Amine separation method

Abstract

The amine separation method comprises a separation step for separating amine by chromatography using a stationary phase that contains at least any of an oligosaccharide, a polysaccharide, and derivatives thereof, and using a mobile phase that contains a solvent, at least one base, and at least one prescribed acid selected from the group consisting of oxalic acid, acetic acid, formic acid, and trifluoroacetic acid. SB / SA in this amine separation method is at least 0.05 and not more than 0.95, where A is the value calculated using formula (1) for each prescribed acid present in the mobile phase, SA is the total value of the individual As calculated for the individual prescribed acids, B is the value calculated using formula (2) for the base present in the mobile phase, and SB is the total value of the individual Bs calculated for the individual bases. Formula (1): A = valence of the prescribed acid × molar concentration of the prescribed acid (mol / L); Formula (2): B = valence of the base × molar concentration of the base (mol / L)

Description

Amine separation method

[0001] The present disclosure relates to a method for separating amines.

[0002] Separators using polysaccharide derivatives as chiral selectors (hereinafter also referred to as "polysaccharide-based CSPs") are known to be highly effective in separating many enantiomers and substances that are difficult to separate with conventional separators due to their similar structures, such as structural isomers. Because these polysaccharide-based CSPs generally use neutral adsorbents, when analyzing samples that are ionic under the analytical conditions, for some reason, such as low affinity with the separator due to solvation of the sample ions, electrostatic repulsion resulting in low loading, or extremely strong interactions between ionic or polar atomic groups (silanols originating from silica gel, which is often problematic) present in very small amounts in the separator, the peaks are often poorly retained or have extremely broad or distorted shapes, making the intended analysis impossible.

[0003] Non-Patent Document 1 discloses a method for solving the above problem by adding an appropriate salt to the mobile phase. Specifically, it is believed to be so-called ion-pair chromatography, in which sample cations and appropriate anions are simultaneously retained on the stationary phase.

[0004] A. Ishikawa, T. Shibata, “Cellulosic Chiral Stationary Phase Under Reversed-Phase Condition”, Journal of Liquid Chromatography, 1993, Vol. 16, 859-878

[0005] However, the separation operation using a mobile phase as disclosed in Non-Patent Document 1 does not provide sufficient separation performance for the substances to be separated, and there is still room for improvement. An object of the present disclosure is to provide a method for separating amines that has excellent separation performance.

[0006] As a result of extensive investigations, the present inventors have found that the above-mentioned problems can be solved by using a stationary phase containing at least one of an oligosaccharide derivative and a polysaccharide derivative and a mobile phase containing specific concentrations of an acid and an amine, and have thus completed the invention according to the present disclosure.

[0007] [1] A method for separating amines, comprising: a separation step of separating amines by chromatography using a stationary phase containing at least one of an oligosaccharide, a polysaccharide, and a derivative thereof; and a mobile phase containing one or more specific acids selected from the group consisting of oxalic acid, acetic acid, formic acid, and trifluoroacetic acid, one or more bases, and a solvent, wherein SB / SA is 0.05 or more and 0.95 or less, where A is the value calculated for each specific acid contained in the mobile phase by the following formula (1), SA is the total of A calculated for each specific acid, B is the value calculated for each base contained in the mobile phase by the following formula (2), and SB is the total of B calculated for each base. Formula (1): A = valence of specific acid × molar concentration of specific acid (mol / L) Formula (2): B = valence of base × molar concentration of base (mol / L) [2] The method for separating amines according to [1], further comprising an analysis step of analyzing the amines separated in the separation step, wherein the analysis step involves analysis using at least a mass spectrometer. [3] The method for separating an amine according to [1] or [2], wherein the base is one or more selected from the group consisting of ammonia, primary amines, secondary amines, and tertiary amines. [4] The method for separating an amine according to [3], wherein the base is one or more selected from the group consisting of ammonia, ethylamine, n-propylamine, 2-propylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, pyrrolidine, and piperidine. [5] The method for separating an amine according to any of [1] to [4], wherein the mobile phase contains at least oxalic acid as the specific acid. [6] The method for separating an amine according to any of [1] to [5], wherein at least one of the oligosaccharide, polysaccharide, and derivative thereof has an optically active site. [7] The method for separating an amine according to any of [1] to [6], wherein the solvent is one or more selected from the group consisting of water, acetonitrile, ethanol, methanol, 2-propanol, hexane, heptane, THF, and liquid carbon dioxide.

[0008] According to the present disclosure, it is possible to provide a method for separating amines with excellent separation performance.

[0009] 1 is a liquid chromatogram obtained by analysis of an amine in Example 1. 2 is a liquid chromatogram obtained by analysis of an amine in Comparative Example 1. 3 is a liquid chromatogram obtained by analysis of an amine in Comparative Example 3. 4 is a liquid chromatogram obtained by analysis of an amine in Comparative Example 4. 5 is a liquid chromatogram obtained by analysis of an amine in Comparative Example 5. 6 is a liquid chromatogram obtained by analysis of an amine in Example 5. 7 is a liquid chromatogram obtained by analysis of an amine in Comparative Example 7. 8 is a liquid chromatogram obtained by analysis of an amine in Comparative Example 8. 9 is a liquid chromatogram obtained by analysis of an amine in Comparative Example 9. 1 is a liquid chromatogram obtained by analysis of an amine in Example 7. 2 is a liquid chromatogram obtained by analysis of an amine in Comparative Example 10. 3 is a liquid chromatogram obtained by analysis of an amine in Example 8. 4 is a liquid chromatogram obtained by analysis of an amine in Comparative Example 11. 5 is a liquid chromatogram obtained by analysis of an amine in Example 9. 6 is a liquid chromatogram obtained by analysis of an amine in Example 10. 7 is a liquid chromatogram obtained by analysis of an amine in Example 11. 8 is a liquid chromatogram obtained by analysis of an amine in Example 12. 9 is a liquid chromatogram obtained by analysis of an amine in Example 13. 10 is a liquid chromatogram obtained by analysis of an amine in Example 14. 11 is a liquid chromatogram obtained by analysis of an amine in Comparative Example 12. 12 is a liquid chromatogram obtained by analysis of an amine in Example 15. 13 is a liquid chromatogram obtained by analysis of an amine in Comparative Example 13. 14 is a liquid chromatogram obtained by analysis of an amine in Example 16.1 is a liquid chromatogram obtained by analysis of an amine in Comparative Example 14. 2 is a liquid chromatogram obtained by analysis of an amine in Example 17. 3 is a liquid chromatogram obtained by analysis of an amine in Example 18. 4 is a liquid chromatogram obtained by analysis of an amine in Example 19. 5 is a mass spectrum obtained by mass analysis of an amine in Example 20. 6 is a mass spectrum obtained by mass analysis of an amine in Example 21. 7 is a mass spectrum obtained by mass analysis of an amine in Example 22. 8 is a mass spectrum obtained by mass analysis of an amine in Example 23. 9 is a mass spectrum obtained by mass analysis of an amine in Example 24.

[0010] The present disclosure will be described below using specific embodiments. However, each configuration and combination thereof in each embodiment is merely an example, and additions, omissions, substitutions, and other modifications of configurations are possible as appropriate within the scope of the present disclosure. The present disclosure is not limited by the embodiments, but only by the scope of the claims. Furthermore, each aspect disclosed in the present disclosure can be combined with any other feature disclosed in the present disclosure. In this disclosure, when a numerical range is described with a lower limit and an upper limit separately, the numerical range can be a combination of any lower limit and any upper limit. In this disclosure, when a numerical value or physical property value is enclosed before and after the "~" symbol, the range is considered to include the lower and upper limits, which are the endpoints. In addition, when a numerical range expressed as "A to B" or "A or greater than B" is described in stages (e.g., in order of preference), the upper and lower limits of each numerical range can be combined in any way. In this disclosure, the expression "A or B" can be interpreted as "at least one selected from the group consisting of A and B."

[0011] <Amine Separation Method> An amine separation method according to one embodiment of the present disclosure (hereinafter also simply referred to as "amine separation method") includes a separation step of separating amines by chromatography using: a stationary phase containing at least one of oligosaccharides, polysaccharides, and derivatives thereof; and a mobile phase containing one or more specific acids selected from the group consisting of oxalic acid, acetic acid, formic acid, and trifluoroacetic acid, one or more bases, and a solvent, wherein, where A is the value calculated for each specific acid contained in the mobile phase using the following formula (1), SA is the total value of A calculated for each specific acid, B is the value calculated for each base contained in the mobile phase using the following formula (2), and SB is the total value of B calculated for each base, SB / SA is 0.05 or more and 0.95 or less. Formula (1): A = valence of specific acid × molar concentration of specific acid (mol / L) Formula (2): B = valence of base × molar concentration of base (mol / L)

[0012] The above-described amine separation method may include other steps besides the separation step, such as an analysis step of analyzing (e.g., identifying) the separated amine, a quantification step of quantifying the separated amine, a step of estimating the structure of the separated amine, or a recovery step of recovering the separated amine. Furthermore, the mobile phase in the above-described embodiment refers to a mobile phase before containing the amine to be separated, and the "amine content in the mobile phase" refers to the amine content in the mobile phase before containing the amine to be separated. Furthermore, in the present disclosure, oligosaccharides are saccharides having five or more sugar residues. Furthermore, in the present disclosure, oligosaccharides and polysaccharides can be distinguished by molecular weight, with those having a molecular weight of 3,000 or less being considered oligosaccharides and those having a molecular weight of more than 3,000 being considered polysaccharides. Furthermore, in the present embodiment, the specific acid includes both free acids and those in the form of acid anions, and the specific base includes both free acids and those in the form of cations. Furthermore, the acid anions and cations may form salts with each other or with other ions.

