Analysis method using lysophosphatidylcholine compound containing stable isotope element in choline moiety

Abstract

An analysis method that comprises a step for detecting, by mass spectrometry, at least one of lysophosphatidylcholine and lysophosphatidic acid in a biological sample and uses a lysophosphatidylcholine compound containing a stable isotope element in a choline moiety as a standard sample can develop a technique capable of accurately analyzing lysophosphatidylcholine and lysophosphatidic acid in a spinal fluid.

Description

Analytical method using lysophosphatidylcholine compounds containing stable isotopes in the choline moiety

[0001] The present invention relates to an analytical method using a lysophosphatidylcholine compound containing a stable isotope in the choline moiety.

[0002] As a method for objectively evaluating pain, which is a subjective measure, for example, Japanese Patent Application Laid-Open No. 2017-187492 proposes a method for detecting at least one of lysophosphatidylcholine (LPC) and lysophosphatidic acid (LPA) in a sample derived from human cerebrospinal fluid. In this method, specific examples of lysophosphatidylcholines include one or more 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, and specific examples of lysophosphatidic acids include one or more selected from 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 and LPA, the first number indicates the number of carbon atoms in the fatty acid chain, and the second number indicates the number of double bonds in the fatty acid chain.

[0003] Conventionally, when detecting lysophosphatidylcholine and lysophosphatidic acid by mass spectrometry (MS), LPA_17_0 (unlabeled with a stable isotope), which is not present in biological samples and has the following structure, has been used as a standard sample.

[0004]

[0005] However, in recent years, it has been discovered that some people may contain a small amount of LPA_17_0, and even when LPA_17_0 is used as a standard sample for quantification, there is a problem with the accuracy. To solve this problem, for example, a lysophosphatidylcholine compound (18:1-d7 Lyso PC / C) represented by the following structure incorporating a stable isotope has been developed. ２６ H４５ D ７ NO ７ It has been proposed to use 1,2,3-trimethylsilyl methylparaben (P) as a standard sample.

[0006]

[0007] However, as shown in Figure 2, when this lysophosphatidylcholine compound is analyzed by MS, it is ionized at a site adjacent to the phosphate group, generating a product ion with m / z = 184. This product ion has the same mass as the LPC product ion and has a similar retention time. Therefore, if the selected product ion is set to m / z = 184, crosstalk (a phenomenon in which ions in the collision cell stagnate, resulting in the erroneous detection of a product ion of the component being measured) occurs, and there is a need to improve the accuracy of quantification.

[0008] JP 2017-187492 A

[0009] An object of the present invention is to solve the above problems and to develop a technique that can accurately analyze lysophosphatidylcholine and lysophosphatidic acid in cerebrospinal fluid.

[0010] A first aspect of the present invention relates to an analytical method comprising a step of detecting at least one of lysophosphatidylcholine and lysophosphatidic acid in a biological sample by mass spectrometry, in which a lysophosphatidylcholine compound containing a stable isotope in the choline moiety is used as a standard sample.

[0011] According to the analytical method of the present invention, the quantitative accuracy can be improved by using a compound that does not exist in a living body as a standard sample.Furthermore, according to the analytical method of the present invention, the quantitative accuracy can be improved by using a lysophosphatidylcholine compound containing a stable isotope in the choline moiety as a standard sample, which avoids contamination of the detection target and crosstalk in mass spectrometry.

[0012] 1 is a diagram showing particularly preferred examples of lysophosphatidylcholine compounds used as labeled samples in the first aspect of the present invention, and FIG. 2 is a diagram showing conventional lysophosphatidylcholine compounds into which stable isotopes have been introduced.

[0013] A first aspect of the present invention is an analytical method comprising a step of detecting at least one of lysophosphatidylcholine and lysophosphatidic acid in a biological sample by mass spectrometry, in which a lysophosphatidylcholine compound containing a stable isotope in the choline moiety is used as a standard sample.

[0014] In the first aspect of the present invention, quantitative accuracy can be improved by using a lysophosphatidylcholine compound containing a stable isotope that is not present in vivo as a standard sample, but the quantitative accuracy may vary greatly depending on where in the lysophosphatidylcholine compound the stable isotope is contained. In the first aspect of the present invention, by labeling the choline moiety adjacent to the phosphate group of lysophosphatidylcholine by introducing a stable isotope, contamination of the detection target and crosstalk in mass spectrometry can be avoided, and quantitative accuracy can be improved.

