CN109358140B

CN109358140B - Rifaximin raw material and detection method of preparation components thereof

Info

Publication number: CN109358140B
Application number: CN201811211328.2A
Authority: CN
Inventors: 赵敬丹; 刘浩; 秦峰; 钱琛
Original assignee: SHANGHAI INSTITUTE FOR FOOD AND DRUG CONTROL
Current assignee: SHANGHAI INSTITUTE FOR FOOD AND DRUG CONTROL
Priority date: 2018-10-17
Filing date: 2018-10-17
Publication date: 2021-07-30
Anticipated expiration: 2038-10-17
Also published as: CN109358140A

Abstract

The invention relates to a method for detecting rifaximin raw material and preparation components thereof, which is a supercritical fluid chromatography and comprises the following steps: dissolving rifaximin raw material or preparation by using organic solvent; by using CO₂Gradient elution with lower alcohol as mobile phase; measuring the components and the content thereof by a detector; wherein, the detected components comprise rifaximin and impurities, and the impurities comprise any one or more of 2-amino-4-methylpyridine, rifamycin B, rifamycin SV, rifaximin Y, rifamycin S, rifamycin O, impurities A, impurities B and hydroxyl rifaximin. The detection method provided by the invention has the advantages of good specificity, high analysis speed, good repeatability and good separation degree of rifaximin and impurity pieces, can be directly combined with a mass spectrometer, is convenient for structure conjecture of impurities, can provide efficient data support for evaluating the quality and production process of rifaximin and rifaximin preparations, and can play an important role in process evaluation and quality consistency evaluation research.

Description

Rifaximin raw material and detection method of preparation components thereof

Technical Field

The invention belongs to the field of analytical chemistry, and particularly relates to a rifaximin raw material and a detection method of components of a preparation thereof.

Background

Rifaximin is a semi-synthetic antibiotic drug belonging to the class of rifamycins, lacking systemic activity, and is mainly an entero-selective antibiotic with minimal systemic absorption. It has broad spectrum activity against both gram-positive and gram-negative pathogens and is well tolerated due to its lack of absorption. The preparation is mainly used for treating intestinal infection caused by pathogenic bacteria sensitive to rifaximin clinically, and is used as an intestinal prophylactic before and after operation and an auxiliary treatment for hepatic encephalopathy.

Currently, the rifaximin related substances and content determination method reported in the literature mainly adopts reversed-phase high performance liquid chromatography, octyl bonded silica gel or octadecyl bonded silica gel is used as a filling agent, and mobile phases are all a methanol-acetonitrile-phosphate/citrate system. Rifaximin is accepted and carried in the current Chinese pharmacopoeia and European pharmacopoeia, the method for measuring related substances and content in the Chinese pharmacopoeia adopts octyl bonded silica gel as a filler, and a mobile phase is a methanol-acetonitrile-phosphate/citrate system and is basically consistent with the literature; the method for measuring related substances and content in European pharmacopoeia adopts octadecyl bonded silica gel as a filler and adopts a methanol-acetonitrile-ammonium formate system as a mobile phase.

After experimental study, the running time of 1 sample is about 120 minutes (see fig. 1) on the premise of meeting the test requirement of the applicability of the current chinese pharmacopoeia system. The operating time of the european pharmacopoeia is about 35 minutes (fig. 2). In addition, the separation of impurities A and rifaximin Y + hydroxyl rifaximin is difficult to realize by the existing method. How to shorten the analysis time of a single sample, improve the chromatographic separation efficiency and improve the quality control efficiency is a problem to be solved.

At present, the document reports that the supercritical fluid chromatography is adopted to analyze related substances in the rifampicin, the separation of the rifampicin and 5 main impurities can be realized within 4 minutes, and compared with the conventional reversed-phase high performance liquid chromatography, the analysis time is greatly shortened.

