A method for separating and determining degradation impurities in cardiolipin-levodopa sustained-release tablets

Abstract

The invention discloses a method for separating and determining degradation impurities in cardiolipin sustained-release tablets, wherein ultra-high performance liquid chromatography is used for separation and determination, wherein the chromatographic column of the ultra-high performance liquid chromatography uses octadecylsilane bonded silica gel as a filler, and mobile phase A and mobile phase B are used for gradient elution, wherein mobile phase A is anhydrous sodium dihydrogen phosphate buffer, and mobile phase B is ethanol, and the total volume of the mobile phase is 100%. The gradient elution procedure is: the degradation impurities are separated and determined within 25 minutes by gradient elution. The method for separating and determining degradation impurities in cardiolipin sustained-release tablets of the invention can realize simultaneous detection of four degradation impurities in the cardiolipin sustained-release tablets.

Description

Technical Field

[0001] The invention relates to the technical field of drug impurity detection, and in particular to a method for separating and determining degradation impurities in cardiolipin-levodopa sustained-release tablets. Background Art

[0002] Carbidopa and Levodopa Extended-Release Tablets is a compound drug mainly used to treat Parkinson's disease and Parkinson's syndrome. It is a compound extended-release tablet made of carbidopa and levodopa, which can relieve symptomatic Parkinson's syndrome caused by primary Parkinson's disease, post-encephalitis Parkinson's syndrome, carbon monoxide or manganese poisoning, etc.

[0003] Carbidopa sustained-release tablets contain two main components, namely levodopa and carbidopa, with a ratio of levodopa to carbidopa of 4:1. The United States Pharmacopoeia discloses that carbidopa sustained-release tablets contain four degradation impurities, namely levodopa (EP) impurity A, methyldopa, 3,4-dihydroxybenzaldehyde (i.e., carbidopa impurity 52), and 3,4-dihydroxypropiophenone (i.e., carbidopa impurity G). In addition, carbidopa sustained-release tablets also contain process impurities of levodopa API and process impurities of carbidopa API. In order to accurately determine the content of degradation impurities in carbidopa sustained-release tablets, it is necessary that process impurities must not interfere with the determination of degradation impurities.

[0004] The detection of degradation impurities in the carbidopa sustained-release tablets included in the United States Pharmacopoeia takes a long time and has low sensitivity. For carbidopa, due to its unstable structure, it is very easy to degrade and produce 3,4-dihydroxypropiophenone, and it is difficult to accurately measure the content of 3,4-dihydroxypropiophenone.

[0005] Therefore, how to establish an analytical method that is simple, fast, efficient, and has good separation, high sensitivity, good durability, and high reproducibility, so as to achieve comprehensive detection and control of the quality of carbidopa sustained-release tablets, is a technical problem that needs to be solved at present. Summary of the invention

[0006] In view of the above-mentioned defects of the prior art, the present invention provides a method for separating and determining degradation impurities in cardiolipin sustained-release tablets, which can realize the simultaneous detection of four degradation impurities in cardiolipin sustained-release tablets.

[0007] In order to solve the above technical problems, the present invention provides a method for separating and determining degradation impurities in cardiolipin sustained-release tablets, which is separated and determined by ultra-high performance liquid chromatography. The chromatographic column of the ultra-high performance liquid chromatography uses octadecylsilane bonded silica gel as a filler, and gradient elution is performed with mobile phase A and mobile phase B. Mobile phase A is anhydrous sodium dihydrogen phosphate buffer, and mobile phase B is ethanol. The total volume of the mobile phase is 100%, and the gradient elution program is:

[0008] Time (min) Mobile phase A (%) Popular phase B (%) 0 97 3 2 97 3 3 96 4 12 96 4 13 92 8 19 92 8 19.1 97 3 25 97 3

[0009] The degradation impurities were separated and determined by gradient elution within 25 min.

[0010] As a preferred embodiment, the chromatographic column is Agilent Proshell 120EC C18.

[0011] As a preferred embodiment, the concentration of the anhydrous sodium dihydrogen phosphate buffer is 2-8 g / L, and the pH is 2-3.

[0012] As a more preferred embodiment, the concentration of the anhydrous sodium dihydrogen phosphate buffer is 6 g / L and the pH is 2.2.

[0013] As a preferred embodiment, the column temperature of the chromatographic column is 25-35°C.

[0014] As a more preferred embodiment, the column temperature of the chromatographic column is 25°C.

[0015] As a preferred embodiment, the flow rate of the mobile phase is 0.5-0.8 ml / min.

[0016] As a more preferred embodiment, the flow rate of the mobile phase is 0.6 ml / min.

[0017] As a preferred embodiment, the injection temperature is 2°C-9°C, and the injection volume is 10 μl-25 μl.

[0018] As a more preferred embodiment, the injection temperature is 6° C. and the injection volume is 20 μl.

[0019] As a preferred embodiment, the detection wavelength is 220nm-300nm.

[0020] As a more preferred embodiment, the detection wavelength is 280 nm.

[0021] As a preferred embodiment, ultra-high performance liquid chromatography detection comprises the following steps:

[0022] S1. Prepare blank solution, sensitivity solution, resolution solution, reference solution and test solution;

[0023] S2, injecting the blank solution, sensitivity solution, resolution solution, reference solution and test solution in step S1 into the chromatograph in sequence, and recording the chromatograms of the blank solution, sensitivity solution, resolution solution, reference solution and test solution in sequence;

[0024] S3. Calculate the content of degradation impurities by external standard method according to the peak area of ​​the chromatogram.

[0025] As a preferred embodiment, the blank solution is prepared by mixing anhydrous sodium dihydrogen phosphate buffer and ethanol in a volume ratio of 96:4, the concentration of the anhydrous sodium dihydrogen phosphate buffer is 6 g / L, and the pH is 2.2; the sensitivity solution is prepared by using the blank solution to prepare a solution containing 0.4 μg / ml of levodopa and 0.1 μg / ml of carbidopa; the separation solution is prepared by using the blank solution to prepare a solution containing 0.1 mg / ml of carbidopa and 0.8 μg / ml of levodopa (EP) impurity C; the reference solution is prepared by using the blank solution to prepare a solution containing 4 μg / ml of levodopa and 1 μg / ml of carbidopa; the test solution is prepared by dissolving the carbidopa-levodopa sustained-release tablets to be tested in the blank solution, wherein the levodopa concentration is 0.4 mg / ml and the carbidopa concentration is 0.1 mg / ml.

