Method for detecting organic impurities in JKN2405

Abstract

The invention provides a method for detecting organic impurities in JKN2405, and relates to the technical field of drug detection, and the method comprises the step of detecting the organic impurities in a JKN2405 test solution by adopting high performance liquid chromatography, a chromatographic column is a phenyl column; the impurity trapping small column is a ghost peak trapping column. Separation of middle and low polarity compounds can be well realized through bonding of the phenyl column and the stationary phase chromatographic column, meanwhile, separation of diastereoisomer compounds with benzene rings is facilitated through the pi-pi conjugation effect of stationary phase filler and sample phenyl, interference substances in a mobile phase can be adsorbed by the ghost peak trapping column, and the detection sensitivity is high. A good baseline background is provided for sample detection, so that a better separation effect is achieved. The technical problem that in the prior art, when impurities in JKN2405 are detected through high performance liquid chromatography, the separation degree is too low, and consequently control is inaccurate is solved.

Description

Technical Field

[0001] This invention relates to the field of drug detection technology, and in particular to a method for detecting organic impurities in JKN2405. Background Technology

[0002] JKN2405 (molecular formula is C) 20 H 21 F2NO3S is a novel, chiral, inhalable small molecule compound that inhibits phosphodiesterase (PDE4), thereby suppressing inflammatory cell activity, reducing immune overexpression, and dilating respiratory smooth muscle to treat asthma, acute cough, and COPD. However, due to the chiral core, many related impurities include diastereomers and other compounds that are difficult to separate, such as C18082796-JIP1, C18082796-JIP2, VIP3156, and VIP3157. Impurities VIP3156 and VIP3157 are diastereomers, making accurate quantitative detection difficult using conventional high-performance liquid chromatography (HPLC). Furthermore, the large number of isomer impurities increases the cost if all are separated and controlled using chiral columns.

[0003] In view of this, the present invention is hereby proposed. Summary of the Invention

[0004] The purpose of this invention is to provide a method for detecting organic impurities in JKN2405, so as to solve the technical problem of inaccurate control caused by low separation in high performance liquid chromatography when detecting impurities in JKN2405.

[0005] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: This invention provides a method for detecting organic impurities in JKN2405, the method comprising using high performance liquid chromatography to detect organic impurities in a JKN2405 test solution; The chromatographic column is a phenyl column; the impurity trapping column is a ghost peak trapping column.

[0006] Furthermore, mobile phase A is phosphate buffer or ammonium acetate solution, and mobile phase B is acetonitrile.

[0007] Furthermore, gradient elution includes: 0~25min: 90%~70% mobile phase A, 10%~30% mobile phase B; 25~40min: 70%~45% mobile phase A, 30%~55% mobile phase B; 40~45min: 45%~30% mobile phase A, 55%~70% mobile phase B; 45~60min: 30% mobile phase A, 70% mobile phase B; 60~65min: 30%~90% mobile phase A, 70%~10% mobile phase B; 65~80 min: 90% mobile phase A, 10% mobile phase B.

[0008] Furthermore, the mobile phase A is an ammonium acetate solution; Preferably, the concentration of the ammonium acetate solution is 0.01~0.05 mol / L, and more preferably 0.01 mol / L.

[0009] Furthermore, the chromatographic column particle size is 3.5 μm; Preferably, the size of the impurity collecting column is 4.6 × 50 mm.

[0010] Furthermore, the phenyl column is preferably XBridge® Phenyl.

[0011] Furthermore, the detection wavelength is 270nm; Preferably, the column temperature is 30°C; Preferably, the flow rate is 0.7 ml / min; Preferably, the injection volume is 10 μl.

[0012] Furthermore, the JKN2405 test solution was prepared by dissolving the JKN2405 sample in a diluent; Each 1 ml of JKN2405 test solution contains 2 mg of JKN2405 sample; Preferably, the diluent is a 40% v / v acetonitrile solution.

[0013] Furthermore, the impurity content was calculated using the principal component external standard method with a correction factor.

[0014] Furthermore, the impurities include at least one of VIP3156, VIP3157, C18082796-JIP1, or C18082796-JIP2.

