Method for detecting related substances in paliperidone

Abstract

The invention relates to a method for detecting related substances in paliperidone, in particular to a method for simultaneously detecting eight known impurities in paliperidone by adopting a high performance liquid chromatography, the method can realize effective separation and determination of the eight known impurities in paliperidone, the separation degree is high, the system durability is strong, and the method is suitable for industrial production. The method is simple in operation, high in detection sensitivity and accuracy, capable of ensuring controllable quality of paliperidone raw material medicines and preparations, simple to operate and suitable for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for detecting related substances in paliperidone. Background Technology

[0002] Paliperidone is the major active metabolite of the atypical antipsychotic drug risperdone. In the liver, risperdone is primarily hydroxylated to 9-hydroxyrisperdone, or paliperidone, via the cytochrome P450 2D6 pathway. Paliperidone possesses pharmacological characteristics and potency comparable to risperdone, but with a longer elimination half-life. The structure of paliperidone is shown below:

[0003]

[0004] CN1022566C discloses the following method for synthesizing paliperidone:

[0005]

[0006] During the synthesis of paliperidone active pharmaceutical ingredient, several synthetic process impurities and degradation impurities are introduced, as shown in the following formulas: impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, and impurity I.

[0007]

[0008] Among them, impurities E and F are both process impurities and degradation impurities. Furthermore, because the structure of impurity E is very similar to that of paliperidone, it is difficult to remove it by simple solvent purification methods. Impurities B and C are unreacted raw materials, and impurity I is an impurity introduced from the raw materials. These impurities in the active pharmaceutical ingredient require quality control.

[0009] CN106018601B discloses a method for determining impurities A, B, C, D, and E in paliperidone raw material using high-performance liquid chromatography (HPLC). The method uses mobile phase A as 0.1 mol / L ammonium formate buffer, mobile phase B as acetonitrile, and mobile phase C as methanol:n-propanol = 1:1. This method employs a ternary mobile phase system, which places high demands on equipment, requiring a quaternary pump HPLC system. Conventional binary pump HPLC systems are incompatible, resulting in low equipment compatibility. Furthermore, the use of n-propanol as the mobile phase leads to high solvent viscosity, affecting solute diffusion and mass transfer, reducing column efficiency, increasing column pressure, and prolonging separation time. This method does not disclose the separation and detection of impurities I and G.

[0010] CN105758972B discloses a liquid chromatography method for separating and detecting impurities B, C, E, and I in paliperidone using a 1% triethylamine aqueous solution as mobile phase A and a detection wavelength of 200-250 nm. This method cannot simultaneously detect other impurities in paliperidone, such as impurities A, D, and F. In addition, triethylamine can bind to the silanol groups of the stationary phase, hindering the interaction between basic compounds in the sample and the silanol groups. Although this can reduce peak tailing, it has a significant impact on the lifespan of the chromatographic column.

[0011] Currently, no literature reports an effective HPLC method for separating and detecting multiple process impurities and degradation impurities, including impurity A, B, C, D, E, F, G, and I, that may be present in paliperidone. Therefore, there is an urgent need to develop a simple, low-cost, and versatile method for accurately and effectively detecting multiple process and degradation impurities in paliperidone to meet the development needs of paliperidone formulations. Summary of the Invention

[0012] The purpose of this invention is to provide a method for detecting related substances in paliperidone, enabling better detection of impurities that may be present in paliperidone raw materials and formulations, thereby improving the quality control of paliperidone raw materials and formulations. This method has one or more advantages, including simple operation, good separation, high specificity, good durability, wide equipment compatibility, and minimal impact on column life.

[0013] Specifically, the present invention provides a method for detecting related substances in paliperidone, the method comprising: detecting the test sample using high performance liquid chromatography (HPLC), wherein the detection conditions of the HPLC include:

[0014] The detection wavelength is 270–280 nm.

[0015] The chromatographic column is a stationary phase composed of octadecylsilane-bonded silica gel.

[0016] Mobile phase A is an aqueous solution of ammonium formate with a concentration of 0.05–0.1 mol / mL;

[0017] Mobile phase B is acetonitrile-methanol-tetrahydrofuran, wherein the tetrahydrofuran does not contain the stabilizer BHT, and the volume ratio of acetonitrile:methanol:tetrahydrofuran is (400-600):(200-450):(150-250);

[0018] Gradient elution is performed using mobile phase A and mobile phase B. The gradient elution process includes:

[0019]

[0020] The relevant substances include one or more of impurities A, B, C, D, E, F, G, and I:

[0021]

[0022] Tetrahydrofuran (THF) is a saturated cyclic ether that forms organic peroxides during long-term storage. This process is typically inhibited by adding butylated hydroxytoluene (BHT) as a stabilizer. During their research, the inventors discovered that using THF containing a small amount of the stabilizer BHT as mobile phase B resulted in high baseline noise, affecting detection performance. Extensive experimental research revealed that this was due to the strong absorption of the antioxidant BHT in THF. Therefore, in the embodiments of this invention, the THF used in mobile phase B does not contain the stabilizer BHT.

[0023] In a preferred embodiment, the method for detecting related substances in paliperidone includes a mobile phase A of 0.05–0.1 mol / ml ammonium formate aqueous solution, with formic acid used to adjust the pH to 4–5. For example, the pH can be adjusted to 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0, or any value between two numbers, preferably with formic acid adjusted to 4.5–4.7.

[0024] In a preferred embodiment, the method for detecting related substances in paliperidone includes a mobile phase B of acetonitrile-methanol-tetrahydrofuran, wherein the tetrahydrofuran is free of the stabilizer BHT, and the volume ratio of acetonitrile:methanol:tetrahydrofuran is (400-600):(200-410):(190-210), for example, 400:390:210, 400:410:190, 400:400:200, or 600:200:200, etc., more preferably 400:(390-410):(190-210).

[0025] In a preferred embodiment, the method for detecting related substances in paliperidone, wherein the gradient elution process includes:

[0026]

[0027] Preferably, the gradient elution process includes:

[0028]

[0029] Further preferably, the gradient elution process includes:

[0030]

[0031] More preferably, the gradient elution process includes:

[0032]

[0033] In a preferred embodiment, the method for detecting related substances in paliperidone, wherein the chromatographic conditions of the high-performance liquid chromatography further include:

[0034] The flow rate is 0.5 to 1.0 ml / min, for example 0.5 ml / min, 0.6 ml / min, 0.7 ml / min, 0.8 ml / min, 0.9 ml / min and 1.0 ml / min, preferably 0.6 ml / min to 0.8 ml / min;

[0035] The column temperature is 20–40℃, such as 20–30℃, 25–35℃, or 30–35℃, with 28–32℃ being the preferred column temperature.

