CN116203172A - Method for detecting 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1 - Google Patents

Abstract

The invention belongs to the technical field of analytical chemistry, and provides a detection method of 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1. The detection method provided by the invention has higher sensitivity and durability, and can be used for effectively detecting whether the dapagliflozin SM-1 contains residual 2-chloro-4-bromobenzoyl chloride and the content thereof.

Description

Method for detecting 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1

Technical Field

The invention relates to the technical field of analytical chemistry, in particular to a detection method of 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1.

Background

Dapagliflozin is a sodium-glucose co-transporter 2 (SGLT 2) selective inhibitor. SGLT2 is selectively expressed in the first segment of the proximal glomerular tubule (S1), regulating the reabsorption of most (90%) of the permeate glucose. Dapagliflozin inhibition of SGLT2 results in the excretion of excess glucose through the urine, thereby improving glycemic control without increasing insulin secretion. In vitro studies have shown that dapagliflozin is effective in selectively inhibiting human (h) SGLT2 relative to closely related selective 1242-1600-folded hsgclt 1. Dapagliflozin was also demonstrated to selectively inhibit SGLT2 in mice (m) and dogs (d) over mGLT1 and dsgclt 1. Clinical research results show that dapagliflozin can obviously reduce HbA1c and fasting blood glucose of type II diabetics by singly using or jointly using medicines such as metformin, pioglitazone, glimepiride, insulin and the like, and has similar adverse reaction incidence rate with placebo and low risk of hypoglycemia.

2-chloro-4-bromobenzoyl chloride is generated during the synthesis of dapagliflozin starting material 1 (SM 1), is a potential genotoxic impurity, and in order to better control the quality of dapagliflozin, it is necessary to provide a measurement method for detecting whether dapagliflozin SM1 contains 2-chloro-4-bromobenzoyl chloride so as to ensure the safety of medication, but no report on the detection method of 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1 is known at present.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for detecting 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1. The detection method provided by the invention can effectively detect whether the dapagliflozin SM-1 contains residual 2-chloro-4-bromobenzoyl chloride and the content thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a detection method of 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1, which comprises the following steps:

dissolving 2-chloro-4-bromobenzoyl chloride reference substance in methanol to obtain reference substance solution;

dissolving dapagliflozin SM-1 to be tested in methanol to obtain a test solution;

respectively performing high performance liquid chromatography detection on the reference substance solution and the sample solution to obtain the peak area of 2-chloro-4-bromobenzoyl chloride in the 2-chloro-4-bromobenzoyl chloride reference substance and the peak area of 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1 to be detected;

obtaining the content of 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1 to be detected based on the peak area of 2-chloro-4-bromobenzoyl chloride in the 2-chloro-4-bromobenzoyl chloride reference substance;

the parameters of the high performance liquid chromatography detection include:

the chromatographic column is octadecylsilane chemically bonded silica reversed phase chromatographic column;

the mobile phase system comprises a mobile phase A and a mobile phase B;

the mobile phase A is water, and the mobile phase B is methanol;

the elution mode is gradient elution;

the procedure for the gradient elution is shown in table 1:

TABLE 1 gradient elution procedure

Detection wavelength: 225nm.

Preferably, the parameters of the high performance liquid chromatography detection further include: the detector is a diode array detector.

Preferably, the flow rate of the mobile phase system is 0.9-1.1 mL-min ^-1 。

Preferably, the parameters of the high performance liquid chromatography detection further include: the column temperature is 25-35 ℃.

Preferably, the parameters of the high performance liquid chromatography detection further include: the sample volume was 10. Mu.L.

Preferably, the size of the chromatographic column is 4.6mm by 250mm,5 μm.

Preferably, the instrument for detecting the high performance liquid chromatography is a Thermo Ultimate 3000 high performance liquid chromatograph.

Preferably, the concentration of the reference solution is 2.7-3.3 mug/mL.

Preferably, the concentration of the sample solution is 18-22 mg/mL.

Preferably, the obtaining the content of the 2-chloro-4-bromobenzoyl chloride in the dapagliflozin SM-1 to be detected based on the peak area of the 2-chloro-4-bromobenzoyl chloride in the 2-chloro-4-bromobenzoyl chloride reference substance comprises:

calculating to obtain the content of 2-chloro-4-bromobenzoyl chloride in the dapagliflozin SM-1 to be detected by adopting a formula 1;

in equation 1:

the unit of the content of the 2-chloro-4-bromobenzoyl chloride is ppm;

A _{feed device} The peak area of 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1 to be detected;

W _{feed device} Mg is the weight of dapagliflozin SM-1 to be tested;

F _{flat plate} Is calculated according to formula 2;

in equation 2:

n is the sample injection times of the reference substance solution;

F _i as a correction factor, according to formula 3;

in formula 3, W _{Pair i} Is the ith2-chloro-4-bromobenzoyl chloride reference substance, mg;

P _{for a pair of} % is the purity of the 2-chloro-4-bromobenzoyl chloride reference substance;

A _{pair i} Peak area for the i-th 2-chloro-4-bromobenzoyl chloride control.

