CN107976489B - Method for determining residual pyridine in pregabalin - Google Patents

Method for determining residual pyridine in pregabalin Download PDF

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CN107976489B
CN107976489B CN201610940235.8A CN201610940235A CN107976489B CN 107976489 B CN107976489 B CN 107976489B CN 201610940235 A CN201610940235 A CN 201610940235A CN 107976489 B CN107976489 B CN 107976489B
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pyridine
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CN107976489A (en
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魏行
刘国柱
杨成
李明
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Guangdong HEC Pharmaceutical
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Sunshine Lake Pharma Co Ltd
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Abstract

The invention provides a method for determining residual pyridine in pregabalin, which comprises the following steps: analyzing residual pyridine in the pregabalin by a high performance liquid chromatography analysis method so as to obtain a chromatogram; and determining the content of residual pyridine in the pregabalin based on the chromatogram, wherein the high performance liquid chromatography adopts the following conditions: the chromatographic column is ZORBAX Eclipse XDB-C18, the thickness of the chromatographic column is 4.6X 250mm, the thickness of the chromatographic column is 5 microns, the detector is DAD, the detection wavelength is 240nm, the column temperature is 33-37 ℃, the mobile phase A phase is 10mmol/L dipotassium phosphate buffer solution, the pH value of the dipotassium phosphate buffer solution is 6.8-7.2, the mobile phase B phase is methanol, the flow rate is 0.8-1.2 mL/min, isocratic elution is adopted, the volume ratio of the phase A to the phase B in the isocratic elution is 68: 32-72: 28, and the running time is 20 min. By utilizing the method, the content of the residual pyridine in the pregabalin bulk drug can be simply, accurately, sensitively and specially determined, so that the drug effect, the quality and the pharmaceutical safety of the pregabalin bulk drug can be effectively controlled.

Description

Method for determining residual pyridine in pregabalin
Technical Field
The invention relates to the field of biological medicines, in particular to a method for determining residual pyridine in pregabalin.
Technical Field
Pregabalin is a novel gamma-aminobutyric acid (GABA) receptor agonist useful for the treatment of neuropathic pain, regional seizure epilepsy and anxiety. The U.S. Food and Drug Administration (FDA) approved in 2004 at 12 months for the treatment of neuropathic pain and postherpetic neuralgia of diabetic peripheral neuropathy, having the structural formula
Figure BDA0001139268130000011
Is white or off-white crystalline solid, and has the characteristics of being easily dissolved in water, alkaline and acidic aqueous solutions.
When pregabalin is synthesized, people often select pyridine as a reaction solvent, and the pyridine residue brings hidden dangers to the safety and effectiveness of products, so that the pyridine residue needs to be controlled.
The boiling point of pyridine is 115.2 ℃, and the residual pyridine in the medicine is generally measured by adopting a gas chromatography headspace sampling method. However, the solubility of the pregabalin bulk drug in an organic solvent is poor, the residual solvent pyridine to be detected is alkalescent, the peak shape is poor when the headspace gas phase analysis is adopted, and the recovery rate is also poor. In addition, when an ion chromatograph is used for analysis, the ionization degree of pregabalin is large, and pyridine peaks of a solvent to be detected are wrapped in, so that the detection of pyridine is interfered.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, the invention aims to provide a method for simply, accurately and sensitively detecting the residual pyridine in the pregabalin.
In a first aspect of the invention, the invention provides a method for determining residual pyridine in pregabalin. According to an embodiment of the invention, the method comprises: analyzing residual pyridine in the pregabalin by a high performance liquid chromatography analysis method so as to obtain a chromatogram; and determining the content of residual pyridine in the pregabalin based on the chromatogram, wherein the high performance liquid chromatography adopts the following conditions: the chromatographic column is ZORBAX Eclipse XDB-C18, the thickness of the chromatographic column is 4.6X 250mm, the thickness of the chromatographic column is 5 microns, the detector is DAD, the detection wavelength is 240nm, the column temperature is 33-37 ℃, the mobile phase A phase is 10mmol/L dipotassium phosphate buffer solution, the pH value of the dipotassium phosphate buffer solution is 6.8-7.2, the mobile phase B phase is methanol, the flow rate is 0.8-1.2 mL/min, isocratic elution is adopted, the volume ratio of the phase A to the phase B in the isocratic elution is 68: 32-72: 28, and the running time is 20 min. By using the detection method provided by the embodiment of the invention, the method has good specificity, and the separation degree of pyridine and adjacent chromatographic peaks is more than 1.5; the precision is good, and the relative standard deviation is less than 2.0 percent; the accuracy is high, the recovery rate is between 90% and 110%, and the requirements of Chinese pharmacopoeia on methods for detecting residual solvents or related substances in the bulk drugs are met. The method can accurately quantify pyridine in the pregabalin bulk drug within the range of 8ppm to 300 ppm.
