Pretreatment method for analysis of metabolite

Abstract

A pretreatment method for the analysis of a metabolite includes: a step for supplying an analysis target sample to a porous carrier to support a metabolite contained in the analysis target sample on the porous carrier; and a step for supplying a vaporized derivatization reagent to the porous carrier to which the metabolite has been supported, thereby derivatizing the metabolite.

Description

Pretreatment method for metabolite analysis

[0001] The present invention relates to a method for pre-analysis of metabolites.

[0002] Metabolomics is a technology for comprehensively analyzing the various metabolites produced by living organisms during their life-sustaining activities, and is applied to areas such as disease biomarker discovery and food development. Despite their biological importance as central metabolic pathways, metabolites such as sugar phosphates and polyvalent organic acids have limited analytical options due to their high polarity and instability. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) are used to measure metabolites in metabolomics. GC-MS, in particular, is often the first choice for metabolomics analysis due to its high resolution and robustness.

[0003] When measuring metabolites by GC-MS, many metabolites are hydrophilic and have low volatility, so they must be derivatized. Conventionally, the sample to be analyzed is dehydrated by freeze-drying to completely remove water, and then derivatized in solution using trimethylsilyl (TMS). Furthermore, when storing or transporting the sample to be analyzed, freeze-drying is performed first (see, for example, Metabolomics Pretreatment Handbook (Non-Patent Document 1) and O. Fiehn, J. Kopka, R. N. Trethewey, and L. Willmitzer, Anal. Chem., 72, 3573-3580 (2000) (Non-Patent Document 2)).

[0004] FIG. 6 shows the conventional procedure for extracting and derivatizing metabolites for GC-MS measurement described in Non-Patent Document 1. In the procedure shown in FIG. 6, a sample to be analyzed containing metabolites is first subjected to vacuum concentration and centrifugation at 16,000 rcf, 4°C, and 3 minutes, then frozen in liquid nitrogen and freeze-dried overnight (16 hours) for dehydration. A derivatization reagent (methoxyamine) is then added and a derivatization reaction is carried out at 30°C for 90 minutes. A derivatization reagent (N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA)) is then added and a derivatization reaction is carried out at 37°C for 30 minutes. The mixture is then centrifuged at 16,000 rcf, 4°C, and 3 minutes, and the supernatant is collected and subjected to GC-MS measurement.

[0005] In addition, in LC-MS, analytical methods that involve derivatization are used to improve the sensitivity and separation of metabolites.

[0006] Metabolomics Pretreatment Handbook, O. Fiehn, J. Kopka, R. N. Trethewey, and L. Willmitzer, Anal. Chem., 72, 3573-3580 (2000)

[0007] Conventional methods require time and effort for extraction and derivatization (for example, in the pretreatment method often used in GC-MS, after sample extraction, dehydration takes overnight (approximately 16 hours) and derivatization takes two hours, requiring a considerable amount of time and effort). In addition, depending on the metabolite, there are problems such as poor derivatization efficiency, poor sensitivity and reproducibility, and the appearance of multiple peaks, resulting in complex chromatograms.

[0008] The present invention solves the above problems and provides a method for pre-analysis of metabolites, which allows for easy collection and storage of metabolites contained in a sample to be analyzed and also enables highly efficient derivatization reactions.

[0009] A first aspect of the present invention relates to a method for pre-analysis of metabolites, comprising the steps of supplying a sample to be analyzed to a porous carrier and retaining metabolites contained in the sample to be analyzed on the porous carrier, and supplying a vaporized derivatization reagent to the porous carrier on which the metabolites have been retained, thereby derivatizing the metabolites.

[0010] According to the metabolite analysis pretreatment method of the present invention, a highly efficient derivatization reaction can be performed, eliminating the influence of solvents, compared to when metabolites are derivatized using a liquid derivatization reagent. Furthermore, according to the metabolite analysis pretreatment method of the present invention, the porous carrier can be stored and transported as is. Furthermore, according to the metabolite analysis pretreatment method of the present invention, water and solvent can be easily removed from the porous carrier holding metabolites by purging with an inert gas. Furthermore, according to the metabolite analysis pretreatment method described in paragraph 1, the porous carrier after purging with an inert gas can be stored and transported as is.

