Multi-specimen analysis method

Abstract

The multi-sample analysis method of the present invention can efficiently perform the analysis of multiple samples having a common main component. The multi-sample analysis method includes: a first analysis step of performing first chromatographic analysis on at least one sample under conditions capable of separating multiple components contained in the at least one sample from each other, thereby obtaining a three-dimensional chromatogram of the at least one sample and extracting spectral data of each of the multiple components contained in the at least one sample; a second analysis step of performing second chromatographic analysis on other samples having the same main component as the at least one sample under conditions for obtaining a three-dimensional chromatogram in a shorter time than the first chromatographic analysis, thereby obtaining a three-dimensional chromatogram of the other samples; and a peak separation step of performing peak separation processing based on the spectral data extracted in the first analysis step on the three-dimensional chromatogram of the other samples obtained in the second analysis step, thereby obtaining peak separation data related to the other samples in which the peaks of the multiple components contained in the other samples are separated from each other.

Description

Technical Field

[0001] The present invention relates to a multi-sample analysis method, which obtains chromatogram data in a state where peaks containing components are separated from each other for a plurality of samples having common main components. Background Art

[0002] In the pharmaceutical industry and the like, it is sometimes necessary to quantify the concentrations of impurities contained in each of a large number of samples having common main components. As a method for quantifying each of the multiple components contained in a sample, liquid chromatography analysis is usually performed (see Patent Document 1).

[0003] [Prior Art Documents]

[0004] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2018 / 027880 Summary of the Invention

[0006] [Problems to be Solved by the Invention]

[0007] In order to quantify each of the multiple components contained in a sample using liquid chromatography analysis, it is necessary to separate the peaks of the multiple components from each other. When multiple components having similar properties are mixed in a sample, in order to completely separate these component peaks, it is necessary to adjust the filler packed in the analytical column, or extend the total length of the analytical column, or make the inner diameter of the analytical column thinner to extremely reduce the flow rate of the mobile phase, etc. to adjust the analysis conditions. In addition, as a result of performing analysis under such analysis conditions, the time required for all components in the sample to elute from the analytical column becomes longer, and it takes a long time until the analysis of one sample is completed. However, if the number of samples to be analyzed is huge, when performing such a long-time analysis on all of these samples, the costs in terms of cost, time, and labor become huge and unrealistic.

[0008] The present invention has been made in view of the above problems, and an object thereof is to provide a multi-sample analysis method capable of efficiently performing analysis of a plurality of samples having common main components.

[0009] [Means for Solving the Problems]

[0010] The multi-sample analysis method of the present invention includes: a first analysis step, for at least one sample, performing first chromatographic analysis using a photodiode array under conditions that can separate multiple components contained in the at least one sample from each other, thereby obtaining a three-dimensional chromatogram of the at least one sample, and extracting spectral data of each of the multiple components contained in the at least one sample from the three-dimensional chromatogram of the at least one sample; a second analysis step, for other samples having the same main component as the at least one sample, performing second chromatographic analysis using a photodiode array under conditions that can obtain a three-dimensional chromatogram in a shorter time than the first chromatographic analysis, thereby obtaining a three-dimensional chromatogram of the other samples; and a peak separation step, for the three-dimensional chromatogram of the other samples obtained in the second analysis step, obtaining peak separation data related to the other samples in which the peaks of the multiple components contained in the other samples are separated from each other by applying peak separation processing based on the spectral data extracted in the first analysis step.

[0011] That is, in the present invention, for a part of multiple samples having a common main component with each other, a first chromatographic analysis with high resolution and long time is performed to obtain spectral data related to multiple components. For the other remaining samples, a second chromatographic analysis with lower resolution but capable of obtaining a three-dimensional chromatogram at high speed compared to the first chromatographic analysis is performed. For the three-dimensional chromatogram with relatively low resolution obtained by the second chromatographic analysis, peak separation processing based on the spectral data obtained by performing the first chromatographic analysis is applied, thereby obtaining peak separation data related to the other remaining samples.

[0012] [Effects of the Invention]

[0013] As described above, according to the multi-sample analysis method of the present invention, only for a part of multiple samples having a common main component with each other, a first chromatographic analysis with high resolution and long time is performed, and for other samples, a second chromatographic analysis with lower resolution but capable of obtaining a three-dimensional chromatogram at high speed compared to the first chromatographic analysis is performed. Thus, even if the first chromatographic analysis that requires a long time is not performed on all of the multiple samples having a common main component, high-precision peak separation data related to all samples can be obtained. Therefore, the analysis of multiple samples having a common main component can be efficiently performed. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a schematic structural diagram showing an example of the structure of a liquid chromatography analysis device.

[0015] Figure 2 It is a conceptual diagram schematically showing an embodiment of the multi-sample analysis method.

