Method for determining impurity compound I and impurity compound II in brexpiprazole key material

Abstract

The invention discloses a method for determining an impurity compound I and an impurity compound II in a brexpiprazole key material, which uses a high performance liquid chromatograph and an ultraviolet detector, uses octadecyl silane bonded silica gel as a chromatographic column filler, uses a phosphoric acid solution as a mobile phase A, uses an acetonitrile-methanol mixed solution as a mobile phase B, and adopts a gradient elution method. And carrying out gradient elution to determine the content of the impurity compound I and the impurity compound II in the brexpiprazole key material. According to the method, the blank that a method for measuring the impurity compound I in the brexpiprazole key material does not exist at present and a method for measuring the impurity compound I and the impurity compound II in the brexpiprazole key material does not exist at the same time is filled up; the method for determining the impurity compound I and the impurity compound II in the brexpiprazole key material is high in selectivity, good in accuracy and high in precision and sensitivity; according to the invention, the quality control of the medicine can be effectively enhanced.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for determining an impurity compound I and an impurity compound II in a key material of brepazine. Background Art

[0002] Brexpiprazole is a new dopamine receptor partial agonist developed by Lundbeck Pharma of Denmark and Otsuka Pharmaceutical Co., Ltd. of Japan. It is used to treat major depressive disorder (MDD) and schizophrenia. Its trade name is Rexulti. It was approved for marketing on July 10, 2015. In 2016, it was approved for maintenance treatment of adult patients with schizophrenia. In 2018, Brexpiprazole was approved to enter the European market. In order to expand the scope of market application, Lundbeck Pharma of Denmark and Otsuka Pharmaceutical Co., Ltd. of Japan have been expanding the indications of Brexpiprazole in recent years. Its research on the treatment of agitation, post-traumatic stress disorder, borderline personality disorder and other aspects of Alzheimer's patients has been deepening.

[0003] The chemical name of breprazol is 7-(4-(4-(benzo[b]thiophen-4-yl)-piperazine-1-yl)butoxy)-1H-quinolin-2-one, and its molecular formula is C 25 H 29 N3O2S, the structural formula is:

[0004]

[0005] In the prior art, 7-hydroxy-2(1H)-quinolinone is a key material for brepirazoline and is of great significance for the synthesis of brepirazoline. Its structural formula is:

[0006]

[0007]

[0008] Among them, the original patent CN200680011923.0 reported the synthesis route of brepazole as follows:

[0009]

[0010] This route uses 7-hydroxy-2(1H)-quinolinone as a starting material, which reacts with 1-bromo-4-chlorobutane and then reacts with 4-piperazine-benzothiophene hydrochloride to produce breprazol.

[0011] Chinese patent CN201610080468.5 reports a method for synthesizing brexpiprazole using 7-hydroxy-2(1H)-quinolinone as a starting material, and the route is as follows:

[0012]

[0013] In addition, Chinese patents CN201410853528.3, CN201510427992.0, CN201510177351.4, CN201510993612.X, CN201610006862.4, CN201610794835.8, and CN202111554063.8 all reported other methods for synthesizing brexpiprazole using 7-hydroxy-2(1H)-quinolinone as a raw material, indicating that 7-hydroxy-2(1H)-quinolinone is a key material for synthesizing brexpiprazole.

[0014] In the prior art, the synthesis of 7-hydroxy-2(1H)-quinolinone mainly includes two methods: oxidation method and cyclization method. The route is as follows:

[0015] Oxidation method:

[0016]

[0017] Cyclomethod:

[0018]

[0019] The oxidants used in the oxidation method include DDQ, sodium persulfate, and palladium carbon / oxygen. The inventors tried the oxidation method route during the research process and found that the sodium persulfate oxidation method produced a lot of small impurities (up to 40 or more) during the reaction process, and the related substances were not easy to control, and the product color was dark, resulting in the appearance of the finished product of brepazole being yellow-brown; the DDQ oxidation method post-treatment requires a large amount of water washing, which is not conducive to environmental protection requirements; and the price of DDQ is high, which is not conducive to cost control. At the same time, the appearance of the product obtained by this method is grayish white, which ultimately leads to the dark color of brepazole; the Pd / O2 method catalyst is expensive, which is not conducive to cost control, and the use of oxygen has safety hazards. Therefore, the inventors ruled out the oxidation method route.