The invention provides a detection method of 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1, which comprises the following steps: dissolving 2-chloro-4-bromobenzoyl chloride reference substance in methanol to obtain reference substance solution; dissolving dapagliflozin SM-1 to be tested in methanol to obtain a test solution; respectively performing high performance liquid chromatography detection on the reference substance solution and the sample solution to obtain the peak area of 2-chloro-4-bromobenzoyl chloride in the 2-chloro-4-bromobenzoyl chloride reference substance and the peak area of 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1 to be detected; obtaining the content of 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1 to be detected based on the peak area of 2-chloro-4-bromobenzoyl chloride in the 2-chloro-4-bromobenzoyl chloride reference substance; the parameters of the high performance liquid chromatography detection include: the chromatographic column is octadecylsilane chemically bonded silica reversed phase chromatographic column; the mobile phase system comprises a mobile phase A and a mobile phase B; the mobile phase A is water, and the mobile phase B is methanol; the elution mode is gradient elution; the procedure for the gradient elution is shown in table 1: detection wavelength: 225nm.

The detection method provided by the invention has higher sensitivity and durability, and can be used for effectively detecting whether the dapagliflozin SM-1 contains residual 2-chloro-4-bromobenzoyl chloride and the content thereof.

Drawings

FIG. 1 is a typical HPLC chromatogram of a solvent in a proprietary verification example;

FIG. 2 is a typical HPLC chromatogram of a control solution in a proprietary verification example;

FIG. 3 is a typical HPLC chromatogram of a test solution in a proprietary verification example;

FIG. 4 is a typical HPLC chromatogram of a labeled test solution in a proprietary verification example;

FIG. 5 is a typical HPLC chromatogram of a proprietary solution (solvent, control solution, and labeled test solution) in a proprietary verification example;

FIG. 6 is a typical HPLC chromatogram of a quantitative limiting solution;

FIG. 7 is a typical HPLC chromatogram of a detection limit solution.

Detailed Description

The invention provides a detection method of 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1, which comprises the following steps:

dissolving 2-chloro-4-bromobenzoyl chloride reference substance in methanol to obtain reference substance solution;

dissolving dapagliflozin SM-1 to be tested in methanol to obtain a test solution;

respectively performing high performance liquid chromatography detection on the reference substance solution and the sample solution to obtain the peak area of 2-chloro-4-bromobenzoyl chloride in the 2-chloro-4-bromobenzoyl chloride reference substance and the peak area of 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1 to be detected;

obtaining the content of 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1 to be detected based on the peak area of 2-chloro-4-bromobenzoyl chloride in the 2-chloro-4-bromobenzoyl chloride reference substance;

the parameters of the high performance liquid chromatography detection include:

the chromatographic column is octadecylsilane chemically bonded silica reversed phase chromatographic column;

the mobile phase system comprises a mobile phase A and a mobile phase B;

the mobile phase A is water, and the mobile phase B is methanol;

the elution mode is gradient elution;

the procedure for the gradient elution is shown in table 1:

detection wavelength: 225nm.

In the present invention, the raw materials used in the present invention are preferably commercially available products unless otherwise specified.

Dissolving 2-chloro-4-bromobenzoyl chloride reference substance in methanol to obtain reference substance solution.

In the invention, the purity of the 2-chloro-4-bromobenzoyl chloride reference substance is preferably more than or equal to 95 weight percent. In the present invention, the concentration of the control solution is preferably 2.7 to 3.3. Mu.g/mL, more preferably 3.0. Mu.g/mL.

According to the invention, dapagliflozin SM-1 to be detected is dissolved in methanol to obtain a sample solution.

In the present invention, the concentration of the sample solution is preferably 18 to 22mg/mL, more preferably 20mg/mL.

After the reference substance solution and the sample solution are obtained, the reference substance solution and the sample solution are respectively subjected to high performance liquid chromatography detection to obtain the peak area of the 2-chloro-4-bromobenzoyl chloride in the 2-chloro-4-bromobenzoyl chloride reference substance and the peak area of the 2-chloro-4-bromobenzoyl chloride in the dapagliflozin SM-1 to be detected.

