A method for detecting related substances of 4-(2,4-difluorobenzoyl)-piperidine hydrochloride, a starting material of paliperidone

Abstract

The present invention provides a detection method for related substances of 4-(2,4-difluorobenzoyl)-piperidine hydrochloride, a starting material of paliperidone. The present invention adopts high performance liquid chromatography, and its chromatographic conditions include: an octadecylsilane bonded silica gel chromatographic column with an embedded pentafluorophenyl functional group, a mixed solution of triethylamine aqueous solution and methanol as mobile phase A, and a mixed solution of acetonitrile and tetrahydrofuran as mobile phase B. The method of the present invention can effectively separate and synchronously determine impurities i-4, i-5, i-8, i-9, i-10, i-11, i-12 and i-14, a total of 8 impurities; the method is simple, does not affect the reproducibility and service life of the chromatographic column, and has low cost; and has the advantages of good specificity, system suitability, linearity, precision and durability, high sensitivity and accuracy.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for detecting related substances of 4-(2,4-difluorobenzoyl)-piperidine hydrochloride, a starting material of paliperidone. Background Art

[0002] Paliperidone is a 5-HT antagonist belonging to the class of benzisoxazole derivatives and is the main metabolite of risperidone. Its sustained-release oral preparation is marketed in the United States under the trade name Invega for the treatment of schizophrenia. Its chemical name is 3-[2-[4-(6-fluoro-1,2-benzisoxazol-3-yl)-1-piperidinyl]ethyl]-6,7,8,9-tetrahydro-9-hydroxy-2-methyl-4H-pyrido[1,2-a]pyrimidin-4-one, and its structural formula is as follows:

[0003]

[0004] As the starting material for the synthesis of paliperidone, 4-(2,4-difluorobenzoyl)-piperidine hydrochloride (DPH) has the following structural formula:

[0005]

[0006] The related substances that may exist in DPH and need to be studied include: impurity i-1, impurity i-3, impurity i-4, impurity i-5, impurity i-8, impurity i-9, impurity i-10, impurity i-11, impurity i-12 and impurity i-14, a total of 10 impurities. The specific information is shown in the following table:

[0007]

[0008]

[0009] 4-(2,4-difluorobenzoyl)-piperidine hydrochloride (DPH) is the starting material for the key step of synthesizing paliperidone. In order to ensure the safety of the drug, it is necessary to study its related substances. However, after researching relevant literature and patents, it was found that the detection content of the above DPH related substances was not included in the pharmacopoeias, patents and literatures of various countries. Summary of the invention

[0010] In order to solve the above problems of the existing detection methods, the present invention provides a detection method for related substances of 4-(2,4-difluorobenzoyl)-piperidine hydrochloride, a starting material of paliperidone. The method of the present invention can effectively separate and synchronously determine impurities i-4, i-5, i-8, i-9, i-10, i-11, i-12 and i-14, a total of 8 impurities; and the method is simple, does not affect the reproducibility and service life of the chromatographic column, and has low cost; at the same time, it has the advantages of good specificity, system applicability, linearity, precision and durability, high sensitivity and accuracy.

[0011] The technical solution of the present invention is as follows:

[0012] A method for detecting related substances of 4-(2,4-difluorobenzoyl)-piperidine hydrochloride, a starting material of paliperidone, adopts a high performance liquid chromatography method, and the chromatographic conditions include: an octadecylsilane bonded silica gel chromatographic column with an embedded pentafluorophenyl functional group, a mixed solution of triethylamine aqueous solution and methanol as mobile phase A, and a mixed solution of acetonitrile and tetrahydrofuran as mobile phase B.

[0013] Preferably according to the present invention, the volume concentration of the triethylamine aqueous solution is 0.45% to 0.55%, preferably 0.5%, and the pH value is 6.8 to 7.2, preferably 7.0.

[0014] Preferably according to the present invention, the volume ratio of triethylamine aqueous solution to methanol in the mobile phase A is 90:10.

[0015] Preferably according to the present invention, the volume ratio of acetonitrile to tetrahydrofuran in the mobile phase B is 90:10.

[0016] Preferably, according to the present invention, the mobile phase is eluted using the following gradient program:

[0017] From 0 to 5 minutes, 100% mobile phase A remains unchanged; from 5 to 45 minutes, the volume ratio of mobile phase A to mobile phase B changes linearly from 100% to 90:10; from 45 to 70 minutes, the volume ratio of mobile phase A to mobile phase B changes linearly from 90:10 to 30:70; from 70 to 80 minutes, the volume ratio of mobile phase A to mobile phase B remains unchanged at 30:70; from 80 to 81 minutes, the volume ratio of mobile phase A to mobile phase B changes linearly from 30:70 to 100% mobile phase A; from 81 to 91 minutes, 100% mobile phase A remains unchanged.

[0018] According to the preferred embodiment of the present invention, the chromatographic column is: ACE Excel 3C18-PFP, 4.6 mm×150 mm, 3 μm.

[0019] According to the preferred embodiment of the present invention, the chromatographic conditions include: using an ultraviolet detector with a detection wavelength of 238 nm; the column temperature is selected from 35 to 45°C, preferably 40°C; the mobile phase flow rate is selected from 0.9 to 1.1 mL / min, preferably 1.0 mL / min; the preferred injection volume is 15 μL.

[0020] Preferably, according to the present invention, a mixed solution of triethylamine aqueous solution and methanol is used as a diluent to prepare a test solution and a control solution; wherein the volume concentration of the triethylamine aqueous solution is 0.5%, the pH value is 7.0, and the volume ratio of the triethylamine aqueous solution to methanol is 27:73; the control solution and the test solution are accurately measured, injected into a liquid chromatograph, and the chromatogram is recorded.

[0021] According to a preferred technical solution of the present invention, the detection method comprises:

[0022] (1) Chromatographic conditions:

[0023] Instrument: High performance liquid chromatography-ultraviolet detector; Chromatographic column: Octadecylsilane bonded silica gel chromatographic column with embedded pentafluorophenyl functional group (ACE Excel 3C18-PFP, 4.6mm×250mm, 3μm); Mobile phase A: by volume ratio, a mixed solution of triethylamine aqueous solution (0.5%, pH 7.0): methanol = 90:10; Mobile phase B: by volume ratio, a mixed solution of acetonitrile: tetrahydrofuran = 90:10; Mobile phase flow rate: 1.0ml / min; Detection wavelength: 238nm; Column temperature: 40℃; Injection volume: 15μl;

[0024] Gradient elution program:

[0025]

[0026] (2) Solution preparation:

[0027] Diluent: a mixed solution of triethylamine aqueous solution (0.5%, pH 7.0) and methanol = 27:73 by volume;

[0028] Stock solution of impurity reference substances: accurately weigh impurity i-4 reference substance, impurity i-5 reference substance, impurity i-8 reference substance, impurity i-9 reference substance, impurity i-10 reference substance, impurity i-11 reference substance, impurity i-12 reference substance and impurity i-14 reference substance, dissolve them ultrasonically and dilute them with a diluent to prepare a solution with a concentration of 50 μg / mL for impurity i-4, impurity i-5, impurity i-8, impurity i-10, impurity i-11, impurity i-12 and impurity i-14 and a concentration of 75 μg / mL for impurity i-9, and shake well;

[0029] System suitability solution: accurately weigh 4-(2,4-difluorobenzoyl)-piperidine hydrochloride (DPH), dissolve it by ultrasonication with a diluent and dilute it to prepare a solution with a concentration of 0.5 mg / mL of 4-(2,4-difluorobenzoyl)-piperidine hydrochloride, 1 μg / mL of impurity i-4, impurity i-5, impurity i-8, impurity i-10, impurity i-11, impurity i-12 and impurity i-14, and 1.5 μg / mL of impurity i-9, and shake well;

[0030] Test solution: accurately weigh 4-(2,4-difluorobenzoyl)-piperidine hydrochloride (DPH), dissolve and dilute with diluent by ultrasonic to make a DPH solution with a concentration of 0.5 mg / mL, and shake well;

