CN112986456A - HPLC analysis detection method of empagliflozin intermediate - Google Patents

Abstract

The invention discloses an HPLC (high performance liquid chromatography) analytical detection method of an engagliflozin intermediate, which uses a high performance liquid system, takes octadecylsilane chemically bonded silica as a chromatographic column, and takes a buffer salt-organic phase as a mobile phase for gradient elution, so that the engagliflozin intermediate and related substances thereof can be quantitatively measured, effective monitoring data is provided for the preparation process of the engagliflozin, the generation of by-products and impurities in the reaction is reduced, and the quality control of the engagliflozin is realized; through perfect methodology verification, the method is proved to have strong specificity, high accuracy and good stability.

Description

HPLC analysis detection method of empagliflozin intermediate

Technical Field

The application relates to the technical field of drug analysis, in particular to an HPLC (high performance liquid chromatography) analysis and detection method of an empagliflozin intermediate.

Background

Engagliflozin is a selective sodium-glucose cotransporter (SGLT) inhibitor useful for treating adult type II diabetes. The engeletin limits the reabsorption of most glucose in vivo through the selective inhibition effect on the sodium-glucose cotransporter, and promotes the massive excretion of glucose from urine to achieve the aim of controlling the blood sugar level.

In the process of synthesizing the empagliflozin, the purity of some key intermediates needs to be controlled so as to reduce the occurrence of side reactions and the generation of impurities, and then the yield and the purity of the empagliflozin are improved.

The establishment of an effective separation method for the empagliflozin intermediate has important significance for the preparation of the empagliflozin product.

Disclosure of Invention

The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the present application.

The synthesis of engagliflozin involves the following intermediate compounds: the chemical name of the compound is (2S,3R,4S,5R,6R) -2- (4-chloro-3- (4- (((S) -tetrahydrofuran-3-yl) oxy) benzyl) phenyl) -3,4, 5-tri ((trimethyl silicon) oxy) -6- (((trimethyl silicon) oxy) methyl) tetrahydro-2H-pyran-2-ol, the structure of which is shown as the following formula (I),

TMS in the formula (I) is trimethylsilyl.

There are 4 related substances (i.e. impurities) used and producible in the process of synthesizing the compound, which are respectively SM1, M1Z1, M1Z2 and M1Z3, and the structural formulas are respectively:

SM1

(S) -3- (4- (2-chloro-5-iodobenzyl) phenoxy) tetrahydrofuran

M1Z1

(S) -3- (4- ((4,4 ' -dichloro-3 ' - (4- ((R) -tetrahydrofuran-3-yl) oxy) benzyl) - [1,1 ' -biphenyl ] -3-yl) methyl) phenoxy) tetrahydrofuran

M1Z2

(S) -3- (4- (2-chlorobenzyl) phenoxy) tetrahydrofuran

M1Z3

(S) -4-chloro-3- (4- ((tetrahydrofuran-3-yl) oxy) benzyl) phenol.

The method can quantitatively determine the engagliflozin intermediate and related substances thereof, provides effective monitoring data for the preparation process of the engagliflozin, reduces the generation of byproducts and impurities in the reaction, and realizes the quality control of the engagliflozin.

The invention provides an analytical detection method of an empagliflozin intermediate, which comprises the following steps:

(1) preparing a test solution: taking a proper amount of the engagliflozin intermediate, adding a diluent to dissolve and quantitatively diluting to prepare a solution containing about 1mg of the engagliflozin intermediate in every 1ml, and taking the solution as a test solution; the diluent is one or more mixed solvents selected from acetonitrile, dimethyl sulfoxide, tetrahydrofuran and methanol;

(2) setting chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler of the chromatographic column; the detection wavelength is 220 nm-230 nm; performing gradient elution by adopting a mobile phase A and a mobile phase B as mobile phases, wherein the mobile phase A is a buffered saline solution, and the mobile phase B is acetonitrile; the flow rate of the mobile phase is 0.5-1.5 ml/min; the column temperature is 25-35 ℃;

(3) and (3) determination: and (2) injecting 5-50 mul of the test solution obtained in the step (1) into a high performance liquid chromatograph, and recording a chromatogram.