[0015] This invention provides a method for detecting organic impurities in JKN2405 chromatographic material. The method utilizes a phenyl-coated stationary phase column, which effectively separates low- to medium-polarity compounds. Simultaneously, the π-π conjugation effect between the stationary phase packing and the phenyl groups in the sample facilitates the separation of diastereomeric compounds containing benzene rings. The ghost peak trapping column adsorbs interfering substances in the mobile phase, providing a good baseline background for sample detection and thus achieving better separation results. This invention solves the technical problem of inaccurate control caused by excessively low resolution in existing high-performance liquid chromatography (HPLC) for detecting impurities in JKN2405 chromatographic material. Attached Figure Description

[0016] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0017] Figure 1 The structural formula of JKN2405 of this invention; Figure 2 This is a flow chart of the JKN2405 preparation process of the present invention; Figure 3 A typical spectrum of the system suitability solution provided in Example 1 of this invention; Figure 4 This is a typical specific spectrum provided in Embodiment 2 of the present invention; Figure 5 The standard curves of JKN2405 and each impurity provided in Embodiment 4 of the present invention; Figure 6 XBridge provided in Embodiment 8 of the present invention ® Typical chromatograms of sample solutions were detected using Phenyl chromatographic columns; Figure 7 The typical chromatogram of the sample solution detected by the ZORBAX SB-CN chromatographic column provided in Example 8 of this invention; Figure 8 This is a comparison of typical spectra before and after using the impurity trapping column, as provided in Embodiment 9 of the present invention. Detailed Implementation

[0018] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0019] Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification.

[0020] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] This invention provides a method for detecting organic impurities in JKN2405. The method includes using high performance liquid chromatography (HPLC) to detect organic impurities in the JKN2405 test solution; the chromatographic column is a phenyl column; and the impurity trapping column is a ghost peak trapping column.

[0022] This invention utilizes a phenyl-coated stationary phase column to effectively separate low- to medium-polarity compounds. Simultaneously, the π-π conjugation effect between the stationary phase packing and the sample phenyl group facilitates the separation of diastereomeric compounds containing benzene rings. The ghost peak trapping column adsorbs interfering substances from the mobile phase, providing a good baseline background for sample detection and thus achieving better separation. It is suitable for controlling process impurities and degradation products in the JKN2405 formulation. It exhibits high sensitivity and can effectively quantify impurities VIP3156, VIP3157, C18082796-JIP1, and C18082796-JIP2, thereby ensuring drug safety. This invention solves the technical problem of inaccurate control caused by low resolution in existing high-performance liquid chromatography (HPLC) for detecting impurities in JKN2405.

[0023] To improve the separation of impurities in JKN2405, in some specific embodiments, gradient elution includes: 0~25min: 90%~70% mobile phase A, 10%~30% mobile phase B; 25~40min: 70%~45% mobile phase A, 30%~55% mobile phase B; 40~45min: 45%~30% mobile phase A, 55%~70% mobile phase B; 45~60min: 30% mobile phase A, 70% mobile phase B; 60~65min: 30%~90% mobile phase A, 70%~10% mobile phase B; 65~80 min: 90% mobile phase A, 10% mobile phase B.

[0024] In some specific embodiments, mobile phase A is phosphate buffer or ammonium acetate solution, and mobile phase B is acetonitrile. Ammonium acetate solution can effectively suppress -NH2 protonation, reduce tailing, and improve separation efficiency. In some specific embodiments, mobile phase A is ammonium acetate solution; in some specific embodiments, the concentration of ammonium acetate solution is 0.01~0.05 mol / L, preferably 0.01 mol / L.

[0025] To achieve higher resolution, in some specific embodiments, the column particle size is 3.5 μm; specifically, the phenyl column is preferably XBridge® Phenyl.

[0026] In some specific embodiments, the size of the impurity trapping column is 4.6 × 50 mm. This effectively adsorbs interfering substances in the mobile phase and system, eliminates background noise, and improves the sensitivity and specificity of the method.

[0027] In some specific embodiments, the detection wavelength is 270 nm; in some specific embodiments, the column temperature is 30 °C; in some specific embodiments, the flow rate is 0.7 ml / min; in some specific embodiments, the injection volume is 10 μl.

[0028] In some specific embodiments, the JKN2405 test solution is prepared by dissolving the JKN2405 sample in a diluent; in some specific embodiments, each 1 ml of the JKN2405 test solution contains 2 mg of the JKN2405 sample; in some specific embodiments, the diluent is a 60% v / v acetonitrile solution.

[0029] In some specific implementations, the impurity content is calculated using the principal component external standard method with a correction factor. Introducing a correction factor improves accuracy.

[0030] In some specific embodiments, the impurity includes at least one of VIP3156, VIP3157, C18082796-JIP1 or C18082796-JIP2.

[0031] The present invention will be further illustrated below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0032] The sample was a self-made JKN2405 inhalation spray, prepared by mixing JKN2405, citric acid, benzalkonium chloride, glycerin, and 65% ethanol solution in a certain proportion, and adjusting the pH to 5.0 with 0.1 mol / L sodium hydroxide solution.

