Method for detecting related substances in naproxen sodium soft capsule by high performance liquid chromatography
阅读说明：本技术 一种高效液相色谱法检测萘普生钠软胶囊中有关物质的方法 (Method for detecting related substances in naproxen sodium soft capsule by high performance liquid chromatography ) 是由 林娟 张宇 李柏园 李琳 沈福星 于 2020-11-26 设计创作，主要内容包括：本发明公开了一种高效液相色谱法检测萘普生钠软胶囊中有关物质的方法,所述方法利用高效液相色谱法对萘普生钠软胶囊样品溶液进行检测；所述流动相为：流动相A：pH为5.0的醋酸铵溶液；流动相B：乙腈；所述稀释剂为：水：乙腈,水与乙腈的比例为60:40；所述空白溶液为稀释剂；所述高效液相色谱法检测中采用梯度法。(The invention discloses a method for detecting related substances in a naproxen sodium soft capsule by using a high performance liquid chromatography, wherein the method is used for detecting a naproxen sodium soft capsule sample solution by using the high performance liquid chromatography; the mobile phase is as follows: mobile phase A: ammonium acetate solution at pH 5.0; mobile phase B: acetonitrile; the diluent is as follows: water: acetonitrile, wherein the ratio of water to acetonitrile is 60: 40; the blank solution is a diluent; the high performance liquid chromatography detection adopts a gradient method.)
1. A method for detecting related substances in a naproxen sodium soft capsule by high performance liquid chromatography is characterized in that: the method utilizes high performance liquid chromatography to detect naproxen sodium soft capsule sample solution; the mobile phase is as follows: the mobile phase A is an ammonium acetate solution with pH of 5.0; the mobile phase B is acetonitrile; the diluent is as follows: water and acetonitrile, wherein the ratio of the water to the acetonitrile is 60: 40; the blank solution is a diluent; the high performance liquid chromatography detection adopts a gradient method, and the gradient table is as follows:
2. The method for detecting related substances in the naproxen sodium soft capsule by the high performance liquid chromatography as claimed in claim 1, which is characterized in that: the preparation method of the mobile phase A comprises the steps of weighing 0.77g of ammonium acetate, dissolving the ammonium acetate in 1000ml of water, filtering the solution by using a 0.2 mu m filter membrane, adjusting the pH value to 5.0 by using glacial acetic acid, and performing ultrasonic degassing to obtain the mobile phase A.
3. The method for detecting related substances in the naproxen sodium soft capsule by the high performance liquid chromatography as claimed in claim 1, which is characterized in that: the concentration of the impurity control solution in the high performance liquid chromatography is 0.88 mu g/mL, and the preparation method comprises the following steps:
(1) precisely weighing 44mg naproxen sodium reference substance in a volumetric flask with 100mL, adding 60mL diluent, carrying out ultrasonic treatment until the reference substance is dissolved, diluting the reference substance to a scale with the diluent, and shaking up to obtain an impurity reference stock solution with the concentration of 440 mug/mL;
(2) precisely transferring 10.0mL (1) of the reference stock solution into a 100mL volumetric flask, diluting the reference stock solution to a scale by using the diluent and shaking up, precisely transferring 2.0mL of the reference stock solution from the shaken solution into the 100mL volumetric flask, diluting the reference stock solution to the scale by using the diluent and shaking up to obtain an impurity reference solution with the concentration of 0.88 mu g/mL.