In the above analytical measurement method, the engletin intermediate is (2S,3R,4S,5R,6R) -2- (4-chloro-3- (4- (((S) -tetrahydrofuran-3-yl) oxy) benzyl) phenyl) -3,4, 5-tris ((trimethylsilyl) oxy) -6- (((trimethylsilyl) oxy) methyl) tetrahydro-2H-pyran-2-ol, and the structure thereof is represented by the following formula (I),

in the above analytical measurement method, the diluent is one or a mixed solvent of two or more selected from acetonitrile, dimethyl sulfoxide, tetrahydrofuran and methanol.

In the above analytical determination method, the diluent is a mixed solvent of dimethyl sulfoxide and acetonitrile, optionally, the volume ratio of dimethyl sulfoxide to acetonitrile in the mixed solvent is (10-30): (70-90), for example, 20: 80.

In the above analytical assay method, the column may be selected from the group consisting of Agilent, Welch brand, and others, preferably Agilent ZORBAX Eclipse Plus C184.6 x 100mm x 3.5 um.

In the above analytical determination method, the buffer salt is selected from one of acetate, phosphate and formate, preferably acetate. The salt may be a sodium, potassium or ammonium salt.

In the above analytical measurement method, the concentration of the buffered saline solution is 4mmol/L to 6 mmol/L; optionally, the pH of the buffered saline solution is 7.3-9.0.

In the above analytical determination method, the buffered saline solution was an ammonium acetate buffer solution having a concentration of 5mmol/L, and the pH was adjusted to 7.5 with aqueous ammonia.

In the above analytical measurement method, the procedure of the gradient elution is:

in the above analytical measurement method, the high performance liquid chromatograph is an Agilent 1260 liquid chromatograph;

a chromatographic column: c18(Agilent ZORBAX Eclipse Plus C184.6 x 100mm x 3.5 um);

the detection wavelength is 225 nm;

the flow rate is 1.0 ml/min;

column temperature: 30 ℃;

sample introduction volume: 10 μ l.

In the above analytical determination method, the mobile phase A was 5mmol/L ammonium acetate buffer solution (pH adjusted to 7.5 with ammonia water); the mobile phase B is acetonitrile; the gradient elution procedure was:

in the above analytical assay method, the preparing a test solution includes: taking appropriate amount of the engagliflozin intermediate, SM1, M1Z1, M1Z2 and M1Z3, adding a diluent to dissolve and quantitatively dilute to prepare a solution containing about 1mg of the engagliflozin intermediate, about 30 mu g of SM1, about 20 mu g M1Z1, about 20 mu g M1Z2 and about 20 mu g M1Z3 in each 1ml of solution as a test solution.

In one of the above analytical detection methods, the analytical detection method includes the steps of:

(1) preparing a test solution: taking a proper amount of the engagliflozin intermediate, adding a diluent to dissolve and quantitatively diluting to prepare a solution containing about 1mg of the engagliflozin intermediate in every 1ml, and taking the solution as a test solution; the diluent is a mixed solvent of dimethyl sulfoxide and acetonitrile in a volume ratio of 20: 80;

(2) setting chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler of the chromatographic column; the detection wavelength is 220 nm-230 nm; the mobile phase adopts a mobile phase A and a mobile phase B to carry out gradient elution; the mobile phase B is acetonitrile; the flow rate of the mobile phase is 0.5-1.5 ml/min; the column temperature is 25-35 ℃;

here, the mobile phase a was an ammonium acetate buffer solution having a concentration of 5mmol/L and adjusted to pH 7.5 with ammonia water;

the procedure for the gradient elution was:

(3) and (3) determination: and (2) taking 10 mu l of the test solution obtained in the step (1), injecting into a high performance liquid chromatograph, and recording a chromatogram.

The method uses octadecylsilane chemically bonded silica as a chromatographic column and uses a buffer salt-organic phase as a mobile phase for gradient elution, so that the method can quantitatively determine the engagliflozin intermediate and related substances, provide effective monitoring data for the preparation process of the engagliflozin, reduce the generation of byproducts and impurities in the reaction, and realize the quality control of the engagliflozin. Through perfect methodology verification, the method is proved to have strong specificity, high accuracy and good stability.

The methodology of the analytical determination method provided by the invention is verified, and the results are as follows:

additional features and advantages of the application will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the application. Other advantages of the present application may be realized and attained by the instrumentalities and combinations particularly pointed out in the specification and the drawings.

