Detection method of aspirin-related substances
1. A method for detecting aspirin-related substances is characterized by comprising the following steps:
quantitatively detecting aspirin related substances by high performance liquid chromatography;
the high performance liquid chromatography conditions include:
a chromatographic column: chromatographic column with octadecylsilane chemically bonded silica as filler;
mobile phase A: phosphoric acid-water;
mobile phase B: acetonitrile;
flow rate: 1.0 ml/min;
column temperature: 25 ℃;
detection wavelength: 237 nm;
diluent agent: mobile phase a-acetonitrile.
2. The method for detecting aspirin-related substance according to claim 1, wherein the elution procedure is as follows:
3. an aspirin-related substance detection method according to claim 1, characterized in that the related substances are 4-hydroxybenzoic acid, 4-hydroxyisophthalic acid, salicylic acid, 2- ((hydroxymethylbenzoyl) oxy) benzoic acid, 2-carboxyphenyl salicylate and 2- (acetoxy) benzoic anhydride.
4. The method for detecting aspirin-related substance according to claim 1, wherein a sample solution and a system suitability solution are separately injected into a liquid chromatograph, and a chromatogram is recorded, the sample solution and the system suitability solution being prepared by:
respectively taking 4-hydroxybenzoic acid, 4-hydroxyisophthalic acid, salicylic acid, 2- ((hydroxymethylbenzoyl) oxy) benzoic acid, salicylic acid-2-carboxyl phenyl ester and 2- (acetoxyl) benzoic anhydride as impurities as reference substances, and adding acetonitrile to dilute into a solution of 0.1g/L, wherein the solution is used as each impurity stock solution;
adding an appropriate amount of an impurity stock solution into raw aspirin, and preparing a mixed solution containing 1g/L of aspirin and 0.001g/L of each impurity by using a diluent to serve as a system applicability solution;
adding a diluent into aspirin, and dissolving by ultrasonic waves to prepare a solution of 1g/L as a test solution.
5. The method for detecting an aspirin-related substance according to claim 1, wherein a volume ratio of the mobile phase A to the acetonitrile in the diluent is 4: 1.
6. The aspirin-related substance detection method according to claim 1, characterized in that aspirin raw material is taken, a diluent is added, ultrasonic treatment is carried out in an ice-water bath, a solution with a constant volume of 1g/L is carried out, and a supernatant is taken as a test solution after centrifugation; the test solution was diluted 1000-fold with a diluent to serve as a 0.1% self-control solution.
7. The method for detecting an aspirin-related substance according to claim 1, wherein in the mobile phase A, the ratio of phosphoric acid to water is 1: 300.
8. the method for detecting aspirin-related substance according to claim 1, wherein a temperature of the sample injection tray is 4 ℃.
9. The method for detecting aspirin-related substance according to claim 1, wherein a color spectrum is recorded, and the content of individual impurities and the content of total impurities are calculated by performing measurement using a self-control method.
10. The method for detecting an aspirin-related substance according to claim 1, wherein the calculation method of the individual impurities and the total impurities is as follows:
single impurity% (% single impurity peak area/0.1% control peak area 0.1 × 100%);
total impurities% (% impurity peak area/0.1% control peak area) (% 0.1) (% 100).
Background
Since 1898, aspirin has been produced as an antipyretic, analgesic and anti-inflammatory drug, it has been known for a century that modern medicine finds it also has: 1) reducing the risk of transient ischemic attacks and secondary sudden brain attacks; 2) reducing the risk of stable and unstable angina; 3) preventing the risk of platelet thrombosis after major surgery; 4) reducing the risk of myocardial infarction of patients with high-risk cardiovascular diseases and other applications. Is one of the typical new medicines used by old medicines.