[0013] The present inventors have found that the separation performance of amines can be improved by using a stationary phase containing at least one of oligosaccharides, polysaccharides, and derivatives thereof (hereinafter, these are also collectively referred to as "oligosaccharide / polysaccharide derivatives"), a mobile phase containing one or more specific acids selected from the group consisting of oxalic acid, acetic acid, formic acid, and trifluoroacetic acid, one or more bases, and a solvent, and further adjusting the contents of these acids and bases. The present inventors speculate that the reason for this is as follows.

[0014] In an environment where the mobile phase is over-acidic (i.e., where there is more acid than base), acid anions are generated, and the analyte (amine) is protonated to generate ammonium cations, forming an ion pair between these acid anions and ammonium cations. Because this ion pair is weakly polar, it adsorbs to oligosaccharide and polysaccharide derivatives, which also have weak polarity, enabling separation of the analyte. After extensive research, the inventors discovered that by adding a base to the mobile phase at a molar concentration lower than the acid molar concentration (content), neutralizing a portion of the acid and converting it to a salt, the concentration of counter anions in the mobile phase can be adjusted to a preferred level, even if the acid is weak. Furthermore, based on the mechanism discovered by the inventors, the inventors further investigated the type of acid, the acid and amine contents, and other factors, and found that the configuration of the present embodiment described above improves amine separation performance, specifically, achieving sufficient analyte retention time in chromatography.

[0015] Furthermore, the present inventors have conducted further intensive research and found that when the stationary phase is acidic and this causes nonspecific adsorption of sample amines, such as when the stationary phase contains silica gel as a carrier, a phenomenon known as peak tailing occurs, which is thought to be caused by the silanol groups. In such cases, the present embodiment can improve the separation performance of amines, specifically, sharpen the peaks in the chromatogram obtained by analysis. If an ionic interaction occurs between the cation and the conjugated anion of the dissociated silanol, the peaks in the chromatogram obtained by analysis will become broad. However, the present inventors speculate that the presence of excess acid in the present embodiment suppresses the dissociation of silanols, thereby suppressing this ionic interaction.

[0016] Another prior art technique involves increasing the pH of the mobile phase and retaining the sample amine as a neutral molecule on a nonionic stationary phase. However, if the stationary phase contains silica gel, such conditions can easily cause the silica gel to dissolve and the column to deteriorate. Therefore, to prevent the pH from rising too high, measures have been taken, such as adding a buffering salt such as ammonium bicarbonate and inserting a guard column, which is more cost-effective than the main column, before the main column to saturate the mobile phase flowing through the main column with silica and thereby suppressing silica elution from the main column. On the other hand, a major advantage of the embodiment is that the pH is lowered by the excess acid, eliminating the need for such measures.

[0017] Furthermore, when a separation operation using the mobile phase disclosed in Non-Patent Document 1 is applied to the separation of amines, a high-boiling, non-volatile salt is added to the mobile phase, and the salt is likely to remain after the solvent in the mobile phase is evaporated, resulting in a decrease in the separation performance of the amine. On the other hand, when an amine is used as the base in the mobile phase of the above-mentioned embodiment, salts containing anions of one or more specific acids selected from the group consisting of oxalic acid, acetic acid, formic acid, and trifluoroacetic acid and amine cations are generated in the mobile phase, but these salts are prone to volatilization or thermal decomposition, and therefore high-boiling, non-volatile salts are unlikely to remain even after the solvent is evaporated.

[0018] [Amine] The separation method according to the present embodiment can separate a mixture of multiple amines into individual amines, or can separate amines from a mixture containing amines and non-amines. In particular, the ability to separate amine enantiomers is of great importance in science and technology.

[0019] The amine to be separated is not particularly limited as long as it is a compound containing an amino group, and may be any of primary amines, secondary amines, and tertiary amines. Specific examples of amines include amino acids such as alanine, cysteine, glutamic acid, methionine, leucine, tyrosine, and tryptophan; amino acid derivatives such as esters and amides of the above amino acids; amino alcohols such as dimethylaminoethanol, propanolamine, methioninol, and norephedrine; phenylethylamine; cyclic amines such as 1-phenyl-1,2,3,4-tetrahydroisoquinoline; and other basic pharmaceuticals containing an amino group, particularly their enantiomers.

[0020] The separation method according to this embodiment can achieve high separation performance that distinguishes between molecular shapes, and can therefore separate mixtures of amines that are difficult to separate due to their similar structures into individual amines. Examples of mixtures of amines with similar structures include mixtures of chain isomers, positional isomers, geometric isomers, and analogs. Furthermore, the greatest feature of this method is that it can separate mixtures of enantiomers into individual enantiomers by using a chiral stationary phase containing an oligosaccharide / polysaccharide derivative having an optically active site as the stationary phase.

[0021] [Stationary Phase] The stationary phase may contain at least one of oligosaccharides, polysaccharides, and derivatives thereof (hereinafter, collectively referred to as "oligosaccharide / polysaccharide derivatives") as an adsorbent (also referred to as "separation agent"), and the form in which the oligosaccharide / polysaccharide derivative is included is not particularly limited. For example, the adsorbent may be a solidified oligosaccharide / polysaccharide derivative or a mixture of oligosaccharide / polysaccharide derivatives and other components, or an oligosaccharide / polysaccharide derivative or a mixture of oligosaccharide / polysaccharide derivatives and other components supported on a carrier. In addition, polysaccharides or oligosaccharide derivatives that have been insolubilized by, for example, establishing a chemical bond between the carrier and the polysaccharide / polysaccharide derivative, establishing a chemical bond between the oligosaccharide / polysaccharide derivative, or providing a polymer-impermeable layer on the surface of the oligosaccharide / polysaccharide derivative are particularly useful.

[0022] The shape of the stationary phase is not particularly limited, and may be, for example, crushed and amorphous, but is preferably spherical. In this case, the particles can be used in column chromatography, packed into a column. The oligosaccharide / polysaccharide derivative itself may be granulated, but from the viewpoint of superior efficiency, pressure resistance, and ease of use, it is preferable to support it on a carrier such as porous silica gel. In the case of a porous carrier, only the surface may be porous, so that when the oligosaccharide / polysaccharide derivative is supported, it is distributed near the surface (variously called core-shell, surface porous, or peripheral).

[0023] When the stationary phase is spherical, its diameter is not particularly limited, but is preferably 0.5 to 100 μm, more preferably 0.5 to 50 μm, and even more preferably 1 to 20 μm (from the viewpoint of affordability in current chromatography devices). A smaller diameter is preferable from the viewpoint of amine separation, but when used in a large-scale device for preparative separation, a larger diameter is preferable in order to reduce flow resistance. In the present disclosure, "spherical" also includes a nearly spherical shape.

[0024] The type of oligosaccharide / polysaccharide derivative is not particularly limited, and may be, for example, an oligosaccharide / polysaccharide in which some or all of the hydroxyl or amino groups of an oligosaccharide or polysaccharide (hereinafter, collectively referred to as "oligosaccharide / polysaccharide") have been substituted with a substituent. Polysaccharide derivatives are obtained, for example, by reacting some or all of the hydroxyl or amino groups of an oligosaccharide / polysaccharide with another compound. For example, oligosaccharide / polysaccharide derivatives in which some or all of the hydroxyl groups have been converted to esters of aliphatic carboxylic acids, benzoic acids, or phenylcarbamic acids, each of which may further contain one or more substituents, are preferred from the viewpoint of separation performance. Among these, oligosaccharides / polysaccharides in which at least one hydrogen atom in the benzene ring has been independently substituted with a methyl group or a halogen atom are preferred. The oligosaccharide / polysaccharide derivatives may be used alone, or two or more may be used in any combination.

[0025] The type of oligosaccharide or polysaccharide that serves as the base for the oligosaccharide or polysaccharide derivative is not particularly limited. The number of sugar residues in the oligosaccharide or polysaccharide is not particularly limited as long as it is 5 or more, and may be, for example, 10 or more, 20 or more, or less than 1,000, 500 or less, 100 or less, 75 or less, or 50 or less.

[0026] The type of monosaccharide constituting the oligosaccharide or polysaccharide is not particularly limited, and examples thereof include aldotetroses such as erythrose or threose; aldopentoses such as ribose, lyxose, xylose, or arabinose; aldohexoses such as allose, talose, gulose, glucose, altrose, mannose, galactose, or idose; ketotetroses such as erythrulose; ketopentoses such as xylose or ribulose; or ketohexoses such as psicose, fructose, sorbose, or tagatose. Among these, glucose, fructose, and glucosamine are preferred as highly pure and easily available raw materials. These monosaccharides may be used alone or in combination.