4. The method for detecting related substances in the naproxen sodium soft capsule by the high performance liquid chromatography as claimed in claim 1, which is characterized in that: the concentration of the sample solution in the high performance liquid chromatography is 440 mug/mL, and the preparation method comprises the following steps:
(1) accurately weighing the total weight of 20 naproxen sodium soft capsules, calculating the average weight, weighing 5 naproxen sodium soft capsules, putting into a 250mL volumetric flask, adding 150mL of the diluent, placing in a 65-DEG water bath and shaking for 20 minutes, shaking for 15 minutes in a mechanical oscillator, taking out and cooling to room temperature, diluting to a scale with the diluent, and shaking up to obtain a sample stock solution with the concentration of 4400 mu g/mL;
(2) precisely transferring 5.0mL of sample stock solution into a 50mL volumetric flask, diluting to a scale with a diluent, shaking up, filtering with a 0.22 μm filter membrane, discarding 4mL of primary filtrate, and taking the clear continuous filtrate as a sample solution to obtain a sample solution with the concentration of 440 μ g/mL.
5. The method for detecting related substances in the naproxen sodium soft capsule by the high performance liquid chromatography as claimed in claim 1, which is characterized in that: the concentration of the impurity identification solution in the high performance liquid chromatography is 0.88 mu g/mL, and the preparation method comprises the following steps: precisely weighing 4.4mg of impurity A, impurity K and impurity L in a 50mL volumetric flask, adding 30mL of diluent into the volumetric flask for ultrasonic dissolution, diluting the solution to a scale with the diluent, shaking up, precisely transferring 1.0mL of the solution into a 100mL volumetric flask, diluting the solution to the scale with the diluent, and shaking up to obtain the impurity identification solution with the concentration of 0.88 mu g/mL.
6. The method for detecting related substances in the naproxen sodium soft capsule by the high performance liquid chromatography as claimed in claim 1, which is characterized in that: the chromatographic conditions of the high performance liquid chromatograph are as follows: a chromatographic column: X-Terra RP 18 with the particle size of 3.5 μm, the column temperature of 37-43 ℃, an ultraviolet absorption detector with the flow rate of 0.8-1.2mL/min, the detection wavelength of 236nm and the sample injection volume of 10 μ L.
7. The method for detecting related substances in the naproxen sodium soft capsule by the high performance liquid chromatography as claimed in claim 1, which is characterized in that: the chromatographic conditions of the high performance liquid chromatograph are as follows: a chromatographic column: X-Terra RP 18 with the particle size of 3.5 μm, the column temperature of 38-42 ℃, the detector which is an ultraviolet absorption detector, the flow rate of 0.8-1.2mL/min, the detection wavelength of 236nm and the sample injection volume of 10 μ L.
8. The method for detecting related substances in the naproxen sodium soft capsule by the high performance liquid chromatography as claimed in claim 6, which is characterized in that: the chromatographic conditions of the high performance liquid chromatograph are as follows: a chromatographic column: X-Terra
Technical Field
The invention belongs to the technical field of medicine quality detection, and particularly relates to a method for detecting related substances in a naproxen sodium soft capsule by using a high performance liquid chromatography.
Background
Naproxen Sodium, English name: Naprofen Sodium, chemical formula: C14H13NaO3Molecular weight: 252.24, structural formula:naproxen sodium is a non-steroidal anti-inflammatory drug, is suitable for relieving various mild to moderate pains such as pains after tooth extraction and other operations, primary dysmenorrheal, headache and the like, is also suitable for rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, juvenile articular bursitis and acute gouty arthritis, and has the effect of relieving symptoms of pain, swelling and activity limitation of arthritis.
The naproxen sodium raw material can produce process impurity A, impurity K, impurity L, impurity E, impurity F, impurity O, impurity L, impurity N, impurity M and impurity I in the production process, and simultaneously, impurities A, K and L are also degradation impurities. The naproxen sodium soft capsule finished product can generate naproxen glyceride, naproxen PEG600 ester, naproxen sorbitol and sorbitol ester, and naproxen propylene ester which are degraded impurities. Through retrieval, no literature report that the 7 impurities in the naproxen sodium soft capsule can be simultaneously detected and 14 impurities can be separated is found. A naproxen sodium soft capsule belongs to antipyretic, anti-inflammatory and analgesic medicine. Therefore, a set of scientific and effective analysis method capable of simultaneously measuring 14 impurities is established, and the method has important application value for more widely and effectively controlling the product quality.
