Method for detecting impurities in trazodone hydrochloride

Abstract

The invention relates to the technical field of medicine detection, in particular to a method for detecting impurities of trazodone hydrochloride. The method for detecting impurities in trazodone hydrochloride provided by the invention comprises the following steps: detecting impurities in a trazodone hydrochloride solution to be detected by adopting high performance liquid chromatography, wherein a solvent in the trazodone hydrochloride solution to be detected is a methanol water solvent; chromatographic conditions of the high performance liquid chromatography comprise that alkylsilane bonded silica gel is used as a filler of a chromatographic column, a monopotassium phosphate aqueous solution is used as a mobile phase A, acetonitrile is used as a mobile phase B, and gradient elution is carried out. The detection method is high in detection efficiency, the service life of the chromatographic column is long, the detection method has remarkable advantages in specificity, quantitation limit, detection limit, linear range, accuracy, precision and durability of trazodone hydrochloride impurity detection, and a very good application prospect is provided for detection of related impurities of trazodone hydrochloride raw material medicines and preparations.

Description

Technical Field

[0001] The present application belongs to the technical field of drug detection, and in particular, relates to a method for detecting impurities of trazodone hydrochloride. Background Art

[0002] Trazodone hydrochloride is a triazole pyridine derivative, and its specific chemical name is 2-[3-[4-(3-chlorophenyl)-1-piperazinyl]propyl]-1,2,4-triazolo[4,3-a]pyridine-3(2H)-hydrochloric acid.

[0003] Trazodone hydrochloride is a white, odorless powder that is easily soluble in water and is a specific 5-hydroxytryptamine reuptake inhibitor. It can induce postural hypotension due to its α1 adrenergic antagonism and antihistamine effects. The drug is not a monoamine oxidase inhibitor and, unlike amphetamines, has no excitatory effect on the central nervous system. It is suitable for the treatment of depression and anxiety disorders associated with depressive symptoms, as well as mood disorders in drug addicts after withdrawal.

[0004] In order to better control the quality of trazodone hydrochloride, it is necessary to control the related substances of trazodone hydrochloride. However, the detection method of impurities in trazodone hydrochloride needs to be improved. Summary of the invention

[0005] The purpose of the present application is to provide a method for detecting impurities in trazodone hydrochloride, aiming to solve the technical problem of how to better detect impurities in trazodone hydrochloride.

[0006] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0007] The present application provides a method for detecting impurities in trazodone hydrochloride, comprising:

[0008] Using high performance liquid chromatography to detect impurities in the trazodone hydrochloride solution to be tested, wherein the solvent in the trazodone hydrochloride solution to be tested is a methanol-water solvent;

[0009] The chromatographic conditions of the high performance liquid chromatography include: using alkylsilane bonded silica gel as the filler of the chromatographic column, using potassium dihydrogen phosphate aqueous solution as mobile phase A, and acetonitrile as mobile phase B, and performing gradient elution.

[0010] The detection method of impurities of trazodone hydrochloride provided by the present application adopts high performance liquid chromatography (HPLC) system to detect impurities in trazodone hydrochloride; Based on the detection process, the trazodone hydrochloride solution to be tested uses methanol water solvent, and the chromatographic conditions use alkylsilane bonded silica gel as filler, potassium dihydrogen phosphate aqueous solution (i.e. mobile phase A) and acetonitrile (i.e. mobile phase B) for gradient elution. Such a detection system not only has high detection efficiency, but also has a long service life of the chromatographic column, and shows significant advantages in the specificity, quantitative limit, detection limit, linear range, accuracy, precision and durability of impurity detection, with strong specificity, high sensitivity, good linearity, high accuracy, good precision and good durability, which provides a good practical application prospect for the raw material drug of trazodone hydrochloride and the relevant impurity detection of the preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0012] Figure 1 is a blank solvent liquid chromatogram of the detection method provided in Example 2 of the present application;

[0013] Figure 2 This is a liquid chromatogram of a blank auxiliary material of the detection method provided in Example 2 of the present application;

[0014] Figure 3 It is a liquid chromatogram of the system applicability solution of the detection method provided in Example 2 of the present application. DETAILED DESCRIPTION

[0015] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0016] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0017] In the present application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0018] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0019] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0020] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

[0021] Trazodone hydrochloride related substances mainly refer to starting materials, intermediates, polymers, side reaction products introduced in the production process of trazodone hydrochloride active pharmaceutical ingredient (API) or its preparations, as well as degradation products during storage. In order to better control the quality of trazodone hydrochloride, it is necessary to control the related substances of trazodone hydrochloride. The currently disclosed detection methods have limited impurity types detected under the same chromatographic conditions and low efficiency.

[0022] Based on this, the present application embodiment combines and optimizes the solvent in the trazodone hydrochloride solution to be tested, the chromatographic column of the chromatographic conditions and the eluent, and the results show that more trazodone hydrochloride related substances can be detected, and it has the advantages of high detection efficiency, strong specificity, high sensitivity, good linearity, high accuracy, good precision and good durability. The specific technical scheme is as follows.

[0023] The present application embodiment provides a method for detecting impurities in trazodone hydrochloride, comprising:

[0024] High performance liquid chromatography is used to detect impurities in the trazodone hydrochloride solution to be tested; wherein the solvent in the trazodone hydrochloride solution to be tested is a methanol-water solvent; the chromatographic conditions of the high performance liquid chromatography include: using alkylsilane bonded silica gel as the filler of the chromatographic column, using potassium dihydrogen phosphate aqueous solution as mobile phase A, and acetonitrile as mobile phase B, and performing gradient elution.