[0033] The reference standard is JKN2405 (molecular formula C). 20 H 21 F2NO3S, structural formula as follows Figure 1 (As shown); Source: Shanghai Hequan Pharmaceutical Co., Ltd. (hereinafter referred to as Hequan Pharmaceutical) (Batch No. JR-C18082796-JFF24001). The key preparation process of JKN2405(QY101) is as follows: Figure 2 As shown.

[0034] Impurities: VIP3156 (molecular formula C20H21F2NO4S), source: Hequan Pharmaceutical Co., Ltd. (batch number PS07938-28-VIP-P1); VIP3157 (molecular formula C20H21F2NO4S), source: Hequan Pharmaceutical Co., Ltd. (batch number PS07938-28-VIP-P2); C18082796-JIP1 (molecular formula C20H21F2NO4S), source: Hequan Pharmaceutical (batch number PS08018-16-JIP1-P2). C18082796-JIP2 (molecular formula C16H15F2NO3), source: Hequan Pharmaceutical (batch number PS08319-14-JIP2-P2).

[0035] In the following examples, peak 1 in the chromatogram is VIP3156, peak 2 is C18082796-JIP2, peak 3 is VIP3157, peak 4 is C18082796-JIP1, and peak 5 is JKN2405.

[0036] Example 1 A method for detecting organic impurities in JKN2405, employing high-performance liquid chromatography (HPLC) to detect organic impurities in the JKN2405 test solution, is disclosed, and the specific operation is carried out according to the following conditions and steps; 1. High Performance Liquid Chromatography Conditions Instrument: Agilent 1260II, detector is an ultraviolet detector; Column: XBridge® Phenyl 4.6×150mm, 3.5μm; Impurity collection column: Ghost-Buster Column 4.6×50mm; Detection wavelength: 270nm; Column temperature: 30℃; Flow rate: 0.7 ml / min; Injection volume: 10 μl; Mobile phase A: 0.01 mol / L ammonium acetate solution; Mobile phase B: Acetonitrile; The elution procedure is shown in Table 1.

[0037] Table 1

[0038] 2. Testing steps: 1) Prepare reagents Preparation of diluent: Measure 800ml of acetonitrile and 1200ml of water and mix them evenly.

[0039] Preparation of the test solution: Accurately take 1 ml of sample (labeled amount of 40 mg / ml) and place it in a 50 ml volumetric flask. Add diluent to dissolve and dilute to the mark, shake well. Accurately measure 2 ml and place it in a 10 ml volumetric flask. Add diluent to the mark, shake well, and the solution is ready.

[0040] Preparation of reference solution: Accurately weigh an appropriate amount of JKN2405 reference standard, add diluent to dissolve and dilute, and prepare a reference solution containing approximately 2 μg JKN2405 per ml.

[0041] Preparation of system suitability solution: Accurately weigh appropriate amounts of impurities VIP3156, VIP3157, C18082796-JIP1, and C18082796-JIP2, respectively, and accurately add them to an appropriate amount of sample solution. Dissolve and dilute with diluent to prepare a solution containing 0.2 mg JKN2405, 1 μg impurity VIP3156, 3 μg impurity VIP3157, 2 μg impurity C18082796-JIP1, and 0.4 μg impurity C18082796-JIP2 per 1 ml.

[0042] 2) Detection Take 10 μL each of blank solution (i.e., diluent), system suitability solution, test solution, and reference solution, and inject them into the high performance liquid chromatograph. The signal is VWD1A. Perform the determination according to the chromatographic conditions described above and record the chromatogram.

[0043] The test results are as follows Figure 3 As shown, the separation of impurities VIP3156, C18082796-JIP2, VIP3157, and C18082796-JIP1 is good, and they do not interfere with the main peak (JKN2405). The positions of the main peak and the peak corresponding to the reference solution are basically the same.

[0044] Example 2 Specificity Verification This embodiment uses the chromatographic conditions of Example 1 for specificity testing. Specific experimental steps: 1. Prepare reagents Blank solution: i.e., diluent.

[0045] System suitability solution: Same as the system suitability solution in Example 1.

[0046] Reference solution: Same as the reference solution in Example 1.

[0047] Positioning solutions: Take appropriate amounts of JKN2405 reference standard, impurity VIP3156, impurity VIP3157, impurity C18082796-JIP1, and impurity C18082796-JIP2, and dissolve and dilute them respectively with diluent to obtain their respective positioning solutions.

