阅读说明：本技术 3-卤代-7-（4-溴苯甲酰基）-1氢-吲哚中异构体杂质的分离检测方法及应用 (Separation and detection method for isomer impurities in 3-halogenated-7- (4-bromobenzoyl) -1-hydro-indole and application thereof ) 是由 路起飞 孔庆兰 郑项元 于 2020-06-30 设计创作，主要内容包括：本发明提供了一种3-卤代-7-(4-溴苯甲酰基)-1氢-吲哚中异构体杂质的分离检测方法及应用,涉及分析化学技术领域。一种3-卤代-7-(4-溴苯甲酰基)-1氢-吲哚中异构体杂质的分离检测方法,采用高效液相色谱法,色谱条件包括：采用正相手性色谱柱；以正己烷-异丙醇混合溶液为流动相；等度洗脱；所述异构体杂质为3-卤代-7-(3-溴苯甲酰基)-1氢-吲哚。本发明的方法经过系统方法学验证,系统适应性好、专属性强、耐用性好、灵敏度高,可用于原料药溴芬酸钠的原料及其异构体杂质的测定和分离,提高了溴芬酸钠滴眼液使用的安全性和有效性。(The invention provides a separation and detection method and application of isomer impurities in 3-halo-7- (4-bromobenzoyl) -1-hydro-indole, and relates to the technical field of analytical chemistry. A separation and detection method of isomer impurities in 3-halo-7- (4-bromobenzoyl) -1-hydro-indole adopts high performance liquid chromatography, and the chromatographic conditions comprise: adopting a normal-phase chiral chromatographic column; taking a n-hexane-isopropanol mixed solution as a mobile phase; isocratic elution; the isomer impurity is 3-halo-7- (3-bromobenzoyl) -1 h-indole. The method is verified by a systematic methodology, has good systematic adaptability, strong specificity, good durability and high sensitivity, can be used for measuring and separating impurities of the raw material of the bulk drug bromfenac sodium and isomers thereof, and improves the safety and effectiveness of the bromfenac sodium eye drops.)

1. A separation and detection method of isomer impurities in 3-halo-7- (4-bromobenzoyl) -1H-indole is characterized in that high performance liquid chromatography is adopted, and the chromatographic conditions comprise: adopting a normal-phase chiral chromatographic column; taking a n-hexane-isopropanol mixed solution as a mobile phase; isocratic elution;

the structural formula of the 3-halo-7- (4-bromobenzoyl) -1-hydro-indole is as follows:

the isomer impurity is 3-halo-7- (3-bromobenzoyl) -1H-indole, and the structural formula is as follows:

wherein X is selected from Cl or Br.

2. The method for separating and detecting the isomeric impurities in 3-halo-7- (4-bromobenzoyl) -1-hydro-indole as claimed in claim 1, wherein X is Cl, i.e. the 3-halo-7- (4-bromobenzoyl) -1-hydro-indole is 3-chloro-7- (4-bromobenzoyl) -1-hydro-indole and the isomeric impurity is 3-chloro-7- (3-bromobenzoyl) -1-hydro-indole.

3. The method for separating and detecting isomer impurities in 3-halo-7- (4-bromobenzoyl) -1 h-indole as claimed in claim 1 or 2, wherein the volume ratio of n-hexane to isopropanol is (92-98): (2-8).

4. The method for separating and detecting isomer impurities in 3-halo-7- (4-bromobenzoyl) -1-hydro-indole as claimed in claim 3, wherein the detector used is an ultraviolet detector with a detection wavelength of 215-225 nm.

5. The method for separating and detecting isomer impurities in 3-halo-7- (4-bromobenzoyl) -1 h-indole according to any one of claims 1, 2 or 4, wherein the column temperature used is 25-35 ℃.

6. The method for separating and detecting the isomer impurities in 3-chloro-7- (4-bromobenzoyl) -1 h-indole as claimed in claim 5, wherein the flow rate used is 0.9-1.1 mL/min.

7. The method for separating and detecting isomer impurities in 3-halo-7- (4-bromobenzoyl) -1-hydro-indole as claimed in any one of claims 1, 2, 4 or 6, wherein the normal phase chiral chromatographic column uses covalently bonded polysaccharide derivative spherical silica gel as a filler.

