Calibrant Used in Mass Spectrometer and Method for Producing the Same

Abstract

A calibrant for use in correction of machine error of a mass spectrometer and a method for producing the calibrant are provided. Provided are a calibrant for use in a mass spectrometer and a method for producing the calibrant, the calibrant containing two or more calibration substances, wherein the ratio of the concentration of one of the two or more calibration substances to the concentration of the other one of the two or more calibration substances is a predetermined value.

Description

Technical Field

[0001] The present invention relates to a calibrant used in mass spectrometry and a method for producing the same.

Background Art

[0002] When it is desired to comparatively analyze the abundance of substances using a mass spectrometer, the method of utilizing the intensity ratio of two signal peaks is most frequently used. For example, a certain amount of an internal standard substance is added to the sample to be compared, and after performing pretreatment if necessary, mass spectrometry is performed on the sample, and the values obtained by calculating the intensity ratio of the measurement target peak to the peak of the internal standard substance are compared (Non-Patent Documents 1, 2, 3).

[0003] Examples of patent documents include International Publication WO2015 / 178398 (U.S. Publication US2017 / 0184573), International Publication WO2017 / 047529 (U.S. Publication US2018 / 0238909), and the like.

[0004] In addition, by using stable isotope elements to give labeled compounds with different masses and labeling the target substances derived from different samples respectively, semi-quantitative comparative analysis can be performed by calculating the intensity ratio between the peaks of the target substances with different masses in mass spectrometry. The ICAT (registered trademark) method and iTRAQ (registered trademark) method used in the field of proteomics fall under this technique (Non-Patent Documents 4, 5).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Literature

[0006]

Non-Patent Literature 1

Non-Patent Literature 2

Non-Patent Literature 3

[0007] When attempting to measure an extremely trace amount of a substance using a mass spectrometer, it has been confirmed that even for the same model, the peak intensity ratios detected for each instrument body are different. As one means of correcting the differences in peak intensity ratios between different instrument bodies, the following absolute quantification method can be cited.

[0008] Prepare a standard of the substance to be quantified and a substance serving as a reference for the intensity ratio (reference substance; generally, a stable isotope-labeled substance of the target substance is used). By measuring a sample in which a reference substance at a certain concentration is mixed with standards at different concentrations, a calibration curve of the peak intensity ratio of the standards with respect to the reference substance is created. Absolute quantification of the target substance can be performed using this calibration curve. Therefore, if a calibration curve is created for each apparatus and each measurement, even if there are differences in peak intensity ratios between different apparatuses, an unknown target substance present in a biological sample can be quantified without being affected by them.

[0009] However, this calibration curve method requires a standard. If the standard cannot be easily synthesized, or if the number of types of target substances is enormous and it is difficult in terms of time and cost to prepare and quality-control all the standards, or if the standard is unstable, a calibration curve cannot be created. As described above, since the peak intensity ratio varies between instrument bodies in a mass spectrometer, if a calibration curve cannot be created, samples to be compared must be measured on one instrument body. However, even then, detector degradation occurs due to the use of the instrument body, causing the peak intensity ratio to fluctuate and making it difficult to obtain consistent data.

[0010] In mass spectrometry, a sample is ionized by irradiating it with a laser. Therefore, the data is affected by the state of the laser of the mass spectrometer (degradation due to the number of uses, etc.). Accordingly, even when the same sample is measured, there are still quite a few instrumental differences between different apparatuses. Therefore, the stability of the data is lacking.

[0011] In order to cancel out such instrumental differences in a mass spectrometer, it is conceivable to correct the measurement data in each mass spectrometer.

[0012] An object of the present invention is to provide a calibrant used for correcting instrumental errors of a mass spectrometer and a method for producing the calibrant.

Means for Solving the Problems

[0013] The present invention includes the following inventions. A calibrant used in a mass spectrometer, wherein the calibrant contains two or more calibration substances, and a ratio of a concentration of one calibration substance among the two or more calibration substances to a concentration of another calibration substance among the two or more calibration substances is a predetermined value.

[0014] The present invention further includes the following inventions. A method for producing a calibrant used in a mass spectrometer, wherein the calibrant contains two or more calibration substances, and a ratio of a concentration of one calibration substance among the two or more calibration substances to a concentration of another calibration substance among the two or more calibration substances is a predetermined value, and the production method includes a pre-step of preparing a solution A containing one calibration substance (S1) among the two or more calibration substances at a predetermined concentration and a solution B containing the one calibration substance (S1) at the same concentration as the predetermined concentration and further containing another calibration substance (S2) among the two or more calibration substances, and a preparation step of obtaining a calibrant in which a ratio of a concentration of the other calibration substance (S2) to a concentration of the one calibration substance (S1) is a predetermined value using the solution A and the solution B. ​A method for producing a calibrant including

[0015] The present invention further includes the following inventions. A calibrant kit used in a mass spectrometer, The kit includes a plurality of calibrants containing two or more calibration substances, The plurality of calibrants are calibrant kits in which the concentration of at least one calibration substance among the calibration substances is different.

[0016] The present invention further includes the following inventions. A calibrant kit used in a mass spectrometer, The kit is a calibrant kit containing two or more calibration substances.

Advantages of the Invention

[0017] According to the present invention, a calibrant used for correcting the instrumental error of a mass spectrometer is provided. By measuring the calibrant of the present invention with a mass spectrometer, a correction formula for the mass spectrometer can be calculated from the measurement results. Using the calculated correction formula, the measurement results in the mass spectrometer can be corrected. As a result, even when the same sample is measured with different instruments, it is possible to obtain equivalent peak intensity ratios.

[0018] Also, according to the present invention, a method for producing a calibrant used for correcting the instrumental error of a mass spectrometer is provided. By using the production method of the present invention, a calibrant containing two or more calibration substances and having a ratio of the concentration of one calibration substance to the concentration of another calibration substance adjusted to a predetermined value can be accurately produced. As a result, the instrumental error of the mass spectrometer can be accurately corrected.

[0019] Further, according to the present invention, there is provided a kit of calibrants used for correcting instrumental errors of a mass spectrometer. Alternatively, there is provided a kit for preparing calibrants. By using the kit of the present invention, a calibrant containing two or more calibration substances can be obtained, in which the ratio of the concentration of one calibration substance to the concentration of another calibration substance is accurately adjusted to a predetermined value. As a result, the instrumental errors of the mass spectrometer can be corrected accurately.

Brief Description of Drawings

[0020]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0021] One embodiment of the calibrant used in the mass spectrometer of the present invention is comprising two or more calibration substances, The ratio of the concentration of one calibration substance among the two or more calibration substances to the concentration of another calibration substance among the two or more calibration substances is a predetermined value.

[0022] The present embodiment will be described in detail below.

[0023] The calibrant of the present embodiment is used for correcting the instrumental error of a mass spectrometer. The calibrant of the present embodiment is measured with a mass spectrometer, and a correction formula in the mass spectrometer is calculated using the measurement result of the calibrant. In the mass spectrometer, a sample containing an analyte is measured, and the measurement data of the sample is corrected using the correction value. The detailed method of correction will be described later.

[0024] [1. Calibration Substance] In the present embodiment, a calibration substance is a substance used for correcting the instrumental error of a mass spectrometer. A solution (calibrant) containing two or more calibration substances is measured with a mass spectrometer. A correction formula in the mass spectrometer is calculated using the measurement result of the calibration substance.

[0025] The calibration substance can be appropriately determined by those skilled in the art. For example, the analyte itself may be used, or a substance different from the analyte may be used. A stable isotope-labeled substance may be used. A pair of a stable isotope-labeled substance and a non-stable isotope-labeled substance may be used.

[0026] Aβ and Aβ-related peptides may be used as the calibration substance. "Aβ and Aβ-related peptides" may also be collectively referred to as simply "Aβ-related peptides". "Aβ and Aβ-related peptides" include Aβ generated by cleavage of amyloid precursor protein (APP) and peptides containing a part of the sequence of Aβ.

[0027] When using Aβ and Aβ-related peptides as substances to be analyzed, for example, Aβ1-38 labeled with a stable isotope (SIL-Aβ1-38) may be used as one of the calibration substances. Here, SIL stands for stable isotope-labeled. For example, the SIL-Aβ1-38 used in the examples has the carbon atoms of phenylalanine (Phe or F) and isoleucine (Ile or I) of Aβ1-38 (SEQ ID NO: 11) 13 substituted with

[0028] The calibration substance may include one compound and the same compound labeled with a stable isotope. In mass spectrometry, the compound is ionized and detected. The ionization efficiency varies depending on the compound, and the difference in ionization efficiency affects the mass spectrometry measurement results. A certain compound and the same compound labeled with a stable isotope have the same ionization efficiency. Therefore, by using a pair of one compound and the same compound labeled with a stable isotope as the calibration substance, a more accurate correction formula can be obtained. For example, Aβ1-38 and Aβ1-38 labeled with a stable isotope (SIL-Aβ1-38) may be used as the calibration substance. Not limited to Aβ1-38, other substances can also be used. For example, other Aβ-related peptides, peptides other than other Aβ-related peptides, proteins, sugar chains, glycopeptides, glycoproteins, lipids, glycolipids, small molecule pharmaceuticals, etc. may be used. In that case, these substances may be combined with the same substance labeled with a stable isotope and used as the calibration substance.

[0029] [2. Calibrant] The calibrant is a solution containing the calibration substance. In this embodiment, the calibrant is a solution containing two or more calibration substances.

[0030] As a calibrant, for example, the sample itself containing the analyte can be used. Also, a sample obtained by adding a calibration substance to the sample itself containing the analyte can be used. A solution containing two or more calibration substances may be prepared and used separately from the sample itself containing the analyte.

[0031] In the calibrant of the present embodiment, the ratio of the concentration of one calibration substance among the two or more calibration substances in the calibrant to the concentration of another calibration substance among the two or more calibration substances in the calibrant is a predetermined value. That is, the ratio of the concentrations in the calibrant is known.

[0032] The calibrant of the present embodiment may be prepared by adjusting the ratio of the concentrations to a desired value. The ratio of the concentrations can be appropriately determined by those skilled in the art. For example, it may be in the range of 1 / 4 to 4. The ratio of the concentrations may be confirmed to be a predetermined value by measuring the concentrations of the respective calibration substances using a method known in advance.

[0033] For example, when the calibrant contains two calibration substances, if the calibration substances are denoted as S1 and S2, in the calibrant, the ratio S2 / S1 of the concentration of one calibration substance (S1) to the concentration of the other calibration substance (S2) is a predetermined value.

[0034] For example, a substance labeled with a stable isotope may be used as the calibration substance (S1), and a substance not labeled with a stable isotope may be used as the calibration substance (S2). As the calibration substance (S1) and the calibration substance (S2), it is preferable to use a combination of a substance labeled with a stable isotope and a substance not labeled with a stable isotope of the same substance. For example, Aβ1-38 labeled with a stable isotope (SIL-Aβ1-38) may be used as the calibration substance (S1), and Aβ1-38 not labeled with a stable isotope may be used as the calibration substance (S2). Further, a substance not labeled with a stable isotope may be used as the calibration substance (S1), and a substance labeled with a stable isotope may be used as the calibration substance (S2).