[0011] 1(a) and 1(b) are diagrams schematically showing the method for pre-treatment of metabolite analysis according to the present invention. FIG. 1(a) is a diagram schematically showing the procedure for the method for pre-treatment of metabolite analysis (in the case of polyvalent organic acids) in Experimental Example 1. FIG. 1(b) is a chromatogram obtained as a result of Experimental Example 1. FIG. 1(b) is a diagram schematically showing the procedure for the method for pre-treatment of metabolite analysis (in the case of glucose 6-phosphate) in Experimental Example 2. FIG. 1(b) is a diagram showing the procedure for extraction and derivatization of metabolites for conventional GC-MS measurement.

[0012] A first aspect of the present invention is a method for pre-analysis of metabolites, comprising the steps of: supplying a sample to be analyzed to a porous carrier and causing metabolites contained in the sample to be analyzed to be retained on the porous carrier; and supplying a vaporized derivatization reagent to the porous carrier on which the metabolites have been retained, thereby derivatizing the metabolites.

[0013] 1(a) and 1(b) are schematic diagrams illustrating a metabolite analysis pretreatment method according to the present invention. In the metabolite analysis pretreatment method according to the present invention, first, as shown in FIG. 1(a), a sample to be analyzed is supplied to a porous carrier 1, and metabolites contained in the sample to be analyzed are retained on the porous carrier 1. In FIG. 1(a), for example, the sample to be analyzed is applied to the porous carrier 1 by injecting the sample to the holes 2 of the porous carrier 1 using a micropipette 3 (loading). A syringe may be used instead of a micropipette to supply the sample to be analyzed.

[0014] The porous carrier 1 is not particularly limited as long as it has a plurality of pores 2 (which may or may not be through-holes) on its surface and is formed from a chemically stable material (in other words, it does not react with the analysis sample containing metabolites or the derivatization reagent). Examples of materials that form the porous carrier 1 include porous ceramics such as silica. The shape of the porous carrier 1 is not particularly limited, and it may be rod-shaped, plate-shaped, columnar, or any other shape.

[0015] The pore size of the pores 2 in the porous carrier 1 is not particularly limited, but it is believed that the pores 2 can hold the sample to be analyzed by interactions such as intermolecular forces, and if the pore size is too small, it may be difficult to hold the sample to be analyzed. The porous carrier 1 may have a monolith structure (a single porous structure having a three-dimensional network-like skeleton structure with through-holes (through-pores, corresponding to pores 2) that are continuous from one end to the other and communicate with each other, and pores (mesopores) that are smaller in diameter than the through-holes and that may or may not communicate with each other within the through-holes), in which case a silica monolith carrier is a suitable example. Silica monolith carriers can be produced by known production methods, for example, by dissolving a water-soluble polymer in an aqueous acetic acid solution, adding silica to this solution, carrying out a hydrolysis reaction, stirring, and then solidifying the resulting solution into a shape that can be used as a carrier, and then immersing the gel in an aqueous ammonia solution, drying, and heating. However, commercially available products (for example, MonoTrap （登録商標）(manufactured by GL Sciences Inc.) (can be used for both solvent extraction and thermal desorption) may also be used. In Experimental Examples 1 and 2 described later, MonoTrap for thermal desorption was used. （登録商標） RSC18TD is used.

[0016] The metabolites contained in the sample to be analyzed may be any known metabolite, such as organic acids, sugar phosphates, sugars, amino acids, and fatty acids. However, the metabolite pretreatment method of the present invention is particularly suitable for ionic polar metabolites, such as polyvalent organic acids and sugar phosphates, which have limited analytical options due to their high polarity and instability. Examples of polyvalent organic acids include, but are not limited to, succinic acid, malic acid, glutaric acid, adipic acid, 2-oxoglutaric acid, cis-aconitic acid, citric acid, isocitric acid, and fumaric acid. Examples of sugar phosphates include, but are not limited to, glucose 6-phosphate (G6P), mannose 6-phosphate (M6P), and fructose 6-phosphate (F6P).

[0017] The sample to be analyzed may be a liquid sample containing such metabolites and a solvent, or an extract obtained by extraction with a solvent. Examples of the solvent include, but are not limited to, water, alcohols such as methanol, and acids such as hydrochloric acid. Note that, since the above-mentioned silica monolithic carrier is water-repellent, if the solvent is water, it is preferable to wet the silica monolithic carrier with methanol or the like before applying the sample to be analyzed.

[0018] As shown in Fig. 1(b), the metabolite pretreatment method according to the present invention involves supplying a vaporized derivatization reagent 5 to a porous carrier 1 carrying metabolites, thereby derivatizing the metabolites. For example, as shown in Fig. 1(b), a liquid derivatization reagent 4 is vaporized by heating, and the porous carrier 1 carrying metabolites is exposed to the vaporized derivatization reagent 5. According to this metabolite pretreatment method according to the present invention, the derivatization reaction can be carried out with high efficiency, eliminating the influence of the solvent, compared to when metabolites are derivatized with a liquid derivatization reagent.