[0016] Figure 3 It is a flowchart showing an example of the sequence of the embodiment.

[0017] [Description of the drawing reference numerals]

[0018] 2: Liquid delivery pump

[0019] 4: Syringe

[0020] 6: Analytical column

[0021] 8: PDA detector

[0022] 10: Oven

[0023] 12: Operation processing device Detailed implementation manner

[0024] Hereinafter, an embodiment of the multi-sample analysis method of the present invention will be described with reference to the drawings.

[0025] First, use Figure 1 To illustrate a structural example of a liquid chromatography analysis device for implementing the multi-sample analysis method.

[0026] The liquid chromatography analysis device includes a liquid delivery pump 2, a syringe 4, an analytical column 6, a photo-diode array (PDA) detector 8, an oven 10, and an operation processing device 12. The liquid delivery pump 2 delivers the mobile phase. Downstream of the liquid delivery pump 2, a syringe 4, an analytical column 6, and a PDA detector 8 are connected in sequence from upstream. The syringe 4 is used to inject a sample into the mobile phase delivered by the liquid delivery pump 2. The analytical column 6 is used to separate the components in the sample injected into the mobile phase by the syringe 4 from each other. The analytical column 6 is housed inside the oven 10 and is controlled at a temperature corresponding to the analysis conditions. The PDA detector 8 measures the time change of the absorbance of the eluate from the analytical column 6 in each wavelength band. That is, analytical data is obtained using the PDA detector 8, and this analytical data includes chromatogram information indicating the time change of the absorbance in each measurement wavelength band and spectral information indicating the spectrum at each time during the analysis.

[0027] The arithmetic processing unit 12 is implemented by a computer device including a Central Processing Unit (CPU) (central arithmetic unit) and a data storage device, etc. Analytical data output from the PDA detector 8 is input to the arithmetic processing unit 12. The arithmetic processing unit 12 has the following functions: using the analytical data output from the PDA detector 8, performing various analysis processes. As the analysis processing functions based on the arithmetic processing unit 12, there are a function of creating a three-dimensional chromatogram combining chromatogram information and spectral information related to a sample, a function of extracting spectral data of each component separated from each other by the analytical column 6 based on the created three-dimensional chromatogram, and further a function of performing peak separation processing on the three-dimensional chromatogram using the extracted spectral data. That is, the arithmetic processing unit 12 has the following functions: accumulating the spectral data extracted from the three-dimensional chromatogram by performing an analysis process once in a database, and using the spectral data accumulated in the database for peak separation processing related to other three-dimensional chromatograms.

[0028] Next, Figure 2 The concept of the multi-sample analysis method will be described.

[0029] Suppose there are multiple samples 1 to n to be analyzed. The main components of these samples 1 to n are common to each other. The purpose of the analysis is to quantify the concentrations of each of the multiple components contained in samples 1 to sample n, and it is implemented by a liquid chromatography device having the Figure 1 structure shown.

[0030] For sample 1, high separation conditions that can obtain a high peak separation degree between the main component peak and the peaks of nearby components are explored, and the analysis is performed under the high separation conditions (first chromatographic analysis). The analysis conditions include elements such as the inner diameter and length of the analytical column 6, the type of packing material of the analytical column 6, the set temperature of the oven 10, the composition of the mobile phase delivered by the liquid delivery pump 2, the liquid delivery flow rate based on the liquid delivery pump 2, and the injection conditions based on the syringe 4. These elements are determined in such a way that at least the peak of the main component of sample 1 and the peaks of other components appearing near the main component are separated individually, preferably in such a way that all components contained in sample 1 are separated.

[0031] Through the first chromatographic analysis, a three-dimensional chromatogram separating the peaks of the multiple components contained in sample 1 from each other is obtained, and spectral data of each component separated in the first chromatographic analysis is obtained from the three-dimensional chromatogram.

[0032] Here, generally, in an analysis under conditions optimized such that the peaks of multiple components in a sample are completely separated as in the first chromatographic analysis, it takes a relatively long time until all components elute from the analytical column 6. Therefore, if such a high-resolution analysis is performed on all of the multiple samples 1 to n, it takes an extremely long time until analytical results related to all of the samples 1 to n are obtained.

[0033] Therefore, for each of the samples 2 to n other than the sample 1, an analysis (second chromatographic analysis) is performed under conditions such that all components elute from the analytical column 6 in a shorter time than in the first chromatographic analysis. In the second chromatographic analysis, an analytical column different from that used in the first chromatographic analysis can be used. That is, the inner diameter, length, and / or type of packing material of the first analytical column used in the first chromatographic analysis and the second analytical column used in the second chromatographic analysis may be different from each other.

