Method for detecting related substances in aripiprazole medicine

Abstract

The invention relates to a method for detecting related substances in an aripiprazole bulk drug or a long-acting injection of the aripiprazole bulk drug, in particular to a method for simultaneously detecting various known impurities in the aripiprazole bulk drug or the long-acting injection of the aripiprazole bulk drug by adopting high performance liquid chromatography. According to the method, effective separation and determination of various known impurities in the aripiprazole bulk drug or the long-acting injection thereof can be realized, the separation degree is high, the system durability is strong, the controllable quality of the aripiprazole bulk drug and the long-acting injection thereof can be ensured, the operation is simple, and the method is suitable for industrial application.

Description

Technical Field

[0001] Aripiprazole is a third-generation antipsychotic drug that has a bidirectional regulatory effect on the dopaminergic nervous system. It acts as a stabilizer of dopaminergic neurotransmitters and has high affinity for D2, D3, 5-HT1A, and 5-HT2A receptors. It can be used to treat schizophrenia, showing significant efficacy for both positive and negative symptoms, improving accompanying affective symptoms, and reducing the relapse rate. Aripiprazole is characterized by rapid onset of action, high safety, and good tolerability. Due to its good efficacy and relatively mild side effects, it is widely used clinically.

[0002] Abilify, a one-month long-acting injectable form of aripiprazole developed in collaboration with Otsuka and Lundbeck. ) and 2-month long-acting injectable (Abilify) Aripiprazole has been approved by the FDA for monotherapy maintenance treatment of adult patients with schizophrenia and bipolar I disorder. Long-acting aripiprazole injections can reduce the frequency of dosing and improve patient adherence. Aripiprazole contains several process impurities and oxidative degradation impurities, which require quality control in the production of aripiprazole.

[0003] CN114689767A discloses a method for detecting related substances in aripiprazole raw material or its preparations. The mobile phases A and B are both mixed solutions of acetonitrile and water, with a certain amount of acid added. The acid is one or more of glacial acetic acid, phosphoric acid, trifluoroacetic acid, and formic acid, with a concentration of 0.01–0.10% v / v. This method has good separation and detection effects on seven impurities in aripiprazole raw material, but it is not suitable for detecting related substances in long-acting aripiprazole injections.

[0004] CN112666267A discloses a method for the separation and detection of various components of aripiprazole, including an Agilent Eclipse Plus C18 chromatographic column; mobile phase A is an ammonium acetate aqueous solution-acetonitrile with a volume ratio of 90:10, and mobile phase B is an ammonium acetate aqueous solution-acetonitrile with a volume ratio of 10:90; wherein the pH of the ammonium acetate aqueous solution is 5.4-6.8. This method can be used for the detection of related substances 7-[4-[4-(2,3-dichlorophenyl)-1-oxopiperazinyl]butoxy]-3,4-dihydroquinolones and 7-[4-[4-(2,3-dichlorophenyl)-1-piperazinyl]butoxy]quinolones in aripiprazole raw materials, oral solutions, tablets, and capsules. However, when this method is used to detect related substances in long-acting aripiprazole injections, the separation effect of impurities A and B is poor, and accurate quantification is not possible.

[0005]

[0006] Currently, there are no literature reports on an effective HPLC method for separating and detecting multiple impurities that may be present in long-acting aripiprazole injections. Therefore, there is an urgent need to develop a simple, low-cost, and versatile method that can accurately and effectively detect multiple impurities in long-acting aripiprazole injections to meet the development needs of these injections. Summary of the Invention

[0007] To address the aforementioned problems in the existing technology, the main objective of this invention is to provide a method for detecting related substances in aripiprazole drugs. This method can effectively determine the main impurities in aripiprazole raw materials and long-acting injections. The method is simple to operate, provides good separation between impurities and between the main component and other impurities, and can be used for the quality control of aripiprazole drugs.

[0008] Firstly, this invention provides a method for detecting related substances in aripiprazole raw material or its long-acting injection, comprising:

[0009] (a) Prepare the test solution;

[0010] (b) The components in aripiprazole raw material or its long-acting injection were separated and detected by high performance liquid chromatography;

[0011] The chromatographic conditions for the high-performance liquid chromatography method include:

[0012] A chromatographic column with octadecylsilane-bonded silica gel as the stationary phase;

[0013] Mobile phase A is ammonium bicarbonate buffer-acetonitrile, and mobile phase B is acetonitrile. Gradient elution is performed using mobile phase A and mobile phase B.

[0014] The pH of the ammonium bicarbonate buffer solution in the mobile phase A is 8-9;

[0015] The relevant substances include one or more of impurities A, B, C, D, and P:

[0016]

[0017] In a preferred embodiment, the method for detecting related substances in the aripiprazole raw material or its long-acting injection, wherein the volume ratio of ammonium bicarbonate buffer to acetonitrile in mobile phase A is (90-98):(2-10), for example 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3 or 98:2, etc., preferably (93-95):(5-7), more preferably 95:5;

[0018] Further preferably, the concentration of the ammonium bicarbonate buffer solution is 5–15 mmol / L, more preferably 10 mmol / L. The ammonium bicarbonate buffer solution can be prepared by the following method: take an appropriate amount of ammonium bicarbonate, add water to dissolve and dilute to a specific concentration, and then adjust the pH to 8–9, preferably 8.6–9.0, with ammonia water.

