Component separation method using supercritical fluid chromatograph

Abstract

To provide a component separation method using a supercritical fluid chromatograph, that is capable of excellently separating an oligonucleotide.SOLUTION: A component separation method using a supercritical fluid chromatograph includes the steps of: infusing a specimen into a moving phase containing a supercritical fluid and a modifier, and introducing the specimen to a column; and separating the components in the specimen during passage in the column. In the component separation method using a supercritical fluid chromatograph, the specimen contains an oligonucleotide as a target component, the supercritical fluid contains carbon dioxide, and the modifier contains a solution including at least one kind selected from a group consisting of ammonium, alkylamine, and amino alcohol, and acid.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for separating components using supercritical fluid chromatography. [Background technology]

[0002] Supercritical fluid chromatography (hereinafter referred to as "SFC") is a type of chromatography that uses a fluid (supercritical fluid) with a temperature and pressure exceeding the critical point (critical temperature, critical pressure) as the main mobile phase, and has the characteristic that the elution power can be controlled by adjusting the temperature and pressure of the mobile phase. In SFC, carbon dioxide is widely used as the supercritical fluid because its critical points for both temperature and pressure are relatively low, it is easy to handle, and it is inexpensive.

[0003] Non-Patent Document 1 discloses an analytical method for human metabolomics using SFC. Patent Document 1 discloses a stationary phase for SFC that exhibits good molecular discrimination ability. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2016 / 152996 [Non-patent literature]

[0005] [Non-Patent Document 1] Vincent Desfontaine et al. Journal of Choromatography A, 1562, 96-107, 2018 Summary of the Invention [Problem to be solved by the invention]

[0006] In recent years, liquid chromatography (LC) and high performance liquid chromatography (HPLC) have been used to analyze oligonucleotides, but the separation characteristics may not be sufficient. For example, in nucleic acid drugs, which are oligonucleotides, phosphorothioate bonds may be used instead of phosphodiester bonds to improve stability in the body. In the synthesis process of oligonucleotides containing phosphorothioate bonds, analogs may be generated that are partially converted to phosphodiester bonds. It is difficult for LC and HPLC to recognize differences of one to several tens of atoms in oligonucleotides with molecular weights of several thousand and separate the main component from the analogs.

[0007] An object of the present invention is to provide a component separation method using supercritical fluid chromatography, which is capable of separating oligonucleotides well. [Means for solving the problem]

[0008] As a result of extensive research, the present inventors have found that oligonucleotides can be effectively separated in supercritical fluid chromatography by using a specific modifier, and have thus completed the present invention.

[0009] A first aspect of the present invention relates to a component separation method using a supercritical fluid chromatograph, comprising the steps of injecting a sample into a mobile phase containing a supercritical fluid and a modifier and introducing the sample into a column, and separating components in the sample while passing through the column, wherein the sample contains an oligonucleotide as a target component, the supercritical fluid contains carbon dioxide, and the modifier contains a solution containing at least one selected from the group consisting of ammonium, alkylamine, and aminoalcohol, and an acid. Effect of the Invention

[0010] According to the present invention, there is provided a component separation method using supercritical fluid chromatography, which is capable of separating oligonucleotides satisfactorily. [Brief description of the drawings]

[0011] [Figure 1] FIG. 2 is a flow path configuration diagram showing an example of an SFC for implementing the component separation method according to the present embodiment. [Diagram 2] FIG. 2 is a chromatogram in the analysis of Example 1. [Diagram 3] FIG. 2 is a chromatogram in the analysis of Example 2. [Figure 4] FIG. 1 shows a chromatogram in the analysis of Example 3. [Diagram 5] FIG. 1 shows a chromatogram in the analysis of Example 4. [Figure 6] FIG. 1 shows a chromatogram in the analysis of Example 5. [Figure 7] FIG. 13 is a chromatogram in the analysis of Example 6. [Figure 8] FIG. 2 is a chromatogram in the analysis of Comparative Example 1. [Figure 9] FIG. 13 is a chromatogram in the analysis of Comparative Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention relates to a component separation method using a supercritical fluid chromatograph (SFC), which includes a step of injecting a sample into a mobile phase containing a supercritical fluid and a modifier and introducing the sample into a column, and a step of separating components in the sample while passing through the column, in which the sample contains an oligonucleotide as a target component, the supercritical fluid contains carbon dioxide (CO2), and the modifier contains a solution containing at least one selected from the group consisting of ammonium, alkylamine, and aminoalcohol, and an acid. The separation method of the present invention can be used in an analysis method for analyzing components of a sample. Hereinafter, one embodiment of the component separation method according to the present invention will be described.

