Analytical methods using lysophosphatidylcholine compounds containing stable isotopes in choline moiety

Abstract

According to the present invention, it is possible to develop a technique whereby lysophosphatidylcholine and lysophosphatidic acid in a cerebrospinal fluid can be analyzed with high accuracy by an analysis method comprising a step for detecting lysophosphatidylcholine and / or lysophosphatidic acid in a biological sample by mass spectrometry, and a lysophosphatidylcholine compound of which the choline part contains stable isotopes is used as a standard sample.

Description

TECHNICAL FIELD

[0001] The present application relates to an analysis method using a lysophosphatidylcholine compound containing a stable isotope in a choline moiety. BACKGROUND

[0002] As a method for objectively evaluating pain, which is a subjective scale, for example, in Japanese Patent Laid-Open No. 2017-187492, a method for detecting at least either of lysophosphatidylcholine (LPC) and lysophosphatidic acid (LPA) in a sample derived from human cerebrospinal fluid is proposed. In this method, specific examples of lysophosphatidylcholine include one or more selected from the group consisting of LPC_14_0, LPC_16_0, LPC_16_1, LPC_18_0, LPC_18_1, LPC_18_2, LPC_20_4, LPC_20_5, and LPC_22_6, and specific examples of lysophosphatidic acid include one or more selected from the group consisting of total LPA, LPA_16_0, LPA_18_0, LPA_18_1, LPA_18_2, LPA_20_4, and LPA_22_6. Here, in the specific examples of LPC, LPA, the first number indicates the number of carbons of the fatty acid chain, and the second number indicates the number of double bonds in the fatty acid chain.

[0003] In the past, when lysophosphatidylcholine and lysophosphatidic acid were detected by mass spectrometry (MS), LPA_17_0 (stable isotope is not labeled) represented by the following structural formula, which is not present in a biological sample, was used as a standard sample.

[0004]

[0005] However, in recent years, it has been found that LPA_17_0 can be present in trace amounts in some human bodies, and even if LPA_17_0 is used as a standard sample for quantification, there is a problem in accuracy. To solve this problem, for example, a lysophosphatidylcholine compound (18:1-d7 Lyso PC / C 26 H 45 D7NO7P) represented by the following structural formula, to which a stable isotope is introduced, is proposed as a standard sample.

[0006]

[0007] However, as Figure 2As shown in the figure, the lysophosphatidylcholine compound is ionized at a portion adjacent to the phosphate group at the time of MS analysis, and a product ion of m / z = 184 is generated. This product ion has the same mass as the product ion of LPC, and the retention time is similar, and thus, if the selected product ion is set to m / z = 184, cross talk (a phenomenon in which ions are stopped in the collision chamber, and the product ion of the measured component is erroneously detected) occurs, and thus, improvement in quantitative accuracy is required.

[0008] Prior Art Documents

[0009] Patent Documents

[0010] Patent Document 1: Japanese Patent Application Publication No. 2017-187492 SUMMARY

[0011] PROBLEMS TO BE SOLVED BY THE INVENTION

[0012] The present application has been made to solve the above problems, and to develop a technique capable of highly accurately analyzing lysophosphatidylcholine and lysophosphatidic acid in cerebrospinal fluid.

[0013] SOLUTION TO PROBLEM

[0014] A first aspect of the present application relates to an analysis method including a step of detecting at least either of lysophosphatidylcholine and lysophosphatidic acid in a biological sample by mass spectrometric analysis, and using a lysophosphatidylcholine compound containing a stable isotope in a choline portion as a standard sample.

[0015] EFFECT OF THE INVENTION

[0016] According to the analysis method of the present application, by using a compound that does not exist in vivo as a standard sample, it is possible to improve quantitative accuracy. Furthermore, according to the analysis method of the present application, by using a lysophosphatidylcholine compound containing a stable isotope in a choline portion as a standard sample, it is possible to avoid contamination of the detection object and cross talk in mass spectrometric analysis, and thus, to improve quantitative accuracy. BRIEF DESCRIPTION OF DRAWINGS

[0017] [ Figure 1 ] A figure showing a particularly preferred example of a lysophosphatidylcholine compound used as a marker sample in the first aspect of the present application.

[0018] [ Figure 2 ] A figure showing a conventional lysophosphatidylcholine compound into which a stable isotope has been introduced. DETAILED DESCRIPTION

[0019] The first aspect of the present invention is an analytical method comprising the steps of detecting at least one of lysophosphatidylcholine and lysophosphatidic acid in a biological sample by mass spectrometry, and using a lysophosphatidylcholine compound containing a stable isotope in the choline moiety as a standard sample.

[0020] In the first aspect of the invention, although using a lysophosphatidylcholine compound containing a stable isotope that is not present in living organisms as a standard sample can improve quantitative accuracy, the location of the stable isotope within the lysophosphatidylcholine compound can significantly affect the quantitative accuracy. In the first aspect of the invention, by labeling the choline portion of the lysophosphatidylcholine adjacent to the phosphate group with a stable isotope, contamination of the analyte and crosstalk in mass spectrometry analysis can be avoided, thereby improving quantitative accuracy.

