CN105319294B - Method for separating and determining canagliflozin and related substances thereof - Google Patents
Method for separating and determining canagliflozin and related substances thereof Download PDFInfo
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- CN105319294B CN105319294B CN201410394363.8A CN201410394363A CN105319294B CN 105319294 B CN105319294 B CN 105319294B CN 201410394363 A CN201410394363 A CN 201410394363A CN 105319294 B CN105319294 B CN 105319294B
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Abstract
The invention discloses a method for separating and determining canagliflozin and related substances thereof. The method can well separate canagliflozin and related substances thereof, has good reproducibility of detection, high sensitivity, strong specificity and simple and convenient operation, and is beneficial to control the quality and the medication safety of the canagliflozin.
Description
Technical Field
The invention belongs to the field of drug analysis methods, and particularly relates to a method for separating and measuring a hypoglycemic drug canagliflozin and related substances thereof by adopting a high performance liquid chromatography.
Background
Canagliflozin is a selective sodium-glucose cotransporter (SGLT) inhibitor useful for treating adult type II diabetes. The canagliflozin limits the reabsorption of most glucose in the body by 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. The urine glucose is increased, and calorie is reduced in vivo, so that the weight is obviously reduced. The systolic pressure decreased slightly due to increased sodium excretion, but there was no significant change in blood sodium.
The chemical structural formula of canagliflozin is as follows:
the chemical name is as follows: (1S) -1, 5-anhydro-1- [3- [ [5- (4-fluorophenyl) -2-thienyl ] methyl ] -4-methylphenyl ] -D-glucitol.
Canagliflozin is a fully synthetic chemical, and a byproduct or residual intermediate possibly generated in the reaction process can affect the purity and quality of the canagliflozin; during the storage process, degradation products may be generated due to environmental factors such as temperature and light, and the purity and quality of the product may be affected, and these residual intermediates, side reaction products and degradation products are related substances in the conventional pharmaceutical quality control.
Canagliflozin has two related substances with known structures, namely impurity A and impurity B, and the chemical structures of the two related substances are as follows:
at present, no report is found on a method for measuring related substances of canagliflozin and a composition thereof, and an effective detection method capable of separating and measuring the canagliflozin and the related substances thereof is urgently needed to ensure the quality controllability of a canagliflozin raw material medicament and a pharmaceutical composition thereof and ensure the effectiveness and the safety of the canagliflozin raw material medicament and the pharmaceutical composition thereof. The inventor discovers a method for rapidly separating and determining canagliflozin and related substances thereof through a large number of scientific experiments and screening of test conditions, can effectively control process impurities and degradation products of the canagliflozin, and ensures that the quality of a raw material drug of the canagliflozin and a pharmaceutical composition of the canagliflozin is controllable, thereby completing the invention.
The inventors have found that the marketed canagliflozin tablet, the pharmaceutical excipients microcrystalline cellulose, croscarmellose sodium, magnesium stearate and the like do not interfere with the determination of the canagliflozin and related substances in the method of the invention.
Disclosure of Invention
The invention aims to provide a method for separating and determining canagliflozin and related substances thereof, which can effectively separate the canagliflozin from the related substances thereof and accurately detect the related substances in a raw medicine of the canagliflozin and a pharmaceutical composition thereof, thereby ensuring the quality control of the canagliflozin and further ensuring the effectiveness and the safety of a canagliflozin medicine.
To achieve the object of the present invention, the following embodiments are provided.
In one embodiment, the method for separating and determining the canagliflozin and related substances comprises the steps of adopting high performance liquid chromatography, taking a butane silane bonded silica gel filler column as a chromatographic column, taking acetonitrile as an organic phase, taking phosphoric acid solution or phosphoric acid buffer solution as a water phase, mixing the acetonitrile and the water phase as a mobile phase, carrying out gradient elution, and detecting by using an ultraviolet detector.
In the above embodiment, in the method of the present invention, the pH of the phosphoric acid solution or the phosphoric acid buffer is 2 to 6, preferably 3.5, wherein the phosphoric acid buffer is a phosphate selected from sodium dihydrogen phosphate, potassium dihydrogen phosphate and ammonium dihydrogen phosphate, the pH is adjusted by the phosphoric acid solution, and the concentration of the phosphate in the aqueous phase is 0 to 0.005 mol/L.