It is needed to develop a method for simply, rapidly and accurately detecting related substances in the naproxen sodium soft capsule, and the peaks can be effectively separated.
Disclosure of Invention
The invention aims to detect a naproxen sodium soft capsule sample by using a high performance liquid chromatography, and provides a method for detecting impurities in a naproxen sodium soft capsule, the method for detecting related substances in the naproxen sodium soft capsule sample provided by the invention is simple to operate and low in cost, 14 related substances in the naproxen sodium soft capsule sample can be quickly and accurately detected, and the impurities can be effectively separated; the impurities detected by the method comprise naproxen sodium raw material medicines and impurities in naproxen sodium tablets, and the method can be simultaneously suitable for detecting naproxen sodium and related substances of preparations of the naproxen sodium, and can control the product quality more widely and effectively.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for detecting related substances in a naproxen sodium soft capsule by using a high performance liquid chromatography method is disclosed, wherein the method uses the high performance liquid chromatography method to detect a naproxen sodium soft capsule sample solution; the mobile phase is as follows: mobile phase A: ammonium acetate solution at pH 5.0; mobile phase B: acetonitrile; the diluent is as follows: water and acetonitrile, wherein the ratio of the water to the acetonitrile is 60: 40; the blank solution is a diluent; the high performance liquid chromatography detection adopts a gradient method, and the gradient table is as follows:
time (min)
Mobile phase A (V%)
Mobile phase B (V%)
0
75
25
5
75
25
13
70
30
24
65
35
50
46
54
51
75
25
56
75
25
Further, the preparation method of the mobile phase A comprises the steps of weighing 0.77g of ammonium acetate, dissolving the ammonium acetate in 1000ml of water, filtering the solution by using a 0.2-micron filter membrane, adjusting the pH value to 5.0 by using glacial acetic acid, and performing ultrasonic degassing to obtain the mobile phase A.
Further, the concentration of the impurity control solution in the high performance liquid chromatography is 0.88 μ g/mL, and the preparation method comprises the following steps: (1) precisely weighing 44mg naproxen sodium reference substance in a volumetric flask with 100mL, adding 60mL diluent, carrying out ultrasonic treatment until the reference substance is dissolved, diluting the reference substance to a scale with the diluent, and shaking up to obtain an impurity reference stock solution with the concentration of 440 mug/mL; (2) precisely transferring 10.0mL (1) of the reference stock solution into a 100mL volumetric flask, diluting the reference stock solution to a scale by using the diluent and shaking up, precisely transferring 2.0mL of the reference stock solution from the shaken solution into the 100mL volumetric flask, diluting the reference stock solution to the scale by using the diluent and shaking up to obtain an impurity reference solution with the concentration of 0.88 mu g/mL.
Further, the concentration of the sample solution in the high performance liquid chromatography is 440 mug/mL, and the preparation method comprises the following steps: (1) accurately weighing the total weight of 20 naproxen sodium soft capsules, calculating the average weight, weighing 5 naproxen sodium soft capsules, putting into a 250mL volumetric flask, adding 150mL of the diluent, placing in a 65-DEG water bath and shaking for 20 minutes, shaking for 15 minutes in a mechanical oscillator, taking out and cooling to room temperature, diluting to a scale with the diluent, and shaking up to obtain a sample stock solution with the concentration of 4400 mu g/mL; (2) precisely transferring 5.0mL of sample stock solution into a 50mL volumetric flask, diluting to a scale with a diluent, shaking up, filtering with a 0.22 μm filter membrane, discarding 4mL of primary filtrate, and taking the clear continuous filtrate as a sample solution to obtain a sample solution with the concentration of 440 μ g/mL.