[0036] The injection volume is 5 μl to 15 μl, preferably 10 μl;

[0037] The detection wavelength is 273-277nm, such as 273nm, 274nm, 275nm, 276nm or 277nm, with 275nm being the preferred detection wavelength.

[0038] In a preferred embodiment, the method for detecting related substances in paliperidone further includes the preparation of a test solution, comprising the following steps:

[0039] Accurately weigh the paliperidone test sample and prepare it with a diluent; preferably, the concentration of paliperidone in the test sample solution is 0.2 mg / ml to 2 mg / ml, for example, 0.2 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1 mg / ml, 1.5 mg / ml or 2 mg / ml, or any value between any two numbers, preferably 0.5 mg / ml to 1.5 mg / ml, more preferably 0.6 mg / ml to 1 mg / ml, for example, 0.8 mg / ml;

[0040] More preferably, the diluent is methanol or a methanol-water solution, wherein the volume ratio of the two in the methanol-water solution is 1 to 2:1, and more preferably, the diluent is a methanol-water solution, wherein the volume ratio of methanol to water is 1:1.

[0041] In a preferred embodiment, the method for detecting related substances in paliperidone further includes the preparation of a reference solution, comprising the following steps:

[0042] Accurately weigh paliperidone reference standard and prepare it with a diluent; preferably, the concentration of paliperidone in the reference standard solution is 0.2 μg / ml to 10 μg / ml, such as 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml, 8 μg / ml, 9 μg / ml or 10 μg / ml, more preferably 0.8 μg / ml to 6 μg / ml, more preferably 3 μg / ml to 5 μg / ml, such as 4 μg / ml;

[0043] The diluent is methanol or a methanol-water solution, wherein the volume ratio of methanol and water in the methanol-water solution is 1 to 2:1, preferably the diluent is a methanol-water solution, wherein the volume ratio of methanol to water is 1:1.

[0044] In a preferred embodiment, the method for detecting related substances in paliperidone further includes the preparation of a system suitability solution, comprising the following steps:

[0045] Accurately weigh paliperidone reference standard and one or more of the following: impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard, impurity E reference standard, impurity F reference standard, impurity G reference standard, impurity I reference standard or their salts, place them in the same volumetric flask, and prepare the solution with diluent.

[0046] The diluent is methanol or a methanol-water solution, wherein the volume ratio of methanol and water in the methanol-water solution is 1 to 2:1, preferably the diluent is a methanol-water solution, wherein the volume ratio of methanol to water is 1:1.

[0047] The method provided by this invention can simultaneously detect impurities A, B, C, D, E, F, G, and I in paliperidone, achieving a detection effect with a resolution greater than 1.5 between the main component and its adjacent impurities, and between known impurities and their adjacent impurities.

[0048] The impurity reference standard added in the preparation of the system suitability solution can be selected from one or more of the following: impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard, impurity E reference standard, impurity F reference standard, impurity G reference standard, impurity I reference standard, or their salts. This mainly depends on the detection purpose. That is, if one or more of the above impurities are to be controlled and their content detected, then only the impurity reference standard to be detected needs to be added during the preparation of the system suitability solution. The impurity reference standard can also be selected in its salt form, such as hydrochloride salt.

[0049] Preferably, the concentration of paliperidone in the system suitability solution is 0.5 mg / ml to 1 mg / ml. Optionally, when the system suitability solution contains one or more of the following reference standards: impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity I, or their salts, their concentrations are respectively:

[0050] The concentration of impurity A reference standard is 0.2 μg / ml to 3 μg / ml, preferably 0.24 μg / ml to 2.4 μg / ml, and more preferably 1.6 μg / ml;

[0051] The concentration of impurity B reference standard is 0.2 μg / ml to 4 μg / ml, preferably 0.24 μg / ml to 4.0 μg / ml, and more preferably 1.6 μg / ml;

[0052] The concentration of impurity C reference standard is 0.2 μg / ml to 2 μg / ml, preferably 0.24 μg / ml to 1.6 μg / ml, and more preferably 1.6 μg / ml;

[0053] The concentration of impurity D reference standard is 0.2 μg / ml to 3 μg / ml, preferably 0.24 μg / ml to 2.4 μg / ml, and more preferably 1.6 μg / ml;

[0054] The concentration of impurity E reference standard is 0.4 μg / ml to 9 μg / ml, preferably 0.40 μg / ml to 8.5 μg / ml, and more preferably 4 μg / ml;

[0055] The concentration of impurity F reference standard is 0.2 μg / ml to 3 μg / ml, preferably 0.24 μg / ml to 2.4 μg / ml, and more preferably 1.6 μg / ml;

[0056] The concentration of impurity G reference standard is 0.2 μg / ml to 3 μg / ml, preferably 0.24 μg / ml to 2.4 μg / ml, and more preferably 1.6 μg / ml;

[0057] The concentration of impurity I reference standard is 0.2 μg / ml to 3 μg / ml, preferably 0.24 μg / ml to 2.4 μg / ml, and more preferably 1.6 μg / ml.

[0058] 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 have many sources, primarily originating from the mobile phase and tubing, such as contaminants in the organic phase, contaminants in the aqueous phase, buffer salts, and those generated during the mobile phase bottle and mixing process. Ghost peaks typically cause problems such as instrument contamination, misjudgment of sample composition, overlap with target peaks, affecting resolution, and increasing workload by requiring more validation work to determine if a substance is indeed the target. In reversed-phase gradient chromatographic analysis, installing a ghost peak trapping column between the gradient mixer and the autosampler not only removes impurities from the mobile phase but also effectively traps impurities from the tubing and mixer.

[0059] Therefore, in a preferred embodiment of the present invention, the method for detecting related substances in paliperidone optionally includes a ghost peak trapping column in the chromatographic conditions; preferably, the ghost peak trapping column is selected from Ghost Sniper Column or a ghost peak trapping column with equivalent performance; in a specific embodiment, the ghost peak trapping column can be, for example, a Ghost Sniper Column 30×4.0mm or a ghost peak trapping column with equivalent performance.