In the above embodiment, in the method of the present invention, the elution initial volume ratio of the aqueous phase to the organic phase is 70: 30 to 50: 50, preferably 70: 30, and the elution termination volume ratio of the aqueous phase to the organic phase is 30: 70 to 15: 85, preferably 30: 70.
In the above embodiments, the method of the present invention, the ultraviolet detection wavelength is 210nm to 215nm or 287nm to 297nm, preferably 210 nm.
In the above embodiment, the method of the present invention, further comprising the steps of:
(1) taking a canagliflozin raw material medicine or a pharmaceutical composition thereof, adding 70% acetonitrile solution to completely dissolve the canagliflozin, diluting to a constant volume, preparing into a solution containing 0.3-0.7 mg, preferably 0.5mg, of the canagliflozin in each 1ml of the solution, and filtering to obtain a test solution;
(2) a butane silane bonded silica gel chromatographic column is used as a chromatographic column, the flow rate is 0.8-1.2 ml/min, preferably 1.0ml/min, the column temperature is 25-35 ℃, preferably 25 ℃, and the wavelength is 210-215 nm or 287-297 nm, preferably 210 nm;
(3) injecting 10-30 mul, preferably 20 mul, of the test solution into a high performance liquid chromatograph;
(4) eluting a phosphoric acid solution with the pH value of 2-6 of an aqueous phase or a phosphoric acid buffer solution and an organic phase acetonitrile at the initial volume ratio of 70: 30-50: 50 until a main peak comes out, gradually increasing the acetonitrile ratio until the volume ratio of the aqueous phase to the organic phase reaches 30: 70-15: 85, and keeping for 20 minutes to finish the determination of related substances of canagliflozin;
optionally, (5) after the relevant substances are completely eluted, the acetonitrile ratio is increased to 95%, the flow rate is increased to 2ml/min, the chromatographic column is washed for 10 minutes, the ratio of the water phase to the acetonitrile is adjusted to 60: 40, and the chromatographic system is balanced;
(6) calculating the purity of the canagliflozin, the content of each single impurity in related substances and the total amount of the related substances by using a self-comparison method of the canagliflozin with a correction factor.
In the above embodiment, in the method of the present invention, the aqueous phase in step (4) is a phosphate solution or phosphate buffer solution having a pH of 2.5; and the initial volume ratio of the aqueous phase to the organic phase is 60: 40, and after the elution is carried out until the main peak comes out, the acetonitrile ratio is gradually increased until the volume ratio of the aqueous phase to the organic phase reaches 30: 70 and is kept for 20 minutes, so that the determination of related substances of canagliflozin is completed.
In the above embodiments, the method of the present invention, the gradient elution comprises: when the time is 0min to 14-20 min, the volume ratio of the water phase to the organic phase in the mobile phase is 55: 45-65: 35; when the time is from 14 th to 20 th min to 26 th to 32 th min, the volume ratio of the water phase to the organic phase is changed to 30: 70-40: 60 at a constant speed; when the time is from 26 th to 32 th min to 33 th to 39 th min, the volume ratio of the water phase to the organic phase is changed to 25: 75-35: 65 at a constant speed; the volume ratio of the water phase to the organic phase is kept constant from 33 th to 39 th min to 48 th to 54 th min.
In the above embodiment, the method of the present invention, preferably, the gradient elution comprises: the volume ratio of the water phase to the organic phase in the mobile phase is 60: 40 from 0min to 16 min; the volume ratio of the water phase to the organic phase is changed to 35: 65 at a constant speed from 16min to 28 min; the volume ratio of the water phase to the organic phase is changed to 30: 70 at a constant speed from 28min to 35 min; the volume ratio of the water phase to the organic phase is kept constant at 35min to 50min at 30: 70.
The invention relates to a method for separating and determining canagliflozin and related substances thereof by using high performance liquid chromatography, which comprises the following steps:
(1) precisely weighing canagliflozin bulk drug or preparation, adding a proper amount of 70% acetonitrile solution for dissolving, diluting with 70% acetonitrile solution to prepare a solution containing 0.5mg of canagliflozin in each 1ml, and filtering if necessary to obtain a test solution; another 1ml sample solution is precisely measured, placed in a 100ml measuring flask, diluted to scale with mobile phase, and shaken up to serve as a control solution.
