Method for detecting related substances in palbosilib or salts thereof

Abstract

The invention relates to a method for detecting related substances in ponsilidone or salts thereof, which comprises the following steps: mixing ponsilidone or salts thereof with a first solvent to prepare a solution to be detected; performing high performance liquid chromatography detection on the solution to be detected, wherein the conditions of the high performance liquid chromatography detection are as follows: (1) silane bonded silica gel is used as a filler of a chromatographic column; (2) a mobile phase A is an aqueous solution containing inorganic salt and / or inorganic acid, a mobile phase B is one or more of acetonitrile, methanol, ethanol and tetrahydrofuran, and gradient elution is adopted. The detection method can be used for separating and detecting the multiple genotoxic impurities in the palbociclib or the salts thereof, and can further realize accurate quantification of the multiple genotoxic impurities.

Description

Technical Field

[0001] The present application relates to the technical field of drug analysis, and in particular to a method for detecting related substances in palbociclib or its salts. Background Art

[0002] Palbociclib (also known as Palbociclib, Palbociclib, Palbociclib) is used for advanced or metastatic ER+ / HER2- breast cancer in menopausal women. Palbociclib is an oral targeted cyclin-dependent kinase 4 and 6 (CDK4 / 6) specific inhibitor, used for first-line treatment of selectively inhibiting CDK4 / 6, restoring cell cycle control, and blocking tumor cell proliferation. It provides an important option for patients with metastatic breast cancer and has broad market prospects.

[0003] However, the synthetic process of Palbociclib is long and prone to produce a variety of genotoxic impurities (as shown in Table 1 below). The toxicity of genotoxic impurities is relatively high, and their residual amount in the drug needs to be strictly controlled. At the same time, there are a variety of impurities with similar structures (such as impurities 1 and 3, impurities 4, 7, 8 and 9, etc.) in the genotoxic impurities, which are difficult to separate. Therefore, how to effectively separate the impurities in Palbociclib and achieve accurate quantitative detection is a problem that needs to be solved urgently.

[0004] Table 1

[0005]

[0006] Summary of the invention

[0007] Based on this, the present application provides a method for detecting related substances in palbociclib or its salts, which can separate and detect various genotoxic impurities in palbociclib or its salts, and can further achieve accurate quantification of various genotoxic impurities.

[0008] The specific technical solutions are as follows:

[0009] A method for detecting related substances in palbociclib or its salts, wherein the related substances include 2-nitro-5-bromopyridine, 4-(6-nitropyridine-3-yl)piperazine-1-carboxylic acid tert-butyl ester, 2-nitro-3,5-dibromopyridine, 2-chloro-8-cyclopentyl-5-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one, di-tert-butyl 4,4'-(diazo-1,2-diylbis(pyridine-6,3-diyl))(E)-di(piperidin-1-carboxylic acid)-1-tert-butyl ester, one or more of 2-nitropyridine-1-carboxylate, 5,5'-(1,4-piperazinediyl)bis(2-nitropyridine), 2,6-dichloro-8-cyclopentylpyrido[2,3-d]pyrimidin-7(8H)-one, 6-bromo-2-chloro-8-cyclopentyl-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one and 6-bromo-2-chloro-8-cyclopentyl-5-bromomethyl-pyrido[2,3-d]pyrimidin-7(8H)-one;

[0010] The detection method comprises the following steps:

[0011] Mixing palbociclib or its salt with a first solvent to prepare a test solution;

[0012] The solution to be tested is subjected to high performance liquid chromatography detection, and the conditions of the high performance liquid chromatography detection include:

[0013] (1) The filler of the chromatographic column is silane-bonded silica gel;

[0014] (2) Mobile phase A is an aqueous solution containing an inorganic salt and / or an inorganic acid, and mobile phase B is one or more of acetonitrile, methanol, ethanol and tetrahydrofuran, and gradient elution is used, including:

[0015] From 0min to 10min, the volume percentage of mobile phase A changes from 90% to 94% to 75%, and the volume percentage of mobile phase B changes from 10% to 6% to 25%;

[0016] From 10 to 35 minutes, the volume percentage of mobile phase A changed from 75% to 30%, and the volume percentage of mobile phase B changed from 25% to 70%;

[0017] 35min-50min, the volume percentage of mobile phase A is maintained at 30%, and the volume percentage of mobile phase B is maintained at 70%;

[0018] From 50 to 51 minutes, the volume percentage of mobile phase A changes from 30% to 90% to 94%, and the volume percentage of mobile phase B changes from 70% to 10% to 6%;

[0019] From 51 min to 60 min, the volume percentage of mobile phase A is maintained at 90% to 94%, and the volume percentage of mobile phase B is maintained at 10% to 6%.

[0020] In some embodiments, mobile phase A has one or more of the following characteristics:

[0021] (1) The inorganic salt includes one or both of dipotassium hydrogen phosphate and disodium hydrogen phosphate;

[0022] (2) The concentration of the inorganic salt in mobile phase A is 0.005 mol / L to 0.02 mol / L;

[0023] (3) The inorganic acid includes one or more of phosphoric acid, sulfuric acid, hydrochloric acid, perchloric acid and trifluoroacetic acid;

[0024] (4) The pH of mobile phase A is 5.5-6.5.

[0025] In some embodiments, mobile phase B is acetonitrile.

[0026] In some of the embodiments, the filler of the chromatographic column is octylsilane bonded silica gel or octadecylsilane bonded silica gel.

[0027] In some embodiments, the conditions for the HPLC detection also include one or more of the following:

[0028] (1) Ultraviolet light detection is used, and the detection wavelength is 225nm~235nm;

[0029] (2) Flow rate: 0.5 mL / min to 1.5 mL / min;

[0030] (3) Column temperature is 30℃~40℃;

[0031] (4) The injection volume is 15 μL to 25 μL.

[0032] In some embodiments, the related substances include 2-nitro-5-bromopyridine, 4-(6-nitropyridin-3-yl)piperazine-1-carboxylic acid tert-butyl ester, 2-nitro-3,5-dibromopyridine, 2-chloro-8-cyclopentyl-5-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one, di-tert-butyl 4,4'-(diazo-1,2-diylbis(pyridine-6,3-diyl))(E)-di(piperazine- 1-carboxylate), 5,5'-(1,4-piperazinediyl)bis(2-nitropyridine), 2,6-dichloro-8-cyclopentylpyrido[2,3-d]pyrimidin-7(8H)-one, 6-bromo-2-chloro-8-cyclopentyl-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one and 6-bromo-2-chloro-8-cyclopentyl-5-bromomethyl-pyrido[2,3-d]pyrimidin-7(8H)-one.

[0033] In some embodiments, the salt of palbociclib includes one or more of palbociclib hydrochloride, palbociclib phosphate, palbociclib carbonate and palbociclib sulfate.

[0034] In some embodiments, the test solution has one or more of the following characteristics:

[0035] (1) The concentration of palbociclib or its salt in the test solution is 1 mg / mL to 20 mg / mL;

[0036] (2) The first solvent is an acetonitrile aqueous solution with a volume concentration of 40% to 60%.

[0037] In some embodiments, the detection method further comprises the following steps:

[0038] Mixing the reference substance of the related substance with the second solvent to prepare reference substance solutions of different concentrations;

[0039] Performing the HPLC test on the reference solution of different concentrations, and constructing a standard curve of the related substances according to the chromatographic peak area in the test result and the concentration of the reference solution;

[0040] Substitute the test result of the high performance liquid chromatography of the test solution into the standard curve of the related substance to calculate the content of the related substance in the test solution.

[0041] In some embodiments, the second solvent is an acetonitrile aqueous solution with a volume concentration of 40% to 60%.