Supercritical Fluid Chromatography (SFC) refers to a type of chromatography in which a supercritical fluid is the mobile phase. In the field of analytical chemistry, it can be used as a powerful complement to gas chromatography and high performance liquid chromatography. For the separation and quantification of a few drugs, the traditional gas chromatography or high performance liquid chromatography has more difficulties such as low separation efficiency, low sensitivity, large consumption of organic reagents and the like, and the SFC method can effectively improve the separation efficiency and sensitivity, reduce the consumption of the organic reagents and greatly shorten the consumptionSeparating time; while the modifier used in the SFC process is CO₂And is more environment-friendly.

In the above method for analyzing rifampicin-related substances, methanol (containing 0.1% ammonium formate and 2% water) -carbon dioxide is used as a mobile phase, and although the analysis of rifampicin-related substances can be rapidly performed, there are the following problems: after multiple sample injections, the system pressure gradually rises until the pressure exceeds the highest pressure endured by the instrument, and the method is not suitable for analysis of a large number of samples. This may be due to the fact that ammonium formate has a low solubility in the organic phase and gradually precipitates in the system.

From the above, the analysis method of the existing rifaximin raw material and the preparation method thereof has the following disadvantages:

1) the analysis time is long, and particularly, the Chinese pharmacopoeia inclusion method comprises the following steps: the analysis time for running 1 sample is about 120 minutes, which is not beneficial to the internal control detection of a production enterprise;

2) impurity A, rifaximin Y + hydroxyrifaximin can not be separated;

3) the mobile phase needs a large amount of methanol and acetonitrile, the analysis cost is high, and the method is not environment-friendly;

4) the mobile phase, particularly the mobile phase adopted by the Chinese pharmacopoeia, contains high-concentration buffer salt, can not be directly connected with a mass spectrometer in series, and is not beneficial to directly guessing the possible structure of impurities.

Therefore, establishing a general, rapid, efficient and effective analysis method with good separation efficiency is necessary for inspecting, evaluating and screening the quality of rifaximin raw materials so as to improve the quality of subsequent preparations thereof.

Disclosure of Invention

In order to overcome the problems of the prior art, the invention develops a method for detecting raw materials of rifaximin and preparation components thereof by taking carbon dioxide and lower alcohol as mobile phases on the basis of analyzing the existing analysis method and combining the production process of the rifaximin and the preparation thereof on the market, and the method adopts a supercritical fluid chromatography method to realize the rapid detection of related substances and contents thereof in the rifaximin preparation.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a method for detecting rifaximin raw materials and preparation components thereof, which is a supercritical fluid chromatography and comprises the following steps:

step 1) dissolving the rifaximin raw material or preparation by using an organic solvent to prepare a sample;

step 2) with CO₂Taking lower alcohol as a mobile phase, and carrying out gradient elution on the sample in a chromatographic column;

step 3) determining the components and the content of the sample by using a detector;

wherein, the components of the sample comprise rifaximin and impurities, and the impurities comprise any one or more of 2-amino-4-methylpyridine, rifamycin B, rifamycin SV, rifaximin Y, rifamycin S, rifamycin O, impurities A, impurities B and hydroxyl rifaximin.

The structures of the rifaximin and impurities are shown in the following table:

TABLE 1 Structure and Source of rifaximin and various known impurities

In order to further optimize the detection method of the rifaximin raw material and the components of the preparation thereof, the technical measures adopted by the invention also comprise the following steps:

further, the rifaximin formulation includes suspension, capsule, injection, tablet of rifaximin, and may be in any suitable formulation form commonly used in the art.

Further, the organic solvent comprises at least one of ethanol, methanol and isopropanol, and can also be usedAny other suitable organic solvent; the lower alcohol comprises at least one of methanol, ethanol and isopropanol; wherein the rifaximin concentration in the sample obtained by dissolving the raw material or the preparation by adopting the organic solvent is 5-10 mg/mL^-1。

Further, the lower alcohol is ethanol or a mixture of ethanol and isopropanol; wherein the mixing proportion of the isopropanol is 0-50 v%. Preferably, the lower alcohol is a mixture of ethanol and isopropanol, wherein the mixing ratio of isopropanol is 10 v%, and the ratio of isopropanol is properly adjusted according to the elution time of rifamycin B in the actual sample.