(2) And (3) taking 20 mu l of each of the test solution and the control solution, injecting into a high performance liquid chromatograph, and performing gradient elution. The chromatographic conditions were as follows: selecting a chromatographic column using butanesilane bonded silica gel as a filler, using a phosphoric acid solution (taking purified water, adjusting the pH value to 2-6 by using phosphoric acid) or a phosphate buffer solution (0.002mol/L sodium dihydrogen phosphate solution, adjusting the pH value to 2-6 by using phosphoric acid) as a mobile phase A, using acetonitrile as a mobile phase B, and performing gradient elution, wherein the column temperature is as follows: 25-35 ℃; the flow rate is 0.8-1.2 ml/min, and the detection wavelength is 210-215 nm or 287-297 nm. The elution conditions were as follows: when the time is 0min to 14-20 min, the volume ratio of the water phase to the organic phase in the mobile phase is 55: 45-65: 35; when the time is from 14 th to 20 th min to 26 th to 32 th min, the volume ratio of the water phase to the organic phase is changed to 30: 70-40: 60 at a constant speed; when the time is from 26 th to 32 th min to 33 th to 39 th min, the volume ratio of the water phase to the organic phase is changed to 25: 75-35: 65 at a constant speed; the volume ratio of the water phase to the organic phase is kept constant from 33 th to 39 th min to 48 th to 54 th min. At this point, the measurement of the relevant substance is completed, and the chromatogram is recorded.
(3) For continuous sample injection, a section of process of washing a chromatographic column and balancing a chromatographic system is arranged, namely the mobile phase A and the mobile phase B change at a constant speed of 60 percent to 40 percent from 48 min to 54min to 60 min to 66min, and then the washing and balancing process is finished after the mobile phase A and the mobile phase B are kept for 13 min.
The method for separating and determining canagliflozin and related substances by using high performance liquid chromatography, which is disclosed by the invention, preferably comprises the following steps of:
(1) precisely weighing canagliflozin bulk drug or preparation, adding a proper amount of 70% acetonitrile solution for dissolving, diluting with 70% acetonitrile solution to prepare a solution containing 0.5mg of canagliflozin in each 1ml, and filtering if necessary to obtain a test solution; another 1ml sample solution is precisely measured, placed in a 100ml measuring flask, diluted to scale with mobile phase, and shaken up to serve as a control solution.
(2) A chromatographic column (Inertsil-C4, 4.6X 250mm, 5 mu m) using butanesilane bonded silica gel as a filler, a phosphoric acid solution (taking purified water, adjusting the pH value to 3.5 with phosphoric acid) as a mobile phase A, acetonitrile as a mobile phase B, and carrying out gradient elution, wherein the column temperature is as follows: 25 ℃; the flow rate was 1.0ml/min and the detection wavelength was 210 nm. The elution conditions were as follows: the volume ratio of the water phase to the organic phase in the mobile phase is 60: 40 from 0min to 16 min; the volume ratio of the water phase to the organic phase is changed to 35: 65 at a constant speed from 16min to 28 min; the volume ratio of the water phase to the organic phase is changed to 30: 70 at a constant speed from 28min to 35 min; the volumes of the aqueous phase and the organic phase are kept constant at a ratio of 30: 70 from 35min to 50min, and chromatograms are recorded.
In the method of the present invention, the term "0 min to 14-20 min" means: starting from 0min to any time in 14-20 min (this time is denoted as time A); when the time is from 14 th to 20 th min to 26 th to 32 th min, the volume of the water phase and the organic phase in the mobile phase is changed to 30: 70-40: 60 at a constant speed, and the expression is that: from the selected 14 th to 20 th min, the volume ratio of the water phase to the organic phase in the mobile phase is changed to any ratio in the range of 30: 70 to 40: 60 through gradient change at the time B from the time A to any time in the range of 26 th to 32 th min (denoted as the time B). The expression with respect to gradient time is analogized.
In the method of the present invention, the peak area correction factor of the impurity a relative to canagliflozin is 0.78, the peak area correction factor of the impurity B relative to canagliflozin is 1.18, the peak area correction factor of each unknown related substance relative to canagliflozin is 1, and the content of each single impurity in the related substance and the total amount of the related substance are calculated by the peak area according to the canagliflozin self-control method with the correction factor added.
