JP2011027501A - Method for simultaneously analyzing negative and positive counter ions - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simultaneous analytical method of negative/positive counter ions which are used frequently for drugs. <P>SOLUTION: The method for simultaneously analyzing negative/positive counter ions includes a separation step for applying a counter-ion containing sample of a drug containing positive ions and negative ions to a hydrophilic interaction chromatography, by using a column having an amphoteric ion type functional group and eluents which are mixed solutions of water, acetonitrile and ammonium acetate buffer, wherein the separation step includes, (i) a step of eluting with an eluent the concentration of which is adjusted so that the mixed solution contains 70-90 v/v% of acetonitrile and 10-40 mmol/L of the buffer solution, and (iii) a step of eluting with an eluent, the concentration of which is adjusted so that the mixed solution contains 40-70 v/v% of acetonitrile and 30-100 mmol/L of the buffer solution, and in the separation step the eluent is concentration adjusted by changing the mutual ratio of water, acetonitrile and the buffer solution in the mixed solution. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for simultaneous analysis of yin and yang counter ions of drugs.

  50% of all drugs used in medical treatment are ionic compounds and exist in salt form. API (Active Pharmaceutical Ingredients) counter ion differences in these compounds are due to API physicochemical properties (eg, solubility, crystallinity and hygroscopicity, and stability against heat, light and hydrolysis), drugs Affects kinetic properties (eg bioavailability, dosage form). Therefore, in drug development, evaluation and selection of various counter ions are regarded as important for the purpose of controlling the property.

  Generally, in the analysis of counter ions, ion chromatography or capillary electrophoresis is used as an analytical instrument, and an evaporative light scattering detector, a charged particle detector, or the like is used as a detector.

  In the method using ion chromatography, analysis is performed by separately determining columns and conditions for each of anions and cations.

On the other hand, hydrophilic interaction chromatography (HILIC) is used for analysis of amino acids and analysis of nonionic surfactants (see Patent Document 1 and Patent Document 2).
There have also been reports of attempts to simultaneously analyze anions and cations and a plurality of ions at the same time using this hydrophilic interaction chromatography (HILIC) (Non-Patent Document 1, Non-Patent Document 2). Non-Patent Document 3). Specifically, isocratic elution method, two-liquid gradient method and the like have been reported.

JP-A-6-331620 Japanese Patent Laid-Open No. 9-43218

32nd The International Symposium on High Performance Liquid Phase Separations, Poster 3 “Current Pharmaceutical Analysis”, Volume 4, p. 25-32, 2008 “LCGC Asia Pacific”, Vol. 9, No. 3, 2006

  Ion chromatographs cannot simultaneously separate anions and cations due to the detection principle. For this reason, at the pharmaceutical development stage in pharmaceutical manufacturers, the development of a simple analytical method to replace this is urgently required.

Several attempts for simultaneous analysis of counter ions in drugs using hydrophilic interaction chromatography have been reported as described above, but they have not been established as simultaneous analysis methods.
For example, the reported method has a problem that monovalent ions (Na ⁺ , Br ⁻ , K ⁺ , Cl ⁻ ) are not sufficiently separated and divalent ions (Ca ²⁺ ) are not detected.

  Accordingly, an object of the present invention is to provide a method capable of analyzing anions and cations simultaneously and a plurality of each ion simultaneously. In particular, an object of the present invention is to provide a method for simultaneously analyzing, for example, about 10 kinds of anion / cation counter ions frequently used in medicine.

The inventors of the present invention use an eluent composed of three kinds of liquids and change the concentration of each of the three kinds of liquids in the eluent to separate ions that could not be separated conventionally. As a result, the inventors have found that the concentration control that enables the above can be realized, and have completed the present invention.
In the present invention, simultaneous analysis and simultaneous analysis are used synonymously.

The present invention includes the following inventions.
(1)
A counter ion-containing sample of a drug containing a cation and an anion is hydrophilic using a column containing a carrier having a zwitterionic functional group and an eluent that is a mixed solution of water, acetonitrile and ammonium acetate buffer. Including a separation step for sex interaction chromatography,
The separation step comprises
(I) a step of elution using an eluent whose concentration is adjusted to be a mixed solution having an acetonitrile concentration of 70 to 90 v / v% and a buffer concentration of 10 to 40 mmol / L;
(Iii) elution using an eluent whose concentration is adjusted so that the acetonitrile concentration is 40 to 70 v / v% and the buffer concentration is 30 to 100 mmol / L,
In the separation step, the eluent is adjusted in concentration by changing the ratio of water, acetonitrile and buffer in the mixed solution.