[0013] [SFC] Fig. 1 is a flow path configuration diagram showing an example of an SFC for carrying out the component separation method according to the present embodiment. The SFC shown in Fig. 1 includes a liquid CO2 cylinder 10, a modifier container 11, a CO2 pump 12, a liquid delivery pump 13, an autosampler 14, a column oven 15, a column 16, a detector 17, and a back pressure regulator 18. The liquid CO2 cylinder 10 is a supply source of liquid CO2 as a supercritical fluid. The modifier container 11 is a container in which a characteristic modifier, which will be described later, is stored and which is added to the supercritical fluid.

[0014] The basic operation of this SFC is as follows. The back pressure regulator 18 adjusts the pressure of the mobile phase in the inlet flow path 20 between the CO2 pump 12 and the liquid delivery pump 13 and the inlet of the column 16, in the column 16, and in the outlet flow path 21 between the outlet of the column 16 and the back pressure regulator 18 to a pressure exceeding the critical pressure of CO2 (7.4 MPa), for example, 10 MPa. The CO2 pump 12 draws liquid CO2 from the liquid CO2 cylinder 10 and delivers it to the inlet flow path 20. Meanwhile, the liquid delivery pump 13 draws modifier from the modifier container 11 and delivers it to the inlet flow path 20. In the inlet flow path 20, the liquid CO2 and the modifier are mixed and flowed to the column 16 as the mobile phase. The autosampler 14 provided in the inlet flow path 20 injects a liquid sample into the mobile phase at a predetermined timing.

[0015] The column oven 15 in which the column 16 is installed is adjusted to, for example, about 35°C, which is higher than the critical temperature (31°C) of CO2, which is the main substance of the mobile phase. Therefore, the mobile phase passing through the column 16 becomes supercritical. The sample introduced into the column 16 by the flow of the mobile phase is separated into individual components by interaction with the stationary phase provided on the inner wall of the column 16. Then, the components are eluted from the column 16 with a time difference and flow into the outlet side flow path 21. The detector 17 provided in the outlet side flow path 21 is an ultraviolet-visible spectroscopic detector (UV detector) or the like, and detects the components in the mobile phase separated in the column 16 while still in a critical state. The mobile phase after component detection passes through the back pressure regulator 18 and is collected in a collection container (not shown) or the like.

[0016] Note that the detector 17 may be an optical detector such as a multi-wavelength detector using a PDA (Photo Diode Array) used in liquid chromatography, an optical rotation detector, a circular dichroism detector, a fluorescence detector, a refractive index detector, or an evaporative light scattering detector. Also, a mass spectrometer using an atmospheric pressure ion source such as an electrospray ion source may be used as the detector 17.

[0017] [Mobile phase] The mobile phase includes a supercritical fluid containing carbon dioxide and a modifier. The modifier includes at least one selected from the group consisting of ammonium, alkylamine, and aminoalcohol (hereinafter also referred to as component (a)), and an acid (hereinafter also referred to as component (b)). The modifier further includes a solvent. By including component (a) and component (b), the modifier can improve the separation performance of the target component, that is, oligonucleotide.

