depending on the mass accuracy, etc. of the mass spectrometer used. Furthermore, this predetermined value is not limited to an absolute value, and may be a value determined based on the ratio to the molecular weight of the target compound (if the molecular weight of the target compound is known), etc. [Explanation of symbols]

[0054] 1...Liquid chromatograph system 10...Liquid chromatograph 11...Component separation section 12...Absorbance detector 13...Mass spectrometer 20...Analysis and processing device 21...Storage section 31...Compound information input reception section 32…Analysis condition setting section 33...Measurement data acquisition section 34...Chromatogram peak extraction section 35...Mass-to-charge ratio acquisition section 36…Molecular weight calculation section 37...Peak matching section 38...Identification section 39...Analysis result display processing section 41...Input section 42...Display section 51...Scroll bar

Claims

1. An apparatus for processing data acquired by a liquid chromatograph equipped with a column for separating compounds contained in a liquid sample, and a mass spectrometer and a main detector other than a mass spectrometer for detecting the compounds separated by the column, comprising: a memory unit in which first chromatogram data obtained by measuring a liquid sample containing a target compound with the main detector under first analytical conditions and first mass analysis data obtained by measuring the liquid sample with the mass spectrometer, and second chromatogram data obtained by measuring the liquid sample with the main detector under second analytical conditions and second mass analysis data obtained by measuring the liquid sample with the mass spectrometer are stored; a chromatogram peak extracting unit that extracts a first peak from the first chromatogram data and a second peak from the second chromatogram data; a mass-to-charge ratio acquiring unit that acquires mass-to-charge ratios of a plurality of ions detected at the position of the first peak from the first mass analysis data, and acquires mass-to-charge ratios of a plurality of ions detected at the position of the second peak from the second mass analysis data; a molecular weight calculation unit that calculates molecular weights of compounds corresponding to the plurality of ions detected at the position of the first peak from the mass-to-charge ratios of the plurality of ions, and calculates molecular weights of compounds corresponding to the plurality of ions from the mass-to-charge ratios of the plurality of ions detected at the position of the second peak; a peak association unit that associates the first peak with the second peak based on the molecular weight calculated for the first peak and the molecular weight calculated for the second peak; A data processing device for a liquid chromatograph, comprising:

2. 2. The data processing device for a liquid chromatograph according to claim 1, wherein the target compound includes any one of a protein, a peptide, and a nucleic acid.

3. 2. The data processing device for a liquid chromatograph according to claim 1, wherein the molecular weight calculation unit calculates the molecular weights of compounds corresponding to the plurality of ions by performing multicharged ion analysis of the mass-to-charge ratios of the plurality of ions detected at the position of the first peak, and calculates the molecular weights of compounds corresponding to the plurality of ions by performing multicharged ion analysis of the mass-to-charge ratios of the plurality of ions detected at the position of the second peak.

4. moreover, a compound information input receiving unit that receives input of information including the molecular weight of the target compound; a compound identification unit that identifies a target compound corresponding to a pair of the first peak and the second peak associated by the peak association unit by comparing the molecular weight calculated by the molecular weight calculation unit with the input molecular weight; and The data processing device for a liquid chromatograph according to claim 1, further comprising:

5. 2. The data processing device for a liquid chromatograph according to claim 1, wherein the peak correlating unit associates the first peak with the second peak when a difference between the molecular weight calculated for the first peak and the molecular weight calculated for the second peak is smaller than a predetermined value.

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