  In the present invention, the composition of the eluent is changed by changing the ratio of water, acetonitrile, and buffer. In the present invention, such a system in which three liquids are involved in the composition change of the eluent may be described as a three-liquid system.

(2)
In the separation step, after the step (i) and before the step (iii),
(Ii) The yin and yang counter ion according to (1), further comprising a step of elution using an eluent having an acetonitrile concentration of 60 to 80 v / v% and a buffer concentration of 20 to 50 mmol / L in the mixed solution. Simultaneous analysis method.

  In the above method (1) or (2), the sample to be subjected to the separation step may contain a pharmaceutical excipient.

(3)
The anion is selected from the group consisting of maleate ion, nitrate ion, bromide ion, chloride ion, phosphate ion and sulfate ion, and the cation is selected from potassium ion, sodium ion, magnesium ion and calcium ion. The simultaneous analysis method of the yin yang counter ion as described in (1) or (2) selected from the group which consists of.

(4)
The method for simultaneous analysis of yin and yang counter ions according to any one of (1) to (3), wherein the counter ions separated in the separation step are detected by an evaporative light scattering detector or a charged particle detector.

  According to the present invention, a method capable of analyzing anions and cations simultaneously and a plurality of each ion at the same time becomes possible. In particular, according to the present invention, it is possible to perform a method for simultaneously analyzing, for example, about 10 kinds of anion / cation counter ions frequently used in medicines.

4 is a graph showing a relationship between acetonitrile concentration in an eluent containing acetonitrile and an ammonium acetate buffer obtained in Reference Example 1 and ion retention behavior. It is the graph which showed the relationship between the ammonium acetate buffer concentration in the eluent containing acetonitrile and the ammonium acetate buffer obtained in Reference Example 2, and the retention behavior of ions. 5 is a graph showing the relationship between the pH of an eluent containing acetonitrile and an ammonium acetate buffer obtained in Reference Example 3 and the ion retention behavior. 6 is a graph showing the relationship between column temperature and ion retention behavior obtained in Reference Example 4. 2 is a chromatogram showing the analysis results of 10 counter ions of a drug obtained in Example 1. FIG. The ion analysis result of the trazodone hydrochloride tablet obtained in Example 2 is represented. The ion analysis result of the ranitidine hydrochloride tablet obtained in Example 3 is represented. The ion analysis result of the dextromethorphan hydrobromide tablet obtained in Example 4 is represented. The ion analysis result of the enalapril maleate tablet obtained in Example 5 is represented. The ion analysis result of the diclofenac sodium tablet obtained in Example 6 is represented. The ion analysis result of the aspara potassium tablet obtained in Example 7 is represented. The anion analysis result of the enalapril maleate tablet obtained by the comparative example 1 is represented. The cation analysis result of the enalapril maleate tablet obtained by the comparative example 1 is represented. It is a calibration curve about each ion created for ion quantification in an example.

[1. sample]
In the present invention, the sample to be analyzed includes a counter ion of the drug.
The counter ion may be any ion that can be a counter ion of API (Active Pharmaceutical Ingredients), and may be an organic ion or an inorganic ion.
Examples of the anion include maleate ion, nitrate ion, bromide ion, chloride ion, phosphate ion and sulfate ion.
Examples of the cation include potassium ion, sodium ion, magnesium ion, and calcium ion.

  The sample may also contain an excipient. The excipient is not particularly limited as long as it can be contained in a drug, that is, pharmaceutically acceptable. Specific examples include lactose, starch, dextrin, sucrose, glucose, fructose and the like. Further, the sample may contain impurities that may be contained in other drugs.

[2. column]
In the present invention, an anion and a cation (hereinafter sometimes simply referred to as an anion) may be simultaneously separated, and therefore, a carrier having a zwitterionic functional group bonded thereto is used for the column.
As the zwitterionic functional group, a betaine group, that is, a functional group having a positive charge and a negative charge at positions not adjacent to each other in the functional group, and a dissociable hydrogen atom is not bonded to the positively charged atom. It is preferable that Examples thereof include a phosphobetaine group, a carboxybetaine group, and a sulfobetaine group.

