Isotope distribution ratio evaluation program, isotope distribution ratio evaluation method, isotope distribution ratio evaluation device, and metabolic flux analyzer

Abstract

To provide an isotope distribution ratio evaluation method capable of performing reliable metabolic flux analysis by evaluating the reliability of an isotope distribution ratio.SOLUTION: There is disclosed an isotope distribution ratio evaluation method. The method includes: quantifying multiple isotopes from a fitting curve that is obtained by fitting measurement values of mass chromatograms of the multiple isotopes with different mass numbers with a predetermined function regarding compound fragments of a measurement target; calculating quantitative reliability indices of the multiple isotopes that indicate the reliability of an isotope distribution ratio of the compound fragments and quantitative results of the isotopes by using the quantitative results of the multiple isotopes; and calculating a reliability index of the isotope distribution ratio and displaying the same on a display screen.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] The present disclosure relates to an isotope distribution ratio evaluation program, an isotope distribution ratio evaluation method, an isotope distribution ratio evaluation apparatus, and a metabolic flux analysis apparatus.

Background Art

[0002] In regenerative medicine and drug discovery support, it is necessary to use high-quality cells cultured under appropriate conditions, and culture quality management is required. Currently, development of methods for managing culture quality using techniques such as component analysis of culture medium supernatant is underway. In component analysis of culture medium supernatant, changes in the concentrations of nutrient components such as amino acids and glucose, and metabolic components such as pyruvic acid and lactic acid are monitored to manage culture quality. However, sufficient knowledge regarding the management of high-level culture quality required during induction of differentiation of stem cells and the like has not been obtained. The inventors have hitherto been promoting the development of a technique for systematically selecting metabolic components to be monitored by identifying metabolic pathways that are key to cell culture quality control and identifying metabolic components involved in those metabolic pathways through metabolic flux analysis. In metabolic flux analysis, first, 13 after culturing cells in a medium containing a substrate labeled with 13 C, metabolites extracted from the cells are mass-analyzed to determine the isotope distribution ratio of the metabolites. Then, based on a metabolic model defined by the network of intracellular metabolic reactions, the state of diffusion of 13 C atoms in the metabolites is calculated to reproduce the isotope distribution ratio of the metabolites evaluated by mass spectrometry. From this result, the reaction rate of the metabolites in each metabolic reaction is estimated, and the metabolic pathway that is key to cell culture quality control is identified. Regarding metabolic flux analysis, the details of the technique are described, for example, in Non-Patent Document 1.

[0003] Regarding the method for evaluating the isotope distribution ratio required for metabolic flux analysis, a plurality of documents have been confirmed. For example, Patent Document 1 discloses that, in order to evaluate the isotope distribution ratio, a plurality of metabolite samples with different concentrations are subjected to gas chromatography-mass spectrometry under the same conditions to obtain data, and data for calculating the isotope distribution ratio is selected from the viewpoints of the signal intensity of the metabolite mass chromatogram (a graph plotting the signal intensity against the elution time), the number of metabolites, and the number of isotopes. Then, each metabolite is quantified from the selected data, and the isotope distribution ratio is calculated.

[0004] In addition, Non-Patent Document 2 discloses a method for quantifying metabolites from a fitting curve obtained by fitting a mass chromatogram with a function and the results of examining a plurality of functions applicable to the fitting of the mass chromatogram.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0007] However, in a sample with a low concentration of metabolites, due to the influence of impurities in the sample, etc., the variation in the isotope distribution ratio evaluated by mass spectrometry is large, and even if the method disclosed in the above-mentioned literature is used, the reliability of the calculated isotope distribution ratio cannot be determined. The isotope distribution ratio is calculated based on the quantitative results of a plurality of isotopes of the metabolite to be evaluated. Therefore, the reliability of the isotope distribution ratio needs to be evaluated based on the reliability of individual isotope quantification.

[0008] However, the above-mentioned literature does not mention anything about such reliability evaluation techniques. Therefore, in order to perform more reliable metabolic flux analysis, it is desirable to be able to evaluate the reliability of the obtained isotope distribution ratio. The present disclosure has been made in view of such a situation, and provides a technique for evaluating the reliability of an isotope distribution ratio.

Means for Solving the Problems

[0009] In order to solve the above problems, the present disclosure is an isotope distribution ratio evaluation method executed by a processor, the processor obtains measurement values of mass chromatograms of a plurality of isotopes with different mass numbers for a compound fragment to be measured, the processor quantifies the plurality of isotopes from a fitting curve obtained by fitting the mass chromatogram with a predetermined function, the processor calculates an isotope distribution ratio of the compound fragment using the quantitative results of the plurality of isotopes, the processor calculates a plurality of isotope quantification reliability indicators indicating the reliability of the quantitative results of the plurality of isotopes, the processor calculates a reliability indicator of the isotope distribution ratio using the plurality of isotope quantification reliability indicators, outputs the reliability indicator of the isotope distribution ratio to an output unit that outputs the calculation result, and proposes an isotope distribution ratio evaluation method including the above.

[0010] Further features related to the present disclosure will become apparent from the description herein and the accompanying drawings. Also, aspects of the present disclosure are achieved and realized by elements and combinations of various elements and the aspects of the following detailed description and the appended claims. It should be understood that the description herein is merely exemplary and is not intended to limit the claims or the scope of any application in any way.

Advantages of the Invention

[0011] According to the technology of the present disclosure, it becomes possible to evaluate the reliability of the isotope distribution ratio. Also, it becomes possible to perform highly reliable metabolic flux analysis using a highly reliable isotope distribution ratio.

Brief Description of the Drawings

[0012]

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Mode for Carrying Out the Invention

[0013] This embodiment relates to a method and program for evaluating the isotope distribution ratio in metabolites, and a method and program for evaluating metabolic flux. Further, this embodiment discloses a method for evaluating a reliability index of the isotope distribution ratio, and a highly reliable metabolic flux analysis method using the reliability index.

[0014] Hereinafter, this embodiment will be described with reference to the accompanying drawings. In the accompanying drawings, functionally identical elements may be denoted by the same reference numerals, and redundant descriptions will be omitted. Note that the accompanying drawings show specific embodiments and examples in accordance with the principles of the present disclosure, but these are for understanding the present disclosure and are not used to limit the interpretation of the present disclosure.

[0015] In this embodiment, although the description is made in sufficient detail for those skilled in the art to implement the present disclosure, other implementations and forms are possible, and it is necessary to understand that changes in configuration and structure and replacement of various elements can be made without departing from the scope and spirit of the technical idea of the present disclosure. Therefore, the following description should not be construed as being limited thereto.