[0035] For example, the calibrant may contain three or more calibration substances. In this case, for the concentration of one calibration substance among the three or more calibration substances, the ratio of the concentration of any other one of the three or more calibration substances is a predetermined value.

[0036] Suppose there are three calibration substances. If the calibration substances are denoted as S1, S2, and S3, for the calibrant, the ratio S2 / S1 of the concentration of any other one calibration substance (for example, S2) to the concentration of one calibration substance (for example, S1) is a predetermined value. In this case, further, the ratio S3 / S1 of the concentration of the calibration substance other than S1 and S2 (in this case, S3) to the concentration of one calibration substance (S1) may also be a predetermined value.

[0037] Using the measurement results of the calibration standard solution containing the two or more calibration substances, a correction formula in the mass spectrometer is calculated as described below. To calculate the correction formula, a plurality of data are required. For example, when calculating the correction formula using the signal peak intensity ratio, a plurality of data with different signal peak intensity ratios are required.

[0038] For example, the calibration standard may be a plurality of calibration standards in which the concentrations of the calibration substances for which the signal peak intensity ratio should be obtained when calculating the correction formula are different. It may be a plurality of calibration standards containing two types of calibration substances and having different ratios of the concentration of one calibration substance to the concentration of the other calibration substance.

[0039] For example, if the calibration substances are denoted as S1 and S2, the calibration standard may be a plurality of calibration standards with different ratios S2 / S1 of the concentration of the other calibration substance (S2) to the concentration of one calibration substance (S1). The concentration ratio S2 / S1 may be, for example, in the range of 1 / 4 to 4. For example, the calibration standard may be five types of calibration standard solutions adjusted so that the concentration ratio S2 / S1 is 1 / 4, 1 / 2, 1, 2, 4.

[0040] For example, a substance labeled with a stable isotope may be used as the calibration substance (S1), and a substance not labeled with a stable isotope may be used as the calibration substance (S2). As the calibration substance (S1) and the calibration substance (S2), a combination of a substance labeled with a stable isotope and a substance not labeled with a stable isotope of that substance may be used. For example, Aβ1-38 labeled with a stable isotope (SIL-Aβ1-38) may be used as the calibration substance (S1), and Aβ1-38 may be used as the calibration substance (S2).

[0041] When using such a plurality of calibrants, for example, it may be used in [5-3-2. Correction for normalizing the signal peak intensity ratio (intensity ratio calibration)] described later. As will be described later, the ratio of the signal peak intensity of one calibration substance to the signal peak intensity of another calibration substance may be calculated for each calibrant, and two or more signal peak intensity ratios may be obtained.

[0042] Alternatively, the calibrant may include three or more types of calibration substances and be a plurality of calibrants in which the ratio of the concentration of any one calibration substance to the concentration of another calibration substance is different. For example, when including three types of calibration substances, if the calibration substances are denoted as S1, S2, and S3, the calibrant may be at least a plurality of calibrants in which the ratio S2 / S1 of the concentration of any one calibration substance (for example, S2) to the concentration of another calibration substance (for example, S1) is different.

[0043] Further, in a plurality of calibrants in which the concentration ratio S2 / S1 is different, for example, the ratio of the concentration of calibration substance S3 to the concentration of calibration substance (S1) (concentration ratio S3 / S1) may be adjusted to the same or different predetermined values in each calibrant.

[0044] When the calibrant contains three or more types of calibration substances, it is also possible to calculate a correction formula in a mass spectrometer using one calibrant. For example, it may be used in [5-3-1. Correction using a correction formula with a standard] described later.

[0045] [3. Method for preparing calibrant] One embodiment of the method for preparing the calibrant of the present invention is a solution A containing one calibration substance (S1) among two or more calibration substances at a predetermined concentration, and Prepare a solution B that contains one calibration substance (S1) at the same concentration as the predetermined concentration and further contains another calibration substance (S2) among the two or more calibration substances. A pre-step of preparing A preparation step of obtaining a calibrant in which the ratio of the concentration of the other calibration substance (S2) to the concentration of the one calibration substance (S1) is a predetermined value by using the solution A and the solution B. The method includes these steps.

[0046] The calibrant is used for correcting the instrumental error of a mass spectrometer. The calibrant produced by the production method of the present embodiment is prepared such that the ratio of the concentration of one calibration substance to the concentration of another calibration substance is a predetermined value.

[0047] Correction of the instrumental error of a mass spectrometer using the calibrant produced by the production method of the present embodiment is For example, it is performed by using the calibration formula (correction value) in the mass spectrometer obtained by using each concentration ratio in calibrants where the ratio of the concentration of one calibration substance to the concentration of another calibration substance is different from each other and the measurement result (for example, signal peak intensity ratio) in the mass spectrometer. Details of the correction method will be described later.

[0048] In order to perform such correction accurately, it is necessary to precisely produce the calibrant. That is, it is necessary to produce the ratio of the concentration of one calibration substance to the concentration of another calibration substance so that it exactly becomes the desired value.

[0049] Hereinafter, the production method of the present embodiment will be described in detail. For the production method, FIGS. 1 to 4 can be referred to. FIG. 1 is a conceptual diagram showing a first embodiment of the preparation step. FIG. 2 is a conceptual diagram showing a second embodiment of the preparation step. FIG. 3 is a schematic diagram showing the production method of the calibrant of Example 1. FIG. 4 is a schematic diagram showing the production method of the calibrant of Example 2.

[0050] Using the production method of the present embodiment, a calibrant is prepared that contains at least two calibration substances (S1 and S2), and the ratio of the concentration of calibration substance (S2) in the calibrant to the concentration of calibration substance (S1) in the calibrant (concentration ratio S2 / S1) is adjusted to a predetermined value.

[0051] As S1, a stable isotope-labeled substance may be used, and as S2, a non-stable isotope-labeled substance may be used. Alternatively, as S1, a non-stable isotope-labeled substance may be used, and as S2, a stable isotope-labeled substance may be used. As calibration substance (S1) and calibration substance (S2), it is preferable to use a combination of a substance labeled with a stable isotope and the same substance without stable isotope labeling. For example, in the examples, as shown in FIGS. 3 and 4, an example is shown in which SIL-Aβ1-38 labeled with a stable isotope is used as calibration substance (S1), and Aβ1-38 is used as calibration substance (S2).

[0052] [3-1. Previous step] A solution A containing calibration substance (S1) at a predetermined concentration, and A solution B containing calibration substance (S1) at the same concentration as the predetermined concentration and further containing calibration substance (S2) are prepared.

[0053] The concentration of (S1) in solution A and solution B may be equal to the concentration of calibration substance (S1) in the calibrant to be prepared. For example, it may be 1 pmol / L to 1 mmol / L.

[0054] The concentration of (S2) in Solution B may be, for example, 0.1 pmol / L to 10 mmol / L. The concentration of (S2) in Solution B may be about 1.1 to 10 times, preferably about 1.5 to 5 times, more preferably 2 times the concentration of the calibration substance (S2) in the calibrant to be prepared. For example, it may be 1.2 to 5 times.

[0055] The solvents used for Solution A and Solution B can be appropriately determined by those skilled in the art. For example, water, acetonitrile, alcohol, etc. may be used, either alone or in combination of multiple types. The solvents used for Solution A and Solution B may be different or the same. Any other substance may be added to the solvent. For example, the solvent may contain bovine serum albumin (BSA), ammonium bicarbonate, etc. BSA functions as an anti-adsorption agent and also as an antioxidant to prevent oxidation of methionine, etc. For example, the solvent may contain an antioxidant, etc. to maintain its quality. As the antioxidant, a protein, an amino acid, or a reducing agent can be used. Examples of the protein include BSA, transferrin, fetuin, etc. Examples of the amino acid include methionine, histidine, cysteine, tryptophan, etc. Examples of the reducing agent include dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), 2-mercaptoethanol (2-ME), tri-n-butylphosphine (TBP), dithioerythritol (DTE), ascorbic acid, polyphenol, sodium pyrosulfite, citric acid, glucose, carotene, tocopherol, thioglycolic acid, N-acetylcysteine, hydroxylamine, reduced glutathione, 2-aminoethylisothiouronium bromide, thioglycerol, etc. These may be used alone or in combination of multiple substances.

[0056] For example, solution A and solution B may be prepared separately. That is, solution A may be prepared using a solvent and a calibration substance (S1). And separately, solution B may be prepared using a solvent, a calibration substance (S1), and a calibration substance (S2).

[0057] Preferably, first, prepare solution A0 containing calibration substance (S1) at a concentration higher than the desired concentration, and solution B0 containing calibration substance (S2).

[0058] The solvent used for solution A0 can be appropriately determined by those skilled in the art. For example, water, acetonitrile, alcohol, etc. may be used. Also, the concentration of (S1) in solution A0 may be about 5 to 500 times the desired concentration of (S1) in the calibrant. Preferably, it can be about 20 to 100 times.

[0059] The solvent used for solution B0 can be appropriately determined by those skilled in the art. For example, water, acetonitrile, alcohol, etc. may be used. It may be used alone or in combination of multiple types. Any other substance may be added to the solvent. For example, it may contain bovine serum albumin (BSA), ammonium bicarbonate, etc. For example, the solvent may contain an antioxidant, etc. to maintain its quality. As the antioxidant, proteins, amino acids, or reducing agents as described above can be used.

[0060] Also, the concentration of (S2) in solution B0 may be about 5 to 500 times the concentration of (S2) in the calibrant. Preferably, it can be about 20 to 100 times.

[0061] Next, a solvent (e.g., water, acetonitrile, alcohol, etc.) is added to solution A0 to prepare solution A containing a calibration substance (S1) at a desired concentration. Further, a solvent (e.g., water, acetonitrile, alcohol, etc.) and solution B0 containing a calibration substance (S2) are added to solution A0 to prepare solution B containing the calibration substance (S1) at the same concentration as the desired concentration in solution A and also containing the calibration substance (S2). The amount of solution B0 added may be appropriately adjusted so that the peak intensity ratio to the ratio of the calibration substance (S2) to the calibration substance (S1) (S2 / S1) becomes a predetermined value.

[0062] Thus, by preparing solution A and solution B from solution A0, the concentration of the calibration substance (S1) in solution A and the concentration of the calibration substance (S1) in solution B can be made more precisely equal. As a result, in the preparation process described later, the concentration of the calibration substance (S1) in the prepared calibrant can be made more precisely constant.

[0063] Alternatively, solution A0 may be diluted in advance with a solvent to prepare solution A0' containing (S1) at a concentration closer to the concentration of (S1) in the calibrant. Solution A and solution B may be prepared using solution A0' and solution B0. By using solution A0', the amount of the solvent added when preparing solution A and solution B can be reduced. As a result, in the preparation process described later, the concentration of the calibration substance (S1) in the prepared calibrant can be made even more precisely constant.

[0064] [3-2. Preparation Process] Prepared in the previous step, solution A containing the calibration substance (S1) at a predetermined concentration, and solution B containing the calibration substance (S1) at the same concentration as the predetermined concentration and further containing the calibration substance (S2), Using this, a calibrant is prepared in which the ratio of the concentration of calibration substance (S2) to the concentration of calibration substance (S1) (concentration ratio S2 / S1) is adjusted to a predetermined value with respect to the concentration of calibration substance (S1).