8. The method for separating and detecting the isomer impurities in 3-halo-7- (4-bromobenzoyl) -1-hydro-indole as claimed in claim 7, wherein the high performance liquid chromatography is adopted, and the chromatographic conditions comprise: using the Japan Daiiol CoAn IA chromatography column; taking a n-hexane-isopropanol mixed solution as a mobile phase, wherein the volume ratio of the n-hexane to the isopropanol is (93-97): (3-7); the detector is an ultraviolet detector, and the detection wavelength is 218-222 nm; the column temperature is 28-32 ℃; the flow rate is 0.95-1.05 mL/min; isocratic elution.

9. The method for separating and detecting the isomer impurities in 3-halo-7- (4-bromobenzoyl) -1-hydro-indole as claimed in claim 8, wherein the high performance liquid chromatography is adopted, and the chromatographic conditions comprise: using the Japan Daiiol CoAn IA chromatography column; taking a mixed solution of n-hexane and isopropanol as a mobile phase, wherein the n-hexane and the isopropanol areThe volume ratio of the alcohol is 95: 5; the detector is an ultraviolet detector, and the detection wavelength is 220 nm; the column temperature used was 30 ℃; the flow rate used was 1.0 mL/min; isocratic elution.

10. Use of the separation and detection method according to any one of claims 1 to 9 for quality control of a bromfenac sodium raw material or a preparation thereof.

Technical Field

The invention relates to the technical field of analytical chemistry, in particular to a separation and detection method and application of isomer impurities in 3-halogenated-7- (4-bromobenzoyl) -1-hydro-indole.

Background

Bromfenac sodium, the chemical name of which is 2-amino-3- (4-bromobenzoyl) sodium phenylacetate, is a non-steroidal anti-inflammatory drug. Bromfenac sodium eye drops were first introduced in japan in 2000 by Senju corporation and used for the treatment of conjunctivitis, blepharitis, panniculitis (including superior panniculitis), postoperative inflammation, and the like.

At present, the mainstream synthesis method of bromfenac sodium comprises the steps of carrying out Friedel-crafts acylation by taking p-bromobenzonitrile and indoline as raw materials and boron trichloride and aluminum trichloride as catalysts, and then preparing the bromfenac sodium by active manganese dioxide oxidation, NBS or NCS halogenation, phosphoric acid hydrolysis and sodium hydroxide hydrolysis salification, wherein the synthesis route is as follows:

3-halo-7- (4-bromobenzoyl) -1 h-indole is the key intermediate of bromfenac sodium, while 3-halo-7- (3-bromobenzoyl) -1 h-indole is the isomer impurity, which is generated after the introduction of the isomer m-bromobenzonitrile in p-bromobenzonitrile as the starting material.

Because the impurity is a positional isomer of 3-halogenated-7- (4-bromobenzoyl) -1H-indole, the two have similar polarities, and the common reversed-phase chromatographic elution system is used for detection, so that the two are difficult to be effectively separated. Considering that the control of the impurities of the starting materials of the raw materials is crucial to the quality of the final product of the bromfenac sodium, the quality control of the raw materials directly influences the quality of the final product of the preparation. At present, the impurities and the detection method thereof have not been reported in documents. Therefore, the invention has important significance on the quality of the bromfenac sodium raw material and the preparation thereof.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The main object of the present invention is to provide a method for separating and detecting isomer impurities in 3-halo-7- (4-bromobenzoyl) -1 h-indole, which is intended to at least partially solve at least one of the above technical problems.

As a first aspect of the invention, the invention provides a separation and detection method of isomer impurities in 3-halo-7- (4-bromobenzoyl) -1-hydro-indole, which adopts high performance liquid chromatography, and the chromatographic conditions comprise: adopting a normal-phase chiral chromatographic column; taking a n-hexane-isopropanol mixed solution as a mobile phase; isocratic elution;

the structural formula of the 3-halo-7- (4-bromobenzoyl) -1-hydro-indole is as follows:

the isomer impurity is 3-halo-7- (3-bromobenzoyl) -1H-indole, and the structural formula is as follows:

wherein X is selected from Cl or Br.

