Nintedanib ethanesulfonate intermediate Z 3 Separation and detection methods of related substances in

Abstract

The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating and detecting related substances in nintedanib ethanesulfonate intermediate Z3. The present invention uses a self-established high performance liquid chromatography method to detect nintedanib ethanesulfonate intermediate Z3, impurity NIN-SM1, impurity NIN-Z 2b , impurity NIN-Z2 and impurity NIN-Z 3d . Octadecylsilane chemically bonded silica gel is used as the stationary phase of the chromatographic column; potassium dihydrogen phosphate buffer solution is used as mobile phase A, and acetonitrile is used as mobile phase B. Separation is carried out by gradient elution and then enters a detector with a detection wavelength of 225 nm for detection to obtain a chromatogram; then the content of each component is calculated using the peak area by the self-control method with correction factors. The high performance liquid chromatography method provided by the present invention can simultaneously detect 4 known impurities and unknown impurities in nintedanib ethanesulfonate intermediate Z3. The method is simple and accurate, and has the characteristics of strong specificity, high sensitivity and short time.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug analysis, and specifically relates to a nintedanib ethanesulfonate intermediate Z 3 Separation and detection method of related substances. Background Art

[0002] Nintedanib ethanesulfonate intermediate Z 3 (Abbreviated as NIN-Z 3 ), chemical name is (3E)-3-[methoxy(phenyl)methylene]-2-oxoindoline-6-carboxylate, English chemical name: Methyl(3E)-3-(Methoxy(phenyl)methylene)-2-oxoindoline-6-carboxylate, molecular formula is C 18 H 15 NO 4 , molecular weight is 309.32, CAS number is 1168150-46-6, and the structural formula is shown in Formula Ⅰ.

[0003]

[0004] NIN-Z 3 It is an important intermediate in the synthesis of nintedanib ethanesulfonate. According to the synthetic process route, we found that NIN-Z 3 Impurities NIN-SM may be present in 1 、Impurity NIN-Z 2b 、Impurity NIN-Z 2 and impurity NIN-Z 3d The presence of these four impurities affects NIN-Z to a certain extent. 3 Quality, such as impurities NIN-Z 2 It is a genotoxic impurity of halogenated hydrocarbons and needs to be added to the intermediate Z 3 Therefore, in order to ensure the quality of nintedanib ethanesulfonate products, it is necessary to detect the impurity NIN-SM 1 、Impurity NIN-Z 2b 、Impurity NIN-Z 2 and impurity NIN-Z 3d Take control.

[0005] Impurity NIN-SM 1 The structural formula of NIN-Z is shown in Formula II. 2b The structural formula is shown in Formula III, impurity NIN-Z 2 The structural formula is shown in Formula IV, and the impurity NIN-Z 3d The structural formula of is shown in Formula V.

[0006]

[0007]

[0008] There are many reports on the analysis methods of related substances in nintedanib ethanesulfonate in the prior art, such as invention patents with publication numbers CN108226314A, CN115888187A, CN106841495A, CN106748960A, CN114426512A, etc. These patents disclose separation and detection methods for various impurities in nintedanib ethanesulfonate, but do not include the impurity NIN-SM 1 、Impurity NIN-Z 2b 、Impurity NIN-Z 2 and impurity NIN-Z 3d , these methods cannot overcome NIN-Z 3 The solution has poor stability and a degradation platform peak will appear in the liquid phase system, so it is difficult to effectively determine the above four impurities. Summary of the invention

[0009] Based on this, one of the purposes of the present invention is to provide a method for separating the intermediate Z of nintedanib ethanesulfonate by high performance liquid chromatography. 3 The method for preparing the intermediate Z of nintedanib ethanesulfonate is highly sensitive and specific. 3 It also provides technical support for the qualitative and quantitative analysis of its four components.

[0010] To achieve the above object, the present invention adopts the following technical solutions:

[0011] Separation of Nintedanib ethanesulfonate intermediate Z by HPLC 3 and methods for related substances thereof, wherein the related substances include the impurity NIN-SM 1 、Impurity NIN-Z 2b 、Impurity NIN-Z 2 Any one or more of, and impurity NIN-Z 3d The high performance liquid chromatography method comprises: the chromatographic column uses octadecylsilane bonded silica gel as the stationary phase; the mobile phase comprises mobile phase A and mobile phase B, the mobile phase A is a potassium dihydrogen phosphate buffer solution, and the mobile phase B is acetonitrile; the nintedanib ethanesulfonate intermediate Z is separated by gradient elution. 3 Separate from the related substances shown; the nintedanib ethanesulfonate intermediate Z 3 The structural formula is shown in Formula I, and the impurity NIN-SM 1 The structural formula is shown in Formula II, and the impurity NIN-Z 2b The structural formula is shown in Formula III, and the impurity NIN-Z2 The structural formula is shown in Formula IV, and the impurity NIN-Z 3d The structural formula is shown in Formula V;

[0012]

[0013]

[0014] Further, the elution order from first to last is impurity NIN-SM 1 、Impurity NIN-Z 3d 、NIN-Z 3 、Impurity NIN-Z 2b 、Impurity NIN-Z 2 .