[0042] The present application is based on high performance liquid chromatography detection, adopts a suitable chromatographic column, mobile phase and gradient elution program, and can achieve the separation and detection of 9 genotoxic impurities contained in palbociclib or its salt. Further combined with the construction of a standard curve, it can also achieve accurate quantification of the 9 genotoxic impurities, thereby effectively controlling the quality of palbociclib or its salt, and more effectively realizing quality monitoring of the products of palbociclib or its salt. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The chromatogram is a chromatogram of the separation and determination of 9 genotoxic impurities in Example 1;

[0044] Figure 2 The chromatogram is a chromatogram of the separation and determination of 9 genotoxic impurities in Example 2;

[0045] Figure 3 The chromatogram is a chromatogram of the separation and determination of 9 genotoxic impurities in Example 3;

[0046] Figure 4 The chromatogram is a chromatogram of the separation and determination of 9 genotoxic impurities in Example 4;

[0047] Figure 5 The chromatogram is a chromatogram of the separation and determination of 9 genotoxic impurities in Example 5;

[0048] Figure 6 The chromatogram is a chromatogram of the separation and determination of 9 genotoxic impurities in Example 6;

[0049] Figure 7 The chromatogram is a chromatogram of the separation and determination of 9 genotoxic impurities in Example 7;

[0050] Figure 8 The chromatogram is a chromatogram of the separation and determination of 9 genotoxic impurities in Example 8;

[0051] Fig. 9 The chromatogram is a chromatogram of the separation and determination of 9 genotoxic impurities in Example 9;

[0052] Fig.10 The chromatogram is a chromatogram of the separation and determination of 9 genotoxic impurities in Example 10;

[0053] Fig.11 The chromatogram is a chromatogram of the separation and determination of 9 genotoxic impurities in Example 11;

[0054] Fig.12 The chromatogram is a chromatogram of the separation and determination of 9 genotoxic impurities in Example 12;

[0055] Fig.13 The chromatogram is a chromatogram of the separation and determination of 9 genotoxic impurities in Example 13;

[0056] Fig.14 The chromatogram is a chromatogram of the separation and determination of 9 genotoxic impurities in Example 14;

[0057] Fig.15 The chromatogram is a chromatogram of the separation and determination of 9 genotoxic impurities in Example 15;

[0058] Fig.16 is the standard curve diagram of impurity 1;

[0059] Fig.17 is the standard curve diagram of impurity 6;

[0060] Fig.18 is the standard curve diagram of impurity 2;

[0061] Fig.19 is the standard curve diagram of impurity 3;

[0062] Fig. 20 is the standard curve diagram of impurity 4;

[0063] Fig.21 is the standard curve diagram of impurity 5;

[0064] Fig. 22 is the standard curve diagram of impurity 7;

[0065] Fig.23 is the standard curve diagram of impurity 8;

[0066] Fig.24 is the standard curve diagram of impurity 9;

[0067] Fig.25 The chromatogram is a chromatogram of the separation and determination of 9 genotoxic impurities in Comparative Example 1;

[0068] Fig.26 This is the chromatogram for the separation and determination of 9 genotoxic impurities in Comparative Example 2. DETAILED DESCRIPTION

[0069] The following is a further detailed description of the detection method of related substances in palbociclib or its salts of the present application in conjunction with specific examples. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0071] As used herein, "one or more" refers to any one, any two, or any two or more of the listed items.

[0072] In the present application, “first”, “second”, “third”, “fourth”, etc. are only used for the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0073] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0074] In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0075] The percentage contents involved in this application, unless otherwise specified, refer to mass percentage for solid-liquid mixing and solid-solid mixing, and refer to volume percentage for liquid-liquid mixing.

[0076] The percentage concentrations mentioned in this application, unless otherwise specified, refer to the final concentration, which refers to the percentage of the added component in the system after the addition of the component.

[0077] The temperature parameters in this application, unless otherwise specified, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range of instrument control.

[0078] The room temperature in the present application generally refers to 4°C to 30°C, preferably 20±5°C.

[0079] In some examples of the present application, a method for detecting related substances in palbociclib or its salt is provided, wherein the related substances include 2-nitro-5-bromopyridine (impurity 1), tert-butyl 4-(6-nitropyridine-3-yl)piperazine-1-carboxylate (impurity 2), 2-nitro-3,5-dibromopyridine (impurity 3), 2-chloro-8-cyclopentyl-5-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (impurity 4), di-tert-butyl 4,4'-(diazo-1,2-diylbis(pyridine-6,3-diyl))(E)- One or more of bis(piperazine-1-carboxylate) (Impurity 5), 5,5'-(1,4-piperazinediyl)bis(2-nitropyridine) (Impurity 6), 2,6-dichloro-8-cyclopentylpyrido[2,3-d]pyrimidin-7(8H)-one (Impurity 7), 6-bromo-2-chloro-8-cyclopentyl-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one (Impurity 8) and 6-bromo-2-chloro-8-cyclopentyl-5-bromomethyl-pyrido[2,3-d]pyrimidin-7(8H)-one (Impurity 9);

[0080] The detection method comprises the following steps:

[0081] Mixing palbociclib or its salt with a first solvent to prepare a test solution;

[0082] The solution to be tested is subjected to high performance liquid chromatography detection, and the conditions of the high performance liquid chromatography detection include:

[0083] (1) The filler of the chromatographic column is silane-bonded silica gel;

[0084] (2) Mobile phase A is an aqueous solution containing an inorganic salt and / or an inorganic acid, and mobile phase B is one or more of acetonitrile, methanol, ethanol and tetrahydrofuran, and gradient elution is used, including:

[0085] From 0min to 10min, the volume percentage of mobile phase A changes from 90% to 94% to 75%, and the volume percentage of mobile phase B changes from 10% to 6% to 25%;

[0086] From 10 to 35 minutes, the volume percentage of mobile phase A changed from 75% to 30%, and the volume percentage of mobile phase B changed from 25% to 70%;

[0087] 35min-50min, the volume percentage of mobile phase A is maintained at 30%, and the volume percentage of mobile phase B is maintained at 70%;

[0088] From 50 to 51 minutes, the volume percentage of mobile phase A changes from 30% to 90% to 94%, and the volume percentage of mobile phase B changes from 70% to 10% to 6%;

[0089] From 51 min to 60 min, the volume percentage of mobile phase A is maintained at 90% to 94%, and the volume percentage of mobile phase B is maintained at 10% to 6%.

[0090] Some examples of gradient elution include:

[0091] From 0 to 10 minutes, the volume percentage of mobile phase A changed from 94% to 75%, and the volume percentage of mobile phase B changed from 6% to 25%;

[0092] From 10 to 35 minutes, the volume percentage of mobile phase A changed from 75% to 30%, and the volume percentage of mobile phase B changed from 25% to 70%;

[0093] 35min-50min, the volume percentage of mobile phase A is maintained at 30%, and the volume percentage of mobile phase B is maintained at 70%;

[0094] From 50 to 51 minutes, the volume percentage of mobile phase A changed from 30% to 94%, and the volume percentage of mobile phase B changed from 70% to 6%;

[0095] From 51 min to 60 min, the volume percentage of mobile phase A was maintained at 94%, and the volume percentage of mobile phase B was maintained at 6%.

[0096] Some examples of gradient elution include:

[0097] From 0 to 10 minutes, the volume percentage of mobile phase A changes from 90% to 75%, and the volume percentage of mobile phase B changes from 10% to 25%;

[0098] From 10 to 35 minutes, the volume percentage of mobile phase A changed from 75% to 30%, and the volume percentage of mobile phase B changed from 25% to 70%;

[0099] 35min-50min, the volume percentage of mobile phase A is maintained at 30%, and the volume percentage of mobile phase B is maintained at 70%;

[0100] From 50 to 51 minutes, the volume percentage of mobile phase A changed from 30% to 90%, and the volume percentage of mobile phase B changed from 70% to 10%;

[0101] From 51 min to 60 min, the volume percentage of mobile phase A was maintained at 90%, and the volume percentage of mobile phase B was maintained at 10%.

[0102] Some examples of gradient elution include:

[0103] From 0 to 10 minutes, the volume percentage of mobile phase A changed from 92% to 75%, and the volume percentage of mobile phase B changed from 8% to 25%;

[0104] From 10 to 35 minutes, the volume percentage of mobile phase A changed from 75% to 30%, and the volume percentage of mobile phase B changed from 25% to 70%;

[0105] 35min-50min, the volume percentage of mobile phase A is maintained at 30%, and the volume percentage of mobile phase B is maintained at 70%;

[0106] From 50 to 51 minutes, the volume percentage of mobile phase A changed from 30% to 92%, and the volume percentage of mobile phase B changed from 70% to 8%;

[0107] From 51 min to 60 min, the volume percentage of mobile phase A was maintained at 92%, and the volume percentage of mobile phase B was maintained at 8%.

[0108] In some examples, the inorganic salt in mobile phase A includes one or both of dipotassium hydrogen phosphate and disodium hydrogen phosphate.

[0109] In some examples, the concentration of the inorganic salt in mobile phase A is 0.005 mol / L to 0.02 mol / L. Specifically, the concentration of the inorganic salt in mobile phase A includes, but is not limited to, 0.005 mol / L, 0.006 mol / L, 0.007 mol / L, 0.008 mol / L, 0.009 mol / L, 0.01 mol / L, 0.015 mol / L, 0.02 mol / L, or a range between any two of the foregoing.