Drawings

The accompanying drawings are included to provide an understanding of the present disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the examples serve to explain the principles of the disclosure and not to limit the disclosure.

FIG. 1 is an HPLC plot of the Engelliflozin intermediate and related substances from example 1;

FIG. 2 is an HPLC plot of the Engelliflozin intermediate and related substances from example 2;

FIG. 3 is an HPLC plot of the Engelliflozin intermediate and related substances at example 3;

fig. 4 is an HPLC profile of the engagliflozin intermediate and related substances in example 4.

Detailed Description

Hereinafter, embodiments of the present invention will be described in detail in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be noted that the embodiments and features of the embodiments in the present application may be arbitrarily combined with each other without conflict.

The present invention will be explained in more detail with reference to examples, which are provided only for illustrating the technical solutions of the present invention and are not intended to limit the spirit and scope of the present invention.

Test instrument for experiments

High performance liquid chromatograph: agilent 1260 liquid chromatograph, DAD detector.

Example 1

Chromatographic conditions

A chromatographic column: c18(Agilent ZORBAX Eclipse Plus C184.6X 100mm, 3.5 μm)

The detection wavelength is 225nm

The flow rate was 1.0ml/min

Column temperature: 30 deg.C

Sample introduction volume: 10 μ l

Mobile phase: taking 5mmol/L ammonium acetate buffer solution (pH is adjusted to 7.5 by ammonia water) as a mobile phase A, taking acetonitrile as a mobile phase B, and carrying out gradient elution according to the following table;

experimental procedure

Appropriate amounts of the engagliflozin intermediate and related substances are taken to prepare a solution containing about 1mg of the engagliflozin intermediate, about 30 μ g of SM1, about 20 μ g of M1Z1, about 20 μ g of M1Z2 and about 20 μ g of M1Z3 in each 1 ml. According to the above chromatographic conditions, 10. mu.l of the resulting mixture was precisely measured, and the resulting mixture was injected into a liquid chromatograph, and the chromatogram was recorded.

The result is shown in figure 1, and the empagliflozin intermediate and related substances can be effectively separated under the chromatographic condition. The retention times and degrees of separation of the empagliflozin intermediate and related materials in figure 1 are shown in the following table:

name (R)	Retention time	Degree of separation
			M1Z3	4.203	14.782
M1Z2	7.586	11.245
			SM1	10.309	21.673
M1Z1	14.979	6.384
			Empagliflozin intermediate 1	25.245	4.660

The detection result of the empagliflozin intermediate under the chromatographic condition is as follows:

YPLJ-M1Z2	0.09％
		YPLJ-SM1	0.149％
unknown impurity (RRT:0.68min)	0.183％
		Unknown impurity (RRT:0.71min)	0.369％
Unknown impurity (RRT:0.92min)	1.192％
		Total miscellaneous%	1.982％
Number of impurities	5

Example 2

Chromatographic conditions

A chromatographic column: c18(Welch Ultimate C184.6X 100mm, 3 μm)

The detection wavelength is 225nm

The flow rate was 1.0ml/min

Column temperature: 30 deg.C

Sample introduction volume: 10 μ l

Mobile phase: taking 5mmol/L ammonium acetate buffer solution (pH is adjusted to 7.5 by ammonia water) as a mobile phase A, taking acetonitrile as a mobile phase B, and carrying out gradient elution according to the following table;

experimental procedure

Appropriate amounts of the engagliflozin intermediate and related substances are taken to prepare a solution containing about 1mg of the engagliflozin intermediate, about 30 μ g of SM1, about 20 μ g of M1Z1, about 20 μ g of M1Z2 and about 20 μ g of M1Z3 in each 1 ml. According to the above chromatographic conditions, 10. mu.l of the resulting mixture was precisely measured, and the resulting mixture was injected into a liquid chromatograph, and the chromatogram was recorded.