[0020] During the research process of the cyclization method, the inventors found that the synthesis of 7-hydroxy-2(1H)-quinolinone would produce a transition state intermediate, whose chemical structure is shown in Formula I:

[0021]

[0022] Its molecular formula is C 15 H 12 ClNO2, molecular weight is 273.72.

[0023] At the same time, the isomeric impurity 5-hydroxy-2(1H)-quinolinone is generated during the cyclization process, and its chemical structure is shown in Formula II:

[0024]

[0025] Its molecular formula is C9H7NO2 and its molecular weight is 161.16.

[0026] According to the reaction mechanism mentioned in the literature Synthesis, 1997, #1, p.87-90, the reaction mechanism for preparing 7-hydroxy-2(1H)-quinolinone is divided into two types (high temperature and low temperature). At high temperature (described in the literature as 120°C), the reaction mechanism is as follows:

[0027]

[0028] The inventors speculated that the mechanism of the generation of impurity I is a reaction mechanism at low temperature (described in the literature as 80° C.), and the reaction uses chlorobenzene as a solvent. The cyclization intermediate 1 reacts with chlorobenzene under the catalysis of aluminum chloride to generate formula I. The detailed mechanism is as follows:

[0029]

[0030] The inventors found in the process of tracking the reaction progress that Formula I will continue to react to generate 7-hydroxy-2(1H)-quinolinone, and speculated that the reaction mechanism is as follows:

[0031]

[0032] However, the structure of formula I cannot be completely reacted in the reaction solution, and about 5% remains in the middle control process of the reaction, and it is no longer converted into the product. Continuing the reaction increases other impurities, the main peak purity decreases, and the yield decreases accordingly. The formula I that cannot be completely reacted becomes an impurity in 7-hydroxy-2 (1H) -quinolinone, so a suitable analytical method is needed to perform middle control and quality control on the reaction to ensure the product quality and yield of 7-hydroxy-2 (1H) -quinolinone.

[0033] The reason for the generation of the impurity of formula II is the side reaction. During the cyclization of intermediate 1, the ortho-ring closure occurs, and the methoxy group removes the methyl group under the action of aluminum chloride. The detailed mechanism is as follows:

[0034]

[0035] In the process of drug development, drug impurities mainly come from reaction by-products and degradation impurities. The impurity content of the drug will directly affect the efficacy and toxic side effects of the drug. Studies have shown that impurity compound I and impurity compound II are inherent impurities in the preparation process of 7-hydroxy-2(1H)-quinolinone. Impurity compound I and impurity compound II also have phenolic hydroxyl structures, which will generate new impurities following subsequent process reactions and even be introduced into the finished product of brepazine.

[0036] During the preparation of 7-hydroxy-2 (1H) -quinolinone, the impurity compound I in the reaction solution cannot be completely converted. The results of the impurity addition experiment show that when the residual content of the impurity compound I in the reaction solution of the preparation of 7-hydroxy-2 (1H) -quinolinone is 8.32%, it can be effectively removed in the dry product. Based on this, the control limit of the impurity compound I is set to no more than 6.0%. In addition, the results of the addition experiment of the impurity compound II show that when the impurity compound II is 3.83% in 7-hydroxy-2 (1H) -quinolinone, it can be effectively removed in the subsequent process. Therefore, the content limit of the impurity compound II in 7-hydroxy-2 (1H) -quinolinone is set to no more than 3.0%.

[0037] Based on the concept of quality by design, 7-hydroxy-2(1H)-quinolinone needs to have a complete detection method to control the content of impurity compound I in this step of the reaction to be below 6.0%; at the same time, the content of impurity compound I in the key material 7-hydroxy-2(1H)-quinolinone is controlled to be below 0.10%, and the content of impurity compound II is controlled to be below 3.0%, so as to ensure the product quality of brepizole, which is more conducive to reducing the research work on related impurities corresponding to subsequent intermediates in the synthesis process of brepizole.

[0038] Currently, there is no reported method for determining impurity compound I and impurity compound II. Therefore, the present invention develops a method for determining impurity compound I and impurity compound II in 7-hydroxy-2(1H)-quinolinone, a key material of breprazol, so as to achieve stricter quality control of the drug. Summary of the invention

[0039] The technical problem to be solved by the present invention is to overcome the technical defects of the background technology and provide a method for determining impurity compound I and impurity compound II in the key material of brepirazole. At present, there is no report on the determination method of impurity compound I and impurity compound II. The present invention designs a new high-performance liquid chromatography method, which can accurately determine the content of impurity compound I and impurity compound II in 7-hydroxy-2 (1H) -quinolinone, a key material of brepirazole, and also verifies the analytical method; the determination method of impurity compound I and impurity compound II in the key material of brepirazole of the present invention has strong selectivity, good accuracy, high precision and high sensitivity; the present invention can effectively enhance the quality control of drugs.