[0034] In the three-dimensional chromatogram obtained by the second chromatographic analysis, the intervals between the peaks become shorter and the resolution decreases compared with the first chromatographic analysis, and it is possible that the peaks of multiple components overlap each other. At this time, if the respective analytical data obtained by the second chromatographic analysis are left as they are, they are incomplete analytical data that cannot be used for quantifying the components contained in the samples 2 to n.

[0035] In order to eliminate the incompleteness of the analytical data obtained by the second chromatographic analysis, a peak separation process is applied to each of the analytical data. The peak separation process is a process for estimating the shapes and sizes of the peaks of multiple components overlapping each other on a chromatogram. In the peak separation process, in addition to an algorithm (for example, refer to International Publication No. 2016 / 035167) for estimating the chromatogram of each component by substituting a model function (peak model) such as an Exponential Modified Gaussian (EMG) function into the waveform of the actual chromatogram, an algorithm for mathematically estimating the chromatogram of each component by applying matrix factorization such as Non-negative Matrix Factorization (NMF) to the original three-dimensional chromatogram data without using a model function, or a principal component analysis algorithm such as Principal Component Analysis (PCA) can also be used.

[0036] In the peak separation process, even in a state where there is completely no information related to the components contained in the sample, the number, shape, and size of the peaks overlapping each other on the three-dimensional chromatogram data can be estimated. However, if spectral data of at least one of the components whose peaks overlap each other exist, the estimation accuracy of the number, shape, and size of the overlapping peaks can be improved by using the spectral data as basic information for the peak separation process.

[0037] In the embodiment, in the peak separation process applicable to each piece of analysis data obtained by the second chromatography, at least a part of the spectral data extracted from the analysis data of the first chromatography is used. Since the main components of Samples 1 to n are common to each other, the spectral data related to the main components extracted from the analysis data of the first chromatography can be used for the peak separation process. As a result, the accuracy of the estimation results obtained by the peak separation process for the analysis data of Samples 2 to n is improved. In addition, when the contained components of Samples 1 to n are all the same, by using the spectral data of all the components isolated in the first chromatography for the peak separation process, high-precision peak separation data related to Samples 2 to n can be obtained.

[0038] Use Figure 3 The flowchart of FIG. is used to illustrate an example of the sequence of the multi-sample analysis method of the embodiment.

[0039] For a part of the majority of samples to be analyzed (as long as there is one or more samples), a high-resolution analysis (first chromatography) is performed such that at least the main component peaks are completely separated from other component peaks (step 101). The analysis data obtained by the first chromatography is analyzed using the arithmetic processing unit 12, and thereby the spectral data related to each component isolated in the high-resolution analysis is extracted (step 102). For a part of the samples, peak separation data required for quantifying each component is obtained by the first chromatography.

[0040] Next, for the remaining samples other than the part of the samples for which the first chromatography has been performed, a low-resolution analysis (second chromatography) is performed in which the resolution between peaks is lower than that of the first chromatography but the analysis data can be obtained in a shorter time than the first chromatography (step 103). Thereby, a three-dimensional chromatogram related to the remaining samples is obtained (step 104).

[0041] For the three-dimensional chromatogram obtained by the second chromatography, a peak separation process based on the spectral data extracted in step 102 is applied (step 105). Through the peak separation process, a plurality of peaks overlapping with each other in the initially incomplete analysis data are separated with high estimation accuracy and become peak separation data that can be used for quantifying each component contained in the sample. That is, by performing the peak separation process of step 105, analysis results equivalent to those obtained by performing a long-time high-resolution analysis for all samples can be obtained.

[0042] As described above, in this embodiment, even in the case where there is a huge number of samples to be analyzed, it is possible to perform the first chromatographic analysis for at least one of these samples under analysis conditions set in such a way that the main component is isolated from other components, which takes a long time (and sometimes is costly). However, for the remaining samples, only the second chromatographic analysis is performed in a short time (and sometimes at low cost), and an analysis result equivalent to the case where a high-resolution analysis is performed for all samples over a long time is obtained. Therefore, the time required to obtain peak separation data related to all samples can be significantly reduced.

[0043] The embodiments described above merely illustrate the implementation modes of the multi-sample analysis method of the present invention. The implementation modes of the multi-sample analysis method of the present invention are as follows.

