JP2018124210A - Method for analyzing test object - Google Patents

Abstract

PROBLEM TO BE SOLVED: To separate an amphoteric compound by reverse-phase liquid chromatography without using an ion pair reagent.SOLUTION: The present invention provides a method for purifying an amphoteric compound with a molecular weight of 250 or more by reverse-phase liquid chromatography, where the reverse-phase liquid chromatography is performed using a phenylalkyl column or pentafluorophenylalkyl column, but not using an ion pair reagent.SELECTED DRAWING: Figure 1

Description

The present invention relates to a method for separating amphoteric compounds.

Since amphoteric compounds have both an acidic functional group and a basic functional group in the molecule, their polarity is high, and when they are to be separated by reverse phase liquid chromatography, they are not retained on the column and are difficult to separate.

As a method for analyzing highly polar components, a method is known in which an ion pair reagent (alkyl sulfonate, organic ammonium salt, etc.) is added to the mobile phase to form an ion pair with the compound to be separated, thereby increasing the retention force on the column. (Non-Patent Document 1)
However, since the ion pair reagent is a contaminant in mass spectrometry and further remains in the mass spectrometer, it also appears as a contaminant in subsequent measurements. For this reason, it was difficult to perform LC-MS (liquid chromatography-mass spectrometer) by the conventional method using an ion pair reagent.

As a method not using an ion-pair reagent, a method using an ethylene-bridged hydrophilic interaction chromatography column (Patent Document 1), a method using a separating agent in which a polysaccharide such as cellulose or amylose is bound to the surface of a carrier (Patent Document 2) ), A method using a polyfunctional stationary phase and a weakly alkaline mobile phase (Patent Document 3) is known.
In order to increase the retention time in the column, a method of decreasing the flow rate of the mobile phase is known, but generally, when the flow rate of the mobile phase is decreased, the peak widens, resulting in a decrease in sensitivity and a decrease in resolution.

JP2011-95165A International Publication No. 2012/033194 JP2009-222421

Anal. Chem. 2006, 78, 6573-6582.

An object of the present invention is to separate an amphoteric compound by reversed-phase liquid chromatography without adding an ion pair reagent to the mobile phase.

As a result of intensive studies on the above problems, the present inventors have found that amphoteric compounds having a molecular weight of 250 or more can be separated and analyzed by reverse phase chromatography using a phenylalkyl column or a pentafluorophenylalkyl column. I found it. A phenylalkyl column or a pentafluorophenylalkyl column and an amphoteric compound having a molecular weight of 250 or more have a high affinity, and without using an ion-pairing reagent, which has been required for the separation analysis of the amphoteric compound, without reducing the flow rate of the mobile phase. A retention time that allows separation analysis can be achieved.
The gist of the present invention is as follows.
[1] A method for purifying an amphoteric compound having a molecular weight of 250 or more by reverse phase liquid chromatography, wherein the reverse phase liquid chromatography is carried out using a phenylalkyl column or a pentafluorophenylalkyl column, and no ion-pairing reagent is used. The method as described above.
[2] The method according to [1], wherein the column according to [1] uses a core-shell column.
[3] The method according to [1], wherein the column according to [1] is a phenylhexyl column or a pentafluorophenylpropyl column.
[4] The method according to any one of [1] to [3], wherein an aqueous solvent to which a volatile acidic substance is added is used as the mobile phase.
[5] The method according to [4], wherein trifluoroacetic acid is used as the acidic substance.

According to the present invention, an amphoteric compound having a molecular weight of 250 or more can be separated and analyzed without the need for adding an ion pair reagent. Although the ion pair reagent becomes a contaminant in mass spectrometry, the analysis method according to the present invention does not require an ion pair reagent, and the separated sample can be used for mass spectrometry as it is.

It is a conceptual diagram for demonstrating a symmetry coefficient (S).

Hereinafter, one embodiment of the present invention will be described in detail.
In the present invention, the amphoteric compound has both an acidic functional group and a basic functional group in the molecule. The acidic functional group means a functional group having a property of ion exchange of hydrogen contained in the functional group, such as a carboxylic acid group, a sulfonic acid group, and a phosphoric acid group. The basic functional group means a functional group having a property of receiving hydrogen ions in a molecule, such as an amino group, a quaternary ammonium salt, and an amide group.