[0019] In a preferred embodiment, the method for detecting related substances in the aripiprazole raw material or its long-acting injection, wherein the gradient elution includes: a volume ratio of mobile phase A to mobile phase B of (90-70):(10-30) during the first 0-5 min, and a volume ratio of mobile phase A to mobile phase B of (60-40):(40-60) during the first 10-50 min;

[0020] Preferably, the gradient elution includes: the volume ratio of mobile phase A to mobile phase B is (90-70):(10-30) during the first 0-3 min, and the volume ratio of mobile phase A to mobile phase B is (60-40):(40-60) during the first 15-45 min;

[0021] Further preferably, the gradient elution includes: a volume ratio of mobile phase A to mobile phase B of (90-70):(10-30) during the first 0-3 min, a volume ratio of mobile phase A to mobile phase B of (60-40):(40-60) during the first 15-45 min, and a volume ratio of mobile phase A to mobile phase B of (30-80):(70-20) during the first 50-65 min;

[0022] Further preferably, the gradient elution includes:

[0023]

[0024] Preferably, the gradient elution includes: a volume ratio of mobile phase A to mobile phase B of (90-70):(10-30) during the first 0-3 min, a volume ratio of mobile phase A to mobile phase B of (60-40):(40-60) during the first 15-45 min, a volume ratio of mobile phase A to mobile phase B of (20-40):(80-60) during the first 50-55 min, and a volume ratio of mobile phase A to mobile phase B of (90-70):(10-30) during the first 56-65 min.

[0025] Further preferably, the gradient elution includes:

[0026]

[0027]

[0028] In a preferred embodiment, the method for detecting related substances in the aripiprazole active pharmaceutical ingredient or its long-acting injection further includes, wherein the chromatographic conditions include:

[0029] Detection is performed using an ultraviolet detector with a detection wavelength of 250nm–260nm, preferably 252–256nm, and more preferably 254nm; and / or,

[0030] The column temperature is 20–40°C, preferably 33–37°C, more preferably 35°C; and / or,

[0031] The injection volume is 5–15 μl, preferably 10 μl; and / or,

[0032] The flow rate is 0.8 to 1.2 ml / min, such as 0.8 ml / min, 0.9 ml / min, 1.0 ml / min, 1.1 ml / min and 1.2 ml / min, preferably 0.9 to 1.1 ml / min, more preferably 1.0 ml / min.

[0033] In a preferred embodiment, the method for detecting related substances in the aripiprazole raw material or its long-acting injection comprises a chromatographic column with the following specifications: column length between 150 mm and 300 mm, column inner diameter between 1 mm and 10 mm, and particle size between 1.9 and 5 μm, preferably 3 μm. The preferred column specifications are 4.6 mm * 150 mm and 3 μm; more preferably, a Shim-pack Scepter C18-120, 4.6 × 150 mm, 3 μm, or a column with equivalent performance.

[0034] During chromatographic separation, especially in gradient elution or after prolonged instrument use, intermittent chromatographic peaks, commonly known as ghost peaks, can easily occur. Ghost peaks originate from various sources, primarily the mobile phase and tubing, such as contaminants in the organic phase, contaminants in the aqueous phase, buffer salts, mobile phase bottles, and the mixing process. Ghost peaks typically cause problems such as instrument contamination, misjudgment of sample composition, overlap with target peaks, affecting resolution, and increasing workload, requiring more validation work to determine if the substance is indeed the target. In reversed-phase gradient chromatographic analysis, a ghost peak trapping column installed between the gradient mixer and the autosampler can not only remove impurities from the mobile phase but also effectively trap impurities from the tubing and mixer. The use of a ghost peak trapping column is not mandatory; for example, if no ghost peaks appear during elution, it is not necessary.

[0035] Optionally, the chromatographic conditions in the method of this invention further include a ghost peak collecting column, preferably selected from a Ghost Sniper Column or a column of equivalent performance; specifically, the ghost peak collecting column may be, for example, a Ghost Sniper Column, 30*4.0 mm, or a column of equivalent performance. In another specific embodiment, the chromatographic conditions in the method of this invention do not include a ghost peak collecting column.

[0036] In a preferred embodiment, the method for detecting related substances in the aripiprazole raw material or its long-acting injection includes the following steps: taking the test sample, accurately weighing it, dissolving it in a diluent, and diluting it to obtain the solution; preferably, the concentration of the test sample solution is about 0.5 mg to 1.5 mg of aripiprazole per 1 ml of solution, preferably 0.8 mg.

[0037] Further preferably, the method further includes the preparation of a reference solution, comprising: accurately weighing aripiprazole raw material or its long-acting injectable reference standard, adding a diluent, dissolving and diluting to obtain the solution; preferably, the concentration of the reference solution is approximately 0.4 to 4 μg of aripiprazole per 1 ml of solution, more preferably approximately 2 μg;

[0038] Optionally, the method further includes the preparation of a system suitability solution, wherein the preparation of the system suitability solution includes: accurately weighing aripiprazole raw material or its long-acting injectable reference standard, and one or more reference standards of impurity A, impurity B, impurity C, impurity D and impurity P, adding an appropriate amount of diluent, dissolving and diluting to prepare the solution; more preferably, the concentration of the system suitability solution is approximately 0.5 to 3 mg of aripiprazole per 1 ml, preferably approximately 1 mg, and approximately 0.4 to 4 μg of each impurity, preferably approximately 2 μg.

[0039] In a preferred embodiment, the diluent is a mixture of sodium sulfate aqueous solution, acetonitrile, methanol, and glacial acetic acid.