According to an embodiment of the present invention, the method for determining residual pyridine in a pregabalin drug substance may further include at least one of the following additional technical features:
according to an embodiment of the invention, the pH of the dipotassium phosphate buffer is 7.0. In experiments, the inventor finds that the pH value of the dipotassium phosphate buffer is 7.0, the buffering capacity of the buffering salt is strong, the pyridine peak symmetry factor is 1.31, and the peak shape is good.
According to an embodiment of the invention, the flow rate is 1.0 mL/min. The inventor finds in experiments that the flow rate is 1.0mL/min, the retention time of pyridine in the substance to be detected is 6.11min, the retention is proper, and the pyridine peak can be effectively separated from other peaks.
According to an embodiment of the invention, the volume ratio of the phase a and the phase B in the isocratic elution is 70: 30. The inventors found in the experiment that the volume ratio of the phase A and the phase B in isocratic elution is 70:30, the separation degree of pyridine is 4.57, the separation degree is high, the symmetry factor is 1.28, the peak shape is good, some impurities in pregabalin are detected, and the condition provides good enough pyridine detection sensitivity.
According to an embodiment of the invention, the column temperature is 35 ℃. The inventor finds out in experiments that the column temperature is 35 ℃, the pyridine peak symmetry factor is 1.33, and the peak shape is better.
According to the embodiment of the invention, the pregabalin drug substance is provided in the form of a test solution, wherein the test solution is a dipotassium hydrogen phosphate-methanol solution of the pregabalin drug substance, and the content of the pregabalin drug substance is 10mg per ml of the test solution, wherein the dipotassium hydrogen phosphate-methanol solution is a mixed solution of the phase A and the phase B, and the volume ratio of the phase A to the phase B is 7: 3. The inventor finds that the solubility of the pregabalin bulk drug in the dipotassium phosphate-methanol solution is good, and meanwhile, when the sampling concentration of the pregabalin bulk drug is 10mg/mL, the pyridine detection sensitivity is good enough.
According to the embodiment of the invention, the dosage of the test solution is 20 microliter, and the determination of the pyridine content is more real, reliable and accurate.
According to an embodiment of the present invention, the determining the content of pyridine in the pregabalin drug substance based on the chromatogram is determined by the following formula:
Figure BDA0001139268130000021
wherein A isiIs the peak area of pyridine in the test solution; a. theSPeak area for pyridine in control solution; wTWeighing sample amount of the sample, mg; wSThe mass of pyridine weighed to prepare a control solution, mg; dTIs the dilution multiple of the test sample;DSis the dilution factor of the control solution; the control solution was a solution of pyridine in dipotassium hydrogen phosphate-methanol and the content of pyridine was 2 micrograms per milliliter of the control solution. According to the embodiment of the invention, the content of the residual pyridine in the pregabalin bulk drug is determined by adopting the method, the accuracy is high, and the result is more real and reliable.
In a second aspect of the present invention, the present invention provides a method for determining residual pyridine in pregabalin. According to an embodiment of the present invention, the method specifically includes:
(1) chromatographic conditions
The chromatographic column is ZORBAX Eclipse XDB-C18, 4.6X 250mm, 5 microns,
the detector is a digital signal detector (DAD),
the detection wavelength is 240nm, and the detection wavelength is 240nm,
the column temperature was 35 c,
the mobile phase A is 10mmol/L dipotassium hydrogen phosphate buffer solution, the pH value of the dipotassium hydrogen phosphate buffer solution is 7.0,
the mobile phase B is methanol, and the mobile phase B is methanol,
the flow rate was 1.0mL/min,
the elution gradient is isocratic elution, the volume ratio of the phase A to the phase B in isocratic elution is 70:30,
the running time is 20min, and the running time is 20min,
(2) preparing a blank solution
A mixed solution of methanol and phase A with the volume ratio of 3:7 is taken as the blank solution,
(3) preparing test solution
Precisely weighing 100mg of a test sample, dissolving the test sample in a 10mL volumetric flask by using a blank solution, performing ultrasonic treatment, diluting the test sample to a scale, shaking up the test sample to obtain the test sample solution,
(4) preparing a control solution
Accurately weighing 20mg of pyridine into a 100mL volumetric flask; dissolving with blank solution, performing ultrasonic treatment, diluting to scale, and shaking to obtain control stock solution;
precisely transferring the control stock solution into a volumetric flask with the volume of 1.0-100 mL, diluting the volumetric flask with the blank solution to a scale, shaking up to obtain a control solution,
(5) injecting 20 microliter of the test solution into a chromatograph to obtain a chromatogram, calculating the content of pyridine in the test solution according to the chromatogram,
wherein, the content of pyridine in the test solution is calculated according to the following formula, and the average value of 2 times of measurement results is taken as the measurement result:
Figure BDA0001139268130000031
in the formula: ai is the peak area of pyridine in the test solution;
ASthe average value of the pyridine peak area in the control solution is continuously fed 6 times;
WTweighing sample amount of the sample, mg;
WSthe mass of pyridine weighed to prepare a control solution, mg;
DTis the dilution multiple of the test sample;
DSis the dilution factor of the control solution.