[0027] More specifically, examples of polysaccharides include amylose, amylopectin, glycogen, cellulose, curdlan, paramylon, chitin, chitosan, dextran, nigeran, agarose, carrageenan, heparin, alginic acid, hyaluronic acid, pectin, xyloglucan, xylan, glucomannan, and levan. Among these, cellulose, amylose, chitin, chitosan, curdlan, schizophyllan, agarose, and the like are preferred from the viewpoints of excellent separation performance and easy availability at high purity. Amylose and amylopectin may be used alone or in a form containing both, such as starch.

[0028] Furthermore, as mentioned above, oligosaccharide / polysaccharide derivatives having an optically active site are preferred from the viewpoint of being able to separate a mixture of enantiomers into each enantiomer.

[0029] The amount of oligosaccharide / polysaccharide derivative in the stationary phase is not particularly limited and can be appropriately set with reference to known methods using oligosaccharide / polysaccharide derivatives as separation agents in the stationary phase. For example, the content of oligosaccharide / polysaccharide derivative in the stationary phase may be 100% by mass, but when the above-mentioned carrier or the like is used, it is preferably 5% by mass or more and 50% by mass or less, more preferably 5% by mass or more and 30% by mass or less, and even more preferably 10% by mass or more and 30% by mass or less.

[0030] As described above, the embodiment containing an oligosaccharide / polysaccharide derivative is not particularly limited, and the oligosaccharide / polysaccharide derivative or the oligosaccharide / polysaccharide derivative itself may be formed into particles, porous particles, or porous monoliths as needed, or a solidified mixture of the oligosaccharide / polysaccharide derivative or the oligosaccharide / polysaccharide derivative with other components may be used, or a carrier may be used in which the oligosaccharide / polysaccharide derivative or the mixture of the oligosaccharide / polysaccharide derivative and other components is supported; however, a carrier in which the oligosaccharide / polysaccharide derivative or the mixture of the oligosaccharide / polysaccharide derivative and other components is supported is preferred, and a carrier in which the oligosaccharide / polysaccharide derivative is supported is more preferred.

[0031] The above-mentioned loading method can be a known method, and can be either physical or chemical loading. For chemical loading, a method in which an oligosaccharide / polysaccharide derivative is loaded onto a support via a chemical bond such as a covalent bond can be preferably used. Specifically, a method can be used in which a reactive group is introduced into an oligosaccharide / polysaccharide derivative, a raw material for the oligosaccharide / polysaccharide derivative, or an intermediate for the oligosaccharide / polysaccharide derivative, and this substituent is reacted with a reactive group present on the support surface. In this case, a treatment to form a chemical bond between the oligosaccharide / polysaccharide derivative may be performed. The reactive group present on the support surface may be a group present on the surface of an untreated support, or a group introduced onto the support surface by surface treating the support with a surface treatment agent, such as a silane coupling agent such as 3-aminopropyltriethoxysilane or 3-glycidyloxypropyltrimethoxysilane. Another method can be used in which a so-called click reaction pair is bound to the stationary phase and the oligosaccharide / polysaccharide derivative, respectively, and then reacted with each other. Alternatively, an insolubilized layer can be formed on the surface of a carrier by forming a bond between the oligosaccharide / polysaccharide derivative and the carrier, or by forming a so-called cross-linking bond between atomic groups containing the oligosaccharide / polysaccharide derivative. Furthermore, as a physical support, the oligosaccharide / polysaccharide derivative may be supported on the carrier by, for example, impregnating silica gel with an oligosaccharide / polysaccharide derivative solution and evaporating the solution to physically adsorb (e.g., coat) the oligosaccharide / polysaccharide derivative onto the carrier. Among these methods, from the viewpoint of suppressing the dissolution of the oligosaccharide / polysaccharide derivative in the solvent, it is preferable to form a chemical bond between the silica gel and the oligosaccharide / polysaccharide derivative backbone, or to form a chemical bond between the oligosaccharide / polysaccharide derivative on the carrier. It should be noted that, in order to suppress the dissolution of the oligosaccharide / polysaccharide derivative in the solvent, it is preferable to form a chemical bond between the oligosaccharide / polysaccharide derivative even when a carrier is not used.

[0032] The carrier is not particularly limited as long as it can immobilize oligosaccharide / polysaccharide derivatives on the carrier. The carrier may be an inorganic carrier or an organic carrier, but an inorganic carrier is preferred. Examples of inorganic carriers include silica gel, alumina, magnesia, glass, kaolin, titanium oxide, silicate, zirconia, and hydroxyapatite. Examples of organic carriers include cross-linked polystyrene, cross-linked poly(meth)acrylamide, cross-linked poly(meth)acrylate, and polysaccharide. Hybrid silica gels in which a polyfunctional silane coupling agent is copolymerized when forming the silica gel can also be used. These organic carriers are preferably insolubilized by cross-linking with a cross-linking agent.

[0033] The shape of the carrier is not particularly limited, and examples thereof include particles and porous cylinders (monoliths) that are liquid-tightly accommodated in a column tube. The carrier can also be the inner wall of a capillary. When the separation factor is sufficiently large, a membrane-type carrier may be used and developed in the thickness direction, or it can be developed in the plane direction as in thin-layer chromatography.

[0034] In this embodiment, the carrier is preferably silica gel. This is because silica gel has the above-mentioned properties, i.e., excellent separation performance, and is hard and durable. As the silica gel, in addition to fully porous silica, so-called core-shell type silica gel may be used, or silica gel with a chemically modified surface may be used.

[0035] [Mobile Phase] The mobile phase contains one or more specific acids selected from the group consisting of oxalic acid, acetic acid, formic acid, and trifluoroacetic acid, one or more bases, and a solvent. The mobile phase may contain components other than the above components as long as the separation performance is not impaired. Furthermore, the mobile phase is preferably a homogeneous system (single-phase system) at least under the conditions for performing the analysis.

[0036] In the present disclosure, the term "mobile phase" refers to a liquid that, in chromatography such as liquid chromatography and supercritical fluid chromatography, is poured into a stationary phase together with the object to be separated, moves together with the object to be separated, and then elutes the separated product from the stationary phase.

[0037] The salt content in the mobile phase is not particularly limited, but from the viewpoint of ensuring the desired separation performance, it is preferably 0.5 mmol / L to 1000 mmol / L, more preferably 1 mmol / L to 200 mmol / L, even more preferably 1 mmol / L to 50 mmol / L, and particularly preferably 1 mmol / L to 20 mmol / L. As mentioned above, when mass spectrometry (MS) is employed, the content of high-boiling salts is preferably low. Examples of high-boiling salts include salts containing metal ions as cations or salts containing quaternary ammonium ions. Specifically, the content of the salt is preferably 5 mM or less, and more preferably substantially absent (below the detection limit).

[0038] (Specific Acid) The mobile phase contains one or more specific acids selected from the group consisting of oxalic acid, acetic acid, formic acid, and trifluoroacetic acid. Among these, it is preferable to contain at least one acid selected from the group consisting of oxalic acid and trifluoroacetic acid due to its relatively high acidity. Furthermore, unlike trifluoroacetic acid, which tends to contaminate other analyses by adsorbing to liquid chromatography components and requires careful attention to prevent fluorine contamination of the environment after disposal, oxalic acid does not have such a tendency, easily decomposes into low-molecular-weight carbon monoxide, carbon dioxide, and water at the temperature of the MS detector, and therefore does not adversely affect MS analysis. It is also particularly preferable to contain at least oxalic acid as the specific acid because it is easily obtained as a non-hygroscopic, easy-to-handle dihydrate crystal at low cost. These acids may be used alone or in combination of any two or more in any ratio. Furthermore, other acids may be contained within the range in which the effects of the present disclosure are obtained. Oxalic acid also has the advantage that it can be precipitated and separated using calcium ions when the eluate is separated. A specific acid contained in the mobile phase preferably provides counterions that promote retention of sample ammonium on the stationary phase in ion-pair chromatography by partially forming a salt, while also remaining partially as an acid to ensure conversion of the sample amine to ammonium ion. Because the sample amine is dilute under chromatographic conditions, there is a high probability that it will exist as an ammonium ion even if the mobile phase is nearly neutral. However, this can be ensured by using an excess of acid.

[0039] The content of the specific acid contained in the mobile phase (total content when two or more types are used in combination) is preferably 1 mmol / L or more, and preferably 1000 mmol / L or less, more preferably 200 mmol / L or less, even more preferably 50 mmol / L or less, and particularly preferably 20 mmol / L or less, from the viewpoint of improving the separation performance of amines. If the content is equal to or greater than the lower limit of the above range, a substantial effect of retaining the sample can be obtained (even at very low concentrations, some effect can be obtained, but a certain concentration is necessary to obtain practical effects). Furthermore, if the content is equal to or less than the upper limit of the above range, there is no adverse effect on detection, particularly detection by MS. In other words, preferred ranges of the content contained in the mobile phase include, for example, 1 to 1000 mmol / L, 1 to 200 mmol / L, 1 to 50 mmol / L, and 1 to 20 mmol / L.

