A method for separating the three components in DOPA CAR-T tablets and determining their content and dissolution rate

Abstract

This invention belongs to the field of chemical analysis technology, specifically relating to a method for separating three components in DOPA / benzisazol tablets and detecting their content and dissolution rate. The method uses a chromatographic column packed with octylsilane-bonded silica gel, employing a mobile phase of a mixed solution of sodium dihydrogen phosphate buffer, methanol, and sodium heptanesulfonate for isocratic elution, sequentially separating levodopa, benzyl hydrochloride, and benzyl hydrochloride impurity B. The separated components are then detected using a UV detector. Specifically, the detection wavelength for levodopa is 280 nm, and the detection wavelengths for benzyl hydrochloride and benzyl hydrochloride impurity B are 220 nm. This method can separate levodopa, benzyl hydrochloride, and benzyl hydrochloride impurity B within 15 minutes, and can achieve quantitative analysis and dissolution rate detection of levodopa and benzyl hydrochloride. This method is highly specific, has a short analysis time, good stability, good accuracy, high repeatability, and is simple and feasible to operate.

Description

Technical Field

[0001] This invention belongs to the field of chemical analysis technology, specifically relating to a method for separating three components in DOPA serotonin tablets and detecting their content and dissolution rate. Background Technology

[0002] The main components of DOPA and benzylhydrazine tablets are levodopa and benzylhydrazine hydrochloride, and the main degradation impurity is benzylhydrazine hydrochloride impurity B. The structural formula of levodopa is shown in Formula I, the structural formula of benzylhydrazine hydrochloride is shown in Formula II, and the structural formula of benzylhydrazine hydrochloride impurity B is shown in Formula III.

[0003]

[0004] Currently, there are no legally recognized or literature-based methods to simultaneously determine the content and dissolution rate of levodopa and benserazide hydrochloride in dopamine tablets. The relevant analytical methods for dopamine tablets are only included in Part II of ChP2020. However, the method for determining the content of levodopa and benserazide hydrochloride in dopamine tablets in ChP2020 takes a long time to analyze.

[0005] Patent CN111812227A discloses an analytical method for benserazide impurity A in a DOPA-carbidopa compound preparation. This method uses a hydrophilic chromatographic column, with an aqueous solution containing sodium heptanesulfonate and potassium dihydrogen phosphate at a pH of 1.7 to 9.0 as mobile phase A, and methanol as mobile phase B. A staged gradient elution method is used to separate and determine the content of benserazide impurity A in the DOPA-carbidopa compound preparation. However, this method can only detect the content of impurity A and cannot simultaneously separate and determine the content of levodopa, benserazide hydrochloride, and benserazide hydrochloride impurity B in DOPA-carbidopa tablets, nor can it simultaneously determine the dissolution rate of levodopa and benserazide hydrochloride. Summary of the Invention

[0006] In view of this, one of the objectives of the present invention is to provide a method for separating levodopa, benserazide hydrochloride, and benserazide hydrochloride impurity B in dopamine tablets using HPLC-UV method. This method has high specificity and short analysis time, providing support for the subsequent simultaneous determination of the content and dissolution of levodopa and benserazide hydrochloride.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for separating levodopa, benzylhydrazine hydrochloride, and benzylhydrazine hydrochloride impurity B using HPLC-UV, wherein the separated components include levodopa, benzylhydrazine hydrochloride, and benzylhydrazine hydrochloride impurity B; the structural formula of levodopa is shown in Formula I, the structural formula of benzylhydrazine hydrochloride is shown in Formula II, and the structural formula of benzylhydrazine hydrochloride impurity B is shown in Formula III; the method is as follows: using a chromatographic column packed with octylsilane-bonded silica gel, and using a mixed solution of sodium dihydrogen phosphate buffer, methanol, and sodium heptanesulfonate as the mobile phase for isocratic elution, sequentially separating levodopa, benzylhydrazine hydrochloride, and benzylhydrazine hydrochloride impurity B; the volume ratio of sodium dihydrogen phosphate buffer to methanol in the mobile phase is 72:28; the molar concentration ratio of sodium heptanesulfonate to sodium dihydrogen phosphate buffer in the mobile phase is 1:10.

[0009]

[0010] The specific preparation method of the sodium dihydrogen phosphate buffer in the mobile phase is as follows: Take 3.9g of sodium dihydrogen phosphate dihydrate and 0.505g of sodium heptanesulfonate, add 1000ml of water to dissolve, and adjust the pH value to 3.5 with phosphoric acid.