Further, X is selected from Cl, i.e., the 3-halo-7- (4-bromobenzoyl) -1 h-indole is 3-chloro-7- (4-bromobenzoyl) -1 h-indole with the isomeric impurity of 3-chloro-7- (3-bromobenzoyl) -1 h-indole.

Further, the volume ratio of the n-hexane to the isopropanol is (92-98): (2-8).

Further, the volume ratio of the n-hexane to the isopropanol is (93-97): (3-7).

Further, the detector used is an ultraviolet detector, and the detection wavelength is 215-225 nm.

Further, the detector used is an ultraviolet detector, and the detection wavelength is 218-222 nm.

Further, the column temperature is 25-35 deg.C

Further, the column temperature is 28-32 deg.C

Further, the flow rate used is 0.9-1.1mL/min

Further, the flow rate used is 0.95-1.05mL/min

Further, the normal-phase chiral chromatographic column takes covalently bonded polysaccharide derivative spherical silica gel as a filler.

Further, high performance liquid chromatography is adopted, and the chromatographic conditions comprise: using the Japan Daiiol CoAn IA chromatography column; taking a n-hexane-isopropanol mixed solution as a mobile phase, wherein the volume ratio of the n-hexane to the isopropanol is (93-97): (3-7); the detector is an ultraviolet detector, and the detection wavelength is 218-222 nm; the column temperature is 28-32 ℃; the flow rate is 0.95-1.05 mL/min; isocratic elution.

Further, high performance liquid chromatography is adopted, and the chromatographic conditions comprise: using the Japan Daiiol CoAn IA chromatography column; taking a n-hexane-isopropanol mixed solution as a mobile phase, wherein the volume ratio of n-hexane to isopropanol is 95: 5; the detector is an ultraviolet detector, and the detection wavelength is 220 nm; the column temperature used was 30 ℃; the flow rate used was 1.0 mL/min; isocratic elution.

As a second aspect of the invention, the invention provides the use of the above separation and detection method in the quality control of bromfenac sodium raw material or preparation.

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

according to the separation and detection method for isomer impurities in 3-halo-7- (4-bromobenzoyl) -1-hydro-indole, provided by the invention, the 3-halo-7- (4-bromobenzoyl) -1-hydro-indole and the isomer impurities thereof are effectively separated through the optimal design of a mobile phase and a chromatographic column, and the separation degree is good and is not interfered by other impurities. Verified by a systematic methodology, the system has good adaptability, strong specificity, good durability and high sensitivity, can be used for separating and detecting isomer impurities in the raw material of the bromfenac sodium, and improves the safety and effectiveness of the bromfenac sodium eye drops.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is an HPLC chromatogram of a LD-Z1 control solution of example 1-1;

FIG. 2 is an HPLC chromatogram of a control solution of chloride from example 1-1;

FIG. 3 is an HPLC chromatogram of the mixed solution of LD-Z1+ chloride in example 1-1;

FIG. 4 is an HPLC chromatogram of a detection limit solution in example 2;

FIG. 5 is an HPLC chromatogram of the quantitation limit solution in example 2;

FIG. 6 is an HPLC chromatogram of a mixed solution of the test sample + LD-Z1 in example 3-1;

FIG. 7 is an HPLC chromatogram of a mixed solution of the test sample + LD-Z1 in example 3-2;

FIG. 8 is an HPLC chromatogram of a mixed solution of the test sample + LD-Z1 in example 3-3;

FIG. 9 is an HPLC chromatogram of a mixed solution of the test sample + LD-Z1 in example 3-4;

FIG. 10 is a spectrum of LD-Z1 of a mixed solution of the test sample + LD-Z1 in example 3-5;

FIG. 11 is an HPLC chromatogram of a mixed solution of the test sample + LD-Z1 in example 3-6;

FIG. 12 is an HPLC chromatogram of a mixed solution of the test sample + LD-Z1 in examples 3 to 7;

FIG. 13 is an HPLC chromatogram of a mixed solution of the test sample + LD-Z1 in examples 3 to 8;