[0065] As a method for preparing a calibrant using Solution A and Solution B, for example, Solution A and Solution B may be respectively measured and mixed so that the concentration ratio S2 / S1 in the calibrant becomes a predetermined concentration ratio.

[0066] As described above, since the concentrations of the calibration substance (S1) in Solution A and Solution B are equal, the concentration of the calibration substance (S1) in the calibrant is constant regardless of the mixing ratio (content ratio) of Solution A and Solution B. On the other hand, the concentration of the calibration substance (S2) in the calibrant can be adjusted to an arbitrary value by the mixing ratio (content ratio) of Solution A and Solution B. That is, the concentration ratio S2 / S1 in the calibrant can be adjusted to a predetermined value.

[0067] Using the production method of the present embodiment, a plurality of calibrants can also be prepared that contain at least two calibration substances (S1) and (S2) and in which the ratio of the concentration of calibration substance (S2) in the calibrant to the concentration of calibration substance (S1) in the calibrant (concentration ratio S2 / S1) is different for each. Here, a case will be described in which a plurality of calibrants with different concentration ratios S2 / S1 in the calibrant are prepared by keeping the concentration of calibration substance (S1) in the calibrant constant and varying the concentration of calibration substance (S2) in the calibrant.

[0068] In this case, the concentration of the calibration substance (S2) in the solution B prepared in the previous step may be about 1.1 to 10 times, for example, 1.2 to 5 times, preferably about 1.5 to 5 times the concentration of the calibration substance (S2) in the calibrant having the highest concentration of the calibration substance (S2) among the plurality of calibrants to be prepared. For example, it may be 2 times the concentration of (S2) in the calibrant having the highest concentration of the calibration substance (S2).

[0069] As described above, since the concentrations of the calibration substance (S1) in the solution A and the solution B are equal, the concentration of the calibration substance (S1) in the calibrant is constant regardless of the mixing ratio (content ratio) of the solution A and the solution B. On the other hand, by changing the mixing ratio (content ratio) of the solution A and the solution B, a plurality of calibrants having different concentrations of the calibration substance (S2) in the calibrant can be prepared. That is, the concentration ratios S2 / S1 in the plurality of calibrants can be adjusted to different predetermined values.

[0070] For example, three calibrants (denoted as IC-1, IC-2, and IC-3) may be prepared. The solution A and the solution B may be mixed so that IC-1, IC-2, and IC-3 have different desired concentration ratios S2 / S1, respectively.

[0071] Alternatively, the solution A and the solution B may be mixed to obtain a calibrant, a part of the obtained calibrant may be separated, and the solution A may be further mixed with the separated part to obtain another calibrant having a ratio of the concentration of the other calibration substance (S2) to the concentration of the one calibration substance (S1) different from the concentration ratio in the calibrant. Further, this step may be sequentially repeated to obtain three or more calibrants.

[0072] That is, IC-1 may be prepared by mixing solution A and solution B, and then, a part of IC-1 and solution A may be mixed to prepare IC-2. Further, a part of the obtained IC-2 and solution A may be mixed to prepare IC-3. In this case, the concentration ratio S2 / S1 is the largest for IC-1 and becomes smaller in the order of IC-2 and IC-3. That is, solution B is sequentially diluted with solution A.

[0073] Alternatively, a calibrant may be obtained by mixing solution A and solution B, a part (for example, half) of the obtained calibrant may be separated, and a mixing operation may be performed in which the same amount of solution A as a part (for example, half) of the calibrant is further mixed with the separated part to obtain another calibrant in which the ratio of the concentration of the other calibration substance (S2) to the concentration of one calibration substance (S1) is 1 / 2 of the concentration ratio in the calibrant. Further, this step may be sequentially repeated (so-called double dilution) to obtain three or more calibrants.

[0074] That is, IC-1 may be prepared by mixing solution A and solution B, and then, a part (for example, half) of IC-1 and the same amount of solution A may be mixed to prepare IC-2. Further, a part (for example, half) of the obtained IC-2 and the same amount of solution A may be mixed to prepare IC-3. In this case, the concentration ratio S2 / S1 is the largest for IC-1, 1 / 2 of that in the case of IC-1 for IC-2, and 1 / 2 of that in the case of IC-2 (1 / 4 of that in the case of IC-1) for IC-3. That is, double dilution is performed using solution A.

[0075] Or, a calibrant may be obtained by mixing solution A and solution B, a part of the obtained calibrant may be separated, and a mixing operation may be performed in which solution B is further mixed with the separated part to obtain another calibrant in which the ratio of the concentration of the other calibration substance (S2) to the concentration of one calibration substance (S1) is a value different from the concentration ratio in the calibrant. Further, this step may be sequentially repeated to obtain three or more calibrants.

[0076] That is, solution A and solution B may be mixed to prepare IC-1, and then a part of IC-1 and solution B may be mixed to prepare IC-2. Further, a part of the obtained IC-2 and solution B may be mixed to prepare IC-3. In this case, the concentration ratio S2 / S1 is the smallest for IC-1, and increases in the order of IC-2 and IC-3.

[0077] Alternatively, solution A and solution B may be mixed to obtain a calibrant, a part (for example, half) of the obtained calibrant may be separated, and a mixing operation of mixing the same amount of the solution B as a part (for example, half) of the calibrant into the separated part may be performed, and another calibrant having a ratio of the concentration of the other calibration substance (S2) to the concentration of the one calibration substance (S1) different from the concentration ratio in the calibrant may be obtained. Further, this step may be repeated to obtain three or more calibrants.

[0078] That is, solution A and solution B may be mixed to prepare IC-1, and then a part (for example, half) of IC-1 and the same amount of solution B may be mixed to prepare IC-2. Further, a part (for example, half) of the obtained IC-2 and the same amount of solution B may be mixed to prepare IC-3.

[0079] FIG. 1 is a conceptual diagram showing a first embodiment of a preparation process in the case of preparing five calibrants (denoted as IC-1, IC-2, IC-3, IC-4, and IC-5). In FIG. 1, the concentration ratios S2 / S1 in IC-1, IC-2, IC-3, IC-4, and IC-5 are shown for the cases where they are 4 (S2:S1 = 4:1), 2 (S2:S1 = 2:1), 1 (S2:S1 = 1:1), 1 / 2 (S2:S1 = 0.5:1), and 1 / 4 (S2:S1 = 0.25:1), respectively.

[0080] As shown in FIG. 1, in the first embodiment, a solution B containing a calibration substance (S1) and a calibration substance (S2) is successively diluted twice with a solution A containing the calibration substance (S1) to prepare IC-1, IC-2, IC-3, IC-4, and IC-5.

[0081] More specifically, solution B and solution A are mixed at a predetermined ratio (for example, the same amount each) to prepare IC-1. Next, a part of the prepared IC-1 (for example, half) is mixed with the same amount of solution A to prepare IC-2. A part of the prepared IC-2 (for example, half) is mixed with the same amount of solution A to prepare IC-3. A part of the prepared IC-3 (for example, half) is mixed with the same amount of solution A to prepare IC-4. Finally, a part of the prepared IC-4 (for example, half) is mixed with the same amount of solution A to prepare IC-5. Using such a successive dilution method, a plurality of calibrants with different ratios of the concentration of the calibration substance (S2) to the concentration of the calibration substance (S1) (concentration ratio S2 / S1) may be prepared.

[0082] FIG. 2 is a conceptual diagram showing a second embodiment of the preparation process in the case of preparing five calibrants (denoted as IC-1, IC-2, IC-3, IC-4, and IC-5). Also in FIG. 2, as in the case of FIG. 1, the concentrations in IC-1, IC-2, IC-3, IC-4, and IC-5 Ratio S 2 / S1 shows the cases where they are 4 (S2:S1 = 4:1), 2 (S2:S1 = 2:1), 1 (S2:S1 = 1:1), 1 / 2 (S2:S1 = 0.5:1), and 1 / 4 (S2:S1 = 0.25:1), respectively.

[0083] In the second embodiment, a solution D containing the calibration substance (S1) and a solution E containing the calibration substance (S2) are prepared.

[0084] In the second embodiment, as shown in FIG. 2, a solution E containing a calibration substance (S2) is successively diluted twice with the solvent of the solution E to prepare five solutions. Then, a solution D containing a calibration substance (S1) is added to each of the five solutions to prepare IC-1, IC-2, IC-3, IC-4, and IC-5.

[0085] More specifically, solution E and the solvent are mixed at a predetermined ratio (for example, in equal amounts) to prepare solution E1. A part of the prepared solution E1 (for example, half of it) and the same amount of the solvent are mixed to prepare solution E2. A part of the prepared solution E2 (for example, half of it) and the same amount of the solvent are mixed to prepare solution E3. A part of the prepared solution E3 (for example, half of it) and the same amount of the solvent are mixed to prepare solution E4. A part of the prepared solution E4 (for example, half of it) and the same amount of the solvent are mixed to prepare solution E5. Solution D is added to solution E1, solution E2, solution E3, solution E4, and solution E5, respectively, to prepare IC-1, IC-2, IC-3, IC-4, and IC-5. Using such a method, a plurality of calibrants with different concentration ratios S2 / S1 may be prepared.

[0086] When using the solution D containing the calibration substance (S1) and the solution E containing the calibration substance (S2) as in the second embodiment, solution D containing the calibration substance (S1) is measured and added to each of the solutions (E1, E2, E3, E4, and E5) with different concentrations of the calibration substance (S2). In such a preparation method, in addition to the error in the concentration of the calibration substance (S2) due to the measurement error when diluting solution E with the solvent, there is also an error in the concentration of the calibration substance (S1) due to the measurement error when mixing each of E1, E2, E3, E4, and E5 with solution D. As a result, there is a concern that the concentration ratio S2 / S1 in each calibrant may vary from the desired value.

[0087] On the one hand, when using the first embodiment, in a plurality of calibrants, the variation in the concentration of the calibration substance (S1) can be minimized. As a result, the concentration ratio S2 / S1 in each calibrant can be prepared to exactly the desired value. By using the calibrant prepared in this way, when calculating the correction formula in the mass spectrometer by the method described later, the accuracy of the correction formula can be made higher.

[0088] The preparation step using solution A and solution B may be performed by measuring by volume. Alternatively, the preparation step using solution A and solution B may be performed by measuring by weight.

[0089] In the preparation step, the required amounts of solution A and solution B are measured and mixed. Alternatively, the required amounts of the calibrant prepared by mixing and solution A are measured and mixed. The measurement at this time may be performed by volume using pipetting or the like. It may also be performed by weight using an electronic balance or the like.

[0090] Particularly when at least one of solution A and solution B is viscous, by using an electronic balance, the variation due to pipettes or the operator's technique is reduced, and the concentration of the calibration substance (S2) in the calibrant can be made to the desired value with higher accuracy. As a result, the concentration ratio S2 / S1 can be adjusted to the desired value with higher accuracy.

[0091] When using an electronic balance, it is possible to leave an accurate work record, which is preferable from the viewpoint of traceability. Also, it is easy to scale up the amount of calibrant produced.