[0060] The solvent effect, also known as solvation, refers to the influence of the physical and chemical properties of the solvent on reaction equilibrium and rate in liquid chromatography. It can cause peak broadening, bifurcation, retention time drift, peak area changes, and double peaks. Simultaneously, earlier eluted peaks may appear forward-delayed or bifurcated, while later eluted peaks maintain their normal shape. A solvent effect eliminator, also called a peak forward delay suppressor, is designed by introducing a specially structured cavity at the front of the column. During chromatographic system equilibrium, this cavity is filled with the mobile phase. Upon injection, the sample solution flows through the cavity, generating minute turbulence within its extremely small volume. This allows the sample solution to mix efficiently with the mobile phase remaining in the cavity before reaching the column, optimizing the surrounding environment of the sample molecules. This ensures that the sample solution and mobile phase are compatible before reaching the column, entering a pre-separation state. Upon reaching the column, the sample can be optimally eluted and separated by the mobile phase, thus eliminating the solvent effect.

[0061] During their research, the inventors discovered that in the method described in this invention, the solvent effect has a certain impact on the detection of impurity I, but has no impact on the detection of several other known impurities. Therefore, when using the method described in this invention to detect impurity I in paliperidone raw material, it is preferable that the chromatographic conditions further include a solvent effect eliminator; more preferably, the solvent effect eliminator is selected from... Solvent-Smoother, Solvs-AB, or equivalent solvent effect eliminators.

[0062] In a preferred embodiment, the method for detecting related substances in paliperidone, wherein in a system suitability solution containing reference standards for impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity I, and paliperidone, the detection results show that impurities I, A, C, B, E, paliperidone, F, G, and D elute sequentially, and the resolution between the main component and its adjacent impurities, and between known impurities and their adjacent impurities, is greater than 1.5.

[0063] In one embodiment, the method for detecting related substances in paliperidone according to the present invention includes chromatographic conditions comprising:

[0064] The chromatographic column used is an octadecylsilane-bonded silica gel column with the following specifications: column length between 150 mm and 300 mm, inner diameter between 1 mm and 10 mm, and particle size between 3 μm and 5 μm. The preferred column size is 4.6 mm × 150 mm with a particle size of 5 μm. More preferably, a Waters XBridge C18 column (4.6 × 150 mm, 3.5 μm) or a column with equivalent performance is used.

[0065] Optionally, the ghost peak collection column may be a Mr. Chromatography Ghost Sniper Column 30×4.0mm or a ghost peak collection column with equivalent performance;

[0066] Optionally, the solvent effect eliminator is selected Solvent-Smoother, Sollvs-AB, or equivalent solvent effect eliminators;

[0067] Mobile phase A is a 0.1 mol / ml ammonium formate aqueous solution, and the pH is adjusted to 4-5 with formic acid, preferably adjusted to 4.5 with formic acid;

[0068] Mobile phase B is acetonitrile-methanol-tetrahydrofuran, wherein the volume ratio of acetonitrile-methanol-tetrahydrofuran is 400:(390~410):(190~210), and the tetrahydrofuran does not contain the stabilizer BHT;

[0069] The column temperature is 20–40℃, preferably 30℃;

[0070] The flow rate is 0.5–1.0 ml / min, preferably 0.8 ml / min;

[0071] The detection wavelength is 273–277 nm, preferably 275 nm;

[0072] The injection volume is 5 μl to 15 μl, preferably 10 μl;

[0073] Furthermore, the method also includes:

[0074] Preparation of the test sample: Accurately weigh the paliperidone test sample and prepare it with a diluent. The concentration of paliperidone in the test sample solution is 0.5 mg / ml to 2 mg / ml, preferably 0.8 mg / ml.

[0075] Preparation of reference standard: Accurately weigh paliperidone reference standard and prepare it with diluent. The concentration of paliperidone in the reference standard solution is 2 μg / ml to 6 μg / ml, preferably 4 μg / ml.

[0076] Preparation of system suitability solution: Accurately weigh paliperidone reference standard and one or more reference standards selected from impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity I, or their salts, and place them in the same volumetric flask. Dissolve in a diluent to prepare the system suitability solution.

[0077] The concentration of paliperidone reference standard is 0.5 mg / ml to 1 mg / ml, preferably 0.8 mg / ml; and / or,

[0078] The concentration of impurity A reference standard is 0.24 μg / ml to 2.4 μg / ml, preferably 1.6 μg / ml; and / or,

[0079] The concentration of impurity B reference standard is 0.24 μg / ml to 4.0 μg / ml, preferably 1.6 μg / ml; and / or,

[0080] The concentration of impurity C reference standard is 0.24 μg / ml to 1.6 μg / ml, preferably 1.6 μg / ml; and / or,

[0081] The concentration of impurity D reference standard is 0.24 μg / ml to 2.4 μg / ml, preferably 1.6 μg / ml; and / or,

[0082] The concentration of impurity E reference standard is 0.40 μg / ml to 8.5 μg / ml, preferably 4 μg / ml; and / or,

[0083] The concentration of impurity F reference standard is 0.24 μg / ml to 2.4 μg / ml, preferably 1.6 μg / ml; and / or,

[0084] The concentration of impurity G reference standard is 0.24 μg / ml to 2.4 μg / ml, preferably 1.6 μg / ml; and / or,

[0085] The concentration of impurity I reference standard is 0.24 μg / ml to 2.4 μg / ml, preferably 1.6 μg / ml;

[0086] The diluent solution is methanol-water in a volume ratio of 1 to 2:1, preferably 1:1;

[0087] Accurately measure the diluent solution, system suitability solution, reference solution, and test solution and inject them into the liquid chromatograph, then record the chromatograms.

[0088] The detection method of the present invention can calculate the content of related substances in paliperidone by calculating the peak area-to-volume ratio or by using the external standard method based on the peak area; more preferably, the formula for calculating the impurity content is as follows:

[0089]

[0090] Total impurities (%) = Known impurities (%) + Unknown impurities (%)

[0091] A T : Peak area of ​​impurities in the test solution;

[0092] A S : The average peak area of ​​the main component in the reference solution for 5 consecutive tests;

[0093] M S Reference standard sample weight, mg;

[0094] W S : Content of reference standard, %;

[0095] V S : Volume of the reference standard stock solution, ml;

[0096] D S The dilution factor of the reference solution;

[0097] V T Volume of the test solution, in ml;

[0098] M T : Sample weight, mg;

[0099] f: Correction factor.