[0019] The derivatization reagent may be any known appropriate derivatization reagent, such as a methylating agent, an acetylating agent, or a silylating agent, and is not particularly limited. In Experimental Example 1 described below, trimethylsilyldiazomethane (TMSD) was used as the derivatization reagent (methylation), and in Experimental Example 2 described below, TMSD was used as the first-stage derivatization reagent, and acetic anhydride and pyridine were used as the second-stage derivatization reagent (two-stage derivatization: methylation and acetylation). The conditions for the derivatization reaction are not particularly limited, and may be selected from known suitable conditions depending on the metabolite and derivatization reagent. For example, in Experimental Example 1 described below, methylation using 500 μL TMSD (0.6 M) and 500 μL methanol was carried out by incubation at 40° C. for 5 minutes, and in Experimental Example 2 described below, methylation using 500 μL TMSD (0.6 M) and 500 μL methanol was carried out by incubation at 60° C. for 5 minutes, and acetylation using 500 μL acetic anhydride and 500 μL pyridine was carried out by incubation at 80° C. for 30 minutes (as described above, since the derivatization reaction can be carried out with high efficiency while eliminating the influence of the solvent, the time required for the derivatization reaction can be significantly reduced compared to conventional methods).

[0020] The metabolite analysis pretreatment method according to the present invention may also include a step of supplying an inert gas to the porous carrier between the step of retaining metabolites on the porous carrier and the step of derivatizing the metabolites, or at least after the step of derivatizing the metabolites. According to the metabolite analysis pretreatment method according to the present invention, by retaining metabolites on the porous carrier, water, solvent, excess derivatization reagents that may affect analysis, and by-products can be easily removed from the porous carrier by purging with an inert gas. Known inert gases such as nitrogen, argon, and helium can be used without particular limitation as the inert gas. Depending on the boiling point of the solvent, purging may be performed while heating. The purging conditions are not particularly limited, and known suitable conditions may be appropriately selected. For example, in Experimental Example 1 described below, purging was performed using helium at 60°C, 200 mL / min, and for 5 minutes. Depending on the derivatization reaction, the step of supplying an inert gas to the porous carrier may be omitted between the step of retaining metabolites on the porous carrier and the step of derivatizing the metabolites. Furthermore, in the case of the step after the step of derivatizing metabolites, if the derivatization reagent and the by-products produced do not affect the analysis, the step of supplying an inert gas to the porous carrier may be omitted.

[0021] In the pretreatment method for analyzing metabolites according to the present invention, the porous carrier holding the metabolites can be stored or transported as is before the step of supplying a vaporized derivatization reagent to the porous carrier holding the metabolites and derivatizing the metabolites. When stored or transported as is, the porous carrier holding the metabolites may or may not have undergone the step of supplying an inert gas to the porous carrier described above.

[0022] The derivatized metabolites can be desorbed from the porous carrier 1 by heating, for example, and then subjected to analysis. For example, in Experimental Examples 1 and 2 described below, the derivatized metabolites are placed in a thermal desorption device 14 connected to a GC-MS device 13, and thermal desorption and GC-MS analysis of the derivatized metabolites are performed. There are no particular restrictions on the GC-MS device 13, and any commercially available product can be used, for example, a GCMS-TQ （商標）8050 NX (Shimadzu Corporation), GCMS-TQ （商標） 8040 NX (Shimadzu Corporation), GCMS-QP （商標） A suitable example is 2020 NX (manufactured by Shimadzu Corporation). The derivatized metabolites may be desorbed from the porous carrier 1 by extracting them into a solvent using ultrasonic extraction, and then subjected to GC-MS analysis or LC-MS analysis.

[0023] FIG. 2 is a diagram schematically illustrating the procedure for the metabolite preanalysis method (for polyvalent organic acids) in Experimental Example 1 described below. First, as described above, the sample to be analyzed is applied to the porous carrier 1 by injecting the sample to be analyzed into the holes 2 of the porous carrier 1 using a micropipette 3. Next, the porous carrier 1 is suspended in the gas phase of a vial 11 containing a liquid derivatization reagent 4 (TMSD in Experimental Example 1), and the vial 11 is capped. The vial 11 is heated with a heater 12 to vaporize the derivatization reagent 4, exposing the porous carrier 1 to the vaporized derivatization reagent 5, and a derivatization reaction is carried out on the porous carrier 1. The porous carrier is then purged with an inert gas, and the porous carrier retaining the derivatized metabolites is placed in a thermal desorption device 14 connected to a GC-MS device 13, where thermal desorption and GC-MS analysis of the derivatized metabolites are performed.