[0110] In some examples, the inorganic acid in the mobile phase A includes one or more of phosphoric acid, sulfuric acid, hydrochloric acid, perchloric acid and trifluoroacetic acid. It can be understood that the inorganic acid is used to adjust the pH of the mobile phase A.

[0111] In some examples, the pH of mobile phase A is 5.5 to 6.5. Specifically, the pH of mobile phase A includes, but is not limited to, 5.5, 5.8, 6, 6.2, 6.5, or a range between any two of the foregoing.

[0112] In some of these examples, mobile phase B was acetonitrile.

[0113] In some examples, the filler of the chromatographic column is C8-C18 alkylsilane bonded silica gel. Further, the filler of the chromatographic column is octylsilane bonded silica gel or octadecylsilane bonded silica gel.

[0114] In some examples, the HPLC detection conditions also include: ultraviolet detection, the detection wavelength is 225nm-235nm. Specifically, the detection wavelength includes but is not limited to: 225nm, 228nm, 230nm, 232nm, 235nm or a range between any two of the foregoing.

[0115] In some examples, the conditions for the HPLC detection also include: a flow rate of 0.5 mL / min to 1.5 mL / min. Specifically, the flow rate includes but is not limited to: 0.5 mL / min, 0.8 mL / min, 0.9 mL / min, 1 mL / min, 1.1 mL / min, 1.2 mL / min, 1.5 mL / min or a range between any two of the foregoing.

[0116] In some examples, the conditions for the HPLC detection also include: a column temperature of 30° C. to 40° C. Specifically, the column temperature includes but is not limited to: 30° C., 33° C., 35° C., 37° C., 40° C., or a range between any two of the foregoing.

[0117] In some examples, the conditions for the HPLC detection also include: the injection volume is 15 μL to 25 μL. Specifically, the injection volume includes but is not limited to: 15 μL, 18 μL, 20 μL, 22 μL, 25 μL or a range between any two of the foregoing.

[0118] In some of the examples, the substances include 2-nitro-5-bromopyridine, tert-butyl 4-(6-nitropyridin-3-yl)piperazine-1-carboxylate, 2-nitro-3,5-dibromopyridine, 2-chloro-8-cyclopentyl-5-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one, di-tert-butyl 4,4'-(diazo-1,2-diylbis(pyridine-6,3-diyl))(E)-di(piperazine- 1-carboxylate), 5,5'-(1,4-piperazinediyl)bis(2-nitropyridine), 2,6-dichloro-8-cyclopentylpyrido[2,3-d]pyrimidin-7(8H)-one, 6-bromo-2-chloro-8-cyclopentyl-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one and 6-bromo-2-chloro-8-cyclopentyl-5-bromomethyl-pyrido[2,3-d]pyrimidin-7(8H)-one. The above detection method can simultaneously detect 9 genotoxic impurities in palbociclib or its salts, and the detection results are accurate, which can improve the detection efficiency.

[0119] Without limitation, the salt of palbociclib includes one or more of palbociclib hydrochloride, palbociclib phosphate, palbociclib carbonate and palbociclib sulfate.

[0120] In some examples, the concentration of palbociclib or its salt in the test solution is 1 mg / mL to 20 mg / mL. Reasonable control of the concentration of palbociclib or its salt in the test solution can improve the accuracy of detection. Specifically, the concentration of palbociclib or its salt in the test solution includes but is not limited to: 1 mg / mL, 2 mg / mL, 4 mg / mL, 5 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg / mL, 15 mg / mL, 18 mg / mL, 20 mg / mL or a range between any two of the foregoing.

[0121] In some examples, the first solvent is an acetonitrile aqueous solution with a volume concentration of 40% to 60%. Specifically, the volume concentration of the acetonitrile aqueous solution includes but is not limited to: 40%, 45%, 48%, 50%, 52%, 55%, 60% or a range between any two of the foregoing.

[0122] In addition, in some of the examples, the detection method further includes the following steps:

[0123] Mixing the reference substance of the related substance with the second solvent to prepare reference substance solutions of different concentrations;

[0124] Performing the HPLC test on the reference solution of different concentrations, and constructing a standard curve of the related substances according to the chromatographic peak area in the test result and the concentration of the reference solution;

[0125] Substitute the test result of the high performance liquid chromatography of the test solution into the standard curve of the related substance to calculate the content of the related substance in the test solution.

[0126] By constructing a standard curve, accurate quantitative detection of various related substances in the solution to be tested can be achieved.

[0127] In some examples, the second solvent is an acetonitrile aqueous solution with a volume concentration of 40% to 60%. Specifically, the volume concentration of the acetonitrile aqueous solution includes but is not limited to: 40%, 45%, 48%, 50%, 52%, 55%, 60% or a range between any two of the foregoing.

[0128] For experimental parameters not specified in the following specific embodiments, reference is made to the instructions given in the present application document, and reference may also be made to experimental manuals in the art or other experimental methods known in the art, or to experimental conditions recommended by manufacturers.

[0129] The raw materials and reagents involved in the following specific examples can be obtained from commercial sources, or can be prepared by those skilled in the art according to known methods.

[0130] Example 1

[0131] Sample preparation: dissolve palbociclib hydrochloride, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9 in acetonitrile aqueous solution with a volume concentration of 50% to prepare a mixed solution containing palbociclib hydrochloride at a concentration of 5 mg / mL and each impurity at a concentration of 4 μg / mL, and inject the mixed solution into a liquid chromatograph;

[0132] Mobile phase A: 0.01 mol / L anhydrous potassium hydrogen phosphate aqueous solution (adjust pH to 6.0 with phosphoric acid);

[0133] Mobile phase B: acetonitrile;

[0134] The gradient elution method is shown in Table 2 below:

[0135] Table 2

[0136]

[0137] High performance liquid chromatograph: waters e2695;

[0138] Chromatographic column: Yuexu XB-C 18 4.6mm×250mm, 5μm;

[0139] Ghost peak trapping column: MORHCHEM ghost Trapping columeⅡ50×4.6mm;

[0140] Flow rate: 1.0 mL / min;

[0141] Detection wavelength: 230nm;

[0142] Column temperature: 35 °C;

[0143] Injection volume: 20 μL.

[0144] HPLC analysis was performed under the above conditions, and the chromatogram was recorded. The retention times were impurity 1, palbociclib (the peaks not integrated before the main peak were other impurity peaks), impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9. The results are shown in Figure 1 It can be seen that baseline separation can be achieved between impurities and palbociclib, and between adjacent impurities, with a separation degree greater than 2.0.

[0145] Example 2

[0146] Sample preparation: dissolve palbociclib hydrochloride, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9 in acetonitrile aqueous solution with a volume concentration of 50% to prepare a mixed solution containing palbociclib hydrochloride at a concentration of 5 mg / mL and each impurity at a concentration of 4 μg / mL, and inject the mixed solution into a liquid chromatograph;

[0147] Mobile phase A: 0.01 mol / L anhydrous potassium hydrogen phosphate (pH adjusted to 6.0 with phosphoric acid);

[0148] Mobile phase B: acetonitrile;

[0149] The gradient elution method is shown in Table 3 below:

[0150] Table 3

[0151]

[0152] High performance liquid chromatograph: waters e2695;

[0153] Chromatographic column: Yuexu XB-C 18 4.6mm×250mm, 5μm;

[0154] Ghost peak trapping column: MORHCHEM ghost Trapping columeⅡ50×4.6mm;

[0155] Flow rate: 1.0 mL / min;

[0156] Detection wavelength: 230nm;

[0157] Column temperature: 30°C;

[0158] Injection volume: 20 μL.

[0159] HPLC analysis was performed under the above conditions, and the chromatogram was recorded. The retention times were impurity 1, palbociclib (the peaks not integrated before the main peak were other impurity peaks), impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9. The results are shown in Figure 2 It can be seen that when the column temperature drops to 30°C, the retention time of the main peak and each impurity peak is slightly delayed, but the separation degree and baseline do not change significantly compared with Example 1.

[0160] Example 3

[0161] Sample preparation: dissolve palbociclib hydrochloride, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9 in an aqueous solution with a volume concentration of 50% acetonitrile to prepare a mixed solution containing palbociclib hydrochloride at a concentration of 5 mg / mL and each impurity at a concentration of 4 μg / mL, and inject the mixed solution into a liquid chromatograph;

[0162] Mobile phase A: 0.01 mol / L anhydrous potassium hydrogen phosphate (pH adjusted to 6.0 with phosphoric acid);

[0163] Mobile phase B: acetonitrile;

[0164] The gradient elution method is shown in Table 4 below:

[0165] Table 4

[0166]

[0167] High performance liquid chromatograph: waters e2695;

[0168] Chromatographic column: Yuexu XB-C 18 4.6mm×250mm, 5μm;

[0169] Ghost peak trapping column: MORHCHEM ghost Trapping columeⅡ50×4.6mm;

[0170] Flow rate: 1.0 mL / min;

[0171] Detection wavelength: 230nm;

[0172] Column temperature: 40°C;

[0173] Injection volume: 20 μL.