[0040] Further preferably, the volume ratio of sodium sulfate aqueous solution - acetonitrile - methanol - glacial acetic acid in the diluent is (50-60):(30-40):(5-15):(0.5-2), more preferably 56:33:11:1; the concentration of the sodium sulfate aqueous solution is 10-30 mmol / L, preferably 20 mmol / L;

[0041] The diluent can be prepared by the following method: Take an appropriate amount of anhydrous sodium sulfate, dissolve it in water and dilute it to the corresponding concentration, then measure an appropriate amount and mix it thoroughly with the corresponding volumes of acetonitrile, methanol and glacial acetic acid. For example, the diluent is a sodium sulfate aqueous solution-acetonitrile-methanol-glacial acetic acid solution, wherein the concentration of the sodium sulfate aqueous solution is 20 mmol / L, and the volume ratio of sodium sulfate aqueous solution-acetonitrile-methanol-glacial acetic acid is 56:33:11:1. Its preparation method is as follows: Take 2.84 g of anhydrous sodium sulfate, dissolve it in water and dilute it to 1000 ml, measure 560 ml of this solution and mix it thoroughly with 330 ml of acetonitrile, 110 ml of methanol and 10 ml of glacial acetic acid.

[0042] In a preferred embodiment, a method for detecting related substances in the aripiprazole raw material or its long-acting injection, wherein the long-acting aripiprazole injection is an aripiprazole injection in the form of sterile lyophilized powder or sterile suspension; preferably, the long-acting aripiprazole injection comprises aripiprazole raw material, a suspending agent, a filler, and a buffer; wherein the suspending agent comprises one or more of carboxymethyl cellulose or its sodium salt, polyvinylpyrrolidone, HPMC, HMC, or polyethylene glycol; the filler is selected from one or more of mannitol, lactose, sucrose, maltose, or sorbitol; and the buffer is selected from a phosphate buffer system; the phosphate buffer system includes, for example, sodium phosphate (such as sodium dihydrogen phosphate, disodium hydrogen phosphate), potassium phosphate (such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate), etc., with the pH adjusted by hydrochloric acid or sodium hydroxide.

[0043] Further preferably, the long-acting aripiprazole injection comprises aripiprazole or its monohydrate, sodium carboxymethyl cellulose, polyethylene glycol 400, polyvinylpyrrolidone, and sodium dihydrogen phosphate monohydrate; particularly preferably, the long-acting aripiprazole injection comprises aripiprazole or its monohydrate, sodium carboxymethyl cellulose, polyethylene glycol 400, polyvinylpyrrolidone, sodium chloride, sodium dihydrogen phosphate monohydrate, sodium hydroxide for pH adjustment, and water for injection; or,

[0044] The aripiprazole long-acting injection contains aripiprazole or its monohydrate, sodium carboxymethyl cellulose, mannitol, sodium dihydrogen phosphate monohydrate, and sodium hydroxide to adjust the pH.

[0045] More preferably, the aripiprazole long-acting injection of the present invention is the same as the one marketed under the name Abilify Maintena or The products are the same or substantially the same; the term substantially similar means that the key excipients of the two formulations are the same or have similar properties, and / or the in vitro dissolution similarity (f2) is greater than 50 when tested using the same method, and / or the in vivo bioavailability is equivalent.

[0046] The aripiprazole long-acting injection applicable to this invention can be prepared by referring to the methods disclosed in patents CN1870980A, CN101801342A, CN1871007A, or CN106389357A, etc., and the entire contents of these patent disclosures are incorporated herein by reference.

[0047] In some embodiments of the present invention, the method for detecting related substances in the aripiprazole active pharmaceutical ingredient or its long-acting injection includes accurately measuring the test solution and injecting it into a liquid chromatograph, and recording the chromatogram. More preferably, the method involves first accurately measuring the diluent solution, optionally the system suitability solution, and the reference solution, injecting them into the liquid chromatograph, and recording the chromatograms, and then accurately measuring the test solution and injecting it into the liquid chromatograph, and recording the chromatogram.

[0048] In some embodiments of the present invention, the method for detecting related substances in the aripiprazole raw material or its long-acting injection involves calculating the impurity content using the external standard method or the principal component external standard method with a correction factor; specifically,

[0049] The formula for calculating the impurity content using the external standard method is as follows:

[0050]

[0051] The formula for calculating the impurity content using the principal component external standard method with correction factors is shown below:

[0052]

[0053] Among them, A T : Peak area of ​​impurities in the test solution;

[0054] A S : The average peak area of ​​aripiprazole in five consecutive reference solutions;

[0055] C S : Concentration of the reference standard;

[0056] C T The concentration of the test sample;

[0057] f: Correction factor for impurities relative to the main peak;

[0058] More preferably, the correction factor of impurity A relative to the main peak is 1.0, the correction factor of impurity B relative to the main peak is 1.2, the correction factor of impurity C relative to the main peak is 0.72, the correction factor of impurity P relative to the main peak is 0.77, and the correction factor of the unknown impurity relative to the main peak is 1.0.

[0059] The detection method of this invention can determine five common related substances in aripiprazole raw material or its long-acting injection, particularly impurities A and B, under the same liquid phase conditions. The method is simple and rapid to operate. Compared with existing methods, this invention can not only detect related substances in aripiprazole raw material, but more importantly, it can detect multiple related substances that may be present in aripiprazole long-acting injection, such as impurities A, B, C, D, and P. Furthermore, the resolution between the main component and its adjacent impurities, and between known impurities and their adjacent impurities, is greater than 1.5. All known impurities can be effectively detected with high resolution, providing a simple and rapid detection method for quality control and impurity research of aripiprazole raw material or its long-acting injection. This invention is applicable to the determination of related substances and sample testing of aripiprazole raw material or its long-acting injection.

[0060] The aripiprazole active pharmaceutical ingredient described in the context of this invention may be aripiprazole in the form of a free base or in its hydrated form, such as aripiprazole in the form of a hydrate, or in the form of a pharmaceutically acceptable salt of aripiprazole. In a preferred embodiment of this invention, the aripiprazole active pharmaceutical ingredient is aripiprazole in the form of a free base or aripiprazole in the form of a hydrate.