[0015] The stable isotope element used in the first aspect of the present invention may be a stable isotope element of an atom constituting the choline moiety adjacent to the phosphate group of lysophosphatidylcholine, and deuterium ( ２ H), heavy carbon ( １３ C) and heavy nitrogen ( １５ Among these, from the viewpoint of comparative ease of production, at least one of deuterium and heavy carbon is preferred, and it is particularly preferred that the compound contains both deuterium and heavy carbon.

[0016] The lysophosphatidylcholine to be used as the 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, and among these, LPC_18_0 or LPC_18_1, which have a relatively high concentration in cerebrospinal fluid, are particularly preferred.

[0017] Particularly preferred examples of the lysophosphatidylcholine compound containing a stable isotope include the lysophosphatidylcholine compound represented by the following structure ([ １３ C3, ２ H4] -18:1 Lyso PC / １３ C3C23D4H48NO7P).

[0018]

[0019] The above-mentioned lysophosphatidylcholine compound is obtained by introducing four deuterium atoms and three heavy carbon atoms into the choline moiety of LPC_18_1 to label it. When such a lysophosphatidylcholine compound is used as a standard sample to detect lysophosphatidylcholine (e.g., LPC_18_1) contained in a biological sample, during mass spectrometry, the standard sample is ionized at a portion adjacent to the phosphate group, as shown in Figure 1, and a product ion of m / z = 191 is generated. Because the product ion from this standard sample has a different mass from the product ion of lysophosphatidylcholine, m / z = 184, crosstalk can be avoided.

[0020] The detection target in the biological sample may be at least one of lysophosphatidylcholine and lysophosphatidic acid. Specifically, the detection target may be one or more lysophosphatidylcholine 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, as disclosed in Japanese Patent Application Laid-Open No. 2017-187492, total LPA, and one or more lysophosphatidic acids selected from LPA_16_0, LPA_18_0, LPA_18_1, LPA_18_2, LPA_20_4, and LPA_22_6. In the case of lysophosphatidic acid, a product ion at m / z = 153 is generated, and crosstalk can be avoided in the first place, so the effect of avoiding crosstalk by including a stable isotope in the choline moiety is particularly useful when the detection target is lysophosphatidylcholine. When lysophosphatidylcholine is the detection target, LPC_18_0 or LPC_18_1 is particularly preferred among the above specific examples.

[0021] Lysophosphatidylcholine compounds containing stable isotopes can be synthesized appropriately by combining known methods, but it is also possible to obtain commercially available lysophosphatidylcholine compounds.

[0022] The biological sample is preferably cerebrospinal fluid (human cerebrospinal fluid). Cerebrospinal fluid tests are commonly performed in clinical settings to diagnose various diseases, and a small amount of cerebrospinal fluid collected before myelography may be used as the biological sample as is. Furthermore, the biological sample may be subjected to appropriate known pretreatment as necessary before being used to measure the concentration of components.

[0023] Mass spectrometry may be, for example, laser desorption / ionization time-of-flight mass spectrometry (LDI-TOF / MS) or electrospray ionization-mass spectrometry (ESI-MS).

[0024] In the TOF / MS method, target lipid molecular species can be detected by pattern analysis of the spectrum obtained by the TOF / MS device. Qualitative detection of whether or not the target lipid molecular species is present in the sample can be performed by detecting whether or not a peak of a specific m / z value is present. Quantitative detection is also possible based on the high and low m / z peak values ​​of the spectrum. LDI-TOF / MS may be, for example, matrix-assisted laser ionization time-of-flight mass spectrometry (MALDI-TOF / MS method: Matrix Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometry) or surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF / MS method: Surface-Enhanced Laser Desorption / Ionization Time-of-Flight Mass Spectrometry).

[0025] MALDI-TOF / MS is an omics analysis method that combines MALDI and TOF / MS. A biological sample is subjected to appropriate pretreatment to prepare an analytical sample, and a matrix solution such as α-cyano-4-hydroxycinnamic acid (CHCA) and dihydroxybenzoic acid (DHB) is added to the sample and applied to a MALDI-TOF / MS system.