[0040] Examples of acids other than the specific acids include methanesulfonic acid, nitric acid, phosphoric acid, glycolic acid, etc., and from the viewpoint of being less likely to form high-boiling, non-volatile salts, organic acids such as nitric acid and glycolic acid are preferred, and from the viewpoint of being less corrosive to the equipment, sulfonic acids, phosphoric acid, and organic acids are preferred. These acids other than the specific acids may be used alone or in combination of any two or more kinds in any ratio.

[0041] (Base) The mobile phase contains a base. To ensure retention in ion pair chromatography using a portion of the specific acid as the conjugate anion, a base is added at a molar concentration lower than that of the specific acid to neutralize a portion of the specific acid. Examples of this base include hydroxides or carbonates of metals or quaternary ammonium. The salt may be a salt of an acid weaker than the specific acid, and is preferably an amine. In the case of an amine, it dissociates into the specific acid and the amine by heat, and volatilizes, thereby not interfering with detection by mass spectrometry (MS). Note that since the mobile phase is in an acid-excess environment, the base basically exists as a salt in the mobile phase.

[0042] With respect to the content of the base in the mobile phase (total content when two or more types are used in combination), from the viewpoint of improving the separation performance of amines, the base is contained so as to satisfy the following condition: SB / SA is 0.05 or more and 0.95 or less, where A is the value calculated for each specific acid in the mobile phase by the following formula (1), SA is the total value of A calculated for each specific acid, B is the value calculated for the base in the mobile phase by the following formula (2), and SB is the total value of B calculated for each base. Formula (1): A = valence of specific acid × molar concentration of specific acid (mol / L) Formula (2): B = valence of base × molar concentration of base (mol / L) Note that the valence of acetic acid is 1, the valence of formic acid is 1, and the valence of trifluoroacetic acid is 1. The valence of oxalic acid can be 1 or 2, but since there is a difference of 10¢3 between the first-stage and second-stage dissociation constants, under the conditions of the present disclosure in which the amount of base is smaller than the number of moles of acid (<0.095), it is estimated that the second-stage dissociation occurs only to a negligible extent, and therefore in the present disclosure, it is treated as 1. As mentioned above, since the valence of all specific acids is 1, the above formula (1) can also be expressed as the following formula (1)'. Formula (1)': A = molar concentration of specific acid (mol / L)

[0043] The SB / SA is not particularly limited as long as it is 0.05 or more and 0.95 or less, but from the viewpoint of improving the separation performance of amines, it is preferably 0.1 or more, more preferably 0.2 or more, and even more preferably 0.4 or more, and from the viewpoint of reducing the acidity of the system and possibly preventing complete ionization of the sample amine, it is preferably 0.9 or less, more preferably 0.7 or less. That is, preferred ranges of the content in the mobile phase for monovalent acids include, for example, ranges of 0.1 to 0.9, 0.2 to 0.7, and 0.4 to 0.7.

[0044] For example, in the mobile phase, acetic acid (valence: 1) is present as a specific acid. １ mol / L, and triethylamine (valence: 1) is contained as a base. １ mol / L, A calculated by the above formula (1) is X １ mol / L (= 1 x X １) and B calculated by the above formula (2) is Y １ mol / L (= 1 x Y １ ) In addition, since there is only one type of acid and one type of base, SA = X １ , SB=Y １ Therefore, SB / SA = Y １ / X １ This becomes:

[0045] An acid having a lower degree of dissociation than the specific acid may be contained at any concentration. However, if an acid having a higher degree of dissociation than the specific acid is contained, this acid will preferentially consume the "base." Therefore, the SB / SA may be calculated by regarding the SB as (the above SB) - (valence of the acid having a higher degree of dissociation than the specific acid contained in the mobile phase) x (concentration (mol / L) of the acid having a higher degree of dissociation than the specific acid contained in the mobile phase). When performing this calculation, if the acid having a higher degree of dissociation than the specific acid is a polyvalent acid, the second term is multiplied by the charge up to the stage having a higher degree of dissociation than the "specific acid." The above dissociation degree is the degree of dissociation in water at 25°C. This dissociation degree is described, for example, in "pKa Data Compiled by R. Williams" published by the American Chemical Society.

[0046] Another embodiment of the present disclosure is an aspect in which the above SB / SA is set taking into consideration only the concentrations of a specific acid and base. In this aspect, the above formulas (1) and (2) are replaced with the following formulas (1)" and (2)", respectively. Other than the replacement of the formulas, the conditions of the above-mentioned embodiment can be similarly applied. Formula (1)": A = molar concentration of specific acid (mol / L) Formula (2)": B = molar concentration of base (mol / L)

[0047] Furthermore, in yet another embodiment of the present disclosure, the valence of oxalic acid is treated as 1 or 2. In this embodiment, when oxalic acid exists as a monovalent acid, the valence is 1, and when oxalic acid exists as a divalent acid, the valence is 2. Other than this, the conditions of the above-described embodiment can be similarly applied.

[0048] These two alternative embodiments differ from the above-described embodiment in terms of the valence of the acid and / or base, but can achieve the same effects as those achieved by the above-described embodiment.

[0049] The amine, which is a preferred embodiment of the base, will be described in detail below.

[0050] The type of amine is not particularly limited, and examples thereof include ammonia, primary amines, secondary amines, and tertiary amines, and is preferably at least one selected from the group consisting of ammonia, primary amines, secondary amines, and tertiary amines. The amine may be a single type or a combination of any two or more types in any ratio.

[0051] Examples of primary amines include primary amines having an alkyl group, and among these, preferred examples include monoalkylamines such as methylamine, ethylamine, n-propylamine, 2-propylamine, and butylamine.

[0052] Examples of secondary amines include secondary amines each independently having two alkyl groups, and among these, preferred examples include acyclic dialkylamines such as dimethylamine, diethylamine, diisopropylamine, and dibutylamine; and cyclic amines such as N-unsubstituted pyrrolidine and N-unsubstituted piperidine.

[0053] Examples of tertiary amines include tertiary amines each independently having three alkyl groups. Among these, preferred examples include acyclic trialkylamines such as trimethylamine, triethylamine, diisopropylethylamine, and tributylamine; and cyclic trialkylamines such as N-substituted pyrrolidine, N-substituted piperidine, and 1,4-diazabicyclo[2.2.2]octane.

[0054] The number of carbon atoms in the alkyl group bonded to each of the above amines is not particularly limited, but those with small molecular weights are prone to high volatility and difficulty in handling, and those with large molecular weights are prone to causing noise in MS detection. Therefore, the number of carbon atoms may be 1 or more, 2 or more, 3 or more, 10 or less, 8 or less, or 6 or less, with 1 to 4 being preferred, and 1 to 2 being more preferred. Primary to tertiary ammonium ions with a small number of carbon atoms in the alkyl group have small molecular weights, so when performing chromatography by LC-MS, ions with masses close to those of the amines to be separated are less likely to be generated, resulting in less noise. However, when analyzing samples with a molecular weight exceeding 200, triethylamine is convenient for ease of handling. On the other hand, when ammonia is used as a base, the solubility of its salts in organic solvents is generally low, so amines with alkyl substituents may be more advantageous.

[0055] Furthermore, the substituent bonded to the N-substituted pyrrolidine, N-substituted piperidine, or N,N-disubstituted pyrrolidine or N,N-disubstituted piperidine is not particularly limited, but is preferably an alkyl group, and the conditions for the alkyl group can be the same as those for the alkyl groups bonded to the above-mentioned amines. This is because, as described above, a smaller molecular weight of the ammonium ion is preferred in LC-MS.

[0056] The amine is preferably one or more selected from the group consisting of ammonia, ethylamine, n-propylamine, 2-propylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, pyrrolidine, and piperidine, for reasons such as its low boiling point, ease of availability, applicability to UV detection, volatility, and small molecular weight, making it less likely to interfere with MS detection, and more preferably one or more selected from the group consisting of ammonia, diethylamine, triethylamine, and 2-propylamine. Note that low-boiling-point amines such as ammonia have high vapor pressures, making it difficult to add them quantitatively. In such cases, an additive substantially equivalent to the addition of ammonia alone can be obtained by using a carbonate or bicarbonate of the amine and reacting it with the acid contained in the salt in a small amount of water to generate carbon dioxide.

[0057] When a metal is used as the base, for example, an alkali metal such as lithium, sodium, or potassium, or other metals can be used.

[0058] Although the method using a specific acid and a base that partially neutralizes the acid is the simplest method for preparing the mobile phase according to this embodiment, an equivalent mobile phase can also be obtained by adding a salt of the specific acid and the specific acid, and the preparation method is not limited to this method. Furthermore, the specific acid added here does not necessarily have to be the specific acid corresponding to the salt, and may be any one or more selected from the group consisting of oxalic acid, acetic acid, formic acid, and trifluoroacetic acid.