[0021] The stable isotopes used in the first aspect of the invention may be any stable isotopes of the choline moiety adjacent to the phosphate group that constitutes lysophosphatidylcholine, such as deuterium (…). 2 H), carbon-13 ( 13 C) and nitrogen-15 ( 15 At least one of N). From the viewpoint of relatively easy manufacturing, at least one of deuterium and carbon-13 is preferred, and it is particularly preferred that both deuterium and carbon-13 are contained.

[0022] Lysophosphatidylcholine, as a lysophosphatidylcholine compound containing a stable isotope, is preferably selected from LPC_14_0, LPC_16_0, LPC_16_1, LPC_18_0, LPC_18_1, LPC_18_2, LPC_20_4, LPC_20_5 and LPC_22_6 contained in cerebrospinal fluid, of which LPC_18_0 or LPC_1, which have relatively high concentrations in cerebrospinal fluid, are particularly preferred.

[0023] As particularly preferred examples of lysophosphatidylcholine compounds containing stable isotopes, examples of lysophosphatidylcholine compounds represented by the following structural formulas can be cited ([ 13 C3, 2 H4]-18:1 Lyso PC / 13 C3C23D4H48NO7P).

[0024]

[0025] The above lysophosphatidylcholine compound is a compound in which four deuterium and three carbon-13 are introduced into the choline portion of LPC_18_1 and labeled. When such a lysophosphatidylcholine compound is used as a standard sample and the lysophosphatidylcholine (e.g., LPC_18_1) contained in a biological sample is detected, as shown in the standard sample, the portion adjacent to the phosphate group is ionized to generate a product ion of m / z = 191. The product ion generated from the standard sample has a mass different from the product ion of lysophosphatidylcholine, m / z = 184, and thus cross talk can be avoided. Figure 1

[0026] As the detection target in the biological sample, at least either of lysophosphatidylcholine and lysophosphatidic acid is acceptable. Specifically, one or more kinds selected from the group consisting of LPC_14_0, LPC_16_0, LPC_16_1, LPC_18_0, LPC_18_1, LPC_18_2, LPC_20_4, LPC_20_5, and LPC_22_6 disclosed in Japanese Patent Application Publication No. 2017-187492, and one or more kinds selected from the group consisting of total LPA, LPA_16_0, LPA_18_0, LPA_18_1, LPA_18_2, LPA_20_4, and LPA_22_6 can be exemplified. In the case of lysophosphatidic acid, a product ion of m / z = 153 is generated and cross talk can be originally avoided, and thus the effect of avoiding cross talk due to the presence of a stable isotope in the choline portion is particularly useful when the detection target is lysophosphatidylcholine. In the case where lysophosphatidylcholine is the detection target, LPC_18_0 or LPC_18_1 is particularly preferable among the above specific examples.

[0027] The lysophosphatidylcholine compound containing a stable isotope can be appropriately synthesized by combining publicly known methods, and of course, a commercially available lysophosphatidylcholine compound can be used.

[0028] The biological sample is preferably cerebrospinal fluid (human cerebrospinal fluid). Cerebrospinal fluid examination is generally performed at a clinical site for the diagnosis of various diseases, and a small amount of cerebrospinal fluid collected before the implementation of myelography or the like can be directly used as a biological sample. In addition, the biological sample can be subjected to a publicly known pretreatment as needed and then used to determine the concentration of the component.

[0029] The mass spectrometry can be, for example, laser desorption / ionization time of flight mass spectrometry (LDI-TOF / MS method) or electro spray ionization mass spectrometry (ESI-MS method).​

[0030] In the TOF / MS method, the lipid molecule species of the subject can be detected by pattern analysis of a spectrum obtained by the TOF / MS device. By detecting the presence or absence of a peak of a specific m / z value, qualitative detection of the presence or absence of the lipid molecule species of the subject in the sample can be performed. Furthermore, according to the height of the m / z peak of the spectrum, quantitative detection can also be performed. The LDI-TOF / MS can be, for example, a matrix assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF / MS method), a surface-enhanced laser desorption / ionization time of flight mass spectrometry (SELDI-TOF / MS method).

[0031] The MALDI-TOF / MS method is an omics analysis method combining MALDI and TOF / MS. A suitable pretreatment is applied to a biological sample to obtain an analysis sample, a matrix solution such as α-cyano-4-hydroxycinnamic acid (CHCA) and dihydroxybenzoic acid (DHB) is added thereto, and the sample is applied to a MALDI-TOF / MS system.

[0032] The SELDI-TOF / MS method is a protein chip technology combining SELDI and TOF / MS. A suitable pretreatment is applied to a biological sample to obtain an analysis sample, the analysis sample is applied to a protein chip, the lipid molecule species is adsorbed to the chip, and then the chip is loaded into a SELDI-TOF / MS system. In addition to chips having a functional group such as a hydrophobic group, an ion exchange group, a metal ion immobilization group, etc. fixed thereto, chips having a specific binding molecule (e.g., an antibody, an antibody fragment, an aptamer, etc.) specific to the lipid molecule species of the subject fixed thereto can also be produced and used.