[0044] In one implementation mode of the multi-sample analysis method of the present invention, it includes: a first analysis step of performing, for at least one sample, a first chromatographic analysis using a photodiode array under conditions capable of separating multiple components contained in the at least one sample from each other, thereby obtaining a three-dimensional chromatogram of the at least one sample, and extracting spectral data of each of the multiple components contained in the at least one sample from the three-dimensional chromatogram of the at least one sample; a second analysis step of performing, for other samples having the same main component as the at least one sample, a second chromatographic analysis using a photodiode array under conditions for obtaining a three-dimensional chromatogram in a shorter time than the first chromatographic analysis, thereby obtaining a three-dimensional chromatogram of the other samples; and a peak separation step of applying peak separation processing based on the spectral data extracted in the first analysis step to the three-dimensional chromatogram of the other samples obtained in the second analysis step, thereby obtaining peak separation data related to the other samples in which the peaks of the multiple components contained in the other samples are separated from each other.

[0045] In the first form of the above-described implementation mode, the inner diameter, overall length, and / or packing material of the first analysis column used in the first chromatographic analysis are different from those of the second analysis column used in the second chromatographic analysis. For example, in the first chromatographic analysis, a relatively expensive analysis column can be used as the first analysis column in order to obtain a high peak separation degree, and in the second chromatographic analysis, an analysis column less expensive than the first analysis column can be used. Thereby, the cost required for analyzing all of the multiple samples can be reduced.

[0046] In the second form of the above-described implementation mode, the flow rate of the mobile phase in the second chromatographic analysis is greater than the flow rate of the mobile phase in the first chromatographic analysis. This second form can be combined with the first form.

[0047] In the third form of the described embodiment, in the case where there are two or more specimens in which the main components are common to each other, the first analysis step is performed on one specimen among the two or more specimens, and spectral data of each of the multiple components contained in the one specimen is extracted. The second analysis step is performed on the remaining specimens among the two or more specimens, three-dimensional chromatograms related to the respective remaining specimens are obtained, and the peak separation step is performed on the obtained three-dimensional chromatograms related to the respective remaining specimens to obtain peak separation data related to the respective remaining specimens. According to this form, only one specimen is subjected to the first chromatographic analysis that requires a long time, and the other remaining specimens are subjected to the second chromatographic analysis that can be completed in a relatively short time. Therefore, the time required until the analysis of all specimens is completed can be significantly shortened. This third form can be combined with the first form and / or the second form.

[0048] In the fourth form of the described embodiment, the first chromatographic analysis and the second chromatographic analysis are liquid chromatography analyses. In addition, in the "chromatographic analysis" of the present invention, not only liquid chromatography analysis is included, but also gas chromatography analysis may be included. This fourth form can be combined with the first form, the second form, and / or the third form.

[0049] In the fifth form of the described embodiment, in the peak separation process, an algorithm for estimating the peaks of each component by substituting a model function is used, or an algorithm for mathematically estimating the peaks of each component by using matrix decomposition without using the model function is used. This fifth form can be combined with the first form, the second form, the third form, and / or the fourth form.

Claims

1. A multi-sample analysis method, characterized in that, Comprising: A first analysis step, for at least one specimen containing a first component, performing a first chromatographic analysis using a photodiode array under conditions capable of separating the multiple components contained in the at least one specimen from each other, thereby obtaining a first three-dimensional chromatogram, and extracting spectral data of the first component whose peak is isolated separately from the peaks of other components from the first three-dimensional chromatogram; A second analysis step, performing a second chromatographic analysis using a photodiode array for other specimens containing the first component, wherein the second chromatographic analysis uses a second analytical column having an inner diameter larger than that of the first analytical column used in the first chromatographic analysis or a shorter overall length, and / or uses a mobile phase flow rate higher than that of the first chromatographic analysis, to obtain a three-dimensional chromatogram in a shorter time with a lower resolution than the first chromatographic analysis, thereby obtaining a second three-dimensional chromatogram, and the second three-dimensional chromatogram includes an unresolved peak formed by the overlap of the peak of the first component and the peaks of other components; And A peak separation step, for the second three-dimensional chromatogram, by applying peak separation processing to the spectral data of the first component, estimating the number, shape or size of the peaks contained in the unresolved peak.

2. The multi-specimen analysis method according to claim 1, wherein In the case where there are two or more specimens in which the main components are common to each other, Performing the first analysis step for one specimen among the two or more specimens, and extracting spectral data of each of the multiple components contained in the one specimen, Performing the second analysis step for the remaining specimens among the two or more specimens, obtaining three-dimensional chromatograms related to each of the remaining specimens, and performing the peak separation step for the obtained three-dimensional chromatograms related to each of the remaining specimens, to obtain peak separation data related to each of the remaining specimens.

3. The multi-specimen analysis method according to claim 1 or 2, wherein The first chromatographic analysis and the second chromatographic analysis are liquid chromatographic analyses.

4. The multi-specimen analysis method according to claim 1 or 2, wherein In the peak separation processing, an algorithm for estimating the peaks of each component by substituting a model function is used, or an algorithm for mathematically estimating the peaks of each component using matrix decomposition without using the model function is used.

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