[0015] Furthermore, the method can achieve the impurity NIN-SM within 32 minutes. 1 、Impurity NIN-Z 3d 、NIN-Z 3 、Impurity NIN-Z 2b and impurity NIN-Z 2 Simultaneous separation and detection.

[0016] Further, the gradient elution program is set as follows:

[0017] At 0 minutes, the volume ratio of the mobile phase A to the mobile phase B is 49-51:49-51;

[0018] At 8 minutes, the volume ratio of the mobile phase A to the mobile phase B is 49-51:49-51;

[0019] At 15 minutes, the volume ratio of the mobile phase A to the mobile phase B is 34-36:64-66;

[0020] At 23 minutes, the volume ratio of the mobile phase A to the mobile phase B is 34-36:64-66;

[0021] At 24 minutes, the volume ratio of the mobile phase A to the mobile phase B is 49-51:49-51;

[0022] At 32 minutes, the volume ratio of the mobile phase A to the mobile phase B was 49-51:49-51.

[0023] Preferably, the gradient elution program is set as follows:

[0024] At 0 minutes, the volume ratio of the mobile phase A to the mobile phase B was 50:50;

[0025] At 8 minutes, the volume ratio of the mobile phase A to the mobile phase B was 50:50;

[0026] At 15 minutes, the volume ratio of the mobile phase A to the mobile phase B was 35:65;

[0027] At 23 minutes, the volume ratio of the mobile phase A to the mobile phase B was 35:65;

[0028] At 24 minutes, the volume ratio of the mobile phase A to the mobile phase B was 50:50;

[0029] At 32 minutes, the volume ratio of the mobile phase A to the mobile phase B was 50:50.

[0030] Furthermore, the pH value of the mobile phase A is 2.8 to 3.2, preferably 3.0, and the concentration of the potassium dihydrogen phosphate buffer solution is 0.01 mol / L to 0.05 mol / L, preferably 0.03 mol / L.

[0031] Furthermore, the flow rate of the mobile phase is 0.9 ml / min-1.1 ml / min, preferably 1.0 ml / min; the column temperature of the chromatographic column is 18°C-22°C, preferably; the temperature of the automatic sampler is ≤5°C, preferably 5°C.

[0032] Preferably, the injection volume is 10 μl.

[0033] The second object of the present invention is to provide a method for detecting the intermediate Z of nintedanib ethanesulfonate based on the aforementioned separation method. 3 A method for determining the content of impurity NIN-SM in 32 minutes or more than 32 minutes. 1 、Impurity NIN-Z 3d 、NIN-Z 3 、Impurity NIN-Z 2b and impurity NIN-Z 2 qualitative and quantitative.

[0034] To achieve the above object, the present invention adopts the following technical solutions:

[0035] Detection of nintedanib ethanesulfonate intermediate Z based on the aforementioned separation method 3 A method for measuring the content of related substances, the method comprising the following steps:

[0036] (1) Separation: Separating the nintedanib ethanesulfonate intermediate Z using the method described in any one of claims 1 to 5 3 and the related substances;

[0037] (2) Detection: After separation, the sample is sent to a detector with a detection wavelength of 225 nm for detection to obtain a chromatogram;

[0038] (3) Content calculation: Based on the measured chromatogram, the content of each component was calculated using the peak area using the self-control method with a correction factor.

[0039] Further, the impurity content calculation formula is as follows:

[0040] (1)

[0041] (2) Total impurities = the sum of the contents of each individual impurity.

[0042] Where: Ar is the peak area of ​​a single impurity peak in the chromatogram of the test solution;

[0043] As—the main peak area in the chromatogram of the reference solution;

[0044] F—Correction factor corresponding to each individual impurity.

[0045] Furthermore, impurity NIN-SM 1 The correction factor is 0.38, impurity NIN-Z 3d The correction factor is 1.0, and the impurity NIN-Z 2b The correction factor is 0.72, impurity NIN-Z 2 The correction factor for impurities is 0.81 and the correction factor for other individual impurities is 1.0.

[0046] Further, the retention time from short to long is as follows: Impurity NIN-SM 1 、Impurity NIN-Z 3d 、Nintedanib ethanesulfonate intermediate Z 3 、Impurity NIN-Z 2b 、Impurity NIN-Z 2 .