[0025] In the embodiment of the present application, methanol water solvent is used based on the trazodone hydrochloride solution to be tested, and alkylsilane bonded silica gel is used as filler in the chromatographic conditions, and potassium dihydrogen phosphate aqueous solution (i.e. mobile phase A) and acetonitrile (i.e. mobile phase B) are used for gradient elution. Such a detection system not only has high detection efficiency, but also has a long service life of the chromatographic column, and shows significant advantages in specificity, quantitative limit, detection limit, linear range, accuracy, precision and durability for impurity detection, has the advantages of strong specificity, high sensitivity, good linearity, high accuracy, good precision and good durability, and provides a good practical application prospect for the detection of related substances of the raw materials of trazodone hydrochloride and preparations.

[0026] In some embodiments, the trazodone hydrochloride solution to be tested is a solution in which trazodone hydrochloride is dissolved, and impurities therein need to be detected. Specifically, the trazodone hydrochloride solution to be tested in the embodiments of the present application can be a test solution containing trazodone hydrochloride, a system suitability solution, and various control solutions. For example, it can be a trazodone hydrochloride test solution, and the trazodone hydrochloride test solution is a sample solution for direct analysis and determination.

[0027] In some embodiments, the trazodone hydrochloride solution to be tested may be a trazodone hydrochloride bulk drug solution or a pharmaceutical preparation solution containing trazodone hydrochloride. For example, it may be a test solution in which a trazodone hydrochloride bulk drug is dissolved, or a test solution in which a trazodone hydrochloride pharmaceutical preparation is dissolved. Among them, the trazodone hydrochloride pharmaceutical preparation involved may be a trazodone hydrochloride tablet or a trazodone hydrochloride sustained-release tablet.

[0028] In some embodiments, the solvent in the trazodone hydrochloride solution to be tested is a methanol-water solvent, and the volume ratio of methanol to water is (40:60) to (60:40). Exemplarily, the volume ratio of methanol to water in the solvent is 40:60, 45:55, 50:50, 55:45, 60:40. For example, the solvent of the embodiment of the present application is a solvent in which methanol and water are combined in a volume ratio of 50:50.

[0029] In some embodiments, the detected impurities include: impurity L, impurity N, impurity C, impurity H, impurity I, impurity K and impurity O. Specifically, see Table 1 for trazodone hydrochloride and impurity types.

[0030] Table 1 Trazodone hydrochloride and impurity types

[0031]

[0032] In some embodiments, in the chromatographic conditions of HPLC detection, based on the total volume of mobile phase A and mobile phase B as 100%, the gradient elution procedure is:

[0033] 0 min, 83-87% mobile phase A and 13-17% mobile phase B;

[0034] 8 min, 50% mobile phase A and 50% mobile phase B;

[0035] 15 min, 40% mobile phase A and 60% mobile phase B;

[0036] 18 min, 20% mobile phase A and 80% mobile phase B;

[0037] 29 min, 15% mobile phase A and 85% mobile phase B;

[0038] After 29 min, 85% mobile phase A and 15% mobile phase B;

[0039] Exemplarily, the mobile phase ratio at the start time (0 min) is: 85% mobile phase A and 15% mobile phase B; after 29 min, for example, from 29.1 to 35 min, 85% mobile phase A and 15% mobile phase B are maintained.

[0040] Performing segmented linear gradient elution in the above-mentioned time periods, i.e. segmented gradient elution, can achieve good elution and separation.

[0041] In some embodiments, in gradient elution, mobile phase A is a salt phase, i.e., potassium dihydrogen phosphate aqueous solution; mobile phase B is an organic phase, i.e., acetonitrile. Among them, the concentration of potassium dihydrogen phosphate in the potassium dihydrogen phosphate aqueous solution is 2.44-2.99 g / L. The pH of the potassium dihydrogen phosphate aqueous solution is 6.2-6.6. Specifically, 2.44-2.99 g of potassium dihydrogen phosphate can be taken, 1000 ml of water can be added, the pH can be adjusted to 6.2-6.6 with triethylamine, and the mobile phase A can be obtained by mixing. Such a mobile phase A can better protect the chromatographic column and further improve the service life of the chromatographic column.

[0042] In the embodiment of the present application, based on the chemical structure and physicochemical properties of trazodone hydrochloride and impurities, and taking into account the better protection of the chromatographic column and the chromatographic system, methanol water solvent is selected as the solvent of the trazodone hydrochloride solution to be tested, and potassium dihydrogen phosphate aqueous solution with a concentration of 2.44 to 2.99 g / L and a pH of 6.2 to 6.6 is used as mobile phase A, and acetonitrile is used as mobile phase B, and gradient elution is performed. This can shorten the time for impurity detection, increase efficiency, and have a better separation effect. The low concentration of mobile phase A can well protect the chromatographic column and extend its service life.

[0043] In some embodiments, the chromatographic conditions of the HPLC detection are as follows: the injection flow rate is 0.7-0.9 ml / min, the column temperature is 30-40° C., and the detection wavelength is 252-256 nm.

[0044] For example, the concentration of potassium dihydrogen phosphate aqueous solution is 2.72 g / L, the pH of potassium dihydrogen phosphate aqueous solution is 6.4, the injection flow rate is 0.78 ml / min, the column temperature is 35°C, and the detection wavelength is 254 nm. The injection volume can be 10 μl.

[0045] In some embodiments, in the chromatographic conditions of HPLC detection, the alkylsilane bonded silica gel in the chromatographic column is octadecylsilane bonded silica gel, which can specifically include: YMC-Pack Pro C18, size 4.6×150mm (i.e., inner diameter 4.6mm, length 150mm), and the particle size of the filler is 3-5μm.