[0100] In one embodiment, the method of the present invention includes impurities A, B, C, D, E, F, G, and I, and the separation degree between the principal component and its adjacent impurities, and between known impurities and their adjacent impurities, is greater than 1.5.

[0101] The method for detecting related substances in paliperidone described in the context of this invention refers to paliperidone as a paliperidone active pharmaceutical ingredient (API) or a paliperidone formulation, preferably a paliperidone sustained-release tablet. More preferably, the method is used to detect related substances in paliperidone API.

[0102] The beneficial effects of this invention are as follows: This invention provides a method for detecting related substances in paliperidone. The method employs a binary mobile phase system, has low equipment requirements, and is widely applicable. Furthermore, the method can simultaneously detect and quantify process impurities and degradation impurities in paliperidone, including impurities A, B, C, D, E, F, G, and I. 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 accuracy and sensitivity, simple operation, and high detection efficiency. In addition, the detection method provided by this invention can shorten detection time and save costs. Other features and beneficial effects of this invention will be further illustrated and explained through subsequent embodiments and accompanying drawings.

[0103] Terminology Definition

[0104] 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.

[0105] 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.

[0106] 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.

[0107] The terms “comprising” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0108] 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%.

[0109] In the context of this invention, the term "active pharmaceutical ingredient" is consistent with the World Health Organization's definition of an active pharmaceutical ingredient, wherein "paliperidone active pharmaceutical ingredient" specifically includes paliperidone or a pharmaceutically acceptable salt thereof.

[0110] The abbreviation mM refers to millimoles per liter (0.001 mol / L). For example, "100 mM ammonium formate aqueous solution" means 0.1 mol / L ammonium formate aqueous solution.

[0111] The impurity reference standards A, B, C, D, E, F, G, and I mentioned in this invention context can also be selected in their corresponding salt forms, such as hydrochloride salts. The molecular weight ratio of the impurity free base to its hydrochloride salt is known, and the amount of salt used can be calculated by converting the molecular weight ratio and the amount of free base disclosed in this invention. For example, impurity reference standard B can be used in its hydrochloride salt form, and the molecular weight ratio of impurity B free base to its hydrochloride salt is 0.86. Attached Figure Description

[0112] Figure 1 : Displays the HPLC chromatogram of the diluent detected in Example 1;

[0113] Figure 2 : Displays the HPLC chromatogram of the system suitability solution tested in Example 1;

[0114] Figure 3 : Displays the HPLC chromatogram of the reference solution detected in Example 1;

[0115] Figure 4 : Displays the HPLC chromatogram of the paliperidone raw material test sample detected in Example 1;

[0116] Figure 5: HPLC chromatograms of the system suitability solutions obtained in the durability test of Example 3 under different mobile phase pH, flow rate, column temperature, and initial conditions with the ratio of mobile phases A and B.

[0117] in, Figure 5A The figure shows HPLC chromatograms of the system suitability solutions obtained under different mobile phase pH conditions. From top to bottom, the pH values ​​of mobile phase A are 4.5 (standard conditions), 4.3, and 4.7.

[0118] Figure 5B The figure shows the HPLC chromatograms of the system suitability solutions obtained under different flow rate conditions in Example 3. The flow rates from top to bottom in the figure are: 0.8 ml / min (standard conditions), 0.7 ml / min, and 0.9 ml / min.

[0119] Figure 5C The figure shows the HPLC chromatograms of the system suitability solutions obtained under different column temperature conditions in Example 3. The column temperatures from top to bottom in the figure are: 30℃ (standard conditions), 28℃, and 32℃.

[0120] Figure 5D The figure shows the HPLC chromatograms of the system suitability solutions obtained under different proportions of mobile phase B in Example 3. From top to bottom, mobile phase B is: acetonitrile-methanol-tetrahydrofuran (400:200:200) (standard conditions), acetonitrile-methanol-tetrahydrofuran (400:390:210), and acetonitrile-methanol-tetrahydrofuran (400:410:190).

[0121] Figure 5E The figure shows the HPLC chromatograms of the system suitability solutions obtained under different initial mobile phase A and B volume ratios in Example 3. The components of mobile phase B from top to bottom are: A:B = 95:5 (standard conditions), A:B = 94:6, and A:B = 96:4.

[0122] Figure 6 : Displays the HPLC chromatogram of the system suitability solution tested by the method in Example 6. Detailed Implementation

[0123] The technical solution and beneficial effects of the present invention will be further explained below with reference to specific embodiments. The present invention provides a method for detecting related substances in paliperidone raw material, the method comprising:

[0124] (a) Preparation of test solution: accurately weigh paliperidone test sample and prepare it with diluent. The concentration of paliperidone in the test solution is 0.5 mg / ml to 1.5 mg / ml, preferably 0.8 mg / ml.

[0125] (b) Preparation of reference solution: Paliperidone reference standard, accurately weighed, and prepared with diluent; the concentration of paliperidone in the reference solution is 0.24 μg / ml to 6 μg / ml, more preferably 4 μg / ml;

[0126] (c) Preparation of system suitability solution: accurately weigh paliperidone reference standard and one or more of impurity reference standards selected from impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity I or their salts, place them in the same volumetric flask, and prepare the solution with diluent.

[0127] In the system adaptability solution, the concentration of paliperidone reference standard is 0.5 mg / ml to 1 mg / ml, preferably 0.8 mg / ml; the concentration of impurity A reference standard is 0.24 μg / ml to 2.4 μg / ml, preferably 1.6 μg / ml; the concentration of impurity B reference standard is 0.24 μg / ml to 4.0 μg / ml, preferably 1.6 μg / ml; the concentration of impurity C reference standard is 0.24 μg / ml to 1.6 μg / ml, preferably 1.6 μg / ml; and the concentration of impurity D reference standard is 0.24 μg / ml. The concentration of impurity E reference standard is 0.4 μg / ml to 2.4 μg / ml, preferably 1.6 μg / ml; the concentration of impurity F reference standard is 0.24 μg / ml to 2.4 μg / ml, preferably 1.6 μg / ml; the concentration of impurity G reference standard is 0.24 μg / ml to 2.4 μg / ml, preferably 1.6 μg / ml; the concentration of impurity I reference standard is 0.24 μg / ml to 2.4 μg / ml, preferably 1.6 μg / ml.

[0128] (d) Detection is performed using high-performance liquid chromatography (HPLC), wherein the HPLC detection conditions include:

[0129] The detection wavelength is 273–277 nm.