As the carrier, known materials such as silica gel and other polymers can be used without particular limitation.
As such a column, for example, ZIC (registered trademark) -pHILIC manufactured by Merk SeQuant can be used. ZIC-pHILIC is a column in which a sulfobetaine group is chemically bonded as a zwitterionic functional group to a porous polymer substrate.

[3. Eluent]
The eluent used in the present invention is a mixed solution composed of water, acetonitrile and a buffer solution. A volatile buffer solution is used as the buffer solution. Specifically, ammonium acetate is used.
In this eluent, the concentrations of water, acetonitrile, and buffer are changed. By setting these three liquids as the objects whose concentration is to be changed, it is possible to realize precise concentration control that enables separation of ions that could not be separated conventionally.

[4. Relationship between various parameters and retention behavior]
[4-1. Relationship between organic solvent ratio and retention behavior]
For both anions and cations, the retention tends to be strong when the acetonitrile ratio in the eluent is high.
In addition, for both anions and cations, the above-mentioned tendency tends to be stronger as multivalent ions.

[4-2. Relationship between buffer concentration and retention behavior]
Anion tends to increase in retention as the buffer concentration increases.
Cations tend to decrease in retention as the buffer concentration increases.

[4-3. Relationship between eluent pH and retention behavior]
The retention of divalent cations, phosphate ions and maleate ions tends to increase as the pH of the eluent increases.

[4-4. Relationship between column temperature and retention behavior]
The column temperature has a small influence on the retention behavior compared to the acetonitrile concentration, buffer concentration, and pH in the eluent.

[5. Separation process]
In the present invention, the separation uses hydrophilic interaction chromatography. Hydrophilic interaction chromatography is a kind of normal phase chromatography that uses a highly polar solvent as a mobile phase for a highly polar stationary phase, and uses an aqueous eluent. The separation step of the present invention includes the following steps (i) and (iii), and may further include step (ii) as appropriate. Preferably, steps (i), (ii) and (iii) are performed in this order.

  In the separation step, step elution is performed in which the concentrations of the three liquids are different from each other in each step. In each step, unless otherwise specified, isocratic elution may be performed at a specific concentration selected from the concentration range shown below, or a specific concentration range selected from the concentration range. Gradient elution may be performed within. Moreover, when selecting a specific density | concentration from the density | concentration range shown below, the tendency described in said 4 can be considered.

[5-1. Step (i)]
This step (i) is preferably applied to elution and separation of monovalent ions in the sample.
In step (i), an eluent whose concentration is adjusted so as to be a mixed solution having an acetonitrile concentration of 70 to 90 v / v% and a buffer solution concentration of 10 to 40 mmol / L is used.
The acetonitrile concentration may further be 75-85 v / v%, for example 80 v / v%. Further, the buffer concentration may be 15 to 30 mmol / L, or 15 to 25 mmol / L, for example 25 mmol / L.

[5-2. Step (ii)]
This step (ii) is preferably applied to separation of these impurities from monovalent ions and multivalent ions when impurities such as excipients can be contained in the sample.

  In step (ii), the eluent may be the same concentration as in step (i) above, or a mixed solution having an acetonitrile concentration of 60-80 v / v% and a buffer concentration of 20-50 mmol / L. The density may be adjusted so that Further, isocratic elution may be performed, or gradient elution may be performed.

  In particular, gradient elution is preferably performed. Gradient elution is carried out in the concentration range of the eluent described in step (i) or preferably from the above eluent (acetonitrile concentration 60 to 80 v / v%, buffer concentration 20 to 50 mmol / L). This can be done by providing a concentration gradient within a concentration range that can be appropriately selected from the concentration range.

[5-3. Step (iii)]
This step (iii) is preferably applied to elution and separation of multivalent ions in the sample.
In step (iii), an eluent whose concentration is adjusted so as to be a mixed solution having an acetonitrile concentration of 40 to 70 v / v% and a buffer solution concentration of 30 to 100 mmol / L is used.
The acetonitrile concentration may further be 50-70 v / v%, alternatively 60-70 v / v%, for example 65 v / v%. Further, the buffer concentration may be 40 to 90 mmol / L, 50 to 90 mmol / L, 60 to 90 mmol / L, 70 to 90 mmol / L, or 80 to 90 mmol / L, for example, 87.5 mmol / L. .