[0046] In some embodiments, the chromatographic conditions also use a ghost peak capture column. The ghost peak capture column can effectively adsorb and remove other impurities with weaker polarity in the system, thereby preventing other impurity peaks in the system from interfering with the target peak. It is installed between the gradient mixer and the injector. For example, the ghost peak capture column used in the embodiment of the present application can be a Welch GhostBuster Column with a size of 4.6×50 mm (i.e., an inner diameter of 4.6 mm and a length of 50 mm).

[0047] In some embodiments, the embodiments of the present application provide a detection method for detecting impurities of trazodone hydrochloride based on an HPLC system. The HPLC method is used, and the solvent is methanol-water (volume ratio 50:50). Chromatographic conditions: octadecylsilane bonded silica gel (YMC-Pack Pro C18, 4.6×150mm, 5μm or a chromatographic column with equivalent performance) is used as a filler, and the recommended ghost peak capture column is Welch Ghost Buster Column 4.6×50mm; potassium dihydrogen phosphate aqueous solution (weigh 2.72g of potassium dihydrogen phosphate, add 1000ml of water, adjust the pH to 6.4 with triethylamine, and mix well) is used as mobile phase A, and acetonitrile is used as mobile phase B, and gradient elution is performed according to Table 2 of the instruction manual; the flow rate is 0.8ml per minute, the column temperature is 35°C, the detection wavelength is 254nm, and the injection volume is 10μl.

[0048] This embodiment uses HPLC to detect impurities in trazodone hydrochloride. When the above detection system is used for detection, the detection time is shorter (about 1 / 3 of the USP pharmacopoeia), which can improve the detection efficiency; at the same time, the chromatographic column can be protected and the service life of the chromatographic column can be improved. It shows significant advantages in specificity, quantitative limit, detection limit, linear range, accuracy, precision, and durability, and has the advantages of strong specificity, high sensitivity, good linearity, high accuracy, good precision, and good durability, providing a detection idea for the related substances of trazodone hydrochloride.

[0049] In some embodiments, the detection method of the present application embodiment was used to conduct a specificity test, and the results showed that: (1) the solvent and the blank auxiliary material did not interfere with the determination of each component; (2) the separation between the main peak and the adjacent peak was at least 2.7, not less than 2.0, and the separation between each known impurity peak and the adjacent chromatographic peak was at least 8.3, not less than 1.5. This shows that the detection method of the present application embodiment has good specificity.

[0050] In some embodiments, the detection method of the embodiment of the present application is used to perform a membrane adsorption test, and a nylon filter (brand: Jinteng and Titan, diameter: 25 mm, pore size: 0.45 μm) is used to filter the spiked test solution. When 5 ml of the volume is discarded, the maximum difference between the percentage of each impurity and the percentage of the centrifuged impurity is 0.008%, which is less than 0.03%, and the maximum difference between the total impurity content and the centrifuged content is 0.026%, which is less than 0.1%, indicating that the membrane adsorption can be ignored. Therefore, the treatment method can be to use a nylon filter (brand: Jinteng and Titan, diameter: 25 mm, pore size: 0.45 μm) to discard 5 ml of the initial filtrate and take the subsequent filtrate for determination of related substances.

[0051] In some embodiments, the detection method of the embodiments of the present application was used to carry out quantitative limit and detection limit tests, and the results showed that impurity K, impurity L, impurity A, impurity I, impurity C, trazodone hydrochloride, impurity N, impurity H and impurity O all met the requirements, indicating that the detection method of the embodiments of the present application has high sensitivity.

[0052] In some embodiments, a linearity test was performed using the detection method of the embodiments of the present application, and the results showed that within the range of the quantitative limit to 200% of the limit concentration, impurity K, impurity L, impurity A, impurity I, impurity C, trazodone hydrochloride, impurity N, impurity H and impurity O all met the requirements, indicating that the detection method of the embodiments of the present application had good linearity.

[0053] In some embodiments, a correction factor test was performed using the detection method of the embodiments of the present application, and the results showed that the correction factors of impurities A, L, and N were in the range of 0.9 to 1.1, and when the related substances were measured, the correction factors were calculated as 1.0; impurity K, impurity I, impurity C, impurity H, and impurity O were calculated according to correction factors of 1.4, 0.9, 0.8, 0.7, and 0.5, respectively.

[0054] In some embodiments, the detection method of the present application is used to perform an accuracy test, and the results show that: (1) the recovery rate of each component is between 80% and 120% within the range of 50% to 150% of the limit concentration; (2) the RSD of the recovery rate should be no greater than 10%, indicating that the detection method of the present application has good accuracy.

[0055] In some embodiments, the detection method of the embodiments of the present application was used to carry out precision tests, and the results showed that the injection precision, repeatability and intermediate precision of the detection method of the embodiments of the present application all met the requirements.

[0056] In some embodiments, the detection method of the embodiment of the present application is used to conduct a solution stability test, and the results show that: (1) the spiked test solution is placed at room temperature for 70 hours, and no new impurities greater than 0.05% are detected. The maximum differences between the contents of impurities K, L, A, I, C, N, H, and O at each time point and those at 0 h are 0.002%, 0.004%, 0.007%, 0.004%, 0.002%, 0.004%, 0.004% and 0.003%, respectively, all less than 0.05%. The maximum difference between the total impurity content and that at 0 h is 0.018%, less than 0.1%, indicating that the spiked test solution is stable within 70 hours at room temperature; (2) the control solution is placed at room temperature for 70 hours, and the ratios of the peak areas at each time point to the peak areas at 0 h are between 98.4% and 100.4%, all within the range of 90% to 110%, indicating that the control solution is stable within 70 hours at room temperature.