[0026] As a preferred embodiment, the percentage of impurities from carbidopa degradation = (Ru / Rs)*(Cs / Cu)*(1 / F)*100

[0027] Ru: Degradation impurity peak area

[0028] Rs: Average peak area of ​​carbidopa reference substance

[0029] Cs: Carbidopa reference concentration mg / mL

[0030] Cu: Carbidopa concentration in the test solution mg / mL

[0031] F: relative response factor, where methyldopa is 1.0, carbidopa impurity G is 1.2, carbidopa impurity 52 is 6.1, and other unknown impurities are calculated as 1.0;

[0032] Impurities from L-DOPA degradation % = (Ru / Rs)*(Cs / Cu)*(1 / F)*100

[0033] Ru: Degradation impurity peak area

[0034] Rs: Average peak area of ​​levodopa reference substance

[0035] Cs: L-dopa reference concentration mg / mL

[0036] Cu: L-DOPA concentration in the test solution mg / mL

[0037] F: Relative response factor, where L-DOPA (EP) impurity A is 0.8 and other unknown impurities are calculated as 1.0.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] (1) The method for separating and determining degradation impurities in carbidopa-levodopa sustained-release tablets of the present invention can achieve simultaneous detection of four degradation impurities in carbidopa-levodopa sustained-release tablets, has excellent separation of degradation impurities, and process impurities in carbidopa and levodopa raw materials do not interfere with degradation impurities. The determination method has the advantages of excellent accuracy, precision and durability.

[0040] (2) The method for separating and determining degradation impurities in cardiolipin sustained-release tablets of the present invention can meet the requirements of system applicability and has good method specificity.

[0041] (3) The method of separating and determining degradation impurities in carbidopa-levodopa sustained-release tablets of the present invention can achieve accurate quantification of levodopa (EP) impurity A above 0.2 μg / ml (0.05%); and can achieve accurate quantification of methyldopa, carbidopa impurity 52, and carbidopa impurity G above 0.1 μg / ml (0.10%).

[0042] (4) The method of separating and determining degradation impurities in carbidopa-levodopa sustained-release tablets of the present invention can detect levodopa (EP) impurity A at above 0.07 μg / ml (0.017%), and can detect methyldopa, carbidopa impurity 52, and carbidopa impurity G at above 0.03 μg / ml (0.033%).

[0043] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is a chromatogram of a blank solution under system suitability in Example 1 of the present invention;

[0045] Figure 2 is a chromatogram of the sensitivity solution under system suitability in Example 1 of the present invention;

[0046] Figure 3 is a chromatogram of the separation solution under system suitability in Example 1 of the present invention;

[0047] Figure 4 It is a chromatogram of a mixed solution of four degradation impurities under the specificity item in Example 1 of the present invention;

[0048] Figure 5 It is a superposition diagram of all impurities and the dual main solution under the specificity item in Example 1 of the present invention;

[0049] Figure 6 is a chromatogram of a blank auxiliary material solution under the specificity item in Example 1 of the present invention;

[0050] Figure 7 is a chromatogram of a 100% limit spiked sample solution under the specificity item in Example 1 of the present invention;

[0051] Figure 8 is a chromatogram of a sample solution destroyed by high temperature under the specificity item in Example 1 of the present invention;

[0052] Fig. 9 is a chromatogram of the acid-destroyed sample solution under the specificity item in Example 1 of the present invention;

[0053] Fig.10 is a chromatogram of the alkali-destroyed sample solution under the specificity item in Example 1 of the present invention;

[0054] Fig.11 is a chromatogram of the oxidatively damaged sample solution under the specificity item in Example 1 of the present invention;

[0055] Fig.12 is a chromatogram of the light-damaged sample solution under the specificity item in Example 1 of the present invention;

[0056] Fig.13 is a linear result graph of levodopa in Example 1 of the present invention;

[0057] Fig.14 is a linear result graph of levodopa (EP) impurity A in Example 1 of the present invention;

[0058] Fig.15 is a linear result graph of carbidopa in Example 1 of the present invention;

[0059] Fig.16 is a linear result graph of methyldopa in Example 1 of the present invention;

[0060] Fig.17 is a linear result graph of carbidopa impurity 52 in Example 1 of the present invention;

[0061] Fig.18 is a linear result graph of carbidopa impurity G in Example 1 of the present invention;

[0062] Fig.19 is the chromatogram corresponding to the chromatographic column 1 in Example 2 of the present invention;

[0063] Fig. 20 is the chromatogram corresponding to the chromatographic column 2 in Example 2 of the present invention;

[0064] Fig.21 is the chromatogram corresponding to gradient 1 in Example 3 of the present invention;

[0065] Fig. 22 is the chromatogram corresponding to gradient 2 in Example 3 of the present invention;

[0066] Fig.23 It is the chromatogram of the sensitivity solution in the USP pharmacopoeia method;

[0067] Fig.24 It is the chromatogram of the sample solution in the USP pharmacopoeia method;

[0068] Fig.25 This is the chromatogram of 5 consecutive injections of the impurity A solution of levodopa (EP) in a common injection vial;

[0069] Fig.26 This is a chromatogram of five consecutive injections of a solution of L-DOPA (EP) impurity A in an Agilent certified glass vial. DETAILED DESCRIPTION

[0070] In order to make the technical means, creative features, objectives and effects of the invention easier to understand, the invention is further described below with reference to specific illustrations. However, the invention is not limited to the following implementation cases.

[0071] It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with this technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantive technical significance. Any structural modification, change in proportion or adjustment of size, without affecting the effects and purposes that can be achieved by the present invention, should still fall within the scope of the technical contents disclosed by the present invention.

[0072] The relevant terms in this application are explained as follows:

[0073] Process impurities: impurities generated during the drug synthesis process. Chemical drugs are usually obtained through multi-step chemical reactions, and a variety of starting materials, intermediates and reagents / solvents may be used in the process. These reactions may produce various by-products, which are process impurities.

[0074] Degradation impurities: impurities generated during the storage of drugs due to external conditions (such as temperature, humidity, light) or microbial action. Drugs may undergo hydrolysis, oxidation, ring opening, polymerization and other reactions during storage and transportation, thus forming degradation impurities.

[0075] Purity angle: refers to the average value of the angles between the spectrum at each point of the peak and the spectrum at the peak apex.

[0076] Purity Threshold: The critical angle used to judge the purity of a peak. If the purity angle of a peak is less than this threshold, the peak is considered pure; if the purity angle is greater than or equal to the threshold, the peak is considered to contain impurities.

[0077] Reference substance: a standard substance used for identification, inspection and content determination.

[0078] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 50" includes ±5% of 50, or from 47.5 to 52.5.

[0079] Carbidopa extended-release tablets contain two main components, namely levodopa and carbidopa, with a ratio of levodopa to carbidopa of 4:1. The process impurities of levodopa are levodopa (EP) impurity 12, levodopa (EP) impurity B, and levodopa (EP) impurity C. The process impurities of carbidopa are carbidopa (USP) impurity A, carbidopa (EP) impurity D, carbidopa (EP) impurity E, carbidopa (EP) impurity F, and carbidopa (EP) impurity H. Carbidopa extended-release tablets contain four degradation impurities, namely levodopa (EP) impurity A, methyldopa, 3,4-dihydroxybenzaldehyde (i.e., carbidopa impurity 52), and 3,4-dihydroxypropiophenone (i.e., carbidopa impurity G). EP is the European Pharmacopoeia, Levodopa (EP) impurity 12 refers to Levodopa impurity 12 named by the European Pharmacopoeia, USP is the United States Pharmacopoeia, Carbidopa (USP) impurity A refers to Carbidopa impurity A named by the United States Pharmacopoeia.