[0033] In the case of the ESI-MS method, it is preferable to use a separation method such as high performance liquid chromatography, gas chromatography, etc. directly connected to a mass spectrometer. The lipid molecule species of the present application has a relatively low molecular weight, and thus when the ESI-MS method is used for detection of the lipid molecule species, it is desirable to use a separation method under conditions suitable for separation of low molecular weight lipid molecule species.

[0034] According to the analysis method of such a first aspect, by using a compound that does not exist in a living organism as a standard sample, it is possible to improve the quantitative accuracy. Furthermore, according to the analysis method of such a first aspect, by using a lysophosphatidylcholine compound in which the choline moiety contains a stable isotope as a standard sample, it is possible to avoid contamination of the detection target and cross talk in mass spectrometric analysis, thereby improving the quantitative accuracy.

[0035] [Aspect]

[0036] The above-described exemplary embodiments are understood by those skilled in the art to be specific examples of the following aspects.

[0037] (1st Aspect)

[0038] An analysis method including a step of detecting at least either of lysophosphatidylcholine and lysophosphatidic acid in a biological sample by mass spectrometric analysis, and using a lysophosphatidylcholine compound in which the choline moiety contains a stable isotope as a standard sample.

[0039] According to the analysis method of the 1st aspect, by using a compound that does not exist in a living organism as a standard sample, it is possible to improve the quantitative accuracy. Furthermore, according to the analysis method of the 1st aspect, by using a lysophosphatidylcholine compound in which the choline moiety contains a stable isotope as a standard sample, it is possible to avoid contamination of the detection target and cross talk in mass spectrometric analysis, thereby improving the quantitative accuracy.

[0040] (2nd Aspect)

[0041] In one mode of the analysis method, the stable isotope is at least either of deuterium and carbon-13.

[0042] According to the analysis method of the 2nd aspect, since the stable isotope is at least either of deuterium and carbon-13, it is possible to easily manufacture a lysophosphatidylcholine compound in which the choline moiety contains a stable isotope, as compared with the case of using nitrogen-15.

[0043] (3rd Aspect)

[0044] In one mode of the analysis method, the lysophosphatidylcholine compound in which the choline moiety contains a stable isotope is LPC_18_0 or LPC_18_1 in which the choline moiety contains a stable isotope.

[0045] According to the analysis method of the 3rd aspect, by using LPC_18_0 or LPC_18_1 in which the choline moiety contains a stable isotope, which does not exist in a living organism, as a standard sample, it is possible to improve the quantitative accuracy. Furthermore, according to the analysis method of the 3rd aspect, by using LPC_18_0 or LPC_18_1 in which the choline moiety contains a stable isotope as a standard sample, it is possible to avoid contamination of the detection target and cross talk in mass spectrometric analysis, thereby improving the quantitative accuracy.

[0046] (4)

[0047] In the analysis method of one embodiment, the lysophosphatidylcholine compound in which the choline moiety contains a stable isotope has the following structure.

[0048]

[0049] According to the analysis method of item 4, the quantitative accuracy can be improved by using, as a standard sample, a lysophosphatidylcholine compound having the above structure, in which the choline moiety contains a stable isotope, which does not exist in vivo. Furthermore, according to the analysis method of item 4, the quantitative accuracy can be improved by using, as a standard sample, a lysophosphatidylcholine compound having the above structure, in which the choline moiety contains a stable isotope, which can prevent contamination of the detection target and crosstalk in mass spectrometric analysis.

[0050] (5)

[0051] In the analysis method of one embodiment, lysophosphatidylcholine in a biological sample is detected.

[0052] According to the analysis method of item 5, lysophosphatidylcholine can be detected with high quantitative accuracy. Furthermore, according to the analysis method of item 5, lysophosphatidylcholine can be detected with high quantitative accuracy, which can prevent contamination of the detection target and crosstalk in mass spectrometric analysis.

[0053] (6)

[0054] In the analysis method of one embodiment, the biological sample is cerebrospinal fluid.

[0055] According to the analysis method of item 6, lysophosphatidylcholine and lysophosphatidic acid in cerebrospinal fluid can be analyzed with high accuracy.

Claims

1. An analytical method comprising the step of detecting at least either one of lysophosphatidylcholine and lysophosphatidic acid in a biological sample by mass spectrometric analysis, and lysophosphatidylcholine compound having a choline moiety containing a stable isotope is used as a standard sample.

2. The method of claim 1, wherein, the stable isotope is at least either one of deuterium and carbon-13.

3. The method of claim 1 or 2, wherein, the lysophosphatidylcholine compound having a choline moiety containing a stable isotope is LPC_18_0 or LPC_18_1 having a choline moiety containing a stable isotope.

4. The method of claim 3, wherein, the lysophosphatidylcholine compound having a choline moiety containing a stable isotope has the following structure: 。 5. The method according to claim 1, which detects lysophosphatidylcholine in a biological sample.

6. The method of claim 1, wherein, the biological sample is cerebrospinal fluid.

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