[0011] Based on the speed at which each component is separated, qualitative identification of levodopa, benserazide hydrochloride, and benserazide hydrochloride impurity B can be achieved.

[0012] Furthermore, the sample is a dopa-carbazine tablet, which contains levodopa, benzylhydrazine hydrochloride, and benzylhydrazine hydrochloride impurity B.

[0013] Furthermore, the HPLC-UV method was used to simultaneously separate levodopa, benzylhydrazine hydrochloride, and benzylhydrazine hydrochloride impurity B, with a separation time of 15 min.

[0014] Furthermore, the concentration of the sodium dihydrogen phosphate buffer solution is 0.025 mol / L, and the pH is 3.5.

[0015] Preferably, the chromatographic column is an Inertsil C8-3, 4.6 mm × 250 mm, 5 μm, or a column with equivalent performance.

[0016] Preferably, the flow rate of the mobile phase is 1.0 ml / min.

[0017] Preferably, the column temperature of the chromatographic column is 30°C.

[0018] Preferably, the injector is set at 5°C.

[0019] As a preferred option, the injection volume is 5 μl.

[0020] The second objective of this invention is to provide a method for simultaneously determining the content and dissolution rate of levodopa and benserazide hydrochloride in dopamine tablets. This method can determine the content and dissolution rate of levodopa and benserazide hydrochloride in dopamine tablets within 15 minutes or more.

[0021] To achieve the above objectives, the present invention adopts the following technical solution:

[0022] Based on the method described in Objective 1, this method simultaneously determines the content and dissolution rate of levodopa and benserazide hydrochloride in dopamine tablets. The method described in Objective 1 is used to separate levodopa, benserazide hydrochloride, and benserazide hydrochloride impurity B from dopamine tablets. After separation, the samples are detected using an ultraviolet detector. The detection wavelength is set to 280 nm for 0-4.3 min and 220 nm for 4.4-15 min.

[0023] Furthermore, the retention times of the three components, from shortest to longest, are as follows: L-DOPA, benzylhydrazine hydrochloride, and benzylhydrazine hydrochloride impurity B.

[0024] Furthermore, the volume ratio of sodium dihydrogen phosphate buffer to methanol in the mobile phase is 72:28, and the molar concentration ratio of sodium heptanesulfonate to sodium dihydrogen phosphate buffer is 1:10; the chromatographic column specifications are 4.6 mm × 250 mm × 5 μm; the mobile phase flow rate is 1.0 ml / min; the column temperature is 30 °C; the detection wavelength for levodopa is 280 nm, and the detection wavelength for benzylhydrazine hydrochloride and benzylhydrazine hydrochloride impurity B is 220 nm; isocratic elution and detection are performed under the above chromatographic conditions, and chromatograms are obtained.

[0025] Furthermore, a retention time of 3.3±0.5 min was identified as levodopa; a retention time of 5.0±0.5 min was identified as benzylhydrazine hydrochloride; and a retention time of 12.6±0.5 min was identified as benzylhydrazine hydrochloride impurity B.

[0026] This retention time allows for qualitative detection of the three components in DOPA tablets.

[0027] Furthermore, a content test solution and a content reference solution were prepared using a 0.1 mol / L phosphoric acid solution as the solvent; a dissolution test solution and a dissolution reference solution were prepared using a 0.1 mol / L hydrochloric acid solution as the solvent.

[0028] Further, standard samples were prepared, and the chromatograms of the standard samples were obtained; the content and dissolution rate of the chromatograms of the test samples were calculated based on the peak area using the external standard method.

[0029] The content of the components in the standard is known, while the content of the components in the test sample is unknown.

[0030] The chromatograms of the standard samples obtained from the tests can be stored in a database and used as chromatographic materials for quality control in smart pharmaceutical factories. This helps robots determine whether their products are qualified and provides quantitative improvement analysis suggestions for unqualified products.

[0031] In this invention, in addition to the two main drugs, benserazide hydrochloride and levodopa, "dopa-carbidopa tablets" also contain other excipients, including mannitol, microcrystalline cellulose, pregelatinized starch, dicalcium phosphate, ethyl cellulose, sodium docusate, magnesium stearate, red iron oxide, colloidal silica, and cross-linked polyvinylpyrrolidone.