[0047] Further, the chromatographic column specifications are 4.6 mm × 250 mm, 5 μm; mobile phase A is a potassium dihydrogen phosphate buffer solution with a concentration of 0.03 mol / L and a pH value of 3.0, and mobile phase B is acetonitrile; the mobile phase flow rate is 1.0 ml / min, the column temperature is 20° C., and the automatic sampler temperature is 5° C.; linear gradient elution is performed according to the gradient elution program and a chromatogram is obtained; the gradient elution program is set as follows:

[0048] At 0 minutes, the volume ratio of the mobile phase A to the mobile phase B was 50:50;

[0049] At 8 minutes, the volume ratio of the mobile phase A to the mobile phase B was 50:50;

[0050] At 15 minutes, the volume ratio of the mobile phase A to the mobile phase B was 35:65;

[0051] At 23 minutes, the volume ratio of the mobile phase A to the mobile phase B was 35:65;

[0052] At 24 minutes, the volume ratio of the mobile phase A to the mobile phase B was 50:50;

[0053] At 32 minutes, the volume ratio of the mobile phase A to the mobile phase B was 50:50.

[0054] Furthermore, the retention time was 3.8±0.5, which was determined to be the impurity NIN-SM 1 ; Retention time is 5.6±0.5, determined to be impurity NIN-Z 3d ; The retention time was 11.0±0.5, which was determined to be the intermediate Z of nintedanib ethanesulfonate 3 ; Retention time is 20.3±0.5, determined to be impurity NIN-Z 2b ; Retention time is 22.9±0.5, determined to be impurity NIN-Z 2 .

[0055] The above retention times can be used for qualitative detection of components.

[0056] Further, the impurity NIN-SM 1 In the linear range of 0.02998 μg / ml to 1.8738 μg / ml, Y=1.6314X-0.0003; the impurity NIN-Z 3d In the linear range of 0.05054 μg / ml to 12.6347 μg / ml, Y=0.6934X-0.014; the nintedanib ethanesulfonate intermediate Z 3 In the linear range of 0.05095 μg / ml to 12.7364 μg / ml, Y=0.6224X+0.0429; the impurity NIN-Z 2b In the linear range of 0.05026 μg / ml to 1.8848 μg / ml, Y=0.8618X+0.0004; the impurity NIN-Z 2 In the linear range of 0.04872 μg / ml to 1.2179 μg / ml, Y=0.7681X+0.0004; wherein Y is the Y-axis, representing the peak area, and X is the X-axis, representing the concentration.

[0057] Furthermore, a standard substance is prepared to obtain a chromatogram of the standard substance; the chromatogram obtained by the test sample is used to calculate the content by peak area according to the self-control method with a correction factor added.

[0058] The content of the components of the standard is known, while the content of the components of the test sample is unknown.

[0059] The obtained standard sample chromatogram can be stored in the database as a chromatographic material for quality control in the smart pharmaceutical factory, to help the robot determine whether the products it produces are qualified and to provide quantitative improvement analysis suggestions for unqualified products.

[0060] Furthermore, before separation, acetonitrile was used as a solvent to prepare the sample solution, the control solution and the system suitability solution. The specific detection method is as follows:

[0061] Step 1. Prepare the test solution:

[0062] Sample solution: Take an appropriate amount of this product, weigh it accurately, add acetonitrile to dissolve it by ultrasonication and dilute it to make a solution with a concentration of 0.5 mg / mL, and store it at 5°C.

[0063] Control solution: Accurately measure 5 ml of the test solution, place it in a 50 ml volumetric flask, dilute it to the scale with acetonitrile, and shake well; accurately measure 1 ml, place it in a 100 ml volumetric flask, dilute it to the scale with acetonitrile, and shake well.

[0064] System suitability solution: Take NIN-Z 3 、Impurity NIN-SM 1 、Impurity NIN-Z 2 、Impurity NIN-Z 2b 、Impurity NIN-Z 3d The reference substances were dissolved and diluted in acetonitrile to make each ml contain about 1 μg of each impurity and NIN-Z. 3 0.5mg of solution.

[0065] Step 2. Take the system suitability solution, control solution and sample solution prepared in step 1, such as 10 μl, and inject them into the high performance liquid chromatograph, record the chromatogram, and calculate the content of each component using the peak area by the self-control method with the correction factor added.

[0066] The beneficial effects of the present invention are:

[0067] 1. The present invention uses octadecylsilane bonded silica gel as a chromatographic column filler to purify NIN-Z 3 The relevant impurities were separated by using potassium dihydrogen phosphate buffer-acetonitrile as the mobile phase system. The injection plate temperature was controlled at 5°C to ensure the stability of the test solution. The column temperature was selected at 20°C to improve the degradation platform peak, and NIN-Z was successfully resolved. 3 The solution stability is poor, and the problem of degradation platform peak will appear in the liquid phase system.