[0016] <Overview of the Isotope Distribution Ratio Evaluation Program> Figure 1A is a diagram showing an overview of an isotope distribution ratio evaluation program according to an embodiment of the present disclosure. The isotope distribution ratio evaluation program of the present embodiment includes a step of measuring, by mass spectrometry, mass chromatograms of a plurality of isotopes having different mass numbers for a compound fragment to be measured (step 1); a step of quantifying a plurality of isotopes from a plurality of fitting curves obtained by fitting the mass chromatograms of the plurality of isotopes with a function (step 2); a step of calculating an isotope distribution ratio of the compound fragment using the quantification results of the plurality of isotopes obtained in the step of quantifying the plurality of isotopes (step 3); and a step of displaying the isotope distribution ratio on a program screen (step 4). Further, the step of quantifying a plurality of isotopes (step 2) includes a step of evaluating a quantification reliability index of the plurality of isotopes. Furthermore, the isotope distribution ratio evaluation program of the present embodiment includes a step of calculating a reliability index of the isotope distribution ratio using the quantification reliability indices of the plurality of isotopes (step 5); and a step of displaying the reliability index of the isotope distribution ratio on a program screen (step 6).

[0017] (i) Step 1 First, a step (step 1) of measuring, by mass spectrometry, mass chromatograms of a plurality of isotopes having different mass numbers for a compound fragment to be measured will be described. Generally, it is known that there is a distribution in the number of neutrons in the constituent atoms of a compound. Therefore, the compound fragment to be measured also has a distribution in mass number. In this specification, following common practice, the isotope with the smallest mass number is denoted as M+0, and the isotope with n more neutrons in the compound compared to M+0 is denoted as M+n.

[0018] In step 1, a measurement sample containing cell metabolites, obtained by pretreating the culture supernatant during cell culture, is mass-analyzed to measure mass chromatograms of a plurality of isotopes having different mass numbers for the compound fragment to be measured. The compound fragment to be measured includes metabolites that have been ionized. Examples of metabolites include pyruvic acid, lactic acid, alanine, glycine, valine, leucine, succinic acid, fumaric acid, serine, threonine, α-ketoglutaric acid, malic acid, aspartic acid, glutamic acid, glutamine, citric acid, and the like.

[0019] The pretreatment of the culture supernatant has an impurity removal process for removing impurities from the culture supernatant as much as possible. Examples of the pretreatment method include solid-phase adsorption method, ultrafiltration method, etc. Further, in order to facilitate mass spectrometry, the sample may be derivatized with a derivatizing agent as necessary. Examples of the derivatizing agent include methoxamine, N-methyl-N-tert-butyldimethylsilyl trifluoroacetamide (MTBSTFA), etc. Also, examples of the mass spectrometry method include gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis chromatography (CE-MS), etc.

[0020] Note that step 1 can also be substituted by a step of importing the data of the measured mask chromatogram into the program of this embodiment. In that case, software for importing the data of the measured mask chromatogram may be implemented in the program of this embodiment.

[0021] (ii) Step 2 Next, the process of quantifying a plurality of isotopes from the fitting curves obtained by fitting a plurality of mass chromatograms with a function (Process 2) will be described. In this process, a plurality of mass chromatograms are fitted using a function according to an algorithm such as the least squares method (for example, it can be fitted using the method disclosed in Non-Patent Document 2). Using the fitting parameters obtained at that time, a plurality of isotopes are quantified, and the quantification reliability index of the plurality of isotopes is evaluated. As the function used for fitting, it is possible to use a Gaussian function, a Bi-Gaussian function, an Exponentially modified Gaussian function, a Fraser-Suzuki function, a Log-normal function, a Harrhoff-van der Lide function, a Cauchy-Gaussian function, a Chesler-Cram function, and the like. Also, the isotope can be quantified from the area value of the mass chromatogram obtained from the fitting curve (the isotope ratio of each isotope can be calculated from the area ratio of the chromatogram). For example, when using the Gaussian function defined by Equation (1), the isotope can be suitably quantified using Equation (2).

[0022]

Number

[0023]

Number

[0024] In addition, the quantitative reliability index for a plurality of isotopes is not particularly limited as long as it is a fitting parameter obtained by fitting a plurality of mass chromatograms with a function, or a value calculated using the fitting parameter. The fitting parameter may be any value obtained as a result of fitting. For example, it includes fitting variables included in the function, the coefficient of determination of the fitting, the sum of squared residuals, and the like. The quantitative reliability index of the isotope can be calculated using Equation (3).

[0025]

Number

[0026] Here, n i is the number of data points of the mass chromatogram of isotope i, Y Di,j is the signal intensity of the j-th data point of the mass chromatogram of isotope i, Y Fi,j is Y Di,j and is the signal intensity of the fitting curve of the mass chromatogram of isotope i at the same elution time as Y, I max,F,i is the maximum value of the signal intensity of the fitting curve of the mass chromatogram of isotope i. By using Equation (3), the quantitative reliability index of the isotope can be calculated based on the deviation of the measurement data from the fitting curve. In a sample with a low metabolite concentration, the influence of impurities in the sample appears as the deviation between the measurement data and the fitting curve. Therefore, the reliability index of the isotope distribution ratio can be preferably calculated by using Equation (3).

[0027] In addition, as another example of the quantitative reliability index, the coefficient of determination (r 2) can be mentioned. The coefficient of determination is an index indicating the goodness of fit of the model of the fitting formula for the measurement data, and is a value that can be easily obtained by fitting the mask chromatogram using a general-purpose programming language. The closer the coefficient of determination is to 1, the more accurately the fitting has been done. Therefore, by using the coefficient of determination as a quantitative reliability index, it becomes possible to reduce the computational amount of the program and contribute to shortening the calculation time. The coefficient of determination is not particularly limited as long as it is an index indicating the goodness of fit of the model of the fitting formula for the measurement data. As an example of the definition formula of the coefficient of determination, Equation (4) is known.

[0028]

Number

[0029] (iii) Step 3 Next, the step of calculating the isotope distribution ratio of the compound fragment using the quantitative results of a plurality of isotopes obtained in Step 2 (Step 3) will be described. The isotope distribution ratio can be calculated using Equation (5).

[0030]

Number

[0031] In formula (5), it is preferable to calculate the isotope distribution ratio considering four or more isotopes. FIG. 2 is a diagram showing the evaluation results of the number of isotopes considered for calculating the isotope distribution ratio and the deviation from the true value of the isotope distribution ratio. In this evaluation, a commercially available compound was measured by GC-MS, and the isotope distribution ratio evaluated considering six isotopes (M+0 to M+5) from the mass chromatogram of each compound fragment was taken as the true value. From FIG. 2, it was found that when evaluating the isotope distribution ratio considering four (M+0 to M+3) or more isotopes, the isotope distribution ratio can be evaluated with an accuracy within an error of 1%. Therefore, it is preferable to calculate the isotope distribution ratio considering four or more isotopes.