[0174] HPLC analysis was performed under the above conditions, and the chromatogram was recorded. The retention times were impurity 1, palbociclib (the peaks not integrated before the main peak were other impurity peaks), impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9. The results are shown in Figure 3 It can be seen that when the column temperature rises to 40°C, the retention time of the main peak and each impurity peak is slightly advanced, but the separation degree and baseline change significantly.

[0175] Example 4

[0176] Sample preparation: dissolve palbociclib hydrochloride, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9 in an aqueous solution with a volume concentration of 50% acetonitrile to prepare a mixed solution containing palbociclib hydrochloride at a concentration of 5 mg / mL and each impurity at a concentration of 4 μg / mL, and inject the mixed solution into a liquid chromatograph;

[0177] Mobile phase A: 0.01 mol / L anhydrous potassium hydrogen phosphate (pH adjusted to 6.0 with phosphoric acid);

[0178] Mobile phase B: acetonitrile;

[0179] The gradient elution method is shown in Table 5 below:

[0180] Table 5

[0181]

[0182] High performance liquid chromatograph: waters e2695;

[0183] Chromatographic column: Yuexu XB-C 18 4.6mm×250mm, 5μm;

[0184] Ghost peak trapping column: MORHCHEM ghost Trapping columeⅡ50×4.6mm;

[0185] Flow rate: 1.1 mL / min;

[0186] Detection wavelength: 230nm;

[0187] Column temperature: 30°C;

[0188] Injection volume: 20 μL.

[0189] HPLC analysis was performed under the above conditions, and the chromatogram was recorded. The retention times were impurity 1, palbociclib (the peaks not integrated before the main peak were other impurity peaks), impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9. The results are shown in Figure 4 It can be seen that when the flow rate is 1.1 ml / min, there is no significant change in the separation degree and baseline except that the retention time of each peak is advanced.

[0190] Example 5

[0191] Sample preparation: dissolve palbociclib hydrochloride, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9 in an aqueous solution with a volume concentration of 50% acetonitrile to prepare a mixed solution containing palbociclib hydrochloride at a concentration of 5 mg / mL and each impurity at a concentration of 4 μg / mL, and inject the mixed solution into a liquid chromatograph;

[0192] Mobile phase A: 0.01 mol / L anhydrous potassium hydrogen phosphate (pH adjusted to 6.0 with phosphoric acid);

[0193] Mobile phase B: acetonitrile;

[0194] The gradient elution method is shown in Table 6 below:

[0195] Table 6

[0196]

[0197] High performance liquid chromatograph: waters e2695;

[0198] Chromatographic column: Yuexu XB-C 18 4.6mm×250mm, 5μm;

[0199] Ghost peak trapping column: MORHCHEM ghost Trapping columeⅡ50×4.6mm;

[0200] Flow rate: 0.9 mL / min;

[0201] Detection wavelength: 225nm;

[0202] Column temperature: 30°C;

[0203] Injection volume: 20 μL.

[0204] HPLC analysis was performed under the above conditions, and the chromatogram was recorded. The retention times were impurity 1, palbociclib (the peaks not integrated before the main peak were other impurity peaks), impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9. The results are shown in Figure 5 It can be seen that changing the wavelength and flow rate within a certain range does not affect the separation and peak shape.

[0205] Example 6

[0206] Sample preparation: dissolve palbociclib hydrochloride, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9 in an aqueous solution with a volume concentration of 50% acetonitrile to prepare a mixed solution containing palbociclib hydrochloride at a concentration of 4 mg / mL and each impurity at a concentration of 3 μg / mL, and inject the mixed solution into a liquid chromatograph;

[0207] Mobile phase A: 0.01 mol / L anhydrous potassium hydrogen phosphate (pH adjusted to 6.0 with phosphoric acid);

[0208] Mobile phase B: acetonitrile;

[0209] The gradient elution method is shown in Table 7 below:

[0210] Table 7

[0211]

[0212] High performance liquid chromatograph: waters e2695;

[0213] Chromatographic column: Yuexu XB-C 18 4.6mm×250mm, 5μm;

[0214] Ghost peak trapping column: MORHCHEM ghost Trapping columeⅡ50×4.6mm;

[0215] Flow rate: 1.0 mL / min;

[0216] Detection wavelength: 230nm;

[0217] Column temperature: 30°C;

[0218] Injection volume: 20 μL.

[0219] HPLC analysis was performed under the above conditions, and the chromatogram was recorded. The retention times were impurity 1, palbociclib (the peaks not integrated before the main peak were other impurity peaks), impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9. The results are shown in Figure 6 It can be seen that by adjusting the initial ratio of the mobile phase within a certain range, the peak shape and separation are relatively good.

[0220] Example 7

[0221] Sample preparation: dissolve palbociclib hydrochloride, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9 in an aqueous solution with a volume concentration of 50% acetonitrile to prepare a mixed solution containing palbociclib hydrochloride at a concentration of 8 mg / mL and each impurity at a concentration of 6 μg / mL, and inject the mixed solution into a liquid chromatograph;

[0222] Mobile phase A: 0.01 mol / L anhydrous potassium hydrogen phosphate (pH adjusted to 6.0 with phosphoric acid);

[0223] Mobile phase B: acetonitrile;

[0224] The gradient elution method is shown in Table 8 below:

[0225] Table 8

[0226]

[0227] High performance liquid chromatograph: waters e2695;

[0228] Chromatographic column: Yuexu XB-C 18 4.6mm×250mm, 5μm;

[0229] Ghost peak trapping column: MORHCHEM ghost Trapping columeⅡ50×4.6mm;

[0230] Flow rate: 1.0 mL / min;

[0231] Detection wavelength: 230nm;

[0232] Column temperature: 30°C;

[0233] Injection volume: 20 μL.

[0234] HPLC analysis was performed under the above conditions, and the chromatogram was recorded. The retention times were impurity 1, palbociclib (the peaks not integrated before the main peak were other impurity peaks), impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9. The results are shown in Figure 7 ,Depend on Figure 6 and Figure 7 It can be seen that after the initial proportion of the mobile phase was adjusted up and down within a certain range, the peak shape was good and the separation degree met the requirements, indicating that the method had good durability.

[0235] Example 8

[0236] Sample preparation: dissolve palbociclib hydrochloride, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9 in an aqueous solution with a volume concentration of 50% acetonitrile to prepare a mixed solution containing palbociclib hydrochloride at a concentration of 5 mg / mL and each impurity at a concentration of 4 μg / mL, and inject the mixed solution into a liquid chromatograph;

[0237] Mobile phase A: 0.02 mol / L anhydrous potassium hydrogen phosphate (adjust pH to 6.0 with trifluoroacetic acid);

[0238] Mobile phase B: acetonitrile;

[0239] The gradient elution method is shown in Table 9 below:

[0240] Table 9

[0241]

[0242] High performance liquid chromatograph: waters e2695;

[0243] Chromatographic column: Yuexu XB-C18 4.6mm×250mm, 5μm;

[0244] Ghost peak trapping column: MORHCHEM ghost Trapping columeⅡ50×4.6mm;

[0245] Flow rate: 1.0 mL / min;

[0246] Detection wavelength: 230nm;

[0247] Column temperature: 30°C;

[0248] Injection volume: 20 μL.

[0249] HPLC analysis was performed under the above conditions, and the chromatogram was recorded. The retention times were impurity 1, palbociclib (the peaks not integrated before the main peak were other impurity peaks), impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9. The results are shown in Figure 8 .

[0250] Example 9

[0251] Sample preparation: dissolve palbociclib hydrochloride, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9 in an aqueous solution with a volume concentration of 50% acetonitrile to prepare a mixed solution containing palbociclib hydrochloride at a concentration of 5 mg / mL and each impurity at a concentration of 4 μg / mL, and inject the mixed solution into a liquid chromatograph;

[0252] Mobile phase A: 0.005 mol / L anhydrous potassium hydrogen phosphate (adjust pH to 6.0 with perchloric acid);

[0253] Mobile phase B: acetonitrile;

[0254] The gradient elution method is shown in Table 10 below:

[0255] Table 10

[0256]

[0257] High performance liquid chromatograph: waters e2695;

[0258] Chromatographic column: Yuexu XB-C 18 4.6mm×250mm, 5μm;

[0259] Ghost peak trapping column: MORHCHEM ghost Trapping columeⅡ50×4.6mm;

[0260] Flow rate: 1.0 mL / min;

[0261] Detection wavelength: 230nm;

[0262] Column temperature: 30°C;

[0263] Injection volume: 20 μL.