FIG. 14 is an HPLC chromatogram of a mixed solution of the test sample + LD-Z1 in examples 3 to 9;

FIG. 15 is an HPLC chromatogram of the LD-Z1 control solution of control example 1;

FIG. 16 is an HPLC chromatogram of a control solution of chloride in control example 1;

FIG. 17 is an HPLC chromatogram of a LD-Z1 control solution in control example 2;

FIG. 18 is an HPLC chromatogram of a control solution of chloride in control example 2;

FIG. 19 is an HPLC chromatogram of a LD-Z1 control solution in control example 3;

FIG. 20 is an HPLC chromatogram of a control solution of chloride in control example 3;

FIG. 21 is an HPLC chromatogram of a LD-Z1 control solution in control example 4;

FIG. 22 is an HPLC chromatogram of a control solution of chloride in control example 4;

FIG. 23 is an HPLC chromatogram of a LD-Z1 control solution in control example 5;

FIG. 24 is an HPLC chromatogram of a control solution of chloride in control example 5.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples were carried out under the conventional conditions, unless otherwise specified. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The invention uses chloro compound to represent 3-chloro-7- (4-bromobenzoyl) -1-hydro-indole, uses LD-Z1 to represent 3-chloro-7- (3-bromobenzoyl) -1-hydro-indole, uses bromo compound to represent 3-bromo-7- (4-bromobenzoyl) -1-hydro-indole, and uses LD-Z2 to represent 3-bromo-7- (3-bromobenzoyl) -1-hydro-indole.

LD-Z1 control, LD-Z2 control, chloride control, and bromide control were from MalladiDrugs & pharmaceuticals Ltd.

Example 1 specificity test

LD-Z1 control solution: precisely weighing an appropriate amount of LD-Z1 reference substance, adding the sample solution to dissolve and dilute to obtain a solution containing 0.4mg per 1mL, and using the solution as LD-Z1 reference substance stock solution. Precisely measuring 0.5mL of LD-Z1 reference stock solution, placing in a 100mL measuring flask, diluting to scale with the sample solution, and shaking to obtain LD-Z1 reference solution.

Chloride control solution: accurately weighing a proper amount of the chloride reference substance, adding the sample solution to dissolve and dilute the chloride reference substance to prepare a solution containing 0.4mg of chloride per 1mL, and using the solution as the chloride reference substance stock solution. Precisely measuring 1mL of the chloride reference stock solution, placing the chloride reference stock solution in a 100mL measuring flask, diluting the chloride reference stock solution to a scale with the sample solution, and shaking up to obtain the chloride reference stock solution.

LD-Z1+ chloride mixed solution: precisely measuring 0.5mL of each of the LD-Z1 reference substance stock solution and the chloride reference substance stock solution, placing the LD-Z1 reference substance stock solution and the chloride reference substance stock solution into a 100mL measuring flask, diluting the LD-Z1 reference substance stock solution to a scale with a sample solution, and shaking up to obtain an LD-Z1+ chloride mixed solution.

Sample dissolving liquid: n-hexane-isopropanol is used as a solvent, and the volume ratio of the n-hexane to the isopropanol is 1: 1.

examples 1 to 1

Chromatographic conditions are as follows:

a chromatographic column:IA(4.6mm×250mm，5μm)；

mobile phase: n-hexane-isopropanol is used as a mobile phase, and the volume ratio of the n-hexane to the isopropanol is 95: 5;

detection wavelength: an ultraviolet detector with the detection wavelength of 220 nm;

flow rate: 1.0 mL/min;

column temperature: 30 ℃;

sample introduction amount: 20 mu L of the solution;

isocratic elution.

The HPLC chromatogram of the LD-Z1 control solution is shown in FIG. 1.

As can be seen from FIG. 1, the retention time of LD-Z1 was 21.185 min.

The HPLC profile of the chlorinated control solution is shown in FIG. 2.

As can be seen from FIG. 2, the retention time of the chloride was 27.860 min.

The HPLC chromatogram of the LD-Z1+ chloride mixed solution is shown in FIG. 3, and the detection data is shown in Table 1.