Examples of amphoteric compounds include ofloxacin, levofloxacin, garenoxacin, moxifloxacin, tosufloxacin, ciprofloxacin, sitafloxacin, pullrifloxacin, pazufloxacin, or 7-[(3S, 4S) -3-{(cyclopropylamino ) Quinolone compounds such as methyl} -4-fluoropyrrolidin-1-yl] -6-fluoro-1- (2-fluoroethyl) -8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid , Aztreonam, cephalexin, fexofenadine, droxidopa, mesalazine and the like, preferably a quinolone compound.
In addition, an amphoteric compound having a molecular weight of 250 or more is preferable in that it has a high affinity for a phenylalkyl column or a pentafluorophenylalkyl column. Examples of amphoteric compounds having a molecular weight of 250 or more include quinolone compounds such as aztreonam, cephalexin, fexofenadine and ofloxacin. More preferable molecular weight is 250 or more and 600 or less, further preferably 300 or more and 550 or less, and particularly preferably 330 or more and 500 or less.

Furthermore, an amphoteric compound having a molecular weight of 250 or more having a carboxylic acid group and an amino group in the molecule is preferred in that it has a high affinity for a phenylalkyl column or a pentafluorophenylalkyl column.

In the present invention, the core-shell column means a column in which particles having a porous membrane bonded are packed on the surface of a non-porous nucleus such as non-porous silica. In the core-shell column, the sample molecules do not diffuse in the core portion, so that the intra-particle diffusion distance becomes short. The core-shell column is preferable in that it realizes a high theoretical plate number and has high detection sensitivity, compared to a fully porous column.
In the present invention, the phenylalkyl column is a column using a filler in which a phenyl group is chemically bonded to silica gel via an alkyl group having 1 to 6 carbon atoms. Phenylalkyl column, phenylethyl column using a filler in which phenylethyl group is chemically bonded to silica gel, phenylpropyl column using a filler in which phenylpropyl group is chemically bonded to silica gel, phenylbutyl group is chemically bonded to silica gel Phenyl butyl column using a filler, phenyl pentyl column using a filler in which a phenyl pentyl group is chemically bonded to silica gel, phenyl hexyl column using a filler in which a phenyl hexyl group is chemically bonded to silica gel, etc. A phenylhexyl column is preferred.

As a phenylhexyl column, for example, a Kinex mini-bore column (Phenyl-Hexyl), a Kinetex mid-bore column (Phenyl-Hexyl), a Kinetex analytical column (Phenyl-Hexyl Cyl-HylIT), an Ascentis Express Phenyl-Hexyl HQ CSH Phenyl-Hexyl Column etc. are mentioned.
Among phenylhexyl columns, a core column such as a Kinetex mini-bore column (Phenyl-Hexyl), a Kinetex mid-bore column (Phenyl-Hexyl), a Kinetex analysis column (Phenyl-Hexyl), an Ascentis Express Phenyl-Hexyl column, etc. Particularly preferred is an Ascentis Express Phenyl-Hexyl column.

In the present invention, the pentafluorophenyl alkyl column is a column using a filler in which a pentafluorophenyl group is chemically bonded to silica gel via an alkyl group having 1 to 6 carbon atoms. For example, a pentafluorophenylethyl column using a filler in which pentafluorophenylethyl groups are chemically bonded to silica gel, a pentafluorophenylpropyl column using a filler in which pentafluorophenylpropyl groups are chemically bonded to silica gel, pentafluorophenylbutyl Pentafluorophenyl butyl column using a filler whose group is chemically bonded to silica gel, Pentafluorophenyl pentyl column using a filler whose pentafluorophenyl pentyl group is chemically bonded to silica gel, Pentafluorophenyl hexyl group is chemically bonded to silica gel A pentafluorophenyl hexyl column and the like using the above filler is exemplified, but a pentafluorophenyl propyl column is preferable.

As a pentafluorophenylpropyl column, for example, a Kinetex minibore column (PFP), a Kinex midbore column (PFP), a Kinex analysis column (PFP), an Ascentis Express F5 column, a COSMOSIL 5PFP Packed Column, and an ACQUITY UPLC CUL FLUCH Fluoro Phenyl Column, XSelect CSH Fluoro Phenyl Column, XSelect HSS Fluoro Phenyl Column, YMC-Triart PFP, TCI Stella PFP, and the like.
Among the pentafluorophenylpropyl columns, core shell columns such as a Kinetex minibore column (PFP), a Kinetex midbore column (PFP), a Kinetex analysis column (PFP), and an Ascentis Express F5 column are preferable. Particularly preferred is an Ascentis Express F5 column.