[0080] Levodopa (EP) impurity A, Levodopa impurity A, and Levorotatory impurity A mentioned in this application document refer to the same content, and both refer to the substance named Levodopa impurity A in the European Pharmacopoeia. Similarly, Levodopa (EP) impurity C and Levodopa impurity C refer to the same content, and both refer to the substance named Levodopa impurity C in the European Pharmacopoeia.

[0081] In order to ensure the therapeutic effect of carboplatin sustained-release tablets, it is necessary to accurately measure the content of degradation impurities therein, which requires that process impurities cannot affect the determination of degradation impurities.

[0082] The present application discloses a method for separating and determining degradation impurities in cardiolipin sustained-release tablets, which is separated and determined by ultra-high performance liquid chromatography. The chromatographic column of the ultra-high performance liquid chromatography uses octadecylsilane bonded silica gel as a filler, and gradient elution is performed with mobile phase A and mobile phase B. Mobile phase A is anhydrous sodium dihydrogen phosphate buffer, and mobile phase B is ethanol. The total volume of the mobile phase is 100%, and the gradient elution procedure is:

[0083]

[0084]

[0085] The degradation impurities were separated and determined by gradient elution within 25 min.

[0086] Ultra-high performance liquid chromatography detection specifically includes the following steps:

[0087] S1. Prepare a blank solution, a sensitivity solution, a resolution solution, a reference solution and a test solution; the blank solution is prepared by mixing anhydrous sodium dihydrogen phosphate buffer and ethanol in a volume ratio of 96:4, the concentration of the anhydrous sodium dihydrogen phosphate buffer is 6 g / L, and the pH is 2.2; the sensitivity solution is prepared by using the blank solution to prepare a solution containing 0.4 μg / ml of levodopa and 0.1 μg / ml of carbidopa; the resolution solution is prepared by using the blank solution to prepare a solution containing 0.1 mg / ml of carbidopa and 0.8 μg / ml of levodopa (EP) impurity C; the reference solution is prepared by using the blank solution to prepare a solution containing 4 μg / ml of levodopa and 1 μg / ml of carbidopa; the test solution is prepared by dissolving the carbidopa-levodopa sustained-release tablets to be tested in the blank solution, wherein the levodopa concentration is 0.4 mg / ml and the carbidopa concentration is 0.1 mg / ml.

[0088] S2. Inject the blank solution, sensitivity solution, resolution solution, reference solution and test solution in step S1 into the chromatograph in sequence, and record the chromatograms of the blank solution, sensitivity solution, resolution solution, reference solution and test solution in sequence.

[0089] Specifically, a blank solution, a sensitivity solution, a resolution solution, a reference solution and a test solution are injected into the chromatograph in the following order, wherein the injection process is as follows: (1) blank solution (i.e., diluent) (injection times ≥ 1 injection), (2) sensitivity solution (injection times 1 injection), (3) resolution solution (injection times 1 injection), (4) reference solution (injection times 6 injections), (5) test solution (injection times ≥ 1 injection; one injection of reference solution is required for each 6 injections of test solution), and (6) reference solution (injection times ≥ 1 injection).

[0090] S3. Calculate the content of degradation impurities according to the peak area of ​​the chromatogram using the external standard method with relative response factor.

[0091] Specifically:

[0092] Impurities from carbidopa degradation % = (Ru / Rs)*(Cs / Cu)*(1 / F)*100

[0093] Ru: Degradation impurity peak area

[0094] Rs: Average peak area of ​​carbidopa reference substance

[0095] Cs: Carbidopa reference concentration mg / mL

[0096] Cu: Carbidopa concentration in the test solution mg / mL

[0097] F: relative response factor, where methyldopa is 1.0, carbidopa impurity G is 1.2, carbidopa impurity 52 is 6.1, and other unknown impurities are calculated as 1.0;

[0098] Impurities from L-DOPA degradation % = (Ru / Rs)*(Cs / Cu)*(1 / F)*100

[0099] Ru: Degradation impurity peak area

[0100] Rs: Average peak area of ​​levodopa reference substance

[0101] Cs: L-dopa reference concentration mg / mL

[0102] Cu: L-DOPA concentration in the test solution mg / mL

[0103] F: Relative response factor, where L-DOPA (EP) impurity A is 0.8 and other unknown impurities are calculated as 1.0.

[0104] The chromatographic column is Agilent Proshell 120EC C18, 3.0×150 mm, 2.7 μm, and the column temperature is 25-35° C., preferably 25° C.

[0105] The concentration of the anhydrous sodium dihydrogen phosphate buffer is 2-8 g / L, preferably 6 g / L. The pH of the anhydrous sodium dihydrogen phosphate buffer is 2-3, preferably 2.2.

[0106] The flow rate of the mobile phase is 0.5-0.8 ml / min, preferably 0.6 ml / min.

[0107] The injection temperature is 2°C-9°C, preferably 6°C.

[0108] The injection volume is 10 μl to 25 μl, preferably 20 μl.

[0109] The detection wavelength is 220nm-300nm, preferably 280nm.

[0110] Example 1

[0111] 1. System suitability test

[0112] 1) Prepare blank solution, sensitivity solution, resolution solution and reference solution.

[0113] Blank solution: prepared by mixing anhydrous sodium dihydrogen phosphate buffer and ethanol in a volume ratio of 96:4. The concentration of anhydrous sodium dihydrogen phosphate buffer is 6 g / L and the pH is 2.2.

[0114] Sensitivity solution: Use blank solution to dissolve levodopa and carbidopa reference substances to prepare solutions containing 0.4 μg / ml levodopa and 0.1 μg / ml carbidopa.

[0115] Separation solution: Use blank solution to dissolve carbidopa and levodopa (EP) impurity C reference substances to prepare solutions containing 0.1 mg / ml carbidopa and 0.8 μg / ml levodopa (EP) impurity C.

[0116] Reference solution: Dissolve the levodopa and carbidopa reference substances in the blank solution to prepare a solution containing 4 μg / ml levodopa and 1 μg / ml carbidopa.

[0117] 2) Sample injection

[0118] Inject blank solution, sensitivity solution, resolution solution and reference solution into the chromatograph in the following order, wherein the injection process is as follows: (1) blank solution (i.e. diluent) (injection times ≥ 1 injection), (2) sensitivity solution (injection times 1 injection), (3) resolution solution (injection times 1 injection), (4) reference solution (injection times 6 injections). Record the chromatograms of the blank solution, sensitivity solution, resolution solution and reference solution in sequence, wherein the chromatograms of the blank solution, sensitivity solution and resolution solution are as follows: Figure 1 , 2 , as shown in Figure 3.

[0119] from Figure 1 It can be seen that the blank solution has no peaks at the positions of carbidopa and levodopa, and has no interference with the test results; Figure 2 It can be seen that the signal-to-noise ratio of carbidopa in the sensitivity solution is 58, which is greater than 10; Figure 3 It can be seen that the separation degree of levodopa (EP) impurity C and carbidopa in the solution is 2.2, which is greater than 1.5.