[0092] In addition to the two calibration substances (S1) and (S2), one or more other substances may be included as calibration substances. For example, when further including a calibration substance S3, the ratio of the concentration of the calibration substance (S2) to the concentration of the calibration substance (S1) (concentration ratio S2 / S1) and the ratio of the concentration of the calibration substance S3 to the concentration of the calibration substance (S1) (concentration ratio S3 / S1) may be prepared so as to be respectively predetermined values.

[0093] The calibrant prepared in the above-mentioned pre-process and preparation process may be directly used for measurement with a mass spectrometer. Alternatively, it may be diluted at a predetermined magnification and used when being subjected to measurement with a mass spectrometer. The dilution magnification may be, for example, about 2 to 50 times. For example, it may be 10 times. The dilution solvent can be appropriately determined by those skilled in the art. The solvent contained in the calibrant may be used.

[0094] [4. Kit] One embodiment of the kit of the present invention includes a plurality of calibrants containing two or more calibration substances, and in the plurality of calibrants, the concentrations of at least one calibration substance among the calibration substances are different from each other.

[0095] For example, when the calibration substances are denoted as S1 and S2, the kit may include a plurality of calibrants having different ratios S2 / S1 of the concentration of the other calibration substance (S2) to the concentration of one calibration substance (S1). The concentration ratio S2 / S1 may be, for example, in the range of 1 / 4 to 4. For example, the kit may include five types of calibrant solutions adjusted so that the concentration ratio S2 / S1 is 1 / 4, 1 / 2, 1, 2, 4. For example, in the five types of calibrant solutions, Aβ1-38 (SIL-Aβ1-38) labeled with a stable isotope may be used as the calibration substance (S1), and Aβ1-38 may be used as the calibration substance (S2).

[0096] The calibrant contained in the kit itself may be directly used for measurement with a mass spectrometer. Alternatively, a calibrant obtained by diluting the calibrant contained in the kit at a predetermined magnification may be used for measurement with a mass spectrometer.

[0097] The concentration ratios S2 / S1 in a plurality of calibrants contained in such a kit are precisely adjusted respectively. By using the kit, an operator can measure a plurality of calibrants in which the concentration ratios S2 / S1 are precisely adjusted respectively with a mass spectrometer. As a result, the calculation of a correction formula in the mass spectrometer described later can be performed with high accuracy.

[0098] Another embodiment of the kit of the present invention includes two or more calibration substances.

[0099] The kit contains materials used to prepare a calibrant in which the ratio S2 / S1 of the concentration of another calibration substance (S2) to the concentration of one calibration substance (S1) is adjusted to a desired value.

[0100] By using such a kit, an operator can prepare a calibrant in which the concentration ratio S2 / S1 is adjusted to a desired concentration when calculating a correction formula in a mass spectrometer. By using such a kit, an operator can suppress the change over time of the concentration ratio S2 / S1 due to storing the calibrant. As a result, the calculation of a correction formula in the mass spectrometer described later can be performed with high accuracy.

[0101] The kit may further contain a solvent used for the calibrant.

[0102] Further, the kit may further contain a container such as an ampoule for containing the calibrant.

[0103] [5. Correction of instrumental error of mass spectrometer] A method for correcting instrumental errors of a mass spectrometer using a calibrant in the present embodiment will be described in detail below. The calibrant is measured with a mass spectrometer, and a correction formula for the mass spectrometer is calculated using the measurement result. A sample containing an analyte is measured with the mass spectrometer, and the measurement result is corrected by the calculated correction formula. By performing this correction, it becomes possible to compare and evaluate the measurement results of the analyte among a plurality of samples regardless of the instrument body of the mass spectrometer.

[0104] [5-1. Analyte] The analyte is not particularly limited, and examples thereof include peptides, glycopeptides, sugar chains, proteins, lipids, glycolipids, low-molecular-weight pharmaceuticals, etc. Peptides, glycopeptides, sugar chains, proteins, lipids, glycolipids, and low-molecular-weight pharmaceuticals can include various ones. More specifically, they may be Aβ and Aβ-related peptides. "Aβ and Aβ-related peptides" may sometimes be collectively referred to simply as "Aβ-related peptides". "Aβ and Aβ-related peptides" include peptides that contain Aβ generated by cleavage of amyloid precursor protein (APP) and also contain at least a part of the sequence of Aβ.

[0105] For example, Aβ-related peptides include the following.

[0106] APP677-709 (Aβ6-38) (SEQ ID NO: 1): HDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGG APP672-704 (Aβ1-33) (SEQ ID NO: 2): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIG APP677-711 (Aβ6-40) (SEQ ID NO: 3): HDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV APP672-706 (Aβ1-35) (SEQ ID NO: 4): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLM APP672 - 708 (Aβ1 - 37) (SEQ ID NO: 5): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG APP674 - 711 (Aβ3 - 40) (SEQ ID NO: 6): EFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV APP672 - 711 (Aβ1 - 40) (SEQ ID NO: 7): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV OxAPP672 - 711 (OxAβ1 - 40) (SEQ ID NO: 8): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL M VGGVV (Met 706 is oxidized) APP672 - 713 (Aβ1 - 42) (SEQ ID NO: 9): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA APP669 - 711 (SEQ ID NO: 10): VKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV APP672 - 709 (Aβ1 - 38) (SEQ ID NO: 11): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGG APP672 - 710 (Aβ1 - 39) (SEQ ID NO: 12): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGV

[0107] Amyloid precursor protein (APP) is a single-pass transmembrane protein consisting of 770 amino acid residues. Amyloid precursor protein (APP) is proteolyzed by β-secretase and γ-secretase, and amyloid-β peptide (Aβ) is produced by proteolysis. APP672-713 and Aβ1-42 represent the same peptide (SEQ ID NO: 9). Also, APP672-711 and Aβ1-40 represent the same peptide (SEQ ID NO: 7).

[0108] Among the Aβ-related peptides as described above, Aβ1-42 (SEQ ID NO: 9), Aβ1-40 (SEQ ID NO: 7), APP669-711 (SEQ ID NO: 10), and Aβ1-39 (SEQ ID NO: 12) are effective biomarkers for Alzheimer's disease. Also, Ratio of APP669-711 level to APP672-713 (Aβ1-42) level: APP669-711 / APP672-713 (Aβ1-42), Ratio of APP672-711 (Aβ1-40) level to APP672-713 (Aβ1-42) level: APP672-711 (Aβ1-40) / APP672-713 (Aβ1-42), and Ratio of APP672-710 (Aβ1-39) level to APP672-713 (Aβ1-42) level: APP672-710 (Aβ1-39) / APP672-713 (Aβ1-42) etc. are also effective biomarkers for Alzheimer's disease.

[0109] Also, the peptide may be a peptide obtained by immunoprecipitation (IP). Alternatively, it may be a peptide generated by digestion of a protein with an enzyme such as a peptidase, or a peptide fractionated by chromatography.

[0110] The analyte may contain an internal standard substance. The internal standard substance can be appropriately selected by those skilled in the art. For example, a substance labeled with a stable isotope may be used. A substance labeled with a stable isotope among one of the analytes may be used. In the examples, an example of using Aβ1-38 labeled with a stable isotope (SIL-Aβ1-38) as the internal standard substance is shown.

[0111] A sample containing the analyte is subjected to mass spectrometry. The sample subjected to mass spectrometry is not particularly limited, but for example, it may be a bio-derived sample. Bio-derived samples include body fluids such as blood, cerebrospinal fluid (CSF), urine, body secretions, saliva, and sputum; and feces. Blood samples include whole blood, plasma, and serum. The blood sample can be prepared by appropriately processing the whole blood collected from an individual. The processing performed when preparing a blood sample from the collected whole blood is not particularly limited, and any clinically acceptable processing may be performed. For example, centrifugation or the like may be performed. In addition, the blood sample subjected to mass spectrometry may be stored at a low temperature such as freezing appropriately at an intermediate stage or a later stage of the preparation process. In the present invention, when a bio-derived sample such as a blood sample is subjected to mass spectrometry, the bio-derived sample is discarded without being returned to the original subject.

[0112] The sample subjected to mass spectrometry may be a sample after various pretreatments. For example, it may be after performing immunoprecipitation (IP). It may be after digestion of a protein with an enzyme such as a peptidase. It may be after performing chromatography. The sample subjected to mass spectrometry may be one to which a certain amount of an internal standard substance is added.

[0113] Before being subjected to mass spectrometry, the sample may be first subjected to immunoprecipitation, and then the eluate obtained by immunoprecipitation may be subjected to mass spectrometry (Immunoprecipitation-mass spectrometry; IP-MS). The immunoprecipitation method may be performed using an antibody-immobilized carrier prepared using an immunoglobulin having an antigen-binding site capable of recognizing the analyte or an immunoglobulin fragment containing an antigen-binding site capable of recognizing the analyte.

[0114] Alternatively, the sample to be subjected to mass spectrometry may be subjected to consecutive immunoprecipitation (cIP), and then the peptides in the sample may be detected using a mass spectrometer (cIP-MS). By performing affinity purification twice in a row, contaminants that could not be completely eliminated by only one affinity purification can be further reduced by the second affinity purification. Therefore, it is possible to prevent the ionization suppression of polypeptides by contaminants, and even trace amounts of polypeptides in biological samples can be measured with high sensitivity by mass spectrometry.

[0115] [5-2. Mass Spectrometry] The mass spectrometry method is not particularly limited, and includes mass spectrometry methods such as matrix-assisted laser desorption ionization (MALDI) mass spectrometry and electrospray ionization (ESI) mass spectrometry. For example, a MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) type mass spectrometer, a MALDI-IT (matrix-assisted laser desorption ionization-ion trap) type mass spectrometer, a MALDI-IT-TOF (matrix-assisted laser desorption ionization-ion trap-time of flight) type mass spectrometer, a MALDI-FTICR (matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance) type mass spectrometer, an ESI-QqQ (electrospray ionization-triple quadrupole) type mass spectrometer, an ESI-Qq-TOF (electrospray ionization-tandem quadrupole-time of flight) type mass spectrometer, an ESI-FTICR (electrospray ionization-Fourier transform ion cyclotron resonance) type mass spectrometer, etc. can be used.

[0116] The matrix and the matrix solvent can be appropriately determined by those skilled in the art according to the analyte substance.

[0117] As the matrix, for example, α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (2,5-DHB), sinapic acid, 3-aminoquinoline (3-AQ), etc. can be used.

[0118] As the matrix solvent, for example, it can be selected from the group consisting of acetonitrile (ACN), trifluoroacetic acid (TFA), methanol, ethanol and water. More specifically, ACN-TFA aqueous solution, ACN aqueous solution, methanol-TFA aqueous solution, methanol aqueous solution, ethanol-TFA aqueous solution, ethanol solution, etc. can be used. The concentration of ACN in the ACN-TFA aqueous solution is, for example, 10 to 90% by volume, and the concentration of TFA is, for example, 0.05 to 1% by volume, preferably 0.05 to 0.1% by volume.

[0119] The matrix concentration can be, for example, 0.1 to 50 mg / mL, preferably 0.1 to 20 mg / mL, or 0.3 to 20 mg / mL, more preferably 0.5 to 10 mg / mL.