The amount of the 1 st control solution was 20. mu.l.
By utilizing the detection method provided by the embodiment of the invention, the content of residual pyridine in the pregabalin bulk drug can be simply, accurately and sensitively determined, so that the quality of the pregabalin bulk drug can be effectively controlled.
Drawings
FIG. 1 is a chromatogram of a sample spiking solution using an Intersil ODS-3 column according to example 1 of the present invention;
FIG. 2 is a chromatogram of a sample spiked solution using a ZORBAX Eclipse XDB-C18 column according to example 1 of the present invention;
FIG. 3 is an ultraviolet absorption spectrum of pyridine according to example 1 of the present invention;
FIG. 4 is an ultraviolet absorption spectrum of pregabalin according to example 1 of the present invention;
FIG. 5 is a chromatogram of a sample-labeled solution at detection wavelengths of 240nm, 245nm and 250nm according to example 1 of the present invention;
FIG. 6 is a chromatogram of a pyridine control solution under different pH (6.8-7.2) buffer conditions according to example 1 of the present invention;
FIG. 7 shows a mobile phase ratio K according to example 1 of the present invention2HPO4The chromatogram of the sample solution under the condition of 70:30 MeOH is shown.
Detailed description of the preferred embodiments
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
In this example, the inventors have detailed the development process of the method for determining residual pyridine in pregabalin and the selection of the system applicability criteria.
1.1 selection of chromatography columns
In the initial stage of the development of the method, the inventor detects that the wavelength is 254nm, the column temperature is 35 ℃, and the mobile phase H2The results of the tests are shown in Table 1, wherein a chromatogram obtained by examining the retention time, the peak shape, the peak height and the like of pregabalin in a sample labeling solution is shown in FIG. 1, a chromatogram obtained by examining an Intersil ODS-3(250 × 4.6mm, 5 microns) column and a chromatogram obtained by examining a ZORBAX Eclipse XDB-C18 column are shown in FIG. 2, and the chromatogram obtained by examining a ZORBAX Eclipse XDB-C18 column is shown in FIG. 1. The preparation of the sample and the standard solution is to take about 100mg of the pregabalin bulk drug, precisely weigh the pregabalin bulk drug into a 10mL volumetric flask, dissolve the pregabalin bulk drug with the contrast solution, fix the volume and shake the pregabalin bulk drug uniformly to obtain 1 part of the reference solution, wherein the preparation of the contrast solution is the same as 2.1 items.
Table 1: examination result of sample added with standard solution
Figure BDA0001139268130000041
Figure BDA0001139268130000051
The results in Table 1 and FIG. 1 show that the retention time of pregabalin was 6.15min, the peak inversion occurred before the main peak of pregabalin, the retention time of pyridine was 9.72min, the peak height of pyridine was 9110, and the peak shape of pyridine was trailing, using Intersil ODS-3 chromatography.
The results in Table 1 and FIG. 2 show that with ZORBAX Eclipse XDB-C18 column, the pregabalin retention time was 4.67min, a peak inversion occurred before the pregabalin main peak, the pyridine retention time was 6.19min, the pyridine peak height was 12425, and the pyridine peak shape was trailing.
Comparing the above results, it can be seen that the peak height of pyridine under the ZORBAX Eclipse XDB-C18 chromatographic column condition is improved compared with that under the Intersil ODS-3 chromatographic column condition, and the sensitivity of the method is better, so the ZORBAX Eclipse XDB-C18 chromatographic column is selected for the subsequent development of the method for analyzing the residual pyridine in the pregabalin.
1.2 determination of the detection wavelength
As shown in FIG. 3, the maximum ultraviolet absorption of pyridine is 256nm, the absorption is strong between 235 nm and 250nm, and the maximum ultraviolet absorption of pregabalin is 196nm, as shown in FIG. 4. Under the condition of the chromatographic column selected in item 1.1, the inventor further inspects the detection wavelength, the inspection results of the detection wavelengths of 240nm, 245nm and 250nm are shown in fig. 5, the inspection results show that the detection wavelengths are 240nm and 245nm, both the front of the pregabalin main peak have inverted peaks, the detection wavelength is 240nm, the peak shape of the pregabalin main peak is normal, no inverted peak occurs, and in consideration of ensuring that the response of pyridine is enough and the occurrence of the inverted peak of the pregabalin is avoided, 240nm is finally selected as the detection wavelength.