[6. Detection process]
A detector capable of detecting anions and cations at the same time can be used for detection of ions separated in the above step without any particular limitation. Examples of such a detector include an evaporative light scattering detector (for example, ELSD-LTII manufactured by Shimadzu Corporation) and a charged particle detector (manufactured by ESA; Corona ^(R) CAD ^(R) ).

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following examples.

  Below, the example which verified about the relation between various parameters and retention behavior is shown as a reference example. In Reference Examples 1 to 4, the following conditions were applied. In addition, all the anion cation standard goods employ | adopted the reagent which has a volatile counter ion (specifically ammonium ion or acetate ion).

Apparatus: High-performance liquid chromatograph Prominence (manufactured by Shimadzu Corporation)
Analytical column: ZIC ^(R) -pHILIC (150mmL. X 4.6mmI.D.) (Merck)
Eluent: Ammonium acetate buffer (2,10,20,30mmol / L)
Acetonitrile aqueous solution (40,50,60,70,80v / v%)
Column temperature: 30,40,50 ℃
Flow rate: 1.0mL / min
Injection volume: 5μL
Detector: Charged particle detector Corona ^(R) CAD ^(R) (manufactured by ESA)
Gas pressure: 35 psi (N ₂ )
Filter: None Range: 500pA
Target component: Anion-chloride ion, sulfate ion, phosphate ion, maleate ion,
Bromide ion, nitrate ion
Cation-sodium ion, potassium ion, calcium ion,
Magnesium ion

[Reference Example 1: Relationship between organic solvent ratio and retention behavior]
Using an eluent composed of 20 mmol / L ammonium acetate buffer (pH 5.0) and acetonitrile, the retention behavior was actually changed depending on the acetonitrile concentration (in other words, water ratio) in the eluent under the condition of a column temperature of 30 ° C. A graph showing how it changes is shown in FIG. In FIG. 1, the vertical axis represents the magnitude of retention (retention coefficient k; the same applies hereinafter), and the horizontal axis represents the water ratio (volume basis).

  As shown in FIG. 1, it can be seen that both the anion and the cation have a large retention when the acetonitrile ratio in the eluent is high, and this tendency is stronger for the polyvalent ions. It can also be seen that in the two-liquid system, the retention can be changed drastically even with a slight difference in acetonitrile concentration. In particular, when the acetonitrile concentration exceeds 70 v / v% (the water ratio falls below 30 v / v%), it can be seen that the retention becomes extremely large. That is, in the two-liquid system, it is extremely difficult to adjust the retention behavior (that is, the retention behavior is likely to fluctuate greatly and peaks with other ions tend to overlap within the range that can be adjusted with two liquids). This suggests that the method has made it impossible to separate ions.

[Reference Example 2: Relationship between buffer concentration and retention behavior]
Using an eluent consisting of ammonium acetate buffer (pH 5.0) and 80 v / v acetonitrile, how the retention behavior actually changes depending on the ammonium acetate buffer concentration under the condition of a column temperature of 30 ° C. The graph shown is shown in FIG. In FIG. 2, the vertical axis represents the retention size, and the horizontal axis represents the buffer solution concentration (mmol / L).

  As shown in FIG. 2, it can be seen that the retention of the anion increases as the buffer concentration increases, and the retention of the cation decreases as the buffer concentration increases. It can also be seen that the retention of the anion is almost constant when it exceeds 10 mmol / L.

[Reference Example 3: Relationship between eluent pH and retention behavior]
Using an eluent consisting of 20 mmol / L ammonium acetate buffer and 80 v / v% acetonitrile, it was shown how the retention behavior actually changes depending on the pH of the ammonium acetate buffer under the condition of a column temperature of 30 ° C. A graph is shown in FIG. In FIG. 3, the vertical axis represents the magnitude of retention, and the horizontal axis represents pH. As shown in FIG. 3, it can be seen that the retention of magnesium ions, calcium ions, phosphate ions and maleate ions increases as the pH of the eluent increases.

[Reference Example 4: Relationship between column temperature and retention behavior]
FIG. 4 shows a graph showing how the retention behavior actually changes depending on the column temperature using an eluent composed of 20 mmol / L ammonium acetate buffer (pH 5.0) and 80 v / v% acetonitrile. In FIG. 4, the vertical axis represents the magnitude of the retention, and the horizontal axis represents the column temperature. As shown in FIG. 4, the influence of the column temperature on the retention behavior is small.