[0068] 2. The chromatographic system established by the present invention, the intermediate Z of nintedanib ethanesulfonate 3 The peaks of each impurity are symmetrical, NIN-Z 3The peaks can be effectively separated from adjacent impurity peaks, and the separation degree between each impurity can meet the requirements. Four known impurities and unknown impurities can be detected at the same time, and the quantitative limit is as low as 0.03-0.05μg / ml. The method is simple, accurate, fast and effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 Chromatogram for system suitability;

[0070] Figure 2 is the chromatogram of sensitivity (quantitation limit);

[0071] Figure 3 is the chromatogram of linear solution 1;

[0072] Figure 4 is the chromatogram of linear solution 2;

[0073] Figure 5 is the chromatogram of linear solution 3;

[0074] Figure 6 is the chromatogram of linear solution 4;

[0075] Figure 7 is the chromatogram of linear solution 5;

[0076] Figure 8 is the chromatogram of the test solution;

[0077] Fig. 9 is the chromatogram of the control solution;

[0078] Fig.10 is the chromatogram of sensitivity (detection limit);

[0079] Fig.11 for ruggedness - chromatograms of normal chromatographic conditions;

[0080] Fig.12 The chromatogram is for durability-column temperature 18°C;

[0081] Fig.13 This is the chromatogram of durability-column temperature 22°C;

[0082] Fig.14 The chromatogram is for durability-flow rate 0.9 ml / min;

[0083] Fig.15 The chromatogram is for durability-flow rate 1.1 ml / min;

[0084] Fig.16 Chromatogram for durability-buffered salt pH 2.8;

[0085] Fig.17Chromatogram for durability-buffered salt pH 3.2;

[0086] Fig.18 The chromatogram is for durability-the initial ratio of the mobile phase is acetonitrile-buffered salt (49:51);

[0087] Fig.19 The chromatogram is of the durability-initial ratio of the mobile phase: acetonitrile-buffered salt (51:49). DETAILED DESCRIPTION

[0088] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0089] In the embodiment of the present invention, this product is nintedanib ethanesulfonate intermediate Z 3 , chemical name is (3E)-3-[methoxy(phenyl)methylene]-2-oxoindoline-6-carboxylic acid methyl ester, referred to as NIN-Z 3 .

[0090] In the embodiment of the present invention, the main instrument is a Thermo Fisher high performance liquid chromatograph.

[0091] Example 1. Specificity

[0092] (1) Solution preparation

[0093] Solvent: acetonitrile.

[0094] Test solution: Take an appropriate amount of the product, weigh it accurately, add acetonitrile to dissolve it by ultrasonication and dilute it to make a solution with a concentration of 0.5 mg / mL, and store it at 5°C.

[0095] Control solution: Accurately measure 5 ml of the test solution, place it in a 50 ml volumetric flask, dilute it to the mark with acetonitrile, shake well, accurately measure 1 ml, place it in a 100 ml volumetric flask, dilute it to the mark with acetonitrile, shake well.

[0096] System suitability solution: Take Nintedanib ethanesulfonate NIN-Z 3 、Impurity NIN-SM 1 、Impurity NIN-Z 2 、Impurity NIN-Z 2b 、Impurity NIN-Z 3d The reference substances were dissolved and diluted in acetonitrile to make each ml contain about 1 μg of each impurity and NIN-Z. 3 0.5mg of solution.

[0097] (2) Chromatographic conditions

[0098] Octadecylsilane bonded silica gel was used as a filler (Shim-pack GIST C18 4.6mm×250mm, 5μm or a chromatographic column of equivalent performance); potassium dihydrogen phosphate solution was used as mobile phase A, acetonitrile was used as mobile phase B, and linear gradient elution was performed according to Table 1; the detection wavelength was 225nm; the flow rate was 1.0ml per minute; the column temperature was 20℃; the injection volume was 10μ1, and the temperature of the automatic sampler was 5℃. The preparation method of potassium dihydrogen phosphate solution is as follows: 2.72g of potassium dihydrogen phosphate was taken, 1000ml of water was added to dissolve it, and the pH value was adjusted to 3.0 with phosphoric acid.

[0099] Table 1. Gradient elution program

[0100] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 49～51 49～51 8 49～51 49～51 15 34～36 64～66 23 34～36 64～66 24 49～51 49～51 32 49～51 49～51

[0101] (3) Determination

[0102] Accurately measure 10 μl of the system suitability solution, inject it into the liquid chromatograph, and record the chromatogram. The peak order is impurity NIN-SM. 1 、Impurity NIN-Z 3d 、NIN-Z 3 Specific impurities (RRT≈0.59), NIN-Z 3 Main peak, impurity NIN-Z 2b 、Impurity NIN-Z 2 , NIN-Z 3 The separation degree between the adjacent impurity peaks should meet the requirements (separation degree should be greater than 2.0). Figure 1 , as shown in Table 2, the separation degree should meet the requirements; Figure 1 The integral results table is shown in Table 3.