[0024] FIG. 3 is a chromatogram obtained as a result of Experimental Example 1 described later. In FIG. 3, it can be seen that a peak 21 of fumaric acid, a peak 22 of succinic acid, a peak 23 of glutaric acid, a peak 24 of adipic acid, a peak 25 of malic acid, a peak 26 of 2-oxoglutaric acid, a peak 27 of cis-aconitic acid, a peak 28 of citric acid, and a peak 29 of isocitric acid are clearly obtained.

[0025] FIG. 4 is a diagram schematically illustrating the procedure for the pretreatment method for metabolite analysis (in the case of glucose 6-phosphate) in Experimental Example 2. First, as described above, the sample to be analyzed is applied to the porous carrier 1 by injecting it into the holes 2 of the porous carrier 1 using a micropipette 3. The porous carrier 1 is then purged with an inert gas to remove the solvent. Next, the porous carrier 1 is suspended in the gas phase of a vial 11 containing a liquid derivatization reagent 4 (TMSD in Experimental Example 2), and the vial 11 is capped. The vial 11 is heated with a heater 12 to vaporize the derivatization reagent 4. The porous carrier 1 is then exposed to the vaporized derivatization reagent 5, and a derivatization reaction is carried out on the porous carrier 1 (first-stage derivatization reaction). After that, the porous carrier 1 is purged with an inert gas, and the porous carrier 1 is suspended in the gas phase of a vial 11 containing a liquid derivatization reagent 31 (acetic anhydride and pyridine in Experimental Example 2), and the vial 11 is then capped. The vial 11 is heated with a heater 12 to vaporize the derivatization reagent 31, and the porous carrier 1 is exposed to this vaporized derivatization reagent 32, thereby carrying out a derivatization reaction on the porous carrier 1 (second-stage derivatization reaction). After that, the porous carrier is purged with an inert gas, and the porous carrier holding the derivatized metabolites is placed in a thermal desorption device 14 connected to a GC-MS device 13, where thermal desorption and GC-MS analysis of the derivatized metabolites are performed.

[0026] In the example shown in FIG. 4, glucose 6-phosphate is derivatized as follows through a two-step derivatization (methylation and acetylation):

[0027]

[0028] FIG. 5 is a chromatogram obtained as a result of Experimental Example 2, and it can be seen from FIG. 5 that a peak 41 for the α isomer and a peak 42 for the β isomer are clearly obtained.

[0029] The present invention will be explained in more detail below by way of experimental examples, but the present invention is not limited to these examples.

[0030] <Experimental Example 1> 10 μL each of fumaric acid, succinic acid, malic acid, glutaric acid, adipic acid, 2-oxoglutaric acid, cis-aconitic acid, citric acid, and isocitric acid (each 0.2 mM) was used as a sample to be analyzed. （登録商標） The porous carrier 1 was suspended in the gas phase of a vial 11 containing 500 μL TMSD (0.6 M) and 500 μL methanol as liquid derivatization reagent 4, and the vial 11 was then capped. The vial 11 was heated with a heater 12 to vaporize the derivatization reagent 4, and the porous carrier 1 was exposed to this vaporized derivatization reagent 5, which was then incubated at 40°C for 5 minutes to carry out a derivatization reaction on the porous carrier 1. Thereafter, the porous carrier was purged with helium at 60°C, 200 mL / min, and the derivatized metabolite-holding porous carrier was analyzed by a GC-MS device (GCMS-TQ （商標） The sample was placed in a thermal desorption apparatus 14 connected to a Shimadzu 8050 NX (Shimadzu Corporation) 13, and thermal desorption and GC-MS analysis of the derivatized metabolites were carried out.

[0031] FIG. 3 is a chromatogram obtained as a result of Experimental Example 1. In FIG. 3, it can be seen that a fumaric acid peak 21, a succinic acid peak 22, a glutaric acid peak 23, adipic acid peak 24, a malic acid peak 25, a 2-oxoglutaric acid peak 26, a cis-aconitic acid peak 27, a citric acid peak 28, and an isocitric acid peak 29 are clearly obtained.