The method has the following beneficial effects or advantages:
the method has strong specificity, not only can provide a method for detecting the total amount of related substances of canagliflozin, but also provides a method for detecting single components of related substances, and impurities cannot interfere with each other;
the sensitivity is high, the known lowest detection concentrations of the impurity A and the impurity B of the canagliflozin are 0.009 mu g/ml and 0.011 mu g/ml respectively, and the detection of trace impurities can be ensured;
the method has strong practicability, and the processes of flushing the chromatographic column and balancing the chromatographic system are designed after the relevant substances are detected, so that the adverse effects of large base line noise, multiple gradient system peaks and the like caused by a large amount of continuous sample introduction can be avoided, and the detection result can be well reproduced;
the durability is good, parameters such as the pH value of the mobile phase, the proportion of the mobile phase, the flow rate, the column temperature, the wavelength and the like in the chromatographic condition fluctuate within the range of the invention, and the detection result is not obviously influenced.
In a word, the method for separating and determining canagliflozin and related substances thereof can realize the separation and analysis of the canagliflozin and the related substances thereof, the peak shapes of the canagliflozin and the peak shapes of the impurities are symmetrical, the number of theoretical plates is high, and all the impurities can be just completely separated; the adoption of proper gradient elution can ensure small baseline noise, can also ensure that small polar impurities are completely eluted, and simultaneously avoids overlong analysis time. Therefore, the method has the advantages of better specificity, sensitivity, accuracy and the like.
Drawings
FIG. 1: high performance liquid chromatogram of solvent blank;
FIG. 2: a high performance liquid chromatogram of a test solution of a canagliflozin bulk drug;
FIG. 3: detecting the high performance liquid chromatogram of a test solution of a crude drug of canagliflozin when the wavelength is 210 nm;
FIG. 4: a high performance liquid chromatogram of a test solution of a canagliflozin bulk drug mixed with the impurity A, B;
FIG. 5: a high performance liquid chromatogram of a test solution of the composition containing no canagliflozin and only the auxiliary material component;
FIG. 6: a high performance liquid chromatogram of a test solution of the pharmaceutical composition containing canagliflozin;
FIG. 7: and (3) a high performance liquid chromatogram of the test solution for acid degradation of the canagliflozin bulk drug.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
Apparatus and conditions
High performance liquid chromatograph: agilent 1260;
a chromatographic column: a chromatographic column (Inertsil-C4, 4.6X 250mm, 5 μm) using a butane silane bonded silica gel as a filler;
mobile phase A: aqueous phase phosphoric acid solution, taking purified water, adjusting the pH value to 3.5 by using phosphoric acid, and mixing the mobile phase B: organic phase acetonitrile;
elution was performed according to the gradient elution table (see table 1) (50 min later, column-wash equilibration stage).
Sample introduction volume: 20 mu l of the mixture;
column temperature: 25 ℃;
detection wavelength: 210 nm.
Experimental procedure
Taking about 25mg of canagliflozin raw material medicine, precisely weighing, placing in a 50ml volumetric flask, adding 70% acetonitrile solution of a solvent to dissolve and dilute to a scale, and shaking up to be used as a test solution. Taking solvent and sample solution, performing high performance liquid chromatography under the above chromatographic conditions, and recording chromatogram, as shown in FIG. 1 and FIG. 2. The result shows that the solvent does not interfere the detection of canagliflozin and related substances thereof, the canagliflozin and impurities thereof can be well separated, and the specificity of the method is good.
Example 2
Apparatus and conditions
High performance liquid chromatograph: agilent 1260;
a chromatographic column: a chromatographic column (Inertsil-C4, 4.6X 250mm, 5 μm) using a butane silane bonded silica gel as a filler;
mobile phase A: phosphoric acid solution, taking purified water, adjusting the pH value to 3.5 by using phosphoric acid, and mixing the mobile phase B: acetonitrile;
elution was performed according to the gradient elution table (see table 2) (50 min later, column-wash equilibration stage).
Sample introduction volume: 20 mu l of the mixture;
column temperature: at 25 ℃.
Experimental procedure
Taking about 25mg of crude drug of canagliflozin, precisely weighing, placing in a 50ml volumetric flask, adding 70% acetonitrile solution of solvent to dissolve and dilute to a scale, shaking up, and taking as a test solution. Taking the sample solution, performing high performance liquid chromatography under the above chromatographic conditions, and recording chromatogram with detection wavelengths of 210nm and 292nm, respectively, as shown in FIG. 3. Test results show that when the detection wavelength is 210nm and 292nm, synthetic intermediates and side reaction products brought in by the canagliflozin crude product can be detected, the canagliflozin and impurities thereof can be well separated, and the specificity of the method is good.