In the present invention, the parameters of the high performance liquid chromatography detection include:

the chromatographic column is octadecylsilane chemically bonded silica reversed phase chromatographic column, preferably Xtimate C18;

the size of the chromatographic column is preferably 4.6mm by 250mm,5 μm;

the column temperature is preferably 25 to 35 ℃, and more preferably 30 ℃;

the mobile phase system comprises a mobile phase A and a mobile phase B;

the flow rate of the mobile phase system is preferably 0.9-1.1 mL.min ^-1 More preferably 1mL/min ^-1 The method comprises the steps of carrying out a first treatment on the surface of the The chromatographic column for high performance liquid chromatography detection preferably comprises octadecylsilane chemically bonded silica reversed phase chromatographic column, and specifically preferably comprises Xtime C18; the inner diameter of the chromatographic column is preferably 4.6mm, the column length is preferably 250mm, and the pore diameter of the sieve plates at the two ends of the chromatographic column is preferably 5 μm.

The mobile phase A is water, and the mobile phase B is methanol;

the elution mode is gradient elution;

the procedure for the gradient elution is shown in table 1:

the sample injection volume is preferably 10 mu L;

the detection wavelength is 225nm;

the detector is preferably a diode array detector.

In the invention, the instrument for detecting the high performance liquid chromatography is preferably a Thermo Ultimate 3000 high performance liquid chromatograph. In the present invention, the Thermo Ultimate 3000 high performance liquid chromatograph preferably includes a quaternary pump, a degas unit, a diode array detector, a column oven, an autosampler, a system monitor, and a Chromeleon7 workstation.

After the peak area of the 2-chloro-4-bromobenzoyl chloride in the 2-chloro-4-bromobenzoyl chloride reference substance and the peak area of the 2-chloro-4-bromobenzoyl chloride in the dapagliflozin SM-1 to be detected are obtained, the content of the 2-chloro-4-bromobenzoyl chloride in the dapagliflozin SM-1 to be detected is obtained based on the peak area of the 2-chloro-4-bromobenzoyl chloride in the 2-chloro-4-bromobenzoyl chloride reference substance.

In the invention, the obtaining of the content of the 2-chloro-4-bromobenzoyl chloride in the dapagliflozin SM-1 to be detected based on the peak area of the 2-chloro-4-bromobenzoyl chloride in the 2-chloro-4-bromobenzoyl chloride reference substance preferably comprises the following steps:

calculating to obtain the content of 2-chloro-4-bromobenzoyl chloride in the dapagliflozin SM-1 to be detected by adopting a formula 1;

in equation 1:

the unit of the content of the 2-chloro-4-bromobenzoyl chloride is ppm;

A _{feed device} The peak area of 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1 to be detected;

W _{feed device} Mg is the weight of dapagliflozin SM-1 to be tested;

F _{flat plate} Is calculated according to formula 2;

in equation 2:

n is the sample injection times of the reference substance solution;

F _i as a correction factor, according to formula 3;

in formula 3, W _{Pair i} Mg of the i-th 2-chloro-4-bromobenzoyl chloride reference substance;

P _{for a pair of} % is the purity of the 2-chloro-4-bromobenzoyl chloride reference substance;

A _{pair i} Peak area for the i-th 2-chloro-4-bromobenzoyl chloride control.

The method for detecting 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1 provided by the invention is described in detail below with reference to examples, but the method is not to be construed as limiting the scope of the invention.

The materials, reagents, instrumentation and HPLC chromatographic conditions used in the following examples and validation examples were:

1. instrument and HPLC chromatographic conditions:

thermo Ultimate 3000 high performance liquid chromatograph (quaternary pump, degas unit, diode array detector, column incubator, autosampler, system monitor and Chromeleon7 workstation) (Thermo Fisher Co., USA); MSA6.6S-0CE parts per million on the day (Sartorius company, germany); model AB265-S analytical balance (METTLETOLIDOPO Co., USA).

Conditions for high performance liquid chromatography detection: chromatographic column: xtimate C18, size 4.6mm×250mm,5 μm, column temperature 30 ℃, mobile phase A water, mobile phase B methanol, gradient elution according to the gradient elution program of Table 1; the flow rate is 1.0mL/min, the sample injection volume is 10 mu L, and the detection wavelength is 225nm.

2. Materials:

2-chloro-4-bromobenzoyl chloride control (source: bolode, lot number: DGLJ 20200701); dapagliflozin SM-1 (source: shandong Lukang pharmaceutical Co., ltd., jinan Sanyuan chemical industry, lot number: C274-strand Z2006001, 20200303, 20200401); methanol (source: new blue scene, lot number: A2219041000).