[0040] The technical solution adopted by the present invention to solve the above technical problems is as follows:

[0041] A method for determining impurity compound I and impurity compound II in a key material of brepirazole, using a high performance liquid chromatograph, an ultraviolet detector, octadecylsilane bonded silica gel as a chromatographic column filler, a phosphoric acid solution as a mobile phase A, an acetonitrile-methanol mixed solution as a mobile phase B, and gradient elution to determine the content of impurity compound I and impurity compound II in the key material of brepirazole;

[0042] The chemical structural formula of the impurity compound I in the key material of breprazol is shown in Formula I:

[0043]

[0044] The chemical structural formula of the impurity compound II in the key material of breprazol is shown in Formula II:

[0045]

[0046] Preferably, the method for determining impurity compound I and impurity compound II in the key material of breprazol comprises the following steps:

[0047] (1) Preparation of system suitability solution: Take the key material of brexpiprazole 7-hydroxy-2(1H)-quinolinone reference substance, impurity compound I reference substance and impurity compound II reference substance, add diluent to prepare a system suitability solution, wherein each 1 ml contains 80 μg 7-hydroxy-2(1H)-quinolinone, 4.8 μg impurity compound I and 3.2 μg impurity compound II;

[0048] (2) preparing a sample solution: taking a 7-hydroxy-2(1H)-quinolinone sample and adding a diluent to prepare a sample solution with a concentration of 80 μg / ml;

[0049] (3) Chromatographic conditions: A high performance liquid chromatograph was used, and a UV detector was used. The chromatographic column packing was octadecylsilane bonded silica gel. Mobile phase A was phosphoric acid solution, and mobile phase B was a mixed solution of acetonitrile-methanol. Gradient elution was performed. The gradient elution parameters were set as follows:

[0050] 0-2min, the volume ratio of mobile phase A is 90%, and the volume ratio of mobile phase B is 10%;

[0051] From 2 to 8 min, the volume proportion of mobile phase A changes from 90% to 80%, and the volume proportion of mobile phase B changes from 10% to 20%;

[0052] From 8 to 15 min, the volume proportion of mobile phase A changes from 80% to 70%, and the volume proportion of mobile phase B changes from 20% to 30%;

[0053] From 15 to 35 min, the volume proportion of mobile phase A changed from 70% to 50%, and the volume proportion of mobile phase B changed from 30% to 50%;

[0054] 35-45 min, the volume proportion of mobile phase A changes from 50% to 10%, and the volume proportion of mobile phase B changes from 50% to 90%;

[0055] 45-53min, mobile phase A volume ratio is 10%, mobile phase B volume ratio is 90%;

[0056] From 53 to 58 min, the volume proportion of mobile phase A changed from 10% to 90%, and the volume proportion of mobile phase B changed from 90% to 10%;

[0057] 58-65min, mobile phase A volume ratio is 90%, mobile phase B volume ratio is 10%;

[0058] Accurately measure the system suitability solution and sample solution, inject them into a liquid chromatograph, record the chromatogram, and calculate the contents of impurity compound I and impurity compound II in the 7-hydroxy-2(1H)-quinolinone sample by peak area normalization method;

[0059] (4) Calculation method

[0060] The impurity content in the 7-hydroxy-2(1H)-quinolinone sample was calculated according to the following formula:

[0061]

[0062] A i = peak area of ​​impurities in sample solution;

[0063] A 总 = The sum of the corrected peak areas in the sample solution chromatogram;

[0064] f = impurity correction factor, the correction factor of impurity compound I in this analytical method is 1.4, and the correction factor of impurity compound II in this analytical method is 2.2;

[0065] The chemical structural formula of the 7-hydroxy-2(1H)-quinolinone is as follows:

[0066]

[0067] The chemical structural formula of the impurity compound I is shown in Formula I:

[0068]

[0069] The chemical structural formula of the impurity compound II is shown in Formula II:

[0070]

[0071] Preferably, in step (1) and step (2), the diluent is 0.1% phosphoric acid solution-acetonitrile (70:30).

