CN111257441B - Method for detecting impurities in parecoxib sodium synthesis process - Google Patents

Method for detecting impurities in parecoxib sodium synthesis process Download PDF

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CN111257441B
CN111257441B CN201911420764.5A CN201911420764A CN111257441B CN 111257441 B CN111257441 B CN 111257441B CN 201911420764 A CN201911420764 A CN 201911420764A CN 111257441 B CN111257441 B CN 111257441B
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prx
parecoxib sodium
impurities
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CN111257441A (en
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隋立朋
王晓雪
马莉艳
石勇志
崔海龙
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Henan Runhong Pharmaceutical Co ltd
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/52—Physical parameters
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
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    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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Abstract

The invention belongs to the field of drug analysis and detection, and particularly relates to a method for detecting impurities in a parecoxib sodium synthesis process. The method for detecting impurities in the parecoxib sodium synthesis process comprises the following steps: dissolving a sample to be detected to obtain a solution to be detected; carrying out high performance liquid chromatography analysis on the solution to be detected to obtain the content of impurities in the synthesis process; the chromatographic column adopted by the high performance liquid chromatography is an octadecylsilane chemically bonded silica chromatographic column, gradient elution is adopted, the mobile phase A is a disodium hydrogen phosphate solution with the pH of 2.5-3.5 and 0.01mol/L, and the mobile phase B is a mixture of a sodium hydrogen phosphate solution and a sodium hydrogen phosphate solution, wherein the volume ratio of the mobile phase A to the mobile phase B is 75-85: 15-25 parts of acetonitrile-methanol mixed solution. The determination method can realize the accurate separation and detection of various impurities in the parecoxib sodium bulk drug or related products by applying the same analysis method, has wide coverage, and especially can realize the detection of 4 excessive sulfonylation impurities; meanwhile, the method has good separation effect and high sensitivity and accuracy, and can effectively evaluate and control the quality of parecoxib sodium.

Description

Method for detecting impurities in parecoxib sodium synthesis process
Technical Field
The invention belongs to the field of drug analysis and detection, and particularly relates to a method for detecting impurities in a parecoxib sodium synthesis process.
Background
Parecoxib sodium (chemical name is N- ((4- (5-methyl-3-phenyl-4-isoxazolyl) phenyl) sulfonyl) propionamide sodium salt), is a specific cyclooxygenase-2 (COX-2) inhibitor, is prepared into a freeze-dried powder injection, and is the first COX-2 inhibitor capable of being administered by intravenous injection and intramuscular injection. The primary medicine is approved to be marketed in China in 2008, and is mainly used for short-term treatment of postoperative pain.
Currently, in the field of drug synthesis, a common synthesis method of parecoxib sodium is as follows:
Figure BDA0002352328680000011
the synthesis method comprises the following steps: the parecoxib sodium product is prepared by taking PRX-A as a starting material through sulfonylation reaction, ammoniation reaction, amidation reaction, salification reaction and necessary refining and purifying processes. In the synthesis process of parecoxib sodium, due to the influence of factors such as process control, product characteristics and the like, synthesis process impurities including incompletely reacted starting raw materials and reaction byproducts such as intermediates, isomers, polymers, excessive sulfonylation products and the like are inevitably introduced into a product, and the existence of the impurities affects the quality of parecoxib sodium medicines, so that the safety and the effectiveness of the product are affected.
The research shows that excessive sulfonylation impurities and reaction raw materials in the parecoxib sodium product obtained by synthesis are very likely to exist, but the current detection method of parecoxib sodium related substances does not relate to the comprehensive detection of the process impurities. For example, the invention patent application with the application publication number of CN108828127A discloses a liquid chromatography method for detecting parecoxib sodium and related substances in a synthetic intermediate, which respectively uses pentafluorophenyl bonded silica gel and octadecylsilane bonded silica gel as chromatographic column packing, and changes elution gradient to determine related substances I and related substances II in parecoxib sodium intermediate valdecoxib and parecoxib twice.
In the quality control of drug development and production, efficient, accurate and sensitive process impurity detection means are used, so that detection resources are occupied as little as possible, and detection control of related substances is realized as complicated as possible.
Disclosure of Invention
The invention aims to provide a method for detecting impurities in a parecoxib sodium synthesis process, which can realize the detection of various parecoxib sodium synthesis process impurities by adopting one method.