[0264] HPLC analysis was performed under the above conditions, and the chromatogram was recorded. The retention times were impurity 1, palbociclib (the peaks not integrated before the main peak were other impurity peaks), impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9. The results are shown in Fig. 9 ,Depend on Figure 8 and Fig. 9 It can be seen that the concentration of dipotassium hydrogen phosphate is in the range of 0.005 to 0.02 mol / mL, the method has good reproducibility, and the separation between the components does not change significantly, indicating that the concentration of dipotassium hydrogen phosphate in this method can be arbitrarily selected within the range of 0.005 to 0.02 mol / mL.

[0265] Example 10

[0266] Sample preparation: dissolve palbociclib hydrochloride, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9 in an aqueous solution with a volume concentration of 50% acetonitrile to prepare a mixed solution containing palbociclib hydrochloride at a concentration of 5 mg / mL and each impurity at a concentration of 4 μg / mL, and inject the mixed solution into a liquid chromatograph;

[0267] Mobile phase A: 0.01 mol / L anhydrous potassium hydrogen phosphate (adjust pH to 6.0 with sulfuric acid);

[0268] Mobile phase B: acetonitrile;

[0269] The gradient elution method is shown in Table 11 below:

[0270] Table 11

[0271]

[0272] High performance liquid chromatograph: waters e2695;

[0273] Chromatographic column: Agilent ZORBAX Eclipse Plus C 18 4.6×250mm, 5μm;

[0274] Ghost peak trapping column: MORHCHEM ghost Trapping columeⅡ50×4.6mm;

[0275] Flow rate: 1.0 mL / min;

[0276] Detection wavelength: 230nm;

[0277] Column temperature: 30°C;

[0278] Injection volume: 20 μL.

[0279] HPLC analysis was performed under the above conditions, and the chromatogram was recorded. The retention times were impurity 1, palbociclib (the peaks not integrated before the main peak were other impurity peaks), impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9. The results are shown in Fig.10 , it can be seen that the method is still applicable after replacing other chromatographic columns.

[0280] Embodiment 11

[0281] Sample preparation: dissolve palbociclib hydrochloride, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9 in an aqueous solution with a volume concentration of 50% acetonitrile to prepare a mixed solution containing palbociclib hydrochloride at a concentration of 5 mg / mL and each impurity at a concentration of 4 μg / mL, and inject the mixed solution into a liquid chromatograph;

[0282] Mobile phase A: 0.01 mol / L anhydrous disodium hydrogen phosphate (adjust pH to 6.0 with phosphoric acid);

[0283] Mobile phase B: acetonitrile;

[0284] The gradient elution method is shown in Table 12 below:

[0285] Table 12

[0286]

[0287] High performance liquid chromatograph: waters e2695;

[0288] Chromatographic column: Yuexu XB-C 18 4.6mm×250mm, 5μm;

[0289] Ghost peak trapping column: MORHCHEM ghost Trapping columeⅡ50×4.6mm;

[0290] Flow rate: 1.0 mL / min;

[0291] Detection wavelength: 230nm;

[0292] Column temperature: 30°C;

[0293] Injection volume: 20 μL.

[0294] HPLC analysis was performed under the above conditions, and the chromatogram was recorded. The retention times were impurity 1, palbociclib (the peaks not integrated before the main peak were other impurity peaks), impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9. The results are shown in Fig.11It can be seen that there is no obvious change in the peak shape, separation, retention time, baseline, etc. in the entire chromatogram, indicating that anhydrous disodium hydrogen phosphate and anhydrous dipotassium hydrogen phosphate can replace each other.

[0295] Example 12

[0296] Sample preparation: dissolve palbociclib hydrochloride, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9 in an aqueous solution with a volume concentration of 50% acetonitrile to prepare a mixed solution containing palbociclib hydrochloride at a concentration of 5 mg / mL and each impurity at a concentration of 4 μg / mL, and inject the mixed solution into a liquid chromatograph;

[0297] Mobile phase A: 0.01 mol / L anhydrous disodium hydrogen phosphate (adjust pH to 6.0 with phosphoric acid);

[0298] Mobile phase B: acetonitrile;

[0299] The gradient elution method is shown in Table 13 below:

[0300] Table 13

[0301]

[0302] High performance liquid chromatograph: waters e2695;

[0303] Chromatographic column: Yuexu XB-C8 4.6mm×250mm, 5μm;

[0304] Ghost peak trapping column: MORHCHEM ghost Trapping columeⅡ50×4.6mm;

[0305] Flow rate: 1.0 mL / min;

[0306] Detection wavelength: 230nm;

[0307] Column temperature: 30°C;

[0308] Injection volume: 20 μL.

[0309] HPLC analysis was performed under the above conditions, and the chromatogram was recorded. The retention times were impurity 1, palbociclib (the peaks not integrated before the main peak were other impurity peaks), impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9. The results are shown in Fig.12 ,It can be seen that the peak shape, separation, retention time, baseline and ,etc. in the entire chromatogram do not change significantly, indicating that the Yuexu XB-C8 4.6mm×250mm, 5μm chromatographic ,column can also reproduce this method.

[0310] Example 13

[0311] Sample preparation: dissolve palbociclib hydrochloride, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9 in an aqueous solution with a volume concentration of 50% acetonitrile to prepare a mixed solution containing palbociclib hydrochloride at a concentration of 5 mg / mL and each impurity at a concentration of 4 μg / mL, and inject the mixed solution into a liquid chromatograph;

[0312] Mobile phase A: 0.01 mol / L anhydrous potassium hydrogen phosphate (pH adjusted to 5.8 with phosphoric acid);

[0313] Mobile phase B: acetonitrile;

[0314] The gradient elution method is shown in Table 14 below:

[0315] Table 14

[0316]

[0317] High performance liquid chromatograph: waters e2695;

[0318] Chromatographic column: Yuexu XB-C18 4.6mm×250mm, 5μm;

[0319] Ghost peak trapping column: MORHCHEM ghost Trapping columeⅡ50×4.6mm;

[0320] Flow rate: 1.0 mL / min;

[0321] Detection wavelength: 230nm;

[0322] Column temperature: 35 °C;

[0323] Injection volume: 20 μL.

[0324] HPLC analysis was performed under the above conditions, and the chromatogram was recorded. The retention times were impurity 1, palbociclib (the peaks not integrated before the main peak were other impurity peaks), impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9. The results are shown in Fig.13 It can be seen that baseline separation can be achieved between impurities and palbociclib, and between adjacent impurities, with a separation degree greater than 1.9.

[0325] Embodiment 14

[0326] Sample preparation: dissolve palbociclib hydrochloride, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9 in acetonitrile aqueous solution with a volume concentration of 50% to prepare a mixed solution containing palbociclib hydrochloride at a concentration of 5 mg / mL and each impurity at a concentration of 4 μg / mL, and inject the mixed solution into a liquid chromatograph;

[0327] Mobile phase A: 0.01 mol / L anhydrous potassium hydrogen phosphate aqueous solution (adjust pH to 6.1 with phosphoric acid);

[0328] Mobile phase B: acetonitrile;

[0329] The gradient elution method is shown in Table 15 below:

[0330] Table 15

[0331]

[0332] High performance liquid chromatograph: waters e2695;

[0333] Chromatographic column: Yuexu XB-C 18 4.6mm×250mm, 5μm;

[0334] Ghost peak trapping column: MORHCHEM ghost Trapping columeⅡ50×4.6mm;

[0335] Flow rate: 1.0 mL / min;

[0336] Detection wavelength: 230nm;

[0337] Column temperature: 35°C;

[0338] Injection volume: 20 μL.

[0339] HPLC analysis was performed under the above conditions, and the chromatogram was recorded. The retention times were impurity 1, palbociclib (the peaks not integrated before the main peak were other impurity peaks), impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9. The results are shown in Fig.14 It can be seen that baseline separation can be achieved between impurities and palbociclib, and between adjacent impurities, with a separation degree greater than 2.4.