[0059] (Solvent) The solvent contained in the mobile phase in this embodiment is not particularly limited as long as it can dissolve the amines and various components to be separated, such as specific acids. Water or a liquid other than water (hereinafter, sometimes referred to as a "nonaqueous solvent") can be used. Examples of nonaqueous solvents include organic solvents (water content: 0% by volume), subcritical or supercritical carbon dioxide, etc. Examples of solvents contained in the mobile phase include water alone; nonaqueous solvent alone; mixed solvents of water and nonaqueous solvents, pressurized carbon dioxide and nonaqueous solvents, and mixed solvents containing trace amounts of water as needed. When the solvent contained in the mobile phase is a mixed solvent consisting of two or more solvents, it is preferable that the two or more solvents are a combination of solvents that do not cause phase separation under the amine separation conditions. Specifically, for example, a solvent with low polarity, a so-called normal phase solvent, can be used. A mixture of a hydrocarbon such as hexane or heptane with a more polar alcohol, ester, ether, nitrile, or halogenated hydrocarbon, or a mixture of these with water to the extent that it dissolves uniformly, is preferably used. More specifically, for example, solvents having a highly polar composition based on an organic solvent, such as those based on acetonitrile and containing a small amount (1 to 20% by volume) of water, those based on water and containing a small amount (usually 1 to 40% by volume) of acetonitrile or methanol as a more polar reverse-phase solvent, or pressurized carbon dioxide, commonly referred to as supercritical fluid chromatography, known as subcritical or supercritical carbon dioxide, can be used as the solvent. Note that in this technical field, "supercritical" does not generally refer to a solvent that is strictly in a supercritical state at all compositions and temperatures, and the term "supercritical" is often used when a solvent primarily composed of carbon dioxide is used. The term "supercritical" is used in the same manner in this disclosure.

[0060] The organic solvent is not particularly limited, but is preferably an organic solvent capable of dissolving the amine to be separated. Suitable organic solvents include, for example, acetonitrile, acetone, tetrahydrofuran (THF), tert-butyl methyl ether, methanol, ethanol, n-propanol, 2-propanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and dimethylacetamide (DMAc). Furthermore, as long as the organic solvent can dissolve various components, such as the amine to be separated and one or more specific acids selected from the group consisting of oxalic acid, acetic acid, formic acid, and trifluoroacetic acid, it may contain hydrocarbons such as hexane, hexane, or heptane; or halogenated hydrocarbons such as dichloromethane; etc. Among these, in terms of high chemical stability, the organic solvent preferably contains one or more selected from the group consisting of acetonitrile, acetone, methanol, ethanol, 2-propanol, hexane, heptane, THF, and tert-butyl methyl ether, and more preferably acetonitrile. It is particularly preferred that the solvent contained in the mobile phase contains 70% or more by volume of acetonitrile. The organic solvent may be one type alone or a combination of any two or more types in any ratio.

[0061] The organic solvent may be a mixture of a hydrocarbon having 5 to 8 carbon atoms and an alcohol, which is commonly used in chromatography. An example of such a mixture is a hexane / ethanol mixture, which is compatible with water. For example, an organic solvent with a hexane / ethanol mixture ratio of 100 / 100 (v / v) can be used to prepare a mixed solvent with a water content of approximately 10% by volume. Addition of water slightly reduces retention, but increases the peak plate number, improving the time efficiency of analysis. Hexane / ethanol / methanol also provides a good peak plate number within a compatible range.

[0062] A mixture of a hydrocarbon having 5 to 8 carbon atoms and an alcohol has low viscosity, which allows for a high flow rate at low pressure in chromatography. In addition, it has low ultraviolet absorption, which allows for the detection of amines by ultraviolet absorption.

[0063] When the non-aqueous solvent contains subcritical or supercritical carbon dioxide, the chromatography is performed using a supercritical fluid chromatograph. In this case, it is preferable to use a mixed solvent of water and an organic solvent, or an organic solvent (water content 0% by volume) in combination with the subcritical or supercritical carbon dioxide as an auxiliary solvent so that the target component can be dissolved at the desired concentration in the mobile phase. The organic solvent here has the same meaning as the organic solvent listed as the non-aqueous solvent. Preferred examples of the mixed solvent of water and an organic solvent as an auxiliary solvent and the organic solvent include a mixed solvent of water and methanol, and methanol, respectively. When the solvent contained in the mobile phase contains subcritical or supercritical carbon dioxide, the volume of the solvent is calculated based on the density of liquid carbon dioxide at -20°C, which is 1.030 g / mL, for convenience.

[0064] The types of solvent have been described above, but from the viewpoints of low viscosity, transparency to ultraviolet light (in the case of ultraviolet detection, which is most commonly used), non-high toxicity and danger, low cost, and tendency of chiral stationary phases made of polysaccharide derivatives to provide good separation, it is preferable to use one or more solvents selected from the group consisting of water, acetonitrile, ethanol, methanol, 2-propanol, hexane, heptane, THF, and liquid carbon dioxide.

[0065] The content of the solvent in the mobile phase (the total content when two or more types are contained) is not particularly limited and can be set appropriately depending on the application. For example, it may be 90% by mass or more, 95% by mass or more, 98% by mass or more, 99% by mass or more, or less than 100% by mass, 99.9% by mass or less, or 99.5% by mass or less. The mobile phase may also contain other components other than the specific acids and bases described above. Examples of the mixing ratios of each component when the solvent is a ternary mixture are described below. Examples of reverse-phase solvents include mixtures of acetonitrile, alcohols, and THF containing 50% by volume or more of water. To achieve good separation, a water content of 70% by volume or more is preferred. Examples of normal-phase solvents include mixtures containing 40% by volume or more of hexane and / or heptane, with the remainder being 2-propanol or ethanol, and a small amount of water.

[0066] (Other Components) The mobile phase may contain components (other components) other than the above-described components as long as the effects of the present disclosure are obtained. However, when detection is performed by mass spectrometry (MS), components that produce non-volatile salts are not preferred.

[0067] (Method for Preparing Mobile Phase) The mobile phase can be prepared by a known solution preparation method or a method based on a known solution preparation method, for example, by mixing the above-mentioned components.

[0068] A known method includes forming a salt from an acid and a base and using the salt to prepare a mobile phase. Although the term "acid and base" is used in this embodiment, both may be literally dissolved in appropriate amounts in a mobile phase solvent. Alternatively, substantially the same mobile phase can be obtained by dissolving the acid and a salt thereof in a mobile phase solvent. This method of preparing a mobile phase by contacting an acid and a base in a solvent, rather than directly contacting them, is advantageous in terms of simplicity and avoidance of problems caused by heat of neutralization, such as evaporation of some components. When a highly volatile amine, such as ammonia, is used as the base, a solvent such as water may be used, and the composition of the mobile phase solvent may be adjusted as necessary. Furthermore, as described above, a salt of a weak acid, such as a carbonate or bicarbonate, can also be used as a precursor. The weak acid by-product does not significantly affect retention, whether it remains in the mobile phase or is vaporized and evaporated.

[0069] If the salt of the cation and the acid anion is commercially available or readily available, the mobile phase can be prepared by dissolving the commercially available product in a solvent and, if necessary, mixing it with an acid to adjust the acidity. Adjusting the acidity becomes necessary when the retention of the sample amine is insufficient or the peak shape is undesirable.

[0070] [Chromatography] The chromatography used in this embodiment is not particularly limited, and examples thereof include liquid chromatography and supercritical fluid chromatography. The type of chromatography is also not particularly limited, and known types can be employed. Not only column chromatography, but also thin-layer chromatography, chromatography using a layer of deposited fibrous adsorbent, or chromatography using a capillary or microchannel in which a thin layer of adsorbent is provided in some form on the inner wall of a thin tube can be employed. Chromatography can be performed using commercially available chromatographs, such as liquid chromatographs and supercritical fluid chromatographs. Column equilibration conditions, flow rate, and other conditions can be appropriately selected depending on the column size, sample volume, type of mobile phase, and the like.

[0071] The amine separation method according to this embodiment may further include an analysis step of analyzing the amine separated in the separation step. The method for detecting the amine in chromatography is not particularly limited, but the method according to this embodiment may include a method using a mass spectrometer. In this embodiment, the formation of a volatile salt can be promoted by appropriately selecting an additive, and therefore, the ability to use a mass spectrometer as the analyzer is a major advantage.

[0072] The separation and analysis of amines may be performed using the same device, or may be performed using separate devices. For example, when liquid chromatography is used as the chromatography, amine separation and mass analysis of the separated amines can be performed consecutively by liquid chromatography-mass spectrometry (LC-MS), which combines liquid chromatography with mass spectrometry (MS). The mass analysis of amines may be for quantitative analysis of the separated amines or for qualitative analysis. A UV detector may be used alone, or even more information can be obtained by using a mass analyzer in combination.

[0073] For mass analysis, known mass analysis methods used in LC-MS can be employed. For example, the ionization method in mass analysis can be appropriately selected from atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), electrospray (ESI), fast atom bombardment (FAB), thermospray (TSP), etc., depending on the type of salt, type of acid, type of amine, and the purpose of analysis. In addition, the mass detector can be appropriately selected from quadrupole mass spectrometers (Q-MS), ion trap mass spectrometers (IT-MS), time-of-flight mass spectrometers (TOF-MS), etc., depending on the required sensitivity, resolution, etc.

[0074] The present disclosure will be explained in more detail below using examples, but the present disclosure is not limited to the following examples as long as it does not deviate from the gist of the disclosure.