TABLE 1 LD-Z1+ chloride mixture solution test results

As can be seen from FIG. 3 and Table 1, the separation degree between LD-Z1 and the chloride is greater than 2, which meets the requirement, and the detection method under the chromatographic condition of the invention can completely separate LD-Z1 from the chloride without interference.

Examples 1 to 2

Chromatographic conditions are as follows:

a chromatographic column:IA(4.6mm×250mm，5μm)；

mobile phase: n-hexane-isopropanol is used as a mobile phase, and the volume ratio of the n-hexane to the isopropanol is 98: 2;

detection wavelength: an ultraviolet detector with the detection wavelength of 215 nm;

flow rate: 1.1 mL/min;

column temperature: 35 ℃;

sample introduction amount: 20 mu L of the solution;

isocratic elution.

The data of the detection of the LD-Z1+ chloride mixed solution are shown in Table 2.

TABLE 2 LD-Z1+ chloride mixture solution test results

As can be seen from Table 2, the separation degree between LD-Z1 and the chloride is more than 2, which meets the requirement, and the detection method under the chromatographic condition of the invention can completely separate LD-Z1 from the chloride without interference.

Examples 1 to 3

Chromatographic conditions are as follows:

a chromatographic column:IA(4.6mm×250mm，5μm)

mobile phase: n-hexane-isopropanol is used as a mobile phase, and the volume ratio of the n-hexane to the isopropanol is 92: 8;

detection wavelength: an ultraviolet detector with the detection wavelength of 225 nm;

flow rate: 0.9 mL/min;

column temperature: 25 ℃;

sample introduction amount: 20 mu L of the solution;

isocratic elution.

The data of the detection of the LD-Z1+ chloride mixed solution are shown in Table 3.

TABLE 3 LD-Z1+ chloride mixture solution test results

As can be seen from Table 3, the separation degree between LD-Z1 and the chloride is more than 2, which meets the requirement, and the detection method under the chromatographic condition of the invention can completely separate LD-Z1 from the chloride without interference.

Examples 1 to 4

Chromatographic conditions are as follows:

a chromatographic column:IA(4.6mm×250mm，5μm)

mobile phase: n-hexane-isopropanol is used as a mobile phase, and the volume ratio of the two is 93: 7;

detection wavelength: an ultraviolet detector with the detection wavelength of 222 nm;

flow rate: 1.05 mL/min;

column temperature: at 32 ℃;

sample introduction amount: 20 mu L of the solution;

isocratic elution.

The data of the LD-Z1+ chloride mixed solution are shown in Table 4.

TABLE 4 LD-Z1+ chloride mixture solution test results

As can be seen from Table 4, the separation degree between LD-Z1 and the chloride is more than 2, which meets the requirement, and the detection method under the chromatographic condition of the invention can completely separate LD-Z1 from the chloride without interference.

Examples 1 to 5

Chromatographic conditions are as follows:

a chromatographic column:IA(4.6mm×250mm，5μm)；

mobile phase: n-hexane-isopropanol is used as a mobile phase, and the volume ratio of the two is 97: 3;

detection wavelength: an ultraviolet detector with the detection wavelength of 218 nm;

flow rate: 0.95 mL/min;

column temperature: 28 ℃;

sample introduction amount: 20 mu L of the solution;

isocratic elution.

The data of the LD-Z1+ chloride mixed solution are shown in Table 5.

TABLE 5 LD-Z1+ chloride mixture solution test results

As can be seen from Table 5, the separation degree between LD-Z1 and the chloride is more than 2, which meets the requirement, and the detection method under the chromatographic condition of the invention can completely separate LD-Z1 from the chloride without interference.

Example 2 sensitivity test

LD-Z1 impurity control stock solution: precisely weighing a proper amount of LD-Z1 impurity reference substance, adding the sample solution to dissolve and dilute the same to prepare a solution containing 0.4mg of LD-Z1 impurity reference substance per 1mL, and using the solution as LD-Z1 impurity reference substance stock solution.

Chloride control stock solution: accurately weighing a proper amount of the chloride reference substance, adding the sample solution to dissolve and dilute the chloride reference substance to prepare a solution containing 0.4mg of chloride per 1mL, and using the solution as the chloride reference substance stock solution.