[0026] The SELDI-TOF / MS method is a protein chip technology that combines SELDI and TOF / MS. A biological sample is subjected to appropriate pretreatment to prepare an analytical sample, which is then applied to a protein chip to adsorb lipid molecular species onto the chip, and then loaded into a SELDI-TOF / MS system. In addition to chips on which functional groups such as hydrophobic groups, ion exchange groups, and metal ion immobilization groups are immobilized, chips on which specific binding molecules (e.g., antibodies, antibody fragments, aptamers, etc.) for target lipid molecular species are immobilized may also be prepared and used.

[0027] In the case of the ESI-MS method, it is preferable to directly connect a separation means such as high performance liquid chromatography or gas chromatography to a mass spectrometer. Since the lipid molecular species of the present invention have a relatively low molecular weight, when detecting lipid molecular species using the ESI-MS method, it is desirable to use a separation means under conditions suitable for separating low molecular weight lipid molecular species.

[0028] According to the analytical method of the first aspect, the use of a compound not present in vivo as a standard sample can improve the accuracy of quantification. Furthermore, the use of a lysophosphatidylcholine compound containing a stable isotope in the choline moiety as a standard sample can avoid contamination of the target substance and crosstalk in mass spectrometry, thereby improving the accuracy of quantification.

[0029] Aspects It will be appreciated by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0030] (Item 1) An analytical method according to one embodiment includes a step of detecting at least one of lysophosphatidylcholine and lysophosphatidic acid in a biological sample by mass spectrometry, and uses a lysophosphatidylcholine compound containing a stable isotope element in the choline moiety as a standard sample.

[0031] According to the analytical method described in paragraph 1, quantitative accuracy can be improved by using a compound that does not exist in vivo as a standard sample. Furthermore, according to the analytical method described in paragraph 1, by using a lysophosphatidylcholine compound containing a stable isotope element in the choline moiety as a standard sample, contamination of the detection target and crosstalk in mass spectrometry can be avoided, and quantitative accuracy can be improved.

[0032] (Item 2) In the analysis method according to one aspect, the stable isotope element is at least one of deuterium and heavy carbon.

[0033] According to the analytical method described in paragraph 2, the stable isotope element is at least one of deuterium and heavy carbon, and therefore, a lysophosphatidylcholine compound containing a stable isotope element in the choline moiety can be easily produced compared to the case where heavy nitrogen is used.

[0034] (Item 3) In the analytical method according to one embodiment, the lysophosphatidylcholine compound containing a stable isotope in the choline moiety is LPC_18_0 or LPC_18_1 containing a stable isotope in the choline moiety.

[0035] According to the analytical method described in paragraph 3, quantitative accuracy can be improved by using LPC_18_0 or LPC_18_1, which does not exist in vivo and contains a stable isotope in the choline moiety, as a standard sample.Furthermore, according to the analytical method described in paragraph 3, by using LPC_18_0 or LPC_18_1, which contains a stable isotope in the choline moiety, as a standard sample, contamination of the detection target and crosstalk in mass spectrometry can be avoided, and quantitative accuracy can be improved.

[0036] (Item 4) In one embodiment of the analytical method, the lysophosphatidylcholine compound containing a stable isotope in the choline moiety has the following structure:

[0037]

[0038] According to the analytical method described in paragraph 4, quantitative accuracy can be improved by using as a standard sample a lysophosphatidylcholine compound that does not exist in vivo and has the above structure and contains a stable isotope element in the choline moiety.Furthermore, according to the analytical method described in paragraph 4, by using as a standard sample a lysophosphatidylcholine compound that has the above structure and contains a stable isotope element in the choline moiety, contamination of the detection target and crosstalk in mass spectrometry can be avoided, and quantitative accuracy can be improved.

[0039] (Item 5) In an analytical method according to one embodiment, lysophosphatidylcholine is detected in a biological sample.

[0040] According to the analytical method described in item 5, lysophosphatidylcholine can be detected with high quantitative accuracy. Furthermore, according to the analytical method described in item 5, contamination of the detection target and crosstalk in mass spectrometry can be avoided, and lysophosphatidylcholine can be detected with high quantitative accuracy.

[0041] (Item 6) In the analytical method according to one aspect, the biological sample is cerebrospinal fluid.

[0042] According to the analytical method described in item 6, lysophosphatidylcholine and lysophosphatidic acid in cerebrospinal fluid can be analyzed with high accuracy.

Claims

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

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

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

4. 2. The method of claim 1, wherein the lysophosphatidylcholine compound containing a stable isotope in the choline moiety has the following structure: 【Chemistry 1】

5. The method of claim 1, wherein lysophosphatidylcholine is detected in a biological sample.

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

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