The control of related substances of the product is of great importance to the quality control of the product, but in the existing detection method for related substances of aspirin, only the detection method of salicylic acid is recorded in Chinese pharmacopoeia, and other impurities cannot be detected. The detection methods of related substances recorded in other foreign pharmacopoeias are the same, but after the method reappearance of the inventor, the foreign pharmacopoeia method is found to be incapable of effectively separating known impurities and unknown impurities and has the phenomena of insufficient sensitivity and peak pattern, and in order to ensure the quality of the preparation in the processes of research, production, storage, sale and use and ensure the safety and effectiveness of medicines, the detection method with high sensitivity and good separation degree among the impurities is urgently needed to be provided.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for detecting aspirin-related substances, and compared with standard chromatographic conditions provided in European pharmacopoeia, the chromatographic conditions provided by the invention are improved compared with the primary color chromatographic conditions, regardless of the peak type or the separation degree of impurities.
Currently, in the process of aspirin bulk drug synthesis, the known impurities that may be contained in the final product are the following 6 impurities:
a method for detecting aspirin-related substances comprises the following steps:
quantitatively detecting aspirin related substances by high performance liquid chromatography;
the high performance liquid chromatography conditions include:
a chromatographic column: chromatographic column with octadecylsilane chemically bonded silica as filler;
mobile phase A: phosphoric acid-water;
mobile phase B: acetonitrile;
flow rate: 1.0 ml/min;
column temperature: 25 ℃;
detection wavelength: 237 nm;
diluent agent: mobile phase a-acetonitrile.
Further, the elution procedure was as follows:
further, the related substances are 4-hydroxybenzoic acid, 4-hydroxyisophthalic acid, salicylic acid, 2- ((hydroxymethylbenzoyl) oxy) benzoic acid, 2-carboxyphenyl salicylate and 2- (acetoxy) benzoic anhydride.
Further, a sample solution and a system applicability solution are respectively injected into a liquid chromatograph, and a chromatogram is recorded, wherein the sample solution and the system applicability solution are prepared by the following method:
respectively taking 4-hydroxybenzoic acid, 4-hydroxyisophthalic acid, salicylic acid, 2- ((hydroxymethylbenzoyl) oxy) benzoic acid, salicylic acid-2-carboxyl phenyl ester and 2- (acetoxyl) benzoic anhydride as impurities as reference substances, and adding acetonitrile to dilute into a solution of 0.1g/L, wherein the solution is used as each impurity stock solution;
adding an appropriate amount of an impurity stock solution into raw aspirin, and preparing a mixed solution containing 1g/L of aspirin and 0.001g/L of each impurity by using a diluent to serve as a system applicability solution;
adding a diluent into aspirin, and dissolving by ultrasonic waves to prepare a solution of 1g/L as a test solution.
Further, the volume ratio of the mobile phase A to the acetonitrile in the diluent is 4: 1.
Further, taking an aspirin raw material medicine, adding a diluent, performing ultrasonic treatment in an ice-water bath, fixing the volume of a solution to 1g/L, and centrifuging to obtain a supernatant serving as a test solution; the test solution was diluted 1000-fold with a diluent to serve as a 0.1% self-control solution.
Further, in the mobile phase a, the ratio of phosphoric acid to water is 1: 300.
further, the temperature of the sample injection tray was 4 ℃.
Further, a color spectrum is recorded, and the content of single impurities and the content of total impurities are calculated by measuring by using a self-contrast method.
Further, the calculation method of the single impurities and the total impurities comprises the following steps:
single impurity% (% single impurity peak area/0.1% control peak area 0.1 × 100%);
total impurities% (% impurity peak area/0.1% control peak area) (% 0.1) (% 100).
A method for detecting aspirin-related substances comprises the following steps:
(1) solution preparation:
weighing 800ml of mobile phase A, adding 200ml of acetonitrile, and shaking up to obtain a blank solution, namely a diluent solution;
an appropriate amount of aspirin raw material medicine is taken, an appropriate amount of stock solution of the impurity A, B, C, D, E, F is added, and a diluent is used for preparing a mixed solution containing 1.0mg of aspirin and 1 microgram of each of the impurity A, B, C, D, E, F in each 1ml of the mixed solution to serve as a system applicability solution.