Further, the concentration of the impurity identifying solution in the high performance liquid chromatography is 0.88 μ g/mL, and the preparation method thereof comprises: precisely weighing 4.4mg of impurity A, impurity K and impurity L in a 50mL volumetric flask, adding 30mL of diluent into the volumetric flask for ultrasonic dissolution, diluting the solution to a scale with the diluent, shaking up, precisely transferring 1.0mL of the solution into a 100mL volumetric flask, diluting the solution to the scale with the diluent, and shaking up to obtain the impurity identification solution with the concentration of 0.88 mu g/mL.
Further, the chromatographic conditions of the high performance liquid chromatograph are as follows: a chromatographic column: X-Terra RP 18 with the particle size of 3.5 μm, the column temperature of 37-43 ℃, an ultraviolet absorption detector with the flow rate of 0.8-1.2mL/min, the detection wavelength of 236nm and the sample injection volume of 10 μ L.
Further, the chromatographic conditions of the high performance liquid chromatograph are as follows: a chromatographic column: X-Terra RP 18 with the particle size of 3.5 μm, the column temperature of 38-42 ℃, the detector which is an ultraviolet absorption detector, the flow rate of 0.8-1.2mL/min, the detection wavelength of 236nm and the sample injection volume of 10 μ L.
Further, the chromatographic conditions of the high performance liquid chromatograph are as follows: a chromatographic column: X-Terra RP 18 with the particle size of 3.5 μm, the column temperature of 40 ℃, an ultraviolet absorption detector with the flow rate of 1.0mL/min, the detection wavelength of 236nm and the sample injection volume of 10 μ L.
Due to the adoption of the technical scheme, the invention has the beneficial effects that:
(1) the method for detecting the related substances in the naproxen sodium soft capsule sample, which is provided by the invention, is simple to operate and low in cost, can quickly and accurately detect 14 related substances in the naproxen sodium soft capsule sample, and can effectively separate impurities;
(2) the impurities detected by the method comprise the naproxen sodium raw material medicine and the impurities in the naproxen sodium tablet, and the method can be suitable for detecting the naproxen sodium and the related substances of the naproxen sodium tablet, and can control the product quality more widely and effectively;
(3) according to the method, the RSD of each impurity is less than 5.0%, and the precision is good;
(4) the method of the invention has good reproducibility;
(5) the invention has strong specificity, high accuracy, good tolerance and high instrument sensitivity.
Drawings
FIG. 1 is a blank solution chromatogram of the present invention;
FIG. 2 is a chromatogram of an impurity control solution (standard solution) according to the present invention;
FIG. 3 is a chromatogram of a sample solution (test solution) according to the present invention;
FIG. 4 is an impurity chromatogram of the present invention;
FIG. 5 is a linear plot of prairie sodium according to the present invention;
FIG. 6 is a linear plot of impurity A of the present invention;
FIG. 7 is a linear plot of impurity L of the present invention;
FIG. 8 is a linear plot of impurity K of the present invention;
FIG. 9 is a linear plot of naproxen glycerides of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The chemical structural formula of the impurities detected by the invention is shown in table 1.
TABLE 1
In the specific embodiment, the following chromatographic conditions are adopted in the high performance liquid chromatography:
a chromatographic column: X-Terra RP 18,3.5 μm, 4.6X 150mm or equivalent, column temperature: 40 ± 2 ℃, detector: UV (ultraviolet absorption detector), flow rate: 1.0mL/min, wavelength: 236nm, sample injection volume: 10 μ L, run time: 56min, needle washing: extension, needle washing liquid: 30% methanol.
The mobile phase comprises a mobile phase A and a mobile phase B, and is prepared by the following steps: mobile phase A: weighing 0.77g of ammonium acetate into 1000ml of water for dissolving, filtering by using a 0.2 mu m filter membrane, adjusting the pH value to 5.0 by using glacial acetic acid, and performing ultrasonic degassing to obtain a mobile phase A; mobile phase B: and (3) acetonitrile.