[0061] The beneficial effects of this invention are:

[0062] This invention provides a method for detecting related substances in aripiprazole raw material or its long-acting injection. The invention scientifically selects anhydrous sodium sulfate aqueous solution-methanol-acetonitrile-acetic acid as a diluent, which provides good sample solubility and does not interfere with the detection of various impurities. The detection method uses a binary mobile phase system, which has low equipment requirements and wide applicability. Furthermore, the method utilizes optimized mobile phases A and B, where mobile phase A is ammonium bicarbonate buffer-acetonitrile and mobile phase B is acetonitrile. The pH of the ammonium bicarbonate buffer in mobile phase A is adjusted to 8-9. This allows for the simultaneous detection and quantification of multiple impurities in aripiprazole raw material or its long-acting injection, including impurity A, impurity B, impurity C, impurity D, and impurity P. System adaptability verification experiments show that the resolution between the main component and its adjacent impurities, and between known impurities and their adjacent impurities, is greater than 1.5, indicating good resolution, strong method specificity, high sensitivity, simple operation, and high detection efficiency. In particular, the method provided by this invention solves the problem that existing methods cannot detect and quantify impurities A and B in long-acting aripiprazole injections. Other features and beneficial effects of this invention will be further illustrated and explained through the following examples and accompanying drawings.

[0063] Terminology Definition

[0064] This invention is intended to cover all alternatives, modifications, and equivalents, all of which are included within the scope of the invention as defined in the claims. Those skilled in the art will recognize that many similar or equivalent methods and materials described herein can be used to practice this invention. This invention is by no means limited to the methods and materials described herein.

[0065] It should be further appreciated that certain features of the invention, for clarity, have been described in multiple independent embodiments, but may also be provided in combination in a single embodiment. Conversely, various features of the invention, for brevity, have been described in a single embodiment, but may also be provided individually or in any suitable sub-combination.

[0066] Unless otherwise stated, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. All patents and publications related to this invention are incorporated herein by reference in their entirety.

[0067] The terms “comprising,” “including,” or “selected from” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0068] In the context of this invention, all figures disclosed herein are approximate values, regardless of whether the words "about" or "approximately" are used. Each figure may vary by less than 10% or by a difference that is considered reasonable by those skilled in the art, such as 1%, 2%, 3%, 4%, or 5%.

[0069] The abbreviations "mM" or "mmol / L" both refer to millimoles per liter (0.001 mol / L). For example, "10mM ammonium bicarbonate buffer" means 0.01 mol / L ammonium bicarbonate buffer, which can also be written as 10 mmol / L ammonium bicarbonate buffer.

[0070] RT(min): Represents the relative retention time in minutes. Attached Figure Description

[0071] Figure 1 : Displays the HPLC chromatogram of the diluent detected in Example 1;

[0072] Figure 2 : Displays the HPLC chromatogram of the system suitability solution tested in Example 1; where the impurity "7-qjklt" with a retention time of 3.377 min refers to impurity C;

[0073] Figure 3 : Displays the HPLC chromatogram of the reference solution tested in Example 1;

[0074] Figure 4: Shows the HPLC chromatogram of the aripiprazole long-acting injection test sample as detected in Example 1;

[0075] Figure 5 The HPLC chromatogram of aripiprazole and various impurities in the system suitability solution detected by the chromatographic method in Comparative Example 2 is shown. It can be seen from the chromatogram that the main peak and the various impurities cannot be completely separated.

[0076] Figure 6 The HPLC chromatogram of aripiprazole and various impurities in the system suitability solution detected by the method of Comparative Example 3 is shown. It can be seen from the figure that the main peak has severe tailing and impurity B and impurity P cannot be separated.

[0077] Figure 7 The figure shows the HPLC chromatogram of aripiprazole and various impurities in the test solution detected by the chromatographic method in Comparative Example 5. It can be seen from the figure that impurity B and impurity P cannot be separated. Detailed Implementation

[0078] The technical solution and beneficial effects of the present invention will be further explained below with reference to specific embodiments.

[0079] This invention provides a method for detecting related substances in aripiprazole long-acting injection, the method comprising:

[0080] (a) Preparation of the test solution: Aripiprazole long-acting injection test sample was prepared by diluting with a diluent; the concentration of aripiprazole in the test solution was about 0.5 mg / ml to 1.5 mg / ml, preferably about 0.8 mg / ml;

[0081] (b) Detection is performed using high-performance liquid chromatography (HPLC), wherein the HPLC detection conditions include:

[0082] Detection is performed using an ultraviolet detector with a wavelength of 250nm–260nm, preferably 254nm.

[0083] The chromatographic column is a stationary phase composed of octadecylsilane-bonded silica gel.

[0084] Mobile phase A is 0.005-0.01 mol / ml ammonium bicarbonate buffer-acetonitrile, preferably the ammonium bicarbonate buffer is adjusted to pH 8-9 with ammonia, more preferably to pH 8.6-9.0; even more preferably, the volume ratio of ammonium bicarbonate buffer to acetonitrile is (90-98):(2-10), more preferably 95:5;

[0085] Mobile phase B is acetonitrile;

[0086] Gradient elution is performed using mobile phase A and mobile phase B. The gradient elution process includes: during the first 0-3 minutes, the volume ratio of mobile phase A to mobile phase B is (90-70):(10-30), and during the first 15-45 minutes, the volume ratio of mobile phase A to mobile phase B is (60-40):(40-60).