[0031] Control solution: Accurately measure the test solution, dilute it with diluent to make a solution with a DPH concentration of 1 μg / mL, and shake well;

[0032] (3) Determination method:

[0033] Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph, and record the chromatogram. In the chromatogram of the system suitability solution, impurity i-11, impurity i-4, impurity i-12, 4-(2,4-difluorobenzoyl)-piperidine, impurity i-14, impurity i-5, impurity i-9, impurity i-10 and impurity i-8 appear in sequence, and the theoretical plate number is not less than 10000 based on the 4-(2,4-difluorobenzoyl)-piperidine peak, and the separation between the impurity i-12 peak and the 4-(2,4-difluorobenzoyl)-piperidine peak should be not less than 1.3. Therefore, the impurity content is calculated according to the principal component self-control method with the correction factor added;

[0034] The formula for calculating the impurity content by the main component self-control method with added correction factor is: impurity content = peak area of ​​the impurity in the test solution × concentration of the control solution × correction factor / peak area of ​​the main peak of the control solution / concentration of the test solution × 100%; the correction factors of the impurity i-4, impurity i-5, impurity i-8, impurity i-9, impurity -10, impurity i-11, impurity i-12 and impurity i-14 are 1.5, 1.0, 0.7, 1.2, 0.7, 1.0, 1.0 and 1.0 respectively.

[0035] In the present invention, the synthesis process route of the starting material DPH of paliperidone is as follows:

[0036]

[0037] Among them, impurity i-5 is an intermediate in the preparation of DPH;

[0038] Impurities i-11 and i-12 are products generated by subsequent reactions of impurities i-15 and i-16 contained in m-difluorobenzene. The generation pathways of impurities i-11 and i-12 are as follows:

[0039]

[0040] Impurities i-4 and i-14 are products of the isomerization reaction between impurity i-3 and m-difluorobenzene during the preparation of DPH. The formation pathways of impurities i-4 and i-14 are as follows:

[0041]

[0042] Impurity i-8 is a product generated by the condensation reaction of DPH and m-difluorobenzene during the preparation of DPH. The generation pathway of impurity i-8 is as follows:

[0043]

[0044] Impurities i-9 and i-10 are products of the intermolecular condensation reaction of DPH under acidic conditions. The generation pathways of impurities i-9 and i-10 are as follows:

[0045]

[0046] The technical features and beneficial effects of the present invention are as follows:

[0047] 1. Through systematic screening of various chromatographic column models, the present invention finally determined that an octadecylsilane bonded silica gel chromatographic column with an embedded pentafluorophenyl functional group (ACE Excel 3C18-PFP, 4.6 mm×150 mm, 3 μm) was selected, which has the best separation efficiency for positional isomer impurities in DPH.

[0048] 2. The method of the present invention achieves the simultaneous determination of positional isomers and other known impurities that may exist in DPH by a single analytical method through screening of chromatographic conditions such as chromatographic columns, mobile phases, and elution procedures. As a whole, the method of the present invention, with the combined effect of various steps and conditions, enables the present invention to effectively separate and simultaneously determine impurities i-4, i-5, i-8, i-9, i-10, i-11, i-12, and i-14, a total of 8 impurities; the method is simple, does not affect the reproducibility and service life of the chromatographic column, and is low in cost; at the same time, it has the advantages of good specificity, system applicability, linearity, precision and durability, and high sensitivity and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1It is the chromatogram of the positioning solution and the test sample solution in Comparative Example 1 (from bottom to top: impurity i-11, diluent, test sample, impurity i-1, impurity i-3, impurity i-14, impurity i-5, impurity i-8, impurity i-4, impurity i-12 positioning solution);

[0050] Figure 2 The chromatograms of the mixed solutions of impurity reference substances with different proportions of mobile phase B in Comparative Example 2 are as follows;

[0051] Figure 3 The chromatograms of the impurity reference substance mixed solution and the test solution in Comparative Example 3;

[0052] Figure 4 The chromatograms of the impurity reference substance mixed solution and the test solution in Comparative Example 4;

[0053] Figure 5 The chromatograms of the impurity 14 localization solution and the test sample spiked solution in Comparative Example 5;

[0054] Figure 6 This is the chromatogram of the spiked solution of the test sample in the ACE Excel 3C18-PFP chromatographic column in Comparative Example 6;

[0055] Figure 7 The chromatogram of the spiked solution of the test sample on the Agilent Eclipse XDB C18 chromatographic column in Comparative Example 6;

[0056] Figure 8 This is the chromatogram of the spiked solution of the Sunniest Biphenyl chromatographic column test sample in Comparative Example 6;

[0057] Fig. 9 The chromatograms of the impurity reference substance mixed solution and the test sample solution in Comparative Example 7;

[0058] Fig.10 This is the chromatogram of the mixed solution of impurity reference substance in Comparative Example 8;

[0059] Fig.11 This is the chromatogram of the mixed solution of impurity reference substance in Comparative Example 9;

[0060] Fig.12 This is the chromatogram of the spiked solution of the test sample in Comparative Example 10;

[0061] Fig.13 This is the chromatogram of the spiked solution of the test sample in Comparative Example 11;

[0062] Fig.14 The chromatograms are of the impurity reference substance mixed solution and the test substance solution in Comparative Example 12. DETAILED DESCRIPTION

[0063] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0064] It should be noted that the terms used herein are for describing specific embodiments only and are not intended to be limiting of exemplary embodiments according to the present invention.

[0065] In the present embodiment, the contents of related substances in DPH samples of batches Y220525351, Y220829351, 220303 and 2003-4001-230502 produced by Baiyin Jingyu New Pharmaceutical Co., Ltd. were detected. The method validation test was conducted using samples provided by Baiyin Jingyu New Pharmaceutical Co., Ltd. All other reagents were commercially available products.

[0066] Comparative Example 1

[0067] 1. Chromatographic conditions

[0068] Chromatographic column: octadecylsilane bonded silica gel chromatographic column (Agilent Eclipse XDB C18, 4.6 mm × 250 mm, 5 μm);

[0069] Detection wavelength: 238nm, UV detector; flow rate: 1.0ml / min; column temperature: 35℃; injection volume: 20μl;

[0070] 0.5% triethylamine aqueous solution (pH adjusted to 7.0 with phosphoric acid)-acetonitrile (volume ratio 90:10) was used as mobile phase A, and methanol was used as mobile phase B;

[0071] Elution procedure:

[0072]

[0073] 2. Solution preparation

[0074] Diluent: a mixed solution of triethylamine aqueous solution (0.5%, pH 7.0) and methanol = 27:73 by volume;

[0075] Positioning solution: Take appropriate amount of impurity i-1, i-3, i-4, i-5, i-8, i-11, i-12, and i-14 reference substances respectively, accurately weigh and place in the same volumetric flask, use appropriate amount of diluent to ultrasonically dissolve and dilute to make a solution containing about 2.5 μg of each impurity in every 1 ml of solvent, and shake well.

[0076] Test solution: Take an appropriate amount of DPH, weigh it accurately, dissolve it by ultrasonication with an appropriate amount of diluent and dilute it to make a solution containing about 0.5 mg of DPH per 1 ml of solvent, and shake well.

[0077] Accurately measure the positioning solution and the test solution and inject them into the liquid chromatograph to record the chromatogram; the results are shown in Table 1 and Figure 1 .

[0078] Table 1

[0079] Impurity Name i-1 i-3 i-11 i-14 i-4 i-12 Main Peak i-5 i-8 Peak time (min) Not yet peaked Not yet peaked 6.073 6.396 6.410 7.528 7.612 22.941 60.328

[0080] Conclusion: Impurities i-1 and i-3 did not elute, and other methods were used to study impurities i-1 and i-3. Impurities i-4 and i-14 were not separated, and impurities i-12 and DPH were not separated. This indicates that the mobile phase ratio under the chromatographic conditions cannot well separate impurities i-4 and i-14, and impurities i-12 and DPH.