1.3 selection and optimization of mobile phase, pH, mobile phase ratio
The inventors further examined the mobile phase, and the examination results showed that the mobile phase a was an aqueous phase, the symmetry factor was 1.68, the mobile phase a was selected to be 10mmol/L dipotassium phosphate solution buffer salt, the symmetry factor was 1.50, the peak shape of pyridine was better and the retention of pyridine on the column was enhanced compared to the mobile phase a being an aqueous phase, and therefore the mobile phase a was selected to be dipotassium phosphate solution buffer salt.
The inventors further examined the pH of the buffer salt, and the examination results are shown in fig. 6, which shows that the pH is 6.8 and the symmetry factor is 1.33; pH 7.0, symmetry factor 1.31; the pH is 7.2, the symmetry factor is 1.34, the buffer capacity of the buffer salt is strong, the pyridine peak shape is good, wherein the pH is 7.0, the pyridine peak symmetry factor is 1.31, the peak shape is better, and therefore the pH is preferably 7.0.
The above examination has confirmed that the column is ZORBAX Eclipse XDB-C18, 10mmol/L dipotassium hydrogen phosphate-methanol with detection wavelength of 240nm and pH 7.0 is used as the mobile phase, under isocratic elution for 20min, the inventor further examined the proportion of the mobile phase, and the examination result shows that the mobile phase is K respectively2HPO4:MeOH＝68:32、K2HPO4:MeOH＝70:30、K2HPO4The peak shapes of pregabalin and pyridine are normal, and the separation effect of pyridine is better. Wherein FIG. 7 shows the mobile phase K2HPO4 MeOH 70 MeOH 30, some impurities in pregabalin were also detected, the separation degree of pyridine was 4.57, the symmetry factor was 1.28, and the sensitivity was better than that, so it is preferable that the subsequent sensitivity examination is performed under the conditions, and the specific results are shown in table 2.
Table 2: examination result of sample added with standard solution
Figure BDA0001139268130000052
Figure BDA0001139268130000061
1.4 investigation of flow Rate and column temperature
Based on the determined conditions and the condition that the mobile phase proportion is preferred, the flow rate is further examined, and the examination result shows that when the flow rate is respectively 0.8mL/min, 1.0mL/min and 1.2mL/min, the peak shapes of the pregabalin and the pyridine are normal, and the separation degree effect of the pyridine is good. Based on the flow rate of 1.0mL/min, the retention time of pyridine to be detected is 6.11min, the retention is more reasonable than that of pyridine, the pyridine peak can be effectively separated from other peaks, and the pyridine peak is not interfered, and the preferred flow rate is 1.0 mL/min.
The inventor further examines the column temperature of 25 ℃, 33 ℃, 35 ℃ and 37 ℃, and the examination result shows that the column temperature is 25 ℃, the pyridine peak symmetry factor is 1.79, the peak shape is not good, the column temperature is respectively 33 ℃, 35 ℃ and 37 ℃, the peak shape is better, wherein the column temperature is 35 ℃, the pyridine peak symmetry factor is 1.33, the peak shape is better than that at other temperatures, and in consideration of the fact that the column temperature is too high, the loss of the stationary phase of the chromatographic column is easy to cause, 35 ℃ is preferred.
1.5 selection of the concentration of the test sample
The ideal sample diluent is a mobile phase solution, and the preferred isocratic elution mobile phase ratio is K2HPO4MeOH 70:30, which has a good dissolution ability for the sample, was selected as methanol/K2HPO4The mixed solution of the buffer solution with the ratio of 30:70 is used as the test sample diluent. Regarding the determination of the preparation concentration and the sample amount of the sample, the inventors finally determined by experiments that the concentration of the sample is 10mg/mL and the sample amount is 20 microliters, and could ensure a sufficiently good pyridine detection sensitivity.
1.6 System suitability criteria selection
Based on the test results of the system applicability that pyridine is retained for 6.15min in ZORBAX Eclipse XDB-C18, the symmetry factor is 1.28 and 3.2, the separation degree of pyridine and adjacent peaks is more than or equal to 1.5, RSD of the area of 6 times of peak injection is less than or equal to 10.0%, and the signal-to-noise ratio of 10% control solution is more than or equal to 10, which are taken as the system applicability standard.
Example 2
In this example, how the inventors obtained a chromatogram based on the chromatographic conditions determined in example 1 and calculated the content of residual pyridine in pregabalin using the chromatogram is described in detail.