[0087] Preferably, the gradient elution process includes:

[0088]

[0089] Further preferably, the gradient elution process includes:

[0090]

[0091] The flow rate is 0.8–1.2 ml / min, preferably 1 ml / min;

[0092] The column temperature is 20–40℃, preferably 35℃;

[0093] The injection volume is 5 μl to 15 μl, preferably 10 μl;

[0094] The diluent is a mixture of sodium sulfate aqueous solution, acetonitrile, methanol, and glacial acetic acid; preferably, the volume ratio of sodium sulfate aqueous solution, acetonitrile, methanol, and glacial acetic acid in the diluent is (50-60):(30-40):(5-15):(0.5-2), more preferably 56:33:11:1; the concentration of the sodium sulfate aqueous solution is preferably 10-30 mmol / L, more preferably 20 mmol / L.

[0095] Accurately measure the sample solution and inject it into the liquid chromatograph, then record the chromatogram.

[0096] In a preferred embodiment, the method further includes:

[0097] Preparation of reference solution: Aripiprazole or its monohydrate reference standard is accurately weighed and diluted with a diluent; the concentration of aripiprazole in the reference solution is approximately 0.4 μg / ml to 4 μg / ml, more preferably 2 μg / ml;

[0098] Accurately measure the diluent solution and the reference solution and inject them into the liquid chromatograph, and record the chromatograms.

[0099] Optionally, the method further includes the preparation of a system suitability solution: accurately weigh aripiprazole or its monohydrate reference standard, and one or more reference standards selected from impurity A, impurity B, impurity C, impurity D, and impurity P, respectively, and place them in the same volumetric flask, and prepare the solution with a diluent; preferably, the concentration of the system suitability solution is about 0.5 to 1.0 mg of aripiprazole per 1 ml, more preferably about 1 mg, and about 0.4 to 4 μg of each impurity, preferably about 2 μg; before testing the test sample, accurately inject the diluent solution, system suitability solution, and reference standard solution into the liquid chromatograph and record the chromatogram, and then accurately inject the test sample solution into the liquid chromatograph and record the chromatogram.

[0100] More preferably, the detection method uses the external standard method or the principal component external standard method with a correction factor to calculate the impurity content. Specifically, the formula for calculating the impurity content using the external standard method is as follows:

[0101]

[0102] The formula for calculating the impurity content using the principal component external standard method with correction factors is shown below:

[0103]

[0104] Among them, A T : Peak area of ​​impurities in the test solution;

[0105] A S : The average peak area of ​​aripiprazole in five consecutive reference solutions;

[0106] C S : Concentration of the reference standard;

[0107] C T The concentration of the test sample;

[0108] f: Correction factor for impurities relative to the main peak;

[0109] The preferred correction factor for impurity A relative to the main peak is 1.0, for impurity B relative to the main peak is 1.2, for impurity C relative to the main peak is 0.72, for impurity P relative to the main peak is 0.77, and for unknown impurities relative to the main peak is 1.0.

[0110] In the above method, the long-acting aripiprazole injection is preferably an aripiprazole injection in the form of sterile lyophilized aripiprazole or sterile suspension, comprising aripiprazole or its monohydrate, a suspending agent, a filler, and a buffer; wherein the suspending agent is selected from one or more of carboxymethyl cellulose or its sodium salt, polyvinylpyrrolidone, HPMC, HMC, or polyethylene glycol; the filler is selected from one or more of mannitol, lactose, sucrose, maltose, or sorbitol; and the buffer is selected from a phosphate buffer system. More preferably, the long-acting aripiprazole injection comprises aripiprazole or its monohydrate, sodium carboxymethyl cellulose, polyethylene glycol 400, polyvinylpyrrolidone, sodium chloride, sodium dihydrogen phosphate monohydrate, sodium hydroxide to adjust pH, and water for injection; or, the long-acting aripiprazole injection comprises aripiprazole or its monohydrate, sodium carboxymethyl cellulose, mannitol, sodium dihydrogen phosphate monohydrate, and sodium hydroxide to adjust pH.

[0111] Example:

[0112] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions or as recommended by the manufacturer. All mobile phase solvents used were chromatographically pure reagents. The aripiprazole long-acting injection used in the following examples is a sterile lyophilized aripiprazole solution (400 mg / vial), containing aripiprazole monohydrate, sodium carboxymethyl cellulose, mannitol, sodium dihydrogen phosphate monohydrate, and sodium hydroxide to adjust the pH.

[0113] Example 1: Detection method for related substances in aripiprazole long-acting injection

[0114] Chromatographic conditions:

[0115] Chromatographic column: SHIMADZU Shim-pack Scepter C18-120, 4.6mm×150mm, 3μm or equivalent column;

[0116] Mobile phase A: 10 mM ammonium bicarbonate buffer (pH 8.8) - acetonitrile (95:5);

[0117] Mobile phase B: Acetonitrile;

[0118] Detection wavelength: 254nm;

[0119] Column temperature: 35℃;

[0120] Flow rate: 1 ml / min;

[0121] Injection volume: 10 μl;

[0122] Perform linear gradient elution according to the table below.

[0123]

[0124]

[0125] Solution preparation:

[0126] (1) Preparation of mobile phase

[0127] Mobile phase A: 10mM ammonium bicarbonate buffer (pH 8.8) - acetonitrile (95:5): Dissolve 0.79g of ammonium bicarbonate in water and dilute to 1000ml. Adjust the pH to 8.8 with ammonia, filter, and use as the buffer solution. Measure 950ml of the buffer solution and mix thoroughly with 50ml of acetonitrile, sonicate to degas, and obtain the final mobile phase.