[0081] Comparative Example 2

[0082] Optimize the situation that impurities i-4, i-11, i-14, i-12 and DPH are difficult to separate. By adjusting the mobile phase elution program, the proportion of methanol in the mobile phase is slowly increased to check the separation. At the same time, because the main peak area of ​​the test sample is large at 20μl, the injection volume is adjusted to 10μl.

[0083] 1. Chromatographic conditions

[0084] Chromatographic column: Octadecylsilane bonded silica column with embedded pentafluorophenyl functional groups (ACE Excel3C18-PFP, 4.6 mm × 250 mm, 3 μm);

[0085] Detection wavelength: 238nm, UV detector; flow rate: 1.0ml / min; column temperature: 35℃; injection volume: 10μl;

[0086] 0.5% triethylamine aqueous solution (pH adjusted to 7.0 with phosphoric acid) was used as mobile phase A, and methanol was used as mobile phase B;

[0087] Elution procedure:

[0088]

[0089] 2. Solution preparation

[0090] Diluent: a mixed solution of triethylamine aqueous solution (0.5%, pH 7.0) and methanol = 27:73 by volume;

[0091] Mixed solution of impurity reference substances: Take appropriate amount of DPH and impurity i-4, i-11, i-12, and i-14 reference substances respectively, weigh accurately and place in the same volumetric flask, use appropriate amount of diluent to ultrasonically dissolve and dilute to make a solution containing DPH and approximately 2.5 μg of each impurity in every 1 ml of solvent, and shake well.

[0092] Accurately measure the impurity reference substance mixed solution and inject it into the liquid chromatograph, and record the chromatogram; the results are shown in Figure 2 .

[0093] Conclusion: After adjusting the methanol ratio in the mobile phase gradient, 4 impurities and the main component reference substance produced 4 peaks in total, and the peak shape and separation were not ideal.

[0094] Comparative Example 3

[0095] Mobile phase B was changed from methanol to acetonitrile, and the elution program was adjusted to optimize the method.

[0096] 1. Chromatographic conditions

[0097] Chromatographic column: Octadecylsilane bonded silica column with embedded pentafluorophenyl functional groups (ACE Excel3C18-PFP, 4.6 mm × 250 mm, 3 μm);

[0098] Detection wavelength: 238nm, UV detector; flow rate: 1.0ml / min; column temperature: 35℃; injection volume: 10μl;

[0099] 0.5% triethylamine aqueous solution (pH adjusted to 7.0 with phosphoric acid) was used as mobile phase A, and acetonitrile was used as mobile phase B;

[0100] Elution procedure:

[0101]

[0102] 2. Solution preparation

[0103] Diluent: a mixed solution of triethylamine aqueous solution (0.5%, pH 7.0) and methanol = 27:73 by volume;

[0104] Mixed solution of impurity reference substances: Take appropriate amount of DPH and impurity i-4, i-11, i-12, and i-14 reference substances respectively, weigh accurately and place in the same volumetric flask, use appropriate amount of diluent to ultrasonically dissolve and dilute to make a solution containing DPH and approximately 2.5 μg of each impurity in every 1 ml of solvent, and shake well.

[0105] Test solution: Take an appropriate amount of 4-(2,4-difluorobenzoyl)-piperidine hydrochloride (DPH), weigh it accurately, dissolve it by ultrasonication with an appropriate amount of diluent and dilute it to make a solution containing about 0.5 mg of DPH per 1 ml of solvent, and shake well.

[0106] Accurately measure the impurity reference substance mixed solution and the test substance solution and inject them into the liquid chromatograph, and record the chromatogram; the results are shown in Figure 3 .

[0107] Conclusion: When mobile phase B was changed to acetonitrile, 4 impurity reference substances and main component reference substances showed 4 peaks in total, and the 4 peak shapes and separations were better than when mobile phase B was methanol, indicating that acetonitrile had better selectivity for peak shape than methanol.

[0108] Comparative Example 4

[0109] Adjust the elution program to optimize the method.

[0110] 1. Chromatographic conditions

[0111] Chromatographic column: Octadecylsilane bonded silica column with embedded pentafluorophenyl functional groups (ACE Excel3C18-PFP, 4.6 mm × 250 mm, 3 μm);

[0112] Detection wavelength: 238nm, UV detector; flow rate: 1.0ml / min; column temperature: 35℃; injection volume: 10μl;

[0113] 0.5% triethylamine aqueous solution (pH adjusted to 7.0 with phosphoric acid) was used as mobile phase A, and acetonitrile was used as mobile phase B;

[0114] Elution procedure:

[0115]

[0116] 2. Solution preparation

[0117] Prepare the diluent, impurity reference substance mixed solution and test sample solution according to the method described in Comparative Example 3.

[0118] Accurately measure the impurity reference substance mixed solution and the test substance solution and inject them into the liquid chromatograph, and record the chromatogram; the results are shown in Figure 4 .

[0119] Conclusion: The change of acetonitrile ratio in mobile phase B still cannot completely separate the five peaks. Combined with the positioning results of comparative example 1, it is inferred that impurities i-12 and DPH are not separated.

[0120] Comparative Example 5

[0121] Tetrahydrofuran was added to mobile phase B, and the elution program was adjusted to optimize the method.

[0122] 1. Chromatographic conditions

[0123] Chromatographic column: Octadecylsilane bonded silica column with embedded pentafluorophenyl functional groups (ACE Excel3C18-PFP, 4.6 mm × 250 mm, 3 μm);

[0124] Detection wavelength: 238nm, UV detector; flow rate: 1.0ml / min; column temperature: 35℃; injection volume: 10μl;

[0125] 0.5% triethylamine aqueous solution (pH adjusted to 7.0 with phosphoric acid) was used as mobile phase A, and acetonitrile-tetrahydrofuran (volume ratio 90:10) was used as mobile phase B;

[0126] Elution procedure:

[0127]

[0128]

[0129] 2. Solution preparation

[0130] Diluent: a mixed solution of triethylamine aqueous solution (0.5%, pH 7.0) and methanol = 27:73 by volume;

[0131] Positioning solution: Take appropriate amount of impurity i-4, i-5, i-8, i-9, i-10, i-11, i-12 and i-14 reference substances respectively, accurately weigh and place in the same volumetric flask, use appropriate amount of diluent to ultrasonically dissolve and dilute to make a solution containing about 2.5 μg of each impurity in every 1 ml of solvent, and shake well.

[0132] Test sample spiked solution: Take appropriate amount of impurities i-4, i-5, i-8, i-9, i-10, i-11, i-12 and DPH, weigh accurately, dissolve and dilute with appropriate amount of diluent by ultrasonication to make a solution containing about 0.5 mg of DPH and about 2.5 μg of each impurity in every 1 ml of solvent, and shake well.

[0133] Accurately measure the positioning solution and the test sample spiked solution and inject them into the liquid chromatograph to record the chromatogram; the results are shown in Table 2 Figure 5 .

[0134] Table 2

[0135] Impurity Name i-11 i-4 i-12 Main Peak i-14 i-5 i-9 i-10 i-8 Peak time (min) 21.886 23.464 26.725 27.274 33.143 52.620 57.881 58.530 64.063 S / N 75.2 49.5 63.5 / 57.1 138.4 293.3 320.5 200.4

[0136] Conclusion: The impurity peaks are well separated; the responses of impurity i-4, impurity i-11, impurity i-12 and impurity i-14 are slightly smaller, and the separation degree between impurity i-12 and the main peak is 1.3, which is slightly smaller.