[0120] When using a detection system by MALDI mass spectrometry, it is preferable to use a matrix additive (co-matrix). The matrix additive can be appropriately selected by those skilled in the art according to the analyte (polypeptide) and / or the matrix. For example, a phosphonic acid group-containing compound can be used as the matrix additive. Specifically, as a compound containing one phosphonic acid group, phosphonic acid, methylphosphonic acid, phenylphosphonic acid, 1-naphthylmethylphosphonic acid, etc. can be mentioned. Also, as a compound containing two or more phosphonic acid groups, methylenediphosphonic acid (MDPNA), ethylenediphosphonic acid, ethane-1-hydroxy-1,1-diphosphonic acid, nitrilotriphosphonic acid, ethylenediaminetetraphosphonic acid, etc. can be mentioned. Among the above phosphonic acid group-containing compounds, a compound having two or more, preferably 2 to 4 phosphonic acid groups in one molecule is preferable.

[0121] The use of a phosphonic acid group-containing compound is useful, for example, when metal ions in the washing solution remaining on the surface of the antibody-immobilized carrier are mixed into the eluate after the dissociation step. These metal ions have an adverse effect on the background in mass spectrometry. The use of a phosphonic acid group-containing compound has the effect of suppressing such an adverse effect.

[0122] In addition to the above matrix additives, more general additives, for example, substances selected from the group consisting of ammonium salts and organic bases, may be used.

[0123] The matrix additive can be prepared into a solution with a concentration of 0.1 to 10 w / v%, preferably 0.2 to 4 w / v% in water or a matrix solvent. The matrix additive solution and the matrix solution can be mixed, for example, at a volume ratio of 1:100 to 100:1, preferably 1:10 to 10:1.

[0124] [5-3. Correction of Measurement Results in Mass Spectrometer] Using the calibrant of the present embodiment, a correction formula for a mass spectrometer can be calculated. The calibrant of the present embodiment contains two or more calibration substances, and the ratio of the concentration of one calibration substance among the two or more calibration substances to the concentration of another calibration substance among the two or more calibration substances is a predetermined value. Using the result of measuring the calibrant of the present embodiment with a mass spectrometer, the correction formula for the mass spectrometer is calculated. Then, using the calculated correction formula, the measurement result of the analyte is corrected. For example, the calibrant of the present embodiment can be used in a method of correction using a correction formula with a standard. Also, in the calibrant of the present embodiment, the measurement result in mass spectrometry can be normalized using the peak intensity ratio of two calibration substances whose concentration ratio is a predetermined value.

[0125] The correction formula is calculated for each mass spectrometer. Even for the same mass spectrometer, when components such as the detector are replaced or when device settings such as the detector voltage are changed, it is preferable to newly calculate the correction formula. Also, the correction formula may be calculated periodically. Thereby, it is possible to detect deterioration or failure of the detector or the like of the mass spectrometer and to obtain a measurement result in which the influence is eliminated. Therefore, regardless of the body or conditions of the mass spectrometer, it is possible to accurately compare and evaluate the abundance ratio of the analyte among a plurality of samples.

[0126] More preferably, when measuring the analyte, the calibration substance is measured under the same apparatus conditions as those for the measurement, and a correction formula is calculated. The correction formula under the same conditions as the measurement of the analyte can be used, and the accuracy of correction is further improved. Therefore, regardless of the body and conditions of the mass spectrometer, it is possible to more accurately compare and evaluate the abundance ratios of the analytes among a plurality of samples.

[0127] [5-3-1. Correction Using Correction Formula with Standard] [5-3-1-1. Calculation of Correction Formula] In the standard, a calibration solution containing two or more calibration substances which are the calibrants of the present embodiment is measured, and the signal peak intensities of the respective calibration substances are obtained, where the ratio of the concentration of one calibration substance among the two or more calibration substances to the concentration of another calibration substance among the two or more calibration substances is a predetermined value. The ratios of the signal peak intensities of one or more other calibration substances to the signal peak intensity of one calibration substance are calculated respectively.

[0128] Two or more ratios of signal peak intensities may be obtained using a plurality of calibration solutions in which the ratio of the concentration of one calibration substance among the two or more calibration substances to the concentration of another calibration substance among the two or more calibration substances is different. Also, two or more ratios of signal peak intensities may be obtained using one calibration solution containing three or more calibration substances.

[0129] As the standard, it is preferable to use a highly reliable mass spectrometer. Each user can arbitrarily set the mass spectrometer to be used as the standard.

[0130] In a mass spectrometer for which a correction formula is to be calculated, the same calibration solution as that measured by a standard device is measured to obtain the signal peak intensity of each calibration substance. The ratio of the signal peak intensity of one or more other calibration substances to the signal peak intensity of one calibration substance is calculated respectively.

[0131] A regression formula is calculated between the signal peak intensity ratio in the standard device and the signal peak intensity ratio in the mass spectrometer for which the correction formula is to be calculated. For the calculation of the regression formula, a known method such as the least squares method may be appropriately used. For the calculation of the regression formula, the logarithm of the signal peak intensity ratio may also be used. The regression formula may be appropriately selected from linear, polynomial, exponential, logarithmic, or power.

[0132] For example, a linear regression formula can be calculated using the logarithmically transformed value of the signal peak intensity ratio. Also, for example, a power regression formula can be calculated using the signal peak intensity ratio. The calculated regression formula is used as the correction formula for the mass spectrometer.

[0133] For example, taking the logarithm of the signal peak intensity ratio in the mass spectrometer for which the correction formula is to be calculated as x and the logarithm of the signal peak intensity ratio in the standard device as y, the correction coefficients a and b of the linear regression formula (y = ax + b) can be calculated. The calculated linear regression formula (y = ax + b) can be used as the correction formula for the mass spectrometer.

[0134] Alternatively, taking the signal peak intensity ratio in the mass spectrometer for which the correction formula is to be calculated as x and the signal peak intensity ratio in the standard device as y, the correction coefficients a and b of the power regression formula (y = ax b ) can be calculated. The calculated power regression formula (y = ax b ) can be used as the correction formula for the mass spectrometer.

[0135] The correction coefficients a and b are considered to be values specific to the mass spectrometer. Therefore, they may be different values depending on the body of the mass spectrometer and its device conditions. Also, since b is a coefficient that is more greatly affected by the body of the mass spectrometer and its device conditions than the correction coefficient a, for example, in the power regression equation (y = ax b ), a fixed value of a = 1 may be used, and only the correction coefficient for which the b value is calculated may be adopted. That is, instead of the power regression equation (y = ax b ), y = x b may be used as the correction formula. Thus, when only b is adopted as the correction coefficient, the b value is referred to as the correction value.

[0136] [5-3-1-2. Measurement and Correction of Analyte] In the mass spectrometer for which the correction formula was calculated in 5-3-1-1., a sample containing the analyte is measured to obtain the signal peak intensity of the analyte. Using one of the analytes (for example, an internal standard substance) as a reference, the ratio of the signal peak intensities of the other analytes to the signal peak intensity of the reference analyte is calculated.

[0137] The calculated signal peak intensity ratio is corrected using the correction formula calculated in 5-3-1-1. above. The corrected signal peak intensity ratio becomes a value in which the difference due to the body from the standard device is canceled. That is, it becomes a value equivalent to the signal peak intensity ratio obtained when measured with the standard device. Therefore, by using the corrected signal peak intensity ratio, it is possible to compare and evaluate the signal peak intensity ratio of the analyte, that is, the abundance ratio of the analyte, among a plurality of samples regardless of the body of the mass spectrometer.

[0138] [5-3-2. Correction for Normalizing Signal Peak Intensity Ratio (Intensity Ratio Calibration)] [5-3-2-1. Calculation of Correction Formula] Using a plurality of calibration solutions, which are calibration substances of the present embodiment and in which the concentration ratios of two calibration substances are different, a correction formula for normalizing the signal peak intensity ratio can be calculated.

[0139] A plurality of calibration solutions (intensity ratio calibrants; IC) with known concentration ratios of two calibration substances are used. A plurality of solutions in which one calibration substance is present at a constant concentration and the concentration of the other calibration substance is different may be used. For example, solutions prepared such that SIL-Aβ1-38 has a constant concentration and Aβ1-38 has concentration ratios of 1 / 4, 1 / 2, 1, 2, and 4 with respect to SIL-Aβ1-38 may be used.

[0140] A plurality of calibration solutions (intensity ratio calibrants; IC) with known concentration ratios of two calibration substances are measured using a mass spectrometer for which a correction formula is to be calculated, and the signal peak intensities of each calibration substance in each solution are obtained. For each solution, the ratio of the signal peak intensity of the other calibration substance to the signal peak intensity of the calibration substance at a constant concentration is calculated.

[0141] A regression formula is calculated between the known concentration ratio of the two calibration substances in each calibration solution and the signal peak intensity ratio calculated above. For the calculation of the regression formula, a known method such as the least squares method may be appropriately used. For the calculation of the regression formula, the logarithm of the signal peak intensity ratio may be used. The regression formula may be appropriately selected from linear, polynomial, exponential, logarithmic, or power.

[0142] For example, a linear regression formula can be calculated using the logarithm of the signal peak intensity ratio. Also, for example, a power regression formula can be calculated using the signal peak intensity ratio. The calculated regression formula is used as the correction formula for the mass spectrometer.

[0143] For example, taking the logarithm of the known concentration ratio of the two calibration substances in each solution as x and the logarithm of the signal peak intensity ratio in the mass spectrometer for which the correction formula is to be calculated as y, Linear regression formula (y = ax + b) The correction coefficients a and b can be calculated. The calculated linear regression equation (y = ax + b) can be used as the correction formula for the mass spectrometer.

[0144] Alternatively, taking the known concentration ratio of two calibration substances in each solution as x and the signal peak intensity ratio in the mass spectrometer for which the correction formula is to be calculated as y, a power regression equation (y = ax b ) the correction coefficients a and b may be calculated. The calculated power regression equation (y = ax b ) can be used as the correction formula for the mass spectrometer.

[0145] The correction coefficients a and b are considered to be values specific to the mass spectrometer. Therefore, they may be different values depending on the body of the mass spectrometer and its device conditions. Also, since b is more affected by the body of the mass spectrometer and its device conditions than the correction coefficient a, for example, in the power regression equation (y = ax b ), a fixed value of a = 1 may be used and only the calculated correction coefficient of the b value may be adopted. That is, instead of the power regression equation (y = ax b ), y = x b may be used as the correction formula. In this way, when correcting using only the correction coefficient b, the b value is referred to as the correction value.

[0146] [5-3-2-2. Measurement and Correction of Analyte] In the mass spectrometer for which the correction formula was calculated in 5-3-2-1, a sample containing the analyte is measured to obtain the signal peak intensity of the analyte. Using one of the analytes (for example, the internal standard substance) as a reference, the ratio of the signal peak intensity of the other analyte to the signal peak intensity of the reference analyte is calculated.