[0057] In some embodiments, the detection method of the embodiment of the present application is used for durability test. By appropriately adjusting the detection flow rate, mobile phase ratio, column temperature, pH value of the salt phase in the mobile phase, salt concentration, chromatographic column and wavelength in the chromatographic conditions, in each system applicability solution, the minimum separation between the main peak and the adjacent peak is 2.3, which is greater than 2.0; the minimum separation between each impurity is 6.4, which is greater than 1.5; the maximum differences between the contents of impurity K, impurity L, impurity A, impurity I, impurity C, impurity N, impurity H, and impurity O measured under standard conditions are 0.021%, 0.040%, 0.016%, 0.020%, 0.004%, 0.025%, 0.013% and 0.035%, respectively, all less than 0.05%; unknown impurities are not detected; the maximum difference between the total impurities and the content measured under standard conditions is 0.038%, which is less than 0.1%; indicating that the detection method of the embodiment of the present application has good durability.

[0058] The room temperature in the examples generally refers to 20 to 30°C.

[0059] Example 1 Chromatographic conditions and chromatographic system of the detection method

[0060] Solvent: methanol-water solvent (wherein the volume ratio of methanol to water is 50:50).

[0061] Preparation of test solution: Take 5 samples of Trazodone Hydrochloride Sustained Release Tablets [produced by AZ.CHIM.RIUN.ANGELINIFRANCESCO ACRAF SPA], place them in a 100ml volumetric flask (Trazodone Hydrochloride Sustained Release Tablets 75mg specification) or a 200ml volumetric flask (Trazodone Hydrochloride Sustained Release Tablets 150mg specification), add 80% of the volume of the volumetric flask with methanol, ultrasonicate for 15 minutes to dissolve the Trazodone Hydrochloride, dilute to the mark with methanol, shake well, and filter. Accurately measure 5ml of the filtrate, place it in a 25ml volumetric flask, dilute to the mark with solvent, and shake well.

[0062] Preparation of control solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with solvent to make a solution containing 1.5 μg of trazodone hydrochloride per 1 ml.

[0063] Preparation of impurity mixed stock solution: Take appropriate amount of impurity L, impurity A, impurity N, impurity C, impurity H, impurity I, impurity K and impurity O reference substances, accurately weigh them, dissolve and dilute them with methanol to make a solution containing approximately 22.5μg impurity A, 15μg impurity L, 15μg impurity N, 15μg impurity C, 15μg impurity H, 15μg impurity I, 15μg impurity K and 15μg impurity O per 1ml.

[0064] System suitability solution: Take an appropriate amount of trazodone hydrochloride reference substance, accurately weigh it, add an appropriate amount of impurity mixed stock solution, dissolve and dilute it with solvent to make a solution containing 0.75 mg trazodone hydrochloride and 2.25 μg impurity A, 1.5 μg impurity L, 1.5 μg impurity N, 1.5 μg impurity C, 1.5 μg impurity H, 1.5 μg impurity I, 1.5 μg impurity K, and 1.5 μg impurity O per 1 ml.

[0065] Chromatographic conditions: Octadecylsilane bonded silica gel (YMC-Pack Pro C18, 4.6×150mm, 5μm or a chromatographic column with equivalent performance) is used as the filler, and the recommended ghost peak trapping column is Welch Ghost Buster Column 4.6×50mm; phosphate buffered saline (weigh 2.72g of potassium dihydrogen phosphate, add 1000ml of water, adjust the pH to 6.4 with triethylamine, and mix well) is used as mobile phase A, acetonitrile is used as mobile phase B, and gradient elution is performed according to Table 2 below; flow rate is 0.8ml per minute, column temperature is 35℃, detection wavelength is 254nm, and injection volume is 10μl.

[0066] Table 2 Gradient elution

[0067] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 85 15 8 50 50 15 40 60 18 20 80 29 15 85 29.1 85 15 35 85 15

[0068] Example 2 Specificity test of detection method

[0069] Solvent: methanol-water solvent (wherein the volume ratio of methanol to water is 50:50, i.e. 50% acetonitrile-water solvent); used as blank solvent.

[0070] Impurity stock solution: Take appropriate amount of Impurity L, Impurity A, Impurity N, Impurity C, Impurity H, Impurity I, Impurity K and Impurity O reference substances, accurately weigh them, dissolve them in methanol and quantitatively dilute them to make a single solution containing approximately 225μg Impurity A, 150μg Impurity L, 150μg Impurity N, 150μg Impurity C, 150μg Impurity H, 150μg Impurity I, 150μg Impurity K and 150μg Impurity O per 1ml.

[0071] Impurity mixed stock solution: Accurately measure 1 ml of each impurity stock solution and place it in the same 10 ml volumetric flask, and dilute to the scale with solvent.

[0072] System suitability solution: Take an appropriate amount of trazodone hydrochloride reference substance, accurately weigh it, add an appropriate amount of impurity mixed stock solution, dissolve and dilute it with solvent to make a solution containing 0.75 mg trazodone hydrochloride and 2.25 μg impurity A, 1.5 μg impurity L, 1.5 μg impurity N, 1.5 μg impurity C, 1.5 μg impurity H, 1.5 μg impurity I, 1.5 μg impurity K, and 1.5 μg impurity O per 1 ml.

[0073] Each impurity location solution: accurately measure 0.2 ml of each impurity stock solution and place it in different 10 ml volumetric bottles, and dilute to the scale with solvent.

[0074] Test solution: Take 5 samples of trazodone hydrochloride sustained-release tablets, place in a 100ml volumetric flask (75mg specification), add 80% of the volume of the volumetric flask with methanol, sonicate for 15 minutes to dissolve the trazodone hydrochloride, dilute to the mark with methanol, shake well, and filter. Accurately measure 5ml of the filtrate, place in a 25ml volumetric flask, dilute to the mark with solvent, and shake well.