[0032] Also, in the evaluation program and evaluation method of this embodiment, in addition to the isotope distribution ratio including the contribution of naturally occurring stable isotopes, an isotope distribution ratio obtained by subtracting and correcting the contribution of naturally occurring stable isotopes may also be output. In metabolic flux analysis, carbon in metabolites is labeled using 13 C, which is a stable isotope of carbon, and 13 the flux of intracellular metabolic reactions is estimated by simulating the isotope distribution ratio of the metabolites after 13 C labeling. In this case, it is difficult to perform the simulation considering the contribution of natural stable isotopes. Therefore, in metabolic flux analysis, it is common to proceed with the analysis using the isotope distribution ratio obtained by subtracting and correcting the contribution of natural stable isotopes present in the metabolites before 13 C labeling. The correction of the isotope ratio can be performed using the matrix transformation described in the literature (R. Nilsson, Mathematical Biosciences, 330, 108481 (2020)) using the ratio of naturally occurring stable isotopes.

[0033] (iv) Step 4 Next, the step of displaying the isotope distribution ratio on the program screen (Step 4) will be described. Step 4 has no particular restrictions as long as it is a step of displaying the isotope distribution ratio evaluated in Step 3 on the program screen. Also, Step 4 may be included in Step 6, which will be described later.

[0034] (v) Step 5 Next, a step of calculating a reliability index of the isotope distribution ratio using the quantitative reliability indices of a plurality of isotopes evaluated in Step 3 (Step 5) will be described. In Step 5, the reliability index of the isotope distribution ratio is calculated according to the defined calculation formula using the quantitative reliability indices of a plurality of isotopes. The calculation formula is not particularly limited as long as it can calculate the reliability index of the isotope distribution ratio using the quantitative reliability index. As an example of the calculation formula, Equation (6) can be used.

[0035]

Equation

[0036] Here, R is the reliability index of the isotope distribution ratio, w i is the weight of the quantitative reliability index of isotope i, and d i is the quantitative reliability index of isotope i. When the coefficient of determination of fitting is used as the quantitative reliability index of isotope i, the higher the reliability index, the higher the reliability. On the other hand, when Equation (3) is used as the quantitative reliability index of isotope i, the lower the reliability index, the higher the reliability.

[0037] The weight of the quantitative reliability index of isotope i in Equation (6) is not particularly limited as long as it indicates the contribution of the quantitative reliability index of isotope i to the reliability index of the isotope distribution ratio. When the contribution of the quantitative reliability index of isotope i to the reliability index is constant regardless of the type of isotope, it is given by Equation (7).

[0038]

Equation

[0039] Here, w i is the weight of the quantitative reliability index of isotope i. For example, in the case of six isotopes (M+0 to M+5), i takes values from 0 to 5. Also, when the contribution of the quantitative reliability index of isotope i to the reliability index is proportional to the ratio of the amount of isotope i to the total amount of the isotope, the weight of the coefficient of determination of isotope i is given by Equation (8).

[0040]

Number

[0041] Here, w i is the weight of the quantitative reliability index of isotope i, and A i is the amount of isotope i. By using the weight of the quantitative reliability index of isotope i calculated using Equation (7) or Equation (8), the reliability index of the isotope distribution ratio can be suitably calculated by Equation (6) according to the contribution of the coefficient of determination of isotope i to the reliability index.

[0042] Based on the reliability index of the isotope distribution ratio obtained in step 5, it is possible not only to obtain the reliability of the isotope distribution ratio but also to gain insights into the measurement conditions of mass spectrometry. For example, if leading or trailing of the mass chromatogram occurs as a result of mass spectrometry, the shape of the mass chromatogram is distorted, so the reliability index of the isotope distribution ratio obtained using a fitting function (such as a Gaussian function) that does not account for the leading or trailing of the mass chromatogram decreases. However, the reliability index of the isotope distribution ratio obtained using a fitting function (such as an Exponentially modified Gaussian function) that accounts for the leading or trailing of the mass chromatogram shows a good value. On the other hand, if a peak of an impurity overlaps with the mass chromatogram of the compound fragment to be measured as a result of mass spectrometry, the reliability index of the isotope distribution ratio takes a low value regardless of whether it corresponds or not to the leading or trailing of the mass chromatogram. From the above examples, by comprehensively evaluating the reliability indices of the isotope distribution ratios obtained using a plurality of different fitting functions (for example, as described later, excluding metabolites with a low isotope distribution ratio from the targets of metabolic flux analysis), it is possible to gain insights into the cause of the decrease in the reliability index. Furthermore, based on the obtained insights, it is also possible to estimate the bad mode of the mass chromatogram. For example, in the metabolic flux analyzer 100 described later, when the reliability index of a specific isotope distribution ratio shows a value equal to or lower than a predetermined threshold, the processor 101 may display a sign (for example, black inversion display in FIG. 6C described later) indicating the possibility (bad mode) of a bad mass chromatogram on the program screen.

[0043] (vi) Step 6 Next, the process of displaying the reliability index of the isotope distribution ratio on the program screen will be described. As step 6, there are no particular restrictions on the form as long as it is a process of displaying the reliability index of the isotope distribution ratio calculated in step 5 on the program screen. Also, step 6 may be performed simultaneously with step 4. Furthermore, in step 4 or step 6, together with the isotope distribution ratio and the reliability index of the isotope distribution ratio, the mass chromatogram measured in step 1 and the fitting curve obtained in step 2 may be displayed on the same screen. This display is effective for visually associating the reliability index with the presence or absence of contaminants in the mass chromatogram. Also, a GUI for selecting the display methods of the isotope distribution ratio, the reliability index of the isotope distribution ratio, the mass chromatogram measured in step 1, and the fitting curve obtained in step 2 may be implemented in this program so that the user of this program can select the display method.

[0044] <Metabolic Flux Analysis> In metabolic flux analysis, first, 13 After culturing cells in a medium containing a substrate labeled with C, the metabolites extracted from the cells are mass-analyzed to determine the isotope distribution ratio. Then, based on a metabolic model defined by the network of metabolic reactions in the cell, 13 The state of diffusion of C atoms in the metabolites is calculated to reproduce the isotope distribution ratio of the metabolite to be analyzed evaluated by mass spectrometry. From this result, the reaction rate (metabolic flux) of the metabolite in each metabolic reaction is estimated. It is possible to estimate the metabolic flux from the isotope distribution ratio of the metabolite using metabolic flux estimation software (such as OpenFLUX and OpenMebius) publicly available on the Internet or the like.

[0045] In metabolic flux analysis, it is necessary to determine (i) the network of metabolic enzyme reactions in the cell (intracellular metabolic model) and (ii) the metabolites to be analyzed at the start of the analysis. In the isotope distribution ratio evaluation program of the present embodiment, first, metabolites included in the intracellular metabolic model are picked up from the metabolites whose isotope distribution ratios are calculated by mass spectrometry. Then, metabolites whose reliability index of the isotope distribution ratio exceeds a preset reliability index threshold are selected. Although it is preferable to have more data on the isotope distribution ratios of the metabolites included in the intracellular metabolic model, isotope distribution ratios with low reliability decrease the estimation accuracy of metabolic fluxes. Therefore, it becomes possible to perform highly reliable metabolic flux analysis using the isotope distribution ratio evaluation program of the present embodiment.