Adding a diluent into aspirin, and ultrasonically dissolving to prepare a solution of 1mg/ml as a test solution;
(2) chromatographic conditions are as follows:
the instrument comprises the following steps: shimadzu LC-2030C-PLUS; a detector: UV (ultraviolet) light
A chromatographic column: welch Utimate AQ-C18(5μm,4.6*250mm)；
Mobile phase A: phosphoric acid-water (1: 300);
mobile phase B: acetonitrile;
flow rate: 1.0 ml/min;
column temperature: 25 ℃;
detection wavelength: 237 nm;
temperature of a sample injection tray: 4 ℃;
the elution procedure is as follows:
(3) and (3) determination: and respectively taking 10 mu l of each of the test solution and the system applicability solution, injecting into a liquid chromatograph, and recording the chromatogram.
Advantageous effects
Compared with the prior art, the chromatographic conditions provided by the invention have the advantages that the separation degree between each impurity and the main peak is good, the peak shape is good, the base line fluctuation is smooth, and the chromatographic conditions are improved compared with the primary color spectrum conditions in terms of the peak shape or the separation degree of the impurities.
Drawings
FIG. 1 is a chromatogram of a system suitability solution under European pharmacopoeia chromatographic conditions;
FIG. 2 is a chromatogram of a system applicability solution and a sample solution under chromatographic conditions of a detection method I;
FIG. 3 is a chromatogram of a system suitability solution and a test sample solution under a condition of a chromatogram in a detection method;
FIG. 4 is a blank solution chromatogram under the second and third chromatographic conditions of the detection method;
FIG. 5 is a chromatogram of a system applicability solution and a sample solution under a four-chromatogram condition of a detection method;
FIG. 6 is a chromatogram of the system suitability solution and the test sample solution under the condition of the five chromatograms in the detection method;
FIG. 7 is a chromatogram of a system suitability solution and a test sample solution under six chromatographic conditions of a detection method;
FIG. 8 is a blank solution chromatogram under chromatographic conditions for detection method two and six;
FIG. 9 is a chromatogram of an aspirin drug substance under acid-destroying conditions;
FIG. 10 is a chromatogram of an aspirin drug substance under base-damaging conditions;
FIG. 11 is a chromatogram of an aspirin drug substance under oxygen destruction conditions;
FIG. 12 is a detection spectrum of related substances of an aspirin enteric-coated tablet sample.
Detailed Description
Example 1
The detection method of aspirin-related substances comprises the following steps:
(1) solution preparation:
weighing 750ml of mobile phase A, adding 250ml of acetonitrile, and shaking up to obtain a blank solution, namely a diluent solution;
taking a proper amount of an impurity A, B, C, D, E, F reference substance respectively, and adding acetonitrile to dilute into solutions containing 0.1mg of the impurity A, B, C, D, E, F in each 1ml respectively to serve as stock solutions of the impurities A, B, C, D, E, F;
an appropriate amount of aspirin raw material medicine is taken, an appropriate amount of stock solution of the impurity A, B, C, D, E, F is added, and a diluent is used for preparing a mixed solution containing 1.0mg of aspirin and 1 microgram of each of the impurity A, B, C, D, E, F in each 1ml of the mixed solution to serve as a system applicability solution.
Adding a diluent into aspirin, and ultrasonically dissolving to prepare a solution of 1mg/ml as a test solution;
(2) chromatographic conditions are as follows:
the instrument comprises the following steps: shimadzu LC-2030C-PLUS; a detector: UV (ultraviolet) light
A chromatographic column: welch Utimate AQ-C18(5μm,4.6*250mm)；
Mobile phase A: phosphoric acid-water (1: 300);
mobile phase B: acetonitrile;
flow rate: 1.0 ml/min;
column temperature: 25 ℃;
detection wavelength: 237 nm;
temperature of a sample injection tray: 4 ℃;
the elution procedure is as follows:
(3) and (3) determination: respectively taking 10 μ l of each of the test solution and the system applicability solution, injecting into a liquid chromatograph, and recording chromatogram, wherein a typical chromatogram is shown in figure 2.