2.1 preparation of the relevant solutions
Mobile phase:
phase A: weighing about 1.74g of anhydrous dipotassium phosphate, weighing into a 1L beaker, adding 1L of ultrapure water for dissolving, uniformly mixing, adjusting the pH value to 7.0 by using 0.1mol/L phosphoric acid solution or 0.1mol/L potassium hydroxide solution, and then filtering with a 0.2 mu m water system filter membrane for ultrasonic treatment to obtain the compound;
phase B: 100% methanol;
dilution (blank solution): phase A is a mixed solution of phase B and phase 7: 3;
control stock solutions: weighing pyridine in a volumetric flask with the volume of about 20mg to 100mL, accurately weighing, diluting with a diluent to a scale, and shaking up to obtain the product;
control solution: transferring 1.0mL of the control stock solution into a 100mL volumetric flask, diluting the volumetric flask to a scale with a diluent, and shaking up to obtain the product;
sensitivity solution: transferring 1.0mL of the control solution into a 10mL volumetric flask, diluting the control solution to a scale with a diluent, and shaking up to obtain the reagent;
test solution: taking about 100mg of a test sample, precisely weighing, putting into a 10mL volumetric flask, diluting to a scale with a diluent, and shaking uniformly to obtain the test sample; 2 parts are prepared in parallel.
2.2 chromatographic conditions
A chromatographic column: ZORBAX Eclipse XDB-C18, 4.6X 250mm, 5 microns,
column temperature: at a temperature of 35 c,
detection wavelength: the particle size of the nano-particles is 240nm,
flow rate: 1.0mL/min of the reaction solution,
sample introduction volume: 20 microliter of the total amount of the active ingredients,
preparing a mobile phase: phase a (0.01mol/L pH 7.0 dibasic potassium phosphate buffer): weighing about 1.74g of anhydrous dipotassium phosphate, weighing into a 1L beaker, adding 1L of ultrapure water for dissolving, uniformly mixing, adjusting the pH value to 7.0 by using 0.1mol/L phosphoric acid solution or 0.1mol/L potassium hydroxide solution, and then filtering and ultrasonically treating by using a 0.2 micron water system filter membrane to obtain the compound;
phase B: the reaction mixture of methanol and water is reacted with methanol,
isocratic elution: phase A and phase B are 70: 30.
2.3 correlation detection operation
After the base line is balanced, under the 2.2 chromatographic conditions, blank solution, sensitivity solution, reference solution and sample solution are sampled according to the sequence in the table 3, and each map is recorded.
Table 3: sample introduction sequence
Name of solution Number of sample introduction needles
Blank solution 1 needle
1 part of sensitivity solution 1 needle
Control solution 1 part 6 needles
2 parts of test solution Each 1 needle
System applicability requirements: the signal-to-noise ratio of the pyridine peak in the sensitivity solution is not less than 10; the separation degree of pyridine and adjacent peaks in the first needle control solution is not less than 1.5; RSD of the peak area of the pyridine with 6 needles for continuous sample injection cannot exceed 10.0%. Wherein, the system is used for detecting a sample and verifying methodology after the applicability of the system meets the requirement.
2.4 chromatogram-based method for calculating residual pyridine content
Calculating the pyridine content of the test sample according to the following formula, and taking the average value of 2 times of measurement results as a detection result:
Figure BDA0001139268130000071
in the formula: a. theiFor pyridine in test solutionPeak area of (a);
ASthe average value of the pyridine peak area in the control solution is continuously fed 6 times;
WTweighing sample amount, mg, of the pregabalin test sample;
WSthe mass of pyridine weighed to prepare a control solution, mg;
DTis the dilution multiple of the test sample;
DSis the dilution multiple of the control solution,
the acceptance criterion is a pyridine content of 200ppm or less.
Example 3
In this example, the inventors examined the methodology of the present invention for determining residual pyridine in pregabalin.
3.1 System applicability
The preparation methods of the blank solution and the control solution are the same as the item 2.1, and 1 part of each solution is prepared.
The operation is as follows: under 2.2 chromatographic conditions, after the system is balanced, taking blank solution for sample injection 1 needle, taking sensitivity solution for sample injection 1 needle, taking contrast solution for sample injection 6 needles, and recording the chromatogram. The test result shows that the peak areas of the continuously fed 6-needle pyridine are 72298, 74131, 74240, 73774, 74888 and 74759 in sequence, and the RSD of the peak area of the continuously fed 6-needle pyridine is 1.27 percent, so that the requirement is met; the pyridine peak signal-to-noise ratio in the sensitivity solution is 32.80, which meets the requirement; the separation degree of pyridine in the first needle of the control solution and an adjacent peak is N/A, based on the peak of only pyridine in the control solution, the N/A is infinite, and the control solution meets the requirement. Therefore, the system suitability meets the detection requirement, so that the subsequent methodology investigation is continued.
3.2 specificity
The preparation method of the blank solution, the reference stock solution, the reference solution and the sample solution is the same as 2.1 items;
preparing a 100% test sample adding standard solution: taking about 100mg of a test article, precisely weighing the test article into a 10mL volumetric flask, dissolving the test article by using a control solution, fixing the volume, and shaking up to obtain 1 part.