[0147] The calculated signal peak intensity ratio is corrected using the correction formula calculated in the above 5-3-2-1. By this correction, the signal peak intensity ratio of the analyte is converted into the signal peak intensity ratio normalized by the calibration substance. The obtained normalized signal intensity has the differences due to the instrument cancelled by the correction formula. Therefore, it is possible to comparatively evaluate the abundance ratio of the analyte among a plurality of samples regardless of the instrument of the mass spectrometer. That is, it becomes possible to directly compare not only with Japan but also with the mass spectrometry measurement results in the United States, France, and other countries. Further, this correction is applicable to research and tests using various mass spectrometers and is a highly versatile technique.

[0148] [5-3-2-3. Correction Value and Instrument Conditions] As described above, in the mass spectrometer used for measuring the analyte and its instrument conditions, by calculating the correction formula and performing the correction, it is possible to comparatively evaluate the abundance ratio of the analyte among a plurality of samples regardless of the instrument body or instrument conditions of the mass spectrometer.

[0149] As described above, for creating the correction formula for intensity ratio calibration, a plurality of calibration solutions with known concentration ratios of two calibration substances can be used. For example, five types of solutions adjusted so that SIL-Aβ1-38 has a constant concentration and the concentration ratio of Aβ1-38 to SIL-Aβ1-38 is 1 / 4, 1 / 2, 1, 2, 4 can be used. In the mass spectrum of each solution, a peak of a certain amount of SIL-Aβ1-38 and a peak of Aβ1-38 corresponding to the concentration appear. Ideally, it should be the peak intensity ratio corresponding to the concentration ratio in each calibration solution. However, since this peak intensity ratio varies depending on the instrument state, the correction formula is calculated so that the peak intensity ratio matches the concentration ratio in each calibration solution, and the measurement results of the specimen are corrected. Thereby, the instrumental error is cancelled.

[0150] The correction formula varies depending on the body of the mass spectrometer. Also, even for the same mass spectrometer, when components such as the detector are replaced or when device settings such as the detector voltage are changed, the state of the mass spectrometer is different, so the correction formula differs depending on the state of the device. Further, the correction formula can also change when the state of the device changes due to detector deterioration or the like.

[0151] For example, when the same sample is measured due to the influence of device conditions such as detector deterioration, the signal peak intensity ratio detected by the mass spectrometer increases. This is considered to be because the signal peak of the substance with a small abundance becomes smaller due to the influence of device conditions such as detector deterioration. In a mass spectrometer, if the signal peak intensity becomes too small, generally, the measurement accuracy decreases from the perspective of the S / N ratio. Therefore, in a mass spectrometer, it is preferable that the signal peak intensity does not become too small. Calibration For example, when the signal peak intensity detected by the mass spectrometer becomes smaller due to the influence of some device conditions when obtaining the power regression formula (y = ax

[0152] ) in 5-3-2-1., the correction value b value in the correction formula becomes larger. Therefore, if this b value is within a certain range, the signal peak intensity does not become too small, the measurement accuracy by the mass spectrometer is further improved, and the correction accuracy is also further improved. b Incidentally, the b value can be varied by changing the detection sensitivity of the mass spectrometer. The b value can be controlled, for example, by changing the detector voltage. Also, for example, the b value can be changed by changing the baseline level of the Analog digital (AD) converter.

[0153]

Example

[0154] Examples are shown below to specifically explain the present invention, but the present invention is not limited to these examples.

[0155] ​[Example 1: Preparation of calibrants based on volume] FIG. 3 is a schematic diagram showing the method for preparing the calibrant of Example 1. In Example 1, as calibration substances, Aβ1-38 (SEQ ID NO: 11) and Aβ1-38 labeled with a stable isotope (SIL-Aβ1-38) were included, and five types of calibrants IC-1, IC-2, IC-3, IC-4, and IC-5 with different ratios of the concentration of Aβ1-38 to the concentration of SIL-Aβ1-38 (concentration ratio Aβ1-38 / SIL-Aβ1-38) were prepared. The calibrants in Example 1 were prepared as follows.

[0156] In Example 1, the concentration of SIL-Aβ1-38 in the five types of calibrants IC-1, IC-2, IC-3, IC-4, and IC-5 was constant, and the concentration of Aβ1-38 was different.

[0157] [1-1 Preparation of Solution A and Solution B] SIL-Aβ1-38 was purchased from AnaSpec (San Jose, CA, USA). In SIL-Aβ1-38, the carbon atoms of Phe and Ile are 13 substituted with

[0158] C. SIL-Aβ1-38 was dissolved in a solvent (a 20% aqueous acetonitrile solution containing 1 mg / mL of BSA and 50 mM of ammonium bicarbonate) to prepare Solution A0 (6-peptide-A solution). The concentration of SIL-Aβ1-38 in Solution A0 (6-peptide-A solution) was 100 nmol / L. Further, Solution A0 also contained Aβ1-33 (300 nmol / L), Aβ1-34 (200 nmol / L), Aβ1-36 (100 nmol / L), Aβ1-37 (50 nmol / L), and APP669-711 (50 nmol / L).

[0159] To 1170 μL of the solvent for IC concentrate (20% aqueous acetonitrile solution containing 0.1 mg / mL of BSA and 50 mM of ammonium bicarbonate), 30 μL of solution A0 (6 - peptide - A solution) was added to prepare solution A (6 - peptide - B). The concentration of SIL - Aβ1 - 38 in solution A (6 - peptide - B) became 2.5 nmol / L. The concentration of SIL - Aβ1 - 38 in solution A (6 - peptide - B) was adjusted to be the desired concentration of SIL - Aβ1 - 38 in the five calibrants IC - 1, IC - 2, IC - 3, IC - 4, and IC - 5 to be prepared.

[0160] The solvent for IC concentrate is a liquid that contains acetonitrile, is volatile, and also contains BSA, so it is viscous.

[0161] To 380 μL of the solvent for IC concentrate (20% aqueous acetonitrile solution containing 0.1 mg / mL of BSA and 50 mM of ammonium bicarbonate), 10 μL of solution A0 (6 - peptide - A solution) and 10 μL of solution B0 (Aβ1 - 38 solution) were added to prepare solution B (IC - 0). The concentration of SIL - Aβ1 - 38 in solution B (IC - 0) became the same as that in solution A (6 - peptide - B), which was 2.5 nmol / L. Also, the concentration of Aβ1 - 38 in solution B (IC - 0) became 20 nmol / L.

[0162] All the measurements in 1 - 1 of Example 1 were performed by volume using pipetting. The pipette used was the Research Plus manufactured by Eppendorf.

[0163] [1 - 2 Preparation of Calibrants] Next, the prepared solution B was successively diluted twice with solution A to prepare IC - 1, IC - 2, IC - 3, IC - 4, and IC - 5 as follows.

[0164] 200 μL of the prepared solution A and 200 μL of solution B were mixed to prepare an IC-1 solution. The ratio of the concentration of Aβ1-38 to the concentration of SIL-Aβ1-38 (concentration ratio Aβ1-38 / SIL-Aβ1-38) in the IC-1 solution was 4. 200 μL of the prepared IC-1 solution and 200 μL of solution A were mixed to prepare an IC-2 solution. The concentration ratio Aβ1-38 / SIL-Aβ1-38 in the IC-2 solution was 2. 200 μL of the prepared IC-2 solution and 200 μL of solution A were mixed to prepare an IC-3 solution. The concentration ratio Aβ1-38 / SIL-Aβ1-38 in the IC-3 solution was 1. 200 μL of the prepared IC-3 solution and 200 μL of solution A were mixed to prepare an IC-4 solution. The concentration ratio Aβ1-38 / SIL-Aβ1-38 in the IC-4 solution was 1 / 2. 200 μL of the prepared IC-4 solution and 200 μL of solution A were mixed to prepare an IC-5 solution. The concentration ratio Aβ1-38 / SIL-Aβ1-38 in the IC-5 solution was 1 / 4.

[0165] All the measurements in 1-2 of Example 1 were performed by pipetting in terms of volume. As the pipette, Research Plus manufactured by Eppendorf was used. Thus, all the measurements in Example 1 were performed by pipetting in terms of volume.

[0166] Three operators prepared IC-1, IC-2, IC-3, IC-4 and IC-5 three times each according to the above procedure. The prepared calibrants were each stored frozen. For the prepared calibrants, those diluted 10-fold each were used for measurement with a mass spectrometer in [3-1 Measurement of Calibrant] described below.

[0167] [Example 2: Preparation of Calibrant Based on Weight] Figure 4 is a schematic diagram showing the method for preparing calibrants in Example 2. Also in Example 2, similar to Example 1, as calibration substances, Aβ1-38 (SEQ ID NO: 11) and Aβ1-38 labeled with a stable isotope (SIL-Aβ1-38) were included, and five types of calibrants IC-1, IC-2, IC-3, IC-4, and IC-5 with different ratios of the concentration of Aβ1-38 to the concentration of SIL-Aβ1-38 (concentration ratio Aβ1-38 / SIL-Aβ1-38) were prepared. The concentrations of SIL-Aβ1-38 in the five types of calibrants in Example 2 are the same as those in Example 1. The concentrations of Aβ1-38 in the five types of calibrants in Example 2 are the same as those in Example 1.

[0168] The calibrants in Example 2 were prepared as follows.

[0169] [Preparation of Solution A and Solution B] Similar to Example 1, SIL-Aβ1-38 was dissolved in a solvent (a 20% aqueous acetonitrile solution containing 1 mg / mL of BSA and 50 mM of ammonium bicarbonate) to prepare Solution A0 (6-peptide-A solution). The concentration of SIL-Aβ1-38 in Solution A0 (6-peptide-A solution) was 100 nmol / L, similar to Example 1. Also, Solution A0 further contained Aβ1-33 (300 nmol / L), Aβ1-34 (200 nmol / L), Aβ1-36 (100 nmol / L), Aβ1-37 (50 nmol / L), and APP669-711 (50 nmol / L), similar to Example 1.

[0170] Furthermore, 220 μL of the prepared Solution A0 (6-peptide-A solution) was mixed with 8140 μL of a solvent for IC concentrate (a 20% aqueous acetonitrile solution containing 0.1 mg / mL of BSA and 50 mM of ammonium bicarbonate) to prepare Solution A0' (6-peptide-A2 solution). Solution A0' is a solution in which Solution A0 is diluted 38-fold. The concentration of SIL-Aβ1-38 in Solution A0' (6-peptide-A2 solution) became approximately 2.63 nmol / L.

[0171] Aβ1-38 was dissolved in a solvent (an aqueous 20% acetonitrile solution containing 1 mg / mL of BSA and 50 mM of ammonium bicarbonate) to prepare Solution B0 (Aβ1-38 solution). The concentration of Aβ1-38 in Solution B0 (Aβ1-38 solution) was 800 nM (nmol / L), the same as in Example 1.

[0172] 3800 μL of Solution A0’ (6-peptide-A2 solution) and 200 μL of the solvent for IC concentrate (an aqueous 20% acetonitrile solution containing 0.1 mg / mL of BSA and 50 mM of ammonium bicarbonate) were mixed to prepare Solution A (6-peptide-B). The concentration of SIL-Aβ1-38 in Solution A (6-peptide-B) was 2.5 nmol / L, the same as in Example 1. As a result, Solution A was a 40-fold dilution of Solution A0. The concentration of SIL-Aβ1-38 in Solution A (6-peptide-B) was adjusted to be the desired concentration of SIL-Aβ1-38 in the five calibrants IC-1, IC-2, IC-3, IC-4, and IC-5 to be prepared.