Further, the pH value of the lower alcohol is 3.1-10.2, and the adopted pH regulator comprises formic acid, glacial acetic acid, trifluoroacetic acid and ammonia solution. Preferably, the pH value of the lower alcohol is 5.0-7.5.

Furthermore, the filler active group of the chromatographic column is diol group, the temperature of the chromatographic column is 35-60 ℃, and the back pressure is 1500-2500 psi. Preferably, the column temperature is 35 ℃ and the back pressure is 2000 psi. The main differences of the component structures in the table 1 of the invention are polar substituent differences such as hydroxymethyl, carboxyl, phenolic hydroxyl and the like; and simultaneously, the separation difficulty is high. On the basis of analyzing the component structure, the invention selects diol group as stationary phase, realizes separation based on the difference of acting force between different polar substituent groups and diol group groups, and has better selectivity.

Further, the sample injection volume of the chromatographic column is 1-4 mu L, and the flow rate is 0.8-1.5 mL/min^-1The detection wavelength is 220-260 nm. Preferably, the sample injection volume of the chromatographic column is 2 mu L, and the flow rate is 1.2 mL-min^-1The detection wavelength is 240nm +/-2 nm.

Further, the mobile phase is: the mobile phase A is carbon dioxide, and the mobile phase B is ethanol-isopropanol; wherein the ratio of ethanol to isopropanol is 90:10 (v/v).

Further, the gradient elution conditions were: 0-1.8min, B10-18%, 1.8-4min, B18-35%, 4-6min, B35-50%, 6-7min, B50%, maintain for 1min, and reach B10% after 7.1 min.

Further, the detector is a UPCC phase chromatograph equipped with an ultraviolet detector, a binary gradient pump, an autosampler.

Further, Torus DIOL (3.0 mm. times.100 mm, 1.7 μm) was used as a column. Other suitable columns may be used and the flow rate may be adjusted to suit the selected column size. The sample injection volume can be adjusted to a sample injection volume adapted to the sample loading of the chromatographic column or the response of the detector.

Further, the limit of detection of the rifaximin is 1 mug.mL^-1。

Compared with the prior art, the invention has at least the following beneficial effects:

the method for rapidly detecting the rifaximin raw material and the related substances and the content thereof in the preparation can shorten the analysis time and improve the speed of internal control detection of enterprises; and can realize the separation of impurities A, rifaximin Y and hydroxyl rifaximin in Chinese pharmacopoeia and the separation of other byproducts and degradation impurities and the separation of rifaximin and adjacent impurities; the method reduces the consumption of organic reagents such as methanol and acetonitrile, and is more environment-friendly; more importantly, the method can be directly connected with a mass spectrometer in series, and structure conjecture of unknown impurities is facilitated.

The detection method provided by the invention has the advantages of good specificity, high analysis speed (within 10 minutes), good repeatability, good separation degree among impurities, process byproducts and degradation impurities possibly introduced in the rifaximin synthesis process and rifaximin and adjacent impurities, and CO as a mobile phase₂And a small amount of alcohol, is environment-friendly, can be directly used together with a mass spectrometer, facilitates the structure speculation of impurities, provides efficient data support for evaluating the quality of rifaximin raw materials and the production of preparations thereof, and can play an important role in process evaluation and quality consistency evaluation research.

Drawings

Figure 1 is a typical chromatogram of a mixed control solution of rifaximin and its impurities under chromatographic system conditions in the item of rifaximin of the chinese pharmacopoeia; wherein, chromatographic peak 1, 2-amino 4-methylpyridine; 2. impurity a + rifaximin Y + hydroxyrifaximin (co-elution); 3. rifamycin B; 4. rifamycin SV; 5. rifaximin; 6. rifamycin S; 7. an impurity B.