The result is shown in figure 2, and the empagliflozin intermediate and related substances can be effectively separated under the chromatographic condition. The retention times and separations of the engagliflozin intermediates and related materials in figure 2 are shown in the following table:

name (R)	Retention time	Degree of separation
			M1Z3	4.364	/
M1Z2	7.931	23.363
			SM1	10.739	16.988
M1Z1	15.438	29.219
			Empagliflozin intermediate 1	26.724	4.900

The detection result of the empagliflozin intermediate under the chromatographic condition is as follows:

YPLJ-M1Z2	0.105％
		YPLJ-SM1	0.184％
unknown impurity (RRT:0.68min)	0.175％
		Unknown impurity (RRT:0.71min)	0.376％
Unknown impurity (RRT:0.92min)	1.261％
		Total miscellaneous%	2.102％
Number of impurities	5

Example 3

Chromatographic conditions

A chromatographic column: c18(Agilent ZORBAX Eclipse Plus C184.6X 100mm, 3.5 μm)

The detection wavelength is 225nm

The flow rate was 1.0ml/min

Column temperature: 30 deg.C

Sample introduction volume: 10 μ l

Mobile phase: taking 5mmol/L ammonium acetate buffer solution (pH is adjusted to 7.3 by ammonia water) as a mobile phase A, taking acetonitrile as a mobile phase B, and carrying out gradient elution according to the following table;

experimental procedure

Appropriate amounts of the engagliflozin intermediate and related substances are taken to prepare a solution containing about 1mg of the engagliflozin intermediate, about 30 μ g of SM1, about 20 μ g of M1Z1, about 20 μ g of M1Z2 and about 20 μ g of M1Z3 in each 1 ml. According to the above chromatographic conditions, 10. mu.l of the resulting mixture was precisely measured, and the resulting mixture was injected into a liquid chromatograph, and the chromatogram was recorded.

The result is shown in figure 3, and the effective separation of the empagliflozin intermediate and related substances can be realized under the chromatographic condition. The retention times and separations of the engagliflozin intermediates and related materials in figure 3 are shown in the following table:

name (R)	Retention time	Degree of separation
			M1Z3	4.081	/
M1Z2	7.399	16.072
			SM1	10.067	12.363
M1Z1	14.682	23.931
			Empagliflozin intermediate 1	25.107	3.420

The detection result of the empagliflozin intermediate under the chromatographic condition is as follows:

YPLJ-M1Z2	0.091％
		YPLJ-SM1	0.163％
unknown impurity (RRT:0.68min)	0.219％
		Unknown impurity (RRT:0.71min)	0.373％
Unknown impurity (RRT:0.92min)	1.412％
		Total miscellaneous%	2.257％
Number of impurities	5

Example 4

Chromatographic conditions

A chromatographic column: c18(Agilent ZORBAX Eclipse Plus C184.6X 100mm, 3.5 μm)

The detection wavelength is 225nm

The flow rate was 1.0ml/min

Column temperature: 35 deg.C

Sample introduction volume: 10 μ l

Mobile phase: taking 5mmol/L ammonium acetate buffer solution (pH is adjusted to 7.5 by ammonia water) as a mobile phase A, taking acetonitrile as a mobile phase B, and carrying out gradient elution according to the following table;

experimental procedure

Appropriate amounts of the engagliflozin intermediate and related substances are taken to prepare a solution containing about 1mg of the engagliflozin intermediate, about 30 μ g of SM1, about 20 μ g of M1Z1, about 20 μ g of M1Z2 and about 20 μ g of M1Z3 in each 1 ml. According to the above chromatographic conditions, 10. mu.l of the resulting mixture was precisely measured, and the resulting mixture was injected into a liquid chromatograph, and the chromatogram was recorded.

The result is shown in figure 4, and the empagliflozin intermediate and related substances can be effectively separated under the chromatographic condition. The retention times and separations of the engagliflozin intermediates and related materials in fig. 4 are shown in the following table:

name (R)	Retention time	Degree of separation
			M1Z3	4.267	/
M1Z2	7.744	21.325
			SM1	10.468	15.583
M1Z1	15.109	27.106
			Empagliflozin intermediate 1	23.446	4.225

The detection result of the empagliflozin intermediate under the chromatographic condition is as follows:

YPLJ-M1Z2	0.088％
		YPLJ-SM1	0.151％
unknown impurity (RRT:0.68min)	0.191％
		Unknown impurity (RRT:0.71min)	0.385％
Unknown impurity (RRT:0.92min)	1.793％
		Total miscellaneous%	2.757％
Number of impurities	5

The present application describes embodiments, but the description is illustrative rather than limiting and it will be apparent to those of ordinary skill in the art that many more embodiments and implementations are possible within the scope of the embodiments described herein.