[0018] Examples of alkylamines include ethylamine and methylamine. Examples of aminoalcohols include aminoethanol and aminomethanol. Examples of acids include formic acid, acetic acid, bicarbonate, and the like. In the modifier, the supply sources of component (a) and component (b) may be the same or different. For example, by adding ammonium formate, ammonium acetate, or ammonium bicarbonate, component (a) and component (b) can be supplied simultaneously. Examples of solvents include various organic solvents, water, and the like. Examples of various organic solvents include methanol, ethanol, isopropyl alcohol, acetonitrile, and the like.

[0019] In the modifier, the concentration of component (a) and component (b) is preferably 10 mM or more and 100 mM or less, more preferably 40 mM or more and 50 mM or less. By being within the above numerical range, the separation performance of the target component, the oligonucleotide, can be further improved. In the modifier, a mixed solvent of water and methanol is preferably used as the solvent, and for example, a mixed solvent of water:methanol at a volume ratio of 5:95 can be used.

[0020] [Stationary phase] The stationary phase is not limited as long as it is a stationary phase commonly used in SFC, and may be, for example, silica gel, a silica carrier whose surface is chemically modified, or polymer beads. An example of a suitable stationary phase is a silica carrier whose surface is modified with an alkyl alcohol.

[0021] [Sample to be separated] The sample to be separated contains an oligonucleotide as a target component. The oligonucleotide is an oligomer having nucleotides as constituent units, and is preferably an oligomer having a length of 2 to 40 bases, more preferably 2 to 20 bases, and further preferably 2 to 10 bases. The nucleotide may be any of ribonucleotides, deoxyribonucleotides, and modified nucleotides. The modified nucleotide refers to a ribonucleotide or deoxyribonucleotide in which the nucleic acid base, sugar moiety, and phosphate bond moiety constituting the ribonucleotide or deoxyribonucleotide are modified in whole or in part.

[0022] Examples of nucleic acid bases include adenine, guanine, cytosine, thymine, uracil, and modified bases thereof. Examples of such modified bases include, but are not limited to, 5-alkylcytosine (e.g., 5-methylcytosine).

[0023] Modifications of the sugar moiety include, for example, modification of the 2'-position of ribose and modification of other parts of the sugar. Modifications of the 2'-position of ribose include, for example, substitution of the -OH group at the 2'-position of ribose with OR, R'OR, or F. Here, R represents alkyl. R' represents alkylene. Modifications of other parts of the sugar include, for example, substitution of O at the 4'-position of ribose or deoxyribose with S, and bridging of the 2'- and 4'-positions of the sugar, such as LNA (Locked Nucleic Acid) or ENA (2'-O,4'-C-Ethylene-bridged Nucleic Acids), but are not limited thereto.

[0024] Modifications of the phosphate bond moiety include, for example, modifications in which the phosphodiester bond is replaced with a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphonate bond, a phosphoramidate bond, or a boranophosphate bond.

[0025] According to the separation method of the present embodiment, even if the sample to be separated contains two or more oligonucleotides and the difference in molecular structure between the two or more oligonucleotides is one to several tens of atoms, or one to 10 atoms, the two or more oligonucleotides can be separated well. Therefore, according to the separation method of the present embodiment, even if the sample to be separated contains an oligonucleotide as a target component and an oligonucleotide different from the target component that was generated in the synthesis process of the target component, the target component can be separated or purified with high accuracy.

[0026] Examples of samples to be separated include oligonucleotides containing the base sequence TAGC, and oligonucleotides containing the base sequence TTTT. EXAMPLES

[0027] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0028] [Sample to be analyzed] Samples containing eight types of oligonucleotides (components a to h) shown in Table 1 were prepared.

[0029] [Table 1]

[0030] In Table 1, T: deoxythymidine, A: deoxyadenine, G: deoxyguanine, C: deoxycytidine, * : indicates that the phosphodiester bonds at the phosphate bonds of the nucleotides on both sides are phosphorothioate bonds.

[0031] [Separation column] An SFC column (Shimadzu Corporation, Shin-pack UC-Diol II) (100 mm×2.0 mm ID, 3 μm) packed with a silica packing material modified with an alkyl alcohol was used.