[0046] By the isotope distribution ratio evaluation program shown above, it becomes possible to evaluate the reliability of the isotope distribution ratio. Also, it becomes possible to perform highly reliable metabolic flux analysis using the highly reliable isotope distribution ratio.

[0047] <Metabolic flux analysis device having an isotope distribution ratio evaluation function> FIG. 1B is a block diagram showing a configuration example of a metabolic flux analysis device 100 having an isotope distribution ratio evaluation function according to the present embodiment.

[0048] The metabolic flux analysis device 100 is a device that executes the isotope distribution ratio evaluation program shown in FIG. 1A and can be realized by a computer. More specifically, the metabolic flux analysis device 100 includes a processor 101, a storage device 102 including a ROM (Read Only Memory), a RAM (Random Access Memory), etc., an input device 103 composed of a keyboard, a mouse, and a touch panel, an output device 104 composed of a display device, a printer, etc., and a communication device 105 for executing communication with the outside of the device. Note that the storage device 102 may be configured to include a drive that starts up a memory (which may be a disk, a USB, etc.) carrying the isotope distribution ratio evaluation program and the metabolic flux analysis program, reads data, and writes data to the memory.

[0049] The processor 101 realizes various functional units 1011 to 1016 by reading the codes of the isotope distribution ratio evaluation program and the metabolic flux analysis program from the storage device 102 and expanding them in an internal memory (not shown). That is, the processor 101 includes, inside thereof, a mass spectrometry unit 1011 that measures mass chromatograms of a plurality of isotopes having different mass numbers by mass spectrometry, an isotope quantification unit 1012 that quantifies a plurality of isotopes and evaluates a quantification reliability index of the plurality of isotopes, an isotope distribution ratio calculation unit 1013 that calculates the isotope distribution ratio of compound fragments using the quantification results of the plurality of isotopes, an isotope distribution ratio reliability index calculation unit 1014 that calculates a reliability index of the isotope distribution ratio using the quantification reliability index of the plurality of isotopes, a display processing unit 1015 that performs processing for outputting the obtained isotope distribution ratio and the reliability index of the isotope distribution ratio to the output device 104, and a metabolic flux analysis unit 1016 that performs metabolic flux analysis reflecting the obtained reliability index (evaluation result) of the isotope distribution ratio. Note that the apparatus may be configured without including the metabolic flux analysis unit 1016, and in this case, it can be referred to as an isotope distribution ratio evaluation apparatus. Further, although the metabolic flux analysis apparatus 100 includes the mass spectrometry unit 1011 as an internal configuration, it may be connected to gas chromatography to obtain the measurement result of the mass chromatogram therefrom.

Example

[0050] Hereinafter, examples and comparative examples according to the present disclosure will be described. In both this example and the comparative example, 13 After metabolizing glucose having 13C into CHO cells with different culture days, the metabolites recovered from the cells were used as measurement samples. Note that the difference between the example and the comparative example is that the former executes metabolic flux analysis in consideration of the above-described isotope distribution ratio reliability data, while the latter executes metabolic flux analysis using all the data obtained from the chromatogram.

[0051] CHO cells were cultured using Dulbecco's Modified Eagle's Medium (DMEM) as the basal medium according to the ATCC manual (temperature: 37 °C, CO2 concentration: 5%). Twenty-four hours before extracting metabolites from the cells, 13 the medium was replaced with a medium containing glucose labeled with 13 C, and the cells were 13 allowed to metabolize 13 C glucose. The ratio of 13 C glucose in the medium was set as [1- 13 C] glucose: [U- 13 C] glucose: unlabeled glucose = 1:1:2. Also, the glucose concentration was 4.5 g / L. The culture days were set as 3 days, 4 days, 5 days, 6 days, and 7 days, and from the change in the number of cells during culture, it was confirmed that the cells underwent logarithmic growth from 3 to 6 days.

[0052] Metabolites in CHO cells were extracted according to the following procedure. First, the medium was removed and the cells were washed three times with 1.0 mL of phosphate buffer. Then, 200 μL of methanol cooled to -20 °C was added and left standing on ice for 10 minutes to immobilize the cells. After that, 300 μL of ultrapure water was added, and while rubbing with the tip of a pipette, the cells were detached from the petri dish and collected in a sample tube together with the ultrapure water. Then, the surface of the petri dish after cell detachment was washed with 300 μL of ultrapure water and also collected in the sample tube. The sample tube was immersed in a vibrating water bath with floating ice and sonicated for 1 minute to disperse the cell collection. Then, 800 μL of chloroform was added to the sample tube, and after vigorously shaking and stirring until it became turbid, it was further shaken and stirred at 1,200 rpm and 4 °C for 30 minutes. Centrifugation was performed at 11,500 rpm and 4 °C for 30 minutes, and the upper layer in which the metabolites were dissolved was collected. Then, the solvent was removed using an evaporator to obtain intracellular metabolites.

[0053] Subsequently, for GC-MS measurement, the extracted intracellular metabolites were derivatized. First, 30 μL of 2% methoxamine hydrochloride (MOX reagent) was added to the intracellular metabolites obtained in the previous section, and the sample tube was immersed in a vibrating water bath with ice floating and sonicated for 5 minutes. The sample was strongly shaken and stirred, and after spinning down the solution in the sample tube, sonication / vibration stirring / spinning down was performed again. Then, it was left standing at 30 °C for 2 hours with vibration stirring at 1,200 rpm to react the metabolites with methoxamine. Then, 45 μL of a mixed solution of N-Methyl-N-tert-butyldimethylsilyltrifluoroacetamide (MTBSTFA) and tert-butyldimethylchlorosilane (TBDMCS; 1%) was added. After vibration stirring / spinning down by Vortex, it was reacted at 55 °C for 1 hour with vibration stirring at 1,200 rpm. Then, it was centrifuged at 13,300 rpm at 4 °C for 3 minutes, and the recovered supernatant was used as a sample for GC-MS measurement.

[0054] The program of this example was created using MatLab (registered trademark) 2021a of MathWorks. Figure 3 is a diagram showing a configuration example of the menu screen 300 of the program of this example. As menus displayed on the menu screen, for example, six types of items such as "0. Settings", "1-1. GC-MS measurement", "1-2. Data Retrieval", "2. Data Processing", "3. Results Display", and "4. Metabolic Flux Analysis" can be included. Also, buttons corresponding to the items of each menu can be arranged on the menu screen. Hereinafter, the steps implemented by clicking each button will be described.

[0055] <0. Settings> For example, an operator (user) can set the detailed conditions of a process started by clicking other buttons by clicking the "0. Settings" button 310 shown in FIG. 3. The detailed conditions that can be set include the format of the data referred to in "1-2. Data Retrieval", the type of function and the mathematical formula for calculating the reliability index of the isotope distribution ratio when fitting a mass chromatogram to data in "2. Data Processing", the type of data to be displayed in "3. Results Display", and the analysis model and the number of simulations used in the metabolic flux analysis performed in "4. Metabolic Flux Analysis". The analysis model includes (i-1) an intracellular metabolic model and (i-2) information on metabolites to be analyzed. In this example and the comparative example, the data format was set to the ms1 format, the type of function was set to the Gaussian function, the mathematical formula for calculating the reliability index was set to the formula described in formula (7), the type of data to be displayed was set to "isotope distribution ratio, mass chromatogram and its fitting curve, reliability index of isotope distribution ratio", the analysis model was set to the model shown in FIG. 4, and the number of simulations was set to 50 times.