The high performance liquid chromatography detection adopts a gradient method, and the gradient table is as follows:
time (min)
Mobile phase A (V%)
Mobile phase B (V%)
0
75
25
5
75
25
13
70
30
24
65
35
50
46
54
51
75
25
56
75
25
The solution was prepared as follows:
diluent agent: water: acetonitrile (600: 400).
Blank solution: a diluent.
Impurity control stock solution (440. mu.g/mL): precisely weighing 44mg naproxen sodium reference substance in a 100mL volumetric flask, adding 60mL diluent, performing ultrasonic treatment until the reference substance is dissolved, diluting the reference substance with the diluent until the reference substance is scaled, and shaking up the reference substance to obtain the naproxen sodium reference substance.
Impurity control solution (0.88. mu.g/mL): precisely transferring 10.0mL of the reference stock solution into a 100mL volumetric flask, diluting the reference stock solution to a scale with a diluent, shaking up, precisely transferring 2.0mL of the reference stock solution into a 100mL volumetric flask, diluting the reference stock solution to a scale with the diluent, and shaking up to obtain the product.
Impurity identifying solution (0.88. mu.g/mL): precisely weighing 4.4mg of impurity A, impurity K and impurity L in a 50mL volumetric flask, adding 30mL of diluent, carrying out ultrasonic treatment until the mixture is dissolved, diluting the mixture to a scale by using the diluent, shaking up the mixture, precisely transferring 1.0mL of the solution in a 100mL volumetric flask, diluting the solution to the scale by using the diluent, and shaking up the solution to obtain the compound.
Stock sample solution (4400. mu.g/mL): the total weight of 20 capsules was accurately weighed and the average weight was calculated. Weighing 5 soft capsules, putting into a 250mL volumetric flask, adding 150mL diluent, placing in a 65-degree water bath and shaking for 20 minutes, and shaking for 15 minutes in a mechanical shaker. Taking out, cooling to room temperature, diluting with diluent to scale, and shaking.
Sample solution (440. mu.g/mL): precisely transferring 5.0mL of the sample stock solution into a 50mL volumetric flask, diluting to the mark with a diluent, and shaking up. The solution was filtered through a 0.22 μm filter, 4mL of the initial filtrate was discarded, and the clear subsequent filtrate was used as the sample solution.
The sample introduction sequence of the high performance liquid chromatography adopts the following sequence:
(1) the mobile phase is fed, and the liquid phase system is balanced until the base line is stable;
(2) adding at least one needle of blank solution and 6 continuous needles of impurity control solution;
(3) one needle for each sample solution, and one standard solution for each 12 sample solutions or samples at most, are introduced within not more than 4 hours (based on the short time).
(4) If necessary, at least 1 needle of blank solution is fed to remove residues.
(5) At the end of the whole sample introduction, at least 1 needle of blank solution is fed and one needle of impurity control solution is returned, so that the system adaptability of all the impurity control solutions is ensured to meet the requirement.
Example 1 systematic suitability test
And (3) the adaptability of the system before sample introduction, wherein the theoretical plate number of the first needle of the working control solution is calculated to be not less than 3000, the tailing factor of the first needle of the working control solution is calculated to be not more than 2.0, and the RSD of the working control solution of 6 needles before naproxen sodium is not more than 5.0%.
The blank solution did not have any interfering peaks at the naproxen retention time. The blank solution chromatogram is shown in figure 1,
good system adaptability, no main peak interference caused by diluent, theoretical plate number of main peak: 22857, tail factor of main peak is 1.0, RSD of main peak area of impurity control solution is 0.9%.
System suitability after injection RSD of all impurity control solutions was not more than 5.0%.
The chromatogram of the impurity control solution (standard solution) is shown in FIG. 2;
the chromatogram of the sample solution (test solution) is shown in FIG. 3;
the chromatogram with impurities is shown in FIG. 4.