[0128] (2) Preparation of diluent solution:

[0129] 20mM sodium sulfate solution-acetonitrile-methanol-glacial acetic acid (560:330:110:10): Dissolve 2.84g of anhydrous sodium sulfate in water and dilute to 1000ml. Measure 560ml of the sodium sulfate solution and mix thoroughly with 330ml of acetonitrile, 110ml of methanol, and 10ml of glacial acetic acid.

[0130] (3) Preparation of test solution:

[0131] Take one vial of aripiprazole long-acting injection (specification: 400mg / vial), add 1.9ml of water and mix. Shake vigorously, take 1.0ml of the suspension and place it in a 200ml volumetric flask. Add an appropriate amount of diluent, sonicate for 10min, dissolve, and then bring the volume to the mark and shake well.

[0132] (4) Preparation of reference solution:

[0133] Accurately weigh aripiprazole monohydrate reference standard, dissolve it in an appropriate amount of diluent, shake well, and prepare a solution containing approximately 2 μg of aripiprazole per 1 ml of solution.

[0134] (5) System suitability solution preparation

[0135] Accurately weigh aripiprazole monohydrate reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard and impurity P reference standard, place them in the same volumetric flask, add an appropriate amount of diluent, and dilute to prepare a solution containing approximately 1 mg of aripiprazole and approximately 2 μg of each impurity per 1 ml.

[0136] Accurately measure 10 μl each of the diluent solution, system suitability solution, reference solution, and test solution and inject them into the liquid chromatograph, then record the chromatograms.

[0137] Test results as follows Figures 1-4 As shown, the HPLC chromatogram of the diluent solution is as follows: Figure 1As shown, the results indicate that the diluent does not interfere with impurity detection.

[0138] The HPLC chromatogram of the system suitability solution is shown below. Figure 2 As shown in Table 1.1, the detection results indicate that impurity C (abbreviation: 7-qjklt), impurity D, impurity A, impurity P, impurity B, and aripiprazole precipitated in that order.

[0139] The HPLC chromatogram of the reference solution is shown below. Figure 3 As shown, the RT for aripiprazole was 35.767 min;

[0140] The HPLC chromatogram of the test solution is shown below. Figure 4 As shown, the peaks that emerge in sequence are impurity A (RT = 13.958 min), impurity B (RT = 28.760 min), and aripiprazole (RT = 35.832 min). The resolutions between impurity A and B, and between impurity B and aripiprazole, are 43.76 and 12.19, respectively. The resolutions between the main peak and each impurity, as well as among the impurities, are very high.

[0141] Table 1.1 System Suitability Solution Test Results

[0142] Compound Name retention time min Resolution Impurity C 3.377 / Impurity D 11.370 49.25 Impurity A 14.310 17.35 impurity P 21.210 22.60 Impurity B 28.517 15.40 aripiprazole 34.380 10.80

[0143] from Figure 1 It can be seen that the diluent solution does not interfere with the detection;

[0144] from Figure 2 As shown in Table 1.1, in the system applicability solution, impurities C, D, A, P, B, and aripiprazole elute in that order. The separation degree between the main component and its adjacent impurities, and between known impurities and their adjacent impurities, is much greater than 1.5, indicating that the separation effect is good.

[0145] Example 2: Durability Test

[0146] The standard conditions were the chromatographic conditions listed in Example 1. While keeping other conditions constant, one of the following was changed each time: wavelength, column temperature, or pH value. The effect of this change on resolution was studied. 10 μl each of the diluent solution and system suitability solution were accurately measured and injected into the liquid chromatograph. Chromatograms were recorded, and the resolution was calculated using the external standard method based on peak area. The preparation methods for the diluent solution and system suitability solution were the same as in Example 1. The detection conditions and results are shown in Table 2.1.

[0147] Table 2.1: Results of Solution Separation Test for System Suitability under Various Durability Conditions

[0148]

[0149] The test results are shown in Table 2.1. Under various changing conditions, the diluent solution did not interfere with the detection. In the system suitability solution, the separation degree between the main component, known impurities and adjacent impurities was much greater than 1.5, indicating good separation. The ratio of the content of the same impurity measured in the same sample solution to the result under standard conditions was between 80% and 120%, which met the requirements.

[0150] Example 3: Limit of Detection and Limit of Quantification Experiment

[0151] The known limits of detection (LOD) and limits of quantitation (LOQ) for impurities are determined using the signal-to-noise ratio (SNR) method. A stock solution of impurities of known concentration is diluted with a diluent to form a low-concentration sample. The measured signal is compared with the signal at the blank, and the lowest concentration or percentage that can be reliably detected is calculated. When S / N≈3, it is the limit of detection, and when S / N≈10, it is the limit of quantitation.

[0152] Table 3.1 Detection Limit

[0153]

[0154] 3.2 Limit of Quantification

[0155]

[0156]

[0157] The results showed that when the concentration of each impurity was 6% of the limit concentration, the signal-to-noise ratio was much greater than 3, and the RSD of the peak areas of the main component and each impurity in the 6 limit solutions were all much less than 10%, indicating that the method of the present invention has high detection sensitivity and can accurately control the content of each impurity.

[0158] Through repeatability experiments, the method provided in this invention was used to detect six 100% spiked test solutions. The results showed that the RSD of impurity C (7-hydroxyquinolineone) was 0.0%; the RSD of impurity D was 2.1%; the RSD of impurity A was 0.0%; the RSD of impurity P was 0.0%; the RSD of impurity B was 1.8%; no other single impurities were detected, and the total impurity content RSD was 0.7%, indicating that the method provided in this invention has good repeatability.