[0103] Table 2. System suitability solution testing

[0104] name Retention time Separation <![CDATA[Impurity NIN-SM 1 > 3.862 8.39 <![CDATA[Impurity NIN-Z 3d > 5.647 3.29 Specific impurities 6.535 12.36 <![CDATA[NIN-Z 3 ]]> 11.055 22.50 <![CDATA[Impurity NIN-Z 2b > 20.345 6.20 <![CDATA[Impurity NIN-Z 2 > 22.943 ——

[0105] Table 3. Figure 1 The integral result table

[0106]

[0107] Accurately measure the test solution and the reference solution and inject them into the liquid chromatograph to record the chromatogram. In the chromatogram of the test solution, except for the solvent peak and the gradient elution peak, the content of each impurity is calculated by the self-reference method with the correction factor added. Among them, the impurity NIN-SM 1 The correction factor is 0.38, impurity NIN-Z 3d The correction factor is 1.0, and the impurity NIN-Z 2bThe correction factor is 0.72, impurity NIN-Z 2 The correction factor for impurities is 0.81, and the correction factor for other single impurities is 1.0. Figure 8-Figure 9 , as shown in Table 4; Figure 8 The integration results are shown in Table 5. Fig. 9 The integration results are shown in Table 6.

[0108] Table 4. Test results of the test products

[0109] batch number <![CDATA[Impurity NIN-SM 1 > <![CDATA[Impurity NIN-Z 3d > <![CDATA[Impurity NIN-Z 2b > <![CDATA[Impurity NIN-Z 2 > Other single impurities Total impurities 01 <0.05％ 0.32％ Not detected Not detected 1.0％ 1.3％

[0110] Table 5. Figure 8 The integral result table

[0111]

[0112] Table 6. Fig. 9 The integral result table

[0113]

[0114] Example 2. Linearity determination

[0115] (1) Preparation of linear solution

[0116] Impurity NIN-SM 1 Mother liquor: Accurately weigh the impurity SM 1 25.06 mg, put into a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and you have it.

[0117] Impurity NIN-Z 3c Mother liquor: Accurately weigh impurity Z 3c 24.37 mg, put in a 50 ml volumetric flask, add solvent, ultrasonically dissolve and dilute to the scale, shake well, and the product is obtained.

[0118] Impurity NIN-Z 3d Mother liquor: Accurately weigh impurity Z 3d 25.32 mg, put in a 50 ml volumetric flask, add solvent, ultrasonically dissolve and dilute to the scale, shake well, and the product is obtained.

[0119] Impurity NIN-Z 2b Mother liquor: Accurately weigh impurity Z 2b 25.41 mg, put in a 50 ml volumetric flask, add solvent, ultrasonically dissolve and dilute to the scale, shake well, and you have it.

[0120] Impurity NIN-Z 2 Mother liquor: Accurately weigh impurity Z 2 25.06 mg, put in a 50 ml volumetric flask, add solvent, ultrasonically dissolve and dilute to the scale, shake well, and you have it.

[0121] NIN-Z 3 Mother liquor: Accurately weigh nintedanib ethanesulfonate Z 3 Reference substance (NIN-Z 3 -210901RS) 25.73 mg, placed in a 50 ml volumetric flask, added solvent, ultrasonically dissolved and diluted to scale, shake well to obtain.

[0122] Linear stock solution: Precise pipetting of impurities NIN-SM 1 Mother liquor 1.5ml, impurity NIN-Z 3c Mother liquor 1.5ml, impurity NIN-Z 2b Mother liquor 1.5ml, impurity NIN-Z 2 Place 1.0 ml of the mother solution in a 50 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the product.

[0123] Linear solution 1: Accurately pipette 1.25 ml of linear stock solution, impurity NIN-Z 3d Mother solution 0.25ml, NIN-Z 3 Place 0.25 ml of the mother solution in a 50 ml volumetric flask, add solvent to the mark, shake well, and you have the product.

[0124] Linear solution 2: Accurately pipette 2.5 ml of linear stock solution, impurity NIN-Z 3d Mother solution 0.5ml, NIN-Z 3 Place 0.5 ml of the mother solution in a 50 ml volumetric flask, add solvent to the mark, shake well, and you have the product.

[0125] Linear solution 3: Accurately pipette 3.75 ml of linear stock solution, impurity NIN-Z 3d Mother solution 0.75ml, NIN-Z 3 Place 0.75 ml of the mother solution in a 50 ml volumetric flask, add solvent to the mark, shake well, and you have the product.

[0126] Linear solution 4: Accurately pipette 5 ml of linear stock solution, impurity NIN-Z 3d Mother solution 1ml, NIN-Z 3 Place 1 ml of the mother solution in a 50 ml volumetric flask, add solvent to the mark, shake well, and you have the product.