In order to realize the purpose, the specific technical scheme of the method for detecting impurities in the parecoxib sodium synthesis process is as follows:
the method for detecting impurities in parecoxib sodium synthesis process comprises the following steps:
1) dissolving a parecoxib sodium sample to be detected to obtain a solution to be detected;
2) and (4) carrying out high performance liquid chromatography analysis on the solution to be detected to obtain the content of the impurities in the synthesis process.
The analysis conditions of the high performance liquid chromatography are as follows: a chromatographic column: octadecylsilane chemically bonded silica chromatographic column; mobile phase A: 0.01mol/L disodium hydrogen phosphate solution with pH of 2.5-3.5, and a mobile phase B: the volume ratio is 75-85: 15-25 of acetonitrile-methanol mixed solution; the elution gradient program included: 0min, 60-70 vol% of mobile phase A, and 78-30 vol% of mobile phase B40; 30min, 23-27 vol% of mobile phase A and 77-73 vol% of mobile phase B; 41min, mobile phase A60-70 vol%, and mobile phase B40-30 vol%.
The parecoxib sodium synthesis process impurities are process impurities generated in the parecoxib sodium synthesis process.
In the step 2), performing high performance liquid chromatography analysis to obtain the content of the impurities in the synthesis process comprises the following specific steps: and performing high performance liquid chromatography analysis on the solution to be detected to obtain retention time and chromatographic peak of the synthetic process impurities, comparing the retention time with the synthetic process impurity standard substance to obtain chromatographic peak corresponding to each synthetic process impurity, substituting the peak area into the standard working curve of the corresponding synthetic process impurity, and calculating to obtain the content of the synthetic process impurities.
The parecoxib sodium sample to be detected comprises parecoxib sodium raw material medicines, preparation products and synthesized crude products.
Regarding the sample pretreatment in the step 1), dissolving a parecoxib sodium sample to be detected by using a solvent to obtain a solution to be detected, wherein the used solvent can dissolve impurities of a synthesis process to be detected. Preferably, an acetonitrile-water mixed solution can be used as the solvent, and the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is preferably 40: 60. In addition, in order to improve the detection accuracy and facilitate quantitative calculation, the parecoxib sodium sample to be detected is contained in an amount of 0.5mg per 1mL of the solution to be detected.
Furthermore, the detection method aims at the impurities of the synthesis process, which are found and summarized by combining the synthesis process of the parecoxib sodium through theoretical analysis and actual detection and need to be researched in the quality research of parecoxib sodium bulk drugs or related products. The impurities of the parecoxib sodium synthesis process can be divided into synthesis reaction raw materials and synthesis reaction byproducts, wherein the synthesis reaction byproducts comprise intermediates, isomers, polymers, over-sulfonylation products and unconventional reaction products. The impurities in the parecoxib sodium synthesis process provided by the invention include, but are not limited to, the compounds shown in table 1:
Table 1 parecoxib sodium synthesis process impurity structure, impurity type and brief description
Figure BDA0002352328680000031
Figure BDA0002352328680000041
The technical scheme of the invention adopts double mobile phases, wherein the mobile phase A adopts a disodium hydrogen phosphate solution with the pH of 2.5-3.5 and 0.01mol/L, and the mobile phase B adopts a solution with the volume ratio of 75-85: 15-25 of acetonitrile-methanol mixed solution, and an octadecylsilane chemically bonded silica chromatographic column is adopted, and gradient elution under specific conditions is matched, so that simultaneous detection of 10 synthetic process impurities in a parecoxib sodium sample to be detected can be realized on the basis of parecoxib sodium detection. When the method is used specifically, the 10 synthesis process impurities can be detected simultaneously by adopting the method, one or part of the synthesis process impurities can be detected, and the method can be used according to actual detection requirements when being used specifically.
The method for detecting the impurities in the parecoxib sodium synthesis process, provided by the invention, can be used for accurately separating and detecting various impurities in the parecoxib sodium synthesis process in a to-be-detected sample by applying the same analysis method, and is wide in coverage range. Particularly, the detection method can realize effective separation of 4 excessive sulfonylation derived impurities PRX-E, PRX-K, PRX-Q, PRX-U, and can detect the raw materials of the synthesis reaction simultaneously. In the synthesis process of parecoxib sodium, a large amount of sulfonylation reagents are required to be used for reaction, so that disubstituted sulfonylation impurities are likely to be generated, and due to similar chemical structures (such as PRX-E and PRX-Q are isomers, and PRX-K and PRX-U are isomers), the simultaneous separation of multiple excessive sulfonylation impurities is necessary for accurately judging and controlling the types of the impurities. The invention further realizes the separation and detection of other process impurities such as reaction raw materials, reaction intermediates and the like and the excessive sulfonylation impurities while realizing the purpose of the separation and detection of the excessive sulfonylation impurities, and has better technical advantages.