The operation is as follows: under 2.2 chromatographic conditions, a blank solution, a test solution, a reference solution and a test solution are respectively sampled for 1 needle, and corresponding chromatograms are recorded, wherein the specific test results are shown in table 4.
Table 4: result of specialization
Name of solution Pyridine retention time (min) Peak area of pyridine Degree of separation of pyridine
Blank solution / / /
Test solution / / /
Control solution 6.11 74482 /
Sample adding solution 6.11 73139 7.24
The examination result in table 4 shows that the blank solution has no interference to pyridine in the control solution and the test solution, and meets the requirement; the minimum value of the separation degree of the pyridine peak and the pregabalin peak in the test solution, the reference solution and the test product adding standard solution is 7.24, which meets the requirement; the retention time of pyridine peak in the sample solution is identical to that of the control solution, and is 6.11 min; the peak area of pyridine in the sample solution is 73139, and compared with the sample solution, the peak area of pyridine in the sample solution is increased, which meets the requirement. Therefore, the method has good specificity.
3.3 precision
Preparation of analytical repetitive solution: taking about 100mg of a test article, precisely weighing the test article into a 10mL volumetric flask, dissolving the test article by using a control solution, fixing the volume, and shaking up to obtain 6 parts in parallel, wherein the solution is prepared by an analyst A;
preparing intermediate precision solution: taking about 100mg of a test article, precisely weighing the test article into a 10mL volumetric flask, dissolving the test article by using a control solution, fixing the volume, shaking up the test article to obtain 6 parts in parallel, and performing the following operations by different analysts in the same laboratory at different times by using different instruments.
And (3) analysis of repeatability: under the condition of 2.2 chromatographs, 6 parts of analysis repeatability solution are taken, 1 needle is injected for each part, and the content of pyridine in the 6 parts of analysis repeatability solution is calculated according to the average value of the control peak area in the system applicability.
Intermediate precision operation: under 2.2 chromatographic conditions, 6 parts of intermediate precision solution is taken, 1 needle is injected for each sample, and the content of pyridine in the 6 parts of intermediate precision solution is calculated according to the average value of the control peak area in the system applicability.
The analysis repeatability test result shows that the RSD value of the pyridine content in 6 analysis repeatability solutions is 0.52 percent, and the analysis repeatability solutions meet the requirements.
The results of the intermediate precision tests show that the RSD value of the pyridine content in 6 parts of intermediate precision solution is 1.24%, the RSD value of the pyridine content in 12 parts of solution with analysis repeatability and intermediate precision is 0.92%, and the absolute difference value of the average values of the analysis repeatability and the intermediate precision pyridine content is 0.60ppm, which both meet the requirements. Therefore, the method is excellent in precision.
3.4 Linear relationship
The preparation methods of the blank solution, the reference stock solution and the reference solution are the same as 2.1 items, and 1 part of the solution is prepared respectively;
preparation of linear stock solution: precisely transferring the control stock solution into a volumetric flask with the volume of 25mL to 100mL, diluting the volumetric flask with a diluent to a scale, and shaking up the volumetric flask to obtain the liquid preparation;
preparing a quantitative limiting solution: and (4) taking 4mL to 100mL of the control solution in a volumetric flask, diluting the control solution to a scale mark with a diluent, and shaking up the control solution to obtain the reagent.
Preparation of a linear solution: precisely transferring linear stock solutions or control solutions with different volumes as shown in the table 5 into volumetric flasks with different volumes, diluting the solutions to scales by using a diluent, shaking up to obtain linear solutions with various concentrations, and preparing 1 part of each solution.
TABLE 5
Concentration level 10％ 50％ 100％ 150％
Linear stock solution (mL) / 1 2 3
Control solution (mL) 1 / / /
Volumetric flask (mL) 10 50 50 50
The operation is as follows: under 2.2 chromatographic conditions, taking a blank solution sample injection needle 1, taking a linear solution and a quantitative limit solution under each concentration level, sequentially carrying out sample injection for 2 times at each concentration from low concentration to high concentration, and recording a chromatogram. The specific test results are shown in table 6, the average value of the pyridine peak area is used for carrying out unary linear regression on the concentration of the pyridine peak area, the linear regression equation is that y is 37784.335x-1294.439, the correlation coefficient r is 1.000, and the method meets the requirements; the peak area ratio of the absolute value of the Y-axis intercept of the pyridine to the 100% limit concentration is 1.74%, which meets the requirement. The pyridine of the method has good linearity in the range of 0.08156 mu g/mL to 3.0585 mu g/mL.