(4) As a result: as can be seen from fig. 2, compared with the system applicability solution chromatogram under the chromatography condition of the european pharmacopoeia, the method obviously improves the separation degree between the impurity A, B and the unknown impurity peak included therein, but the impurity a is wrapped with other impurities, and the method needs to be continuously optimized.
Example 2
And a second detection method of aspirin-related substances:
(1) solution preparation:
weighing 800ml of mobile phase A, adding 200ml of acetonitrile, and shaking up to obtain a blank solution, namely a diluent solution;
an appropriate amount of aspirin raw material medicine is taken, an appropriate amount of stock solution of the impurity A, B, C, D, E, F is added, and a diluent is used for preparing a mixed solution containing 1.0mg of aspirin and 1 microgram of each of the impurity A, B, C, D, E, F in each 1ml of the mixed solution to serve as a system applicability solution.
Adding a diluent into aspirin, and ultrasonically dissolving to prepare a solution of 1mg/ml as a test solution;
(2) chromatographic conditions are as follows:
the chromatographic conditions of example 1 were followed, except that the elution procedure was adjusted accordingly (see table below).
(3) And (3) determination: respectively taking 10 μ l of each of the test solution and the system applicability solution, injecting into a liquid chromatograph, and recording chromatogram, wherein a typical chromatogram is shown in figure 3.
4) As a result: as can be seen from fig. 3, in the second method, compared with the system-applicable solution chromatogram under the condition of the method chromatogram, the impurity a and the unknown impurity wrapped by the impurity a reach baseline separation, but the baseline fluctuation at the main peak position is large, and the method needs to be continuously optimized.
Example 3
And a third detection method of aspirin-related substances:
(1) solution preparation: see example 2.
(2) Chromatographic conditions are as follows:
the chromatographic conditions of example 1 were followed, except that the elution procedure was adjusted accordingly (see table below).
(3) And (3) determination: injecting 10 μ l blank solution into liquid chromatograph, and recording chromatogram, wherein typical chromatogram is shown in figure 4.
4) As a result: as can be seen from fig. 4, in the third method, the baseline fluctuation problem is not improved compared with the blank solution chromatogram under the second method, and the method needs to be further optimized.
Example 4
The detection method of aspirin-related substances is as follows:
(1) solution preparation:
weighing 700ml of mobile phase A, adding 300ml of acetonitrile, and shaking up to obtain a blank solution, namely a diluent solution;
an appropriate amount of aspirin raw material medicine is taken, an appropriate amount of stock solution of the impurity A, B, C, D, E, F is added, and a diluent is used for preparing a mixed solution containing 1.0mg of aspirin and 1 microgram of each of the impurity A, B, C, D, E, F in each 1ml of the mixed solution to serve as a system applicability solution.
Adding a diluent into aspirin, and ultrasonically dissolving to prepare a solution of 1mg/ml as a test solution;
(2) chromatographic conditions are as follows:
the chromatographic conditions of example 1 were followed, except that the elution procedure was adjusted accordingly (see table below).
(3) And (3) determination: respectively taking 10 μ l of each of the test solution and the system applicability solution, injecting into a liquid chromatograph, and recording chromatogram, wherein a typical chromatogram is shown in figure 5.
(4) As a result: compared with the system applicability solution chromatogram under the condition of the method dichroism, the method has stable base line, and as can be seen from fig. 5, the impurity C is wrapped by the impurity peak, and the method needs to be continuously optimized.
Example 5
And a fifth detection method of aspirin-related substances:
(1) solution preparation: see example 1.
(2) Chromatographic conditions are as follows:
a chromatographic column: SHIMADZU shim-pack GIST C18(5μm,4.6*150mm)；
The chromatographic conditions of example 2 were followed, except that the column was adjusted accordingly.