[0075] Control solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with solvent to make a solution containing 1.5 μg of trazodone hydrochloride per 1 ml.

[0076] Blank excipient solution: weigh 69 mg of the remaining excipients after removing trazodone hydrochloride from the trazodone hydrochloride sustained-release tablets as blank excipients and place in a 20 ml volumetric flask, add 80% of the volume of the volumetric flask with methanol, sonicate for 15 minutes, dilute to scale with methanol, shake well, and filter. Accurately measure 2 ml of the filtrate, place in a 10 ml volumetric flask, dilute to scale with solvent, and shake well.

[0077] Accurately measure 10 μl of each of the above solutions, inject into the liquid chromatograph, run according to the chromatographic conditions of Example 1, and record the chromatogram. The test results are as follows: Figure 1-3 As shown in Tables 3 and 4: the solvent and blank auxiliary material do not interfere with the determination of each component, the separation between the main peak and the adjacent peak is at least 2.7, not less than 2.0, the separation between each known impurity peak and the adjacent chromatographic peak is at least 8.3, not less than 1.5; the retention time of each impurity in the positioning solution is consistent with that in the system suitability solution. This shows that the detection method of the embodiment of the present application has good specificity.

[0078] Table 3 System suitability solution results

[0079]

[0080]

[0081] Table 4 Specificity test results

[0082]

[0083] Note: “N / A” means not detected.

[0084] Example 3 Detection Method Filter Adsorption Test

[0085] Solvent: methanol-water solvent (wherein the volume ratio of methanol to water is 50:50).

[0086] Impurity stock solution: Take appropriate amount of impurity L, impurity A, impurity N, impurity C, impurity H, impurity I, impurity K and impurity O reference substances, accurately weigh them, dissolve them in methanol and quantitatively dilute them to make a mixed solution containing approximately 225μg impurity A, 150μg impurity L, 150μg impurity N, 150μg impurity C, 150μg impurity H, 150μg impurity I, 150μg impurity K and 150μg impurity O per 1ml.

[0087] Spiked test solution: Take 5 samples of trazodone hydrochloride sustained-release tablets, place them in a 100ml volumetric flask (75mg specification), add 5ml of impurity stock solution, add 80% of the volume of the volumetric flask with methanol, sonicate for 15 minutes to dissolve the trazodone hydrochloride, dilute to the mark with methanol, and shake well. Take an appropriate amount of solution and treat it by filtration and centrifugation. Accurately measure 5ml of the treated solution, place it in a 25ml volumetric flask, dilute to the mark with solvent, and shake well.

[0088] The centrifugation was performed at 10000 rpm for 5 min, and the supernatant was taken. Two filter membranes (brand 1: Titan, material: nylon, diameter: 25 mm, pore size: 0.45 μm; brand 2: Jinteng, material: nylon, diameter: 25 mm, pore size: 0.45 μm) were used for filtration, and 3 ml and 5 ml of the initial filtrate were discarded for filtration with each filter membrane, and the subsequent filtrate was taken.

[0089] Table 5 Filter membrane adsorption test results

[0090]

[0091]

[0092] Note: “N / A” means not detected.

[0093] Conclusion: The results are shown in Table 5. When the spiked test solution was filtered using a nylon filter (brand: Jinteng and Titan, diameter: 25 mm, pore size: 0.45 μm), the maximum difference between the percentage of each impurity and the percentage of the centrifuged impurity was 0.008%, which was less than 0.03%, and the maximum difference between the total impurity content and the centrifuged content was 0.026%, which was less than 0.1%, indicating that the membrane adsorption can be ignored. Therefore, the treatment method can be to use a nylon filter (brand: Jinteng and Titan, diameter: 25 mm, pore size: 0.45 μm) to discard 5 ml of the initial filtrate and take the subsequent filtrate for the determination of related substances.

[0094] Example 4 Quantitation Limit and Detection Limit of Detection Method

[0095] Solvent: methanol-water solvent (wherein the volume ratio of methanol to water is 50:50).

[0096] Impurity stock solution 1: Take appropriate amounts of impurity L, impurity A, impurity N, impurity C, impurity H, impurity I, impurity K and impurity O reference substances, accurately weigh them, dissolve them in methanol and quantitatively dilute them to make a mixed solution containing approximately 225 μg impurity A, 150 μg impurity L, 150 μg impurity N, 150 μg impurity C, 150 μg impurity H, 150 μg impurity I, 150 μg impurity K and 150 μg impurity O per 1 ml.

[0097] Impurity stock solution 2: Accurately measure 1 ml of impurity stock solution 1 into a 10 ml volumetric flask and add solvent to dilute to the mark.

[0098] API reference substance stock solution: Take an appropriate amount of trazodone hydrochloride reference substance, accurately weigh it, dissolve it in methanol and dilute it to make a solution containing approximately 15 μg of trazodone hydrochloride per 1 ml.

[0099] Quantitation limit solution: Accurately measure 0.8 ml of impurity stock solution 2 and API reference stock solution into a 50 ml volumetric flask, and dilute to the mark with solvent.

[0100] Detection limit solution: Accurately measure 3 ml of the quantitation limit solution and place it in a 10 ml volumetric flask, then add solvent to dilute to the mark.

[0101] Accurately measure 10 μl of the quantitative limit solution and the detection limit solution, respectively, inject them into the liquid chromatograph, run according to the chromatographic conditions of Example 1, and record the chromatogram.