Example 3
Apparatus and conditions
High performance liquid chromatograph: agilent 1260;
a chromatographic column: a chromatographic column (Inertsil-C4, 4.6X 250mm, 5 μm) using a butane silane bonded silica gel as a filler;
a set of mobile phases a: phosphoric acid solution, taking purified water, adjusting the pH value to 2.0 by using phosphoric acid, and adding another group of mobile phases A: phosphoric acid solution, taking purified water, adjusting the pH value to be 6.0 by using phosphoric acid, and carrying out mobile phase B: acetonitrile; the two groups of mobile phase A and mobile phase B are respectively combined to be eluted once (the column washing balance system stage is after 50 min) according to a gradient elution table (see table 3).
Sample introduction volume: 20 mu l of the mixture;
column temperature: at 25 ℃.
Experimental procedure
Taking about 25mg of crude drug of canagliflozin, precisely weighing, placing in a 50ml volumetric flask, adding 70% acetonitrile solution of solvent to dissolve and dilute to a scale, shaking up, and taking as a test solution. Taking the sample solution, performing high performance liquid chromatography analysis on the sample solution by using a mobile phase with a pH value of 2.0 and a mobile phase with a pH value of 6.0 under the chromatographic conditions, and recording chromatograms. The results show that the mobile phases with the pH values of 2.0 and 6.0 are basically consistent with the detection result of the pH value of 3.5, and the mobile phase of the method has wide pH value application range.
Example 4
Apparatus and conditions
High performance liquid chromatograph: agilent 1260;
a chromatographic column: a chromatographic column (Inertsil-C4, 4.6X 250mm, 5 μm) using a butane silane bonded silica gel as a filler;
mobile phase A: phosphate solution, 0.005mol/L NaH2PO4Solution, pH adjusted to 2.0 with phosphoric acid, mobile phase B: acetonitrile;
eluting according to gradient elution table (see Table 4) (50 min later, column-flushing equilibrium system stage)
Sample introduction volume: 20 mu l of the mixture;
column temperature: at 25 ℃.
Experimental procedure
Taking about 25mg of crude drug of canagliflozin, precisely weighing, placing in a 50ml volumetric flask, adding 70% acetonitrile solution of solvent to dissolve and dilute to a scale, shaking up, and taking as a test solution. Taking the sample solution, performing high performance liquid chromatography under the chromatographic conditions, and recording the chromatogram. The results show that the detection effect of the phosphate solution as the mobile phase is basically consistent with that of the phosphate solution as the mobile phase.
Example 5
Apparatus and conditions
High performance liquid chromatograph: agilent 1260;
a chromatographic column: a chromatographic column (Inertsil-C4, 4.6X 250mm, 5 μm) using a butane silane bonded silica gel as a filler;
mobile phase A: phosphoric acid solution, taking purified water, adjusting the pH value to 3.5 by using phosphoric acid, and mixing the mobile phase B: acetonitrile;
elution was performed according to the gradient elution table (see table 5) (50 min later, column-wash equilibration stage).
Sample introduction volume: 20 mu l of the mixture;
column temperature: at 25 ℃.
Experimental procedure
Taking about 25mg of crude drug of canagliflozin, precisely weighing, placing in a 50ml volumetric flask, adding 70% acetonitrile solution of solvent to dissolve and dilute to a scale, shaking up, and taking as a test solution. Taking the sample solution, performing high performance liquid chromatography under the chromatographic conditions, and recording the chromatogram. The results show that the initial proportion of the organic phase in the mobile phase is reduced moderately, and the detection result of the method is not influenced.
Example 6
Apparatus and conditions
High performance liquid chromatograph: agilent 1260;
a chromatographic column: a chromatographic column (Inertsil-C4, 4.6X 250mm, 5 μm) using a butane silane bonded silica gel as a filler;
mobile phase A: phosphoric acid solution, taking purified water, adjusting the pH value to 3.5 by using phosphoric acid, and mixing the mobile phase B: acetonitrile;
elution was performed according to the gradient elution table (see table 6) (50 min later, column-wash equilibration stage).
Sample introduction volume: 20 mu l of the mixture;
column temperature: at 25 ℃.