“Kinetex” is a registered trademark of Phenomenex, “COSMOSIL” is a registered trademark of Nakarai-Tex Corporation, and “Ascentis” is a trade name of Sigma-Aldrich.

A C1-C6 alkyl group is a linear or branched C1-C6 alkyl group. Examples of the alkyl group having 1 to 6 carbon atoms include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, and 1-ethylpropyl group. , 2-ethylpropyl group, hexyl group and the like.

An aqueous solvent and an organic solvent can be used for the mobile phase of the present invention.
It is preferable to add an acidic substance to the aqueous solvent. By adding an acidic substance, the basic functional group in the amphoteric compound receives hydrogen ions, and the molecule as a whole is positively charged. The amphoteric compound in a positively charged state has good affinity for a phenylalkyl column or a pentafluorophenylalkyl column, and the column retention time becomes long. Examples of the acidic substance added to the mobile phase include trifluoroacetic acid, formic acid, phosphoric acid and the like. In the present invention, since an ion pair reagent is not used, LC-MS is possible. In consideration of performing LC-MS, a volatile acidic substance such as trifluoroacetic acid, formic acid, or acetic acid in which the liquid becomes a gas at normal temperature is preferable. More preferred is trifluoroacetic acid.

Examples of the organic solvent include, but are not limited to, alcohol solvents such as methanol, ethanol, and propanol, tetrahydrofuran, or acetonitrile. Preferably, acetonitrile or methanol is used.
Regarding the mixing ratio of the aqueous solvent and the organic solvent, the value (C) represented by the following formula (1) is 1% or more and 100% or less, more preferably 30% or more and 100% or less, and further preferably 50% or more and 100%. % Or less, still more preferably 65% or more and 95% or less, and still more preferably 65% or more and 92% or less.

本発明においては、イオンペア試薬を用いないため、ＬＣ−ＭＳが可能である。ＬＣ−ＭＳとは液体クロマトグラフィーと質量分析装置を連結したシステムである。測定対象試料を、液体クロマトグラフィー（ＬＣ）を用いて分離を行い、次に、得られたＬＣ溶出液を質量分析装置（ＭＳ）に導入し、イオン化させたのち、そのイオンを検出する。ＬＣ溶液中に含まれていた、化合物由来のイオン（親イオン）が生成するので、当該イオンのｍ／ｚ比を検出することで、化合物の存在の有無が確認できる。この際、適切なスキャン法を選択することができる。

In the present invention, since an ion pair reagent is not used, LC-MS is possible. LC-MS is a system in which liquid chromatography and a mass spectrometer are connected. The sample to be measured is separated using liquid chromatography (LC), and then the obtained LC eluate is introduced into a mass spectrometer (MS) and ionized, and then the ions are detected. Since the compound-derived ion (parent ion) contained in the LC solution is generated, the presence / absence of the compound can be confirmed by detecting the m / z ratio of the ion. At this time, an appropriate scanning method can be selected.

  Examples of the mass spectrometer (MS) include a quadrupole mass spectrometer, an ion trap mass spectrometer, a time-of-flight mass analyzer, a magnetic field mass analyzer, and a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR). However, the type of apparatus used in the present invention is not particularly limited. As the ionization method, there is an electrospray ionization method (ESI), an atmospheric pressure ionization method (APCI), or the like, but the type is not limited.

In the purification method described in the present invention, the flow rate of the mobile phase to be used can be equal to or faster than the conventional analysis method to which an ion pair reagent is added.
The equivalent flow rate is the flow rate ± 0.2 mL / min used in the conventional analysis method, more preferably ± 0.1 mL / min, and still more preferably ± 0.05 mL / min.

For the purification method according to the present invention, the flow rate of the mobile phase is preferably 0.5 mL / min or more, more preferably 0.5 mL / min or more and 3.0 mL / min or less, and further preferably 0.6 mL / min. It is 1.5 mL / min or more, more preferably 0.7 mL / min or more and 1.0 mL / min or less.
Since the purification method described in the present invention can use a flow rate that is equal to or faster than the flow rate of an analysis method to which a conventional ion pair reagent is added, tailing and reading of the target amphoteric compound peak can be suppressed.