LD-Z1+ chloride mixed stock solution: precisely measuring 1mL of each of the LD-Z1 reference substance stock solution and the chloride reference substance stock solution, placing the LD-Z1 reference substance stock solution and the chloride reference substance stock solution into a 100mL measuring flask, diluting the LD-Z1+ chloride reference substance stock solution to a scale by using the sample solution, and shaking up to obtain the mixed stock solution of the LD-Z1+ chloride.

And (3) quantitative limiting solution, namely precisely measuring 0.5mL of LD-Z1+ chloride mixed stock solution, placing the LD-Z1+ chloride mixed stock solution into a 20mL measuring flask, diluting the LD-Z1+ chloride mixed stock solution to a scale with a sample solution, and shaking the mixture uniformly to obtain the quantitative limiting solution.

And (3) measuring 1.6mL of LD-Z1+ chloride mixed stock solution precisely, placing the mixed stock solution into a 200mL measuring flask, diluting the mixed stock solution to a scale by using a sample solution, and shaking the mixed stock solution uniformly to serve as a detection limiting solution.

Sample dissolving liquid: n-hexane-isopropanol is used as a solvent, and the volume ratio of the n-hexane to the isopropanol is 1: 1.

chromatographic conditions are as follows:

a chromatographic column:IA(4.6mm×250mm，5μm)；

mobile phase: n-hexane-isopropanol is used as a mobile phase, and the volume ratio of the n-hexane to the isopropanol is 95: 5;

detection wavelength: an ultraviolet detector with the detection wavelength of 220 nm;

flow rate: 1.0 mL/min;

column temperature: 30 ℃;

sample introduction amount: 20 mu L of the solution;

isocratic elution.

The HPLC chromatogram of the detection limit solution is shown in FIG. 4, and the detection data is shown in Table 6.

TABLE 6 detection limit solution detection results

As can be seen from FIG. 4 and Table 6, when the concentration of LD-Z1 is 0.0316 μ g/mL, it is equivalent to 0.008% of the concentration of the test sample, and the signal-to-noise ratio is greater than 3, and when the concentration of chloride is 0.0301 μ g/mL, it is equivalent to 0.008% of the concentration of the test sample, and the signal-to-noise ratio is greater than 3, which meets the requirement of detection limit.

The HPLC spectrum of the quantitative limiting solution is shown in FIG. 5, and the detection data is shown in Table 7.

TABLE 7 quantitative limiting solution test results

As can be seen from FIG. 5 and Table 7, when the concentration of LD-Z1 is 0.1054. mu.g/mL, the concentration is equivalent to 0.025% of the concentration of the sample, and the signal-to-noise ratio is greater than 10, and when the concentration of chloride is 0.1005. mu.g/mL, the concentration is equivalent to 0.025% of the concentration of the sample, and the signal-to-noise ratio is greater than 10, which meets the requirement of the limit of quantitation.

EXAMPLE 3 durability test

LD-Z1 control stock solution: precisely weighing an appropriate amount of LD-Z1 impurity reference substance, adding the sample solution to dissolve and dilute to prepare a solution containing 0.4mg per 1mL, and using the solution as LD-Z1 reference substance stock solution.

Chloride control stock solution: accurately weighing a proper amount of the chloride reference substance, adding the sample solution to dissolve and dilute the chloride reference substance to prepare a solution containing 0.4mg of chloride per 1mL, and using the solution as the chloride reference substance stock solution.

LD-Z1+ chloride mixed solution: precisely measuring 1mL of each of the LD-Z1 reference substance stock solution and the chloride reference substance stock solution, placing the LD-Z1 reference substance stock solution and the chloride reference substance stock solution into a 100mL measuring flask, diluting the LD-Z1+ chloride reference substance stock solution to a scale by using the sample solution, and shaking up to obtain a mixed solution of the LD-Z1+ chloride.

Test sample + LD-Z1 mixed solution: precisely weighing 10mg of chloride, placing the chloride in a 25mL measuring flask, ultrasonically dissolving the chloride by using a sample solution, adding 2.5mLLD-Z1 reference substance solution, diluting the sample solution to a scale by using the sample solution, and shaking up to be used as a mixed solution of a test sample and LD-Z1.