[0072] Preferably, in step (3), the detection wavelength of the ultraviolet detector is 218-222 nm, more preferably 220 nm.

[0073] Preferably, in step (3), the chromatographic column is Agilent ZORBAX Eclipse Plus C18, with specifications of 4.6 mm×250 mm and 5 μm.

[0074] Preferably, in step (3), the column temperature of the chromatographic column is 30-40°C, more preferably 35°C.

[0075] Preferably, in step (3), the mobile phase A is a 0.1% phosphoric acid solution, and the mobile phase B is acetonitrile-methanol ((78-82):(22-18)), more preferably 80:20.

[0076] Preferably, in step (3), the flow rate of the gradient elution is 0.9-1.1 ml / min, more preferably 1.0 ml / min.

[0077] Preferably, in step (3), the gradient elution parameters are set to:

[0078] 0min, the volume proportion of mobile phase A is 90%, and the volume proportion of mobile phase B is 10%;

[0079] 2 min, mobile phase A volume ratio is 90%, mobile phase B volume ratio is 10%;

[0080] 8 min, mobile phase A volume ratio is 80%, mobile phase B volume ratio is 20%;

[0081] 15 min, mobile phase A volume ratio is 70%, mobile phase B volume ratio is 30%;

[0082] 35 min, mobile phase A volume ratio is 50%, mobile phase B volume ratio is 50%;

[0083] 45 min, mobile phase A volume ratio is 10%, mobile phase B volume ratio is 90%;

[0084] 53min, mobile phase A volume ratio is 10%, mobile phase B volume ratio is 90%;

[0085] 58min, mobile phase A volume ratio is 90%, mobile phase B volume ratio is 10%;

[0086] 65min, the volume proportion of mobile phase A is 90%, and the volume proportion of mobile phase B is 10%.

[0087] Preferably, in step (3), 10 μl of each of the system suitability solution and the sample solution are accurately measured.

[0088] Compared with the prior art, the present invention has the following beneficial effects:

[0089] The present invention fills the gap that there is no method for determining impurity compound I in the key material of brepirazoline, and there is no method for simultaneously determining impurity compound I and impurity compound II in the key material of brepirazoline; the method of the present invention has strong specificity, good repeatability and high accuracy, can ensure the controllability of impurity compound I and impurity compound II, and can be applied to the quality control of brepirazoline. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 is the impurity compound I in the key material of breprazol of the present invention 1 H-NMR spectrum;

[0091] Figure 2 is the impurity compound I in the key material of breprazol of the present invention 13 C-NMR spectrum;

[0092] Figure 3 is the mass spectrum of the impurity compound I in the key material of breprazol of the present invention, wherein the peak retention time of the impurity compound I is 19.281 min;

[0093] Figure 4 is the impurity compound II in the key material of breprazol of the present invention 1 H-NMR spectrum;

[0094] Figure 5 The mass spectrum of the impurity compound II in the key material of breprazol of the present invention;

[0095] Figure 6 The chromatogram is a system applicability solution of the impurity determination method of the present invention (containing 80 μg of brepirazole key material 7-hydroxy-2 (1H) -quinolinone, 4.8 μg of impurity compound I and 3.2 μg of impurity compound II per 1 ml), wherein the peak retention time of 7-hydroxy-2 (1H) -quinolinone is 11.748 min, the peak retention time of impurity compound I is 31.984 min, and the peak retention time of impurity compound II is 9.560 min;

[0096] Figure 7 It is a localization chromatogram of impurity compound I in the key material of breprazol of the present invention;

[0097] Figure 8It is a localization chromatogram of impurity compound II in the key material of breprazol of the present invention;

[0098] Fig. 9 The chromatogram of the sample solution of 7-hydroxy-2(1H)-quinolinone in the key material of brepirazole of the present invention;

[0099] Fig.10 The chromatogram is a spiked solution of impurities in the key material of breprazol of the present invention. DETAILED DESCRIPTION

[0100] In order to better understand the content of the present invention, further description is given below in conjunction with specific embodiments and accompanying drawings. It should be understood that these embodiments are only used to further illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content described in the present invention, those skilled in the art may make some non-essential changes or adjustments to the present invention, which still belong to the protection scope of the present invention.