Table 6: linear result
Figure BDA0001139268130000101
3.5 degree of accuracy
The preparation method of the blank solution and the sample solution is the same as 2.1 items, wherein 1 part of the blank solution is prepared, and 2 parts of the sample solution is prepared;
preparation of linear stock solution: precisely transferring the control stock solution into a volumetric flask with the volume of 25mL to 100mL, diluting the volumetric flask to the scale with a diluent, and shaking up to obtain 1 part;
preparation of an accuracy solution: taking about 500mg of a sample to be tested, precisely weighing, placing in a 50mL volumetric flask, and weighing 9 parts in parallel; after weighing, 30-40 mL of diluent is added into each volumetric flask, the sample is ultrasonically dissolved, and then linear stock solutions with different volumes are transferred into the 9 volumetric flasks according to the following table 7, wherein 3 parts of each concentration is prepared. Diluting with diluent to scale, and shaking.
TABLE 7
Concentration level 50% spiking solution 100% spiking solution 150% labeling solution
Linear stock solution (mL) 1.0 2.0 3.0
Volumetric flask (mL) 50 50 50
The operation is as follows: under 2.2 chromatographic conditions, taking a blank solution and injecting a sample into 1 needle, taking 2 parts of sample solution and injecting a sample into 1 needle respectively, and recording a chromatogram map, wherein the sample is accurately injected into 1 needle respectively at each concentration level.
Wherein, the pyridine content and the solvent recovery rate in the sample solution before and after the standard addition are respectively calculated according to the following formulas:
Figure BDA0001139268130000102
theoretical quantity ═ CS×V
Figure BDA0001139268130000103
Figure BDA0001139268130000111
In the formula: cSAdding the concentration of pyridine linear stock solution in the standard solution into the sample, wherein the concentration is mg/mL;
v is the volume of pyridine linear stock solution added into the standard solution added into each sample, mL;
CS+Tadding the measured pyridine residual quantity in the standard solution to each sample;
Cithe residual pyridine amount measured in the test solution is the pyridine residual amount;
Figure BDA0001139268130000112
the average value of the pyridine residual quantity measured in the test solution is shown;
Aithe peak area of pyridine in the test solution is shown;
AS2 against the average peak area of pyridine in control solution;
AS+Tadding the peak area of pyridine in the standard solution to each sample;
WTweighing the sample amount of the sample in the sample solution, namely mg;
WSweighing pyridine in the control solution in mg;
WS+Tadding the sample weighing and mg of the sample in the standard solution to each sample;
DTis the dilution multiple of the test solution;
DSis the dilution factor of the control solution;
DS+Tand adding the dilution times of the test samples in the standard solution to each test sample.
The test results are shown in table 8, and show that the single value range of the pyridine recovery rate is 91.20% -104.23%, and between 90% -110% under the limit concentration of 50%, 100%, 150%, and meets the requirements; the single value RSD of the pyridine recovery rate is 4.50 percent, which meets the requirement. The method is therefore accurate.
Table 8: accuracy results
Figure BDA0001139268130000113
3.6 durability examination
3.6.1 solution stability test
The preparation method of the contrast solution is the same as that of the item 2.1, and 1 part of the contrast solution is prepared;
preparing a 100% test sample adding standard solution: taking about 100mg of a test article, precisely weighing the test article into a 10mL volumetric flask, dissolving the test article by using a control solution, fixing the volume, and shaking up to obtain 1 part.
The operation is as follows: under 2.2 chromatographic conditions, taking a control solution and a 100% sample solution, standing at room temperature, injecting samples of 1 needle at 0h, 7h, 12h, 19h, 24h, 48h and 72h respectively, and recording a chromatogram. At room temperature, the ratio of the pyridine peak area to the 0h peak area in the control solution at each time point is 0.94-1.00 (between 0.90-1.10) within 72h under investigation; the ratio of the pyridine peak area to the 0h peak area in the standard solution added with 100 percent of the sample is 0.94-0.99 (between 0.90-1.10), which meets the requirement. The control and test solutions were stable at room temperature for 72 hours.
3.6.2 chromatographic Condition Change test
The preparation methods of the blank solution, the sensitivity solution and the control solution are the same as 2.1 items, and 1 part of each of the blank solution, the sensitivity solution and the control solution is prepared.
The operation is as follows: the chromatographic conditions under item 2.2 were used as the base conditions for single factor changes, as shown in Table 9. Under each condition, after the base line is balanced, 1 needle is respectively injected into a blank solution, a sensitivity solution and a contrast solution, and the chromatogram is recorded.
TABLE 9
Figure BDA0001139268130000121
The test results are shown in table 10, and show that the minimum value of the pyridine peak signal-to-noise ratio of the sensitivity solution under the changed chromatographic conditions is 22.85; the maximum value of RSD of the peak area of the continuous sample injection 3 needles of pyridine is 1.15 percent; the minimum value of the separation degree of pyridine and an adjacent peak in the first needle of the contrast solution is more than or equal to 1.5, the minimum value meets the requirement, and the durability of the method is good. Wherein the degree of separation of pyridine from adjacent peaks in the first needle of the control solution is infinite based on the pyridine-only peak in the control solution.