Sample dissolving liquid: n-hexane-isopropanol is used as a solvent, and the volume ratio of the n-hexane to the isopropanol is 1: 1.

chromatographic conditions are as follows:

a chromatographic column:IA(4.6mm×250mm，5μm)；

the conditions of detection wavelength, column temperature, flow rate and mobile phase proportion are as follows:

sample introduction amount: 20 mu L of the solution;

isocratic elution.

Example 3-1

The data of the LD-Z1+ chloride mixed solution are shown in Table 8.

TABLE 8 detection results of LD-Z1+ chloride mixed solution

The HPLC chromatogram of the test sample + LD-Z1 mixed solution is shown in FIG. 6, and the detection data is shown in Table 9.

TABLE 9 test results of test sample + LD-Z1 mixed solution

Examples 3 to 2

The data of the LD-Z1+ chloride mixed solution are shown in Table 10.

TABLE 10 LD-Z1+ chloride mixture solution test results

The HPLC chromatogram of the test sample + LD-Z1 mixed solution is shown in FIG. 7, and the detection data is shown in Table 11.

TABLE 11 test results of test article + LD-Z1 Mixed solution

Examples 3 to 3

The data for the detection of the LD-Z1+ chloride mixed solution are shown in Table 12.

TABLE 12 LD-Z1+ chloride mixture solution test results

The HPLC chromatogram of the test sample + LD-Z1 mixed solution is shown in FIG. 8, and the detection data is shown in Table 13.

TABLE 13 test results of test article + LD-Z1 Mixed solution

Examples 3 to 4

The data of the LD-Z1+ chloride mixed solution are shown in Table 14.

TABLE 14 LD-Z1+ chloride mixture solution test results

The HPLC chromatogram of the test sample + LD-Z1 mixed solution is shown in FIG. 9, and the detection data is shown in Table 15.

TABLE 15 test results of test article + LD-Z1 mixed solution

Examples 3 to 5

The data of the LD-Z1+ chloride mixed solution are shown in Table 16.

TABLE 16 LD-Z1+ chloride mixture solution test results

The HPLC chromatogram of the test sample + LD-Z1 mixed solution is shown in FIG. 10, and the detection data is shown in Table 17.

TABLE 17 test results of test sample + LD-Z1 mixed solution

Examples 3 to 6

The data of the LD-Z1+ chloride mixed solution are shown in Table 18.

TABLE 18 LD-Z1+ chloride mixture solution test results

The HPLC chromatogram of the test sample + LD-Z1 mixed solution is shown in FIG. 11, and the detection data is shown in Table 19.

TABLE 19 test results of test article + LD-Z1 mixed solution

Examples 3 to 7

The data of the LD-Z1+ chloride mixed solution are shown in Table 20.

TABLE 20 LD-Z1+ chloride mixture solution test results

The HPLC chromatogram of the mixed solution of the test sample + LD-Z1 is shown in FIG. 12, and the detection data is shown in Table 21.

TABLE 21 test results of test sample + LD-Z1 mixed solution

Examples 3 to 8

The data of the LD-Z1+ chloride mixed solution are shown in Table 22.

TABLE 22 LD-Z1+ chloride mixture solution test results

The HPLC chromatogram of the mixed solution of the test sample + LD-Z1 is shown in FIG. 13, and the detection data is shown in Table 23.

TABLE 23 test results of test article + LD-Z1 mixed solution

Examples 3 to 9

The data of the LD-Z1+ chloride mixed solution are shown in Table 24.

TABLE 24 LD-Z1+ chloride mixture solution test results

The HPLC chromatogram of the test sample + LD-Z1 mixed solution is shown in FIG. 14, and the detection data is shown in Table 25.

TABLE 25 test results of test sample + LD-Z1 Mixed solution

As is clear from FIGS. 6 to 14 and tables 8 to 25, LD-Z1 was detected well even when the detection wavelength, the column temperature, and the flow rate ratio of the mobile phase were slightly changed, indicating that the durability of the detection method under the chromatographic conditions of the present invention was good.