As a further optimized scheme, the detection method provided by the invention is adopted to simultaneously detect 4 excessive sulfonylation products.
As a further optimized scheme, the detection method provided by the invention is adopted to simultaneously detect 4 excessive sulfonylation products and synthesis reaction raw materials.
As a further optimized scheme, the detection method provided by the invention is adopted to simultaneously detect the 10 synthesis process impurities.
In the detection method, a mobile phase A adopts a disodium hydrogen phosphate solution with the pH of 2.5-3.5 and 0.01mol/L, and the pH of the disodium hydrogen phosphate solution is adjusted to 2.5-3.5 by using phosphoric acid. Preferably, the pH of the disodium hydrogen phosphate solution is 3.
Preferably, the volume ratio of the acetonitrile-methanol mixed solution is acetonitrile: methanol 80: 20.
the flow rate of the mobile phase adopted during the high performance liquid chromatography analysis is 0.8-1.2 mL/min, and the preferred flow rate is 1.0 mL/min. The detector adopted by the high performance liquid chromatography is an ultraviolet detector, and the detection wavelength is 215 nm.
The specification of the octadecylsilane chemically bonded silica chromatographic column is 4.6mm x 250mm, and the particle size of the filler is 5 mu m; in particular, selected columns are commercially available, column types include YMC-Pack ODS-AQ (4.6 x 250mm, 5 μm), Welch Ultimate XB-C18(4.6 x 250mm, 5 μm) and the like, and selection of different columns does not preclude the practice of the methods provided herein. The column temperature of the chromatographic column during high performance liquid chromatography is 25-35 ℃, and more preferably 30 ℃.
The detection method has the advantages of good separation effect, high sensitivity and high accuracy. The quantitative limit of impurities in each synthesis process is 0.10-0.30 ng and is lower than 0.01% of the concentration of a test sample; the detection limit of impurities in each synthesis process is 0.05-0.15 ng and is lower than 0.005% of the concentration of a test sample; meanwhile, in the concentration range of 0.01-5.0 mug/mL, the linearity of each impurity concentration and peak area is good, and the correlation coefficient is larger than 0.999; the sample adding recovery rate of 9 samples with the concentration of 3 samples in high, medium and low is measured, the sample adding recovery rate is within the range of 90-110%, the RSD of a measurement result is less than 5%, and the precision and the accuracy are good; in addition, the method has better durability by inspecting the impurity detection capability and separation capability under the conditions of changing the flow rate, the column temperature, the mobile phase proportion, replacing chromatographic columns and chromatographs of different manufacturers and the like without obvious change under each condition.
When the method is used for detecting parecoxib sodium raw material medicine, preparation products or crude synthetic products, the impurity content of the synthetic process in parecoxib sodium samples is detected, so that the production process and the product quality of parecoxib sodium can be effectively evaluated and controlled, the accurate quality control of parecoxib sodium is realized, and the perfect safety evaluation and quality controllability evaluation of medicines are guaranteed.
Drawings
FIG. 1 is a liquid chromatogram of example 1 of the present invention;
FIG. 2 is a liquid chromatogram of example 2 of the present invention;
FIG. 3 is a liquid chromatogram of example 3 of the present invention;
FIG. 4 is a liquid chromatogram of example 4 of the present invention;
FIG. 5 is a liquid chromatogram of comparative example 1 of the present invention.
Detailed Description
The invention is further described with reference to the following specific embodiments and the accompanying drawings. In the following examples, the purity of each synthetic process impurity standard was: PRX-A (purity 99.93%), PRX-B (purity 99.07%), PRX-C (purity 92.95%), PRX-E (purity 98.10%), PRX-F (purity 99.30%), PRX-I (purity 99.50%), PRX-K (purity 99.50%), PRX-N (purity 99.70%), PRX-Q (purity 97.40%), PRX-U (purity 99.30%).