[0032] In this invention, dissolution refers to the rate and extent to which a drug dissolves from a solid dosage form, such as a tablet, in a specified solvent. Dissolution is an important indicator for tablet quality control, and it should generally be tested for poorly soluble drugs.

[0033] The beneficial effects of this invention are as follows:

[0034] 1. This invention establishes an HPLC-UV method by screening different mobile phases and ratios, column temperatures, and other chromatographic conditions. This method can rapidly and simultaneously determine the content and dissolution rate of the main components levodopa and benserazide hydrochloride in dopa / carbidopa tablets, and ensures that the main degradation impurity, benserazide hydrochloride impurity B (limit 3%), does not interfere with the determination of the content and dissolution rate of the two main components.

[0035] 2. The HPLC-UV method provided by this invention uses octylsilane-bonded silica gel as the packing material. Utilizing the polarity of each substance and the strength of hydrogen bonding, a mobile phase system of sodium dihydrogen phosphate buffer salt-methanol-sodium heptanesulfonate ion pairs was selected. The addition of sodium heptanesulfonate significantly improves the separation of the main component and major degradation impurities, enabling the effective separation of levodopa, benserazide hydrochloride, and benserazide hydrochloride impurity B within 15 minutes, and allowing for quantitative analysis of levodopa and benserazide hydrochloride. This method exhibits high specificity, short analysis time, good stability, good accuracy, high repeatability, and simple and feasible operation. Attached Figure Description

[0036] Figure 1 Chromatogram of specificity-content blank solvent;

[0037] Figure 2 The chromatogram of the specific excipient mixture (blank excipient);

[0038] Figure 3 Chromatogram of the specificity / accuracy-content recovery test solution;

[0039] Figure 4 Chromatogram of a specificity-dissolution blank solvent;

[0040] Figure 5 Chromatogram of dissolution specificity of test solution / dissolution rate - room temperature stability of test solution - 0 h;

[0041] Figure 6 The chromatogram of impurity B- in benzylhydrazine hydrochloride under normal conditions;

[0042] Figure 7 This is a chromatogram of a mixture of excipients with dissolution specificity. Detailed Implementation

[0043] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0044] In this embodiment of the invention, the preparation method of the content test solution is as follows: Take an appropriate amount of this product (dopa and benzylhydrazine tablets), accurately weigh it, add an appropriate amount of 0.1 mol / L phosphoric acid solution, sonicate for 5 min to dissolve, cool, and dilute to prepare a solution containing approximately 0.8 mg of levodopa and 0.228 mg of benzylhydrazine hydrochloride per 1 ml, shake well, filter, and use as the content test solution.

[0045] In this embodiment of the invention, the preparation method of the content reference solution is as follows: take appropriate amounts of levodopa and benserazide hydrochloride reference standards, weigh them accurately, add 0.1 mol / L phosphoric acid solution, sonicate to dissolve and quantitatively dilute to prepare a solution containing approximately 0.8 mg of levodopa and 0.228 mg of benserazide hydrochloride per 1 ml, which is used as the content reference solution.

[0046] In this embodiment of the invention, the method for preparing the dissolution test solution is as follows: Take this product (dopacaridine tablets), use 0.1 mol / L hydrochloric acid solution as the dissolution medium, rotate at 50 rpm, operate according to the method, take an appropriate amount of the dissolution solution after 30 minutes, filter it, and take the filtrate as the dissolution test solution.

[0047] In this embodiment of the invention, the dissolution reference solution is prepared as follows: appropriate amounts of levodopa and benserazide hydrochloride reference standards are accurately weighed, dissolved in a dissolution medium, and quantitatively diluted to prepare a solution containing approximately 0.22 mg of levodopa and 63 μg of benserazide hydrochloride per 1 ml, which is used as the dissolution reference solution.

[0048] In this embodiment of the invention, the excipient mixture solution (blank excipient) refers to the components in the formulation of this product other than the active pharmaceutical ingredient (benzirsine hydrochloride and levodopa), and includes 10 excipients such as mannitol, microcrystalline cellulose, pregelatinized starch, and dicalcium phosphate.

[0049] In this embodiment of the invention, the chromatographic conditions are as follows:

[0050] The experiment was conducted according to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512). Octylsilane-bonded silica gel was used as the stationary phase (Inertsil C8-3, 4.6 mm × 250 mm, 5 μm, or a column with equivalent performance). The mobile phase was 0.025 mol / L sodium dihydrogen phosphate buffer (pH 3.5) (containing 2.5 mmol / L heptanesulfonate sodium)-methanol (72:28). The flow rate was 1.0 mL / min. The column temperature was 30 °C. The injector temperature was 5 °C. The detection wavelength for levodopa was 280 nm. After the levodopa peak had completely eluted, the detection wavelength was changed to 220 nm. The injection volume was 5 μL.