Peak tailing is a phenomenon in which the slope of the latter half becomes gentler than the slope of the first half of the peak, and the tailing peak has a symmetry coefficient (S) of 1 or more. The peak reading is a phenomenon in which the slope of the first half of the peak becomes gentler than the slope of the second half, and the symmetry coefficient (S) is 1 or less. From the viewpoint of purification effect, a symmetrical peak without tailing or reading is preferable.

The symmetry coefficient is a coefficient indicating the degree of symmetry of the peak, and is a value (S) represented by the following formula (2). As the symmetry coefficient is closer to 1, it means that the peak shape is closer to a normal distribution, preferably 0.7 to 1.3, more preferably 0.8 to 1.2, and still more preferably 0.9. Above 1.1 is mentioned.

（式（２）中、Ｗ_{０．０５ｈ}は、ピークのベースラインからピーク高さの１／２０の高さにおけるピーク幅を意味し、ｆは、Ｗ_{０．０５ｈ}のピーク幅におけるピーク開始点から、当該ピーク開始点を含む水平線と、ピーク頂点から横軸へ下ろした垂線との交点までの距離を意味する）。シンメトリー係数を説明するための概念図を図１に示す。
本発明に記載の精製方法について、被検対象物である両性化合物の保持時間は、１０分以上であることが好ましい。より好ましい保持時間として１５分以上、さらに好ましくは２０分以上５０分以下、より好ましくは２０分以上４０分以下が挙げられる。

(In Formula (2), W _0.05h means the peak width at a height of 1/20 of the peak height from the peak baseline, and f is the peak starting point at the peak width of W _0.05h. , Meaning the distance from the horizontal line including the peak start point to the intersection of the peak vertex and the vertical line drawn down to the horizontal axis). A conceptual diagram for explaining the symmetry coefficient is shown in FIG.
In the purification method according to the present invention, it is preferable that the retention time of the amphoteric compound as the test object is 10 minutes or more. More preferable holding time is 15 minutes or more, further preferably 20 minutes or more and 50 minutes or less, more preferably 20 minutes or more and 40 minutes or less.

(General analysis method)
1. A sample solution is prepared by dissolving an amphoteric compound having a molecular weight of 250 or more in one or more solvents selected from the group consisting of water, alcohol solvents, tetrahydrofuran, or acetonitrile.
2. Select the mobile phase. As the mobile phase, a liquid for mixing an aqueous solvent and an organic solvent is used. As the aqueous solvent, water that may contain an acidic substance such as trifluoroacetic acid, formic acid, and acetic acid can be used. As the organic solvent, one or two or more organic solvents selected from the group selected from alcohol solvents, tetrahydrofuran, or acetonitrile can be used.
3. The sample solution is charged into a phenylalkyl column or a pentafluorophenylalkyl column, the mobile phase is allowed to flow, and the retention time of the amphoteric compound is measured.

EXAMPLES The present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited by these examples.
Example 1
Aztreonam (molecular weight: 435.43) was dissolved in a 0.5% trifluoroacetic acid aqueous solution / HPLC methanol mixture (7: 3) to a concentration of 5 μg / mL, and the measurement was performed under the following conditions. The retention time of aztreonam was 28.1 minutes (symmetry coefficient: 0.9).