(3) And (3) determination: respectively taking 10 μ l of each of the test solution and the system suitability solution, injecting into a liquid chromatograph, and recording a chromatogram, wherein a typical chromatogram is shown in FIG. 6.
(4) As a result: compared with the system applicability solution chromatogram under the condition of the method dichroism, the method has the advantages that the problems of large baseline fluctuation and impurity A wrapping impurity peaks cannot be solved by replacing the chromatographic column, and the method needs to be continuously optimized.
Example 6
And a sixth detection method of aspirin-related substances:
(1) solution preparation: the grade and manufacturer of phosphoric acid in example 2 were changed.
(2) Chromatographic conditions are as follows: the same chromatographic conditions as in example 2.
(3) And (3) determination: respectively taking 10 μ l of each of the test solution and the system suitability solution, injecting into a liquid chromatograph, and recording chromatogram, wherein a typical chromatogram is shown in figure 7.
(4) As a result: method six chromatographic conditions of the system applicability solution chromatogram, the separation degree between each impurity and the main peak is good and the peak pattern is good, as shown in fig. 8, compared with the chromatographic conditions of example 2, the base line fluctuation is gentle.
Example 7
Aspirin solution stability experiments:
(1) solution preparation:
weighing 800ml of mobile phase A, adding 200ml of acetonitrile, and shaking up to obtain a blank solution, namely a diluent solution;
taking a proper amount of an impurity A, B, C, D, E, F reference substance respectively, and adding acetonitrile to dilute into solutions containing 0.1mg of the impurity A, B, C, D, E, F in each 1ml respectively to serve as stock solutions of the impurities A, B, C, D, E, F;
an appropriate amount of aspirin raw material medicine is taken, an appropriate amount of stock solution of the impurity A, B, C, D, E, F is added, and a diluent is used for preparing a mixed solution containing 1.0mg of aspirin and 1 microgram of each of the impurity A, B, C, D, E, F in each 1ml of the mixed solution to serve as a system applicability solution.
Grinding 10 aspirin enteric-coated tablets into fine powder, adding a proper amount of diluent, performing ultrasonic treatment in an ice-water bath, then fixing the volume to 1mg/ml of solution, and centrifuging to obtain supernatant serving as a test solution;
0.1ml of the test solution was taken in a 100ml volumetric flask and the volume was fixed with a diluent to obtain a 0.1% self-control solution.
(2) Chromatographic conditions are as follows:
same as example 2
(3) And (3) determination: respectively measuring 10 mul of system applicability solution, test solution and 0.1% self-contrast solution, injecting into a liquid chromatograph, and recording chromatogram, wherein the data are shown in tables 1-3.
TABLE 1 System suitability solution stability
TABLE 2 stability of test solutions
Impurities
0h peak area
Peak area of 1.5h
RD/％
RT＝11.6min
3424
3552
1.83
ASPI
16702193
16689684
0.04
IMPC
22712
29783
13.47
IMPD
7893
7906
0.08
TABLE 30.1% stability of self-control solution
Time
0h
3.0h
4.0h
5.0h
6.5h
7.5h
9.0h
11.5h
Peak area
20698
20905
20720
20768
20700
20804
20645
20499
RD/％
/
0.50
0.05
0.17
0.00
0.26
0.13
0.48
(4) As a result: as can be seen from the above table, the 0.1% self-control solution has good stability and is stable within 11.5h, and the system applicability solution and the test solution are not stable within 1.6h, and the method needs to be further optimized.