Comparative example 1

LD-Z1 control solution: precisely weighing an appropriate amount of LD-Z1 reference substance, adding the sample solution, dissolving, and diluting to obtain a solution containing 0.5mg per 1mL as LD-Z1 reference substance stock solution. Precisely measuring 1mL of LD-Z1 reference stock solution, placing in a 10mL measuring flask, diluting to scale with the sample solution, and shaking to obtain LD-Z1 reference stock solution.

Chloride control solution: accurately weighing a proper amount of the chloride reference substance, adding the sample solution to dissolve and dilute the chloride reference substance to prepare a solution containing 0.5mg of chloride per 1mL, and using the solution as the chloride reference substance solution.

Sample dissolving liquid: and (3) acetonitrile.

Chromatographic conditions are as follows:

a chromatographic column: agela XBP C18(L) (4.6 mm. times.250 mm, 5 μm);

mobile phase: the mobile phase is methanol-water, and the volume ratio of the methanol to the water is 70: 30, of a nitrogen-containing gas;

detection wavelength: an ultraviolet detector with the detection wavelength of 220 nm;

column temperature: 30 ℃;

flow rate: 1.0 mL/min;

sample introduction amount: 20 mu L of the solution;

isocratic elution.

The HPLC chromatogram of the LD-Z1 control solution is shown in FIG. 15, and the HPLC chromatogram of the chloride control solution is shown in FIG. 16.

As can be seen from FIG. 15, the retention time of LD-Z1 is 38.114 min; as is clear from FIG. 16, the retention time of the chlorinated substance was 37.997min, and the two retention times were coincident with each other and could not be separated.

Comparative example 2

The chromatographic conditions of this comparative example differ from those of comparative example 1 in that the mobile phase: the mobile phase is methanol-water, and the volume ratio of the methanol to the water is 80: 20; the other conditions were the same.

The HPLC chromatogram of the LD-Z1 control solution is shown in FIG. 17, and the HPLC chromatogram of the chloride control solution is shown in FIG. 18.

As can be seen from FIG. 17, the retention time of LD-Z1 is 13.604 min; as is clear from FIG. 18, the retention time of the chlorinated substance was 13.487min, and the two retention times were coincident with each other and could not be separated.

Comparative example 3

The chromatographic conditions of this comparative example were different from those of comparative example 1 in that the column: agilent Pursuit 5 PFP (4.6 mm. times.250 mm, 5 μm); the other conditions were the same.

The HPLC chromatogram of the LD-Z1 control solution is shown in FIG. 19, and the HPLC chromatogram of the chloride control solution is shown in FIG. 20.

As can be seen from FIG. 19, the retention time of LD-Z1 is 27.184 min; as is clear from FIG. 20, the retention time of the chlorinated substance was 27.961min, and the two retention times were coincident with each other and could not be separated.

Comparative example 4

The chromatographic conditions of this comparative example differ from those of comparative example 1 in that the mobile phase: the mobile phase is acetonitrile-water, and the volume ratio of the acetonitrile to the water is 75: 25; the other conditions were the same.

The HPLC chromatogram of the LD-Z1 control solution is shown in FIG. 21, and the HPLC chromatogram of the chloride control solution is shown in FIG. 22.

As can be seen from FIG. 21, the retention time of LD-Z1 is 10.829 min; as is clear from FIG. 22, the retention time of the chlorinated substance was 10.955min, and the two retention times were coincident with each other and could not be separated.

Comparative example 5

The chromatographic conditions of this comparative example differ from those of comparative example 1 in that the mobile phase: the mobile phase is acetonitrile-water, and the volume ratio of the acetonitrile to the water is 60: 40; the other conditions were the same.

The HPLC chromatogram of the LD-Z1 control solution is shown in FIG. 23, and the HPLC chromatogram of the chloride control solution is shown in FIG. 24.

As can be seen from FIG. 23, the retention time of LD-Z1 is 29.663 min; as is clear from FIG. 24, the retention time of the chlorinated substance was 30.306min, and the two retention times were coincident with each other and could not be separated.

The separation effect of the invention on LD-Z2 in bromide is similar to that of LD-Z1 in chloride, and is not repeated here.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.