Experimental procedure
Taking about 25mg of crude drug of canagliflozin, precisely weighing, placing in a 50ml volumetric flask, adding 70% acetonitrile solution of solvent to dissolve and dilute to a scale, shaking up, and taking as a test solution. Taking the sample solution, performing high performance liquid chromatography under the chromatographic conditions, and recording the chromatogram. The results show that the detection result of the method is not influenced by moderately increasing the initial proportion and the termination proportion of the organic phase in the mobile phase.
Example 7
Apparatus and conditions
High performance liquid chromatograph: agilent 1260;
a chromatographic column: a chromatographic column (Inertsil-C4, 4.6X 250mm, 5 μm) using a butane silane bonded silica gel as a filler;
mobile phase A: phosphoric acid solution, taking purified water, adjusting the pH value to 3.5 by using phosphoric acid, and mixing the mobile phase B: acetonitrile;
elution was performed according to the gradient elution table (see table 7) (50 min later, column-wash equilibration stage).
Sample introduction volume: 20 mu l of the mixture;
column temperature: 25 ℃;
detection wavelength: 210 nm.
Experimental procedure
Taking about 25mg of canagliflozin raw material medicine, precisely weighing, placing in a 50ml volumetric flask, adding 1ml of solution containing A, B impurity with the concentration of 37.5 mu g/ml, adding 70% acetonitrile solution of solvent to dissolve and dilute to a scale, shaking up to be used as a test solution. Taking the sample solution, performing high performance liquid chromatography under the above chromatographic conditions, and recording chromatogram, as shown in FIG. 4. The results indicate that canagliflozin impurity A, B can be detected, the impurity response is high, canagliflozin is well separated from the impurity, the method specificity is good, and the sensitivity is high.
Example 8
Apparatus and conditions
High performance liquid chromatograph: agilent 1260;
a chromatographic column: a chromatographic column (Inertsil-C4, 4.6X 250mm, 5 μm) using a butane silane bonded silica gel as a filler;
mobile phase A: phosphoric acid solution, taking purified water, adjusting the pH value to 3.5 by using phosphoric acid, and mixing the mobile phase B: acetonitrile;
elution was performed according to the gradient elution table (see Table 8) (50 min later, column-wash equilibration stage).
Sample introduction volume: 20 mu l of the mixture;
column temperature: 25 ℃;
detection wavelength: 210 nm.
Experimental procedure
Taking a proper amount of canagliflozin pharmaceutical composition containing 25mg of canagliflozin, precisely weighing, placing in a 50ml volumetric flask, adding 70% acetonitrile solution of a solvent, carrying out ultrasonic treatment for 30 minutes to completely dissolve the canagliflozin, diluting to a scale, and shaking up to be used as a test solution. And precisely weighing an equal amount of composition which does not contain canagliflozin and only contains auxiliary materials, placing the composition into a 50ml volumetric flask, adding 70% acetonitrile solution of a solvent, carrying out ultrasonic treatment for 30 minutes, diluting to a scale, and shaking up to obtain a blank solution. Taking blank solution and sample solution, performing high performance liquid chromatography under the above chromatographic conditions, and recording chromatogram, as shown in FIG. 5 and FIG. 6. The result shows that the blank composition does not interfere the detection of canagliflozin and related substances thereof, and the method has good specificity.
Example 9
Apparatus and conditions
High performance liquid chromatograph: agilent 1260;
a chromatographic column: a chromatographic column (Inertsil-C4, 4.6X 250mm, 5 μm) using a butane silane bonded silica gel as a filler;
mobile phase A: phosphoric acid solution, taking purified water, adjusting the pH value to 3.5 by using phosphoric acid, and mixing the mobile phase B: acetonitrile;
elution was performed according to the gradient elution table (see table 9) (50 min later, column equilibration stage).
Sample introduction volume: 20 mu l of the mixture;
column temperature: 25 ℃;
detection wavelength: 210 nm.
Experimental procedure
Taking about 25mg of canagliflozin raw material medicine, precisely weighing, placing in a 50ml volumetric flask, adding 3ml of 1mol/L hydrochloric acid solution and 3ml of acetonitrile, standing at room temperature for 24h, neutralizing with sodium hydroxide solution and cooling, adding 70% acetonitrile solution of a solvent to dissolve and dilute to a scale, shaking up, and taking the solution as an acid degradation test sample solution.