[0127] Linear solution 5: Accurately pipette 1.25 ml of linear stock solution, impurity NIN-Z 3d Mother solution 0.25ml, NIN-Z 3 Place 0.25 ml of the mother solution in a 10 ml volumetric flask, add solvent to the mark, shake well, and you have the product.

[0128] (2) Determination

[0129] Take the linear solutions 1-5 prepared in (1) and inject them in order of concentration from low to high (chromatographic conditions are the same as in Example 1), record the chromatogram, and use the least squares method to linearly regress the concentration X (μg / ml) against the peak area Y to calculate the regression equation and correlation coefficient. The test results are as follows Figure 3-Figure 7 , as shown in Table 7; Figure 3-Figure 7 The integral results are shown in Table 8; the correction factor calculation results are shown in Table 9.

[0130] Table 7. Linearity test results

[0131] name Linear concentration Regression equation Correlation coefficient <![CDATA[Impurity NIN-SM 1 > 0.02998μg / ml~1.8738μg / ml Y=1.6314X-0.0003 r＝0.9997 <![CDATA[Impurity NIN-Z 3d > 0.05054μg / ml~12.6347μg / ml Y=0.6934X-0.014 r＝0.9998 <![CDATA[NIN-Z 3 ]]> 0.05095μg / ml~12.7364μg / ml Y=0.6224X+0.0429 r＝0.9998 <![CDATA[Impurity NIN-Z 2b > 0.05026μg / ml~1.8848μg / ml Y=0.8618X+0.0004 r＝0.9997 <![CDATA[Impurity NIN-Z 2 > 0.04872μg / ml~1.2179μg / ml Y=0.7681X+0.0004 r＝0.9997

[0132] Table 8. Figure 3-Figure 7 The integral results table

[0133]

[0134]

[0135]

[0136] Calculation of impurity correction factor:

[0137]

[0138] Where: K Z3 ——Nintedanib Ethylate Z 3 The slope of

[0139] K 杂质 ——The slope of impurities.

[0140] Table 9. Correction factor calculation results

[0141]

[0142] Example 3. Sensitivity

[0143] (1) Solution preparation

[0144] Impurity SM 1 Mother liquor: Accurately weigh the impurity SM 1 25.06 mg, put into a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and you have it.

[0145] Impurity Z 3c Mother liquor: Accurately weigh impurity Z 3c 24.37 mg, put in a 50 ml volumetric flask, add solvent, ultrasonically dissolve and dilute to the scale, shake well, and the product is obtained.

[0146] Impurity Z 3d Mother liquor: Accurately weigh impurity Z 3d25.32 mg, put in a 50 ml volumetric flask, add solvent, ultrasonically dissolve and dilute to the scale, shake well, and obtain.

[0147] Impurity Z 2b Mother liquor: Accurately weigh impurity Z 2b 25.41 mg, put in a 50 ml volumetric flask, add solvent, ultrasonically dissolve and dilute to the scale, shake well, and you have it.

[0148] Impurity Z 2 Mother liquor: Accurately weigh impurity Z 2 25.06 mg, put in a 50 ml volumetric flask, add solvent, ultrasonically dissolve and dilute to the scale, shake well, and you have it.

[0149] Z 3 Mother liquor: Accurately weigh nintedanib ethanesulfonate Z 3 Place 25.73 mg of reference substance in a 50 ml volumetric flask, add solvent, dissolve by ultrasonication and dilute to the scale, shake well, and the product is obtained.

[0150] Quantitation limit stock solution: Precise pipetting of impurities SM 1 Mother liquor 0.3ml, impurity Z 3c Mother liquor 0.5ml, impurity Z 3d Mother liquor 0.5ml, impurity Z 2b Mother liquor 0.5ml, impurity Z 2 Mother solution 0.5ml, Z 3 Place 0.5 ml of each mother solution in the same 50 ml volumetric flask and dilute to the mark with solvent.

[0151] Quantitation limit solution: Accurately pipette 0.5 ml of the quantitation limit stock solution into a 50 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0152] Test line solution: Accurately pipette 3.7 ml of the quantitative limit solution into a 10 ml volumetric flask, dilute to the scale with solvent, shake well, and obtain.

[0153] (2) Determination

[0154] The quantitative limit solution prepared in (1) was injected (chromatographic conditions were the same as those in Example 1), the chromatogram was recorded, and the ratio of peak height to noise (signal-to-noise ratio) was calculated. Figure 2 , Fig.10 and Tables 10-13.