[0075] <Experiment I-1> <Example 1> [Preparation of Mobile Phase] 1260 mg of oxalic acid dihydrate was weighed and transferred to a 1000 mL volumetric flask. Approximately 20 mL of a mixed solvent (80 / 20 by volume) was added to this volumetric flask, weighing out 800.0 g and 156.6 g of water and acetonitrile, respectively, and then 506 mg of triethylamine was added. The mixture was then filled to capacity with the water / acetonitrile (80 / 20 (v / v)) mixed solvent and stirred until no undissolved material remained, yielding a mobile phase. The solvent composition in the mobile phase was water / acetonitrile = 80 / 20 (v / v), and the oxalic acid (OXA) and triethylamine (TEA) contents in the mobile phase were 10.0 mmol / L and 5.0 mmol / L, respectively. The amount of water contained in oxalic acid dihydrate was small, so there was no effect on the solvent composition. Furthermore, 100 μL of the mobile phase was dropped onto a stainless steel plate heated to 200° C., but no residue was observed. This result was similar to that observed in the other examples below.

[0076] Similarly, for the preparation of the mobile phase below, the solvent was weighed to the nearest 0.1 g, and the additives were weighed to the nearest 1 mg, and the concentrations were calculated based on this. The density of water was 1.00 g / mL, and the density of acetonitrile was 0.783 g / mL.

[0077] [Amine Separation and Analysis] A column packed with a chiral stationary phase of cellulose tris(3,5-dimethylphenylcarbamate) supported on silica gel (CHIRALPAK IB-N (manufactured by Daicel Corporation, registered trademark), average packing diameter 5 μm, length 250 mm, inner diameter 4.6 mmφ) was used, and amines were separated using a high-performance liquid chromatograph. The amine to be separated, represented by the following formula (3) ("Pindolol" manufactured by USP), was dissolved in a water / acetonitrile (1 / 1 (v / v)) mixed solvent to a concentration of approximately 1 mg / mL, and 2 μL of the resulting solution was injected into the column using an autosampler. The mobile phase was pumped through the column at a flow rate of 0.43 mL / min, the temperature of which was adjusted to 30°C. The details of the amine separation and analysis methods are described below.

[0078] (Separation Method / Analysis Method) An SPD-M20A (Shimadzu Corporation, diode array UV-Vis detector) was used as the detector for high-performance liquid chromatography (HPLC). After pumping the mobile phase through the column at 1.0 mL / min for at least one hour, 5 μL of a sample solution prepared by dissolving the sample in the mobile phase at approximately 1 mg / mL was injected using an autosampler. The column temperature was set to 30°C at the same flow rate, and amines were separated and analyzed. Details of the equipment used in the HPLC and data processing device are shown below. All of the following equipment was manufactured by Shimadzu Corporation. HPLC equipment: system controller CBM-20A, pump LC-20AD, autosampler SIL-20AC, column oven SPD-M20A. Data processing device: LabSolutions Version 5.97. The results are shown in Figure 1.

[0079]

[0080] Comparative Example 1 Separation and analysis of the amine represented by formula (3) was again carried out in the same manner as in Example 1, except that a water / acetonitrile (80 / 20 (v / v)) mixed solvent was used as the mobile phase through the column after separation and analysis in Example 1, and the solution was passed through the column for one hour or more without sample injection. Note that, because no acid or amine was added to the mobile phase used, the acid and amine contents in the mobile phase were 0 mmol / L and 0 mmol / L, respectively. The results are shown in Figure 2.

[0081] Comparative Example 2 The same procedure as in Example 1 was carried out, except that oxalic acid dihydrate was not used and that ammonium bicarbonate (AHC) and diethylamine (DEA) were used instead of triethylamine, to prepare a mobile phase containing 10.0 mmol / L of AHC and 15.5 mmol / L of DEA, respectively. Separation and analysis of the amine represented by formula (3) were carried out in the same manner as in Example 1, except that the above mobile phase was used and a guard column described in the footnote of Table 1 was inserted before the CHIRALPAK IB-N column (this comparative example only). The results are shown in Figure 3.

[0082] Comparative Example 3 Separation and analysis of the amine represented by formula (3) was carried out in the same manner as in Example 1, except that a water / acetonitrile (80 / 20 (v / v)) mixed solvent was used as the mobile phase through the column after separation and analysis in Comparative Example 2, and the solution was passed through the column for one hour or more without sample injection. Note that, because no acid or amine was added to the mobile phase used, the acid and amine contents in the mobile phase were 0 mmol / L and 0 mmol / L, respectively. The results are shown in FIG. 4.

[0083] Example 2 The same procedure as in Example 1 was carried out, except that the solvent was changed from water and acetonitrile to hexane and ethanol (hexane / ethanol = 50 / 50 (v / v)), and a mobile phase containing OXA and TEA at concentrations of 10.0 mmol / L and 5.0 mmol / L, respectively, was prepared. Separation and analysis of the amine represented by formula (3) were carried out in the same manner as in Example 1, except that the above mobile phase was used. The results are shown in Figure 5.

[0084] Comparative Example 4 A mobile phase with a DEA content of 10.0 mmol / L was prepared by the same procedure as in Example 2, except that oxalic acid dihydrate was not used and that diethylamine ("DEA" in Table 1) was used instead of triethylamine. Separation and analysis of the amine represented by formula (3) were carried out in the same manner as in Example 1, except that the above mobile phase was used as the mobile phase. The results are shown in Figure 6.

[0085] Example 3 A column packed with a chiral stationary phase in which cellulose tris(3-chloro-4-methylphenylcarbamate) was supported on silica gel (CHIRALPAK IM (registered trademark, manufactured by Daicel Corporation), average packing diameter 5 μm, length 250 mm, inner diameter 4.6 mmφ) was used, and the amine to be analyzed was separated and analyzed in the same manner as in Example 2, except that the amine to be analyzed was changed to an amine represented by the following formula (4). The results are shown in FIG. 7.

[0086]

[0087] Comparative Example 5 The amine represented by formula (4) was separated and analyzed in the same manner as in Comparative Example 4, except that the column used in Example 3 was used. The results are shown in Figure 8.

[0088] Example 4 The amine to be analyzed was separated and analyzed in the same manner as in Example 2, except that the object to be analyzed was changed to the amide of phenylglycine represented by the following formula (5): C ６ H ５ -CH(NH ２ )-CONH ２ (Tokyo Chemical Industry Co., Ltd.) (5) The results are shown in FIG.

[0089] Comparative Example 6 Separation and analysis of the amine to be analyzed were carried out in the same manner as in Comparative Example 5, except that the object to be analyzed was changed to the amide of phenylglycine represented by the above formula (5). The results are shown in Figure 10.

[0090] Example 5 The same procedure as in Example 3 was carried out, except that the solvent was changed from hexane and ethanol to hexane, ethanol, and methanol (hexane / ethanol / methanol = 70 / 20 / 19 (v / v / v)), and a mobile phase containing OXA and TEA at concentrations of 10.0 mmol / L and 5.0 mmol / L, respectively, was prepared. Separation and analysis of the amine represented by formula (4) was carried out in the same manner as in Example 3, except that the above mobile phase was used and the column used was a chiral stationary phase containing amylose tris(3-chlorophenylcarbamate) supported on silica gel (CHIRALPAK ID (manufactured by Daicel Corporation, registered trademark), average packing diameter 5 μm, length 250 mm, inner diameter 4.6 mmφ). The results are shown in FIG. 11 .

[0091] Comparative Example 7 A mobile phase containing 0 mmol / L of OXA and 0 mmol / L of TEA was prepared by the same procedure as in Example 5, except that oxalic acid dihydrate and triethylamine were not added. Separation and analysis of the amine represented by formula (4) were carried out in the same manner as in Example 5, except that the above mobile phase was used. The results are shown in Figure 12.

[0092] Example 6 Separation and analysis of the amine represented by formula (4) was carried out in the same manner as in Example 5, except that a column packed with a chiral stationary phase in which amylose tris(3,5-dichlorophenylcarbamate) was supported on silica gel (CHIRALPAK IE (registered trademark, manufactured by Daicel Corporation), average packing diameter 5 μm, length 250 mm, inner diameter 4.6 mmφ) was used. Separation and analysis of the amine represented by formula (4) was carried out in the same manner as in Example 5, except that the mobile phase was used. The results are shown in FIG. 13.

[0093] Comparative Example 8 A mobile phase containing 0 mmol / L of OXA and 0 mmol / L of TEA was prepared by the same procedure as in Example 6, except that oxalic acid dihydrate and triethylamine were not added. Separation and analysis of the amine represented by formula (4) were carried out in the same manner as in Example 6, except that the above mobile phase was used. The results are shown in Figure 14.

[0094] The separation conditions for Examples 1 to 4 and Comparative Examples 1 to 6 are summarized in Table 1 below.

[0095] (Resolution) The resolution in each example and comparative example is a USP resolution automatically calculated using LabSolutions Version 5.97, a chromatography data analysis program from Shimadzu Corporation. The results are shown in Table 1.

[0096] In the following tables, "AN" stands for acetonitrile, "OxA" stands for oxalic acid, "TEA" stands for triethylamine, "AHC" stands for ammonium bicarbonate, and "DEA" stands for diethylamine.