Taking about 25mg of canagliflozin bulk drug, precisely weighing, placing in a 50ml volumetric flask, adding 3ml of 1mol/L sodium hydroxide solution and 3ml of acetonitrile, standing at room temperature for 24h, neutralizing with hydrochloric acid solution and cooling, adding 70% acetonitrile solution of a solvent to dissolve and dilute to a scale, shaking up, and taking the solution as an alkali degradation test sample solution.
Taking about 25mg of canagliflozin raw material medicine, precisely weighing, placing in a 50ml volumetric flask, heating at 105 ℃ for 30h, cooling, adding 70% acetonitrile solution of a solvent, dissolving and diluting to a scale, shaking up, and taking the solution as a thermal degradation test sample solution.
Taking about 25mg of canagliflozin raw material medicine, precisely weighing, placing in a 50ml white glass volumetric flask, irradiating for 48 hours at the temperature of 4500Lx +/-500 Lx and 25 ℃, adding 70% acetonitrile solution of a solvent to dissolve and dilute to a scale, shaking up, and taking the solution as photodegradation test sample solution.
Taking about 25mg of canagliflozin raw material medicine, precisely weighing, placing in a 50ml volumetric flask, adding 15% H2O2And standing the solution 3ml and acetonitrile 3ml at room temperature for 24h, adding a solvent of 70% acetonitrile solution to dissolve and dilute the solution to a scale, and shaking up the solution to be used as an oxygen degradation test sample solution.
Taking each sample solution, performing high performance liquid chromatography under the above chromatographic conditions, and recording chromatogram, as shown in FIG. 7. The result shows that a series of related substances generated by forced degradation (acid, alkali, light, heat and oxygen) of canagliflozin can be separated and detected, the canagliflozin can be well separated from impurities thereof, and the specificity of the method is good.
Example 10
Apparatus and conditions
High performance liquid chromatograph: agilent 1260;
a chromatographic column: a chromatographic column (Inertsil-C4, 4.6X 250mm, 5 μm) using a butane silane bonded silica gel as a filler;
mobile phase A: phosphoric acid solution, taking purified water, adjusting the pH value to 3.5 by using phosphoric acid, and mixing the mobile phase B: acetonitrile;
elution was performed according to the gradient elution table (see table 10) (50 min later, column-wash equilibration stage).
Sample introduction volume: 20 mu l of the mixture;
column temperature: 25 ℃;
detection wavelength: 210 nm.
Experimental procedure
Taking about 25mg of crude drug of canagliflozin, precisely weighing, placing in a 50ml volumetric flask, adding 70% acetonitrile solution of solvent to dissolve and dilute to a scale, shaking up, and taking as a test solution. Taking sample solution, performing high performance liquid chromatography under the chromatographic conditions, recording chromatogram, and changing chromatograph parameters such as pH value of mobile phase, proportion of mobile phase, flow rate, column temperature, wavelength, etc. within a certain range, and performing single factor investigation, wherein specific chromatographic conditions are shown in Table 11, and results are shown in Table 12. The result shows that when the chromatographic parameters are changed in a certain range, the detection result of related substances of canagliflozin is basically stable, the number of the theoretical plates of the main peak of canagliflozin is still high, the separation degrees of the canagliflozin and impurities thereof are higher than 1.5, and the method has good durability.
b: the main peak is the canagliflozin peak.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (6)
1. A method for separating and determining canagliflozin and related substances thereof comprises the steps of performing gradient elution by adopting high performance liquid chromatography, taking a butane silane bonded silica gel filler column as a chromatographic column, taking acetonitrile as an organic phase, taking a phosphoric acid solution or a phosphate buffer solution as a water phase, mixing the acetonitrile and the phosphoric acid solution or the phosphate buffer solution as a mobile phase, and detecting by using an ultraviolet detector, wherein the pH value of the phosphoric acid solution or the phosphate buffer solution is 3.5, and the gradient elution comprises the following steps: when the time is 0min to 14-20 min, the volume ratio of the water phase to the organic phase in the mobile phase is 55: 45-65: 35; when the time is from 14 th to 20 th min to 26 th to 32 th min, the volume ratio of the water phase to the organic phase is changed to 30: 70-40: 60 at a constant speed; when the time is from 26 th to 32 th min to 33 th to 39 th min, the volume ratio of the water phase to the organic phase is changed to 25: 75-35: 65 at a constant speed; keeping the volume ratio of the water phase to the organic phase unchanged from 33 th to 39 th min to 48 th to 54 th min; wherein the related substances comprise impurities A and B, and the chemical structural formula is as follows:
2. the method of claim 1, wherein the phosphate buffer is selected from the group consisting of sodium dihydrogen phosphate, potassium dihydrogen phosphate, and ammonium dihydrogen phosphate, and the pH is adjusted with a phosphoric acid solution.