[0101] Example 1

[0102] A method for determining impurity compound I and impurity compound II in a key material of breprazol comprises the following steps:

[0103] (1) Preparation of system suitability solution: Take the key materials of brexpiprazole 7-hydroxy-2(1H)-quinolinone reference substance, impurity compound I reference substance and impurity compound II reference substance, add diluent to prepare a system suitability solution, wherein each 1 ml contains about 80 μg 7-hydroxy-2(1H)-quinolinone, 4.8 μg impurity compound I, and 3.2 μg impurity compound II; the diluent is 0.1% phosphoric acid solution-acetonitrile (70:30);

[0104] (2) Preparation of sample solution: Take about 16 mg of 7-hydroxy-2(1H)-quinolinone sample, accurately weigh it, place it in a 200 ml volumetric flask, add a diluent, ultrasonically dissolve it and dilute it to the scale, shake it well, and the concentration is 80 μg / ml; the diluent is 0.1% phosphoric acid solution-acetonitrile (70:30);

[0105] (3) Chromatographic conditions: A high performance liquid chromatograph Agilent 1260 (VWD) was used, with a UV detector, and the chromatographic column was filled with Agilent ZORBAX Eclipse Plus C18 (4.6 mm × 250 mm, 5 μm). The mobile phase A was 0.1% phosphoric acid solution, and the mobile phase B was acetonitrile-methanol (80:20), with gradient elution; the flow rate was 1.0 ml / min, the column temperature was 35°C, the wavelength was 220 nm, and the injection volume was 10 μl; the gradient elution parameters were set as follows:

[0106] 0min, the volume proportion of mobile phase A is 90%, and the volume proportion of mobile phase B is 10%;

[0107] 2 min, mobile phase A volume ratio is 90%, mobile phase B volume ratio is 10%;

[0108] 8 min, mobile phase A volume ratio is 80%, mobile phase B volume ratio is 20%;

[0109] 15 min, mobile phase A volume ratio is 70%, mobile phase B volume ratio is 30%;

[0110] 35 min, mobile phase A volume ratio is 50%, mobile phase B volume ratio is 50%;

[0111] 45 min, mobile phase A volume ratio is 10%, mobile phase B volume ratio is 90%;

[0112] 53min, mobile phase A volume ratio is 10%, mobile phase B volume ratio is 90%;

[0113] 58min, mobile phase A volume ratio is 90%, mobile phase B volume ratio is 10%;

[0114] 65 min, mobile phase A volume ratio is 90%, mobile phase B volume ratio is 10%;

[0115] Accurately measure the system suitability solution and sample solution, inject them into a liquid chromatograph, record the chromatogram, and calculate the contents of impurity compound I and impurity compound II in the 7-hydroxy-2(1H)-quinolinone sample by peak area normalization method;

[0116] (4) Calculation method

[0117] The impurity content in the 7-hydroxy-2(1H)-quinolinone sample was calculated according to the following formula:

[0118]

[0119] A i = peak area of ​​impurities in sample solution;

[0120] A 总 = The sum of the corrected peak areas in the sample solution chromatogram;

[0121] f = impurity correction factor, the correction factor of impurity compound I in this analytical method is 1.4, and the correction factor of impurity compound II in this analytical method is 2.2;

[0122] The chemical structural formula of the 7-hydroxy-2(1H)-quinolinone is as follows:

[0123]

[0124] The chemical structural formula of the impurity compound I is shown in Formula I:

[0125]

[0126] The chemical structural formula of the impurity compound II is shown in Formula II:

[0127]

[0128] Effect Example

[0129] 1. Determination of the chemical structure of the impurity compound I of the present invention

[0130] The impurity compound I of the present invention 1 H-NMR spectrum, 13 C-NMR spectrum and mass spectrum are shown in Figure 1 to Figure 3 As shown, the structure of the impurity compound I can be confirmed, and its structure is shown in Formula I:

[0131]

[0132] 2. Determination of the Chemical Structure of Impurity Compound II of the Present Invention

[0133] The impurity compound II of the present invention 1 H-NMR spectra and mass spectra are shown in Figure 4-5 As shown, the structure of the impurity compound II can be confirmed, and its structure is shown in Formula II:

[0134]

[0135] III. Methodological Verification of the Determination Method of Impurity Compound I and Impurity Compound II in the Key Materials of Brepirapazole of the Present Invention

[0136] 1. Specificity - sample matrix and known impurities

[0137] Impurity Compound I stock solution: Take about 2.4 mg of the impurity compound I reference substance, accurately weigh it, put it in a 50 ml volumetric flask, add the diluent, ultrasonically dissolve it and dilute it to the scale, and shake it well.