[0032] [Examples 1 to 6, Comparative Examples 1 and 2] The above separation column was attached to a supercritical fluid chromatography system (Shimadzu Corporation, Nexera UC), and the above analysis samples were analyzed by SFC-MS / MS. The analysis conditions were as follows:

[0033] Mobile phase: Mobile phase A (CO2): Mobile phase B (modifiers listed in Table 2) = 50:50 (volume ratio) Mobile phase flow rate: 1.5mL / min Column oven temperature: 35°C Back pressure valve pressure: 10.0MPa (pressure at 50℃) Sample injection volume: 5 μL Detector: Photodiode array detector (260 nm)

[0034] [Table 2]

[0035] In Table 2, water:methanol=5:95 indicates a volume ratio.

[0036] 2 to 9 show chromatograms obtained by the analysis of Examples 1 to 6 and Comparative Examples 1 and 2. In Figures 2 to 9, peaks a to h correspond to components a to h, respectively. From the results shown in Figures 2 to 9, it can be seen that components a to h were separated in Examples 1 to 6, whereas components a to h could not be separated in Comparative Examples 1 and 2.

[0037] [Aspects] It will be appreciated by those skilled in the art that the exemplary embodiments and examples described above are examples of the following aspects. (Item 1) A component separation method using a supercritical fluid chromatograph according to one embodiment includes the steps of: A component separation method using a supercritical fluid chromatograph, comprising the steps of injecting a sample into a mobile phase containing a supercritical fluid and a modifier and introducing the sample into a column, and separating components in the sample while passing through the column, wherein the sample contains an oligonucleotide as a target component, the supercritical fluid contains carbon dioxide, and the modifier contains a solution containing at least one selected from the group consisting of ammonium, alkylamine, and aminoalcohol, and an acid.

[0038] (Item 2) In the component separation method described in item 1, the acid is at least one selected from the group consisting of formic acid, acetic acid, and bicarbonate.

[0039] (Item 3) In the component separation method according to item 1 or 2, the oligonucleotide comprises the base sequence TAGC.

[0040] (Item 4) In the component separation method according to any one of items 1 to 3, at least one of the phosphodiester bonds in the phosphate binding portion of the oligonucleotide is substituted with a phosphorothioate bond.

[0041] (Item 5) In the component separation method according to any one of items 1 to 4, the column has a stationary phase containing an alkyl alcohol.

[0042] Although the embodiments and examples of the present invention have been described above, it is also intended from the beginning that the configurations of the above-described embodiments and examples may be appropriately combined.

[0043] The embodiments and examples disclosed herein are illustrative in all respects and are not restrictive. The scope of the present invention is not limited to the above-mentioned embodiments and examples. All claims within the scope of the claims and their equivalent meanings and scope are included. Modifications are intended to be included. [Explanation of symbols]

[0044] 10 liquid CO2 cylinder, 11 modifier container, 12 CO2 pump, 13 solvent delivery pump, 14 autosampler, 15 column oven, 16 column, 17 detector, 18 back pressure regulator.

Claims

1. introducing the sample into the column by injecting the sample into a mobile phase comprising a supercritical fluid and a modifier; and separating components in the sample while passing through the column, The sample contains an oligonucleotide as a target component, the supercritical fluid comprises carbon dioxide; The component separation method using supercritical fluid chromatography, wherein the modifier comprises a solution containing at least one selected from the group consisting of ammonium, alkylamine, and aminoalcohol, and an acid.

2. The component separation method according to claim 1 , wherein the acid is at least one selected from the group consisting of formic acid, acetic acid, and bicarbonate.

3. The component separation method according to claim 1 or 2, wherein the oligonucleotide comprises the base sequence TAGC.

4. 3. The component separation method according to claim 1, wherein at least one phosphodiester bond in the phosphate binding portion of the oligonucleotide is substituted with a phosphorothioate bond.

5. The component separation method according to claim 1 or 2, wherein the column has a stationary phase containing an alkyl alcohol.

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