  Examples in which simultaneous analysis of counter ions is performed by the method of the present invention are shown below. In the examples, the following conditions were applied.

Apparatus: High-performance liquid chromatograph Prominence (manufactured by Shimadzu Corporation)
Analytical column: ZIC ^(R) -pHILIC (150mmL. X 4.6mmI.D.) (Merck)
Eluent: (A) Water
(B) 250 mmol / L ammonium acetate buffer (pH 5.0)
(C) Acetonitrile elution method: Three-component column temperature: 30 ° C
Flow rate: 1.0mL / min
Injection volume: 5μL
Detector: Charged particle detector Corona ^(R) CAD ^(R) (manufactured by ESA)
Gas pressure: 35 psi (N ₂ )
Filter: None Range: 500pA
Countermeasure component: anion-chloride ion, sulfate ion, phosphate ion, maleate ion,
Bromide ion, nitrate ion
Cation-sodium ion, potassium ion, calcium ion,
Magnesium ion

[Example 1: Simultaneous analysis of 10 kinds of counter ions by 3 liquid system]
Simultaneous analysis was conducted on 10 counter ions frequently used for drugs. In addition, all the anion cation standard goods employ | adopted the reagent which has a volatile counter ion (specifically ammonium ion or acetate ion). Anthracene was used as a void marker (t ₀ ).

The composition of the eluent was controlled by a three-liquid system.
In step (i) for the purpose of elution and separation of monovalent ions, isocratic elution was performed with the acetonitrile concentration in the eluent adjusted to 80 v / v% and the ammonium acetate buffer concentration adjusted to 20 mmol / L.
In step (ii) aimed at elution of impurities such as excipients, the gradient of acetonitrile in the eluent is from 80 v / v% to 65 v / v%, and the concentration of ammonium acetate buffer is from 20 mmol / L. Gradient elution adjusted to provide a gradient of 35 mmol / L was performed.
In the step (iii) for the purpose of elution and separation of multivalent ions, isocratic elution was performed with the acetonitrile concentration in the eluent adjusted to 65 v / v% and the ammonium acetate buffer concentration adjusted to 87.5 mmol / L.

  The result of simultaneous analysis is shown in FIG. As FIG. 5 shows, all 10 ions were completely separated and detected.

[Example 2: Analysis of preparation (trazodone hydrochloride tablets)]
The preparation was pretreated as follows.
One tablet containing 25.0 mg of the active ingredient trazodone hydrochloride is crushed, 5 mL of water is added, and after ultrasonic irradiation for 30 minutes, the mixture is centrifuged, and the supernatant is a mixed solvent of water / acetonitrile = 5/5 (volume ratio). Diluted 5 times.
The obtained sample was subjected to the same conditions as in Example 1. The obtained analysis results are shown in FIG.

[Example 3: Analysis of preparation (ranitidine hydrochloride tablets)]
The preparation was pretreated as follows.
Crush one tablet containing 168 mg of the active ingredient ranitidine hydrochloride, add 50 mL of water, irradiate with ultrasonic waves for 30 minutes, centrifuge, and remove the supernatant with water / acetonitrile = 5/5 (volume ratio) mixed solvent. Diluted twice.
The obtained sample was subjected to the same conditions as in Example 1. The obtained analysis results are shown in FIG.

[Example 4: Analysis of formulation (dextromethorphan hydrobromide tablets)]
The preparation was pretreated as follows.
Crush one tablet containing 15 mg of the active ingredient dextromethorphan hydrobromide, add 5 mL of water, irradiate with ultrasonic waves for 30 minutes, then centrifuge, and supernatant the water / acetonitrile = 5/5 (volume ratio) The mixture was diluted 5 times with a mixed solvent.
The obtained sample was subjected to the same conditions as in Example 1. The obtained analysis results are shown in FIG.

[Example 5: Analysis of formulation (enalapril maleate tablets)]
The preparation was pretreated as follows.
Crush one tablet containing 2.5 mg of the active ingredient enalapril maleate, add 3 mL of water, irradiate with ultrasonic waves for 30 minutes, centrifuge, and mix the supernatant with water / acetonitrile = 5/5 (volume ratio) Diluted 5 times with solvent.
The obtained sample was subjected to the same conditions as in Example 1. The obtained analysis results are shown in FIG.