Example 8
Aspirin solution stability experiments:
(1) solution preparation:
acetonitrile is used as a blank solution, namely a diluent solution;
taking a proper amount of an impurity A, B, C, D, E, F reference substance respectively, and adding acetonitrile to dilute into solutions containing 0.1mg of the impurity A, B, C, D, E, F in each 1ml respectively to serve as stock solutions of the impurities A, B, C, D, E, F;
10 aspirin enteric-coated tablets are taken and ground into fine powder, a proper amount of diluent is taken and added in an ice-water bath for ultrasonic treatment, a proper amount of stock solution of the impurity A, B, C, D, E, F is added, and the diluent is used for preparing mixed solution containing 1.0mg of aspirin and 1 microgram of the impurity A, B, C, D, E, F in each 1ml as system applicability solution.
Taking a proper amount of aspirin enteric-coated tablet powder, adding a diluent, carrying out ultrasonic treatment in an ice-water bath, fixing the volume to a solution of 1mg/ml, centrifuging, and taking supernatant as a test solution;
0.1ml of the test solution was taken in a 100ml volumetric flask and the volume was fixed with a diluent to obtain a 0.1% self-control solution.
(2) Chromatographic conditions are as follows:
same as example 2
(3) And (3) determination: respectively measuring 10 mul of system applicability solution and 0.1% of self-contrast solution, injecting the solutions into a liquid chromatograph, and recording a chromatogram, wherein data are shown in tables 4-5 below.
Table 4 system applicability solution stability data
TABLE 50.1% stability data for self-control solutions
Time
0h
7.0h
12.0h
15.5h
18.5h
20.5h
23.5h
Peak area
18647
18339
18182
18063
18002
18026
18069
RD/％
/
0.83
1.26
1.59
1.76
1.69
1.57
(4) As a result: as can be seen from the above table, the system suitability solution stability is good, stable within 16h, the 0.1% self control solution stability is good, stable within 23.5h, so the diluent is changed to acetonitrile.
Example 8
Forced degradation experiment of aspirin bulk drug solution:
(1) solution preparation:
acetonitrile is used as a blank solution, namely a diluent solution;
taking a proper amount of an impurity A, B, C, D, E, F reference substance respectively, and adding acetonitrile to dilute into solutions containing 0.1mg of the impurity A, B, C, D, E, F in each 1ml respectively to serve as stock solutions of the impurities A, B, C, D, E, F;
taking a proper amount of aspirin raw material medicine, adding a proper amount of stock solution of the impurity A, B, C, D, E, F, and preparing mixed solution containing 1.0mg of aspirin and 1 microgram of the impurity A, B, C, D, E, F in each 1ml of the mixed solution by using a diluent to serve as system applicability solution;
the samples were not destroyed: taking a proper amount of aspirin raw material medicine, adding a diluent, and fixing the volume to a solution of 1mg/ml to obtain the aspirin composition;
acid-base blank: taking 1.0ml of 0.1M sodium hydroxide solution into a 10ml volumetric flask, adding 1.0ml of 0.1M hydrochloric acid solution for neutralization, and fixing the volume to obtain the sodium chloride solution;
10% oxidation blank: 1.0ml of 10% H was taken2O2Putting the mixture into a 10ml volumetric flask, and fixing the volume to obtain the product;
acid destruction of the sample: weighing about 10mg of aspirin in a 10ml volumetric flask, adding 1.0ml of 0.1M hydrochloric acid solution, standing at room temperature for 18h, adding 1.0ml of 0.1M sodium hydroxide solution for neutralization, and fixing the volume to obtain the aspirin-aspirin composite solid preparation;
and (3) sample alkali destruction: weighing about 10mg aspirin in a 10ml volumetric flask, adding 0.5ml of 0.1M sodium hydroxide solution, standing at room temperature for 10min, adding 0.5ml of 0.1M hydrochloric acid solution for neutralization, and fixing the volume to obtain the aspirin-aspirin composite solid preparation;
sample oxidative destruction: approximately 10mg of aspirin was weighed into a 10ml volumetric flask, and 1.0ml of 10% H was added2O2Standing for 4h, and fixing the volume to obtain the product.
(2) Chromatographic conditions are as follows:
same as example 2
(3) And (3) determination: respectively measuring 10 μ l of the above solutions, injecting into a liquid chromatograph, recording chromatogram, and showing typical chromatogram in fig. 9-11, and showing data in table 6 below.