[0051] Example 1. Comparison of self-developed method with ChP2020 dopamine and phosphatidylcholine tablet method

[0052] The content determination method for levodopa and benserazide hydrochloride in Part II of ChP2020 and the self-developed HPLC-UV method of this invention were used to locate levodopa and benserazide hydrochloride. Levodopa and benserazide hydrochloride eluted sequentially. In the self-developed method of this invention, the retention times of levodopa and benserazide hydrochloride were 3.324 min and 5.049 min, respectively, while the retention times of levodopa and benserazide hydrochloride in the ChP2020 method were 7.363 min and 30.410 min, respectively. The self-developed method of this invention significantly reduces the analysis time compared to the ChP2020 method.

[0053] In addition, the method developed in this invention is also applicable to the determination of dissolution in dopaminergic tablets, and can simultaneously determine the content and dissolution of multiple components in dopaminergic tablets.

[0054] Example 2. Specificity

[0055] The tablets contain excipients such as mannitol, microcrystalline cellulose, and pregelatinized starch, as well as levodopa and benzylhydrazine hydrochloride and their main degradation impurities. Specificity was investigated in this embodiment.

[0056] Take 5 μl each of blank solvent (content blank solvent: 0.1 mol / L phosphoric acid solution; dissolution blank solvent: 0.1 mol / L hydrochloric acid solution), excipient mixture solution, test solution (containing levodopa and benserazide hydrochloride), and benserazide hydrochloride impurity B, and inject them according to the method. Record the chromatograms. The results are shown in Table 1 and [Table data missing]. Figure 1-7 As shown.

[0057] Table 1 Results of specificity determination

[0058]

[0059] Table 2 Figure 6 Integral Results Table

[0060]

[0061] Conclusion: Blank solvent, excipients, and benserazide hydrochloride impurity B do not interfere with the determination of levodopa and benserazide hydrochloride under this chromatographic system. The resolution of the two main component peaks is 8.80, indicating that this method has good specificity for the determination of levodopa and benserazide hydrochloride.

[0062] Example 3. Accuracy

[0063] Preparation of the content recovery test solution: Take about 0.275 g of this product, accurately add about 100 mg of levodopa reference standard and about 28.5 mg of benserazide hydrochloride reference standard, place in a 250 ml volumetric flask, add an appropriate amount of solvent, sonicate for 5 minutes to dissolve levodopa and benserazide hydrochloride, cool, dilute to the mark with solvent, shake well, filter, and take the filtrate to obtain a test solution with 100% accuracy equivalent to the test concentration. Prepare three parallel aliquots.

[0064] Preparation of dissolution recovery test solution: Accurately weigh approximately 0.15 g of intermediate product granules, add an appropriate amount of 0.1 mol / L hydrochloric acid solution, sonicate for 5 minutes to dissolve levodopa and benserazide hydrochloride, cool, dilute to the mark with 0.1 mol / L hydrochloric acid, shake well, filter, and collect the filtrate to obtain a test solution with an accuracy equivalent to 100% final dissolution concentration. Prepare three parallel aliquots.

[0065] Accurately measure 5 μl of each of the above test solutions and inject them into the liquid chromatograph. Record the chromatograms. Calculate the amounts of levodopa and benserazide hydrochloride based on peak area using the external standard method. Calculate the recovery rate and the corresponding RSD. The experimental results are shown in Tables 3-4 and 4. Figure 3 As shown, Figure 3 The corresponding integration results are shown in Table 5.

[0066] Table 3. Accuracy test results (content)

[0067]

[0068] Table 4. Accuracy test results (dissolution rate)

[0069]

[0070] Table 5 Figure 3 Integral Results Table

[0071]

[0072] Conclusion: The recoveries of levodopa and benserazide hydrochloride ranged from 98.0% to 101.0%, with average recoveries of 100.0% and 100.4% and 99.8% and 99.8%, respectively, and RSDs of 0.1% and 0.6% and 0.9% and 0.9%, respectively, meeting the requirements. This test method has good accuracy and meets the requirements for content and dissolution determination by high-performance liquid chromatography.