[0097] The above-mentioned SB / SA values ​​are shown in the following Tables 1 to 5. Note that, when calculated from the degree of dissociation, most of the oxalic acid exists as a monovalent acid.

[0098]

[0099] By comparing Example 1 with Comparative Examples 1 and 3, Example 5 with Comparative Example 7, and Example 6 with Comparative Example 8, it can be seen that even when the same solvent is used, if nothing is added, peak tailing is generally severe and the degree of resolution is low, or the chromatogram obtained is unstable due to the column history before and after (Comparative Examples 1 and 3 use the same mobile phase, but are sandwiched between Example 1 and Comparative Example 2, respectively), whereas stable retention and separation can be obtained by using the mobile phase according to an embodiment of the present disclosure.

[0100] Furthermore, compared to the mobile phase of Comparative Example 9, which contains only acid, Example 2, which uses a partially neutralized mobile phase, achieves good separation. Specifically, Comparative Example 9 uses a mobile phase containing only oxalic acid, but the peak shape and separation are clearly poorer than those of Example 2.

[0101] Furthermore, in the chromatography of amine samples, a method of adding a low-molecular-weight amine to the mobile phase is often employed. Silica gel, which is often used as a chromatography packing or carrier, contains acidic atomic groups called silanols, which are thought to form salts with the sample, causing significant peak tailing. In this case, adding an amine can prevent the sample from forming salts with silanols, thereby suppressing abnormal sample retention. Comparative Examples 4, 5, and 6 were prepared by adding diethylamine, which is often used for this purpose. Comparative Examples 4, 5, and 6 correspond to Examples 2, 3, and 4, respectively. While peak tailing was almost nonexistent in Comparative Examples 4 and 5, the resolution was lower than in Examples 2 and 3, respectively.

[0102] In addition, a conventional method for aqueous-based mobile phases involves shifting the mobile phase to an alkaline state to suppress protonation of amine samples and retain them as neutral molecules in the stationary phase. Comparative Example 2 is one such example, using an alkaline mobile phase. In this case, the peak width is larger and the time efficiency of separation is poorer than in Example 1. Furthermore, when using a more basic mobile phase with a stationary phase containing silica gel, it is necessary to prevent column deterioration by inserting a guard column (pre-column) containing silica gel, and this comparative example employs the same technique. However, the peak shape became abnormal in Comparative Example 3 after this analysis, indicating that the column had a history.

[0103] <Experiment I-2> <Comparative Example 9> A mobile phase was prepared in the same manner as in Example 1, except that the solvent was changed from a water / acetonitrile = 80 / 20 (v / v) mixed solution to a water / acetonitrile = 2.5 / 97.5 (v / v) mixed solution, and trifluoroacetic acid (TFA) was used instead of OxA so that the TFA content in the mobile phase was 10.0 mol / L, and TEA was not used. Separation and analysis of the amine represented by formula (3) above were carried out in the same manner as in Example 1, except that the above mobile phase was used as the mobile phase. The results are shown in Figure 15.

[0104] Example 7 A mobile phase (TEA content: 5.0 mol / L) was prepared in the same manner as in Example 1, except that the solvent was changed from a 50 / 50 (v / v) water / acetonitrile mixed solution to a 2.5 / 97.5 (v / v) water / acetonitrile mixed solution, and trifluoroacetic acid (TFA) was used instead of OxA so that the TFA content in the mobile phase was 10.0 mol / L. Separation and analysis of the amine represented by formula (3) were carried out in the same manner as in Example 1, except that the above mobile phase was used as the mobile phase. The results are shown in FIG. 16 .

[0105] Comparative Example 10 A mobile phase was prepared in the same manner as in Comparative Example 9, except that the solvent was changed from a water / acetonitrile = 2.5 / 97.5 (v / v) mixed solution to a hexane / ethanol = 50 / 50 (v / v) mixed solution. Separation and analysis of the amine represented by formula (3) above were carried out in the same manner as in Example 1, except that the above mobile phase was used as the mobile phase. The results are shown in Figure 17.

[0106] Example 8 A mobile phase was prepared in the same manner as in Example 7, except that the solvent was changed from a water / acetonitrile = 2.5 / 97.5 (v / v) mixed solution to a hexane / ethanol = 50 / 50 (v / v) mixed solution. Separation and analysis of the amine represented by formula (3) above were carried out in the same manner as in Example 1, except that the above mobile phase was used. The results are shown in Figure 18.

[0107] The separation conditions for Examples 7 and 8 and Comparative Examples 9 and 10 are summarized in Table 2 below.

[0108] (Resolution) The resolution in each example and comparative example is a USP resolution automatically calculated using LabSolutions Version 5.97, a chromatography data analysis program from Shimadzu Corporation. The results are shown in Table 1.

[0109] In the following tables, "TFA" stands for trifluoroacetic acid.

[0110]

[0111] By comparing Example 7 with Comparative Example 9, and Example 8 with Comparative Example 10, it can be seen that the degree of separation is improved when SB / SA is 0.05 or more and 0.95 or less.

[0112] <Experiment I-3> <Comparative Example 11> A mobile phase was prepared in the same manner as in Example 1, except that AcOH was used instead of OxA so that the content of acetic acid (AcOH) in the mobile phase was 10.0 mol / L, and TEA was not used. Separation and analysis of the amine represented by formula (3) above were carried out in the same manner as in Example 1, except that the above mobile phase was used as the mobile phase. The results are shown in Figure 19.

[0113] Example 9 A mobile phase (TEA content: 5.0 mol / L) was prepared in the same manner as in Example 1, except that AcOH was used instead of OxA so that the AcOH content in the mobile phase was 10.0 mol / L. Separation and analysis of the amine represented by formula (3) were carried out in the same manner as in Example 1, except that the above mobile phase was used. The results are shown in Figure 20.

[0114] Example 10: Ammonium acetate (NH ４ OAc) was replaced with NH 4 in place of TEA so that the content was 5.0 mol / L. ４ A mobile phase was prepared in the same manner as in Example 9, except that OAc was used. Separation and analysis of the amine represented by formula (3) were carried out in the same manner as in Example 1, except that the above mobile phase was used. The results are shown in Figure 21.

[0115] Example 11 A mobile phase was prepared in the same manner as in Example 9, except that the solvent was changed from a water / acetonitrile = 80 / 20 (v / v) mixed solution to a hexane / ethanol = 50 / 50 (v / v) mixed solution. Separation and analysis of the amine represented by formula (3) above were carried out in the same manner as in Example 1, except that the above mobile phase was used. The results are shown in Figure 22.

[0116] Example 12 A mobile phase was prepared in the same manner as in Example 10, except that the solvent was changed from a water / acetonitrile = 80 / 20 (v / v) mixed solution to a hexane / ethanol = 50 / 50 (v / v) mixed solution. Separation and analysis of the amine represented by formula (3) above were carried out in the same manner as in Example 1, except that the above mobile phase was used as the mobile phase. The results are shown in Figure 23.

[0117] Example 13 The amine to be analyzed was separated and analyzed in the same manner as in Example 12, except that the amine to be analyzed was changed to an amine represented by the following formula (6) (1-phenyl-1,2,3,4-tetrahydroisoquinoline). The results are shown in Figure 24.

[0118]

[0119] Example 14: AcOH was used so that the content of AcOH in the mobile phase was 10.0 mol / L. ４ NH so that the OAc content was 10.0 mol / L. ４ A mobile phase was prepared in the same manner as in Example 12, except that OAc was used. Separation and analysis of the amine represented by formula (3) were carried out in the same manner as in Example 1, except that the above mobile phase was used. The results are shown in Figure 25.

[0120] The separation conditions for Examples 9 to 14 and Comparative Example 11 are summarized in Table 3 below.

[0121] (Resolution) The resolution in each example and comparative example is a USP resolution automatically calculated using LabSolutions Version 5.97, a chromatography data analysis program from Shimadzu Corporation. The results are shown in Table 3.

[0122] In the following tables, "AcOH" means acetic acid, "NH ４ "OAc" stands for ammonium acetate, and "Ph-β-isoq" stands for phenyl-β-isoquinoline.

[0123] In Examples 10, 12, 13, and 14, ammonium acetate was added as a base instead of ammonia. Therefore, in addition to the acetic acid added as an acid, the mobile phase also contains acetic acid generated by dissociation of ammonium acetate. Therefore, when a molar ratio of acetic acid to ammonium acetate is used at 1:1, the molar ratio of acetic acid to ammonia in the mobile phase is 2:1, and the SB / SA is 0.5.

[0124]

[0125] By comparing Examples 9 to 14 with Comparative Example 11, it can be seen that the degree of separation is improved when SB / SA is 0.05 or more and 0.95 or less.

[0126] <Experiment I-4> <Comparative Example 12> A mobile phase was prepared in the same manner as in Example 1, except that the solvent was changed from a mixed solution of water / acetonitrile = 80 / 20 (v / v) to a mixed solution of hexane / ethanol = 50 / 50 (v / v), and that formic acid (FA) was used instead of OxA so that the content of FA in the mobile phase was 10.1 mol / L, and TEA was not used. Separation and analysis of the amine represented by formula (3) above were carried out in the same manner as in Example 1, except that the above mobile phase was used as the mobile phase. The results are shown in Figure 26.