TABLE 6 forced degradation experimental data of crude drugs
Name (R)
Is not destroyed
0.1M HCL
0.1M NaOH
10％H2O2
Weighing (mg)
20.46
10.19
10.08
9.85
Concentration (mg/ml)
1.023
1.019
1.008
0.985
Impurity A (%)
/
/
/
/
Impurity B (%)
/
/
/
/
Impurity C (%)
0.023
7.526
11.715
5.492
Impurity D (%)
0.044
0.038
0.016
0.041
Impurity E (%)
/
/
0.028
/
Impurity F (%)
/
/
/
/
ASPI(％)
99.934
92.437
88.241
94.394
Purity of main peak
0.999999
0.999999
0.999999
0.999999
Number of unknown impurities
0
0
0
3
Maximum single impurity (%)
0.044
7.526
11.715
5.492
Maximum unknown simple impurity (%)
/
/
/
0.044
Total peak area
16324763
16652916
16886379
15675060
Conservation of Material (%)
/
102.41
104.98
99.72
(4) As a result: from the above table, after the aspirin is destroyed by acid, alkali and oxygen, the main peak purity is 0.999999, and the main degradation impurity is impurity C. Under the acid and oxidation conditions, the impurity C is obviously increased, no other known impurities are increased, but under the oxidation conditions, 3 new unknown impurities are added; under alkaline conditions, impurity C increased from 0.023% to 11.715%, impurity E increased from undetectable to 0.028%, and no unknown impurity was produced.
Under the condition of acid, alkali and oxygen destruction, the separation degree between each impurity and the main peak meets the requirement, and the materials are basically conserved, which shows that the method can effectively detect each degradation impurity.
Example 9
And (3) detecting related substances of the aspirin enteric-coated tablet:
(1) solution preparation:
acetonitrile is used as a blank solution, namely a diluent solution;
taking a proper amount of an impurity A, B, C, D, E, F reference substance respectively, and adding acetonitrile to dilute into solutions containing 0.1mg of the impurity A, B, C, D, E, F in each 1ml respectively to serve as stock solutions of the impurities A, B, C, D, E, F;
taking a proper amount of aspirin raw material medicine, adding a proper amount of stock solution of the impurity A, B, C, D, E, F, and preparing mixed solution containing 1.0mg of aspirin and 1 microgram of the impurity A, B, C, D, E, F in each 1ml of the mixed solution by using a diluent to serve as system applicability solution;
taking a proper amount of aspirin enteric-coated tablet powder, adding a diluent, carrying out ultrasonic treatment in an ice-water bath, fixing the volume to a solution of 1mg/ml, centrifuging, and taking supernatant as a test solution;
0.1ml of the test solution was taken in a 100ml volumetric flask and the volume was fixed with a diluent to obtain a 0.1% self-control solution.
(2) Chromatographic conditions are as follows:
same as example 2
(3) And (3) determination: injecting 10 μ l of each solution into a liquid chromatograph, recording chromatogram, and obtaining a typical map as shown in figure 12, wherein the calculation formula is as follows:
single impurity% (% single impurity peak area/0.1% control peak area 0.1 × 100%);
total impurities% (% impurity peak area/0.1% control peak area) (% 0.1) (% 100).
The results are shown in Table 7 below.
TABLE 7 results of sample examination
Tablet formulation
Peak area
Test results (%)
0.1% self control
18902
/
RT＝11.959min
3214
0.017
RT as 21.917min (impurity C)
11302
0.060
RT＝22.517min
1146
0.006
RT＝25.235min
1002
0.005
RT＝26.079min
938
0.005
RT as 28.845min (impurity D)
7587
0.040
Maximum unknown single impurity (less than or equal to 0.1%)
/
0.017
Total peak area
15874245
/
Total impurities (impurity removal C)
25189
0.073
ASPI
15849056
99.867
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