Example 1

Preparing a reference substance solution: accurately weighing 2-chloro-4-bromobenzoyl chloride reference substance, adding methanol to dissolve and dilute into solution containing 3.0 μg 2-chloro-4-bromobenzoyl chloride reference substance per 1mL, and taking as reference substance solution.

Sample solution preparation: taking a proper amount of dapagliflozin SM-1, precisely weighing, adding methanol for dissolving and quantitatively diluting to prepare a solution containing dapagliflozin SM-120mg per 1mL serving as a test sample solution.

Respectively measuring 10 mu L of each solution precisely, injecting into a Thermo Ultimate 3000 high performance liquid chromatograph, recording peak area, and calculating the content of 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1 by adopting a method of formulas 1-3, wherein the result is shown in Table 2.

TABLE 2 determination of 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1

Verification example methodology verification

1. Specialization of

Solvent: methanol.

Control stock solution: taking about 3mg of 2-chloro-4-bromobenzoyl chloride reference substance, precisely weighing, placing into a 100mL volumetric flask, adding methanol to dissolve, fixing volume to scale, and shaking uniformly to obtain the final product.

Control solution: precisely measuring 1mL of the reference substance stock solution, placing into a 10mL volumetric flask, adding methanol for dilution, fixing the volume to a scale, and shaking uniformly to obtain the product.

Test solution: taking dapagliflozin SM-1200mg, precisely weighing, placing into a 10mL volumetric flask, adding methanol for dissolution, fixing the volume to a scale, and shaking uniformly to obtain the dapagliflozin.

Adding a labeled test sample solution: taking dapagliflozin SM-1200mg, precisely weighing, precisely measuring 1mL of control stock solution, placing into a 10mL volumetric flask, adding methanol for dissolution, fixing the volume to a scale, and shaking uniformly to obtain the dapagliflozin.

Precisely measuring 10 μl of each of the solvent, the reference solution, the sample solution and the labeled sample solution, injecting into a Thermo Ultimate 3000 high performance liquid chromatograph, and recording the chromatograms, wherein the results are shown in fig. 1-4.

Fig. 1 is a typical HPLC chromatogram of a solvent in a specific verification example, fig. 2 is a typical HPLC chromatogram of a reference solution in a specific verification example, fig. 3 is a typical HPLC chromatogram of a test solution in a specific verification example, fig. 4 is a typical HPLC chromatogram of a labeled test solution in a specific verification example, and fig. 5 is a typical HPLC chromatogram of a specific solution (solvent, reference solution, and labeled test solution) in a specific verification example; as can be seen from fig. 1 to 5: the solvent has no interference to the detection of the 2-chloro-4-bromobenzoyl chloride, and the separation degree of the 2-chloro-4-bromobenzoyl chloride peak and the main peak in the chromatogram of the solution of the standard sample solution is in accordance with the requirement.

2. Quantitative limit and detection limit

The detection Limit (LOD) and the quantification Limit (LOQ) are determined according to the signal-to-noise ratio method. 2-chloro-4-bromobenzoyl chloride with known concentration is diluted in a reference stock solution, the concentration at S/N is about 10 is taken as a quantitative limit concentration, and the concentration at S/N is about 3 is taken as a detection limit concentration.

According to the conditions of the high performance liquid chromatography detection, precisely measuring each quantitative limit solution and each detection limit solution by 10 mu L, respectively injecting into a Thermo Ultimate 3000 high performance liquid chromatograph, and recording the chromatograms. The results are shown in Table 3, the spectra are shown in FIGS. 6-7, FIG. 6 is a typical HPLC chromatogram of a quantitative limiting solution, and FIG. 7 is a typical HPLC chromatogram of a detection limiting solution.

TABLE 3 quantitative limit and limit results of the detection method

As can be seen from table 3, the detection limit S/n=6.8 >3, the detection limit concentration is 0.18 μg/mL less than 20% of the limit concentration; the minimum S/N=11.3 in the six-needle quantitative limit is larger than 10, the concentration of the quantitative limit is 0.6 mug/mL and smaller than 30% of the concentration limit, and the quantitative limit is continuous six-needle RSD=3.25% and smaller than 6.0% and meets the regulations. According to the specification, the detection method provided by the invention has higher sensitivity.

3. Durability of

Solvent: methanol.

Control stock solution: taking 3mg of 2-chloro-4-bromobenzoyl chloride reference substance, precisely weighing, placing into a 100mL volumetric flask, adding methanol to dissolve, fixing volume to scale, and shaking uniformly to obtain the final product.