Figure 2 is a typical chromatogram of a mixed control solution of rifaximin and its impurities under chromatographic system conditions according to the european pharmacopoeia rifaximin entry; wherein, chromatographic peak 1, 2-amino 4-methylpyridine; 2. rifamycin B; 3. rifamycin SV; 4. impurity a + rifaximin Y + hydroxyrifaximin (co-elution); 5. rifaximin; 6. rifamycin S; 7. rifamycin O; 8. an impurity B.

Figure 3 is a typical UPCC chromatogram of a mixed control solution of rifaximin and its impurities according to one embodiment of the present invention; wherein, chromatographic peak 1, rifamycin S; 2. rifamycin O; 3. 2-amino-4-methylpyridine; 4. an impurity B; 5. rifaximin; 6. impurity A; 7. rifamycin SV; 8. rifamycin B.

FIGS. 4-6 are typical chromatograms of rifaximin starting material from different sources (A, B and C, respectively) in one embodiment of the invention; among them, in fig. 4 (source a), chromatographic peak 1, impurity B; 2. rifaximin; 3. impurities a, 4, rifaximin Y + hydroxyrifaximin; 5. a source A process-specific byproduct; among them, in fig. 5 (source B), chromatographic peak 1, impurity B; 2. rifaximin; 3. rifaximin Y + hydroxyrifaximin; wherein in FIG. 6 (Source C, original research), chromatographic Peak 1, impurity B; 2. rifaximin; 3. rifaximin Y + hydroxyrifaximin.

FIGS. 7-8 are typical chromatograms of rifaximin assay in another embodiment of the present invention.

FIGS. 9-11 are typical chromatograms of rifaximin tablets from different sources, respectively, in yet another embodiment of the invention; in fig. 9 (rifaximin tablet from raw material a), chromatographic peak 1, impurity B; 2. rifaximin; 3. rifaximin Y + hydroxyrifaximin; 4. a source A process-specific byproduct; in fig. 10 (rifaximin tablet derived from raw material B), chromatographic peak 1, impurity B; 2. rifaximin; 3. rifaximin Y + hydroxyrifaximin; among them, in fig. 11 (rifaximin tablet of raw material source C), chromatographic peak 1, impurity B; 2. rifaximin; 3. rifaximin Y + hydroxyrifaximin.

Figure 12 is a typical chromatogram for rifaximin capsules in yet another embodiment of the invention; wherein, chromatographic peak 1, unknown impurity; 2. an impurity B; 3. rifaximin; 4. rifaximin Y + hydroxyrifaximin.

Figure 13 is a typical chromatogram for a rifaximin soft capsule in yet another embodiment of the invention; wherein, chromatographic peak 1, impurity B; 2. rifaximin; 3. rifaximin Y + hydroxyrifaximin; 4. unknown impurities.

FIG. 14 is a rifamycin B chromatogram at pH3.1 of an embodiment of the invention.

FIG. 15 is a rifamycin B chromatogram at pH10.2 in an example of the invention.

FIG. 16 is a chromatogram for verifying detection limits in an embodiment of the present invention.

Detailed Description

The invention relates to a method for detecting rifaximin raw materials and preparation components thereof, which is a supercritical fluid chromatography and comprises the following steps: step 1) dissolving the rifaximin raw material or preparation by using an organic solvent to prepare a sample; step 2) with CO₂Taking lower alcohol as a mobile phase, and carrying out gradient elution on the sample in a chromatographic column; step 3) determining the components and the content of the sample by using a detector; wherein, the components of the sample comprise rifaximin and impurities, and the impurities comprise any one or more of 2-amino-4-methylpyridine, rifamycin B, rifamycin SV, rifaximin Y, rifamycin S, rifamycin O, impurities A, impurities B and hydroxyl rifaximin.

The following description of the embodiments of the present invention will be made with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.

Example 1

The instrument adopted in this example is: a Waters UPCC phase-combining chromatograph equipped with an ultraviolet detector, a binary gradient pump and an automatic sample injector; electronic balance (CP225D, Sartorius, Germany).