[0102] Table 6 Detection limit test results

[0103]

[0104] Table 7 Quantitation limit test results

[0105]

[0106]

[0107] Conclusion: The test results are shown in Tables 6 and 7. The relative standard deviations (RSDs) of the retention times of impurity K, impurity L, impurity A, impurity I, impurity C, trazodone hydrochloride, impurity N, impurity H and impurity O were all 0.1%, less than 1%; the peak area RSDs were 1.4%, 1.1%, 1.3%, 0.9%, 3.2%, 1.5%, 2.9%, 2.5% and 1.6%, respectively, all less than 10%; the signal-to-noise ratios were 32.0-35.5, 37.8-42.4, 65.7-83.9, 73.9-90.8, 64.7-83.9, 66.6-83.8, 68.8-93.9, 69.9-83.8, 77.7-83.9, 78.9-83.9, 79.9-93.9, 80.9-83.9, 81.9-83.9, 82.9-83.9, 83 ... .6~74.0, 45.5~51.4, 35.9~40.8, 34.0~38.2, 27.5~31.1, 30.6~35.0 and 46.1~52.1, all greater than 10; the maximum quantitative limit concentration of each component is 0.048%, less than 0.05%; the detection limit signal-to-noise ratio is 8.6, 9.7, 17.3, 11.9, 9.8, 10.4, 7.3, 8.0 and 12.5, all greater than 3. It shows that the detection method of the embodiment of the present application has good sensitivity.

[0108] Example 5 Linearity test of detection method

[0109] Impurity stock solution 1: Take appropriate amounts of impurity L, impurity A, impurity N, impurity C, impurity H, impurity I, impurity K and impurity O reference substances, accurately weigh them, dissolve them in methanol and quantitatively dilute them to make a mixed solution containing approximately 225 μg impurity A, 150 μg impurity L, 150 μg impurity N, 150 μg impurity C, 150 μg impurity H, 150 μg impurity I, 150 μg impurity K and 150 μg impurity O per 1 ml.

[0110] Impurity stock solution 2: Accurately measure 1 ml of impurity stock solution 1 into a 10 ml volumetric flask and add solvent to dilute to the mark.

[0111] API reference substance stock solution: Take an appropriate amount of trazodone hydrochloride reference substance, accurately weigh it, dissolve it in methanol and dilute it to make a solution containing approximately 15 μg of trazodone hydrochloride per 1 ml.

[0112] Linear solution: Accurately measure the impurity stock solution 2 and the API reference stock solution and place them in the same volumetric flask. Dilute to the scale with solvent. The corresponding preparation ratio is shown in Table 8.

[0113] Table 8 Linear preparation process

[0114]

[0115] Linear regression was performed with concentration (μg / ml) as the abscissa and peak area (A) as the ordinate. The test results of trazodone hydrochloride and various impurities are shown in Tables 9 to 17 below.

[0116] Table 9 Impurity K Linear Regression

[0117]

[0118] Table 10 Impurity L linear regression

[0119]

[0120] Table 11 Impurity A linear regression

[0121]

[0122] Table 12 Impurity I Linear Regression

[0123]

[0124] Table 13 Impurity C linear regression

[0125]

[0126] Table 14 Trazodone hydrochloride linear regression

[0127]

[0128] Table 15 Impurity N Linear Regression

[0129]

[0130] Table 16 Impurity H Linear Regression

[0131]

[0132] Table 17 Impurity O Linear Regression

[0133]

[0134] Based on the above test results, the correction factor (impurity correction factor = slope of the principal component linear equation / slope of the impurity linear equation) was calculated. The results are shown in Table 18 below.

[0135] Table 18 Correction factor calculation

[0136]

[0137] Conclusion: The above data show that the peak area and concentration of trazodone hydrochloride and each impurity show a good linear relationship. The correction factor of each impurity is calculated by the slope of the linear equation. The correction factors of impurities A, L and N are in the range of 0.9 to 1.1. When determining related substances, the correction factor is calculated as 1.0; impurity K, impurity I, impurity C, impurity H and impurity O are calculated according to correction factors of 1.4, 0.9, 0.8, 0.7 and 0.5, respectively.

[0138] Example 6 Accuracy test of detection method

[0139] Add each known impurity at 50%, 100%, and 150% of the impurity limit, and prepare a recovery solution with solvent. Prepare 3 portions for each concentration point. Test according to the proposed method, and the specific preparation is as follows:

[0140] Solvent: methanol-water solvent (wherein the volume ratio of methanol to water is 50:50).

[0141] Impurity stock solution: Take appropriate amount of impurity L, impurity A, impurity N, impurity C, impurity H, impurity I, impurity K and impurity O reference substances, accurately weigh them, dissolve them in methanol and quantitatively dilute them to make a mixed solution containing approximately 225μg impurity A, 150μg impurity L, 150μg impurity N, 150μg impurity C, 150μg impurity H, 150μg impurity I, 150μg impurity K and 150μg impurity O per 1ml.

[0142] Test solution: Take 1 trazodone hydrochloride sustained-release tablet sample, place it in a 20ml volumetric flask (75mg specification), add 80% of the volume of the volumetric flask methanol, ultrasonicate for 15 minutes to dissolve the trazodone hydrochloride, dilute to the mark with methanol, shake well, and filter. Accurately measure 5ml of the filtrate, place it in a 25ml volumetric flask, dilute to the mark with solvent, and shake well (prepare 2 portions in parallel).

[0143] 50% recovery solution: Take 1 sample of Trazodone Hydrochloride Sustained Release Tablets, place it in a 20ml volumetric flask (75mg specification), add 0.5ml of impurity stock solution, add 80% of the volume of the volumetric flask with methanol, sonicate for 15 minutes to dissolve the Trazodone Hydrochloride, dilute to the mark with methanol, shake well, and filter. Accurately measure 5ml of the filtrate, place it in a 25ml volumetric flask, dilute to the mark with solvent, and shake well (prepare 3 copies in parallel).