In the following examples, the content of impurities in the parecoxib sodium synthesis process in the sample is measured by an external standard method, namely a standard curve method. The preparation of the standard working solution comprises the preparation of a single standard stock solution of impurities in the synthesis process and the preparation of a series of standard working solutions, and the standard working solution is prepared into a grade 7 standard working solution as shown in Table 2.
TABLE 2 concentration of the series of standard working solutions (. mu.g/mL)
Impurities 1 2 3 4 5 6 7
PRX-A 0.030 0.254 0.508 0.762 1.016 2.539 5.079
PRX-B 0.015 0.258 0.516 0.774 1.032 2.580 5.160
PRX-C 0.019 0.234 0.467 0.701 0.935 2.337 4.674
PRX-E 0.010 0.245 0.490 0.735 0.981 2.451 4.903
PRX-F 0.014 0.234 0.468 0.702 0.936 2.340 4.679
PRX-I 0.015 0.248 0.496 0.744 0.992 2.481 4.961
PRX-K 0.016 0.262 0.524 0.786 1.048 2.621 5.242
PRX-N 0.014 0.237 0.474 0.711 0.948 2.369 4.739
PRX-Q 0.010 0.240 0.480 0.720 0.960 2.401 4.801
PRX-U 0.010 0.247 0.493 0.740 0.986 2.466 4.932
And (3) carrying out high performance liquid chromatography analysis on the series of standard working solutions, and carrying out regression analysis on the concentration of the target substance according to the chromatographic peak area of the target substance to obtain a regression equation of a standard curve. And substituting the high performance liquid chromatography analysis result of the solution to be detected into the standard curve in the subsequent determination, and calculating to obtain the content of the target object in the solution to be detected.
Example 1
The method for detecting impurities in the parecoxib sodium synthesis process adopts the following steps:
(1) taking a proper amount of parecoxib sodium sample to be detected, precisely weighing, adding acetonitrile-water (40:60, v/v) for dissolving, and quantitatively diluting to prepare a solution containing 0.5mg of parecoxib sodium sample to be detected in every 1mL, wherein the solution is used as a solution to be detected;
(2) and (3) injecting 10 mu L of solution to be detected into a liquid chromatograph, recording a chromatogram, and calculating the content of impurities in each process according to a standard curve method.
The chromatographic conditions for liquid phase detection are as follows: a chromatographic column: YMC-Pack ODS-AQ (4.6X 250mm, 5 μm); mobile phase A: 0.01mol/L disodium hydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid); mobile phase B: acetonitrile-methanol (75:25, v/v); detection wavelength: 215 nm; column temperature: 30 ℃; flow rate: 0.8 mL/min; sample introduction volume: 10 μ L. The elution procedure is shown in the following table 3, wherein 0-41 min is the elution gradient of impurities in the synthesis process, and 41-55 min is isocratic elution increased for keeping the cleanliness of the chromatographic column.
Table 3 example 1 elution procedure
Figure BDA0002352328680000071
In order to effectively illustrate the separation effect of example 1, a proper amount of parecoxib sodium (PRX), PRX-A, PRX-B, PRX-C, PRX-E, PRX-F, PRX-I, PRX-K, PRX-N, PRX-Q, PRX-U standard substance is respectively taken, acetonitrile-water (40:60, v/v) is added to dissolve and dilute the solution to prepare a solution containing 0.5mg of parecoxib sodium and 2.5 mug of impurities in each 1mL, and the solution is used as a test solution. A sample solution (10. mu.L) was taken and injected into a liquid chromatograph, and the measurement was carried out under the liquid chromatography conditions of example 1, and a chromatogram was recorded. The chromatogram of the test sample of example 1 is shown in FIG. 1, and the experimental data are shown in Table 4.