Wherein the chromatographic column used was Torus DIOL (3.0 mm. times.100 mm, 1.7 μm); the detection wavelength is 240 nm; flow rate: 1.2 mL/min^-1(ii) a Sample introduction volume: 2 mu L of the solution; column temperature: 35 ℃; back pressure: 2000 psi.

Mobile phase a is carbon dioxide and mobile phase B is ethanol-isopropanol (90: 10); the gradient elution conditions were: 0-1.8min, B10-18%, 1.8-4min, B18-35%, 4-6min, B35-50%, 6-7min, B50% (maintaining for 1min), after 7.1min, B10%;

and (4) detecting related substances of the rifaximin raw material.

1. Preparation of mixed control solution: respectively taking proper amounts of impurity A, impurity B, rifaximin, 2-amino-4-methylpyridine, rifamycin B, rifamycin SV, rifamycin S and rifamycin O, dissolving with ethanol to obtain rifaximin with concentration of 5 mg/mL^-1The concentration of other impurities is about 50. mu.g/mL^-1The mixed solution of (1).

2. Preparation of sample solution: dissolving rifaximin raw materials from 3 sources (source A, B, C) with ethanol respectively to prepare 5 mg/mL^-1The solution of (1).

According to the above-mentioned instrument and test method, sample analysis is carried out.

The results of the rifaximin feedstock experiments with the mixed control solution and 3 sources are shown in figures 3-6.

Comparative example 1

The control example is the detection of rifaximin raw material related substances of a mixed control solution of rifaximin and impurities thereof under the condition of a chromatographic system recorded in Chinese pharmacopoeia.

The mixed reference solution in example 1 was used, and the mixed reference solution was tested according to the chromatographic system conditions described in the chinese pharmacopoeia, and a typical chromatogram obtained by the test is shown in fig. 1.

Comparative example 2

The control example is the detection of rifaximin raw material related substances of a mixed control solution of rifaximin and impurities thereof under the condition of a chromatographic system recorded in European pharmacopoeia.

The mixed control solution in example 1 was used, and the mixed control solution was tested under the conditions of the chromatography system described in the european pharmacopoeia, and a typical chromatogram obtained by the test is shown in fig. 2.

According to the above examples 1 and comparative examples 1 to 2, it can be seen from FIGS. 4 to 5 that the impurity spectra of the raw material derived from A and the raw material derived from B, C are different. As shown in fig. 1, the separation degree between impurities eluted prior to rifaximin is poor in the item of the chinese pharmacopoeia analysis method; co-eluting impurity A and rifaximin Y + hydroxyrifaximin; the elution time of impurity B is too long. As can be seen from fig. 2, the single sample run time of the european pharmacopoeia chromatography system is significantly shortened compared to the chinese pharmacopoeia chromatography system; but still has the problem of co-elution of impurity a and rifaximin Y + hydroxyrifaximin. As shown in fig. 3 and 4, in the supercritical fluid chromatography system, the single sample running time is greatly shortened to within 10 minutes; separation between rifaximin and known impurities can be compromised; from figure 4, it is known that rifaximin Y + hydroxyrifaximin (co-elution chromatographic peak), the main process impurity, can be well separated from impurity a. And the sample from the source A detects a special process byproduct with higher content, and the impurities are well separated from rifaximin Y + hydroxyl rifaximin.

From the above example 1, the chromatographic separation system established by the invention can realize the separation and analysis of rifaximin rapidly and in high throughput, can distinguish raw materials from different process sources, provides technical support for monitoring the production process of a production enterprise, and provides basis for the evaluation and research of the preparation quality consistency.

Example 2

This example is the rifaximin raw material content test under the same conditions as example 1.

Preparation of sample solution: dissolving rifaximin raw material with ethanol respectively to prepare 0.5 mg/mL^-1The solution of (1).

Analysis was performed by injection according to the test method described above.