HPLC condition injection volume: 10 μL
Measurement wavelength: 254nm
Column: Phenyl column (product name Ascentis Express Phenyl-Hexyl, Sigma-Aldrich) with an inner diameter of 4.6 mm x length of 15 cm and a particle size of 2.7 μm
Mobile phase A: 0.5% trifluoroacetic acid aqueous solution Mobile phase B: methanol for HPLC Liquid transfer: Mobile phase A: Mobile phase B = 90: 10
Flow rate: 0.80mL / min
Column temperature: 35 ° C
(Example 2)
Cephalexin (molecular weight: 347.39) was dissolved in a 0.5% trifluoroacetic acid aqueous solution / HPLC methanol mixture (7: 3) so as to have a concentration of 5 μg / mL, and was measured under the following conditions. Cephalexin was strongly retained and had a retention of over 35 minutes.
HPLC condition injection volume: 10 μL
Measurement wavelength: 254nm
Column: Phenyl column (product name Ascentis Express Phenyl-Hexyl, Sigma-Aldrich) with an inner diameter of 4.6 mm x length of 15 cm and a particle size of 2.7 μm
Mobile phase A: 0.5% trifluoroacetic acid aqueous solution Mobile phase B: methanol for HPLC Liquid transfer: Mobile phase A: Mobile phase B = 80: 20
Flow rate: 0.80mL / min
Column temperature: 35 ° C
(Example 3)
Fexofenadine (molecular weight: 538.12) was dissolved in a 0.5% trifluoroacetic acid aqueous solution / HPLC acetonitrile mixture (7: 3) to a concentration of 5 μg / mL, and measured under the following conditions. . The retention time of fexofenadine was 25.9 minutes (symmetry coefficient: 1.0).
HPLC condition injection volume: 10 μL
Measurement wavelength: 220nm
Column: Phenyl column (product name Ascentis Express Phenyl-Hexyl, Sigma-Aldrich) with an inner diameter of 4.6 mm x length of 15 cm and a particle size of 2.7 μm
Mobile phase A: 0.5% aqueous trifluoroacetic acid mobile phase B: acetonitrile for HPLC Liquid transfer: mobile phase A: mobile phase B = 70: 30
Flow rate: 0.80mL / min
Column temperature: 35 ° C
Example 4
Aztreonam (molecular weight: 435.43) was dissolved in a 0.5% trifluoroacetic acid aqueous solution / HPLC methanol mixture (7: 3) to a concentration of 5 μg / mL, and the measurement was performed under the following conditions. The retention time of aztreonam was 23.7 minutes (symmetry coefficient: 0.9).
HPLC condition injection volume: 10 μL
Measurement wavelength: 254nm
Column: Fluorophenyl column with an inner diameter of 4.6 mm x length of 15 cm and a particle size of 2.7 μm (trade name Ascentis Express F5, Sigma-Aldrich)
Mobile phase A: 0.5% trifluoroacetic acid aqueous solution Mobile phase B: methanol for HPLC Liquid transfer: Mobile phase A: Mobile phase B = 90: 10
Flow rate: 0.80mL / min
Column temperature: 35 ° C
(Example 5)
Ofloxacin (molecular weight: 361.37) was dissolved in a 0.5% aqueous solution of trifluoroacetic acid / acetonitrile for HPLC (7: 3) so as to have a concentration of 5 μg / mL, and was measured under the following conditions. The retention time of ofloxacin was 21.2 minutes (symmetry factor: 1.6).
HPLC condition injection volume: 10 μL
Measurement wavelength: 294nm
Column: Fluorophenyl column with an inner diameter of 4.6 mm x length of 15 cm and a particle size of 2.7 μm (trade name Ascentis Express F5, Sigma-Aldrich)
Mobile phase A: 0.5% aqueous trifluoroacetic acid mobile phase B: acetonitrile for HPLC Liquid transfer: mobile phase A: mobile phase B = 85: 15
Flow rate: 0.80mL / min
Column temperature: 35 ° C
(Example 6)
Cephalexin was dissolved in a 0.5% trifluoroacetic acid aqueous solution / HPLC methanol mixture (7: 3) to a concentration of 5 μg / mL, and the sample solution was measured under the following conditions. The retention time of cephalexin was 24.6 minutes (symmetry coefficient: 1.0).
HPLC condition injection volume: 10 μL
Measurement wavelength: 254nm
Column: Fluorophenyl column with an inner diameter of 4.6 mm x length of 15 cm and a particle size of 2.7 μm (trade name Ascentis Express F5, Sigma-Aldrich)
Mobile phase A: 0.5% trifluoroacetic acid aqueous solution Mobile phase B: methanol for HPLC Liquid transfer: Mobile phase A: Mobile phase B = 80: 20
Flow rate: 0.80mL / min
Column temperature: 35 ° C
(Example 7)
Fexofenadine (molecular weight: 538.12) was dissolved in a 0.5% trifluoroacetic acid aqueous solution / HPLC acetonitrile mixture (7: 3) to a concentration of 5 μg / mL, and measured under the following conditions. . The retention time of fexofenadine was 21.9 minutes (symmetry coefficient: 1.0).
HPLC condition injection volume: 10 μL
Measurement wavelength: 220nm
Column: Fluorophenyl column with an inner diameter of 4.6 mm x length of 15 cm and a particle size of 2.7 μm (trade name Ascentis Express F5, Sigma-Aldrich)
Mobile phase A: 0.5% aqueous trifluoroacetic acid mobile phase B: acetonitrile for HPLC Liquid transfer: mobile phase A: mobile phase B = 70: 30
Flow rate: 0.80mL / min
Column temperature: 35 ° C
(Example 8)
Ofloxacin (molecular weight: 361.37) was dissolved in a 0.5% aqueous solution of trifluoroacetic acid / acetonitrile for HPLC (7: 3) to a concentration of 5 μg / mL to prepare a sample solution. Under the following conditions, LC-MS / MS was measured to confirm MS of ofloxacin with a molecular weight of +1. The apparatus used was Waters AQUITY UPLC H-Class / TQD.
HPLC condition injection volume: 10 μL
Column: Fluorophenyl column with an inner diameter of 4.6 mm x length of 15 cm and a particle size of 2.7 μm (trade name Ascentis Express F5, Sigma-Aldrich)
Mobile phase A: 0.5% aqueous trifluoroacetic acid mobile phase B: acetonitrile for HPLC Liquid transfer: mobile phase A: mobile phase B = 85: 15
Flow rate: 0.80mL / min
Column temperature: 35 ° C