[0138] Impurity Compound II stock solution: Take about 3.2 mg of the impurity Compound II reference substance, accurately weigh it, place it in a 100 ml volumetric flask, add diluent, ultrasonically dissolve it and dilute it to the scale, and shake it well.

[0139] Spiked sample solution: Take about 8 mg of 7-hydroxy-2(1H)-quinolinone sample and place it in a 100 ml volumetric flask. Accurately measure 10 ml of impurity compound I stock solution and 10 ml of impurity compound II stock solution and add them. Add diluent and ultrasonically dissolve and dilute to the scale, and shake well.

[0140] Take the prepared diluent, 7-hydroxy-2(1H)-quinolinone sample solution, each impurity stock solution, and spiked sample solution, inject them into the liquid chromatograph respectively, and record the chromatogram. The key spectrum is shown in Figure 6 to Figure 10 ,in Figure 6 is the chromatogram of the system suitability solution, Figure 7 is the localization chromatogram of impurity compound I, Figure 8 is the localization chromatogram of impurity compound II, Fig. 9 is the chromatogram of 7-hydroxy-2(1H)-quinolinone sample solution, Fig.10 Chromatogram of the solution spiked with impurities.

[0141] The results showed that the diluent had no interference. In the 7-hydroxy-2(1H)-quinolinone sample solution, the separation between the main peak and the adjacent impurity peaks was not less than 1.5 (13.91), and the separation between the impurity compound II peak and the adjacent impurity peaks was not less than 1.5 (13.91). In the spiked sample solution, the separation between the main peak and the adjacent impurity peaks was not less than 1.5 (13.93), the separation between the compound I peak and the adjacent impurity peaks was not less than 1.5 (2.72), and the separation between the impurity compound II peak and the adjacent impurity peaks was not less than 1.5 (13.93). The purity angles of the above main peak, impurity compound I peak, and impurity compound II peak were all less than the purity threshold. The peak elution time was moderate, the peak shape was symmetrical, and the separation effect was good, meeting the requirements.

[0142] 2. Detection limit and quantification limit test

[0143] The signal-to-noise ratio method was used to determine the detection limit and quantification limit. 7-Hydroxy-2(1H)-quinolinone reference stock solution, impurity compound I reference stock solution, and impurity compound II reference stock solution were prepared, diluted to a certain concentration, injected, and the chromatogram was recorded. The sample detection amount with a signal-to-noise ratio S / N ≥ 10 is the quantification limit, and the sample detection amount with a signal-to-noise ratio S / N ≥ 3 is the detection limit. The results are shown in Table 1.

[0144] Table 1 Detection limit and quantification limit test results of the present invention

[0145]

[0146] The results show that the method is sensitive to both impurity compound I and impurity compound II, and can accurately control the content of impurity compound I and the content of impurity compound II.

[0147] 3. Linearity test

[0148] Take 7-hydroxy-2(1H)-quinolinone reference substance, impurity compound I reference substance, and impurity compound II reference substance, add diluent to dilute into a linear stock solution containing 48 μg 7-hydroxy-2(1H)-quinolinone, 48 μg impurity compound I, and 32 μg impurity compound II per 1 ml.

[0149] Take the linear stock solution and prepare a total of 7 linear solutions with diluent according to Table 2. Inject the samples into different chromatographs and record the chromatograms. Draw the standard curve with concentration as the horizontal axis (X) and peak area as the vertical axis (Y). The results are shown in Table 3.

[0150] Table 2 Linear test solution preparation of the present invention

[0151]

[0152]

[0153] Table 3 Linear test results of the present invention

[0154]

[0155] The results showed that the method showed good linearity in the concentration range of 0.0237-9.4798 μg / ml for 7-hydroxy-2(1H)-quinolinone, in the concentration range of 0.0223-8.9261 μg / ml for impurity compound I, and in the concentration range of 0.0316-6.3200 μg / ml for impurity compound II.