Typical chromatogram of the control solution is shown in FIG. 7, and typical chromatogram of rifaximin raw material is shown in FIG. 8 (taking source B as an example.)

According to the embodiment, the chromatographic separation system established by the invention can be used for rapidly detecting the content of the raw materials, providing data support for quality monitoring, and providing a basis for the production of subsequent preparations and the like.

Example 3

In this example, the test conditions are the same as those in example 1, which is the detection of the related substances of rifaximin tablets, capsules and soft capsules.

Preparation of sample solution:

rifaximin tablets: taking rifaximin tablets, grinding, taking a proper amount, dissolving with ethanol to prepare 5 mg/mL^-1Filtering the solution to obtain a subsequent filtrate.

Rifaximin capsule: dissolving appropriate amount of rifaximin capsule content in ethanol to obtain 5 mg/mL^-1Filtering the solution to obtain a subsequent filtrate.

And (3) rifaximin soft capsules: taking the rifaximin soft capsule, washing the content into a proper volumetric flask by using ethanol, and quantitatively diluting to prepare 5 mg/mL^-1Filtering the solution to obtain a subsequent filtrate.

Typical chromatograms are shown in FIGS. 9-13.

Fig. 9-11 respectively adopt rifaximin tablets of a raw material source a, a raw material source B and a raw material source C, wherein in fig. 9, a chromatographic peak 1 is impurity B, 2 is rifaximin, 3 is rifaximin Y and hydroxyrifaximin, and 4 is a byproduct unique to the process of the source a; in fig. 10 to 11, peak 1 is impurity B, 2 is rifaximin, 3 is rifaximin Y and hydroxyrifaximin. Fig. 12 shows rifaximin capsules from raw material source B, in which chromatographic peak 1 is unknown impurity, 2 is impurity B, 3 is rifaximin, and 4 is rifaximin Y and hydroxyrifaximin. Fig. 13 shows rifaximin soft capsules from raw material source B, wherein chromatographic peak 1 is impurity B, 2 is rifaximin, 3 is rifaximin Y and hydroxyrifaximin, and 4 is unknown impurity.

As can be seen from figures 9-13, the main impurities in the chromatogram of the related substances of the rifaximin preparation are basically consistent with those in the raw materials of the corresponding sources. The impurities which are not detected in the raw materials appear before the impurities B in the rifaximin capsules, and the impurities which are not detected in the raw materials appear after the rifaximin Y and the hydroxyl rifaximin in the rifaximin soft capsules, and can be further confirmed by connecting with a mass spectrometer in series.

Example 4

This example investigates the effect of the type of modifier on the degree of separation between rifaximin and impurity a and the chromatographic retention behavior of rifamycin B.

As can be seen from fig. 3, rifamycin B is a strong retention impurity among all known impurities. Different modifiers mainly affect the degree of separation between rifaximin and impurity a and the chromatographic retention behavior of rifamycin B. The rifaximin concentration is 5 mg/mL^-1The amount of impurities a and rifamycin B was 1% as an example, and the effect of different classes of modifiers on the degree of separation between rifaximin and impurities a and on the chromatographic retention of rifamycin B was examined.

TABLE 2 Effect of different modifiers on the degree of separation and Retention behavior of rifamycin B

The study of this example shows that: the proper modifier can be selected according to the impurity types in rifaximin to be detected and the preparation thereof and the concentration of the rifaximin. The chromatographic separation system used in example 1 of the present invention uses ethanol-isopropanol (90:10) as a modifier and rifaximin concentration is 5 mg/mL^-1The separation degree of the difficult-to-separate substance pair and the strong retention of impurities are ensured to be eluted at a proper time.

Example 5

This example investigated the rifamycin B peak retention behavior at different pH conditions, which was the same as example 1, using only different pH values. The experimental results of this example are shown in fig. 14 and 15. As shown in FIG. 14, the rifamycin B has a broad peak profile, which is probably caused by the strong interaction between rifamycin B and the diol group under the pH condition. As can be seen from FIG. 15, the rifamycin B peak pattern is sharper.