[0056] <1-1. GC-MS measurement> For example, an operator (user) can start the measurement program of the GC-MS apparatus by clicking the "1-1. GC-MS measurement" button 320 shown in FIG. 3 (related to the above step 1). In this example and the comparative example, Chromeleon (registered trademark), which is software for operating a GC-MS apparatus (ISQ7000EI) manufactured by Thermo Fisher Science, was implemented. Also, the GC-MS measurement was carried out in selected ion monitoring (SIM) mode. The injection volume was 1 μL, and the helium flow rate was 1 mL / min. Also, the split ratio at the time of sample injection was 10, and the temperatures of the sample inlet, sample transfer line, and ion source were 250°C, 250°C, and 200°C, respectively. The measurement temperature range was 100°C to 300°C, and the temperature rising rate of the column was 3.5°C / min. Also, the measurement interval was 0.5 seconds. After the data of the measured mass chromatogram was output in raw format, it was converted to ms1 format. Then, the data in ms1 format was converted into a table of elution time × m / z.

[0057] <1-2. Data Retrieval> For example, an operator (user) can select the data of the mass chromatogram to be analyzed by clicking the "1-2. Data Retrieval" button 330 shown in FIG. 3 (related to the above step 1). By clicking this button, it is possible to read not only the mass chromatogram measured by clicking the "1-1. GC-MS measurement" button but also the data of separately measured mass chromatograms. Also, the selected data in ms1 format was converted into a csv file format in which signal intensities were arranged in a table of elution time × m / z to facilitate subsequent analysis. Note that by pressing the "1-2. Data Retrieval" button 330, the acquired mass chromatogram data may be converted so that it can be processed by the above isotope distribution ratio evaluation program.

[0058] <2. Data Processing> For example, an operator (user) starts data analysis of the mask chromatogram to be analyzed by clicking the "2. Data Processing" button 340 shown in FIG. 3 (related to steps 2, 3, and 5 above). In the programs of this example and the comparative example, after clicking the "2. Data Processing" button 340, first, an analysis model for metabolic flux analysis is loaded, and the metabolite to be analyzed (default) is determined. The metabolites (default) analyzed in this example and the comparative example are shown in FIG. 5. Next, the m / z and elution time of the isotopes (6 types: M+0 to M+5) of the metabolite to be analyzed (default) are read from the database, and using this information, the mask chromatogram of the isotopes of the metabolite to be analyzed (default) is extracted from the data of the mask chromatogram to be analyzed. Then, for each metabolite, the extracted mask chromatogram is fitted with a function to obtain the isotope amount and the quantitative reliability index of the isotope. In this example and the comparative example, the mask chromatogram was fitted with a Gaussian function (Equation (1)) using the built-in function (fit) of MatLab (registered trademark) 2021, and the isotope amount was obtained using Equation (2). Then, the quantitative reliability index of the isotope was obtained using Equation (3) or Equation (4). After that, the isotope distribution ratio was obtained from the isotope amount using Equation (5), and the evaluation index of the isotope distribution was obtained from the quantitative reliability index using Equations (6) and (7). Also, an isotope distribution ratio with the contribution of naturally occurring stable isotopes subtracted and corrected was obtained using the matrix transformation described in the literature (R. Nilsson, Methematical Biosciences, 330, 108481 (2020)). Each metabolite can be determined by the time output from the column of the gas chromatography. Therefore, it can be said that each row (horizontal cell) in a table such as FIG. 6C indicates the time domain.

[0059] <3. Results Display> For example, by clicking the "3. Results Display" button 350 shown in Fig. 3, the operator (user) can display the analysis results according to the type of display data set in "0. Settings". In this example and the comparative example, the types of display data are "isotope distribution ratio, mass chromatogram and its fitting curve, reliability index of isotope distribution ratio". Note that as the display form of the analysis results displayed on the program, for example, the forms shown in Figs. 6A to 6C described below can be adopted.

[0060] (Example of reliability index of isotope distribution ratio: form presenting index value based on deviation from measurement data) Figs. 6A and 7A are diagrams showing the isotope ratios of metabolites to be analyzed in a bar graph. In Figs. 6A and 7A, the isotope distribution ratios of the metabolites to be analyzed can be confirmed. Fig. 6B is a diagram showing the chromatogram of metabolite LAC123 in measurement sample 0421 and the fitting result with a Gaussian function. Fig. 6C is a diagram showing the reliability index values of the isotope distribution ratios of each metabolite contained in a plurality of measurement samples (such as 04101, 04201, 05101, 05201, 06101, 06201, etc.). Fig. 7B is a diagram showing the chromatogram of metabolite SUC1234 in measurement sample 05101 and the fitting result with a Gaussian function. Fig. 7C is, similar to Fig. 6C, a diagram showing the reliability index values of the isotope distribution ratios of each metabolite contained in a plurality of measurement samples (such as 04101, 04201, 05101, 05201, 06101, 06201, etc.). Note that in Fig. 6C and Fig. 7C (and also in Fig. 8C and Fig. 9C described below), taking 04101 as an example, the first two digits (04) of the measurement sample indicate the culture days of CHO cells, and the subsequent digits (101) indicate the sample lot number.

[0061] In this example, the isotope quantitative reliability index was obtained using Equation (3), and the reliability index was calculated. In the table of FIG. 6C, the reliability index of the isotope distribution ratio is displayed as the logarithmic value of the reciprocal, and the larger the displayed value, the higher the reliability. Further, in this example, cells with values where the evaluation index (logarithmic value) in the table is less than 1.5 were colored (black inverted display) so that metabolites with low-reliability isotope distribution ratios could be easily identified. Furthermore, by clicking (selecting) any cell in the table, measurement data points and fitting curves of chromatograms of corresponding metabolites (six types: M+0 to M+5) are displayed as shown in FIGS. 6B and 7B. For example, in the table of FIG. 6C, the state where metabolite LAC123 of sample 04201 is selected is shown. In FIG. 7C, different from FIG. 6C, the state where metabolite SUC1234 of sample 05101 is selected is shown.