Control solution: precisely measuring 1mL of the reference substance stock solution, placing into a 10mL volumetric flask, adding methanol for dilution, fixing the volume to a scale, and shaking uniformly to obtain the product.

Adding a labeled test sample solution: and (3) taking dapagliflozin SM-1200mg, precisely weighing, placing into a 10mL volumetric flask, adding the reference substance solution for dissolution, fixing the volume to a scale, and shaking uniformly to obtain a sample solution.

The flow rate (+ -0.1 mL/min), the initial proportion of mobile phase (+ -2%) and the chromatographic column of different batches were changed respectively. Precisely measuring 10 mu L of each of the solvent, the reference substance solution and the standard sample solution, injecting into a Thermo Ultimate 3000 high performance liquid chromatograph, and recording the chromatograms, wherein the results are shown in Table 4.

Table 4 durability results of the test method

As can be seen from table 4, the solvent and the test solution do not interfere with the detection of 2-chloro-4-bromobenzoyl chloride under each durability condition; compared with the content of the 2-chloro-4-bromobenzoyl chloride measured under normal conditions, the relative deviation is less than +/-5%, which indicates that the detection method provided by the invention has good durability.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. The method for detecting 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1 is characterized by comprising the following steps:

dissolving 2-chloro-4-bromobenzoyl chloride reference substance in methanol to obtain reference substance solution;

dissolving dapagliflozin SM-1 to be tested in methanol to obtain a test solution;

respectively performing high performance liquid chromatography detection on the reference substance solution and the sample solution to obtain the peak area of 2-chloro-4-bromobenzoyl chloride in the 2-chloro-4-bromobenzoyl chloride reference substance and the peak area of 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1 to be detected;

obtaining the content of 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1 to be detected based on the peak area of 2-chloro-4-bromobenzoyl chloride in the 2-chloro-4-bromobenzoyl chloride reference substance;

the parameters of the high performance liquid chromatography detection include:

the chromatographic column is octadecylsilane chemically bonded silica reversed phase chromatographic column;

the mobile phase system comprises a mobile phase A and a mobile phase B;

the mobile phase A is water, and the mobile phase B is methanol;

the elution mode is gradient elution;

the procedure for the gradient elution is shown in table 1:

TABLE 1 gradient elution procedure

Detection wavelength: 225nm.

2. The method according to claim 1, wherein the parameters of the high performance liquid chromatography detection further comprise: the detector is a diode array detector.

3. The method according to claim 1, wherein the flow rate of the mobile phase system is 0.9 to 1.1 mL/min ^-1 。

4. The method according to claim 1, wherein the parameters of the high performance liquid chromatography detection further comprise: the column temperature is 25-35 ℃.

5. The method according to claim 1, wherein the parameters of the high performance liquid chromatography detection further comprise: the sample volume was 10. Mu.L.

6. The method of claim 1, wherein the chromatographic column has dimensions of 4.6mm x 250mm,5 μm.

7. The method according to any one of claims 1 to 6, wherein the high performance liquid chromatography detection instrument is a Thermo Ultimate 3000 high performance liquid chromatograph.

8. The method according to claim 1, wherein the concentration of the control solution is 2.7 to 3.3. Mu.g/mL.

9. The method according to claim 1, wherein the concentration of the sample solution is 18 to 22mg/mL.

10. The detection method according to claim 1, wherein the obtaining the content of 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1 to be detected based on the peak area of 2-chloro-4-bromobenzoyl chloride in the 2-chloro-4-bromobenzoyl chloride control comprises:

calculating to obtain the content of 2-chloro-4-bromobenzoyl chloride in the dapagliflozin SM-1 to be detected by adopting a formula 1;

in equation 1:

the unit of the content of the 2-chloro-4-bromobenzoyl chloride is ppm;

A _{feed device} The peak area of 2-chloro-4-bromobenzoyl chloride in dapagliflozin SM-1 to be detected;

W _{feed device} For dapagliflozin SM-1 to be testedWeight, mg;

F _{flat plate} Is calculated according to formula 2;

in equation 2:

n is the sample injection times of the reference substance solution;

F _i as a correction factor, according to formula 3;

in formula 3, W _{Pair i} Mg of the i-th 2-chloro-4-bromobenzoyl chloride reference substance;

P _{for a pair of} % is the purity of the 2-chloro-4-bromobenzoyl chloride reference substance;

A _{pair i} Peak area for the i-th 2-chloro-4-bromobenzoyl chloride control.

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