[0159] Comparative Example 1: The Influence of Flow Relative Detection Results

[0160] Chromatographic conditions:

[0161] Chromatographic column: Shim-pack Scepter C18-120 3μm 4.6×150mm LC079;

[0162] Mobile phase A: 10 mM potassium dihydrogen phosphate (pH 8.8);

[0163] Mobile phase B: Acetonitrile;

[0164] Detection wavelength: 254nm;

[0165] Column temperature: 35℃;

[0166] Flow rate: 1.0 ml / min;

[0167] Injection volume: 10 μl;

[0168] Perform linear gradient elution according to the table below.

[0169]

[0170] Solution preparation:

[0171] Diluent: 20mM anhydrous sodium sulfate-acetonitrile-acetic acid (660:340:10), prepared in the same way as in Example 1;

[0172] System suitability solution: Preparation method is the same as in Example 1;

[0173] Precisely inject 10 μl each of the diluent solution and the system suitability solution into the liquid chromatograph and record the chromatograms. Perform five injections of the same solution (labeling the results as SST-1 to SST-5), and the results are shown in Table D1.1 below.

[0174] Table D1.1 Repeatability Test Results

[0175] SST-1 SST-2 SST-3 SST-4 SST-5 Impurity Name RT(min) RT(min) RT(min) RT(min) RT(min) Impurity C 2.210 2.207 2.207 2.205 2.203 Impurity D 4.282 4.267 4.253 4.243 4.237 Impurity A 9.665 9.608 9.568 9.523 9.503 impurity P 22.978 22.922 22.880 22.843 22.818 Impurity D 24.825 24.775 24.738 24.708 24.687 Impurity B 25.827 25.773 25.735 25.705 25.685 aripiprazole 28.702 28.650 28.612 28.582 28.562

[0176] The above experimental results show that the chromatographic conditions have poor repeatability, and the main peak and impurity peaks are shifted.

[0177] Comparative Example 2:

[0178] Chromatographic conditions:

[0179] Chromatographic column: Shim-pack GIST C18-AQ 5μm 4.6×250mm LC073;

[0180] Mobile phase A: 0.05% TFA;

[0181] Mobile phase B: Acetonitrile;

[0182] Detection wavelength: 254nm;

[0183] Column temperature: 30℃;

[0184] Flow rate: 1.2 ml / min;

[0185] Injection volume: 20 μl;

[0186] Perform linear gradient elution according to the table below.

[0187]

[0188] Solution preparation:

[0189] The diluent preparation method is the same as in Example 1;

[0190] Preparation of system suitability solution: Accurately weigh aripiprazole monohydrate reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard and impurity P reference standard, place them in the same volumetric flask, add an appropriate amount of diluent, and dilute to prepare a solution containing approximately 0.5 mg of aripiprazole and approximately 2 μg of each impurity per 1 ml.

[0191] Precisely measure 20 μl each of the diluent solution and the system suitability solution and inject them into the liquid chromatograph, then record the chromatograms.

[0192] Test results as follows Figure 5 As shown, the separation before and after the main peak is poor, and the impurities after the main peak are almost completely encapsulated.

[0193] Comparative Example 3:

[0194] Chromatographic conditions:

[0195] Column: Shim-pack Scepter Phenyl-120 3μm 4.6×150mm LC078;

[0196] Mobile phase A: 20 mM ammonium acetate - acetonitrile - acetic acid (660:340:10);

[0197] Mobile phase B: Acetonitrile;

[0198] Detection wavelength: 254nm;

[0199] Column temperature: 30℃;

[0200] Flow rate: 1.0 ml / min;

[0201] Injection volume: 20 μl;

[0202] Isocratic elution for 40 min;

[0203] Solution preparation:

[0204] The diluent preparation method is the same as in Example 1;

[0205] Preparation of system suitability solution: Accurately weigh aripiprazole monohydrate reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard, impurity P reference standard, as well as impurity C1 (3-dechloroaripiperazole) reference standard, impurity D1 (2-dechloroaripiperazole) reference standard, and impurity F reference standard. Place them in the same volumetric flask, add an appropriate amount of diluent, and dilute to prepare a solution containing approximately 1 mg of aripiprazole and approximately 2 μg of each impurity per ml. Accurately inject 20 μl each of the diluent solution and the system suitability solution into the liquid chromatograph and record the chromatogram.

[0206] like Figure 6 As shown, the relative retention time of aripiprazole was 11.083 min. Figure 6 As can be seen above, the main peak has severe tailing, and the separation between impurity B and impurity P is less than 1.5, indicating poor separation.

[0207] Comparative Example 4: Diluent Investigation

[0208] The study investigated the preparation of aripiprazole long-acting injection test solutions using methanol-acetonitrile-water-glacial acetic acid in different volume ratios as diluents. During the experiment, it was found that the diluent could not dissolve the preparation sample.

[0209] Comparative Example 5:

[0210] Chromatographic conditions:

[0211] Chromatographic column: Agilent Eclipse Plus C18 column, 4.6mm × 250mm, 5μm;

[0212] Mobile phase A: 0.01 mol / L sodium dodecyl sulfate citrate buffer (pH 5.2) - acetonitrile (55:45);

[0213] Mobile phase B: 0.01 mol / L sodium dodecyl sulfate citrate buffer (pH 5.2) - acetonitrile (38:62);

[0214] Detection wavelength: 254nm;

[0215] Column temperature: 40℃;

[0216] Flow rate: 1.0 ml / min;

[0217] Injection volume: 20 μl;

[0218] Perform linear gradient elution according to the table below.

[0219]

[0220]

[0221] Solution preparation:

[0222] The diluent preparation method is the same as in Example 1;

[0223] Preparation of system suitability solution: Accurately weigh aripiprazole monohydrate reference standard, impurity A reference standard, impurity B reference standard and impurity P reference standard, place them in the same volumetric flask, add an appropriate amount of diluent, and dilute to prepare a solution containing approximately 0.5 mg of aripiprazole and approximately 2 μg of each impurity per 1 ml.