[0062] As shown in FIG. 6B, as a result of displaying the chromatogram of LAC123 (measurement sample: 4201), which is a metabolite with an evaluation index (logarithmic value) of 1.5 or more, it can be seen that all measurement data points from M+0 to M+5 are well fitted by a Gaussian function. On the other hand, as shown in FIG. 7B, as a result of displaying the chromatogram of SUC1234 (measurement sample: 05101), which is a metabolite with an evaluation index (logarithmic value) of less than 1.5, it can be seen that in the measurement data with a molecular weight of M+5, a small peak overlaps the shoulder of the main peak, and the measurement data points are not well fitted by a Gaussian function. In the case of FIG. 7B, at M+5 in terms of molecular weight, the concentration of the metabolite is low and the influence of contaminants is large. Therefore, it affects the fitting, and the value of r 2 (the coefficient of determination of the fitting) deteriorates. By checking the parameters (r 2 ) shown in FIGS. 6B and 7B, the fitting status of the entire metabolite can be monitored, and the presence or absence of the reliability of the fitting can be determined.

[0063] From the above, it was found that by displaying the reliability index of the isotope distribution ratio on the program screen, it is possible to judge the quality of fitting. The quality of fitting affects the quantification of the amount of isotope used in the calculation of the isotope distribution ratio. Therefore, it was found that by displaying the reliability index of the isotope distribution ratio on the program screen, the reliability of the isotope distribution ratio can be judged.

[0064] (Example of the reliability index of the isotope distribution ratio: the form presenting the index value based on the coefficient of determination) Next, an example will be described in which an isotope quantitative reliability index is obtained using the coefficient of determination of fitting to calculate a reliability index, and the analysis result is displayed in the table at the lower right of the screen. FIGS. 8A and 9A are diagrams showing the isotope ratios of metabolites to be analyzed in the form of bar graphs, similar to FIGS. 6A and 7A. FIG. 8B is a diagram showing the chromatogram of metabolite LAC123 in measurement sample 0421 and the fitting result by a Gaussian function, similar to FIG. 6B. FIG. 8C is a diagram showing the reliability index values of the isotope distribution ratios of each metabolite contained in a plurality of measurement samples (such as 04101, 04201, 05101, 05201, 06101, 06201, etc.) based on the coefficient of determination of fitting. In FIG. 8C, the state where metabolite LAC123 of sample 04201 is selected is shown. FIG. 9B is a diagram showing the chromatogram of metabolite SUC1234 in measurement sample 05101 and the fitting result by a Gaussian function, similar to FIG. 7B. FIG. 9C is a diagram showing the reliability index values of the isotope distribution ratios of each metabolite contained in a plurality of measurement samples (such as 04101, 04201, 05101, 05201, 06101, 06201, etc.), similar to FIG. 8C. However, in FIG. 9C, different from FIG. 8C, the state where metabolite SUC1234 of sample 05101 is selected is shown. In the tables of FIGS. 8C and 9C, the reliability evaluation index of the isotope distribution ratio is normalized and displayed so that it becomes 100 when the quantitative reliability index of all fittings reaches the full score (1.000). In this example, cells with values less than 90 in the evaluation index in the table are colored (black reversed display) so that metabolites with low-reliability isotope distribution ratios can be easily confirmed. Further, similar to FIGS. 6C and 7C, by clicking on any cell in the table, the measurement data points and fitting curves of the chromatograms (6 types: M+0 to M+5) of the corresponding metabolites are displayed as in FIGS. 8B and 9B. For example, in the table of FIG. 8C, the state where metabolite LAC123 of sample 04201 is selected is shown. In the table of FIG. 9C, different from FIG. 8C, the state where metabolite SUC1234 of sample 05101 is selected is shown.

[0065] From Figure 8B showing the chromatogram of LAC123 (measurement sample: 4201), which is a metabolite with an evaluation index of 90 or higher, it can be seen that all measurement data points from M+0 to M+5 are well fitted by the Gaussian function. On the other hand, from Figure 9B showing the chromatogram of SUC1234 (measurement sample: 05101), which is a metabolite with an evaluation index of less than 90, it can be seen that in the measurement data of M+5, a small peak overlaps with the shoulder of the main peak, indicating that the measurement data points are not well fitted by the Gaussian function.

[0066] From the above, it was also found that even when the isotope quantitative reliability index is obtained using the determination coefficient of fitting, the goodness or badness of the fitting can be judged by displaying the reliability index of the isotope distribution ratio on the program screen. The goodness or badness of the fitting affects the quantification of the isotope amount used in the calculation of the isotope distribution ratio. Therefore, it was found that the reliability of the isotope distribution ratio can be judged by displaying the reliability index of the isotope distribution ratio on the program screen.

[0067] <4. Metabolic Flux Analysis> When the "4. Metabolic Flux Analysis" button 360 is clicked, the metabolic flux analysis can be started using the input results and calculation results obtained so far. In the program of this example, OpenFLUX, which is a metabolic flux estimation software publicly available on the Internet, was implemented to perform the metabolic flux analysis. Also, metabolites with low-reliability isotope distribution ratios, which were colored (black reverse display) in Figure 6C, were excluded from the analysis targets for the metabolic flux analysis.

[0068] On the other hand, as a comparative example, the metabolic flux analysis was performed without excluding the metabolites colored (black reverse display) in Figure 6C from the analysis targets. The metabolic flux analysis was performed on the intracellular metabolites of CHO cells on the 7th day of culture. Also, the value of the metabolic flux was taken as the average value of a plurality of metabolic fluxes obtained from the measurement sample group on the 7th day of culture, and the width of the error bar was taken as its standard deviation.

[0069] FIG. 10 is a diagram showing the result of metabolic flux analysis (Example: (a)) excluding metabolites with a low-reliability isotope distribution ratio from the analysis target, and the result of metabolic flux analysis (Comparative Example: (b)) using all the measured isotope distribution ratios as the analysis target. From FIG. 10, it can be seen that the width of the error bar in the example (the standard deviation of the results obtained by performing metabolic flux analysis N times (e.g., N = 50) is displayed as the error bar) is reduced compared to the comparative example. Therefore, it can be understood that by selecting the compound fragments to be analyzed using the reliability index of the isotope distribution ratio, it is possible to perform a highly reliable metabolic flux analysis.

[0070] <Summary of the Embodiment> (i) In the metabolic flux analysis device 100 having the isotope distribution ratio evaluation function according to this embodiment, the computer (processor 101) acquires measurement values of mass chromatograms of a plurality of isotopes with different mass numbers for a compound fragment (sample) to be measured. At this time, the processor 101 may read the values of the already measured mass chromatograms from the storage device 102, or the processor 101 may operate the gas chromatography connected to the metabolic flux analysis device 100 to acquire the values of the mass chromatograms immediately after measurement. Further, the processor 101 quantifies a plurality of isotopes from a fitting curve obtained by fitting the acquired mass chromatogram with a predetermined function (for example, a Gaussian function, etc.), and calculates the isotope distribution ratio of the compound fragment using the quantification results of the plurality of isotopes. Then, the processor 101 calculates a plurality of isotope quantification reliability indicators indicating the reliability of the quantification results of the plurality of isotopes, uses this to calculate a reliability indicator of the isotope distribution ratio, and displays (outputs) this on the display screen of the display device (output device 104). Further, the calculated value of the isotope distribution ratio (see FIG. 6C, etc.) may be similarly displayed on the display screen. By doing so, an operator (user) who has seen the value of the reliability indicator of the isotope distribution ratio can exclude metabolites of a sample corresponding to a reliability indicator of the isotope distribution ratio having a value smaller than a predetermined threshold and execute metabolic flux analysis. Therefore, it becomes possible to obtain a more reliable metabolic flux analysis result.