Table 4 statistical data table for example 1
Attribution Retention time Area of Degree of separation Tailing factor Number of theoretical plate
PRX-C 8.778 78080 -- 1.127 26041
PRX-E 10.060 41886 5.723 1.084 30469
PRX-Q 10.542 18645 2.099 1.058 33953
PRX-B 19.479 46084 36.217 1.045 86675
PRX-U 20.524 37074 3.944 1.007 95813
PRX-I 21.463 37354 3.559 1.035 106722
PRX-K 22.060 30115 2.260 1.031 110311
PRX-N 22.529 46095 1.757 1.026 113376
PRX 25.550 23243036 11.013 1.043 132189
PRX-F 28.573 74851 10.512 0.995 151191
PRX-A 38.693 28756 33.112 1.002 237832
Example 2
The method for detecting impurities in the parecoxib sodium synthesis process provided by the embodiment adopts the following steps:
(1) taking a proper amount of parecoxib sodium sample to be detected, precisely weighing, adding acetonitrile-water (40:60, v/v) for dissolving, and quantitatively diluting to prepare a solution containing 0.5mg of parecoxib sodium sample to be detected in every 1mL, wherein the solution is used as a solution to be detected;
(2) and (3) injecting 10 mu L of solution to be detected into a liquid chromatograph, recording a chromatogram, and calculating the content of impurities in each process according to a standard curve method.
The chromatographic conditions for liquid phase detection are as follows: a chromatographic column: welch Ultimate XB-C18 (4.6X 250mm, 5 μm); mobile phase A: 0.01mol/L disodium hydrogen phosphate solution (pH adjusted to 3.2 with phosphoric acid); mobile phase B: acetonitrile-methanol (80:20, v/v); detection wavelength: 215 nm; column temperature: 25 ℃; flow rate: 1.0 mL/min; sample introduction volume: 10 μ L. The elution procedure is shown in the following table 5, wherein 0-41 min is the elution gradient of impurities in the synthesis process, and 41-55 min is isocratic elution increased for keeping the cleanliness of the chromatographic column.
Table 5 example 2 elution procedure
Figure BDA0002352328680000081
In order to effectively illustrate the separation effect of example 2, a proper amount of parecoxib sodium (PRX), PRX-A, PRX-B, PRX-C, PRX-E, PRX-F, PRX-I, PRX-K, PRX-N, PRX-Q, PRX-U standard substance is respectively taken, acetonitrile-water (40:60, v/v) is added to dissolve and dilute the solution to prepare a solution containing 0.5mg of parecoxib sodium and 2.5 mug of impurities in each 1mL, and the solution is used as a test solution. A sample solution (10. mu.L) was taken and injected into a liquid chromatograph, and the measurement was carried out under the liquid chromatography conditions of example 2, and a chromatogram was recorded. The chromatogram of the test sample of example 2 is shown in FIG. 2, and the experimental data are shown in Table 6.
Table 6 statistical data table for example 2
Attribution Retention time Area of Degree of separation Tailing factor Number of theoretical plate
PRX-C 8.870 13871 -- -- 20770
PRX-E 10.074 32581 4.797 1.009 24833
PRX-Q 10.558 35616 1.881 1.006 26516
PRX-B 19.505 27920 31.522 0.989 64024
PRX-U 20.495 30156 3.240 0.983 73518
PRX-I 21.435 29309 3.127 0.984 82247
PRX-K 22.030 22947 1.967 0.991 82750
PRX-N 22.525 37590 1.603 0.986 83861
PRX 25.523 20410945 9.445 0.999 99501
PRX-F 28.833 22798 10.479 0.982 140196
PRX-A 38.687 27151 28.857 0.985 170319
Example 3
The method for detecting impurities in the parecoxib sodium synthesis process provided by the embodiment adopts the following steps:
(1) taking a proper amount of parecoxib sodium sample to be detected, precisely weighing, adding acetonitrile-water (40:60, v/v) for dissolving, and quantitatively diluting to prepare a solution containing 0.5mg of parecoxib sodium sample to be detected in every 1mL, wherein the solution is used as a solution to be detected;
(2) and (3) injecting 10 mu L of solution to be detected into a liquid chromatograph, recording a chromatogram, and calculating the content of impurities in each process according to a standard curve method.
The chromatographic conditions for liquid phase detection are as follows: a chromatographic column: YMC-Pack ODS-AQ (4.6X 250mm, 5 μm); mobile phase A: 0.01mol/L disodium hydrogen phosphate solution (pH adjusted to 2.8 with phosphoric acid); mobile phase B: acetonitrile-methanol (85:15, v/v); detection wavelength: 215 nm; column temperature: 35 ℃; flow rate: 1.2 mL/min; sample introduction volume: 10 μ L. The elution procedure is shown in Table 7 below, wherein 0-41 min is the elution gradient of impurities in the synthesis process, and 41-55 min is the isocratic elution increased for maintaining the cleanliness of the chromatographic column.