[0130] The chromatographic column is a stationary phase composed of octadecylsilane-bonded silica gel.

[0131] Mobile phase A is an aqueous solution of ammonium formate with a concentration of 0.05–0.1 mol / mL, and the pH is adjusted to 4–5 using formic acid, preferably to 4.5–4.7.

[0132] Mobile phase B is acetonitrile-methanol-tetrahydrofuran, wherein the tetrahydrofuran does not contain the stabilizer BHT, and the volume ratio of acetonitrile:methanol:tetrahydrofuran is (400-600):(200-410):(190-210), preferably 400:(390-410):(190-210) or 600:200:200;

[0133] Gradient elution is performed using mobile phase A and mobile phase B. The gradient elution process includes:

[0134]

[0135] Preferably, the gradient elution process includes:

[0136]

[0137] The flow rate is 0.6 ml / min to 0.8 ml / min;

[0138] The column temperature is 20–40℃, preferably 28–32℃;

[0139] The injection volume is 5 μl to 15 μl, preferably 10 μl;

[0140] The diluent solution is a methanol-water solution, wherein the volume ratio of methanol to water is 1:1;

[0141] Accurately inject 10 μl each of the diluent solution, reference solution, system suitability solution and test solution into the liquid chromatograph and record the chromatogram.

[0142] The aforementioned detection method can calculate the content of related substances in paliperidone raw material by calculating the peak area-to-volume ratio or by using the external standard method based on peak area; preferably, the formula for calculating impurity content is as follows:

[0143]

[0144] Total impurities (%) = Known impurities (%) + Unknown impurities (%)

[0145] A T : Peak area of ​​impurities in the test solution;

[0146] A S : The average peak area of ​​the main component in the reference solution for 5 consecutive tests;

[0147] M S Reference standard sample weight, mg;

[0148] W S : Content of reference standard, %;

[0149] V S : Volume of the reference standard stock solution, ml;

[0150] D S The dilution factor of the reference solution;

[0151] V T Volume of the test solution, in ml;

[0152] M T : Sample weight, mg;

[0153] f: Correction factor;

[0154] The above method can simultaneously detect related substances in paliperidone raw material, including impurities A, B, C, and D.

[0155] Impurity E, impurity F, impurity G, and impurity I;

[0156] More preferably, in the above method, the preparation method of the system suitability solution includes adding impurity A reference standard, impurity B hydrochloride reference standard, impurity C reference standard, impurity D reference standard, impurity E reference standard, impurity F reference standard, impurity G reference standard, impurity I reference standard and paliperidone reference standard, wherein impurities I, A, C, B, E, F, G and D elute in sequence, and the resolution between the main component and its adjacent impurities, and between known impurities and their adjacent impurities is greater than 1.5.

[0157] 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. Liquid chromatograph: Ultimate 3000, purchased from Thermo Fisher Scientific; Solvent effect eliminator: Solvent-Smoother (Part: Solvs-AB) was purchased from Puning Technology; Ghost Sniper Column (30×4.0mm) was purchased from Chromatography Master. Impurity E reference standard had a purity of 78.7%, Impurity D reference standard had a purity of 89.42%, Impurity F reference standard had a purity of 93.01%, Impurity A reference standard had a purity of 97.4%, and the purities of Impurities B, C, G, and I reference standards were all above 99%. These impurity reference standards could be obtained commercially or synthesized according to publicly available methods. Paliperidone reference standard had a purity of 100.0% and was prepared by Suzhou Enhua Biomedical Technology Co., Ltd. The tetrahydrofuran used in mobile phase B was BHT-free and obtained commercially.

[0158] Example 1: Detection method for related substances in paliperidone raw material

[0159] Solution preparation:

[0160] (1) Preparation of diluent (methanol:water = 1:1):

[0161] Measure 200ml of methanol and 200ml of ultrapure water, mix and shake well, and use after cooling to room temperature.

[0162] (2) System suitability solution preparation

[0163] Impurity stock solution 1: Take about 2 mg each of impurity I reference standard, impurity A reference standard, impurity G reference standard, impurity D reference standard and impurity F reference standard, take about 2.4 mg of impurity B hydrochloride reference standard and about 5 mg of impurity E reference standard and place them in the same 20 ml volumetric flask, add an appropriate amount of methanol to dissolve and dilute to the mark with diluent, and shake well.

[0164] Impurity stock solution 2: Take about 2 mg of impurity C reference standard into a 20 ml volumetric flask, dissolve and dilute with acetonitrile to the mark, and shake well.

[0165] System suitability solution: Take about 20 mg of paliperidone reference standard and place it in a 25 ml volumetric flask. Add 10 ml of methanol and sonicate to dissolve. Then add 10 ml of water and let it cool to room temperature. Add 0.4 ml of impurity stock solution 1 and dilute to the mark with diluent. Shake well. Take 1 ml of this solution and add 16 μl of impurity stock solution 2. Mix well to prepare a solution containing 0.8 mg of paliperidone, 4 μg of impurity E, and 1.6 μg of other impurities per 1 ml.

[0166] (3) Preparation of reference solution

[0167] Accurately weigh approximately 20 mg of paliperidone reference standard into a 50 ml volumetric flask. Dissolve the solution in methanol by sonication and dilute to the mark. Shake well. Accurately measure 1 ml of this solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well. Prepare a solution containing approximately 4 μg of paliperidone per ml.

[0168] (4) Preparation of test solution

[0169] Weigh approximately 20 mg of paliperidone raw material into a 25 ml volumetric flask, add 10 ml of methanol and sonicate to dissolve, then add 10 ml of water, bring to room temperature and dilute to the mark with diluent, shake well to prepare a solution containing approximately 0.8 mg of paliperidone per ml.

[0170] Chromatographic conditions

[0171] Column: Waters XBridge C18, 4.6 × 150 mm, 3.5 μm or equivalent column;

[0172] Ghost Sniper Column: Mr. Chromatography Ghost Sniper Column 30×4.0mm or equivalent ghost peak collection column;

[0173] Solvent effect eliminator: Solvent-Smoother, Sollvs-AB, or equivalent solvent effect eliminators;

[0174] Mobile phase A: 100 mM ammonium formate aqueous solution (pH adjusted to 4.5 with formic acid);

[0175] Mobile phase B: Acetonitrile-methanol-tetrahydrofuran (400:400:200); (Tetrahydrofuran does not contain the stabilizer BHT)

[0176] Column temperature: 30℃;

[0177] Flow rate: 0.8 ml / min;

[0178] Detection wavelength: 275nm;

[0179] Injection volume: 10 μl;

[0180] Perform linear gradient elution according to the table below:

[0181]

[0182] 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.