[0120] The reference solution was injected 6 times, and the measured carbidopa peak area and levodopa peak area are shown in Table 1.

[0121] Table 1

[0122] Reference solution Carbidopa Peak Area L-DOPA peak area 1 24569 113560 2 24511 113486 3 24352 113551 4 24338 113427 5 24244 113513 6 24207 113495 Average of 6 peak areas 24370 113505 RSD% (n=6) 0.6 0.0

[0123] As shown in Table 1, the RSD of the peak areas of carbidopa and levodopa in the reference solution after 6 injections were 0.6% and 0.0%, respectively, which were much less than 3.0%. The chromatograms of the blank solution, sensitivity solution, and resolution solution proved that the system applicability of this method met the requirements.

[0124] 2. Method specificity test

[0125] 1) Prepare blank solution, blank excipient solution, reference substance solution, 4 degradation impurity solutions, 8 process impurity solutions, 4 degradation impurity mixed solutions and impurity 100% limit spike solution.

[0126] Blank solution: prepared by mixing anhydrous sodium dihydrogen phosphate buffer and ethanol in a volume ratio of 96:4. The concentration of anhydrous sodium dihydrogen phosphate buffer is 6 g / L and the pH is 2.2.

[0127] Blank excipient solution: Use blank solution to prepare mixed excipients (hydroxypropyl methylcellulose, hydroxypropyl cellulose, magnesium stearate, allura red aluminum lake, indigo aluminum lake according to the proportion of self-developed prescription) into 0.06 mg / mL excipient solution, filter with 0.22um PVDF filter membrane and then inject.

[0128] Reference solution: Dissolve the levodopa and carbidopa reference substances in the blank solution to prepare a solution containing 4 μg / ml levodopa and 1 μg / ml carbidopa.

[0129] 4 degradation impurity solutions: Use blank solution to dissolve levodopa (EP) impurity A, methyldopa, carbidopa impurity 52, and carbidopa impurity G reference substances respectively, and prepare solutions with a concentration of 5 μg / mL for levodopa (EP) impurity A, methyldopa, carbidopa impurity 52, and carbidopa impurity G, for a total of 4 solutions.

[0130] 8 process impurity solutions: Use blank solution to dissolve levodopa (EP) impurity B, levodopa (EP) impurity C, levodopa (EP) impurity 12, carbidopa (USP) impurity A, carbidopa (EP) impurity D, carbidopa (EP) impurity E, carbidopa (EP) impurity F, and carbidopa (EP) impurity H reference substances, and prepare them into 5 μg / mL solutions respectively, for a total of 8 solutions.

[0131] 4 mixed solutions of degradation impurities: Use blank solution to dissolve reference substances of levodopa (EP) impurity A, methyldopa, carbidopa impurity 52, and carbidopa impurity G to prepare mixed solutions with a levodopa (EP) impurity A concentration of 0.4 μg / mL, a methyldopa concentration of 0.5 μg / mL, a carbidopa impurity 52 concentration of 0.2 μg / mL, and a carbidopa impurity G concentration of 1 μg / mL.

[0132] 100% limit spiked sample solution: 100% limit sample spiked accuracy solution under the same accuracy test item.

[0133] The above solutions were sampled and analyzed separately, and the chromatograms were recorded. The chromatograms of the 4 degraded impurity mixed solutions, the superposition of all impurities and the two main solutions, the blank auxiliary material solution, and the 100% limit spiked sample solution are shown in Figure 1. Figure 4 , 5 , 6, and 7. Figure 5 In which Blank refers to blank solution, L-dopa is the abbreviation of L-dopa, L-impurity A is the abbreviation of L-dopa (EP) impurity A, Tyrosine is another name for L-dopa (EP) impurity B, L-impurity C is the abbreviation of L-dopa (EP) impurity C, Carbidopa is the abbreviation of carbidopa, Carbidopa 52 is the abbreviation of carbidopa impurity 52, Carbidopa E is the abbreviation of carbidopa (EP) impurity E, Carbidopa D is the abbreviation of carbidopa (EP) impurity D, Carbidopa G is the abbreviation of carbidopa impurity G, Carbidopa A is the abbreviation of carbidopa (USP) impurity A, Carbidopa H is the abbreviation of carbidopa (EP) impurity H, Carbidopa F is the abbreviation of carbidopa (EP) impurity F, and L-impurity 12 is the abbreviation of L-dopa (EP) impurity 12.

[0134] from Figure 6 It can be seen that the blank auxiliary material solution has no UV absorption and no interference; Figure 7 It can be seen that the 100% limit spiked sample solution only has an unknown peak adjacent to the L-dopa (EP) impurity A, but it can achieve baseline separation with the L-dopa (EP) impurity A, and the resolution is 4.6.

[0135] The peak results of 4 degradation impurities and 8 process impurities are shown in Table 2.

[0136] Table 2

[0137]

[0138]

[0139] From the peak time and Figure 5 It can be seen that the peak positions of degradation impurities and process impurities are separated, and process impurities do not affect the determination of degradation impurities. The detection method of the present application has good specificity.

[0140] An appropriate amount of Carbidopa sustained-release tablets was subjected to forced degradation tests under high temperature, acid, alkali, oxidation and light.

[0141] High temperature damage experiment: Take an appropriate amount of carbidopa sustained-release tablets and place them in a 105℃ oven for 24 hours. Simultaneously place the levodopa API, carbidopa API and mixed excipients in a 105℃ oven for 24 hours to determine the ownership of the impurities in the sample degradation. Take out the samples after 24 hours, prepare them into a solution containing 0.4mg / mL of levodopa and 0.1mg / mL of carbidopa, prepare the levodopa API into a solution of 0.4mg / mL, prepare the carbidopa API into a solution of 0.1mg / mL and the excipients into a solution of 0.06mg / mL.

[0142] Acid destruction experiment: Take an appropriate amount of carbidopa sustained-release tablets (containing about 40 mg of levodopa and 10 mg of carbidopa) in a 100 mL volumetric flask, add 10 mL of 2 mol / L hydrochloric acid and place in a 65°C water bath for 24 hours. Simultaneously, place 40 mg of levodopa API, 11 mg of carbidopa API and 6 mg of mixed excipients in different 100 mL volumetric flasks, add 10 mL of 2 mol / L hydrochloric acid and place in a 65°C water bath for 24 hours to determine the impurities of sample degradation. Take out after 24 hours, add 10 mL of 2 mol / L sodium hydroxide solution to each volumetric flask, neutralize and add diluent (blank solution) to make up to volume.

[0143] Alkali destruction experiment: Take an appropriate amount of carbidopa sustained-release tablets sample (containing about 40 mg of levodopa and 10 mg of carbidopa) in a 100 mL volumetric flask, add 10 mL of 0.02 mol / L sodium hydroxide, and simultaneously place 40 mg of levodopa API, 11 mg of carbidopa API and 6 mg of mixed excipients in different 100 mL volumetric flasks, add 10 mL of 0.02 mol / L sodium hydroxide to determine the impurities of sample degradation. Take out after 1 hour, add 10 mL of 0.02 mol / L hydrochloric acid solution to each volumetric flask, neutralize and add diluent (blank solution) to the fixed volume.