Table 10: durability results
Figure BDA0001139268130000131
Figure BDA0001139268130000141
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims (8)

1. A method for determining residual pyridine in pregabalin is characterized in that the residual pyridine in pregabalin is analyzed by a high performance liquid chromatography analysis method so as to obtain a chromatogram; and determining the content of residual pyridine in the pregabalin based on the chromatogram, wherein the high performance liquid chromatography adopts the following conditions:
the chromatographic column is ZORBAX Eclipse XDB-C18, 4.6X 250mm, 5 microns,
the detector is a digital signal detector (DAD),
the detection wavelength is 240nm, and the detection wavelength is 240nm,
the column temperature is 33-37 ℃,
the mobile phase A is 10mmol/L dipotassium phosphate buffer solution, the pH value of the dipotassium phosphate buffer solution is 6.8-7.2,
the mobile phase B is methanol, and the mobile phase B is methanol,
the flow rate is 0.8mL/min to 1.2mL/min,
the elution gradient is isocratic elution, the volume ratio of the phase A to the phase B in isocratic elution is 68: 32-72: 28,
the running time is 20 min;
the test solution is a dipotassium hydrogen phosphate-methanol solution of a pregabalin bulk drug, and the content of the pregabalin bulk drug is 10mg based on each milliliter of the test solution, wherein the dipotassium hydrogen phosphate-methanol solution is a mixed solution of the phase A and the phase B, and the volume ratio of the phase A to the phase B is 7: 3.
2. The method of claim 1, wherein the dipotassium phosphate buffer has a pH of 7.0.
3. The method of claim 1, wherein the flow rate is 1.0 mL/min.
4. The method of claim 1, wherein the volume ratio of the phase A and the phase B in the isocratic elution is 70: 30.
5. The method of claim 1, wherein the column temperature is 35 ℃.
6. The method of claim 1, wherein the sample solution is present in an amount of 20 μ l.
7. The method of claim 1, wherein the determining residual pyridine in the pregabalin based on the chromatogram is determined by the following formula:
Figure FDA0002461926910000011
wherein: a. theiIs the peak area of pyridine in the test solution;
ASpeak area for pyridine in control solution;
WTweighing sample amount of the sample, mg;
WSthe mass of pyridine weighed to prepare a control solution, mg;
DTis the dilution multiple of the test sample;
DSis the dilution factor of the control solution;
the control solution was a solution of pyridine in dipotassium hydrogen phosphate-methanol and the content of pyridine was 2 micrograms per milliliter of the control solution.
8. A method for determining residual pyridine in pregabalin, which is characterized by comprising the following steps:
(1) chromatographic conditions
The chromatographic column is ZORBAX Eclipse XDB-C18, 4.6X 250mm, 5 microns,
the detector is a digital signal detector (DAD),
the detection wavelength is 240nm, and the detection wavelength is 240nm,
the column temperature was 35 c,
the mobile phase A is 10mmol/L dipotassium hydrogen phosphate buffer solution, the pH value of the dipotassium hydrogen phosphate buffer solution is 7.0,
the mobile phase B is methanol, and the mobile phase B is methanol,
the flow rate was 1.0mL/min,
the elution gradient is isocratic elution, the volume ratio of the phase A to the phase B in isocratic elution is 70:30,
the running time is 20min, and the running time is 20min,
(2) preparing a blank solution
A mixed solution of methanol and phase A with the volume ratio of 3:7 is taken as the blank solution,
(3) preparing test solution
Precisely weighing 100mg of a test sample, dissolving the test sample in a 10mL volumetric flask by using a blank solution, performing ultrasonic treatment, diluting the test sample to a scale, shaking up the test sample to obtain the test sample solution,
(4) preparing a control solution
Precisely weighing 20mg of pyridine, dissolving the pyridine in a 100mL volumetric flask by using a blank solution, performing ultrasonic treatment, diluting the solution to a scale, and shaking up the solution to obtain a control stock solution;
precisely transferring the control stock solution into a volumetric flask with the volume of 1.0-100 mL, diluting the volumetric flask with the blank solution to a scale, shaking up to obtain a control solution,
(5) injecting 20 microliter of the test solution into a chromatograph to obtain a chromatogram, calculating the content of pyridine in the test solution according to the chromatogram,
wherein, the content of pyridine in the test solution is calculated according to the following formula, and the average value of 2 times of measurement results is taken as the measurement result:
Figure FDA0002461926910000021
wherein: a. theiIs the peak area of pyridine in the test solution;
ASthe average value of the pyridine peak area in the control solution is continuously fed 6 times;
WTweighing sample amount of the sample, mg;
WSthe mass of pyridine weighed to prepare a control solution, mg;
DTis the dilution multiple of the test sample;
DSis the dilution factor of the control solution;
the amount of 1 to control solution was 20. mu.l.
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