3. The process of claim 1, wherein the phosphate concentration in the aqueous phase is: 0 to 0.005 mol/L.
4. The method of claim 1, the gradient elution comprising: the volume ratio of the water phase to the organic phase in the mobile phase is 60: 40 from 0min to 16 min; the volume ratio of the water phase to the organic phase is changed to 35: 65 at a constant speed from 16min to 28 min; the volume ratio of the water phase to the organic phase is changed to 30: 70 at a constant speed from 28min to 35 min; the volume ratio of the water phase to the organic phase is kept constant at 35min to 50min at 30: 70.
5. The method of claim 1, further comprising the steps of:
(1) taking a canagliflozin raw material medicine or a pharmaceutical composition thereof, adding 70% acetonitrile solution to completely dissolve the canagliflozin, diluting to a constant volume, preparing into a solution containing 0.3-0.7 mg of canagliflozin in each 1ml of solution, and filtering to obtain a test solution;
(2) a butane silane bonded silica gel chromatographic column is used as a chromatographic column, the flow rate is 0.8-1.2 ml/min, the column temperature is 25-35 ℃, and the wavelength is 210-215 nm or 287-297 nm;
(3) injecting 10-30 mul of test solution into a high performance liquid chromatograph;
(4) a phosphate solution or phosphate buffer at ph3.5 in the aqueous phase was mixed with acetonitrile in the organic phase at a ratio of 60: after the initial volume ratio of 40 is eluted until a main peak comes out, gradually increasing the acetonitrile ratio until the volume ratio of the water phase to the organic phase reaches 30: 70 and keeping for 20 minutes to finish the determination of related substances of canagliflozin;
(5) calculating the purity of the canagliflozin, the content of each single impurity in related substances and the total amount of the related substances by using a self-comparison method of the canagliflozin with a correction factor.
6. The method according to claim 5, wherein in step (1), each 1ml of the solution contains 0.5mg of canagliflozin, or in step (2), the flow rate is 1.0ml/min, the column temperature is 25 ℃, the wavelength is 210nm, or in step (3), 20 μ l of the sample solution is taken, or in step (4), the aqueous phase is phosphoric acid solution or phosphate buffer solution with pH3.5, the initial volume ratio of the aqueous phase to the organic phase is 60: 40, and after the elution is carried out until the main peak comes out, the acetonitrile ratio is gradually increased until the volume ratio of the aqueous phase to the organic phase reaches 30: 70.
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| CN107515255B (en) * | 2016-06-17 | 2022-04-08 | 中美华世通生物医药科技(武汉)股份有限公司 | Method for measuring dapagliflozin and related substances thereof by using high performance liquid chromatograph |
| CN106226426B (en) * | 2016-07-18 | 2019-03-08 | 江苏德源药业股份有限公司 | A kind of method that high performance liquid chromatography splits canagliflozin five-membered ring impurity enantiomer |
| CN111077235B (en) * | 2018-10-19 | 2023-09-08 | 重庆医药工业研究院有限责任公司 | Method for determining 2- [ (2-methyl-5-bromophenyl) methyl ] -5- (4-fluorophenyl) thiophene impurity |
| CN111077234B (en) * | 2018-10-19 | 2023-09-15 | 重庆医药工业研究院有限责任公司 | Method for separating and measuring canagliflozin and impurities thereof by liquid chromatography |
| CN109374783B (en) * | 2018-12-21 | 2022-02-01 | 安徽联创生物医药股份有限公司 | Method for separating and determining related substances of canagliflozin bulk drug by using HPLC (high performance liquid chromatography) |
| CN111398457A (en) * | 2020-04-01 | 2020-07-10 | 上海应用技术大学 | Method for detecting substances related to Eagliflozin influence factors under degradation condition |
| CN112697932A (en) * | 2020-12-03 | 2021-04-23 | 湖南泰新医药科技有限公司 | Method for determining canagliflozin concentration in human plasma by LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) method |
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