[0156] 4. Accuracy test

[0157] Impurity compound I and impurity compound II were added to the 7-hydroxy-2(1H)-quinolinone sample for sample recovery determination. A 7-hydroxy-2(1H)-quinolinone sample solution (unspiked, concentration 80 μg / ml) was prepared for determination, and three portions were prepared in parallel to obtain the content of impurity compound I in the sample. The limit of impurity compound I was set at 6.0%, and the research concentrations were 50%, 100%, and 150% of the relative limit. The limit of impurity compound II during method validation was set at 4.0%, and the research concentrations were 50%, 100%, and 150% of the relative limit. Three portions were prepared in parallel for each concentration and the measured value was obtained. The recovery was calculated by dividing the difference between the measured value and the content in the sample by the amount of reference substance added. The results are shown in Table 4.

[0158] Table 4 Accuracy test results of the present invention

[0159]

[0160] The results showed that the recovery rates of individual samples were between 95.0% and 102.0%, indicating that the method had good accuracy.

[0161] 5. Precision test

[0162] (1) Repeatability: The precision of the results obtained by the same analyst under the same conditions is determined by preparing six spiked sample solutions of 7-hydroxy-2(1H)-quinolinone in parallel and measuring them under the same conditions as much as possible. The measured amount and RSD are calculated.

[0163] (2) Intermediate precision: The effect of random variation on precision was investigated. The intermediate precision was calculated by another analyst who prepared six spiked sample solutions of 7-hydroxy-2(1H)-quinolinone in parallel on different days. The measured amount and RSD were calculated and combined with the results of 12 measurements in the repeatability test.

[0164] The results showed that: for the repeatability measurement results of 6 spiked samples, the RSD of the impurity compound I content was 0.50%, and the RSD of the impurity compound II content was 0.28%; for the intermediate precision measurement results of 6 samples, the RSD of the impurity compound I content was 2.3%, and the RSD of the impurity compound II content was 1.3%; for the repeatability and intermediate precision results calculated together, the RSD of the impurity compound I content of 12 samples was 1.3%, and the RSD of the impurity compound II content was 2.0%, indicating that the precision of this method is good.

[0165] 6. Durability test

[0166] (1) Solution stability: Take a sample solution of 7-hydroxy-2(1H)-quinolinone and inject it for testing at 0, 6, 12, 20, 25, 35, 41 and 48 hours, record the chromatogram, and calculate the difference between the content of each impurity and the total impurity and that at 0 hour.

[0167] The results show that the sample solution is stable within 48 hours when placed in a sealed condition at room temperature.

[0168] (2) Durability study: The ability of the method to be unaffected by changes in the method parameters was investigated by deliberately changing the method parameters. In this study, the following parameters were independently investigated: column temperature (35 ± 5 °C), column flow rate (1.0 ± 0.1 ml / min), mobile phase ratio (B phase changed 2% absolute value), and the difference between the measured amount of the determined component and the measured amount under the initial conditions was investigated. The spiked sample solution (two parallel copies) was injected into the liquid chromatograph and the chromatogram was recorded. The results are shown in Table 5.

[0169] Table 5 Durability study test results of the present invention

[0170]

[0171] The results showed that after changing the method parameters, the difference between the determination results of impurity compound I and impurity compound II and those under normal conditions was 0.00% to 0.18%, indicating that the method had good durability.

[0172] From the above results, it can be seen that the method of the present invention can effectively detect impurity compound I and impurity compound II in 7-hydroxy-2 (1H) -quinolinone, a key material of brepirazole. The method has strong specificity, good repeatability and high accuracy, can ensure the controllability of impurity compound I and impurity compound II, and can be applied to the quality control of brepirazole.