[0340] Embodiment 15

[0341] Sample preparation: dissolve palbociclib hydrochloride, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9 in acetonitrile aqueous solution with a volume concentration of 50% to prepare a mixed solution containing palbociclib hydrochloride at a concentration of 5 mg / mL and each impurity at a concentration of 4 μg / mL, and inject the mixed solution into a liquid chromatograph;

[0342] Mobile phase A: 0.01 mol / L anhydrous potassium hydrogen phosphate aqueous solution (adjust pH to 6.0 with phosphoric acid);

[0343] Mobile phase B: acetonitrile;

[0344] The gradient elution method is shown in Table 16 below:

[0345] Table 16

[0346]

[0347] High performance liquid chromatograph: waters e2695;

[0348] Chromatographic column: Waters symmetry C18, 4.6×250 mm, 5 μm;

[0349] Ghost peak trapping column: MORHCHEM ghost Trapping columeⅡ50×4.6mm;

[0350] Flow rate: 1.0 mL / min;

[0351] Detection wavelength: 230nm;

[0352] Column temperature: 35°C;

[0353] Injection volume: 20 μL.

[0354] HPLC analysis was performed under the above conditions, and the chromatogram was recorded. The retention times were impurity 1, palbociclib (the peaks not integrated before the main peak were other impurity peaks), impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8 and impurity 9. The results are shown in Fig.15 It can be seen that baseline separation can be achieved between impurities and palbociclib, and between adjacent impurities, with a separation degree greater than 1.2.

[0355] It can be seen from Examples 1 to 15 that by adjusting the detection conditions within a certain range, baseline separation can be achieved between impurities and palbociclib, and between adjacent impurities, with a separation degree greater than 1.2, indicating that the detection method of the present application has good durability.

[0356] Methodological investigation

[0357] 1. Exclusiveness Investigation

[0358] The preparation of each solution is shown in Table 17 below:

[0359] Table 17

[0360]

[0361]

[0362] Acceptable standards are as follows:

[0363] 1. The blank solution does not interfere with the detection of each substance; S22102-2-IM1, S22102-SM1, impurity 2-IM1 and S22102-SM1-IM4, impurity C, impurity G, impurity I, S22102-6 do not interfere with the detection of known impurities; 2. In the system suitability solution, the separation degree between impurity 6 and its adjacent impurities should be no less than 1.2, and the separation degree between other known impurities and their adjacent impurities should be no less than 1.5; 3. The retention time of the main peak of the positioning solution of impurity 1, impurity 4, impurity 5, impurity 2, impurity 3, impurity 8, impurity 9, impurity 7 and impurity 6 should be consistent with the retention time of each known impurity in the system suitability solution, and the RD of the retention time is ≤2.0%; 4. The reference solution was injected 6 times continuously, and the retention time RSD was ≤1.0%, and the peak area RSD was ≤10%.

[0364] The detection was carried out according to the chromatographic conditions of Example 1, and the detection results are shown in Tables 18 to 20 below:

[0365] Table 18

[0366] Material composition RT(min) Minimum separation Impurity 1 17.048 3.29 Impurity 6 23.555 2.41 Impurity 2 29.463 4.74 Impurity 3 31.238 2.26 Impurity 4 34.165 1.80 Impurity 5 36.450 2.25 Impurity 7 38.658 3.17 Impurity 8 39.654 3.17 Impurity 9 43.086 10.80

[0367] Table 19

[0368]

[0369]

[0370] Table 20

[0371]

[0372] The conclusion is as follows:

[0373] 1. The blank solution does not interfere with the detection of various substances; S22102-2-IM1, S22102-SM1, impurity 2-IM1 and S22102-SM1-IM4, impurity C, impurity G, impurity I, S22102-6 do not interfere with the detection of known impurities;

[0374] 2. In the system suitability solution, the separation degrees of impurities 1, 2, 3, 4, 5, 7, 8, 9 and their adjacent impurities are 3.29, 4.74, 2.26, 1.80, 2.25, 3.17, 3.17 and 10.80, respectively, all not less than 1.5; the separation degree of impurity 6 and its adjacent impurities is 2.41, not less than 1.2;

[0375] 3. The retention time of the main peak of the positioning solution of impurity 1, impurity 6, impurity 2, impurity 3, impurity 4, impurity 5, impurity 7, impurity 8, and impurity 9 should be 1.0%, 0.1%, 0.1%, 0.1%, 0.1%, 0.1%, 0.1%, 0.1%, 0.1%, and 0.1% respectively, and the RD of each of them should not be greater than 2.0% with the retention time of each known impurity in the system suitability solution.

[0376] 4. The reference solution was injected 6 times continuously. The RSDs of the retention times of impurity 1, impurity 6, impurity 2, impurity 3, impurity 4, impurity 5, impurity 7, impurity 8 and impurity 9 were all 0.1% and not more than 1.0%. The RSDs of the peak areas were 0.8%, 1.7%, 0.9%, 1.0%, 1.1%, 3.1%, 0.6%, 0.7% and 0.8% respectively, and the RSDs were all not more than 10%.

[0377] 2. Investigation of detection limit and quantification limit

[0378] The preparation of each solution is shown in Table 21 below:

[0379] Table 21

[0380]

[0381] Acceptable standards are as follows:

[0382] 1. The signal-to-noise ratio of the quantitative limit solution is S / N ≥ 10, and the S / N of the detection limit solution is ≥ 3-8; 2. For 6 consecutive injections of the quantitative limit, the RSD of the retention time is ≤ 2%, and the RSD of the peak area is ≤ 15%; 3. The quantitative limit should be less than 30% of the limit concentration.

[0383] The detection was carried out according to the chromatographic conditions of Example 1, and the detection results are shown in Tables 22 to 24 below:

[0384] Table 22

[0385] name Concentration (μg / mL) Percentage of test sample concentration (%) S / N Impurity 1 0.0418 0.0008 37.3 Impurity 6 0.0384 0.0008 38.2 Impurity 2 0.0407 0.0008 20.7 Impurity 3 0.0417 0.0008 20.9 Impurity 4 0.0427 0.0009 39.9 Impurity 5 0.0794 0.0016 26.4 Impurity 7 0.0397 0.0008 45.2 Impurity 8 0.0388 0.0008 40.7 Impurity 9 0.0398 0.0008 24.2

[0386] Table 23

[0387] name Concentration (μg / mL) Percentage of test sample concentration (%) S / N Impurity 1 0.0125 0.0003 5.3 Impurity 6 0.0115 0.0002 6.2 Impurity 2 0.0122 0.0002 3.8 Impurity 3 0.0125 0.0003 3.3 Impurity 4 0.0128 0.0003 6.3 Impurity 5 0.0238 0.0005 4.1 Impurity 7 0.0119 0.0002 7.5 Impurity 8 0.0116 0.0002 6.7 Impurity 9 0.0119 0.0002 4.5

[0388] Table 24

[0389]

[0390]

[0391] The conclusion is as follows:

[0392] 1. The signal-to-noise ratios of the quantitative limit solutions of impurity 1, impurity 6, impurity 2, impurity 3, impurity 4, impurity 5, impurity 7, impurity 8 and impurity 9 were 37.3, 38.2, 20.7, 20.9, 39.9, 26.4, 45.2, 40.7 and 24.2, respectively, all greater than 10; the signal-to-noise ratios of the detection limit solutions were 5.3, 6.2, 3.8, 3.3, 6.3, 4.1, 7.5, 6.7 and 4.5, all between 3 and 8;

[0393] 2. For the quantitative limit solutions of impurity 1, impurity 6, impurity 2, impurity 3, impurity 4, impurity 5, impurity 7, impurity 8 and impurity 9, the RSDs of retention time were all 0.1% and no more than 2%, and the RSDs of peak areas were 5.1%, 5.2%, 4.1%, 6.4%, 5.8%, 2.8%, 2.6%, 2.3% and 7.8%, respectively, all no more than 15%;

[0394] 3. The limits of quantification of impurity 1, impurity 6, impurity 2, impurity 3, impurity 4, impurity 5, impurity 7, impurity 8 and impurity 9 were 0.0418μg / ml, 0.0384μg / ml, 0.0407μg / ml, 0.0417μg / ml, 0.0427μg / ml, 0.0794μg / ml, 0.0397μg / ml, 0.0388μg / ml and 0.0398μg / ml, respectively, and their percentages in the test sample concentration were 0.0008%, 0.0008%, 0.0008%, 0.0008%, 0.0009%, 0.0016%, 0.0008%, 0.0008 and 0.0008%, respectively, which were all less than 30% limit concentration (0.0024%).