[0144] 100% recovery solution: Take 1 sample of Trazodone Hydrochloride Sustained Release Tablets, place it in a 20ml volumetric flask (75mg specification), add 1ml of impurity stock solution, add 80% of the volume of the volumetric flask with methanol, sonicate for 15 minutes to dissolve the Trazodone Hydrochloride, dilute to the mark with methanol, shake well, and filter. Accurately measure 5ml of the filtrate, place it in a 25ml volumetric flask, dilute to the mark with solvent, and shake well (prepare 3 copies in parallel).

[0145] 150% recovery solution: Take 1 sample of Trazodone Hydrochloride Sustained Release Tablets, place it in a 20ml volumetric flask (75mg specification), add 1.5ml of impurity stock solution, add 80% of the volume of the volumetric flask with methanol, sonicate for 15 minutes to dissolve the Trazodone Hydrochloride, dilute to the mark with methanol, shake well, and filter. Accurately measure 5ml of the filtrate, place it in a 25ml volumetric flask, dilute to the mark with solvent, and shake well (prepare 3 copies in parallel).

[0146] Control solution: Accurately measure an appropriate amount of each test solution and recovery solution, and quantitatively dilute with solvent to prepare a solution containing 1.5 μg of trazodone hydrochloride per 1 ml.

[0147] Determination steps: Take 10 μl of the above-mentioned test solution, recovery solution, and control solution, respectively, inject them into the chromatograph, run according to the chromatographic conditions of Example 1, and record the chromatogram; the test results are shown in Table 19 below.

[0148] Calculation formula:

[0149] Table 19 Recovery test results

[0150]

[0151]

[0152] Conclusion: The data in the above table show that the recoveries of impurity K, impurity L, impurity A, impurity I, impurity C, impurity N, impurity H and impurity O are in the range of 88.6% to 96.8%, 92.6% to 101.6%, 95.1% to 104.7%, 102.7% to 111.3%, 90.3% to 97.3%, 95.7% to 103.9%, 91.7% to 100.8% and 96.6% to 105.8%, respectively, all within the range of 80% to 120%, and the RSDs are 2.8%, 2.9%, 2.7%, 2.4%, 2.1%, 2.5%, 2.8% and 2.8%, respectively, all less than 10%, indicating that the detection method of the embodiment of the present application has good accuracy.

[0153] Example 7 Repeatability test of the detection method

[0154] Solvent: methanol-water solvent (wherein the volume ratio of methanol to water is 50:50).

[0155] System suitability solution: prepare the system suitability solution as in Example 2 under the “Specificity Test”.

[0156] Spiked test solution: Prepare the same 100% recovery solution as in Example 6 under the "Accuracy Test".

[0157] Control solution: Prepare the control solution as in the "Accuracy Test" of Example 6.

[0158] Accurately measure the solvent, the spiked test solution and the control solution and inject them into the liquid chromatograph, run according to the chromatographic conditions of Example 1, and record the chromatogram; the test results are shown in Table 20.

[0159] Table 20 Repeatability test results

[0160]

[0161] Note: “N / A” means not detected.

[0162] Conclusion: The data show that in the 6 spiked test solutions, the RSDs of impurity K, impurity L, impurity A, impurity I, impurity C, impurity N, impurity H, impurity O and total impurities were 0.8%, 0.9%, 0.9%, 0.9%, 0.8%, 0.7%, 0.4%, 1.0% and 0.7%, respectively, all less than 10%, and the maximum unknown impurity and the sum of unknown impurities were not detected, indicating that the detection method of the embodiments of the present application has good repeatability.

[0163] Example 8 Intermediate precision test of the detection method

[0164] Solvent: methanol-water solvent (wherein the volume ratio of methanol to water is 50:50).

[0165] System suitability solution: prepare the system suitability solution as in Example 2 under the “Specificity Test”.

[0166] Spiked test solution: Prepare the same 100% recovery solution as in Example 6 under the "Accuracy Test".

[0167] Control solution: Prepare the control solution as in the "Accuracy Test" of Example 6.

[0168] Accurately measure the solvent, the spiked test solution and the control solution and inject them into the liquid chromatograph, run according to the chromatographic conditions of Example 1, and record the chromatogram; the test results are shown in Table 21.

[0169] Table 21 Intermediate precision test results

[0170]

[0171]

[0172] Note: “N / A” means not detected.

[0173] Conclusion: In the 12 spiked test solutions, the RSDs of impurity K, impurity L, impurity A, impurity I, impurity C, impurity N, impurity H, impurity O and total impurities were 7.8%, 1.3%, 2.6%, 1.7%, 6.0%, 1.9%, 2.6%, 7.3% and 2.9%, respectively, all less than 10%, indicating that the detection method of the embodiment of the present application has good repeatability.

[0174] Example 9 Solution Stability Test of Detection Method

[0175] Solvent: methanol-water solvent (wherein the volume ratio of methanol to water is 50:50).

[0176] System suitability solution: prepare the system suitability solution as in Example 2 under the “Specificity Test”.

[0177] Spiked test solution: Prepare the same 100% recovery solution as in Example 6 under the "Accuracy Test".

[0178] Control solution: Prepare the control solution as in the "Accuracy Test" of Example 6.

[0179] Under room temperature conditions, the spiked test solution and the control solution were injected into the chromatograph at a certain time, and the chromatographic conditions of Example 1 were used to run the chromatograms. The test results are shown in Tables 22 to 24.

[0180] Table 22 Stability test results of spiked test solution-1

[0181]

[0182]

[0183] Table 23 Stability test results of spiked test solution-2

[0184]

[0185] Note: “N / A” means not detected.