[0137] Comparative Example 6

[0138] 1. Chromatographic conditions

[0139] Chromatographic columns: ACE Excel 3C18-PFP (4.6 mm × 250 mm, 3 μm), Agilent Eclipse XDB C18 (4.6 mm × 250 mm, 5 μm), Sunniest Biphenyl (4.6 mm × 250 mm, 5 μm);

[0140] Detection wavelength: 238nm, UV detector; flow rate: 1.0ml / min; column temperature: 40℃; injection volume: 15μl;

[0141] A mixed solution of triethylamine aqueous solution (0.5%, pH 7.0): methanol = 90:10 was used as mobile phase A, and acetonitrile-tetrahydrofuran (volume ratio 90:10) was used as mobile phase B;

[0142] Elution procedure:

[0143]

[0144]

[0145] 2. Solution preparation

[0146] Diluent: a mixed solution of triethylamine aqueous solution (0.5%, pH 7.0) and methanol = 27:73 by volume;

[0147] Test sample spiked solution: Take appropriate amount of impurities i-4, i-5, i-8, i-9, i-10, i-11, i-12, i-14 and DPH, weigh accurately, dissolve and dilute with appropriate amount of diluent by ultrasonication to make a solution containing about 0.5 mg of DPH and about 2.5 μg of each impurity in every 1 ml of solvent, and shake well.

[0148] Accurately measure the test sample spiked solution and inject it into the liquid chromatograph to record the chromatogram; the test results of ACE Excel 3C18-PFP (4.6mm×250mm, 3μm) chromatographic column are shown in Table 3 and Figure 6 , Agilent Eclipse XDB C18 (4.6mm×250mm, 5μm) chromatographic column test results are shown in Figure 7 , Sunniest Biphenyl (4.6mm×250mm, 5μm) chromatographic column test results are shown in Figure 8 .

[0149] Table 3

[0150] Impurity Name i-11 i-4 i-12 Main Peak i-14 i-5 i-9 i-10 i-8 Peak time (min) 19.369 20.073 23.755 24.701 29.612 56.853 62.009 62.931 70.932 S / N 190.3 157.9 179.7 / 174.4 705.2 1437.8 995.6 860.3

[0151] Conclusion: Combined with the impurity localization of comparative example 5, the test results show that the ACE Excel 3C18-PFP (4.6mm×250mm, 3μm) chromatographic column is used for detection, and the impurity peaks are well separated, and the separation degree between impurity i-12 and the main peak is 1.7; the signal-to-noise ratio of each impurity peak height meets the requirements and has high sensitivity. The Agilent Eclipse XDB C18 (4.6mm×250mm, 5μm) chromatographic column is used for detection, and the impurity i-12 is wrapped in the main peak and is not separated. The Sunniest Biphenyl (4.6mm×250mm, 5μm) chromatographic column is used for detection, and the number of impurity peaks is significantly less than 8, which cannot be used for detection.

[0152] Comparative Example 7

[0153] 1. Chromatographic conditions

[0154] Chromatographic column: Octadecylsilane bonded silica column with embedded pentafluorophenyl functional groups (ACE Excel3C18-PFP, 4.6 mm × 250 mm, 3 μm);

[0155] Detection wavelength: 238nm, UV detector; flow rate: 1.0ml / min; column temperature: 40℃; injection volume: 20μl;

[0156] 20mmol / L potassium dihydrogen phosphate solution (pH adjusted to 3.5 with phosphoric acid) was used as mobile phase A, and acetonitrile was used as mobile phase B;

[0157] Elution procedure:

[0158]

[0159]

[0160] 2. Solution preparation

[0161] Prepare the diluent, impurity reference substance mixed solution and test sample solution according to the method described in Comparative Example 3.

[0162] Accurately measure the impurity reference substance mixed solution and the test substance solution and inject them into the liquid chromatograph, and record the chromatogram; the results are shown in Fig. 9 .

[0163] Conclusion: Two impurities overlapped with the main peak and could not be separated. The tailing factor of the main peak under the acidic mobile phase condition was 4.2, while triethylamine was added to the mobile phase A to form an alkaline mobile phase, and the tailing factor of the main peak was 1.8, which made it easier to separate the main peak and the adjacent impurities behind the main peak, so alkaline conditions were preferred as the mobile phase.

[0164] Comparative Example 8

[0165] 1. Chromatographic conditions

[0166] Chromatographic column: Octadecylsilane bonded silica column with embedded pentafluorophenyl functional groups (ACE Excel3C18-PFP, 4.6 mm × 250 mm, 3 μm);

[0167] Detection wavelength: 238nm, UV detector; flow rate: 1.0ml / min; column temperature: 40℃; injection volume: 20μl;

[0168] 20mmol / L potassium dihydrogen phosphate solution (pH adjusted to 3.5 with phosphoric acid) was used as mobile phase A, and methanol was used as mobile phase B;

[0169] Elution procedure:

[0170]

[0171] 2. Solution preparation

[0172] Prepare a mixed solution of diluent and impurity reference substance according to the method described in Comparative Example 3.

[0173] Accurately measure the impurity reference substance mixed solution and inject it into the liquid chromatograph, and record the chromatogram; the results are shown in Fig.10 .

[0174] Conclusion: Impurities overlap with the main peak and cannot be separated, indicating that the addition of phosphate cannot improve the separation of impurities.

[0175] Comparative Example 9

[0176] 1. Chromatographic conditions

[0177] Chromatographic column: Octadecylsilane bonded silica column with embedded pentafluorophenyl functional groups (ACE Excel3C18-PFP, 4.6 mm × 250 mm, 3 μm);

[0178] Detection wavelength: 238nm, UV detector; flow rate: 1.0ml / min; column temperature: 40℃; injection volume: 20μl;

[0179] 0.1% phosphoric acid aqueous solution was used as mobile phase A, and methanol was used as mobile phase B;

[0180] Elution procedure:

[0181]

[0182] 2. Solution preparation

[0183] Prepare a mixed solution of diluent and impurity reference substance according to the method described in Comparative Example 3.

[0184] Accurately measure the impurity reference substance mixed solution and inject it into the liquid chromatograph, and record the chromatogram; the results are shown in Fig.11 .

[0185] Conclusion: Impurities overlap with the main peak and cannot be separated. The separation between impurities is poor.

[0186] Comparative Example 10

[0187] 1. Chromatographic conditions

[0188] Chromatographic column: Octadecylsilane bonded silica column with embedded pentafluorophenyl functional groups (ACE Excel3C18-PFP, 4.6 mm × 250 mm, 3 μm);

[0189] Detection wavelength: 238nm, UV detector; flow rate: 1.0ml / min; column temperature: 40℃; injection volume: 10μl;

[0190] A mixed solution of triethylamine aqueous solution (0.5%, pH 7.0): methanol = 90:10 was used as mobile phase A, and acetonitrile was used as mobile phase B;

[0191] Elution procedure:

[0192]

[0193] 2. Solution preparation

[0194] Prepare the diluent and test sample spike solution according to the method described in Comparative Example 6.

[0195] Accurately measure the test sample spiked solution and inject it into the liquid chromatograph, and record the chromatogram; the results are shown in Fig.12 .

[0196] Conclusion: Combined with the experimental results of Comparative Example 6, it can be inferred that under the chromatographic conditions of this comparative example, impurity i-9 and impurity i-10 completely overlap and are not separated.

[0197] Comparative Example 11

[0198] 1. Chromatographic conditions

[0199] Chromatographic column: Octadecylsilane bonded silica column with embedded pentafluorophenyl functional groups (ACE Excel3C18-PFP, 4.6 mm × 250 mm, 3 μm);

[0200] Detection wavelength: 238nm, UV detector; flow rate: 1.0ml / min; column temperature: 40℃; injection volume: 10μl;

[0201] A mixed solution of triethylamine aqueous solution (0.5%, pH 7.0): methanol = 90:10 was used as mobile phase A, and a mixed solution of methanol: acetonitrile = 50:50 was used as mobile phase B;

[0202] Elution procedure:

[0203]

[0204] 2. Solution preparation

[0205] Prepare the diluent and test sample spike solution according to the method described in Comparative Example 6.

[0206] Accurately measure the test sample spiked solution and inject it into the liquid chromatograph, and record the chromatogram; the results are shown in Fig.13 .