[0395] 3. Linearity and Range

[0396] The preparation of each solution is shown in Table 25 below:

[0397] Table 25

[0398]

[0399]

[0400] Acceptable standards are as follows:

[0401] Each substance is within the concentration range of the limit of quantification to 200% limit:

[0402] 1. Linear correlation coefficient r≥0.990; 2. Y-axis intercept accounts for 100% of the concentration response value should be ≤25%. According to the chromatographic conditions of Example 1, the test results are shown in Table 26 below:

[0403] Table 26

[0404]

[0405]

[0406]

[0407] The conclusion is as follows:

[0408] 1. Impurity 1 is in the range of 0.0418μg / ml to 0.8359μg / ml, the linear equation is y=53.406-0.0506; the correlation coefficient r is 1.0000, not less than 0.990; the y-axis intercept accounts for 0.23% of the 100% limit concentration peak area, not more than 25%;

[0409] 2. Impurity 6 is in the range of 0.0384 μg / ml to 0.7671 μg / ml, and the linear equation is y=56.032x-0.0887; the correlation coefficient r is 0.9999, not less than 0.990; the y-axis intercept accounts for 0.41% of the 100% limit concentration peak area, not more than 25%;

[0410] 3. Impurity 2 is in the range of 0.0407μg / ml to 0.8148μg / ml, the linear equation is y=30.623x+0.1009; the correlation coefficient r is 0.9999, not less than 0.990; the y-axis intercept accounts for 0.80% of the 100% limit concentration peak area, not more than 25%;

[0411] 4. Impurity 3 is in the range of 0.0417μg / ml to 0.8340μg / ml, the linear equation is y=36.105x+0.342; the correlation coefficient r is 0.9996, not less than 0.990; the y-axis intercept accounts for 2.22% of the 100% limit concentration peak area, not more than 25%;

[0412] 5. Impurity 4 is in the range of 0.0427μg / ml to 0.8549μg / ml, the linear equation is y=67.550x+0.0087; the correlation coefficient r is 1.0000, not less than 0.990; the y-axis intercept accounts for 0.03% of the 100% limit concentration peak area, not more than 25%;

[0413] 6. Impurity 5 is in the range of 0.0794 μg / ml to 0.7940 μg / ml, the linear equation is y=21.758x-0.0692; the correlation coefficient r is 0.9999, not less than 0.990; the y-axis intercept accounts for 0.81% of the 100% limit concentration peak area, not more than 25%;

[0414] 7. Impurity 7 is in the range of 0.0397 μg / ml to 0.7944 μg / ml, the linear equation is y=80.713x+0.3275; the correlation coefficient r is 1.0000, not less than 0.990; the y-axis intercept accounts for 1.01% of the 100% limit concentration peak area, not more than 25%;

[0415] 8. Impurity 8 is in the range of 0.0388 μg / ml to 0.7759 μg / ml, and the linear equation is y=85.962x+0.2294; the correlation coefficient r is 0.9999, not less than 0.990; the y-axis intercept accounts for 0.67% of the 100% limit concentration peak area, not more than 25%;

[0416] 9. Impurity 9 is in the range of 0.0398 μg / ml to 0.7953 μg / ml, and the linear equation is y=61.734x+0.1837; the correlation coefficient r is 1.0000, not less than 0.990; the y-axis intercept accounts for 0.74% of the 100% limit concentration peak area, not more than 25%.

[0417] 4. Accuracy test

[0418] The preparation of each solution is shown in Table 27 below:

[0419] Table 27

[0420]

[0421] Acceptable standards are as follows:

[0422] 1. The recovery rate should be between 80% and 120% at each concentration level; 2. The RSD of the recovery rate of three samples at each level is ≤15%, and the RSD% of the recovery rate at each level is ≤15%.

[0423] The detection was carried out according to the chromatographic conditions of Example 1, and the detection results are shown in Table 28 below:

[0424] Table 28

[0425]

[0426]

[0427]

[0428] The conclusion is as follows:

[0429] Taking the impurity limit concentration as 100%, three concentrations of 50%, 100% and 150% were used to quantitatively add impurities to the sample. At each concentration level:

[0430] 1. The recovery rate of impurity 1 was in the range of 88.23% to 118.16%, the recoveries of 3 samples were 14.5%, 0.9% and 0.3% respectively, and the RSD of the recoveries of 9 samples was 8.6%;

[0431] 2. The recovery rate of impurity 6 was in the range of 100.56% to 103.84%, the recoveries of 3 samples were 1.5%, 1.5% and 0.7% respectively, and the RSD of the recoveries of 9 samples was 1.2%;

[0432] 3. The recovery rate of impurity 2 was in the range of 98.14% to 105.12%, and the recoveries of the three samples were 3.1%, 0.9% and 1.4% respectively, and the RSD of the recoveries of the nine samples was 2.0%;

[0433] 4. The recovery rate of impurity 3 was in the range of 88.40% to 100.21%, with 3 recoveries of 4.0%, 2.3% and 5.7% respectively, and the RSD of the 9 recoveries was 4.0%;

[0434] 5. The recovery rate of impurity 4 was in the range of 95.99% to 102.25%, the recoveries of 3 samples were 3.3%, 1.1% and 1.2% respectively, and the RSD of the recoveries of 9 samples was 1.9%;

[0435] 6. The recovery rate of impurity 5 was in the range of 97.86% to 115.53%, with 3 recoveries of 8.5%, 1.1% and 1.4% respectively, and the RSD of 9 recoveries was 5.7%;

[0436] 7. The recovery rate of impurity 7 was in the range of 97.11% to 108.93%, with 3 recoveries of 4.4%, 1.3% and 1.3% respectively, and the RSD of the 9 recoveries was 4.1%;

[0437] 8. The recovery rate of impurity 8 was in the range of 95.97% to 101.58%, the recoveries of 3 samples were 1.3%, 1.7% and 1.5% respectively, and the RSD of the recoveries of 9 samples was 1.9%;

[0438] 9. The recovery rate of impurity 9 was in the range of 91.48% to 103.50%, and the recoveries of the three samples were 2.4%, 1.4% and 1.2% respectively. The RSD of the 9 recoveries was 4.2%;

[0439] The recovery rates of various impurities were all in the range of 80% to 120%, and the RSDs were all less than 15%.

[0440] 5. Repeatability Study

[0441] The preparation of each solution is shown in Table 29 below:

[0442] Table 29

[0443] blank solution 50% acetonitrile aqueous solution Reference stock solution Take the reference stock solution under the accuracy item Impurity 6 stock solution Take the stock solution of impurity 6 under the accuracy item Reference solution Take two reference solutions under the accuracy item Spiked test solution Take 6 portions of 100% recovery solution under accuracy

[0444] Acceptable standards are as follows:

[0445] For the 6 test samples, the detection content of each genotoxic impurity should be RSD ≤ 20.0%.

[0446] The detection was carried out according to the chromatographic conditions of Example 1, and the detection results are shown in Table 30 below:

[0447] Table 30

[0448]

[0449] The conclusion is as follows:

[0450] In the 6 spiked test solutions, the average detection amounts of impurity 1, impurity 6, impurity 2, impurity 3, impurity 4, impurity 5, impurity 7, impurity 8 and impurity 9 were 0.008%, 0.009%, 0.008%, 0.008%, 0.008%, 0.008%, 0.008%, 0.008%, and 0.008%, respectively, and the RSDs were 2.0%, 1.0%, 1.4%, 1.9%, 1.0%, 2.2%, 1.1%, 1.8% and 1.0%, respectively, all of which were less than 20%.

[0451] 6. Intermediate Precision

[0452] The preparation of each solution is shown in Table 31 below:

[0453] Table 31

[0454]

[0455]

[0456] Acceptable standards are as follows:

[0457] For 6 spiked test solutions, the detection content of each genotoxic impurity should be RD≤20%; for 6 spiked test solutions under the repeatability item and 6 spiked test solutions under the intermediate precision item, a total of 12 spiked test solutions, the detection content of each genotoxic impurity should be RD≤15%.

[0458] The detection was carried out according to the chromatographic conditions of Example 1, and the detection results are shown in Table 32 below:

[0459] Table 32

[0460]

[0461] The conclusion is as follows:

[0462] 1. In the 6 spiked test solutions, the average detection amounts of impurity 1, impurity 6, impurity 2, impurity 3, impurity 4, impurity 5, impurity 7, impurity 8, and impurity 9 were 0.008%, 0.007%, 0.007%, 0.008%, 0.008%, 0.008%, 0.008%, 0.008%, and 0.008%, respectively, and the RSDs were 3.3%, 1.4%, 1.1%, 1.2%, 1.4%, 2.7%, 1.3%, 1.4%, and 1.1%, respectively, all of which were not more than 20%;

[0463] 2. In the 12 spiked test solutions, the RD% of impurity 1, impurity 6, impurity 2, impurity 3, impurity 4, impurity 5, impurity 7, impurity 8 and impurity 9 were detected to be 3.8%, 10.2%, 4.1%, 1.1%, 0.7%, 2.1%, 0.9%, 2.3% and 0.2%, respectively, all of which were less than 15%.