[0127] Example 15 A mobile phase (TEA content: 5.0 mol / L) was prepared in the same manner as in Example 1, except that the solvent was changed from a water / acetonitrile = 80 / 20 (v / v) mixed solution to a hexane / ethanol = 50 / 50 (v / v) mixed solution, and formic acid (FA) was used instead of OxA so that the FA content in the mobile phase was 10.0 mol / L. Separation and analysis of the amine represented by formula (3) were carried out in the same manner as in Example 1, except that the above mobile phase was used as the mobile phase. The results are shown in Figure 27.

[0128] Comparative Example 13 The amine to be analyzed was separated and analyzed in the same manner as in Comparative Example 12, except that the amine to be analyzed was changed to the amine represented by the above formula (6) (phenyl-β-isoquinoline). The results are shown in Figure 28.

[0129] Example 16 The amine to be analyzed was separated and analyzed in the same manner as in Example 15, except that the amine to be analyzed was changed to the amine represented by the above formula (6) (phenyl-β-isoquinoline). The results are shown in Figure 29.

[0130] The separation conditions for Examples 15 and 16 and Comparative Examples 12 and 13 are summarized in Table 4 below.

[0131] (Resolution) The resolution in each example and comparative example is a USP resolution automatically calculated using LabSolutions Version 5.97, a chromatography data analysis program from Shimadzu Corporation. The results are shown in Table 4.

[0132] In the tables below, "FA" stands for formic acid.

[0133]

[0134] By comparing Example 15 with Comparative Example 12 and Example 16 with Comparative Example 13, it can be seen that the degree of separation is improved when SB / SA is 0.05 or more and 0.95 or less.

[0135] <Experiment I-5> <Comparative Example 14> A mobile phase containing 10.0 mmol / L of OxA was prepared by the same procedure as in Example 2, except that TEA was not added. Separation and analysis of the amine represented by formula (3) were carried out in the same manner as in Example 2, except that the above mobile phase was used. The results are shown in Figure 30.

[0136] Example 17 A mobile phase was prepared in the same manner as in Example 2, except that OxA was used so that the OxA content in the mobile phase was 2.50 mol / L, and TEA was used so that the TEA content in the mobile phase was 1.25 mol / L. Separation and analysis of the amine represented by formula (3) were carried out in the same manner as in Example 1, except that the above mobile phase was used as the mobile phase. The results are shown in Figure 31.

[0137] Example 18 A mobile phase was prepared in the same manner as in Example 2, except that OxA was used so that the OxA content in the mobile phase was 5.0 mol / L, and TEA was used so that the TEA content in the mobile phase was 2.5 mol / L. Separation and analysis of the amine represented by formula (3) were carried out in the same manner as in Example 1, except that the above mobile phase was used as the mobile phase. The results are shown in Figure 32.

[0138] Example 19 The same procedure as in Example 1 was carried out, except that the solvent was changed from a mixed solution of water and acetonitrile to a mixed solution of hexane, ethanol, and water (hexane / ethanol / water=50 / 50 / 2 (v / v / v)), and a mobile phase containing OXA and TEA at concentrations of 10.0 mmol / L and 5.0 mmol / L, respectively, was prepared. Separation and analysis of the amine represented by formula (3) were carried out in the same manner as in Example 1, except that the above mobile phase was used. The results are shown in FIG. 33.

[0139] The separation conditions for Examples 17 to 19 and Comparative Example 14 are summarized in Table 5 below.

[0140] (Resolution) The resolution in each example and comparative example is a USP resolution automatically calculated using LabSolutions Version 5.97, a chromatography data analysis program from Shimadzu Corporation. The results are shown in Table 5.

[0141]

[0142] By comparing Examples 17 and 18 with Comparative Example 14, it can be seen that the degree of separation is improved when SB / SA is 0.05 or more and 0.95 or less.

[0143] <Experiment II> <Example 20> [Preparation of mobile phase] A mobile phase was prepared in the same manner as in Example 1 above.

[0144] [Separation of Amine] Separation and analysis of the amine represented by the above formula (3) were carried out in the same manner as in Example 1, except that the following method was used for separation and analysis of the amine.

[0145] (Separation Method / Analysis Method) An ACQUITY Qda (a mass detector manufactured by Nihon Waters Co., Ltd.) was used as the detector for high-performance liquid chromatography (HPLC). After pumping the mobile phase through the column at 0.75 mL / min for 1 hour, 1 μL of sample solution prepared by dissolving the sample in the mobile phase at approximately 1-2 mg / mL (filtered) was injected using an autosampler. The column temperature was set to room temperature (approximately 25°C) at the same flow rate, and amine separation and analysis were performed. Details of the equipment used for HPLC are as follows: HPLC equipment: ACQUITY UPLC H-Class PLUS; Ionization method: ESI; Measurement mode: Positive; Probe temperature: 400°C; Source temperature: 120°C. The results are shown in Figure 34. The vertical axis of the graph in the figure represents the maximum value of the highest peak as 100%. The same applies to the following Figures 35 to 38. The detected mass m / z (value obtained by dividing the relative mass of the ion by its charge) obtained by the above analysis is shown in Table 6 below.

[0146] Example 21 A mobile phase was prepared in the same manner as in Example 1. Separation and analysis of the amine to be analyzed were carried out in the same manner as in Example 20, except that the method shown in Example 20 was used for separation and analysis of the amine, and the analyte was changed to the amine represented by formula (6) above (1-phenyl-1,2,3,4-tetrahydroisoquinoline). The results are shown in Figure 35 and Table 6.

[0147] Example 22 A mobile phase was prepared in the same manner as in Example 2. Separation and analysis of the amine represented by formula (3) was carried out in the same manner as in Example 2, except that the method shown in Example 20 was used for amine separation and analysis. The results are shown in Figure 36 and Table 6.

[0148] Example 23 A mobile phase was prepared in the same manner as in Example 3. Separation and analysis of the amine represented by formula (3) was carried out in the same manner as in Example 3, except that the method shown in Example 20 was used for separation and analysis of the amine. The results are shown in Figure 37 and Table 6.

[0149] Example 24 A mobile phase was prepared in the same manner as in Example 3. Separation and analysis of amines were carried out in the same manner as in Example 3, except that the method shown in Example 20 was used for separation and analysis, and the analyte was changed to the amine represented by the following formula (7) (nicardipine). The results are shown in Figure 38 and Table 6.

[0150]

[0151] The above-mentioned SB / SA is shown in Table 2 below. In calculating the SB / SA, the valence of oxalic acid was set to 1 because most of the oxalic acid exists as a monovalent acid.

[0152]

[0153] As is clear from Figures 34 to 38 and Table 6, good mass chromatograms (chromatograms obtained with a mass detector) were obtained. This indicates that the method according to the embodiment of the present disclosure is not only effective for separating amines, particularly their enantiomers, but also suitable for use in a detection method known as LC-MS. LC-MS is a technique that has seen increasing use in recent years because it can distinguish between multiple overlapping elution peaks in chromatography based on mass, and the ability to apply this method is of great industrial significance.

[0154] From the above, it can be seen that the amine separation method according to the embodiment of the present disclosure can improve the separation performance of amines. Furthermore, by appropriately selecting components contained in the mobile phase that can avoid or reduce adverse effects on detection by a mass spectrometer, it is believed that the method can also be applied to LC-MS. Therefore, the amine separation method can be widely applied to analysis and purification by various liquid chromatography methods, and is expected to be used in fields such as organic chemistry, medicine, and pharmacology.

Claims

1. A method for separating amines, comprising: a stationary phase containing at least one of oligosaccharides, polysaccharides, and derivatives thereof; and a mobile phase containing one or more specific acids selected from the group consisting of oxalic acid, acetic acid, formic acid, and trifluoroacetic acid, one or more bases, and a solvent, wherein, when a value calculated for each specific acid contained in the mobile phase by the following formula (1) is A, the total value of A calculated for each specific acid is SA, a value calculated for each base contained in the mobile phase by the following formula (2) is B, and the total value of B calculated for each base is SB, SB / SA is 0.05 or more and 0.95 or less. Formula (1): A = valence of specific acid × molar concentration of specific acid (mol / L) Formula (2): B = valence of base × molar concentration of base (mol / L) 2. The method for separating an amine according to claim 1, further comprising an analysis step of analyzing the amine separated in the separation step, wherein the analysis step involves analysis using at least a mass spectrometer.

3. The method for separating amines according to claim 1 or 2, wherein the base is one or more selected from the group consisting of ammonia, primary amines, secondary amines, and tertiary amines.

4. The method for separating amines according to claim 3, wherein the base is one or more selected from the group consisting of ammonia, ethylamine, n-propylamine, 2-propylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, pyrrolidine, and piperidine.

5. The method for separating amines according to claim 1 or 2, wherein the mobile phase contains at least oxalic acid as the specific acid.

6. The method for separating amines according to claim 1 or 2, wherein at least one of the oligosaccharides, polysaccharides, and derivatives thereof has an optically active site.

7. The method for separating amines according to claim 1 or 2, wherein the solvent is at least one selected from the group consisting of water, acetonitrile, ethanol, methanol, 2-propanol, hexane, heptane, THF, and liquid carbon dioxide.

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