[0144] Oxidative damage experiment: Take an appropriate amount of carbidopa sustained-release tablets sample (containing about 40 mg of levodopa and 10 mg of carbidopa) in a 100 mL volumetric flask, add 10 mL of 6% hydrogen peroxide solution, and simultaneously place 40 mg of levodopa API, 11 mg of carbidopa API and 6 mg of mixed excipients in different 100 mL volumetric flasks, add 10 mL of 6% hydrogen peroxide solution to determine the ownership of the impurities degraded from the sample. Take out after 40 hours and add diluent to make up to volume.

[0145] Light damage experiment: Take an appropriate amount of carbidopa sustained-release tablets and place them in a light box, one of which is wrapped in tin foil and the other is not wrapped in tin foil. Simultaneously place levodopa API, carbidopa API and mixed excipients in the light box to determine the ownership of the impurities degraded from the samples. Take out the samples after 7 days, prepare them into a solution containing 0.4 mg / mL levodopa and 0.1 mg / mL carbidopa, prepare 0.4 mg / mL levodopa API, 0.1 mg / mL carbidopa API and 0.06 mg / mL excipients.

[0146] The solutions prepared after high temperature, acid, alkali, oxidation and light damage experiments were sampled and analyzed to determine the content of levodopa and carbidopa, their degradation amount and total impurity content. Figure 8-12 They are the chromatograms obtained after high temperature, acid, alkali, oxidation and light destruction experiments, the statistical results of the content of levodopa and carbidopa and their degradation amount and total impurity content are shown in Table 3, and the peak purity results are shown in Table 4.

[0147] Table 3

[0148]

[0149]

[0150] Table 4

[0151]

[0152] It can be seen from Tables 3 and 4 that the quality is conserved before and after the high temperature, acid, alkali, oxidation and light destruction experiments, and the peak purity also meets the requirements.

[0153] pass Figure 8-Figure 12 It can be seen that the impurities produced by degradation are well separated from the main peak and the four degradation impurities, and the specificity of the present invention can meet the requirements.

[0154] 3. Linearity and range tests

[0155] Standard solutions of five concentration levels were prepared, with levodopa and levodopa (EP) impurity A being 0.05% (LOQ), 0.10%, 0.25%, 0.50% and 1.00% of the target level (target level concentration: 0.4 mg / ml), and carbidopa, methyldopa, carbidopa impurity 52 and carbidopa impurity G being 0.10% (LOQ), 0.20%, 0.50%, 1.00% and 2.00% of the target level (target level concentration: 0.1 mg / ml).

[0156] Take one injection of each of the above solutions and record the chromatogram, with concentration as the horizontal axis and peak area as the vertical axis, and make a linear recovery equation. The linear correlation coefficient r should be no less than 0.990; the y-axis intercept should be within 20% of the target concentration. The results are shown in Table 5 and Figure 13-18 shown.

[0157] Table 5

[0158]

[0159]

[0160] From Table 5 and Figure 13-18 It can be seen that the linear relationship of levodopa is good in the concentration range of 0.228μg / ml to 4.560μg / ml; the linear relationship of levodopa (EP) impurity A is good in the concentration range of 0.207μg / ml to 4.139μg / ml; the linear relationship of carbidopa is good in the concentration range of 0.105μg / ml to 2.103μg / ml; the linear relationship of methyldopa is good in the concentration range of 0.108μg / ml to 2.160μg / ml; the linear relationship of carbidopa impurity 52 is good in the concentration range of 0.106μg / ml to 2.117μg / ml; the linear relationship of carbidopa impurity G is good in the concentration range of 0.107μg / ml to 2.141μg / ml.

[0161] 4. Limit of Quantitation

[0162] Quantitation limit solution: the lowest concentration solution that satisfies the linear relationship, that is, the concentration of levodopa is 0.228 μg / ml, the concentration of levodopa (EP) impurity A is 0.207 μg / ml, the concentration of carbidopa is 0.105 μg / ml, the concentration of methyldopa is 0.108 μg / ml, the concentration of carbidopa impurity 52 is 0.106 μg / ml, and the concentration of carbidopa impurity G is 0.107 μg / ml.

[0163] The quantitative limit solution was tested 6 times in parallel, and the results are shown in Table 6.

[0164] Table 6

[0165]

[0166] As can be seen from Table 6, the signal-to-noise ratios of levodopa, carbidopa, levodopa (EP) impurity A, methyldopa, carbidopa impurity 52, and carbidopa impurity G are all not less than 10, the RSD% of the peak area is not greater than 15.0, and the quantification limit meets the requirements. Therefore, the impurities of levodopa and levodopa (EP) impurity A above 0.2 μg / ml (0.05%) can be accurately quantified; the impurities of carbidopa, methyldopa, carbidopa impurity 52, and carbidopa impurity G above 0.1 μg / ml (0.10%) can be accurately quantified.

[0167] 5. Detection limit

[0168] Detection limit solution: The quantification limit solution was diluted 3 times and tested 3 times in parallel. The test results are shown in Table 7.

[0169] Table 7

[0170]

[0171] As can be seen from Table 7, the signal-to-noise ratios of levodopa, carbidopa, levodopa (EP) impurity A, methyldopa, carbidopa impurity 52, and carbidopa impurity G are all not less than 3. Therefore, levodopa and levodopa (EP) impurity A can be detected above 0.07 μg / ml (0.017%), and carbidopa and methyldopa, carbidopa impurity 52, and carbidopa impurity G can be detected above 0.03 μg / ml (0.033%).

[0172] 6. Accuracy test

[0173] Prepare the reference solution, unspiked sample solution, LOQ spiked sample accuracy solution, 100% limit sample spiked accuracy solution, and 200% limit sample spiked accuracy solution.

[0174] Levodopa (EP) impurity A stock solution: Weigh an appropriate amount of Levodopa (EP) impurity A reference substance into a volumetric bottle to prepare an impurity stock solution containing Levodopa (EP) impurity A at a concentration of 8 μg / mL.

[0175] Mixed stock solution of methyldopa, carbidopa impurity 52, and carbidopa impurity G 1: Weigh appropriate amounts of methyldopa, carbidopa impurity 52, and carbidopa impurity G reference substances into the same volumetric bottle to prepare a mixed impurity stock solution containing 10 μg / mL of methyldopa, 4 μg / mL of carbidopa impurity 52, and 20 μg / mL of carbidopa impurity G.

[0176] Methyldopa, Carbidopa Impurity 52, and Carbidopa Impurity G mixed stock solution 2: Weigh appropriate amounts of methyldopa, Carbidopa Impurity 52, and Carbidopa Impurity G reference substances into the same volumetric bottle to prepare an impurity mixed stock solution containing 10 μg / mL of methyldopa, 10 μg / mL of Carbidopa Impurity 52, and 10 μg / mL of Carbidopa Impurity G.