[0464] 7. Solution stability

[0465] The preparation of each solution is shown in Table 33 below:

[0466] Table 33

[0467]

[0468]

[0469] Acceptable standards are as follows:

[0470] 1. The peak area RSD of the control solution should be ≤10.0%; 2. For the spiked test solution, the content of each genotoxic impurity should change by ≤30%.

[0471] The detection was carried out according to the chromatographic conditions of Example 1, and the detection results are shown in Table 34 below:

[0472] Table 34

[0473]

[0474] The conclusion is as follows:

[0475] 1. The spiked test solution was placed at 4°C for 30 hours. Compared with 0 hour, the maximum change rates of impurity 1, impurity 6, impurity 2, impurity 3, impurity 4, impurity 5, impurity 7, impurity 8, and impurity 9 at each time point were 4.64%, 4.75%, 2.95%, 2.09%, 1.25%, 3.97%, 1.67%, 1.92%, and 6.20%, respectively, all of which were no more than 30%;

[0476] 2. When the reference solution was placed at 4°C for 36 hours, the peak area RSDs of impurity 1, impurity 6, impurity 2, impurity 3, impurity 4, impurity 5, impurity 7, impurity 8 and impurity 9 were 0.72%, 0.51%, 1.90%, 1.63%, 1.10%, 2.09%, 1.60%, 0.86% and 0.92%, respectively, which were not more than 10%.

[0477] Therefore, at 4°C, the reference solution is stable for 36 hours, and the spiked test solution is stable for 30 hours.

[0478] Comparative Example 1

[0479] In this comparative example 1, the chromatographic conditions of Example 1 in CN111239299A were followed to inject the sample of Example 1. The test results are shown in Tables 35 and Fig.25 As shown:

[0480] Table 35

[0481] Components Retention time (min) Impurity 1 7.667 Palbociclib 20.539 Impurity 2 30.082 Impurity 3 29.418 Impurity 4 40.734 Impurity 5 46.130 Impurity 6 20.335 Impurity 7 47.620 Impurity 8 45.123 Impurity 9 44.171

[0482] It can be seen that it is difficult to achieve effective separation of the 9 impurities in this application under this chromatographic condition.

[0483] Comparative Example 2

[0484] In this comparative example 1, the chromatographic conditions of Example 1 in CN 116754683 A were followed to inject the sample of Example 1. The test results are shown in Tables 36 and 37. Fig.26 As shown:

[0485] Table 36

[0486] Components Retention time (min) Impurity 1 8.897 Palbociclib 31.498 Impurity 2 49.066 Impurity 3 51.193 Impurity 4 55.484 Impurity 5 58.128 Impurity 6 31.626 Impurity 7 69.263 Impurity 8 66.882 Impurity 9 65.801

[0487] It can be seen that it is difficult to achieve effective separation of the 9 impurities in this application under this chromatographic condition.

[0488] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of the present specification. The above-mentioned embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, several deformations and improvements can be made without departing from the concept of the present application, which all belong to the scope of protection of the present application. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent of the present application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A method for detecting related substances in palbociclib or its salts, characterized in that: The related substances include 2-nitro-5-bromopyridine, 4-(6-nitropyridine-3-yl)piperazine-1-carboxylic acid tert-butyl ester, 2-nitro-3,5-dibromopyridine, 2-chloro-8-cyclopentyl-5-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one, di-tert-butyl 4,4'-(diazo-1,2-diylbis(pyridine-6,3-diyl))(E)-di(piperazine-1-carboxylate), 5 , one or more of 5'-(1,4-piperazinediyl)bis(2-nitropyridine), 2,6-dichloro-8-cyclopentylpyrido[2,3-d]pyrimidin-7(8H)-one, 6-bromo-2-chloro-8-cyclopentyl-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one and 6-bromo-2-chloro-8-cyclopentyl-5-bromomethyl-pyrido[2,3-d]pyrimidin-7(8H)-one; The detection method comprises the following steps: Mixing palbociclib or its salt with a first solvent to prepare a test solution; The solution to be tested is subjected to high performance liquid chromatography detection, and the conditions of the high performance liquid chromatography detection include: (1) The filler of the chromatographic column is silane-bonded silica gel; (2) Mobile phase A is an aqueous solution containing an inorganic salt and / or an inorganic acid, and mobile phase B is one or more of acetonitrile, methanol, ethanol and tetrahydrofuran, and gradient elution is used, including: From 0min to 10min, the volume percentage of mobile phase A changes from 90% to 94% to 75%, and the volume percentage of mobile phase B changes from 10% to 6% to 25%; From 10 to 35 minutes, the volume percentage of mobile phase A changed from 75% to 30%, and the volume percentage of mobile phase B changed from 25% to 70%; 35min-50min, the volume percentage of mobile phase A is maintained at 30%, and the volume percentage of mobile phase B is maintained at 70%; From 50 to 51 minutes, the volume percentage of mobile phase A changes from 30% to 90% to 94%, and the volume percentage of mobile phase B changes from 70% to 10% to 6%; From 51 min to 60 min, the volume percentage of mobile phase A is maintained at 90% to 94%, and the volume percentage of mobile phase B is maintained at 10% to 6%.

2. The method for detecting related substances in palbociclib or its salt according to claim 1, characterized in that: Mobile phase A has one or more of the following characteristics: (1) The inorganic salt includes one or both of dipotassium hydrogen phosphate and disodium hydrogen phosphate; (2) The concentration of the inorganic salt in mobile phase A is 0.005 mol / L to 0.02 mol / L; (3) The inorganic acid includes one or more of phosphoric acid, sulfuric acid, hydrochloric acid, perchloric acid and trifluoroacetic acid; (4) The pH of mobile phase A is 5.5-6.

5.

3. The method for detecting related substances in palbociclib or its salt according to claim 1, characterized in that: Mobile phase B was acetonitrile.

4. The method for detecting related substances in palbociclib or its salt according to claim 1, characterized in that: The filler of the chromatographic column is C8-C18 alkylsilane bonded silica gel.

5. The method for detecting related substances in palbociclib or its salt according to any one of claims 1 to 4, characterized in that: The conditions for the HPLC detection also include one or more of the following: (1) Ultraviolet light detection is used, and the detection wavelength is 225nm~235nm; (2) Flow rate: 0.5 mL / min to 1.5 mL / min; (3) Column temperature is 30℃~40℃; (4) The injection volume is 15 μL to 25 μL.

6. The method for detecting related substances in palbociclib or its salt according to any one of claims 1 to 4, characterized in that: The related substances include 2-nitro-5-bromopyridine, 4-(6-nitropyridin-3-yl)piperazine-1-carboxylic acid tert-butyl ester, 2-nitro-3,5-dibromopyridine, 2-chloro-8-cyclopentyl-5-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one, di-tert-butyl 4,4'-(diazo-1,2-diylbis(pyridine-6,3-diyl))(E)-di(piperazine-1-carboxylate ), 5,5'-(1,4-piperazinediyl)bis(2-nitropyridine), 2,6-dichloro-8-cyclopentylpyrido[2,3-d]pyrimidin-7(8H)-one, 6-bromo-2-chloro-8-cyclopentyl-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one and 6-bromo-2-chloro-8-cyclopentyl-5-bromomethyl-pyrido[2,3-d]pyrimidin-7(8H)-one.

7. The method for detecting related substances in palbociclib or its salt according to any one of claims 1 to 4, characterized in that: The salt of palbociclib includes one or more of palbociclib hydrochloride, palbociclib phosphate, palbociclib carbonate and palbociclib sulfate.

8. The method for detecting related substances in palbociclib or its salt according to any one of claims 1 to 4, characterized in that: The solution to be tested has one or more of the following characteristics: (1) The concentration of palbociclib or its salt in the test solution is 1 mg / mL to 20 mg / mL; (2) The first solvent is an acetonitrile aqueous solution with a volume concentration of 40% to 60%.

9. The method for detecting related substances in palbociclib or its salt according to any one of claims 1 to 4, characterized in that: The detection method further comprises the following steps: Mixing the reference substance of the related substance with the second solvent to prepare reference substance solutions of different concentrations; Performing the HPLC test on the reference solution of different concentrations, and constructing a standard curve of the related substances according to the chromatographic peak area in the test result and the concentration of the reference solution; Substitute the test result of the high performance liquid chromatography of the test solution into the standard curve of the related substance to calculate the content of the related substance in the test solution.

10. The method for detecting related substances in palbociclib or its salt according to claim 9, characterized in that: The second solvent is an acetonitrile aqueous solution with a volume concentration of 40% to 60%.

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