[0173] 3800 μL of Solution A0’ (6-peptide-A2 solution), 100 μL of the solvent for IC concentrate (an aqueous 20% acetonitrile solution containing 0.1 mg / mL of BSA and 50 mM of ammonium bicarbonate), and 100 μL of Solution B0 (Aβ1-38 solution) were mixed to prepare Solution B (IC-0). The concentration of SIL-Aβ1-38 in Solution B (IC-0) was 2.5 nmol / L, the same as the concentration of SIL-Aβ1-38 in Solution A (6-peptide-B), as in Example 1. Also, the concentration of Aβ1-38 in Solution B (IC-0) was 20 nmol / L, the same as in Example 1.

[0174] As described above, in Example 2, for 3800 μL of Solution A0', a clearly smaller amount of solvent was added to prepare Solution A. Also, for 3800 μL of Solution A0', a clearly smaller amount of solvent and Solution B0 were added to prepare Solution B. It is considered that the concentrations of SIL-Aβ1-38 in Solution A and Solution B of Example 2 could be made more precisely a certain predetermined value than the concentrations of SIL-Aβ1-38 in Solution A and Solution B of Example 1.

[0175] In 2-1 of Example 2, all the measurements were carried out by volume using pipetting. As the pipette, Research Plus manufactured by Eppendorf was used.

[0176] [Preparation of Calibrants] Next, the prepared Solution B was sequentially diluted with Solution A to prepare IC-1, IC-2, IC-3, IC-4, and IC-5 as follows.

[0177] 750 mg of the prepared Solution A and 750 mg of Solution B were mixed to prepare the IC-1 solution. The ratio of the concentration of Aβ1-38 to the concentration of SIL-Aβ1-38 (concentration ratio Aβ1-38 / SIL-Aβ1-38) in the IC-1 solution is 4. 750 mg of the prepared IC-1 solution and 750 mg of Solution A were mixed to prepare the IC-2 solution. The concentration ratio Aβ1-38 / SIL-Aβ1-38 in the IC-2 solution is 2. 750 mg of the prepared IC-2 solution and 750 mg of Solution A were mixed to prepare the IC-3 solution. The concentration ratio Aβ1-38 / SIL-Aβ1-38 in the IC-3 solution is 1. 750 mg of the prepared IC-3 solution and 750 mg of Solution A were mixed to prepare the IC-4 solution. The concentration ratio Aβ1-38 / SIL-Aβ1-38 in the IC-4 solution is 1 / 2. 750 mg of the prepared IC-4 solution and 750 mg of Solution A were mixed to prepare the IC-5 solution. The concentration ratio Aβ1-38 / SIL-Aβ1-38 in the IC-5 solution is 1 / 4.

[0178] In Example 2 2-2All measurements were performed by weight using an electronic balance. The electronic balance used was the AUX-220 manufactured by Shimadzu Corporation. The repeatability error of this electronic balance is 0.1 mg in terms of standard deviation. The weighing error in the electronic balance is considered to be very small.

[0179] Two operators prepared IC-1, IC-2, IC-3, IC-4, and IC-5 once each according to the above procedure. The prepared calibrants were each stored frozen. For the measurement with the mass spectrometer, the prepared calibrants were each diluted 10-fold and used.

[0180] [Example 3: Measurement of Calibrant and Calculation of Correction Value] [3-1 Measurement of Calibrant] Five types of concentrated solutions (IC-1 to 5) of the intensity ratio calibrant (IC) reagent prepared in Example 1 and Example 2 are shown in Table 1. Before MS measurement, IC-1 to 5 were thawed and diluted 10-fold with an IP eluent (5 mM HCl, 0.1 mM Methionine, 70% (v / v) acetonitrile) that can be used for immunoprecipitation to prepare diluted solutions (IC-1 to 5 diluted solutions) of IC-1 to 5. First, 0.5 μL of 0.5 mg / mL CHCA / 0.2% (w / v) MDPNA was dropped onto a μFocus MALDI plate and dried. TM 1 μL of each of IC-1 to 5 was dropped into 4 wells on it and dried. The protein and peptide compositions of the IC-1 to 5 diluted solutions are shown in Table 2. The quantity ratios of Aβ1-38 to SIL-Aβ1-38 in IC-1 to 5 were 4, 2, 1, 1 / 2, and 1 / 4, respectively. The quantity ratio of Aβ1-38 to SIL-Aβ1-38 is not affected by dilution. The quantity ratios of Aβ1-38 to SIL-Aβ1-38 in the IC-1 to 5 diluted solutions were also 4, 2, 1, 1 / 2, and 1 / 4, respectively.

[0181]

Table 1

[0182]

Table 2

[0183] Mass spectrometry data were acquired in positive ion mode with Linear TOF using AXIMA Performance (Shimadzu / KRATOS, Manchester, UK). The m / z values in Linear TOF were displayed as the average mass of the peaks. The m / z values were calibrated using human angiotensin II, human ACTH fragment 18-39, bovine insulin oxidized beta-chain, and bovine insulin as external standards. The mass spectra were obtained by integrating 40 shots at each of 400 points in raster mode. The quantitative value used was the average of the peak intensity ratios of Aβ1-38 to SIL-Aβ1-38 in the spectra measured in 4 wells. The detection limit was set at S / N = 3, and peaks below the detection limit were considered undetectable.

[0184] The above measurements were performed for IC-1 to 5 dilutions.

[0185] A power approximation formula was created from the intensity ratio of Aβ1-38 to SIL-Aβ1-38 in the mass spectra of IC-1 to 5 dilutions and the concentration ratio (quantity ratio) of Aβ1-38 to SIL-Aβ1-38 in IC-1 to 5 dilutions.

[0186] Power approximation formula: y = ax b Here, x is the quantity ratio of Aβ1-38 / SIL-Aβ1-38, y is the peak intensity ratio of Aβ1-38 / SIL-Aβ1-38, and a and b are the coefficients in the approximation formula.

[0187] The a value of the power approximation formula does not change depending on the state of the aircraft, but the b value is greatly affected by the conditions of the aircraft. Therefore, by performing correction using only the b value, it is possible to accurately correct the instrumental error of the mass spectrometer.

[0188] In addition, if the b value is adjusted within a certain range, the signal peak intensity ratio of the analyte can be corrected more accurately. For example, the range of the b value may be adjusted to 0.9 to 1.1.

[0189] The results when using the calibrant of Example 1 are shown in Table 3. For each of the calibrants prepared three times each by three persons, the above IC-1 to 5 diluents were measured and the b value was calculated.

[0190]

Table 3

[0191] The IC3 int.ratio in Table 3 indicates the peak intensity ratio of Aβ1-38 to SIL-Aβ1-38 in the IC-3 diluent. The IC1 int.ratio indicates the peak intensity ratio of Aβ1-38 to SIL-Aβ1-38 in the IC-1 diluent. a and b are the coefficients of the above power approximation formula (y = ax b ). Average indicates the average value of the b values for the calibrants prepared three times by the same operator. STDEV.P indicates the standard deviation of the b values for the calibrants prepared three times by the same operator. CV(%) indicates the coefficient of variation of the b values for the calibrants prepared three times by the same operator.

[0192] When the b value was calculated using the calibrant of Example 1, the b value was in the range of 1.0063 to 1.0801. In Example 1, in all cases, the b value was within the range of 0.9 to 1.1.

[0193] The results when using the calibrants of Example 2 are shown in FIG. 5. For each of the calibrants prepared by Operator 1 and Operator 2 once each, the measurement of the above IC-1 to 5 diluents was performed three times in total, and the b value in each measurement was calculated. FIG. 5 is a bar graph showing the average value of the b values calculated using the calibrants prepared by each operator. The vertical axis represents the b value. The error bar indicates the standard deviation of the three b values obtained by measuring the calibrants prepared by each operator three times.

[0194] When calculating the b value using the calibrant of Example 2, the average values of the b values calculated using the calibrants prepared by two operators were 0.9231 and 0.9213, respectively. Regardless of the operator, almost equivalent b values were obtained. Thus, the b value calculated using the calibrant of Example 2 had even less variation than the b value calculated using the calibrant of Example 1.

[0195] In Example 2, as a factor for the smaller variation of the b value, it is considered that the ratio of the concentration of Aβ1-38 to the concentration of SIL-Aβ1-38 (concentration ratio Aβ1-38 / SIL-Aβ1-38) in each calibrant of Example 2 was more accurately adjusted.

[0196] In Example 2, when sequentially diluting Solution B with Solution A, it was weighed by weight using an electronic balance. Since the solvents used in the calibrants in Example 1 and Example 2 contain BSA and acetonitrile, there is an influence on pipetting due to the viscosity and the volatilization of acetonitrile. When weighing such a solution, it is considered that the weighing error is reduced compared to the case of pipetting by weighing by weight using an electronic balance. Also, it is considered that the weighing with an electronic balance has less difference due to repetition and less difference due to the operator than pipetting.

[0197] Therefore, in Example 2, it is considered that in each of IC-1, IC-2, IC-3, IC-4, and IC-5, Aβ1-38 was more accurately prepared at a predetermined concentration than in Example 1. That is, it is considered that it was prepared at a more accurate concentration ratio of Aβ1-38 / SIL-Aβ1-38. Also, the variation by the operator was small, and it is considered that a calibrant prepared at an accurate concentration ratio of Aβ1-38 / SIL-Aβ1-38 could be produced with good reproducibility.

[0198] In Example 2, solution A0 was diluted 38-fold to prepare solution A0'. Then, using the same amount of the prepared solution A0', solutions A and B were prepared. Therefore, in Example 2, it is considered that in each of IC-1, IC-2, IC-3, IC-4, and IC-5, SIL-Aβ1-38 was more accurately prepared at a predetermined constant concentration than in Example 1. That is, it is considered that it was prepared at a more accurate concentration ratio of Aβ1-38 / SIL-Aβ1-38.

[0199] The present invention includes, for example, the following forms.

[0200] (1) A calibrant used in a mass spectrometer, The calibrant contains two or more calibration substances, A calibrant in which the ratio of the concentration of one calibration substance among the two or more calibration substances to the concentration of another calibration substance among the two or more calibration substances is a predetermined value.

[0201] (2) The calibrant according to (1) above, wherein the calibration substances include a stable isotope-labeled substance and a non-stable isotope-labeled substance.

[0202] (3) The calibrant according to (1) or (2) above, wherein the calibration substance is an Aβ-related peptide.

[0203] (4) The calibrant according to any one of (1) to (3) above, wherein the calibration substance is Aβ1-38 and Aβ1-38 labeled with a stable isotope.

[0204] (5) The calibrant according to any one of (1) to (4) above, wherein the calibrant contains a plurality of types of calibrants in which the concentrations of at least one of the two or more calibration substances are different from each other.

[0205] (6) The calibrant according to any one of (1) to (5) above, wherein the ratio of the concentration of one of the two or more calibration substances to the concentration of another one of the two or more calibration substances is in the range of 1 / 4 to 4.