[0183] Test results as follows Figures 1-4 As shown, the HPLC chromatogram of the diluent is as follows: Figure 1 As shown, from Figure 1 It can be seen that the diluent does not interfere with the detection of impurities;

[0184] The HPLC chromatogram of the system suitability solution is shown below. Figure 2 As shown in Table 1.1, impurities I, A, C, B, E, paliperidone, F, G and D eluted in that order.

[0185] The HPLC chromatogram of the reference solution is shown below. Figure 3 As shown, paliperidone RT = 22.483 min;

[0186] The HPLC chromatogram of the test solution is shown below. Figure 4 As shown, the peaks that appeared in sequence were impurity E (RT = 20.704 min, peak area 0.1126%), paliperidone (RT = 22.180 min, peak area 99.78%), impurity F (RT = 24.720 min, peak area 0.0096%), and impurity G (RT = 25.977 min, peak area 0.0474%).

[0187] Table 1.1 System Suitability Solution Test Results

[0188] Compound Name retention time min Resolution Tail factor Impurity I 6.123 / 1.29 Impurity A 12.633 35.10 1.10 Impurity C 17.140 22.20 1.02 Impurity B 17.827 2.70 1.07 Impurity E 20.633 7.18 1.66 Paliperidone 22.113 2.93 2.53 impurity F 24.630 6.11 1.02 Impurity G 25.873 3.79 1.07 Impurity D 28.060 6.78 1.07

[0189] from Figure 2 As shown in Table 1.1, in the system suitability solution, impurities I, A, C, B, E, paliperidone, F, G, and D precipitate peaks in sequence. The minimum resolution between the main component and its adjacent impurities, and between known impurities and their adjacent impurities, is 2.70, which is greater than 1.5 and meets the requirements.

[0190] Example 2: Specificity Experiment

[0191] The diluent was prepared in the same manner as in Example 1;

[0192] Preparation of stock solution

[0193] Accurately weigh appropriate amounts of paliperidone reference standard, impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard, impurity E reference standard, impurity F reference standard, impurity G reference standard, and impurity I reference standard, and prepare paliperidone reference standard stock solution (0.24 mg / ml), impurity A stock solution (0.24 mg / ml), impurity B stock solution (0.16 mg / ml), impurity D stock solution (0.24 mg / ml), impurity E stock solution (0.4 mg / ml), impurity F stock solution (0.16 mg / ml), impurity G stock solution (0.24 mg / ml), impurity I stock solution (0.24 mg / ml), and impurity C stock solution (0.16 mg / ml) respectively.

[0194] Preparation of positioning solution

[0195] Take separate amounts of paliperidone reference standard stock solution (0.24 mg / ml), impurity A stock solution (0.24 mg / ml), impurity-B stock solution (0.16 mg / ml), impurity D stock solution (0.24 mg / ml), impurity E stock solution (0.4 mg / ml), impurity F stock solution (0.16 mg / ml), impurity G stock solution (0.24 mg / ml), impurity I stock solution (0.24 mg / ml), and impurity C stock solution (0.16 mg / ml). The impurities were diluted with diluent to prepare paliperidone positioning solution (12 μg / ml), impurity A positioning solution (12 μg / ml), impurity B positioning solution (8 μg / ml), impurity D positioning solution (12 μg / ml), impurity E positioning solution (20 μg / ml), impurity F positioning solution (8 μg / ml), impurity G positioning solution (12 μg / ml), impurity I positioning solution (12 μg / ml), and impurity C positioning solution (8 μg / ml).

[0196] Preparation of separation solution

[0197] Accurately weigh 19.66 mg of paliperidone reference standard into a 25 ml volumetric flask, dissolve in 10 ml of methanol by sonication, add 10 ml of water, and allow to cool to room temperature. Add 260 μl of impurity-B stock solution (0.16 mg / ml), 170 μl each of impurity A stock solution (0.24 mg / ml), impurity D stock solution (0.24 mg / ml), impurity G stock solution (0.24 mg / ml), and impurity I stock solution (0.24 mg / ml), and 250 μl each of impurity E stock solution (0.4 mg / ml) and impurity F stock solution (0.16 mg / ml). Dilute to the mark with diluent and mix well. Take 1 ml of this solution and add 10 μl of impurity C stock solution (0.16 mg / ml), mix well, and the solution is ready.

[0198] Measurement

[0199] The chromatographic conditions were the same as in Example 1. 10 μl each of the above-mentioned diluent solution, positioning solution, and resolution solution were accurately injected into the liquid chromatograph, and the chromatograms were recorded. The results of the resolution solution detection are shown in Table 2.1.

[0200] Table 2.1: Results of solution separation test:

[0201]

[0202] The test results show that the minimum separation degree between the principal component, known impurities and adjacent impurities in the separation solution is 2.70, which is greater than 1.5, indicating that the method provided by the present invention has good specificity.

[0203] Example 3: Durability Test

[0204] The standard conditions were the chromatographic conditions listed in Example 1. While keeping other conditions constant, the wavelength, column temperature, pH value, mobile phase ratio, and one of mobile phase B were changed each time to study the effect of these changes on resolution. 10 μl each of the diluent solution, system suitability solution, and resolution 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 diluent solution and system suitability solution were prepared using the same method as in Example 1, and the resolution solution was prepared using the same method as in Example 2.

[0205] Table 3.1: Results of Solution Separation Test for System Suitability under Various Durability Conditions

[0206]

[0207]

[0208] Test results as follows Figures 5A-5E As shown in Table 3.1, under all varying conditions, the diluent solution did not interfere with the detection. In the system suitability solution, the resolution of the main component, known impurities, and adjacent impurities was greater than 1.5. In the resolution solution, the resolution of the main component, known impurities, and adjacent impurities was greater than 2.60, indicating good resolution. The ratio of the content of the same impurity measured in the same sample solution to the measurement result under standard conditions met the requirements.

[0209] Example 4: Limit of Detection and Limit of Quantification Experiment

[0210] Mixed impurity detection limit solution: Accurately weigh paliperidone reference standard, impurity A reference standard, impurity B reference standard, impurity D reference standard, impurity E reference standard, impurity F reference standard, impurity G reference standard, and impurity I reference standard, place them in the same volumetric flask, and dilute with diluent to prepare solutions with the LOD concentrations listed in Table 4.1.