[0155] Table 10. Sensitivity (quantitation limit) test results

[0156] name Quantitation limit solution concentration (μg / ml) Signal-to-Noise Ratio Expressed as concentration in sample (%) <![CDATA[Impurity NIN-SM 1 > 0.02998 72.2 0.006％ <![CDATA[Impurity NIN-Z 3d > 0.05054 36.6 0.01 <![CDATA[NIN-Z 3 ]]> 0.05095 19.8 0.01 <![CDATA[Impurity NIN-Z 2b > 0.05026 28.8 0.01 <![CDATA[Impurity NIN-Z 2 > 0.04872 21.4 0.01

[0157] Table 11. Sensitivity (detection limit) determination results

[0158] name Detection limit solution concentration (μg / ml) Signal-to-Noise Ratio Expressed as concentration in sample (%) <![CDATA[Impurity NIN-SM 1 > 0.01109 19.9 0.002％ <![CDATA[Impurity NIN-Z 3d > 0.01870 10.9 0.004％ <![CDATA[NIN-Z 3 ]]> 0.01885 6.0 0.004％ <![CDATA[Impurity NIN-Z 2b > 0.01860 8.2 0.004％ <![CDATA[Impurity NIN-Z 2 > 0.01803 6.4 0.004％

[0159] Table 12. Figure 2 The integral result table

[0160] Serial number Retention time min Peak name Peak area mAU*min Peak height mAU Signal-to-Noise Ratio Tailing Factor Separation Number of plates 1 3.777 <![CDATA[NIN-SM 1 ]]> 0.052 0.518 72.2 1.24 9.40 9674 2 5.485 <![CDATA[NIN-Z 3d ]]> 0.035 0.262 36.6 1.15 17.78 10821 3 10.683 <![CDATA[NIN-Z 3 ]]> 0.034 0.142 19.8 1.04 17.00 12957 4 16.815 - 0.027 0.122 17.1 0.96 1.83 37420 5 17.393 <![CDATA[NIN-Z 3c ]]> 0.037 0.209 29.2 1.12 7.95 58703 6 19.897 <![CDATA[NIN-Z 2b ]]> 0.044 0.206 28.8 1.03 6.67 53371 7 22.355 <![CDATA[NIN-Z 2 ]]> 0.038 0.153 21.4 1.12 na 51238 sum: 225.04

[0161] Table 13. Fig.10 The integral result table

[0162]

[0163]

[0164] Example 4. Chromatographic Condition Variation Robustness

[0165] (1) Solution preparation

[0166] Mixed solution: weigh NIN-Z 3 、Impurity NIN-SM 1 、Impurity NIN-Z 3d 、NIN-Z 3c 、Impurity NIN-Z 2b 、Impurity NIN-Z 2 Appropriate amounts of each, add solvent to dissolve and dilute to make each ml contain about NIN-Z 3 0.5mg, impurity NIN-SM 1 0.75μg, impurity NIN-Z 3d 3μg, NIN-Z 3c 0.75μg, impurity NIN-Z 2b 0.75μg, impurity NIN-Z 2 0.5 μg of mixed solution.

[0167] (2) Determination

[0168] The mixed solution prepared in (1) was injected and tested under normal chromatographic conditions (the chromatographic conditions were the same as those in Example 1) and adjusted chromatographic conditions (column temperature ±2°C, flow rate ±0.1 ml / min, buffer salt pH ±0.2, initial ratio of mobile phase ±1%). After the instrument system was stable, the tests were performed separately and the separation between the peaks was recorded. The test results are shown in Tables 14 to 23. Figure 11-Figure 19 .

[0169] Table 14. Results of the durability test for changes in chromatographic conditions (resolution)

[0170]

[0171] Table 15. Fig.11 The integral result table

[0172]

[0173]

[0174] Table 16. Fig.12 The integral result table

[0175]

[0176] Table 17. Fig.13 The integral result table

[0177]

[0178] Table 18. Fig.14 The integral result table

[0179]

[0180] Table 19. Fig.15 The integral results table

[0181]

[0182]

[0183] Table 20. Fig.16 The integral results table

[0184]

[0185] Table 21. Fig.17 The integral result table

[0186]

[0187] Table 22. Fig.18 The integral result table

[0188]

[0189] Table 23. Fig.19 The integral results table

[0190]