[0071] (ii) When outputting the reliability index of the isotope distribution ratio, the processor 101 displays, on the display screen, the measurement results of the mass chromatograms of a plurality of isotopes (see, for example, FIG. 6A) and the fitting curve obtained by fitting the mass chromatogram with a predetermined function (see, for example, FIG. 6B), together with the reliability index of the isotope distribution ratio. At this time, the processor 101 displays, on the display screen, the measured value of the mass chromatogram and the fitting curve corresponding to the metabolite of the sample selected in the table of the reliability index of the isotope distribution ratio (see, for example, FIG. 6C) (see, for example, FIG. 6B). When the user selects the metabolite of another sample in the display such as FIG. 6C, in response thereto, the display of the measured value of the mass chromatogram and the fitting curve is also changed. For example, when the display target is changed from the metabolite LAC123 of sample 04201 (selected in FIG. 6C) to the metabolite SUC1234 of sample 05101 (selected in FIG. 7C), the display of the measured value of the mass chromatogram and the fitting curve is also changed from the display in FIG. 6B to the display in FIG. 7B in conjunction therewith. By doing so, when the value of the reliability index of the isotope distribution ratio is low, the user can check whether the coefficient of determination in the fitting is good only by selecting (clicking) that value. At this time, when the value of the reliability index is low despite the good coefficient of determination in the fitting, the user can know that there may be another cause rather than low reliability due to impurities.

[0072] In addition, based on the reliability index of the isotope distribution ratio, a display indicating the possibility of defects in the mass chromatogram may be displayed on the display screen, or a selection target specific display that enables the selection of the compound fragments to be analyzed may be performed on the reliability index of the isotope distribution ratio. The selection target specific display can be, for example, a display form that distinguishes and displays the compound fragments with the value of the reliability index of the isotope distribution ratio less than a predetermined threshold from the compound fragments with the value of the reliability index of the isotope distribution ratio greater than or equal to the predetermined threshold value (see the black inversion display in FIG. 6C, etc.).

[0073] (iii) When calculating the reliability index of the isotope distribution ratio, the processor 101 preferably uses the quantitative reliability indices of four or more isotopes. By using four or more isotopes, the error of the isotope distribution ratio can be reduced (for example, within 1% error), and as a result, the accuracy of the reliability index of the isotope distribution ratio can be improved.

[0074] (iv) The functions of this embodiment and each example can also be realized by software program codes. In this case, a storage medium recording the program codes is provided to the system or device, and the computer (or CPU or MPU) of the system or device reads the program codes stored in the storage medium. In this case, the program codes themselves read from the storage medium realize the functions of the foregoing embodiments, and the program codes themselves and the storage medium storing them constitute this disclosure. As the storage medium for supplying such program codes, for example, flexible disks, CD-ROMs, DVD-ROMs, hard disks, optical disks, magneto-optical disks, CD-Rs, magnetic tapes, non-volatile memory cards, ROMs, etc. are used.

[0075] Also, based on the instructions of the program codes, an OS (operating system) running on the computer or the like may perform part or all of the actual processing, and the functions of the foregoing embodiments may be realized by such processing. Further, after the program codes read from the storage medium are written into the memory on the computer, based on the instructions of the program codes, the CPU of the computer or the like may perform part or all of the actual processing, and the functions of the foregoing embodiments may be realized by such processing.

[0076] Furthermore, by distributing the program code of the software that realizes the functions of the embodiments and each example via a network, it can be stored in a storage means such as a hard disk or memory of a system or device, or a storage medium such as a CD-RW or CD-R, and when in use, a computer (or CPU or MPU) of the system or device reads and executes the program code stored in the storage means or the storage medium.

[0077] The processes and technologies described herein are not essentially related to any specific device and can also be implemented by a combination of each component. Also, various types of general-purpose devices can be added. To execute the functions of this embodiment and each example, a dedicated device may be constructed. Also, by appropriately combining a plurality of components disclosed in this embodiment and each example, various functions can be formed. For example, some components may be deleted from all the components shown in the embodiment and each example, or components from different examples may be appropriately combined.

[0078] In this disclosure, specific examples are described, but these are for explanation (understanding of the technology of this disclosure) rather than limitation in all aspects. It is considered that those with ordinary knowledge in this technical field can understand that there are many combinations of hardware, software, and firmware suitable for implementing the technology of this disclosure. For example, the described software can be implemented in a wide range of programs or script languages such as assembler, C / C++, perl, Shell, PHP, Java (registered trademark), etc.

[0079] Furthermore, in the above-described embodiment, control lines and information lines show those considered necessary for explanation, and not necessarily all control lines and information lines are shown on the product. All components may be interconnected.

[0080] In addition, those with ordinary knowledge in the relevant technical field can clarify other implementations of the present disclosure from the considerations of this embodiment and each example. The specification and specific examples are merely typical, and the scope and spirit of the technology of the present disclosure are indicated in the subsequent claims.

Explanation of Reference Numerals

[0081] 100 Metabolic flux analysis device 101 Processor 102 Memory device 103 Input device 104 Output device 105 Communication device 1011 Mass spectrometry unit 1012 Isotope quantification unit 1013 Isotope distribution ratio calculation unit 1014 Isotope distribution ratio reliability index calculation unit 1015 Display processing unit 1016 Metabolic flux analysis unit 300 Program menu screen 310 "0. Settings" button 320 "1-1. GC-MS measurement" button 330 "1-2. Data Retrieval" button 340 "2. Data Processing" button 350 "3. Results Display" button 360 "4. Metabolic Flux Analysis" button

Claims

1. A program for causing a computer to execute an isotope distribution ratio evaluation method, wherein the isotope distribution ratio evaluation method includes: measuring mass chromatograms of a plurality of isotopes having different mass numbers for a compound fragment to be measured by mass spectrometry; quantifying the plurality of isotopes from a fitting curve obtained by fitting the mass chromatogram with a predetermined function; calculating an isotope distribution ratio of the compound fragment using the quantification results of the plurality of isotopes; calculating a quantification reliability index for each of the plurality of isotopes indicating the reliability of the quantification results of the plurality of isotopes; calculating a reliability index of the isotope distribution ratio using the quantification reliability indexes of the plurality of isotopes; causing an output unit that outputs an operation result of the program to output the reliability index of the isotope distribution ratio; and the program includes the above steps.

2. The program according to claim 1, wherein the isotope distribution ratio evaluation method further includes outputting the isotope distribution ratio to the output unit.

3. The program according to claim 1, wherein outputting the reliability index of the isotope distribution ratio includes causing the output unit to output, together with the reliability index of the isotope distribution ratio, a measurement result of the mass chromatogram of the plurality of isotopes and a fitting curve obtained by fitting the mass chromatogram with a predetermined function.