[0211] Impurity C detection limit solution: Accurately weigh impurity C reference standard, place it in a volumetric flask, dilute to the mark with diluent, shake well, and prepare a solution with a concentration of 0.12 μg / ml.

[0212] Accurately measure 10 μl of the detection limit solution and inject it into the liquid chromatograph. Record the chromatogram. The chromatographic conditions are the same as in Example 1, with three consecutive injections. The detection results are shown in Table 4.1 below:

[0213] Table 4.1: Detection limit solution concentration and results of three consecutive injections

[0214]

[0215] 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.

[0216] The results above show that the method provided by this invention achieves a signal-to-noise ratio (S / N) of over 10 for impurities A, B, D, E, F, G, and I at a concentration of approximately 0.12 μg / ml, which is much greater than 3.

[0217] Limit of quantitation:

[0218] Preparation of mixed impurity limit of quantitation solution: Accurately weigh paliperidone reference standard, impurity A reference standard, impurity B reference standard, impurity D reference standard, impurity E reference standard, impurity F reference standard, impurity G reference standard, and impurity I reference standard, place them in the same volumetric flask, and dilute with diluent to prepare limit of quantitation solution (LOQ).

[0219] Impurity C Quantitative Limit Solution: Accurately weigh impurity C reference standard, place it in a volumetric flask, dilute to the mark with diluent, shake well, and prepare the impurity C quantitative limit solution.

[0220] Accurately measure 10 μl of each limit-of-quantity solution and inject it into the liquid chromatograph. Record the chromatogram and inject 6 times consecutively. The detection results are shown in Table 4.2 below.

[0221] Table 4.2: Limit of Quantitation Solution Concentration and Detection Results

[0222]

[0223] Note: The test sample in "Ratio relative to test sample concentration" in Table 4.2 is the same as the test sample solution prepared by the method in Example 1, wherein the concentration of paliperidone is 0.8 mg / ml.

[0224] The results showed that when the concentration of each impurity was 0.03% relative to the concentration of the test sample, the signal-to-noise ratio was much greater than 10. The RSD of the peak areas of the main component and each impurity in the six limit-of-quantitation solutions was 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.

[0225] Example 5: Linearity and Range

[0226] Accurately weigh paliperidone reference standard, impurity A reference standard, impurity B reference standard, impurity D reference standard, impurity E reference standard, impurity F reference standard, impurity G reference standard, and impurity I reference standard, place them in the same volumetric flask, and prepare 8 solutions with diluent. The solution names are L500%, L300%, L200%, L150%, L100%, L80%, L50%, and LOQ solution, respectively.

[0227] Accurately weigh each impurity C reference standard and place them in volumetric flasks. Prepare 8 solutions with diluent, named L500%, L300%, L200%, L150%, L100%, L80%, L50%, and LOQ solutions, respectively. These solutions serve as linear solutions for impurity C.

[0228] Table 5.1: Concentrations of paliperidone and known impurities in various solutions:

[0229]

[0230] Accurately measure 10 μl of the diluent and each linear solution and inject them into the liquid chromatograph. Record the chromatograms. Chromatographic conditions are the same as in Example 1. Perform linear regression analysis with concentration on the x-axis and peak area on the y-axis to plot a standard curve. The curve ranges are as follows: LOQ to L200% for impurities A, D, E, G, and I; LOQ to L300% for impurities C and F; and LOQ to L500% for impurities B and paliperidone. The linearity results are shown in Table 5.2.

[0231] Table 5.2: Linear concentration range and linear regression equation for each impurity

[0232] Impurity Name Concentration range (μg / ml) Relative concentration of the test sample (%) Linear regression equation Correlation coefficient r Impurity A 0.2390～2.3902 0.03～0.3 y = 0.3444x - 0.0005 0.9998 Impurity B 0.2341～3.9019 0.03～0.5 y = 0.2012x - 0.0020 0.9998 Impurity C 0.2412～1.6547 0.03～0.2 y = 0.3026x - 0.0014 1.0000 Impurity D 0.2395～2.3947 0.03～0.3 y = 0.2622x - 0.0000 0.9997 Impurity E 0.4236～8.4713 0.05～1.0 y = 0.1463x - 0.0064 0.9998 impurity F 0.2412～2.4122 0.03～0.3 y = 0.2316x + 0.0053 0.9998 Impurity G 0.2384～2.3836 0.03～0.3 y = 0.2995x + 0.0018 0.9998 Impurity I 0.2436～2.4359 0.03～0.3 y = 0.3243x - 0.0067 0.9997

[0233] Note: The test sample in "Relative test sample concentration" in Table 5.2 is the same as the test sample solution prepared by the method in Example 1, wherein the concentration of paliperidone is 0.8 mg / ml.

[0234] Experimental results show that, within the concentration ranges shown in Table 5.2, the linear correlation coefficient r between the concentrations of the principal component and each impurity and the response is the smallest at 0.9997, which is greater than 0.990; the absolute value of the linear intercept between the concentrations of the principal component and each impurity and the response is the largest at 2.76%, which is less than 25% of the 100% response value; and the RSD of the response factor at the concentration levels of the principal component and each impurity is the largest at 4.0%, which is less than 10%, indicating that the method provided by this invention has good linearity.

[0235] Example 6: Detection method for related substances in paliperidone raw material:

[0236] The diluent preparation method is the same as in Example 1;

[0237] System suitability solution preparation:

[0238] Impurity stock solution 1: Take about 2 mg each of impurity I reference standard, impurity A reference standard, impurity G reference standard, impurity H, impurity D reference standard and impurity F reference standard, take about 2.4 mg of impurity B reference standard and about 5 mg of impurity E reference standard into the same 20 ml volumetric flask, add an appropriate amount of methanol to dissolve and dilute to the mark with diluent, and shake well.

[0239] Impurity stock solution 2: Take about 2 mg of impurity C reference standard into a 20 ml volumetric flask, dissolve and dilute with acetonitrile to the mark, and shake well.

[0240] System suitability solution: Take about 20 mg of paliperidone reference standard into a 25 ml volumetric flask, add 10 ml of methanol and sonicate to dissolve, then add 10 ml of water, let it stand at room temperature, add 0.4 ml of impurity stock solution 1, dilute to the mark with diluent, and shake well; take 1 ml of this solution, add 16 μl of impurity stock solution 2, mix well, and prepare a solution containing 0.8 mg of paliperidone, 4 μg of impurity E, and 1.6 μg of other impurities per 1 ml.