[0207] Conclusion: Combined with the experimental results of Comparative Example 6, it can be inferred that under the chromatographic conditions of this comparative example, impurity i-9 and impurity i-10 completely overlap and are not separated.

[0208] Comparative Example 12

[0209] 1. Chromatographic conditions

[0210] Chromatographic column: Octadecylsilane bonded silica column with embedded pentafluorophenyl functional groups (ACE Excel3C18-PFP, 4.6 mm × 250 mm, 3 μm);

[0211] Detection wavelength: 238nm, UV detector; flow rate: 1.0ml / min; column temperature: 30℃; injection volume: 10μl;

[0212] A mixed solution of triethylamine aqueous solution (0.5%, pH 7.0): methanol = 90:10 was used as mobile phase A, and acetonitrile-tetrahydrofuran (volume ratio 70:30) was used as mobile phase B;

[0213] Elution procedure:

[0214]

[0215] 2. Solution preparation

[0216] Prepare the diluent, impurity reference substance mixed solution and test sample solution according to the method described in Comparative Example 3.

[0217] Accurately measure the impurity reference substance mixed solution and the test substance solution and inject them into the liquid chromatograph, and record the chromatogram; the results are shown in Fig.14 .

[0218] Conclusion: Under the conditions of this comparative example, the impurity reference mixed solution has three peaks out of five components, and the main peak may overlap with the impurity, and the impurity and the main component are not separated. This shows that it is necessary to add tetrahydrofuran to mobile phase B. At the same time, if the amount of tetrahydrofuran added is too large, the separation degree of impurities will be reduced.

[0219] Example 1

[0220] Detection method of related substances of 4-(2,4-difluorobenzoyl)-piperidine hydrochloride, the starting material of paliperidone

[0221] (1) Chromatographic conditions:

[0222] Instrument: High performance liquid chromatography-ultraviolet detector; Chromatographic column: Octadecylsilane bonded silica gel chromatographic column with embedded pentafluorophenyl functional group (ACE Excel 3C18-PFP, 4.6mm×250mm, 3μm); Mobile phase A: by volume ratio, a mixed solution of triethylamine aqueous solution (0.5%, pH value adjusted to 7.0 with phosphoric acid): methanol = 90:10; Mobile phase B: by volume ratio, a mixed solution of acetonitrile: tetrahydrofuran = 90:10; Mobile phase flow rate: 1.0ml / min; Detection wavelength: 238nm; Column temperature: 40℃; Injection volume: 15μl;

[0223] Gradient elution program:

[0224]

[0225] (2) Solution preparation:

[0226] Diluent: a mixed solution of triethylamine aqueous solution (0.5%, pH value adjusted to 7.0 with phosphoric acid): methanol = 27:73 by volume;

[0227] Stock solution of impurity reference substances: accurately weigh impurity i-4 reference substance, impurity i-5 reference substance, impurity i-8 reference substance, impurity i-9 reference substance, impurity i-10 reference substance, impurity i-11 reference substance, impurity i-12 reference substance and impurity i-14 reference substance, dissolve them ultrasonically and dilute them with diluent to make solutions with concentrations of 50 μg / mL for impurity i-4, impurity i-5, impurity i-8, impurity i-10, impurity i-11, impurity i-12 and impurity i-14 and 75 μg / mL for impurity i-9, and shake well;

[0228] System suitability solution: accurately weigh 4-(2,4-difluorobenzoyl)-piperidine hydrochloride (DPH), dissolve it by ultrasonication with a diluent and dilute it to make a solution with a DPH concentration of 0.5 mg / mL, an impurity i-4 reference substance, an impurity i-5 reference substance, an impurity i-8 reference substance, an impurity i-10 reference substance, an impurity i-11 reference substance, an impurity i-12 reference substance and an impurity i-14 reference substance concentration of 1 μg / mL, and an impurity i-9 reference substance concentration of 1.5 μg / mL, and shake well;

[0229] Test solution: accurately weigh DPH, dissolve and dilute with diluent by ultrasonic to make a DPH solution of 0.5 mg / mL, and shake well;

[0230] Control solution: Accurately measure the test solution, dilute it with diluent to make a solution with a DPH concentration of 1 μg / mL, and shake well;

[0231] (3) Determination method:

[0232] Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph, and record the chromatogram. Calculate the content of each impurity according to the principal component self-reference method with added correction factor.

[0233] Table 4. Sample test results

[0234]

[0235] Example 2: Methodological Validation

[0236] 2.1 System suitability and specificity test

[0237] Solvent: 0.5% triethylamine solution (pH adjusted to 7.0 with phosphoric acid)-methanol (27:73).

[0238] Blank solution: solvent.

[0239] Impurity i-4 reference substance stock solution: Take about 5 mg of impurity i-4 reference substance, accurately weigh it, place it in a 100 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0240] Impurity i-4 positioning solution: Accurately measure 1 ml of impurity i-4 reference stock solution, place it in a 50 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0241] Refer to the impurity i-4 reference stock solution and impurity i-4 positioning solution to prepare the reference stock solutions and positioning solutions of impurity i-5, impurity i-8, impurity i-10, impurity i-11, impurity i-12 and impurity i-14.

[0242] Impurity i-9 reference substance stock solution: Take about 7.5 mg of impurity i-9 reference substance, accurately weigh, place in a 100 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0243] Impurity i-9 positioning solution: Accurately measure 1 ml of impurity i-9 reference stock solution, place it in a 50 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0244] System suitability solution: Take about 25 mg of this product, weigh accurately, place in a 50 ml volumetric flask, accurately add 1 ml of each of the above impurity reference stock solutions, add solvent to dissolve and dilute to the scale, and shake well.

[0245] Test solution: Take about 25 mg of the product, weigh accurately, place in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale and shake well.

[0246] Interference stock solution: Take about 5 mg of impurity i-1 reference substance and impurity i-3 reference substance respectively, place in a 100 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0247] Mixed solution: Take about 25 mg of this product, weigh accurately, place in a 50 ml volumetric flask, accurately add 1 ml of the above-mentioned impurity reference stock solution and interference stock solution, place in a 50 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0248] Take blank solution, system suitability solution, test solution, mixed solution and each impurity location solution and inject them into the liquid chromatograph respectively, record the chromatogram, and the test results are shown in Table 5.

[0249] Table 5. DPH related substances specificity verification results

[0250]

[0251] Verification conclusion: The system suitability solution was injected 5 times continuously, the theoretical plate number of the DPH main peak was at least 22967, which was not less than 10000, the separation between the impurity i-12 peak and the DPH main peak was at least 1.5, not less than 1.3, the RSD of the retention time of each known impurity peak was less than 1%, the tailing factor of each known impurity peak was less than 2.0, and the signal-to-noise ratio of the peak height of each known impurity peak was greater than 100, which met the requirements, indicating that the method system suitability was good. The blank solution and the impurities studied in other methods (impurity i-1, impurity i-3) did not interfere with the detection of the impurity peaks studied in this method, indicating that the method specificity was good.

[0252] 2.2 Linearity and range tests

[0253] Reference substance stock solution ②: Take approximately 5 mg each of DPH reference substance, impurity i-4 reference substance, impurity i-5 reference substance, impurity i-8 reference substance, impurity i-10 reference substance, impurity i-11 reference substance, impurity i-12 reference substance and impurity i-14 reference substance, accurately weigh, place in the same 100 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0254] Linear solutions 3, 4, 5, 6 (50%, 100%, 150%, 250%): Accurately measure 1, 2, 3, 5 ml of reference stock solution ② respectively, place in different 20 ml volumetric flasks, dilute to the scale with solvent, and shake well.