Example 2 good specificity
The table below shows the retention time and relative retention time of the main peak and each impurity peak, and it can be seen that the main peak and each impurity peak do not interfere with each other and impurities can be effectively separated.
Name (R)
Retention time
Relative retention time
Impurity A
4.792
0.38
Impurity I
6.519
0.52
Impurity O
7.444
0.59
Naproxen sorbitol and sorbitol esters
8.801
0.70
Naproxen sodium
12.572
-
Naproxen glyceride 1
14.706
1.17
Naproxen glyceride 2
15.726
1.25
Impurity K
16.918
1.35
Impurity L
25.003
1.99
Naproxen ethyl propylene ester
27.132
2.16
PEG600 esters
28.08～31.08
2.23～2.47
Impurity M
33.710
2.68
Impurity E
37.260
2.96
Impurity F
43.260
3.44
Impurity N
46.923
3.73
TABLE 2
Example 3: test for Strong degradation
Strong degradation test: a. any impurity peaks produced in the strong degradation test do not interfere with the main peak. b. The main peak purity threshold is greater than the peak purity angles of the reference and degraded samples.
Sample (I)
The recovery rate is high
Pure angle
Threshold of purity
Control
-
-
-
Illumination of light
96.1
0.344
1.363
High temperature
100.2
0.316
1.324
Oxidation by oxygen
91.1
0.330
1.371
Alkali
104.3
0.241
1.336
Acid(s)
99.0
0.314
1.341
TABLE 3
Example 4: linearity and range
Composition (I)
Equation of standard curve
R
Naproxen sodium
Y＝116049.7679x+194.2069
R＝1.0000
Impurity A
Y＝111311.6433x-521.0616
R＝1.0000
Impurity L
Y＝74475.2585x-289.8192
R＝0.9999
Impurity K
Y＝100188.4567x+104.1250，
R＝1.0000
Naproxen glycerides
Y＝93878.3468x+626.3477
R＝1.0000
TABLE 4
And (4) conclusion: in the LOQ-200% concentration range, the peak area has a good linear relationship with the concentration.
Example 5: precision of the method
The sample solution is injected repeatedly for 6 times, the RSD of each impurity is less than 5.0 percent, and the result shows that the method has good precision.
Example 6: intermediate precision
Different people and different days adopt different chromatographic columns and reagents of different batches to prepare 6 sample solutions, sample introduction is carried out, the RSD of each impurity is less than 5.0%, the difference between the impurities does not exceed 0.1%, and the result shows that the reproducibility is good.
Example 7: stability of solution
The stability of the control and sample solutions was examined and the results showed that the control and sample solutions were stable within 96 hours.
Example 8: durability
By changing the flow rate to 1.0 plus or minus 0.2ml/min, the column temperature to 40 plus or minus 3 ℃, the pH of the mobile phase to 5.0 plus or minus 0.2 and the initial proportion of the organic phase to 25 plus or minus 2 percent, the sample solution is measured, and compared with the result of the solution measured under the normal condition, the absolute difference of the recovery rate is within 10 percent, and the result shows that the method has good tolerance.
Example 9: detection limit and quantification limit
The detection limit and the quantification limit of the main peak are inspected, and the result shows that the detection limit concentration is equivalent to the level of 0.006 percent, the signal-to-noise ratio S/N is not less than 3, the quantification limit concentration is equivalent to the level of 0.02 percent, the signal-to-noise ratio S/N is not less than 10, the% RSD is less than 5.0 percent, and the sensitivity of a result display instrument is good.
Example 10: filter membrane filtration test
The results of the investigation on the interference of the filter membrane filtration on the sample show that the recovery rates of the samples with different volumes filtered by the filter membrane and the sample solution processed by the centrifuge are similar and are all between 95.0 percent and 105.0 percent, which indicates that the filter membrane filtration does not have the interference on the sample.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.