Table 7 example 3 elution procedure
Figure BDA0002352328680000091
In order to effectively illustrate the separation effect of example 3, a proper amount of parecoxib sodium (PRX), PRX-A, PRX-B, PRX-C, PRX-E, PRX-F, PRX-I, PRX-K, PRX-N, PRX-Q, PRX-U standard substance is respectively taken, and acetonitrile-water (40:60, v/v) is added to dissolve and dilute the solution to prepare a solution containing 0.5mg of parecoxib sodium and 2.5 mug of impurities in each 1mL, and the solution is used as a test solution. A sample solution (10. mu.L) was taken and injected into a liquid chromatograph, and the measurement was carried out under the liquid chromatography conditions of example 3, and a chromatogram was recorded. The chromatogram of the test sample of example 3 is shown in FIG. 3, and the experimental data are shown in Table 8.
Table 8 statistical table of data of example 3
Attribution Retention time Area of Degree of separation Tailing factor Number of theoretical plate
PRX-C 8.833 13543 -- -- 21798
PRX-E 10.072 32363 5.074 1.016 26193
PRX-Q 10.556 35331 1.930 1.011 27946
PRX-B 19.507 27641 32.479 0.992 68284
PRX-U 20.496 29969 3.342 0.988 78299
PRX-I 21.438 29025 3.231 0.984 87089
PRX-K 22.036 22764 2.030 0.992 87400
PRX-N 22.534 37290 1.660 0.990 89354
PRX 25.527 20224773 9.737 1.001 106249
PRX-F 28.751 22807 10.539 0.983 148350
PRX-A 38.694 26997 30.001 0.993 180508
Example 4
The method for detecting impurities in the parecoxib sodium synthesis process provided by the embodiment adopts the following steps:
(1) taking a proper amount of parecoxib sodium sample to be detected, precisely weighing, adding acetonitrile-water (40:60, v/v) for dissolving, and quantitatively diluting to prepare a solution containing 0.5mg of parecoxib sodium sample to be detected in every 1mL, wherein the solution is used as a solution to be detected;
(2) and (3) injecting 10 mu L of solution to be detected into a liquid chromatograph, recording a chromatogram, and calculating the content of impurities in each process according to a standard curve method.
The chromatographic conditions for liquid phase detection are as follows: a chromatographic column: YMC-Pack ODS-AQ (4.6X 250mm, 5 μm); mobile phase A: 0.01mol/L disodium hydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid); mobile phase B: acetonitrile-methanol (80:20, v/v); detection wavelength: 215 nm; column temperature: 30 ℃; flow rate: 1.0 mL/min; sample introduction volume: 10 μ L. The elution procedure is shown in Table 9 below, wherein 0-41 min is the elution gradient of impurities in the synthesis process, and 41-55 min is the isocratic elution increased for maintaining the cleanliness of the chromatographic column.
Table 9 example 4 elution procedure
Figure BDA0002352328680000101
To effectively illustrate the separation effect of example 4, a proper amount of parecoxib sodium (PRX), PRX-A, PRX-B, PRX-C, PRX-E, PRX-F, PRX-I, PRX-K, PRX-N, PRX-Q, PRX-U standard substance is respectively taken, and acetonitrile-water (40:60, v/v) is added to dissolve and dilute the solution to prepare a solution containing 0.5mg of parecoxib sodium and 2.5 mug of impurities in each 1mL, and the solution is used as a test solution. A sample solution (10. mu.L) was taken and injected into a liquid chromatograph, and the measurement was carried out under the liquid chromatography conditions of example 4, and a chromatogram was recorded. The chromatogram of the test sample of example 4 is shown in FIG. 4, and the experimental data are shown in Table 10.