[0177] Reference solution: Pipette 5.0mL of levodopa (EP) impurity A stock solution and 5.0mL of methyldopa, carbidopa impurity 52, and carbidopa impurity G mixed stock solution 1 into the same 100mL volumetric flask, dilute to scale with blank solution, and shake well. Prepare a reference solution containing levodopa (EP) impurity A at a concentration of 0.4μg / mL, methyldopa at 0.5μg / mL, carbidopa impurity 52 at 0.2μg / mL, and carbidopa impurity G at 1.0μg / mL.

[0178] Unspiked sample solution: Take an appropriate amount of test sample powder (obtained by grinding carbidopa sustained-release tablets) in a volumetric flask, add an appropriate amount of blank solution, sonicate for a certain period of time, and mix with the blank solution to make a solution containing 0.4 mg / ml levodopa and 0.1 mg / ml carbidopa. Prepare 3 copies in parallel and inject one injection for each sample.

[0179] LOQ spiked sample accuracy solution: Take an appropriate amount of the test sample powder in a 100mL volumetric flask, add 2.5mL of levodopa (EP) impurity A stock solution, 1.0mL of methyldopa, carbidopa impurity 52, and carbidopa impurity G mixed stock solution 2 to the volumetric flask, dilute to the scale with blank solution, filter with a 0.22um PVDF filter membrane and inject. The concentration of levodopa (EP) impurity A (0.05%) is 0.2ug / mL, the concentration of methyldopa (0.1%) is 0.1ug / mL, the concentration of carbidopa impurity 52 (0.1%) is 0.1ug / mL, the concentration of carbidopa impurity G (0.1%) is 0.1ug / mL, the concentration of levodopa is 0.4mg / ml, and the concentration of carbidopa is 0.1mg / ml. Prepare 3 copies in parallel and inject one injection for each sample.

[0180] 100% limit sample spiked accuracy solution: Take an appropriate amount of the test sample powder in a 100mL volumetric flask, add 5.0mL of levodopa (EP) impurity A stock solution, 5.0mL of methyldopa, carbidopa impurity 52, and carbidopa impurity G mixed stock solution 1 to the volumetric flask, dilute to the scale with blank solution, filter with a 0.22um PVDF filter membrane and inject. Prepare a solution containing levodopa (EP) impurity A at a concentration of 0.4μg / mL, methyldopa at 0.5μg / mL, carbidopa impurity 52 at 0.2μg / mL, carbidopa impurity G at 1.0μg / mL, levodopa at a concentration of 0.4mg / ml, and carbidopa at a concentration of 0.1mg / ml. Prepare three copies in parallel and inject one injection for each sample.

[0181] 200% limit sample spiked accuracy solution: Take an appropriate amount of the test sample powder in a 100mL volumetric flask, add 10.0mL of levodopa (EP) impurity A stock solution, 10.0mL of methyldopa, carbidopa impurity 52, and carbidopa impurity G mixed stock solution 1 to the volumetric flask, dilute to the scale with blank solution, filter with a 0.22um PVDF filter membrane and inject. Prepare a solution containing levodopa (EP) impurity A concentration of 0.8μg / mL, methyldopa 1.0μg / mL, carbidopa impurity 52 0.4μg / mL, carbidopa impurity G 2.0μg / mL, levodopa concentration of 0.4mg / ml, and carbidopa concentration of 0.1mg / ml, prepare 3 copies in parallel, and inject one injection for each sample.

[0182] The above solutions were sampled and analyzed respectively and the chromatograms were recorded. The recoveries of levodopa (EP) impurity A, methyldopa, carbidopa impurity 52 and carbidopa impurity G were calculated by the external standard method. The results are shown in Table 8.

[0183] Table 8

[0184]

[0185]

[0186] As can be seen from Table 8, the recoveries of levodopa (EP) impurity A, methyldopa, carbidopa impurity 52, and carbidopa impurity G at LOQ concentration levels were all between 70% and 130%, with RSDs no greater than 15.0%, and the recoveries at 100% and 200% concentration levels were all between 80% and 120%, with RSDs no greater than 10.0%, indicating that the accuracy of this method is good.

[0187] 7. Precision test

[0188] Repeatability sample solution: Prepare the solution in the same manner as the 100% limit sample spiked accuracy solution. Prepare six replicates for injection and analysis. The results are shown in Table 9.

[0189] Table 9

[0190]

[0191] As can be seen from Table 9, the test was repeated 6 times, and the maximum RSD of the content of each degradation impurity obtained was 1.9%, which was no more than 15.0%, indicating that the precision of this method was good.

[0192] 8. Stability test of spiked test and reference solutions

[0193] Spiked test solution: Prepare in the same manner as the 100% limit sample spiked accuracy solution.

[0194] Reference solution: Dissolve the levodopa and carbidopa reference substances in the blank solution to prepare a solution containing 4 μg / ml levodopa and 1 μg / ml carbidopa.

[0195] The reference solution and the spiked test solution were placed at 6°C for inspection. The stability results of the reference solution are shown in Table 10, and the stability results of the spiked test solution are shown in Table 11.

[0196] Table 10

[0197] Time - Hours 0 6 24 L-DOPA peak area 113058 113330 113637 L-DOPA recovery % / 100.2 100.5 Carbidopa Peak Area 25231 24887 24925 Carbidopa recovery % / 98.6 99.2

[0198] Table 11

[0199]

[0200] As can be seen from Tables 10 and 11, the reference solution was placed at 6°C for less than 24 hours, and the recoveries of levodopa and carbidopa were between 95.0% and 105.0%, indicating good stability within 24 hours at 6°C; the spiked test solution was placed for less than 75 minutes, and no new impurities greater than LOQ appeared except RRT=1.22. For impurities with a content below 0.2%, the impurity content changed within 0.05%, and for impurities above 0.2%, the impurity content changed within 0.10%. At zero point, the content of RRT=1.22 was 0.078%, close to LOQ (0.01%). At 75 minutes, the content of RRT=1.22 was 0.124%, and the impurity content changed within 0.05%, indicating good stability within 75 minutes at 6°C.

[0201] 9. Durability test

[0202] By making slight changes in the column temperature or using columns from different batches, the variation in results under different test conditions was investigated, as shown in Table 12.

[0203] Table 12

[0204]

[0205] It can be seen from Table 12 that by changing the test parameters, for impurities with a content below 0.2%, the change in impurity content is within ±0.05%; for impurities with a content above 0.2%, the change in impurity content is within ±0.1%, indicating that this method has good durability.

[0206] Example 2 Chromatographic column screening experiment

[0207] Test condition 1:

[0208] - Column 1: Agilent Proshell 120AQ C18 2.7um 2.1*100mm

[0209] -Mobile phase A: 6 g / L anhydrous sodium dihydrogen phosphate (pH = 2.2)

[0210] -Mobile phase B: ethanol

[0211] -Mobile phase gradient as shown in Table 13

[0212] Table 13

[0213] Time (min) Mobile phase A (%) Popular phase B (%) 0 96 4 25 96 4

[0214] -Mobile phase flow rate: 0.6 ml / min;

[0215] - Column temperature: 25°C;

[0216] - Autosampler temperature: 6°C;

[0217] - Detection wavelength: 280nm;

[0218] -Injection volume: 20 μl.