[0173] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A method for determining impurity compound I and impurity compound II in a key material of breprazol, characterized in that: The steps include: (1) Preparation of system suitability solution: Take the key materials of brexpiprazole, 7-hydroxy-2(1H)-quinolinone reference substance, impurity compound I reference substance and impurity compound II reference substance, and add diluent to prepare a system suitability solution, wherein each 1 ml contains 80 μg 7-hydroxy-2(1H)-quinolinone, 4.8 μg impurity compound I and 3.2 μg impurity compound II; (2) preparing a sample solution: taking a 7-hydroxy-2(1H)-quinolinone sample and adding a diluent to prepare a sample solution with a concentration of 80 μg / ml; (3) Chromatographic conditions: A high performance liquid chromatograph was used, and a UV detector was used. The chromatographic column packing was octadecylsilane bonded silica gel. Mobile phase A was phosphoric acid solution, and mobile phase B was a mixed solution of acetonitrile-methanol. Gradient elution was performed. The gradient elution parameters were set as follows: 0-2min, the volume ratio of mobile phase A is 90%, and the volume ratio of mobile phase B is 10%; From 2 to 8 min, the volume proportion of mobile phase A changes from 90% to 80%, and the volume proportion of mobile phase B changes from 10% to 20%; From 8 to 15 min, the volume proportion of mobile phase A changes from 80% to 70%, and the volume proportion of mobile phase B changes from 20% to 30%; From 15 to 35 min, the volume proportion of mobile phase A changed from 70% to 50%, and the volume proportion of mobile phase B changed from 30% to 50%; 35-45 min, the volume proportion of mobile phase A changes from 50% to 10%, and the volume proportion of mobile phase B changes from 50% to 90%; 45-53min, mobile phase A volume ratio is 10%, mobile phase B volume ratio is 90%; From 53 to 58 min, the volume proportion of mobile phase A changed from 10% to 90%, and the volume proportion of mobile phase B changed from 90% to 10%; 58-65min, mobile phase A volume ratio is 90%, mobile phase B volume ratio is 10%; Accurately measure the system suitability solution and sample solution, inject them into a liquid chromatograph, record the chromatogram, and calculate the contents of impurity compound I and impurity compound II in the 7-hydroxy-2(1H)-quinolinone sample by peak area normalization method; (4) Calculation method The impurity content in the 7-hydroxy-2(1H)-quinolinone sample was calculated according to the following formula: A i = peak area of ​​impurities in sample solution; A 总 = The sum of the corrected peak areas in the sample solution chromatogram; f = impurity correction factor, the correction factor of impurity compound I in this analytical method is 1.4, and the correction factor of impurity compound II in this analytical method is 2.2; The chemical structural formula of the 7-hydroxy-2(1H)-quinolinone is as follows: The chemical structural formula of the impurity compound I is shown in Formula I: The chemical structural formula of the impurity compound II is shown in Formula II:

2. The method for determining impurity compound I and impurity compound II in a key material of breprazol according to claim 1, characterized in that: In step (1) and step (2), the ratio of the diluent by volume is 0.1% phosphoric acid solution: acetonitrile = 70:

30.

3. The method for determining impurity compound I and impurity compound II in a key material of breprazol according to claim 1, characterized in that: In step (3), the detection wavelength of the ultraviolet detector is 218-222 nm.

4. The method for determining impurity compound I and impurity compound II in a key material of breprazol according to claim 3, characterized in that: The detection wavelength of the ultraviolet detector is 220 nm.

5. The method for determining impurity compound I and impurity compound II in a key material of breprazol according to claim 1, characterized in that: In step (3), the column temperature of the chromatographic column is 30-40°C.

6. The method for determining impurity compound I and impurity compound II in a key material of breprazol according to claim 5, characterized in that: The column temperature of the chromatographic column is 35°C.

7. The method for determining impurity compound I and impurity compound II in a key material of breprazol according to claim 1, characterized in that: In step (3), the mobile phase A is a 0.1% phosphoric acid solution, and the volume ratio of the mobile phase B is acetonitrile:methanol=(78-82):(22-18).

8. The method for determining impurity compound I and impurity compound II in a key material of breprazol according to claim 1, characterized in that: In step (3), the flow rate of the gradient elution is 0.9-1.1 ml / min.

9. The method for determining impurity compound I and impurity compound II in a key material of breprazol according to claim 8, characterized in that: The flow rate of the gradient elution was 1.0 ml / min.

10. The method for determining impurity compound I and impurity compound II in a key material of breprazol according to claim 1, characterized in that: In step (3), the gradient elution parameters are set as: 0min, the volume proportion of mobile phase A is 90%, and the volume proportion of mobile phase B is 10%; 2 min, mobile phase A volume ratio is 90%, mobile phase B volume ratio is 10%; 8 min, mobile phase A volume ratio is 80%, mobile phase B volume ratio is 20%; 15 min, mobile phase A volume ratio is 70%, mobile phase B volume ratio is 30%; 35 min, mobile phase A volume ratio is 50%, mobile phase B volume ratio is 50%; 45 min, mobile phase A volume ratio is 10%, mobile phase B volume ratio is 90%; 53min, mobile phase A volume ratio is 10%, mobile phase B volume ratio is 90%; 58min, mobile phase A volume ratio is 90%, mobile phase B volume ratio is 10%; 65min, the volume proportion of mobile phase A is 90%, and the volume proportion of mobile phase B is 10%.

Images