Claims

1. Separation of Nintedanib ethanesulfonate intermediate Z by HPLC 3 and methods for substances related thereto, It is characterized in that The related substances include impurity NIN-SM 1 、Impurity NIN-Z 2b 、Impurity NIN-Z 2 and impurity NIN-Z 3d The high performance liquid chromatography method comprises: the chromatographic column uses octadecylsilane bonded silica gel as the stationary phase; the mobile phase comprises mobile phase A and mobile phase B, the mobile phase A is a potassium dihydrogen phosphate buffer solution, and the mobile phase B is acetonitrile; the pH value of the mobile phase A is 2.8 to 3.2; the nintedanib ethanesulfonate intermediate Z is separated by gradient elution 3 Separate from the related substances shown; the gradient elution program is set as follows: At 0 minutes, the volume ratio of the mobile phase A to the mobile phase B is 49-51:49-51; At 8 minutes, the volume ratio of the mobile phase A to the mobile phase B is 49-51:49-51; At 15 minutes, the volume ratio of the mobile phase A to the mobile phase B is 34-36:64-66; At 23 minutes, the volume ratio of the mobile phase A to the mobile phase B is 34-36:64-66; At 24 minutes, the volume ratio of the mobile phase A to the mobile phase B is 49-51:49-51; At 32 minutes, the volume ratio of the mobile phase A to the mobile phase B is 49-51:49-51; The nintedanib ethanesulfonate intermediate Z 3 has the structural formula shown in Formula I, and the impurity NIN-SM 1 has the structural formula shown in Formula II, and the impurity NIN-Z 2b has the structural formula shown in Formula III, and the impurity NIN-Z 2 has the structural formula shown in Formula IV, and the impurity NIN-Z 3d has the structural formula shown in Formula V; 2. The method according to claim 1, It is characterized in that The concentration of the potassium dihydrogen phosphate buffer solution is 0.01 mol / L to 0.05 mol / L.

3. The method according to claim 1, It is characterized in that The flow rate of the mobile phase is 0.9 ml / min-1.1 ml / min, the column temperature of the chromatographic column is 18° C.-22° C.; the temperature of the automatic sample injector is ≤5° C.

4. Detection of nintedanib ethanesulfonate intermediate Z based on the method described in any one of claims 1 to 3 3 and the method for determining the content of related substances, It is characterized in that The method comprises the following steps: (1) Separation: Separating the nintedanib ethanesulfonate intermediate Z by the method described in any one of claims 1 to 3. 3 and the related substances; (2) Detection: After separation, the sample enters a detector with a detection wavelength of 225 nm for detection to obtain a chromatogram; (3) Content calculation: Based on the measured chromatogram, the content of each component is calculated using the peak area using the self-control method with a correction factor.

5. The method according to claim 4, It is characterized in that The retention times from short to long are: Impurity NIN-SM 1 、Impurity NIN-Z 3d 、Nintedanib ethanesulfonate intermediate Z 3 、Impurity NIN-Z 2b 、Impurity NIN-Z 2 .

6. The method according to claim 4, It is characterized in that The specifications of the chromatographic column are 4.6 mm × 250 mm, 5 μm; the mobile phase A is a potassium dihydrogen phosphate buffer solution with a concentration of 0.03 mol / L and a pH value of 3.0, and the mobile phase B is acetonitrile; the mobile phase flow rate is 1.0 ml / min, the column temperature is 20°C, and the automatic sampler temperature is 5°C; linear gradient elution is performed according to the gradient elution program and a chromatogram is obtained; the gradient elution program is set as follows: At 0 minutes, the volume ratio of the mobile phase A to the mobile phase B was 50:50; At 8 minutes, the volume ratio of the mobile phase A to the mobile phase B was 50:50; At 15 minutes, the volume ratio of the mobile phase A to the mobile phase B was 35:65; At 23 minutes, the volume ratio of the mobile phase A to the mobile phase B was 35:65; At 24 minutes, the volume ratio of the mobile phase A to the mobile phase B was 50:50; At 32 minutes, the volume ratio of the mobile phase A to the mobile phase B was 50:

50.

7. The method according to claim 6, It is characterized in that The retention time is 3.8±0.5, which is determined to be impurity NIN-SM 1 ; Retention time is 5.6±0.5, determined to be impurity NIN-Z 3d ; The retention time was 11.0±0.5, which was determined to be the intermediate Z of nintedanib ethanesulfonate 3 ; Retention time is 20.3±0.5, determined to be impurity NIN-Z 2b ; Retention time is 22.9±0.5, determined to be impurity NIN-Z 2 .

8. The method according to claim 6, It is characterized in that The impurity NIN-SM 1 In the linear range of 0.02998 μg / ml to 1.8738 μg / ml, Y=1.6314X-0.0003; the impurity NIN-Z 3d In the linear range of 0.05054µg / ml to 12.6347µg / ml, Y=0.6934X-0.014; the nintedanib ethanesulfonate intermediate Z 3 In the linear range of 0.05095µg / ml~12.7364µg / ml, Y=0.6224X+0.0429; the impurity NIN-Z 2b In the linear range of 0.05026µg / ml~1.8848µg / ml, Y=0.8618X+0.0004; the impurity NIN-Z 2 In the linear range of 0.04872µg / ml to 1.2179µg / ml, Y=0.7681X+0.0004; Y is the Y-axis, representing the peak area, and X is the X-axis, representing the concentration.

9. The method according to claim 4, It is characterized in that Prepare standard products and obtain their chromatograms; calculate the content of the chromatograms obtained from the test samples by using the peak area according to the self-control method with the addition of correction factors.

Images