The study of this example shows that: the modifier pH mainly affects the retention behavior and peak pattern of rifamycin B. The influence of different pH values (3.1-10.2) on the retention time and peak type of the rifamycin B is respectively examined. The results show that the rifamycin B retention is reduced and the peak profile is sharp with increasing pH. Therefore, the pH range of the chromatographic packing material is 3.1-10.2, and preferably 5.0-7.5 according to the property of the chromatographic packing material.

Example 6

This example verifies the limit of detection of rifaximin by the detection method of the present invention, and the experimental conditions are the same as those of example 1, and the experimental results are shown in fig. 16.

As is clear from the examples, the sample concentration in the measurement of the substance concerned was 5 mg/mL^-1The rifaximin detection limit is about 1 mug.mL^-1Corresponding to 0.00002%, the sensitivity was high (fig. 16). The sample injection amount of the invention is 2 mu L, and the sample concentration and the sample injection amount can be adjusted according to the size of a quantitative ring of a selected instrument.

According to the embodiment, the chromatographic separation system established by the invention takes the lower alcohol and the carbon dioxide as the mobile phase, so that the use of a large amount of organic solvents is avoided, the analysis cost is low, and the harm of the organic solvents to the body and the environment is reduced; importantly, the main byproducts and the degradation impurities related to the raw material production process and the main process impurities related to the preparation are well separated, the response is high, and the rifaximin and the impurities are well separated; the analysis time is short, the analysis efficiency is greatly improved, and the quality of the rifaximin preparation in the preparation process and the storage process can be better reflected and monitored; can be directly connected with a mass spectrum in series, identifies unknown impurities and analyzes the correlation with the production process, and provides a basis for the improvement of the process.

The detection method provided by the embodiment of the invention can be used for detecting rifaximin raw material and related substances and content of preparation thereof by supercritical fluid chromatography, and has the advantages of high speed and high flux.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims

1. A method for detecting rifaximin raw materials and preparation components thereof is characterized in that the detection method is a supercritical fluid chromatography method, and comprises the following steps:

step 1) dissolving the rifaximin raw material or preparation by using ethanol to prepare a sample;

step 2) subjecting the sample to gradient elution in a chromatographic column, wherein the mobile phase used is: the mobile phase A is carbon dioxide, and the mobile phase B is ethanol-isopropanol, wherein the ethanol-isopropanol is mixed according to a volume ratio of 90: 10;

wherein the components of the sample comprise rifaximin and impurities comprising rifaximin Y + hydroxyrifaximin, impurity a and impurity B;

wherein the chromatographic column used is Torus DIOL with specification of 3.0mm × 100mm and 1.7 μm; the detection wavelength is 240 nm; the flow rate is 1.2mL min-1; the sample injection volume is 2 mu L; the column temperature was 35 ℃; back pressure 2000 psi;

the conditions of the gradient elution are as follows: 0-1.8min, B10-18%, 1.8-4min, B18-35%, 4-6min, B35-50%, 6-7min, B50%, maintain for 1min, and reach B10% after 7.1 min.

2. The method for detecting rifaximin raw material and rifaximin preparation components according to claim 1, wherein the rifaximin concentration in a sample obtained by dissolving raw material or preparation with ethanol is 5-10 mgmL^-1。

3. The method for detecting rifaximin raw material and the components of the rifaximin preparation according to any one of claims 1 to 2, wherein the pH value of the ethanol is 3.1 to 10.2, and the pH regulator used in the method comprises formic acid, glacial acetic acid, trifluoroacetic acid and ammonia solution.

4. The method of claim 1, wherein the detector is a UPCC phase chromatograph equipped with UV detector, binary gradient pump, and autosampler.

5. The method for detecting rifaximin raw material and preparation components thereof according to claim 1, wherein the limit of detection of rifaximin is 1 μ gmL^-1。