4. The program according to claim 3, wherein the isotope distribution ratio evaluation method further includes causing the output unit to display, in response to a value of the reliability index of the isotope distribution ratio being selected, a measured value of the mass chromatogram of the corresponding compound fragment and the corresponding fitting curve.

5. The program according to claim 1, wherein calculating the reliability index of the isotope distribution ratio includes calculating the reliability index of the isotope distribution ratio using quantification reliability indexes of four or more isotopes.

6. The program according to claim 1, wherein the predetermined function is a Gaussian function.

7. The program according to claim 1, wherein the reliability index of the isotope distribution ratio is calculated by the following formula. where R is the reliability index of the isotope distribution ratio, w i is the weight of the quantitative reliability index of isotope i, and d i is the quantitative reliability index for isotope i.

8. The program according to claim 1, wherein the quantification reliability index is calculated by the following formula. Here, d i is the quantitative reliability index of isotope i, n i is the number of data points of the mass chromatogram of isotope i, Y Di,j is the signal intensity of the j-th signal of the mass chromatogram of isotope i, Y Fi,j is Y Di,j and is the signal intensity of the fitting curve of the mass chromatogram of isotope i at the same elution time as Y, I max,F,i is the maximum value of the signal intensity of the fitting curve of the mass chromatogram of isotope i.

9. The program according to claim 1, A program in which the quantitative reliability index is represented by the coefficient of determination when the mass chromatogram is fitted with the predetermined function.

10. In claim 1, The isotope distribution ratio evaluation method further includes outputting, to the output unit, a display indicating the possibility of defect in the mass chromatogram based on the reliability index of the isotope distribution ratio.

11. In claim 1, The isotope distribution ratio evaluation method further includes outputting, to the output unit, a selection target specific display that enables selection of a compound fragment to be analyzed using the reliability index of the isotope distribution ratio.

12. In claim 11, The selection target specific display is a display form that distinguishes a compound fragment having a value of the reliability index of the isotope distribution ratio less than a predetermined threshold from a compound fragment having a value of the reliability index of the isotope distribution ratio equal to or greater than the predetermined threshold value and displays them.

13. An isotope distribution ratio evaluation method executed by a processor, wherein the processor acquires measurement values of mass chromatograms of a plurality of isotopes having different mass numbers for a compound fragment to be measured; the processor quantifies the plurality of isotopes from a fitting curve obtained by fitting the mass chromatogram with a predetermined function; the processor calculates an isotope distribution ratio of the compound fragment using the quantification results of the plurality of isotopes; the processor calculates a quantitative reliability index of the plurality of isotopes indicating the reliability of the quantification results of the plurality of isotopes; the processor calculates a reliability index of the isotope distribution ratio using the quantitative reliability index of the plurality of isotopes; outputting the reliability index of the isotope distribution ratio to an output unit that outputs calculation results; An isotope distribution ratio evaluation method comprising:

14. In claim 13, further, the processor outputs the isotope distribution ratio to the output unit. An isotope distribution ratio evaluation method.

15. In claim 13, When outputting the reliability index of the isotope distribution ratio, the processor outputs to the output unit, together with the reliability index of the isotope distribution ratio, the measurement result of the mass chromatogram of the plurality of isotopes and the fitting curve obtained by fitting the mass chromatogram with a predetermined function. An isotope distribution ratio evaluation method.

16. In claim 15, further, In response to the value of the reliability index of the isotope distribution ratio being selected, the processor displays on the output unit the measured value of the mass chromatogram of the corresponding compound fragment and the corresponding fitting curve. An isotope distribution ratio evaluation method.

17. In claim 13, Calculating the reliability index of the isotope distribution ratio includes the processor calculating the reliability index of the isotope distribution ratio using the quantitative reliability indices of four or more isotopes. An isotope distribution ratio evaluation method.

18. In claim 13, The predetermined function is a Gaussian function. An isotope distribution ratio evaluation method.

19. In claim 13, The processor calculates the reliability index of the isotope distribution ratio by the following formula. An isotope distribution ratio evaluation method. Here, R is the reliability index of the isotope distribution ratio, w i is the weight of the quantitative reliability index of isotope i, and d i is the quantitative reliability index of isotope i.

20. In claim 13, The processor calculates the quantitative reliability index by the following formula. An isotope distribution ratio evaluation method. Here, d i is the quantitative reliability index of isotope i, n i is the number of data points of the mass chromatogram of isotope i, Y Di,j is the signal intensity of the j-th signal of the mass chromatogram of isotope i, Y Fi,j is Y Di,j and is the signal intensity of the fitting curve of the mass chromatogram of isotope i at the same elution time as Y, I max,F,i is the maximum value of the signal intensity of the fitting curve of the mass chromatogram of isotope i.

21. In claim 13, The processor represents the quantitative reliability index by the coefficient of determination when the mass chromatogram is fitted with the predetermined function. An isotope distribution ratio evaluation method.

22. In claim 13, further, The processor outputs to the output unit a display indicating the possibility of defect of the mass chromatogram based on the reliability index of the isotope distribution ratio. An isotope distribution ratio evaluation method.

23. In claim 13, further, The processor outputs to the output unit a selection target specific display that enables selection of a compound fragment to be analyzed using the reliability index of the isotope distribution ratio. An isotope distribution ratio evaluation method.

24. In claim 23, The isotope distribution ratio evaluation method is a display form in which the compound fragments with the value of the reliability index of the isotope distribution ratio less than a predetermined threshold are distinguished from the compound fragments with the value of the reliability index of the isotope distribution ratio being equal to or greater than the predetermined threshold value and displayed.

25. A storage device that holds an evaluation program for causing a computer to execute an isotope distribution ratio evaluation method, A processor that reads and executes the evaluation program from the storage device, and The processor Performs a process of acquiring measurement values of mass chromatograms of a plurality of isotopes with different mass numbers for a compound fragment to be measured, Performs a process of quantifying the plurality of isotopes from a fitting curve obtained by fitting the mass chromatogram with a predetermined function, Performs a process of calculating an isotope distribution ratio of the compound fragment using the quantification results of the plurality of isotopes, Performs a process of calculating quantification reliability indices of the plurality of isotopes indicating the reliability of the quantification results of the plurality of isotopes, Performs a process of calculating a reliability index of the isotope distribution ratio using the quantification reliability indices of the plurality of isotopes, Performs a process of outputting the reliability index of the isotope distribution ratio to an output unit that outputs calculation results, An isotope distribution ratio evaluation device that executes.

26. A metabolic flux analysis device having the configuration and functions of the isotope distribution ratio evaluation device according to claim 25, The storage device further holds an analysis program for causing a computer to execute metabolic flux analysis, The processor reads the analysis program from the storage device and executes the metabolic flux analysis for the compound fragment selected based on the reliability index of the isotope distribution ratio. A metabolic flux analysis device.

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