[0219] -The diluent is prepared by mixing 6g / L anhydrous sodium dihydrogen phosphate buffer (pH 2.2) with ethanol at a volume ratio (V / V) of 96:4

[0220] The preparation of the sensitivity solution and the resolution solution is the same as that of the system suitability test in Example 1.

[0221] - The sample was prepared into a test solution containing 0.4 mg / ml of levodopa and 0.1 mg / ml of carbidopa, filtered with a 0.22 μm PVDF filter membrane and then injected. The corresponding chromatogram is shown in FIG19 .

[0222] Test condition 2:

[0223] -Chromatographic column 2Agilent Proshell 120EC C18 2.7um 3.0*150mm

[0224] -Mobile phase A: 6 g / L anhydrous sodium dihydrogen phosphate (pH = 2.2)

[0225] -Mobile phase B: ethanol

[0226] -The mobile phase gradient is shown in Table 14.

[0227] Table 14

[0228] Time (min) Mobile phase A (%) Popular phase B (%) 0 96 4 25 96 4

[0229] -Mobile phase flow rate: 0.7 ml / min;

[0230] - Column temperature: 25°C;

[0231] - Autosampler temperature: 6°C;

[0232] - Detection wavelength: 280nm;

[0233] -Injection volume: 20 μl.

[0234] -The diluent is prepared by mixing 6g / L anhydrous sodium dihydrogen phosphate buffer (pH 2.2) with ethanol at a volume ratio (V / V) of 96:4

[0235] The preparation of the sensitivity solution and the resolution solution is the same as that of the system suitability test in Example 1.

[0236] - The sample was prepared into a test solution containing approximately 0.4 mg / ml of levodopa and 0.1 mg / ml of carbidopa, filtered through a 0.22 μm PVDF filter membrane and injected. The corresponding chromatogram is shown in Fig. 20 shown.

[0237] By comparison Fig.19 , Fig. 20 It can be seen that chromatographic column 2 can separate the two main peaks further, therefore, chromatographic column 2 is selected as the chromatographic column used in this application.

[0238] Example 3 Screening test of mobile phase gradient

[0239] -Chromatographic column: Agilent Proshell 120EC C18 2.7um 3.0*150mm

[0240] -Mobile phase A: 6 g / L anhydrous sodium dihydrogen phosphate (pH = 2.2)

[0241] -Mobile phase B: ethanol

[0242] - Gradient 1 is shown in Table 15.

[0243] Table 15

[0244] Time (min) Mobile phase A (%) Popular phase B (%) 0 97 3 2 97 3 3 96 4 12 96 4 13 92 8 16 92 8 16.1 97 3 22 97 3

[0245] - Gradient 2 is shown in Table 16.

[0246] Table 16

[0247] Time (min) Mobile phase A (%) Popular phase B (%) 0 97 3 2 97 3 3 96 4 12 96 4 13 92 8 19 92 8 19.1 97 3 25 97 3

[0248] -Mobile phase flow rate: 0.7 ml / min;

[0249] - Column temperature: 25°C;

[0250] - Autosampler temperature: 6°C;

[0251] - Detection wavelength: 280nm;

[0252] -Injection volume: 20 μl.

[0253] -The diluent is prepared by mixing 6g / L anhydrous sodium dihydrogen phosphate buffer (pH 2.2) with ethanol at a volume ratio (V / V) of 96:4

[0254] The preparation of the sensitivity solution and the resolution solution is the same as that of the system suitability test in Example 1.

[0255] - The sample was prepared into a test solution containing approximately 0.4 mg / ml of levodopa and 0.1 mg / ml of carbidopa, filtered through a 0.22 μm PVDF filter membrane and injected. The chromatogram corresponding to gradient 1 is shown in Fig.21 As shown, the chromatogram corresponding to gradient 2 is as follows Fig. 22 shown.

[0256] By comparison Fig.21 and Fig. 22 It can be seen that the retention time of carbidopa in gradient 2 (5.1 minutes) is longer than that of carbidopa in gradient 1 (4.5 minutes), which is more conducive to the separation of levodopa (EP) impurity C and carbidopa, and is also more conducive to the separation of impurities between levodopa and carbidopa. Therefore, gradient 2 is preferred.

[0257] Example 4 Determination of relevant impurities in the United States Pharmacopoeia Carbidopa sustained-release tablets

[0258] The USP chromatographic conditions are as follows:

[0259] - Chromatographic system: Waters ARC;

[0260] -Chromatographic column: chromatographic column with octadecylsilane bonded silica as filler (YMC-Pack ODS-A C184.6×150mm, 5μm);

[0261] -Mobile phase A: 6 g / L anhydrous sodium dihydrogen phosphate (pH = 2.2);

[0262] -Mobile phase B: ethanol

[0263] - Isocratic elution MPA (mobile phase A): MPB (mobile phase B) = 95:5 at least 6 times the retention time of carbidopa;

[0264] -Mobile phase flow rate: 1.0ml / min

[0265] - Column temperature: 25℃;

[0266] -Autosampler temperature: 2~8℃;

[0267] - Detection wavelength: 280nm;

[0268] -Injection volume: 20 μl.

[0269] -The diluent is prepared by mixing 6g / L anhydrous sodium dihydrogen phosphate buffer (pH 2.2) with ethanol at a volume ratio (V / V) of 95:5

[0270] - The sensitivity solution is prepared with a diluent to have a levodopa concentration of approximately 0.5 μg / ml and a carbidopa concentration of 0.125 μg / ml.

[0271] -The separation solution is prepared with a diluent to have a carbidopa concentration of approximately 0.125 mg / ml and a levodopa (EP) impurity C concentration of 1.0 ug / ml.

[0272] -The reference solution is prepared with a diluent to have a levodopa concentration of approximately 5 μg / ml and a carbidopa concentration of approximately 1.25 μg / ml.

[0273] - The sample solution is prepared with a diluent to a solution containing approximately 0.5 mg / ml of levodopa and 0.125 mg / ml of carbidopa, filtered with a 0.45 μm PVDF filter membrane and injected. The chromatogram of the sensitivity solution in the USP method is as follows Fig.23 As shown, the chromatogram of the sample solution is shown in Figure 24, and the results corresponding to the method in the United States Pharmacopoeia and the method of the present application (the corresponding test conditions are the same as the system suitability test in Example 1, the levodopa concentration in the sensitivity solution is 0.4 μg / ml, and the carbidopa concentration is 0.1 μg / ml) are compared as shown in Table 17.

[0274] Table 17

[0275]

[0276] It can be seen from Table 17 that the sensitivity solution concentration of the method of the present invention is lower than that of the pharmacopoeia, but the peak area is larger than that of the pharmacopoeia, indicating that the method has better response, higher sensitivity, shorter running time, better separation between levodopa (EP) impurity A and levodopa, and higher stability.