Claims (9)

1. An analytical detection method of an empagliflozin intermediate comprises the following steps:

(1) preparing a test solution: taking a proper amount of the engagliflozin intermediate, adding a diluent to dissolve and quantitatively diluting to prepare a solution containing about 1mg of the engagliflozin intermediate in every 1ml, and taking the solution as a test solution;

(2) setting chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler of the chromatographic column; the detection wavelength is 220 nm-230 nm; performing gradient elution by adopting a mobile phase A and a mobile phase B as mobile phases, wherein the mobile phase A is a buffered saline solution, and the mobile phase B is acetonitrile; the flow rate of the mobile phase is 0.5-1.5 ml/min; the column temperature is 25-35 ℃;

(3) and (3) determination: and (2) injecting 5-50 mul of the test solution obtained in the step (1) into a high performance liquid chromatograph, and recording a chromatogram.

2. The analytical detection method according to claim 1, wherein the diluent is one or a mixed solvent of two or more selected from acetonitrile, dimethylsulfoxide, tetrahydrofuran and methanol;

optionally, the diluent is a mixed solvent of dimethyl sulfoxide and acetonitrile, and optionally, the volume ratio of the dimethyl sulfoxide to the acetonitrile in the mixed solvent is (10-30) to (70-90).

3. The analytical detection method according to claim 1, wherein the diluent is a mixed solvent of dimethyl sulfoxide and acetonitrile in a volume ratio of 20: 80.

4. The assay of claim 1, wherein the buffer salt is selected from one of acetate, phosphate and formate, optionally acetate;

optionally, the concentration of the buffered saline solution is 4mmol/L to 6 mmol/L; optionally, the pH of the buffered saline solution is 7.3-9.0.

5. The analytical detection method according to claim 4, wherein the buffered saline solution is an ammonium acetate buffer solution having a concentration of 5mmol/L and the pH is adjusted to 7.5 with aqueous ammonia.

6. The analytical detection method according to any one of claims 1 to 5, wherein the gradient elution is performed by:

7. the analytical detection method according to any one of claims 1 to 5, wherein the high performance liquid chromatograph is an Agilent 1260 liquid chromatograph;

a chromatographic column: c18(Agilent ZORBAX Eclipse Plus C184.6 x 100mm x 3.5 um);

the detection wavelength is 225 nm;

the flow rate is 1.0 ml/min;

column temperature: 30 ℃;

sample introduction volume: 10 μ l.

8. The assay detection method of any one of claims 1 to 5, wherein said preparing a test solution comprises: taking appropriate amount of the engagliflozin intermediate, SM1, M1Z1, M1Z2 and M1Z3, adding a diluent to dissolve and quantitatively dilute to prepare a solution containing about 1mg of the engagliflozin intermediate, about 30 mu g of SM1, about 20 mu g M1Z1, about 20 mu g M1Z2 and about 20 mu g M1Z3 in each 1ml of solution as a test solution.

9. An analytical detection method of an empagliflozin intermediate, comprising the following steps:

(1) preparing a test solution: taking a proper amount of the engagliflozin intermediate, adding a diluent to dissolve and quantitatively diluting to prepare a solution containing about 1mg of the engagliflozin intermediate in every 1ml, and taking the solution as a test solution; the diluent is a mixed solvent of dimethyl sulfoxide and acetonitrile in a volume ratio of 20: 80;

(2) setting chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler of the chromatographic column; the detection wavelength is 220 nm-230 nm; the mobile phase adopts a mobile phase A and a mobile phase B to carry out gradient elution; the mobile phase B is acetonitrile; the flow rate of the mobile phase is 0.5-1.5 ml/min; the column temperature is 25-35 ℃;

here, the mobile phase a was an ammonium acetate buffer solution having a concentration of 5mmol/L and adjusted to pH 7.5 with ammonia water;

the procedure for the gradient elution was:

(3) and (3) determination: and (2) taking 10 mu l of the test solution obtained in the step (1), injecting into a high performance liquid chromatograph, and recording a chromatogram.

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