[0206] (7) A method for preparing a calibrant used in a mass spectrometer, wherein the calibrant contains two or more calibration substances, the ratio of the concentration of one of the two or more calibration substances to the concentration of another one of the two or more calibration substances is a predetermined value, and the preparation method includes a pre-step of preparing a solution A containing one of the two or more calibration substances (S1) at a predetermined concentration and a solution B containing the one calibration substance (S1) at the same concentration as the predetermined concentration and further containing another one of the two or more calibration substances (S2), and a pre-step of preparing A preparation step of obtaining a calibrant in which the ratio of the concentration of the other calibration substance (S2) to the concentration of the one calibration substance (S1) is a predetermined value using the solution A and the solution B; A method for producing a calibrant, comprising:

[0207] (8) The method for producing a calibrant according to (7) above, wherein in the preparation step, a plurality of types of calibrants are obtained in which the concentration of the one calibration substance (S1) is constant and at least the concentration of the other calibration substance (S2) is different.

[0208] (9) In the preparation step, a calibrant in which the ratio of the concentration of the other calibration substance (S2) to the concentration of the one calibration substance (S1) is a predetermined value is obtained using the solution A and the solution B, a part of the calibrant is separated, and a mixing operation of further mixing the solution A with the separated part is performed, The method for producing a calibrant according to (7) above, wherein another calibrant having a ratio of the concentration of the other calibration substance (S2) to the concentration of the one calibration substance (S1) different from the predetermined value is obtained.

[0209] (10) In the preparation step, a calibrant in which the ratio of the concentration of the other calibration substance (S2) to the concentration of the one calibration substance (S1) is a predetermined value is obtained using the solution A and the solution B, a part (for example, half) of the calibrant is separated, and a mixing operation of further mixing the solution A with the separated part in the same amount as the part of the calibrant is performed, The method for producing a calibrant according to (7) above, wherein another calibrant having a ratio of the concentration of the other calibration substance (S2) to the concentration of the one calibration substance (S1) of 1 / 2 of the predetermined value is obtained.

[0210] (11) The method for producing a calibrant according to (9) or (10) above, wherein in the preparation step, the mixing operation is performed sequentially a plurality of times.

[0211] (12) In the preparation step, using the solution A and the solution B, a calibrant is obtained in which the ratio of the concentration of the other calibration substance (S2) to the concentration of one calibration substance (S1) is a predetermined value. A part of the calibrant is separated, and a mixing operation of further mixing the solution B with the separated part is performed. The method for producing a calibrant according to (7) above, wherein a calibrant having a ratio of the concentration of the other calibration substance (S2) to the concentration of one calibration substance (S1) different from the predetermined value is obtained.

[0212] (13) In the preparation step, using the solution A and the solution B, a calibrant is obtained in which the ratio of the concentration of the other calibration substance (S2) to the concentration of one calibration substance (S1) is a predetermined value. A part (for example, half) of the calibrant is separated, and a mixing operation of further mixing the solution B in the same amount as the separated part with the separated part is performed. The method for producing a calibrant according to (7) above, wherein a calibrant having a ratio of the concentration of the other calibration substance (S2) to the concentration of one calibration substance (S1) different from the predetermined value is obtained.

[0213] (14) The method for producing a calibrant according to (12) or (13) above, wherein in the preparation step, the mixing operation is performed sequentially a plurality of times.

[0214] (15) The method for preparing the calibrant according to any one of (7) to (14) above, wherein the preparation step using the solution A and the solution B is performed by volumetric measurement.

[0215] (16) The method for preparing the calibrant according to any one of (7) to (14) above, wherein the preparation step using the solution A and the solution B is performed by weight measurement.

[0216] (17) One of the calibration substances (S1) is a stable isotope-labeled substance, The method for preparing the calibrant according to any one of (7) to (16) above, wherein the other calibration substance (S2) is a non-stable isotope-labeled substance.

[0217] (18) One of the calibration substances (S1) is stable isotope-labeled Aβ1-38, The method for preparing the calibrant according to any one of (7) to (17) above, wherein the other calibration substance (S2) is non-stable isotope-labeled Aβ1-38.

[0218] (19) A calibrant kit for use in a mass spectrometer, The kit includes a plurality of calibrants containing two or more calibration substances, The plurality of calibrants are calibrant kits in which the concentrations of at least one calibration substance among the calibration substances are different from each other.

[0219] (20) A calibrant kit for use in a mass spectrometer, The kit is a calibrant kit containing two or more calibration substances.

[0220] (21) Furthermore, the calibrant kit according to (20) above, which contains a solvent.

[0221] (22) Furthermore, the calibrant kit according to (20) or (21) above, which contains a container for holding the calibrant.

Claims

1. A method for preparing a calibrant used in a mass spectrometer, wherein the calibrant contains two or more calibration substances, and the ratio of the concentration of one calibration substance among the two or more calibration substances to the concentration of another calibration substance among the two or more calibration substances is a predetermined value, and the preparation method includes a pre-step of preparing a solution A containing one calibration substance (S1) among the two or more calibration substances at a predetermined concentration, a solution B containing the one calibration substance (S1) at the same concentration as the predetermined concentration and further containing another calibration substance (S2) among the two or more calibration substances, and a calibration substance is obtained by using the solution A and the solution B such that the ratio of the concentration of the other calibration substance (S2) to the concentration of the one calibration substance (S1) is a predetermined value, and a mixing operation is performed in which a part of the calibrant is separated and the solution A is further mixed with the separated part, and a preparation step of obtaining another calibrant in which the ratio of the concentration of the other calibration substance (S2) to the concentration of the one calibration substance (S1) is a value different from the predetermined value, and the preparation step includes performing the mixing operation sequentially a plurality of times to obtain a plurality of types of calibrants in which the concentration of the one calibration substance (S1) is constant and at least the concentrations of the other calibration substances (S2) are different from each other. A method for preparing a calibrant. A method for preparing a calibrant used in a mass spectrometer,

2. A method for preparing a calibrant used in a mass spectrometer, wherein the calibrant contains two or more calibration substances, The ratio of the concentration of one calibration substance among the two or more calibration substances to the concentration of another calibration substance among the two or more calibration substances is a predetermined value, The preparation method is Solution A containing one calibration substance (S1) among the two or more calibration substances at a predetermined concentration, and Solution B containing the one calibration substance (S1) at the same concentration as the predetermined concentration and further containing another calibration substance (S2) among the two or more calibration substances, A pre-step of preparing, Using the solution A and the solution B, obtaining a calibrant in which the ratio of the concentration of the other calibration substance (S2) to the concentration of the one calibration substance (S1) is a predetermined value, Separating a part of the calibrant and performing a mixing operation of mixing the solution A in the same amount as the separated part with the separated part, A preparation step of obtaining another calibrant in which the ratio of the concentration of the other calibration substance (S2) to the concentration of the one calibration substance (S1) is 1 / 2 of the predetermined value, Including, In the preparation step, performing the mixing operation a plurality of times sequentially, A method for preparing a calibrant, in which the concentration of the one calibration substance (S1) is constant, and obtaining a plurality of types of calibrants in which at least the concentrations of the other calibration substances (S2) are different.

3. A method for preparing a calibrant used in a mass spectrometer, The calibrant is Containing two or more calibration substances, The ratio of the concentration of one calibration substance among the two or more calibration substances to the concentration of another calibration substance among the two or more calibration substances is a predetermined value. The preparation method is Solution A containing one calibration substance (S1) among the two or more calibration substances at a predetermined concentration, and Solution B containing the one calibration substance (S1) at the same concentration as the predetermined concentration and further containing another calibration substance (S2) among the two or more calibration substances, and A pre-step of preparing them, Using the solution A and the solution B, obtaining a calibrant in which the ratio of the concentration of the other calibration substance (S2) to the concentration of the one calibration substance (S1) is a predetermined value, Separating a part of the calibrant and performing a mixing operation of further mixing the solution B with the separated part, A preparation step of obtaining another calibrant in which the ratio of the concentration of the other calibration substance (S2) to the concentration of the one calibration substance (S1) is a value different from the predetermined value, including In the preparation step, performing the mixing operation sequentially a plurality of times, A method for preparing a calibrant, in which the concentration of the one calibration substance (S1) is constant, and obtaining a plurality of types of calibrants in which at least the concentrations of the other calibration substances (S2) are different.

4. A method for preparing a calibrant used in a mass spectrometer, The calibrant is composed of two or more calibration substances, The ratio of the concentration of one calibration substance among the two or more calibration substances to the concentration of another calibration substance among the two or more calibration substances is a predetermined value. The preparation method is Solution A containing one calibration substance (S1) out of the two or more calibration substances at a predetermined concentration, and Solution B containing the one calibration substance (S1) at the same concentration as the predetermined concentration and further containing another one calibration substance (S2) out of the two or more calibration substances, and A pre-step of preparing them, Using the solution A and the solution B, obtaining a calibrant in which the ratio of the concentration of the other calibration substance (S2) to the concentration of the one calibration substance (S1) is a predetermined value, Separating a part of the calibrant, and performing a mixing operation of further mixing the solution B in the same amount as the separated part with the separated part, A preparation step of obtaining another calibrant in which the ratio of the concentration of the other calibration substance (S2) to the concentration of the one calibration substance (S1) is a value different from the predetermined value, including In the preparation step, performing the mixing operation a plurality of times sequentially, A method for preparing a calibrant, in which the concentration of the one calibration substance (S1) is constant, and a plurality of types of calibrants in which at least the concentrations of the other calibration substances (S2) are different from each other are obtained.

5. The method for preparing a calibrant according to any one of claims 1 to 4, wherein the preparation step using the solution A and the solution B is performed by measuring by volume.

6. The method for preparing a calibrant according to any one of claims 1 to 4, wherein the preparation step using the solution A and the solution B is performed by measuring by weight.

7. The one calibration substance (S1) is a substance labeled with a stable isotope, The method for preparing a calibrant according to any one of claims 1 to 4, wherein the other calibration substance (S2) is a substance not labeled with a stable isotope.

8. The one calibration substance (S1) is Aβ1-38 labeled with a stable isotope, The method for preparing a calibrant according to any one of claims 1 to 4, wherein the other calibration substance (S2) is Aβ1-38 not labeled with a stable isotope.

9. The method for preparing a calibrant according to any one of claims 1 to 4, wherein the calibrant is used for correcting the instrumental error of a mass spectrometer.

10. A method for correcting the instrumental error of a mass spectrometer, measuring a plurality of types of calibrants prepared by the method for preparing a calibrant according to any one of claims 1 to 4 with a mass spectrometer, obtaining the signal peak intensities of the one calibration substance (S1) and the other calibration substance (S2) in each of the plurality of types of calibrants, for each of the plurality of types of calibrants, calculating a signal peak intensity ratio of the signal peak intensity of the other calibration substance (S2) to the signal peak intensity of the one calibration substance (S1), using the calculated signal peak intensity ratios in the plurality of types of calibrants to calculate a correction formula for the mass spectrometer, measuring a sample containing two or more analyte substances in the mass spectrometer, obtaining the signal peak intensities of the two or more analyte substances, calculating a signal peak intensity ratio of the signal peak intensity of one analyte substance to the signal peak intensity of another analyte substance, and correcting the calculated signal peak intensity ratio in the sample using the correction formula.

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