[0255] Linear solution 2 (20%): Accurately measure 45 ml of the linear solution, place it in a 25 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0256] Impurity i-4, impurity i-11, impurity i-12, impurity i-14 and DPH quantitative limit solution (linear solution 1): Accurately measure 2-3 ml of linear solution, place in a 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0257] Impurity i-5, impurity i-8 and impurity i-10 quantitative limit solution (linear solution 1): Accurately measure 1 ml of linear solution 2, place it in a 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0258] Reference substance stock solution ①: Take about 7.5 mg of DPH reference substance and impurity i-9 reference substance respectively, weigh accurately, place in the same 100 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well.

[0259] Linear solutions ③, ④, ⑤, ⑥ (50%, 100%, 150%, 250%): Accurately measure 1, 2, 3, 5 ml of reference stock solution ② respectively, place in different 20 ml volumetric flasks, dilute to the scale with solvent, and shake well.

[0260] Linear solution ② (20%): Accurately measure 5 ml of linear solution ④, place in a 25 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0261] Impurity i-9 quantitative limit solution (linear solution ①): Accurately measure 1 ml of linear solution ②, place in a 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0262] Accurately measure 15 μl of each of the above linear solutions, inject them into the liquid chromatograph, and record the chromatogram. Perform linear regression with the concentration C (μg / ml) of each component as the abscissa and the peak area A as the ordinate. The test results are shown in Table 6.

[0263] Table 6 Test results of linear relationship between impurities and main components

[0264] name Linear concentration range (μg / ml) Linear equations Correlation coefficient r DPH 0.0592～2.4655 A=31499.2718C-18.6023 0.9998 Impurity i-4 0.0578～2.4074 A=21299.2710C-217.7569 0.9998 Impurity i-5 0.0188～2.3558 A=35381.4053C+64.7812 0.9999 Impurity i-8 0.0202～2.5301 A=43736.7981C+103.5705 0.9998 Impurity i-10 0.0202～2.5253 A=45096.9066C-237.2361 0.9999 Impurity i-11 0.0605～2.5219 A=34250.0796C+368.9604 0.9998 Impurity i-12 0.0569～2.3710 A=31078.8859C-365.6230 0.9999 Impurity i-14 0.0587～2.4443 A=34592.6148C-439.5381 0.9998 DPH(Group 2) 0.0592～3.7423 A=30843.4794C-1159.6537 0.9997 Impurity i-9(Group 2) 0.0299～3.7362 A=26246.7567C+115.3709 0.9999

[0265] The correction factors of each known impurity were calculated by the ratio of the slope of the DPH linear relationship curve to the slope of each known impurity linear relationship curve. The results are shown in Table 7.

[0266] Table 7. Results of correction factors for various impurities

[0267] Impurity Name Impurity i-4 Impurity i-5 Impurity i-8 Impurity i-9 Impurity i-10 Impurity i-11 Impurity i-12 Impurity i-14 Correction Factor 1.5 0.9 0.7 1.2 0.7 0.9 1.0 0.9

[0268] Verification conclusion: Each impurity has a good linear relationship within the designed linear range. The correction factors of impurity i-4, impurity i-5, impurity i-8, impurity i-9, impurity -10, impurity i-11, impurity i-12 and impurity i-14 are 1.5, 0.9, 0.7, 1.2, 0.7, 0.9, 1.0 and 0.9 respectively, among which the correction factors of impurity i-5, impurity i-11, impurity i-12 and impurity i-14 are all in the range of 0.9 to 1.1, and are calculated as 1.0.

[0269] 2.3. Limit of Quantitation and Limit of Detection Test

[0270] Prepare 6 portions of the quantitative limit concentration solution and inject them sequentially, inject the detection limit solution once, and record the chromatogram. Report the quantitative limit and detection limit concentration (μg / ml), S / N, peak area, RSD of peak area, percentage of test sample concentration and percentage of limit concentration respectively. The test results are shown in Table 8.

[0271] Table 8 Results of quantitative limits and detection limits of each impurity and main component

[0272]

[0273] Verification conclusion: The quantitative limit of each impurity is not greater than 0.02% of the test sample concentration, and is less than 10% of the limit concentration. The detection limit of each impurity is not greater than 0.01% of the test sample concentration, and is less than 5% of the limit concentration. The RSD of the peak area of ​​the quantitative limit repeatability test of each impurity is less than 20%; all meet the requirements.

[0274] 2.4 Solution stability test

[0275] Stock solution of impurity reference substances: Take approximately 5 mg each of impurity i-4 reference substance, impurity i-5 reference substance, impurity i-8 reference substance, impurity i-10 reference substance, impurity i-11 reference substance, impurity i-12 reference substance and impurity i-14 reference substance, and approximately 7.5 mg of impurity i-9 reference substance, accurately weigh, place in the same 100 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0276] Reference solution: Accurately measure 1 ml of the impurity reference stock solution, place it in a 50 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0277] Spiked test solution: Take about 25 mg of the product, accurately weigh it, put it in a 50 ml volumetric flask, accurately add 1 ml of the impurity reference stock solution, add solvent to dissolve and dilute to the scale, and shake well.

[0278] Test solution: Take about 25 mg of the product, weigh accurately, place in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale and shake well.

[0279] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the mark with solvent, and shake well. Accurately measure 2 ml, place it in a 10 ml volumetric flask, dilute it to the mark with solvent, and shake well.

[0280] The reference solution, spiked test solution, test solution and control solution were placed at room temperature, and injected once at 0h, 6h, 12h, 24h, 36h and 48h after preparation, and the chromatograms were recorded. The test results are shown in Tables 9 to 11.

[0281] Table 9. Results of the ratio of the main peak area and the peak area at 0 h for the reference substance and the reference solution at each time point (%)

[0282] Impurity Name 0h 6h 12h 24h 36h 48h Impurity i-4 — 100.7 106.0 101.3 106.1 102.6 Impurity i-5 — 97.6 98.5 97.4 98.8 97.7 Impurity i-8 — 100.8 102.6 102.7 105.1 102.0 Impurity i-9 — 98.5 99.9 101.1 102.8 102.1 Impurity i-10 — 100.9 101.7 103.6 102.0 102.0 Impurity i-11 — 97.2 98.3 99.2 102.1 102.2 Impurity i-12 — 99.0 98.2 101.6 100.8 103.6 Impurity i-14 — 103.5 101.5 103.3 101.1 100.0 DPH — 98.9 97.7 99.6 101.1 101.0

[0283] Table 10. Absolute value of the difference between the content of each impurity in the test solution at each time point and the content at 0h (%)

[0284] Impurity Name 0h 6h 12h 24h 36h 48h Impurity i-4 — 0.01 0.01 0.01 0 0 Impurity i-9 — 0 0.01 0.01 0.02 0.01 Other single miscellaneous — — — — — — Total impurities — 0.01 0 0.02 0.02 0.01

[0285] Table 11. Absolute value of the difference between the content of each impurity in the spiked test solution at each time point and the content at 0h (%)

[0286]

[0287]

[0288] Verification conclusion:

[0289] The test solution was injected with one injection at each time point from 0 to 48 h. The absolute value of the difference between the impurity i-4 and impurity i-9 at each time point and the 0 h content did not exceed 0.02%, the absolute value of the difference between the total impurity content and the 0 h content did not exceed 0.05%, other known impurities were not detected, and during the storage process, no impurities that affected the detection of known impurities and other impurities exceeding 0.02% were generated, indicating that the test solution had good stability within 48 h at room temperature.

[0290] The reference solution and the control solution were injected with one injection each at time points from 0 to 48 h, and the ratios of the peak areas of each known impurity and DPH at each time point to the peak area at 0 h were all in the range of 90 to 108%, indicating that the reference solution and the control solution had good stability within 48 h at room temperature.

[0291] The spiked test solution was injected with one injection at each time point from 0 to 36 h. The absolute value of the difference between the known impurities and other single impurities at each time point and the 0 h content did not exceed 0.03%, and the absolute value of the difference between the total amount of impurities and the 0 h content did not exceed 0.10%. In addition, no impurities that affected the detection of known impurities and other impurities exceeding 0.02% were produced during the storage process, indicating that the spiked test solution had good stability within 36 hours at room temperature.