Table 10 statistical table of data of example 4
Attribution Retention time Area of Degree of separation Tailing factor Number of theoretical plate
PRX-C 8.752 13359 -- -- 22969
PRX-E 10.060 32092 5.551 1.028 28036
PRX-Q 10.545 34922 2.010 1.025 30217
PRX-B 19.486 27391 33.984 1.003 75564
PRX-U 20.473 29350 3.511 0.991 86301
PRX-I 21.414 28918 3.389 0.993 95844
PRX-K 22.010 22694 2.127 0.998 96341
PRX-N 22.516 37065 1.771 0.997 98588
PRX 25.510 20024094 10.224 1.010 116444
PRX-F 28.600 22674 10.616 0.998 163336
PRX-A 38.706 26577 32.101 0.993 199609
Comparative example 1
The method is characterized in that the following conditions are selected to detect and separate impurities by referring to a detection method disclosed in a parecoxib sodium imported drug registration standard (JX20110120) for injection, and the operation steps are as follows:
(1) taking a proper amount of parecoxib sodium (PRX) and PRX-A, PRX-B, PRX-C, PRX-E, PRX-F, PRX-I, PRX-K, PRX-N, PRX-Q, PRX-U standard products respectively, adding acetonitrile-water (40:60, v/v) to dissolve and dilute the standard products to prepare solutions containing 0.5mg of parecoxib sodium and 2.5 mu g of impurities respectively in each 1mL of standard products, and taking the solutions as test solution.
(2) And (4) injecting the test solution into a liquid chromatograph, and recording the chromatogram.
The liquid chromatography conditions were: a chromatographic column: YMC-Pack ODS-AQ (4.6X 150mm, 5 μm); mobile phase A: 0.01mol/L disodium hydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid); mobile phase B: acetonitrile; detection wavelength: 215 nm; column temperature: 30 ℃; flow rate: 1.0 mL/min; sample introduction volume: 20 μ L. Elution procedure is as in table 11:
table 11 comparative example elution procedure
Time/minute Mobile phase A (%) Mobile phase B (%)
0.01 60 40
7.00 60 40
55.00 20 80
56.00 60 40
65.00 60 40
The chromatogram of the solution to be tested in the comparative example is shown in FIG. 5, and the experimental data are shown in Table 12.
TABLE 12 statistics of comparative examples
Attribution Retention time 1 Positioning time 2 Area of Degree of separation Tailing factor Number of theoretical plate
PRX-C 2.979 2.943 141253 -- 1.453 4948
PRX-E 4.230 4.221 126355 6.837 -- 7410
PRX-Q 4.478 4.479 77015 1.227 -- 7396
PRX-B 10.416 10.334 403227 16.338 0.931 6452
PRX-U 11.053 11.001 154348 1.414 -- 13390
PRX-I 12.255 12.405 108885 2.803 -- 10632
PRX-K 12.554 12.044 90784 0.603 -- 9504
PRX-N 13.223 13.005 185897 1.421 1.052 15372
PRX 17.367 / 60351134 9.700 1.046 26361
PRX-F 20.158 19.819 86718 6.802 -- 42148
PRX-A 31.98 32.02 97472 5.030 1.019 85634
Note: retention time 1: retention time in the test solution; positioning time 2: single needle positioning time of each impurity.
According to the data, the detection method disclosed by the invention can be used for realizing good separation of the 10 synthetic process impurities, so that the quality of parecoxib sodium can be better researched and controlled; in the comparative examples, the separation of the impurity PRX-Q and the impurity PRX-E, the impurity PRX-U and the impurity PRX-B, the impurity PRX-K and the impurity PRX-I, and the impurity PRX-N and the impurity PRX-K was poor.
Examples of the experiments
The chromatographic conditions of example 4 are used for carrying out methodology validation research, the methodology validation test process refers to the methodology validation guiding principle of Chinese pharmacopoeia, the detection limit (S/N is 3) and the quantification limit (S/N is 10) are calculated by using the standard working solution with the lowest concentration, the linear concentration of each process impurity is repeatedly injected for 6 times, the precision of the measurement result is calculated, meanwhile, the recovery rate of a low, medium and high concentration gradient measurement method is set, and the column temperature, the instrument brand, the initial proportion of an organic phase and the durability of a chromatographic column test method are changed. The detection limit, the quantification limit, the precision, the recovery rate and the durability of the method are measured and the results are shown in tables 13-16.