The mass spectrometric ionization method used electrospray ionization and was measured in the positive ion mode. The capillary voltage was set to 3.50 kV, the source temperature was set to 150 ° C., and the desolvation gas temperature was set to 450 ° C.
As a result of Examples 1 to 8, it can be seen that the amphoteric compound having a molecular weight of 250 or more is subjected to reverse phase liquid chromatography purification using a phenylalkyl column or a pentafluorophenylalkyl column and exhibits a retention time of 20 minutes or more. Even without using an ion-pairing reagent, it is sufficiently retained on a phenylhexyl column or a pentafluorophenylpropyl column, and separation analysis is possible.

Since amphoteric compounds have a structural property of having an acidic functional group and a basic functional group, it is difficult to separate and analyze them. In general, when separating a compound having an acidic functional group in the molecule, the mobile phase is adjusted to be acidic, and when separating a compound having a basic functional group in the molecule, the mobile phase is Adjust to basic. Thereby, the acidic functional group or the basic functional group in the molecule is not ionized and becomes a molecular type (non-dissociation type). Molecular form compounds have low polarity and are easy to hold in the column.
However, when separating the amphoteric compounds, if the mobile phase is acidified, the acidic functional groups in the amphoteric compounds will not ionize, but the basic functional groups will receive and ionize hydrogen ions, and the molecule as a whole will be positively charged dissociated. Become a mold. In addition, when the mobile phase is made basic, the reverse phenomenon occurs, resulting in a negatively charged dissociation type. The dissociative compound is highly polar and is not easily retained on the column.

In the conventional method, an ion pair reagent is added to the mobile phase to form an ion pair with a dissociated compound, thereby increasing the retention force on the column and performing chromatographic separation analysis. However, since the ion pair reagent is a contaminant in mass spectrometry and further remains in the mass spectrometer, it also appears as a contaminant in subsequent measurements. For this reason, it was difficult to perform LC-MS (liquid chromatography-mass spectrometer) by the conventional method using an ion pair reagent.
The present invention has found that amphoteric compounds having a molecular weight of 250 or more and a phenylalkyl column or a pentafluorophenylalkyl column have high affinity, and have found that amphoteric compounds can be separated and analyzed without using an ion pair reagent. It is a feature. Since no ion-pair reagent is used, LC-MS is possible, and this method is very useful for the purpose of evaluating the efficacy of drugs and identifying degradation products.