[0292] 2.5 Repeatability test

[0293] Solvent: 0.5% triethylamine solution (pH adjusted to 7.0 with phosphoric acid)-methanol (27:73).

[0294] Blank solution: solvent.

[0295] Stock solution of impurity reference substances: Take approximately 5 mg each of impurity i-4 reference substance, impurity i-5 reference substance, impurity i-8 reference substance, impurity i-10 reference substance, impurity i-11 reference substance, impurity i-12 reference substance and impurity i-14 reference substance, and approximately 7.5 mg of impurity i-9 reference substance, accurately weigh, place in the same 100 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0296] System suitability solution: Take about 25 mg of this product, weigh accurately, place in a 50 ml volumetric flask, accurately add 1 ml of impurity reference stock solution, add solvent to dissolve and dilute to the scale, and shake well.

[0297] Reference solution: Accurately measure 1 ml of the impurity reference stock solution, place it in a 50 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0298] Spiked test solution: Take about 25 mg of the product, weigh accurately, place in a 50 ml volumetric flask, accurately add 1 ml of impurity reference stock solution, dissolve with solvent and dilute to scale, shake well. Prepare 6 copies in parallel.

[0299] Control solution: Accurately measure 1 ml of the spiked test solution, place it in a 100 ml volumetric flask, dilute to the mark with solvent, shake well, accurately measure 2 ml, place it in a 10 ml volumetric flask, dilute to the mark with solvent, shake well. Prepare 6 copies in parallel.

[0300] Take blank solution, system suitability solution, reference solution, and spiked test solution, inject them into the liquid chromatograph, and record the chromatogram. Calculate the content of each known impurity in the 6 spiked test solutions by peak area using the main component self-reference method and the impurity reference external standard method with correction factors, calculate the content of other single impurities according to the main component self-reference method, and calculate the total amount of impurities. The test results are shown in Tables 12-13.

[0301] Table 12. Repeatability test results-1 (main component self-control method with correction factor, %)

[0302] Impurity Name 1 2 3 4 5 6 RSD (n=6) Impurity i-4 0.28 0.29 0.29 0.28 0.28 0.29 2.0 Impurity i-5 0.22 0.24 0.24 0.23 0.24 0.24 3.6 Impurity i-8 0.23 0.23 0.23 0.23 0.23 0.24 1.8 Impurity i-9 0.34 0.34 0.35 0.33 0.36 0.36 3.5 Impurity i-10 0.18 0.18 0.18 0.18 0.19 0.19 2.9 Impurity i-11 0.22 0.22 0.23 0.22 0.23 0.23 2.5 Impurity i-12 0.20 0.21 0.21 0.20 0.21 0.20 2.7 Impurity i-14 0.22 0.22 0.23 0.21 0.22 0.21 3.5 Other single impurities ND ND ND ND ND ND — Total impurities 1.89 1.93 1.96 1.88 1.96 1.96 2.0

[0303] Table 13, Repeatability test results-2 (impurity reference substance external standard method, %)

[0304] Impurity Name 1 2 3 4 5 6 RSD (n=6) Impurity i-4 0.27 0.28 0.27 0.28 0.27 0.28 2.0 Impurity i-5 0.18 0.19 0.19 0.19 0.19 0.20 3.4 Impurity i-8 0.20 0.19 0.19 0.20 0.19 0.20 2.9 Impurity i-9 0.32 0.32 0.32 0.32 0.33 0.33 1.6 Impurity i-10 0.20 0.20 0.20 0.20 0.20 0.21 2.1 Impurity i-11 0.20 0.19 0.20 0.20 0.20 0.20 2.1 Impurity i-12 0.20 0.20 0.21 0.21 0.21 0.20 2.7 Impurity i-14 0.20 0.20 0.20 0.20 0.20 0.19 2.1

[0305] Verification conclusion: The RSD of each known impurity content calculated by the external standard method of impurity reference substance in 6 spiked test solutions was less than 10%; the RSD of each known impurity content calculated by the main component self-control method with correction factor was less than 10%, and other single impurities were not detected. The RSD of the total impurity content was 2.0%, less than 5%, which met the requirements, indicating that the method had good repeatability. The impurity content was calculated by the main component self-control method with correction factor and the external standard method, respectively. The results showed that the contents of impurities i-4, impurity i-8, impurity i-9, impurity i-10, impurity i-11, impurity i-12 and impurity i-14 calculated by the two methods were basically the same; the content of impurity i-5 calculated by the main component self-control method with correction factor was about 0.04% higher than that of the external standard method. The calculation of the content of impurity i-5 by the main component self-control method with correction factor was equivalent to strict control; considering the detection cost and simple operation, the main component self-control method with correction factor was selected to control the known impurities in the test sample.

[0306] 2.6 Intermediate precision test

[0307] In order to verify the intermediate precision of the method, the same batch of samples were used, and on different dates, different analysts prepared 6 spiked test solutions and 6 self-control solutions in the same way according to the repeatability test operation steps, and the same method was used to measure on different instruments. The 6 results of the repeatability test were combined, and a total of 12 measurement results were analyzed. The test results are shown in Table 14.

[0308] Table 14. Intermediate precision test results (%)

[0309]

[0310] Verification conclusion: The RSD of the known impurity content in the 6 spiked test solutions was less than 10%, and other single impurities were not detected. The RSD of the total impurity content was 4.4%, which was less than 5%, which met the requirements; the RSD of the known impurity content in the 12 spiked test solutions in the combined repeatability test was less than 10%, and other single impurities were not detected. The RSD of the total impurity content was 3.7%, which was less than 5%, which met the requirements, indicating that the intermediate precision of the method is good.

[0311] 2.7 Accuracy test

[0312] Solvent: 0.5% triethylamine solution (pH adjusted to 7.0 with phosphoric acid)-methanol (27:73).

[0313] Stock solution of impurity reference substances: Take approximately 5 mg each of impurity i-4 reference substance, impurity i-5 reference substance, impurity i-8 reference substance, impurity i-10 reference substance, impurity i-11 reference substance, impurity i-12 reference substance and impurity i-14 reference substance, and approximately 7.5 mg of impurity i-9 reference substance, accurately weigh, place in the same 100 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0314] Reference solution: Accurately measure 1 ml of the impurity reference stock solution, place it in a 50 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0315] Test solution: Take about 25 mg of the product, weigh accurately, place in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0316] Low, medium and high concentration accuracy solutions (40%, 100%, 200%): Take about 25 mg of this product respectively, weigh accurately, put into 3 50 ml volumetric bottles, accurately add 0.4, 1, 2 ml of impurity reference substance stock solution for each concentration, dissolve with solvent and dilute to scale, shake well. Prepare 3 copies for each concentration in parallel.

[0317] Take the reference solution, the test solution and each concentration accuracy solution, inject them into the liquid chromatograph respectively, and record the chromatogram. Calculate the measured amount of each impurity by peak area according to the impurity reference external standard method, and calculate the recovery rate and average recovery rate of each impurity.

[0318] Verification results and conclusion: Three limit concentrations of 40%, 100% and 200% were selected for recovery rate, and 3 solutions were prepared for each concentration. The recovery rates of impurity i-4, impurity i-5, impurity i-8, impurity i-9, impurity i-10, impurity i-11, impurity i-12 and impurity i-14 in a total of 9 accuracy solutions were 98.21%~107.23%, 92.80%~99.88%, 97.07%~103.28%, 99.48%~104.09%, 96.90%~100.46%, 92.14%~101.64%, 99.33%~107.65% and 92.83%~104.82%, respectively, all within the range of 90%~108%, which met the requirements, indicating that the method has high accuracy.

[0319] 2.8 Durability test

[0320] Solvent: 0.5% triethylamine solution (pH adjusted to 7.0 with phosphoric acid)-methanol (27:73).

[0321] Blank solution: solvent.