TABLE 13 sensitivity test data for the detection method of the present invention
Figure BDA0002352328680000121
TABLE 14 precision experimental data of the detection method of the present invention
Name of impurity Measured value% Mean value of RSD％
PRX-C 102.11～103.17 102.5 0.36
PRX-E 102.20～108.97 103.6 2.56
PRX-Q 100.23～100.84 100.5 0.21
PRX-U 99.85～100.57 100.1 0.27
PRX-K 100.73～101.56 101.1 0.27
PRX-N 100.11～102.22 101.3 0.33
PRX-I 103.32～104.57 103.9 0.41
PRX-F 100.40～101.27 100.8 0.37
PRX-A 100.48～101.33 100.8 0.33
TABLE 15 Experimental data on recovery rates of the detection methods of the present invention
Impurities in the product Test range The recovery rate is high Mean value of RSD％
PRX-C 50％～150％ 101.91～104.37 102.7 0.86
PRX-E 50％～150％ 102.00～105.29 103.0 1.14
PRX-Q 50％～150％ 99.74～101.92 100.7 0.61
PRX-U 50％～150％ 99.41～101.27 100.2 0.57
PRX-K 50％～150％ 100.73～103.13 101.4 0.67
PRX-N 50％～150％ 99.87～103.11 101.2 1.12
PRX-I 50％～150％ 103.32～108.54 104.8 1.80
PRX-F 50％～150％ 100.40～101.94 101.0 0.42
PRX-A 50％～150％ 100.48～101.43 101.0 0.33
TABLE 16 results of durability measurement of the measuring method of the present invention
Figure BDA0002352328680000131
As is clear from the above-mentioned results, in the method of the present invention,
1) the quantitative limit of each impurity is 0.10-0.30 ng, which is lower than 0.01% of the concentration of the test sample; the detection limit of each impurity is 0.05-0.15 ng, which is lower than 0.005% of the concentration of the test sample;
2) linearity and range: in the concentration range of 0.01-5.0 mu g/mL, the linearity of each impurity concentration and peak area is good, and the correlation coefficient is more than 0.999;
3) precision and accuracy: the sample adding recovery rate of 9 samples with the concentration of 3 samples in high, medium and low are measured, the sample adding recovery rate is in the range of 90-110%, and the RSD of the measurement result is less than 5%, which shows that the method has good precision and accuracy;
4) durability: the method has the advantages that the impurity detection capability and separation capability are inspected by changing the conditions of flow rate, column temperature, mobile phase proportion, replacement of chromatographic columns and chromatographs of different manufacturers and the like, and no obvious change exists under all conditions, so that the method is good in durability.
Therefore, the method has good sensitivity, precision, accuracy and durability.

Claims (5)

1. The method for detecting impurities in parecoxib sodium synthesis process is characterized by comprising the following steps:
1) dissolving a parecoxib sodium sample to be detected to obtain a solution to be detected;
2) carrying out high performance liquid chromatography analysis on the solution to be detected to obtain the content of impurities in the synthesis process;
the analysis conditions of the high performance liquid chromatography are as follows: a chromatographic column: octadecylsilane chemically bonded silica chromatographic column; mobile phase A: 0.01mol/L disodium hydrogen phosphate solution with pH of 2.5-3.5, and a mobile phase B: the volume ratio is 75-85: 15-25 of acetonitrile-methanol mixed solution; the elution gradient program included: 0min, 60-70 vol% of mobile phase A and 40-30 vol% of mobile phase B; 30min, 23-27 vol% of mobile phase A and 77-73 vol% of mobile phase B; 41min, 60-70 vol% of mobile phase A and 40-30 vol% of mobile phase B;
the parecoxib sodium synthesis process impurities comprise parecoxib sodium synthesis reaction raw materials and synthesis reaction byproducts, wherein the synthesis reaction raw materials are as follows:
Figure FDA0003646179970000011
the synthesis reaction by-products include the following compounds:
Figure FDA0003646179970000012
the model of the octadecylsilane chemically bonded silica chromatographic column is YMC-Pack ODS-AQ or Welch Ultimate XB-C18; the size of the octadecylsilane bonded silica chromatographic column was 4.6mm x 250mm, and the filler particle size was 5 μm.
2. The method for detecting impurities in parecoxib sodium synthesis process according to claim 1, wherein in step 1), a sample to be detected of parecoxib sodium is dissolved by using an acetonitrile-water mixed solution.
3. The method for detecting impurities in the parecoxib sodium synthesis process according to claim 1, wherein in step 1), the solution to be detected is a solution containing 0.5mg of parecoxib sodium sample to be detected in every 1 mL.
4. The method for detecting impurities in parecoxib sodium synthesis process according to claim 1, wherein the flow rate of the mobile phase adopted during high performance liquid chromatography is 0.8-1.2 mL/min.
5. The method for detecting impurities in the parecoxib sodium synthesis process according to claim 1, wherein the column temperature of a chromatographic column during high performance liquid chromatography is 25-35 ℃.
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