In addition, the aztreonam of Examples 1 and 4 has a peak with a symmetry coefficient of 0.9, the fexofenadine of Examples 3 and 7 and the peak of a symmetry coefficient of 1.0 with Cephalexin of Example 6. According to the method of the present invention, since the retention time can be secured without lowering the flow rate of the mobile phase, peak tailing and reading can be suppressed, and a symmetrical peak can be obtained.
(Comparative Example 1)
Ofloxacin (molecular weight: 361.37) was dissolved in a water / HPLC acetonitrile mixture (6: 1) to a concentration of 1 μg / mL to obtain a standard solution, and measurement was performed under the following conditions. The retention time of ofloxacin was 20.4 minutes.
HPLC condition injection volume: 10 μL
Measurement wavelength: 294nm
Column: Octadecylsilylated silica gel for HPLC with 4.6mm ID x 25cm length and 5μm particle size (trade name Inertsil ODS-3)
Mobile phase: Dissolve 7.0 g of sodium perchlorate and 4.0 g of ammonium acetate in 1300 mL of water, adjust to pH 2.2 by adding phosphoric acid, and add 240 mL of acetonitrile Flow rate: 0.85 mL / min
Column temperature: 45 ° C
(Comparative Example 2)
Ofloxacin (molecular weight: 361.37) was dissolved in a water / HPLC acetonitrile mixture (6: 1) to a concentration of 1 μg / mL to obtain a standard solution, and measurement was performed under the following conditions. The retention time of ofloxacin was 9.7 minutes and the retention on the column was weak.
HPLC condition injection volume: 10 μL
Measurement wavelength: 294nm
Column: Octadecylsilylated silica gel for HPLC with 4.6mm ID x 25cm length and 5μm particle size (trade name Inertsil ODS-3)
Mobile phase: Dissolve 4.0 g of ammonium acetate in 1300 mL of water, add phosphoric acid to adjust to pH 2.2, and add 240 mL of acetonitrile Flow rate: 0.85 mL / min
Column temperature: 45 ° C
Comparative Examples 1 and 2 are analyzes using an ODS column. When an ion pair reagent is used for the mobile phase, a retention time of 20.4 minutes is shown (Comparative Example 1). However, if the ion pair reagent is not used, it is not well retained on the column, and the retention time is 9.7 minutes and halved. End up. It can be seen that an ion pair reagent is necessary when an ODS column is used.
(Comparative Example 3)
Droxidopa (molecular weight: 213.19) was dissolved in a 0.5% aqueous solution of trifluoroacetic acid / acetonitrile for HPLC (7: 3) so as to be 50 μg / mL, and the sample solution was measured under the following conditions. The retention time of droxidopa was 2.3 minutes and was not sufficiently retained on the column. A compound having a small molecular weight is presumed to be hardly retained even under this condition.
HPLC condition injection volume: 10 μL
Measurement wavelength: 220nm
Column: Fluorophenyl column with an inner diameter of 4.6 mm x length of 15 cm and a particle size of 2.7 μm (trade name Ascentis Express F5, Sigma-Aldrich)
Mobile phase A: 0.5% aqueous trifluoroacetic acid mobile phase B: acetonitrile for HPLC Liquid transfer: mobile phase A: mobile phase B = 95: 5
Flow rate: 0.80mL / min
Column temperature: 35 ° C
(Comparative Example 4)
Mesalazine (molecular weight: 153.14) was dissolved in a 0.5% trifluoroacetic acid aqueous solution / HPLC acetonitrile mixture (7: 3) to a concentration of 50 μg / mL, and the sample solution was measured under the following conditions. The retention time of mesalazine was 3.7 minutes and was not sufficiently retained on the column. A compound having a small molecular weight is presumed to be hardly retained even under this condition.
HPLC condition injection volume: 10 μL
Measurement wavelength: 235nm
Column: Fluorophenyl column with an inner diameter of 4.6 mm x length of 15 cm and a particle size of 2.7 μm (trade name Ascentis Express F5, Sigma-Aldrich)
Mobile phase A: 0.5% aqueous trifluoroacetic acid mobile phase B: acetonitrile for HPLC Liquid transfer: mobile phase A: mobile phase B = 95: 5
Flow rate: 0.80mL / min
Column temperature: 35 ° C
Comparative Examples 3 and 4 are analysis results of amphoteric compounds (droxidopa or mesalazine) having a molecular weight of 250 or less. It can be seen that the affinity with the fluorophenyl column is weak and the retention time is short.

According to this embodiment, the amphoteric compound having a molecular weight of 250 or more can be separated and analyzed without requiring the addition of an ion pair reagent, which is industrially useful.

Claims (5)

  A method for purifying an amphoteric compound having a molecular weight of 250 or more by reverse-phase liquid chromatography, wherein the reverse-phase liquid chromatography is carried out using a phenylalkyl column or a pentafluorophenylalkyl column, and no ion-pairing reagent is used. And the above method.   The method according to claim 1, wherein the column according to claim 1 uses a core-shell column.   The method according to claim 1, wherein the column according to claim 1 is a phenylhexyl column or a pentafluorophenylpropyl column.   The method according to any one of claims 1 to 3, wherein an aqueous solvent to which a volatile acidic substance is added is used as the mobile phase.   The method according to claim 4, wherein trifluoroacetic acid is used as the acidic substance.

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