[Example 6: Analysis of preparation (diclofenac sodium tablets)]
The preparation was pretreated as follows.
Crush one tablet containing 25 mg of the active ingredient diclofenac sodium, add 5 mL of water, irradiate with ultrasonic waves for 30 minutes, centrifuge, and supernatant 5 times with water / acetonitrile = 5/5 (volume ratio) mixed solvent Diluted.
The obtained sample was subjected to the same conditions as in Example 1. The obtained analysis results are shown in FIG.

[Example 7: Analysis of preparation (aspara potassium tablet)]
The preparation was pretreated as follows.
One tablet containing 300 mg of active ingredient L-potassium potassium aspartate was pulverized, 200 mL of water was added, ultrasonic irradiation was performed for 30 minutes, followed by centrifugation. Diluted 5 times.
The obtained sample was subjected to the same conditions as in Example 1. The obtained analysis results are shown in FIG.

[Comparative Example 1: Analysis of preparation by ion chromatography]
[Analysis of enalapril maleate tablets]
To a sample prepared by pretreatment by the method described in Example 5, sodium bicarbonate was added for the purpose of antacid, diluted 10 to 20 times with purified water, and ion chromatograph Prominence HIC-NS (Shimadzu Corporation) For ion chromatography.
The conditions for ion chromatography are as follows.

(For anion analysis)
Column: Shim-pack IC-A3 (150mmL. X 4.6mm ID)
Eluent: 8mmol / L p-hydroxybenzoic acid
3.2mmol / L Bis-tris, 50mmol / L boric acid Column temperature: 40 ℃
Flow rate: 1.2mL / min
Injection volume: 50 μL

(For positive ion analysis)
Column: Shim-pack IC-A4 (150mmL. X 4.6mm ID)
Eluent: 2.5mmol / L Oxalic acid Column temperature: 40 ° C
Flow rate: 1.0mL / min
Injection volume: 50 μL

  A chromatograph showing the anion analysis results is shown in FIG. 12, and a chromatograph showing the cation analysis results is shown in FIG.

[Analysis of other preparations]
Trazodone hydrochloride tablets, ranitidine hydrochloride tablets, dextromethorphan hydrobromide tablets, diclofenac sodium tablets, and aspara potassium tablets were also subjected to the same analysis as described above.

<< Formulation analysis results >>
Table 1 shows the analysis results of all the preparations obtained in Examples 2 to 7 (simultaneous analysis method) and the analysis results of all the preparations obtained in Comparative Example 1 (ion chromatography) together with their preparation information. Shown in A calibration curve shown in FIG. 14 was created, and quantification was performed based on this calibration curve.

  From Table 1, as a result of analyzing the preparation using the method of the present invention, the recovery rate was 94 to 103%. Further, as a result of the analysis by the method of the present invention, it was confirmed that a quantitative value equivalent to the method of the comparative example using ion chromatography was obtained.

Claims (4)

A counter ion-containing sample of a drug containing a cation and an anion is hydrophilic using a column containing a carrier having a zwitterionic functional group and an eluent that is a mixed solution of water, acetonitrile and ammonium acetate buffer. Including a separation step for sex interaction chromatography,
The separation step comprises
(I) a step of elution using an eluent whose concentration is adjusted to be a mixed solution having an acetonitrile concentration of 70 to 90 v / v% and a buffer concentration of 10 to 40 mmol / L;
(Iii) elution using an eluent whose concentration is adjusted so that the acetonitrile concentration is 40 to 70 v / v% and the buffer concentration is 30 to 100 mmol / L,
In the separation step, the eluent is adjusted in concentration by changing the ratio of water, acetonitrile and buffer in the mixed solution. In the separation step, after the step (i) and before the step (iii),
(Ii) The yin and yang counter ion according to claim 1, further comprising a step of eluting with an eluent having an acetonitrile concentration of 60 to 80 v / v% and a buffer concentration of 20 to 50 mmol / L in the mixed solution. Simultaneous analysis method.   The anion is selected from the group consisting of maleate ion, nitrate ion, bromide ion, chloride ion, phosphate ion and sulfate ion, and the cation is selected from potassium ion, sodium ion, magnesium ion and calcium ion. The simultaneous analysis method of the yin yang counter ion of Claim 1 or 2 selected from the group which consists of. The simultaneous analysis method of the yin and yang counter ion of any one of Claims 1-3 with which the counter ion isolate | separated by the said separation process is detected by an evaporative light scattering detector or a charged particle detector.