[0322] Stock solution of impurity reference substances: Take approximately 5 mg each of impurity i-4 reference substance, impurity i-5 reference substance, impurity i-8 reference substance, impurity i-10 reference substance, impurity i-11 reference substance, impurity i-12 reference substance and impurity i-14 reference substance, and approximately 7.5 mg of impurity i-9 reference substance, accurately weigh, place in the same 100 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0323] System suitability solution: Take about 25 mg of this product, weigh accurately, place in a 50 ml volumetric flask, accurately add 1 ml of impurity reference stock solution, add solvent to dissolve and dilute to the scale, and shake well.

[0324] Spiked test solution: Take about 25 mg of the product, accurately weigh it, put it in a 50 ml volumetric flask, accurately add 1 ml of the impurity reference stock solution, add solvent to dissolve and dilute to the scale, and shake well.

[0325] Control solution: Accurately measure 1 ml of the spiked test solution, place it in a 100 ml volumetric flask, dilute it to the mark with solvent, shake well; accurately measure 2 ml, place it in a 10 ml volumetric flask, dilute it to the mark with solvent, shake well.

[0326] Chromatographic columns of the same brand, model, specification and different batches were replaced respectively, and the column temperature (35°C and 45°C), flow rate (0.9 ml / min and 1.1 ml / min), pH value of mobile phase A (pH 6.8 and pH 7.2), and concentration of triethylamine in mobile phase A (0.45% and 0.55%) were adjusted. Blank solution, system suitability solution, spiked test solution and control solution were taken under each condition, injected into the liquid chromatograph respectively, and the chromatograms were recorded. The test results are shown in Table 15.

[0327] Table 15. Durability test results of different chromatographic conditions on the impurity content in the spiked test solution

[0328]

[0329]

[0330] Verification conclusion: Under each condition, the system suitability solution was injected 5 times continuously, the theoretical plate number of the DPH main peak was not less than 10,000, the separation between the impurity i-12 peak and the DPH main peak was not less than 1.3, the retention time RSD of each known impurity peak was less than 1%, the peak area RSD was less than 5%, the tailing factor of each known impurity peak was not greater than 2.0, and the signal-to-noise ratio of each known impurity peak height was greater than 100. The absolute value of the difference between the content of impurities i-4, impurity i-5, impurity i-8, impurity i-9, impurity i-10, impurity i-11, impurity i-12 and impurity i-14 in the spiked test solution under each condition and the content under standard conditions was no more than 0.04%, and other single impurities were not detected under each condition, and the absolute value of the difference between the total amount of impurities and the total amount of impurities under standard conditions was no more than 0.10%. The method has good durability.

[0331] Under each condition, the relative retention times of impurities i-4, i-11, i-12 and i-14 (0.82, 0.79, 0.97 and 1.20, respectively) all changed slightly, and the relative retention times of impurities i-4, i-11 and i-12 differed slightly, making it difficult to identify the above three impurities; the relative retention times of impurities i-5, i-8, i-9 and i-10 (2.17-2.34, 2.70-2.92, 2.37-2.57 and 2.41-2.61, respectively) all changed significantly, and the relative retention times of impurities i-5, i-8, i-9 and i-10 differed slightly, making it difficult to identify the above four impurities. In summary, when formulating the standard, a system suitability solution is added to locate each known impurity.

Claims

1. A method for detecting related substances of 4-(2,4-difluorobenzoyl)-piperidine hydrochloride, a starting material of paliperidone, characterized in that: The paliperidone starting material 4-(2,4-difluorobenzoyl)-piperidine hydrochloride related substance is (2,6-difluorophenyl)(piperidin-4-yl)methanone hydrochloride, 1-(4-(2,4-difluorophenyl)piperidinyl)ethanone, (2,4-difluorophenyl)(1-(3-fluorophenyl)piperidin-4-yl)methanone, (4-(4-(2,4-difluorophenyl)piperidin-1-yl)-2-fluorophenyl)(piperidin-4-yl)methanone hydrochloride, (2-(4-(2,4-difluorophenyl)piperidin-1-yl)-4-fluorophenyl)(piperidin-4-yl)methanone hydrochloride, (2-fluorophenyl)(piperidin-4-yl)methanone hydrochloride, (2,5-difluorophenyl)(piperidin-4-yl)methanone hydrochloride or (3,5-difluorophenyl)(piperidin-4-yl)methanone hydrochloride; The detection method comprises the following specific steps: (1) Chromatographic conditions: Instrument: High performance liquid chromatography-ultraviolet detector; Chromatographic column: Octadecylsilane bonded silica gel chromatographic column with embedded pentafluorophenyl functional group; Mobile phase A: volume ratio, a mixed solution of triethylamine aqueous solution: methanol = 90:10; Mobile phase B: volume ratio, a mixed solution of acetonitrile: tetrahydrofuran = 90:10; Mobile phase flow rate: 1.0 ml / min; Detection wavelength: 238 nm; Column temperature: 40°C; injection volume: 15 μl; Wherein, the octadecylsilane bonded silica gel chromatography column with embedded pentafluorophenyl functional group is ACEExcel 3 C18-PFP, 4.6mm×250mm, 3μm; The volume concentration of the triethylamine aqueous solution is 0.5%, and the pH value is 7.0; Gradient elution program: (2) Solution preparation: Diluent: a mixed solution of triethylamine aqueous solution (0.5%, pH 7.0) and methanol = 27:73 by volume; Stock solution of impurity reference substances: accurately weigh impurity i-4 reference substance, impurity i-5 reference substance, impurity i-8 reference substance, impurity i-9 reference substance, impurity i-10 reference substance, impurity i-11 reference substance, impurity i-12 reference substance and impurity i-14 reference substance, dissolve them ultrasonically and dilute them with diluent to make solutions with concentrations of 50 μg / mL for impurity i-4, impurity i-5, impurity i-8, impurity i-10, impurity i-11, impurity i-12 and impurity i-14 and 75 μg / mL for impurity i-9 reference substance, and shake well; System suitability solution: accurately weigh 4-(2,4-difluorobenzoyl)-piperidine hydrochloride (DPH), dissolve it by ultrasonication with a diluent and dilute it to make a solution with a DPH concentration of 0.5 mg / mL, impurity i-4, impurity i-5, impurity i-8, impurity i-10, impurity i-11, impurity i-12 and impurity i-14 concentrations of 1 μg / mL, and impurity i-9 concentration of 1.5 μg / mL, and shake well; Test solution: accurately weigh 4-(2,4-difluorobenzoyl)-piperidine hydrochloride, dissolve and dilute with diluent by ultrasonication to make a solution of 4-(2,4-difluorobenzoyl)-piperidine hydrochloride with a concentration of 0.5 mg / mL, and shake well; Control solution: Accurately measure the test solution, dilute it with diluent to make a solution of 4-(2,4-difluorobenzoyl)-piperidine hydrochloride with a concentration of 1 μg / mL, and shake well; (3) Determination method: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph, and record the chromatogram. In the chromatogram of the system suitability solution, impurity i-11, impurity i-4, impurity i-12, 4-(2,4-difluorobenzoyl)-piperidine, impurity i-14, impurity i-5, impurity i-9, impurity i-10 and impurity i-8 appear in sequence, and the theoretical plate number is not less than 10,000 based on the 4-(2,4-difluorobenzoyl)-piperidine peak. The separation between the impurity i-12 peak and the 4-(2,4-difluorobenzoyl)-piperidine peak should be not less than 1.

3. Calculate the impurity content according to the principal component self-reference method with the correction factor added; The formula for calculating the impurity content by the main component self-control method with added correction factor is: impurity content = peak area of ​​the impurity in the test solution × concentration of the control solution × correction factor / peak area of ​​the main peak of the control solution / concentration of the test solution × 100%; the correction factors of the impurity i-4, impurity i-5, impurity i-8, impurity i-9, impurity i-10, impurity i-11, impurity i-12 and impurity i-14 are 1.5, 1.0, 0.7, 1.2, 0.7, 1.0, 1.0 and 1.0 respectively.

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