[0032] Experimental Example 2 10 μL of glucose-6 phosphate was used as a sample to be analyzed, and the sample was poured into the porous carrier 1 (MonoTrap) using a micropipette 3. （登録商標）The solution was applied to the hole 2 of a RSC18TD (manufactured by GL Sciences Inc.). Then, helium was purged at 60 ° C., 200 mL / min, for 5 minutes to remove the solvent. Porous carrier 1 was suspended in the gas phase of vial 11, which contained 500 μL TMSD (0.6 M) and 500 μL methanol as liquid derivatization reagent 4. The vial 11 was then capped and heated with heater 12 to vaporize the derivatization reagent 4. The porous carrier 1 was then exposed to the vaporized derivatization reagent 5 and incubated at 40 ° C. for 5 minutes to carry out a derivatization reaction. Then, helium was purged at 60 ° C., 200 mL / min, for 5 minutes to remove excess derivatization reagent and by-products that could affect the analysis. The porous carrier 1 was suspended in the gas phase of a vial 11 containing 500 μL of acetic anhydride and 500 μL of pyridine as liquid derivatization reagent 31, and the vial 11 was then capped. The vial 11 was heated with a heater 12 to vaporize the derivatization reagent 31, and the porous carrier 1 was exposed to this vaporized derivatization reagent 32, thereby carrying out a derivatization reaction. After that, the porous carrier 1 was purged with helium at 60°C, 200 mL / min, for 5 minutes to remove excess derivatization reagent and by-products that would affect the analysis. The porous carrier holding the derivatized metabolites was analyzed by a GC-MS device (GCMS-TQ). （商標） The sample was placed in a thermal desorption apparatus 14 connected to a Shimadzu 8050 NX (Shimadzu Corporation) 13, and thermal desorption and GC-MS analysis of the derivatized metabolites were carried out.

[0033] FIG. 5 is a chromatogram obtained as a result of Experimental Example 2, and it can be seen from FIG. 5 that a peak 41 for the α isomer and a peak 42 for the β isomer are clearly obtained.

[0034] Aspects It will be understood by those skilled in the art that the exemplary embodiments and experimental examples described above are examples of the following aspects.

[0035] (Item 1) A method for pre-analyzing metabolites according to one embodiment includes the steps of supplying a sample to be analyzed to a porous carrier and retaining metabolites contained in the sample to be analyzed on the porous carrier, and supplying a vaporized derivatization reagent to the porous carrier retaining the metabolites, thereby derivatizing the metabolites.

[0036] According to the metabolite analysis pretreatment method described in paragraph 1, a highly efficient derivatization reaction can be carried out without the influence of a solvent, compared to when a metabolite is derivatized using a liquid derivatization reagent. Furthermore, according to the metabolite analysis pretreatment method described in paragraph 1, the porous carrier holding the metabolite can be stored and transported as is.

[0037] (Item 2) The method for pre-processing metabolite analysis described in item 1 includes a step of supplying an inert gas to the porous carrier at least either between the step of retaining the metabolite on the porous carrier and the step of derivatizing the metabolite, or after the step of derivatizing the metabolite.

[0038] According to the metabolite analysis pretreatment method described in paragraph 2, water and solvent can be easily removed from the metabolite-retaining porous carrier by purging with an inert gas. Furthermore, according to the metabolite analysis pretreatment method described in paragraph 1, the metabolite-retaining porous carrier after purging with an inert gas can be stored and transported as is.

[0039] 1 Porous carrier, 2 Hole, 3 Micropipette, 4 Derivatization reagent (liquid), 5 Derivatization reagent (gas), 11 Vial, 12 Heater, 13 GC-MS, 14 Thermal desorption apparatus, 21 Fumaric acid peak, 22 Succinic acid peak, 23 Glutaric acid peak, 24 Adipic acid peak, 25 Malic acid peak, 26 2-oxoglutaric acid peak, 27 Cis-aconitic acid peak, 28 Citric acid peak, 29 Isocitric acid peak, 31 Derivatization reagent (liquid), 32 Derivatization reagent (gas), 41 α isomer peak, 42 ​​β isomer peak.

Claims

1. A method for pre-analysis of metabolites, comprising the steps of: supplying a sample to be analyzed to a porous carrier and causing metabolites contained in the sample to be analyzed to be retained in the porous carrier; and supplying a vaporized derivatization reagent to the porous carrier on which the metabolites have been retained, thereby derivatizing the metabolites.

2. The method for pre-analysis of metabolites according to claim 1, comprising a step of supplying an inert gas to the porous carrier at least either between the step of retaining the metabolites in the porous carrier and the step of derivatizing the metabolites, and after the step of derivatizing the metabolites.

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