[0277] Example 5 Effect of different injection vials on the stability of levodopa (EP) impurity A and reference solution Test conditions:

[0278] -Mobile phase A: 6 g / L anhydrous sodium dihydrogen phosphate (pH = 2.2);

[0279] -Mobile phase B: ethanol

[0280] -The mobile phase gradient is shown in Table 18:

[0281] Table 18

[0282]

[0283]

[0284] -Mobile phase flow rate: 0.6 ml / min;

[0285] - Column temperature: 25°C;

[0286] - Autosampler temperature: 6°C;

[0287] - Detection wavelength: 280nm;

[0288] -Injection volume: 20 μl.

[0289] -Injection vials: Agilent certified clear vials, blue caps, PTFE / red silicone septa, 2mL, vial dimensions: 12 x 32 mm (12 mm cap).

[0290] -The diluent is prepared by mixing 6 g / L anhydrous sodium dihydrogen phosphate buffer (pH 2.2) and ethanol at a volume ratio (V / V) of 95:5.

[0291] The preparation of the sensitivity solution and the resolution solution is the same as that of the system suitability test in Example 1.

[0292] -Levodopa (EP) impurity A was prepared into a 0.4 μg / mL solution with diluent and injected into Agilent certified clear glass vials and other vials respectively.

[0293] -The reference solution was prepared with the diluent to contain a solution with a levodopa concentration of approximately 4 μg / ml and a carbidopa concentration of approximately 1 μg / ml, and the samples were injected and compared using a Thermo Fisher clear screw-mouth sample vial (model: CHSV9-10P), a Thermo Fisher amber screw-mouth sample vial (model: CHSV9-20P), a Waters clear glass screw-neck sample vial (model: certified, 12×32 mm, 2 mL), a Waters amber glass screw-neck sample vial (model: uncertified, 12×32 mm, 2 mL), and an Agilent clear sample vial (model: certified, 12×32 mm, 2 mL). The results are shown in Tables 19-24. Among them, Table 19 shows the stability results of the reference solution injected by Thermo Fisher clear threaded vials, Table 20 shows the stability results of the reference solution injected by Thermo Fisher amber threaded vials, Table 21 shows the stability results of the reference solution injected by Waters amber glass threaded neck vials; Table 22 shows the stability results of the reference solution injected by Waters clear glass threaded neck vials; Table 23 shows the stability results of the reference solution injected by Agilent clear vials; Table 24 compares the effects of different injection vials on the stability of levodopa (EP) impurity A. The chromatogram of 5 consecutive injections of levodopa (EP) impurity A solution injected by ordinary injection vials is shown in the figure. Fig.25 As shown in Figure 1, the chromatogram of 5 consecutive injections of L-DOPA (EP) impurity A solution injected using Agilent certified glass vials is shown in Figure 1. Fig.26 shown.

[0294] Table 19

[0295]

[0296]

[0297] Table 20

[0298]

[0299] Table 21

[0300]

[0301]

[0302] Table 22

[0303]

[0304] Table 23

[0305]

[0306]

[0307] Table 24

[0308]

[0309] Combined with Tables 19-24, it can be seen that using Agilent certified glass sample bottles can make the levodopa (EP) impurity A solution and the reference solution more stable.

[0310] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A method for separating and determining degradation impurities in cardiolipin sustained-release tablets, characterized in that: Ultra-high performance liquid chromatography was used for separation and determination. The chromatographic column of ultra-high performance liquid chromatography was filled with octadecylsilane bonded silica gel. Mobile phase A and mobile phase B were used for gradient elution. Mobile phase A was anhydrous sodium dihydrogen phosphate buffer, and mobile phase B was ethanol. The total volume of the mobile phase was 100%. The gradient elution procedure was: The degradation impurities were separated and determined by gradient elution within 25 min.

2. The method for separating and determining degradation impurities in cardiolipin sustained-release tablets according to claim 1, characterized in that: The chromatographic column is Agilent Proshell 120EC C18.

3. The method for separating and determining degradation impurities in cardiolipin sustained-release tablets according to claim 1, characterized in that: The concentration of the anhydrous sodium dihydrogen phosphate buffer is 2-8 g / L, and the pH is 2-3.

4. The method for separating and determining degradation impurities in cardiolipin sustained-release tablets according to claim 1, characterized in that: The column temperature of the chromatographic column is 25-35°C.

5. The method for separating and determining degradation impurities in cardiolipin sustained-release tablets according to claim 1, characterized in that: The flow rate of the mobile phase is 0.5-0.8 ml / min.

6. The method for separating and determining degradation impurities in cardiolipin sustained-release tablets according to claim 1, characterized in that: The injection temperature is 2℃-9℃, and the injection volume is 10μl~25μl.

7. The method for separating and determining degradation impurities in cardiolipin sustained-release tablets according to claim 1, characterized in that: The detection wavelength is 220nm-300nm.

8. The method for separating and determining degradation impurities in cardiolipin sustained-release tablets according to claim 1, characterized in that: Ultra-high performance liquid chromatography detection includes the following steps: S1. Prepare blank solution, sensitivity solution, resolution solution, reference solution and test solution; S2, injecting the blank solution, sensitivity solution, resolution solution, reference solution and test solution in step S1 into the chromatograph in sequence, and recording the chromatograms of the blank solution, sensitivity solution, resolution solution, reference solution and test solution in sequence; S3. Calculate the content of degradation impurities by external standard method according to the peak area of ​​the chromatogram.

9. The method for separating and determining degradation impurities in cardiolipin sustained-release tablets according to claim 1, characterized in that: The blank solution is prepared by mixing anhydrous sodium dihydrogen phosphate buffer and ethanol in a volume ratio of 96:4, the concentration of the anhydrous sodium dihydrogen phosphate buffer is 6 g / L, and the pH is 2.2; the sensitivity solution is prepared by using the blank solution to prepare a solution containing 0.4 μg / ml of levodopa and 0.1 μg / ml of carbidopa; the separation solution is prepared by using the blank solution to prepare a solution containing 0.1 mg / ml of carbidopa and 0.8 μg / ml of levodopa impurity C; the reference solution is prepared by using the blank solution to prepare a solution containing 4 μg / ml of levodopa and 1 μg / ml of carbidopa; the test solution is prepared by dissolving the carbidopa-levodopa sustained-release tablet to be tested in the blank solution, wherein the levodopa concentration is 0.4 mg / ml and the carbidopa concentration is 0.1 mg / ml.

10. The method for separating and determining degradation impurities in cardiolipin sustained-release tablets according to claim 1, characterized in that: Impurities from carbidopa degradation % = (Ru / Rs)*(Cs / Cu)*(1 / F)*100 Ru: Degradation impurity peak area Rs: Average peak area of ​​carbidopa reference substance Cs: Carbidopa reference concentration mg / mL Cu: Carbidopa concentration in the test solution mg / mL F: relative response factor, where methyldopa is 1.0, carbidopa impurity G is 1.2, and carbidopa impurity 52 is 6.1; Impurities from L-DOPA degradation % = (Ru / Rs)*(Cs / Cu)*(1 / F)*100 Ru: Degradation impurity peak area Rs: Average peak area of ​​levodopa reference substance Cs: L-dopa reference concentration mg / mL Cu: L-DOPA concentration in the test solution mg / mL F: Relative response factor, where L-DOPA impurity A is 0.8.

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