[0224] Precisely measure 20 μl each of the diluent solution and the system suitability solution and inject them into the liquid chromatograph, then record the chromatograms.

[0225] During the detection process, it was found that impurity B (relative retention time RT = 25.917 min) and photodegradation impurity P (relative retention time RT = 26.272 min) were indistinguishable, so the detection was terminated. The HPLC chromatogram is shown below. Figure 7 As shown.

Claims

1. A method for detecting related substances in aripiprazole raw material or its long-acting injection, comprising: (a) Prepare the test solution; (b) The components in aripiprazole raw material or its long-acting injection were separated and detected by high performance liquid chromatography; The chromatographic conditions for the high-performance liquid chromatography method include: A chromatographic column with octadecylsilane-bonded silica gel as the stationary phase; Mobile phase A is ammonium bicarbonate buffer-acetonitrile, and mobile phase B is acetonitrile. Gradient elution is performed using mobile phase A and mobile phase B. The pH of the ammonium bicarbonate buffer solution in the mobile phase A is 8-9; The relevant substances include one or more of impurities A, B, C, D, and P:

2. The method according to claim 1, characterized in that, The volume ratio of ammonium bicarbonate buffer to acetonitrile in mobile phase A is (90-98):(2-10), preferably (93-95):(5-7), and more preferably 95:5; More preferably, the concentration of the ammonium bicarbonate buffer solution is 5–15 mmol / L, more preferably 10 mmol / L.

3. The method according to any one of claims 1 to 2, characterized in that, The gradient elution includes: during the first 0-5 min, the volume ratio of mobile phase A to mobile phase B is (90-70):(10-30), and during the first 10-50 min, the volume ratio of mobile phase A to mobile phase B is (60-40):(40-60). Preferably, the volume ratio of mobile phase A to mobile phase B is (90-70):(10-30) during the first 0-3 min, and (60-40):(40-60) during the last 15-45 min. Further preferably, the gradient elution includes: Further preferably, the gradient elution includes: a volume ratio of mobile phase A to mobile phase B of (90-70):(10-30) during the first 0-3 min, a volume ratio of mobile phase A to mobile phase B of (60-40):(40-60) during the first 15-45 min, a volume ratio of mobile phase A to mobile phase B of (20-40):(80-60) during the first 50-55 min, and a volume ratio of mobile phase A to mobile phase B of (90-70):(10-30) during the first 56-65 min. Further preferably, the gradient elution includes:

4. The method according to any one of claims 1 to 3, characterized in that, The chromatographic conditions also include: Detection is performed using an ultraviolet detector with a detection wavelength of 250 nm to 260 nm, preferably 252 nm to 256 nm; and / or, The column temperature is 20–40°C, preferably 33–37°C; and / or, The injection volume is 5–15 μl, preferably 10 μl; and / or, The flow rate is 0.8–1.2 ml / min, preferably 0.9–1.1 ml / min.

5. The method according to any one of claims 1 to 4, characterized in that, The chromatographic column is a SHIMADZU Shim-pack Scepter C18-120 or a column with equivalent performance; Optionally, the chromatographic conditions further include a ghost peak trapping column, preferably selected from Ghost Sniper Column or a column with equivalent performance.

6. The method according to any one of claims 1 to 5, characterized in that, The preparation of the test sample solution includes: taking the test sample, accurately weighing it, dissolving and diluting it with a diluent; Further preferably, the method also includes the preparation of a reference solution, comprising: accurately weighing aripiprazole raw material or its long-acting injectable reference standard, adding a diluent, dissolving and diluting to prepare the solution; Optionally, the method further includes the preparation of a system suitability solution, wherein the preparation of the system suitability solution includes: accurately weighing aripiprazole raw material or its long-acting injectable reference standard, and one or more reference standards of impurity A, impurity B, impurity C, impurity D and impurity P, adding an appropriate amount of diluent, and dissolving and diluting to prepare the solution.

7. The method according to claim 6, characterized in that, The diluent is a mixture of sodium sulfate aqueous solution, acetonitrile, methanol, and glacial acetic acid. Preferably, the volume ratio of sodium sulfate aqueous solution-acetonitrile-methanol-glacial acetic acid in the diluent is (50-60):(30-40):(5-15):(0.5-2), more preferably 56:33:11:1; Further preferably, the concentration of the sodium sulfate aqueous solution is 10–30 mmol / L, preferably 20 mmol / L.

8. The method according to any one of claims 1 to 7, characterized in that, The aripiprazole long-acting injection is an aripiprazole injection in the form of sterile lyophilized aripiprazole or sterile suspension. Preferably, the aripiprazole long-acting injection comprises aripiprazole active pharmaceutical ingredient, a suspending agent, a filler, and a buffer; wherein the suspending agent is selected from one or more of carboxymethyl cellulose or its sodium salt, polyvinylpyrrolidone, HPMC, HMC, or polyethylene glycol; the filler is selected from one or more of mannitol, lactose, sucrose, maltose, or sorbitol; and the buffer is selected from a phosphate buffer system.

9. The method according to any one of claims 1 to 8, characterized in that, The content of impurities was calculated using the principal component external standard method with correction factors, where the correction factor for impurity A relative to the main peak was 1.0, and the correction factor for impurity B relative to the main peak was 1.

2.

10. The method according to any one of claims 1 to 9, characterized in that, The method can simultaneously detect impurities A, B, C, D and P in aripiprazole raw material or its long-acting injection, and the separation degree between the main component and its adjacent impurities, and between known impurities and their adjacent impurities is greater than 1.5.

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