[0073] Example 4. Repeatability

[0074] The results of Example 3 were evaluated, and three samples were tested in parallel. The recoveries of levodopa and benserazide hydrochloride were between 98.0% and 101.0%, with average recoveries of 100.0% and 99.8%, respectively, and RSDs of 0.1% and 0.9%, respectively. The test method showed good repeatability.

[0075] Example 5. Solution stability

[0076] Take the test solution and control solution separately, place them at 5℃, and take samples at different investigation times. Accurately measure 5 μl of each sample and inject them into the chromatograph. Calculate the RSD of the peak areas of levodopa and benserazide hydrochloride at each time point and the deviation from the peak areas of levodopa and benserazide hydrochloride at 0 hours. The experimental results are shown in Tables 6-7 and 8. Figure 5 , Figure 5 The corresponding integration results are shown in Table 8.

[0077] Table 6. Results of Stability Test (Content)

[0078]

[0079] Table 7. Results of stability test (dissolution rate)

[0080]

[0081] Table 8 Figure 5 Integral Results Table

[0082]

[0083]

[0084] Conclusion: The standard solution and test solution were stable at 5℃ for 20 and 17 hours, respectively, with peak area RSDs of 0.4% and 0.5%, and 0.1% and 0.3%, respectively. The dissolution standard solution and test solution were stable at 5℃ for 24 hours, with peak area RSDs of 0.4% and 0.8%, and 0.3% and 0.7%, respectively. The deviations of the main peak areas of the stability of the standard solution and test solution from the 0-hour peak area were all within ±2.0%, indicating that the standard solution and test solution had good stability at 5℃.

Claims

1. A method for separating levodopa, benzylhydrazine hydrochloride, and benzylhydrazine hydrochloride impurity B using HPLC-UV, characterized in that, The separated components include levodopa, benserazide hydrochloride, and benserazide hydrochloride impurity B; the structural formula of levodopa is shown in Formula I, the structural formula of benserazide hydrochloride is shown in Formula II, and the structural formula of benserazide hydrochloride impurity B is shown in Formula III; the method is as follows: using a chromatographic column packed with octylsilane-bonded silica gel, isocratic elution is performed with a mobile phase of a mixed solution of sodium dihydrogen phosphate buffer, methanol, and sodium heptanesulfonate, to sequentially separate levodopa, benserazide hydrochloride, and benserazide hydrochloride impurity B; the volume ratio of sodium dihydrogen phosphate buffer to methanol in the mobile phase is 72:28; the molar concentration ratio of sodium heptanesulfonate to sodium dihydrogen phosphate buffer in the mobile phase is 1:10; 。 2. The method according to claim 1, characterized in that, The chromatographic column has dimensions of 4.6 mm × 250 mm × 5 μm; the mobile phase flow rate is 1.0 ml / min; and the column temperature is 30 °C.

3. The method according to claim 1, characterized in that, The separation time is 15 minutes.

4. The method according to claim 1, characterized in that, The sodium dihydrogen phosphate buffer solution has a concentration of 0.025 mol / L and a pH of 3.

5.

5. A method for simultaneously determining the content and dissolution rate of levodopa, benserazide hydrochloride, and impurity B of benserazide hydrochloride in DOPA tablets based on the method according to any one of claims 1-4, characterized in that, After separation, the samples were detected using an ultraviolet detector.

6. The method according to claim 5, characterized in that, The retention times of the three components, from shortest to longest, are: levodopa, benserazide hydrochloride, and impurity B of benserazide hydrochloride.

7. The method according to claim 5, characterized in that, The detection wavelength for the levodopa is 280 nm, and the detection wavelength for the benzylhydrazine hydrochloride and the benzylhydrazine hydrochloride impurity B is 220 nm.

8. The method according to claim 6, characterized in that, The retention time was 3.3 ± 0.5 min, which was identified as levodopa; the retention time was 5.0 ± 0.5 min, which was identified as benzylhydrazine hydrochloride; and the retention time was 12.6 ± 0.5 min, which was identified as impurity B of benzylhydrazine hydrochloride.

9. The method according to claim 7, characterized in that, Prepare the content test solution and content reference solution using 0.1 mol / L phosphoric acid solution as solvent; prepare the dissolution test solution and dissolution reference solution using 0.1 mol / L hydrochloric acid solution as solvent.

10. The method according to claim 7, characterized in that, Prepare standards and obtain chromatograms of standard samples; calculate the content and dissolution rate of test samples based on peak area using the external standard method.

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