[0186] Table 24 Control solution stability test results

[0187]

[0188] Conclusion: After the spiked test solution was placed at room temperature for 70 hours, no new impurities greater than 0.05% were detected. The maximum differences between the contents of impurity K, impurity L, impurity A, impurity I, impurity C, impurity N, impurity H and impurity O at each time point and those at 0 h were 0.002%, 0.004%, 0.007%, 0.004%, 0.002%, 0.004%, 0.004% and 0.003%, respectively, all less than 0.05%. The maximum difference between the total impurity content and that at 0 h was 0.018%, less than 0.1%, indicating that the spiked test solution was stable within 70 hours at room temperature. When the control solution was placed at room temperature for 70 hours, the ratios of the peak area at each time point to the peak area at 0 h were between 98.4% and 100.4%, all within the range of 90% to 110%, indicating that the control solution was stable within 70 hours at room temperature.

[0189] Example 10 Durability test of the detection method

[0190] Solvent: methanol-water solvent (wherein the volume ratio of methanol to water is 50:50).

[0191] System suitability solution: prepare the system suitability solution as in Example 2 under the “Specificity Test”.

[0192] Spiked test solution: Prepare the same 100% recovery solution as in Example 6 under the "Accuracy Test".

[0193] Control solution: Prepare the control solution as in the "Accuracy Test" of Example 6.

[0194] Appropriately adjust the detection flow rate, mobile phase ratio, column temperature, pH value of the salt phase in the mobile phase, salt concentration, and chromatographic column in the chromatographic conditions, and examine the changes in the system suitability parameters after the chromatographic conditions change.

[0195] Table 25 Durability Chromatographic Conditions

[0196]

[0197] Note: Column 1: YMC-Pack Pro C18, 4.6×150mm, 5μm S / N: 105ZB20036; Column 2: YMC-Pack Pro C18, 4.6×150mm, 3μm S / N: 107ZB20380.

[0198] The durability test results are shown in Tables 26 to 28 below.

[0199] Table 26 System suitability solution results

[0200]

[0201] Table 27 Impurity test results of spiked test solution-1

[0202]

[0203] Table 28 Impurity test results of spiked test solution-2

[0204]

[0205] Note: “N / A” means not detected.

[0206] Conclusion: The data show that by appropriately adjusting the detection flow rate, mobile phase ratio, column temperature, pH value of the salt phase in the mobile phase, salt concentration, chromatographic column and wavelength in the chromatographic conditions, in each system suitability solution, the minimum separation between the main peak and the adjacent peak is 2.3, which is greater than 2.0; the minimum separation between each impurity is 6.4, which is greater than 1.5; the maximum differences between the contents of impurity K, impurity L, impurity A, impurity I, impurity C, impurity N, impurity H, and impurity O measured under standard conditions are 0.021%, 0.040%, 0.016%, 0.020%, 0.004%, 0.025%, 0.013% and 0.035%, respectively, all less than 0.05%; unknown impurities were not detected; the maximum difference between the total impurities and the contents measured under standard conditions is 0.038%, which is less than 0.1%; it shows that the detection method of the embodiment of the present application has good durability.

[0207] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for detecting impurities in trazodone hydrochloride, characterized in that: include: Using high performance liquid chromatography to detect impurities in the trazodone hydrochloride solution to be tested, wherein the solvent in the trazodone hydrochloride solution to be tested is a methanol-water solvent; The chromatographic conditions of the high performance liquid chromatography include: using alkylsilane bonded silica gel as the filler of the chromatographic column, using potassium dihydrogen phosphate aqueous solution as mobile phase A, and acetonitrile as mobile phase B, and performing gradient elution.

2. The detection method according to claim 1, characterized in that The impurities include substances with the following chemical structures:

3. The detection method according to claim 1, characterized in that Taking the total volume of the mobile phase A and the mobile phase B as 100%, the procedure of the gradient elution is: 0 min, 83-87% mobile phase A and 13-17% mobile phase B; 8 min, 50% mobile phase A and 50% mobile phase B; 15 min, 40% mobile phase A and 60% mobile phase B; 18 min, 20% mobile phase A and 80% mobile phase B; 29 min, 15% mobile phase A and 85% mobile phase B; After 29 min, 85% mobile phase A and 15% mobile phase B.

4. The detection method according to claim 1, characterized in that In the methanol-water solvent, the volume ratio of methanol to water is (40:60) to (60:40).

5. The detection method according to claim 4, characterized in that: In the methanol-water solvent, the volume ratio of methanol to water is 50:

50.

6. The detection method according to claim 1, characterized in that The concentration of the potassium dihydrogen phosphate aqueous solution is 2.44-2.99 g / L; and / or the pH of the potassium dihydrogen phosphate aqueous solution is 6.2-6.

6.

7. The detection method according to claim 1, characterized in that The chromatographic conditions also include: an injection flow rate of 0.7 to 0.9 ml / min, a column temperature of 30 to 40° C., and a detection wavelength of 252 to 256 nm.

8. The detection method according to any one of claims 1 to 7, characterized in that: The chromatographic conditions include: the concentration of the potassium dihydrogen phosphate aqueous solution is 2.72 g / L, the pH of the potassium dihydrogen phosphate aqueous solution is 6.4; the injection flow rate is 0.78 ml / min, the column temperature is 35° C., and the detection wavelength is 254 nm.

9. The detection method according to any one of claims 1 to 7, characterized in that: The alkylsilane bonded silica gel includes: YMC-Pack Pro C18, size 4.6×150 mm, particle size 3-5 μm; The chromatographic conditions also used a ghost peak trapping column: Welch Ghost Buster Column, with a size of 4.6×50 mm.

10. The detection method according to any one of claims 1 to 7, characterized in that: The trazodone hydrochloride solution to be tested includes a trazodone hydrochloride bulk drug solution or a pharmaceutical preparation solution containing trazodone hydrochloride.

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