[0241] Chromatographic conditions:

[0242] Column: Waters XBridge C18, 4.6 × 150 mm, 3.5 μm or equivalent column;

[0243] Ghost Sniper Column: Mr. Chromatography Ghost Sniper Column 30×4.0mm or equivalent ghost peak collection column;

[0244] Solvent effect eliminator: Solvent-Smoother, Sollvs-AB, or equivalent solvent effect eliminators;

[0245] Mobile phase A: 100 mM ammonium formate aqueous solution (pH adjusted to 4.2 with formic acid);

[0246] Mobile phase B: Acetonitrile-methanol-tetrahydrofuran (600:200:200); (Tetrahydrofuran without stabilizer BHT)

[0247] Column temperature: 30℃;

[0248] Flow rate: 0.8 ml / min;

[0249] Detection wavelength: 275nm;

[0250] Injection volume: 10 μl;

[0251] Perform linear gradient elution according to the table below:

[0252]

[0253] Precisely measure 10 μl each of the diluent solution and the system suitability solution and inject them into the liquid chromatograph, then record the chromatograms.

[0254] Test results as follows Figure 6 As shown, the separation degree of each impurity is greater than 2.3, indicating that this method can simultaneously detect all the above-mentioned impurities in paliperidone raw material and has good separation degree.

[0255] The accuracy of a known impurity is determined by adding a known amount of the impurity and then measuring the ratio between the measured result and the theoretical value of the known impurity in the spiked sample (recovery rate), expressed as a percentage (%). The method of this invention was used to detect the recoveries of impurities A, B, C, D, E, F, G, and I at the limit of quantitation (LOQ) level and at 40%, 100%, and 200% levels. Experimental results show that the recoveries of each known impurity are between 86.02% and 115.48%, indicating that the method provided by this invention has good accuracy.

Claims

1. A method for detecting related substances in paliperidone, characterized in that, The method includes: The test sample was detected using high-performance liquid chromatography (HPLC), and the detection conditions for HPLC included: The chromatographic column is stationary using octadecylsilane-bonded silica gel as the packing material. Mobile phase A is an aqueous solution of ammonium formate with a concentration of 0.05–0.10 mol / mL; Mobile phase B is acetonitrile-methanol-tetrahydrofuran, wherein the tetrahydrofuran does not contain the stabilizer BHT, and the volume ratio of acetonitrile:methanol:tetrahydrofuran is (400-600):(200-450):(150-250); Gradient elution is performed using mobile phase A and mobile phase B, and the gradient elution process includes: The relevant substances include one or more of impurities A, B, C, D, E, F, G, and I:

2. The method according to claim 1, characterized in that, The mobile phase A is adjusted to pH 4-5 using formic acid, preferably to pH 4.5-4.7; Further preferred, the volume ratio of acetonitrile:methanol:tetrahydrofuran in mobile phase B is (400-600):(390-400):(190-210).

3. The method according to claim 1, characterized in that, The gradient elution process includes: Preferably, the gradient elution process includes:

4. The method according to any one of claims 1 to 3, characterized in that, The chromatographic conditions for the high-performance liquid chromatography method also include: The flow rate is 0.5–1.0 ml / min, preferably 0.6–0.8 ml / min; The column temperature is 20–40℃, preferably 28–32℃; The injection volume is 5 μl to 15 μl, preferably 10 μl; The detection wavelength is 270–280 nm, preferably 273–277 nm.

5. The method according to any one of claims 1 to 4, characterized in that, It also includes the preparation of the test solution, comprising the following steps: Accurately weigh paliperidone sample and prepare it with diluent; Preferably, the concentration of paliperidone in the test solution is 0.2 mg / ml to 2 mg / ml, more preferably 0.5 mg / ml to 1.5 mg / ml.

6. The method according to any one of claims 1 to 5, characterized in that, It also includes the preparation of the reference solution, comprising the following steps: Accurately weigh paliperidone reference standard and prepare it with diluent; Preferably, the concentration of paliperidone in the reference solution is 0.2 μg / ml to 10 μg / ml, more preferably 0.8 μg / ml to 6 μg / ml.

7. The method according to any one of claims 1 to 6, characterized in that, It also includes the preparation of system-adaptive solutions, comprising the following steps: Take paliperidone reference standard and one or more of the following: impurity A reference standard, impurity B reference standard, impurity C reference standard, impurity D reference standard, impurity E reference standard, impurity F reference standard, impurity G reference standard, impurity I reference standard or their salts, accurately weigh them, place them in the same volumetric flask, and prepare them with diluent. Preferably, in the system adaptability solution, the concentration of paliperidone is 0.5 mg / ml to 1 mg / ml. Optionally, the concentration of impurity A reference standard is 0.2 μg / ml to 3 μg / ml, and / or the concentration of impurity B reference standard is 0.2 μg / ml to 4 μg / ml, and / or the concentration of impurity C reference standard is 0.2 μg / ml to 2 μg / ml, and / or the concentration of impurity D reference standard is 0.2 μg / ml to 3 μg / ml, and / or the concentration of impurity E reference standard is 0.4 μg / ml to 9 μg / ml, and / or the concentration of impurity F reference standard is 0.2 μg / ml to 3 μg / ml, and / or the concentration of impurity G reference standard is 0.2 μg / ml to 3 μg / ml, and / or the concentration of impurity I reference standard is 0.2 μg / ml to 3 μg / ml.

8. The method according to any one of claims 5 to 7, characterized in that, The diluent is methanol or a methanol-water solution, wherein the volume ratio of methanol to water is 1 to 2:1, preferably the diluent is a methanol-water solution with a volume ratio of 1:

1.

9. The method according to any one of claims 1 to 8, characterized in that, The chromatographic conditions also include: using a ghost peak trapping column and / or a solvent effect eliminator; Preferably, the ghost peak capture column is selected from the Ghost Sniper Column or a ghost peak capture column with equivalent performance; The solvent effect eliminator is selected from... Solvent-Smoother, Solvs-AB, or equivalent solvent effect eliminators.

10. The method according to any one of claims 1 to 8, characterized in that, The method can simultaneously detect impurities A, B, C, D, E, F, G and I in paliperidone, and the resolution 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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