[0048] 2. Verify specificity Accurately inject 10 μL each of blank solution, system suitability solution, reference solution, and each localization solution into the ion chromatograph, and perform the determination according to the chromatographic conditions described above, and record the chromatogram.

[0049] The test results are as follows Figure 4 As shown, A is the blank solution and B is the system suitability solution, indicating that the impurities VIP3156, C18082796-JIP2, VIP3157, and C18082796-JIP1 are effectively separated; and there are no other peaks interfering with each impurity peak, indicating that the method of this invention has good specificity.

[0050] Example 3 Linear Range Verification This embodiment uses the chromatographic conditions of Example 1 to perform a linearity range test. The linearity range test method in this embodiment follows the analytical method validation guidelines of Chinese Pharmacopoeia 9101.

[0051] The results showed that JKN2405 underwent linear regression with concentration as the x-axis and peak area as the y-axis within the concentration range of L-200% (0.1-4 ug / ml), and the linear correlation coefficient r was 0.9999. This indicates that the method of the present invention has a good linear relationship within the concentration range of L-200% (0.1-4 ug / ml) and is suitable for the quantitative detection of organic impurities in JKN2405.

[0052] Example 4 This embodiment uses the chromatographic conditions of Example 1 to determine the correction factor. Specific experimental steps: 1. Take appropriate amounts of JKN2405 reference standard, impurity VIP3156, impurity VIP3157, impurity C18082796-JIP1, and impurity C18082796-JIP2, dissolve them in diluent, and then dilute them stepwise to obtain their respective series of concentration solutions.

[0053] 2. Accurately inject 10 μL each of the blank solution and solutions of each concentration series into the ion chromatograph, and perform the determination according to the chromatographic conditions described above, recording the chromatograms. Plot a linear regression with concentration on the x-axis and peak area on the y-axis, using the ratio of the slope of each impurity to the slope of JKN2405 as the correction factor. The results are shown in Table 2, and the corresponding standard curve is shown below. Figure 5 As shown.

[0054] Table 2

[0055] Example 5 In this embodiment, the chromatographic conditions of Example 1 were used to conduct limit of detection (LOD) and limit of quantitation (LOQ) tests. The methods used for both LOD and LOQ tests in this embodiment followed the analytical method validation guidelines of Chinese Pharmacopoeia 9101. The results are shown in Table 3.

[0056] Table 3

[0057] The results show that the method of the present invention has high sensitivity and can effectively and quantitatively detect impurities VIP3156, VIP3157, C18082796-JIP1, and C18082796-JIP2, thereby ensuring drug safety.

[0058] Example 6 This embodiment uses the chromatographic conditions of Example 1 to conduct an accuracy test. Specific experimental steps: 1. Preparation of reference solution: Accurately weigh an appropriate amount of JKN2405 reference standard, add diluent to dissolve and dilute, and prepare a reference solution containing approximately 2 μg JKN2405 per ml.

[0059] 2. Preparation of test solution: Accurately take 1 ml of sample (labeled amount is 40 mg / ml) and place it in a 50 ml volumetric flask. Add diluent to dissolve and dilute to the mark, shake well. Accurately measure 2 ml and place it in a 10 ml volumetric flask. Add diluent to the mark, shake well, and the solution is ready.

[0060] 3. Preparation of spiked sample solutions: Prepare 6 spiked sample solutions according to the preparation method of the system suitability solution in Example 1.

[0061] 4. The reference solution was injected 6 times consecutively, and the test solution and 6 spiked sample solutions were each injected once. The content of impurities in the test solution and spiked sample solutions was calculated using the external standard method with correction factor. The results are shown in Table 4.

[0062] Table 4

[0063] The results showed that the recovery rate of each impurity was between 92.6% and 97.9% in the six determinations, and the accuracy RSD% was less than 2.0%, indicating that the detection method of the present invention has good accuracy.

[0064] Example 7 This embodiment uses the chromatographic conditions of Example 1 to conduct repeatability and intermediate precision tests.

[0065] For the same batch of samples, the accuracy was determined by another experimenter. The precision test method in this embodiment follows the analytical method validation guidelines of Chinese Pharmacopoeia 9101. The test results of the two experimenters are shown in Table 5.

[0066] Table 5

[0067] The results showed that the RSD of the impurity content in the six measurements taken by each of the two researchers was less than 2.0%, and the RSD% of the impurities in the twelve measurements taken by the two researchers was less than 2.0%, indicating that the detection method of the present invention has good precision.

[0068] Example 8 This embodiment conducts a comparative experiment on the selection of chromatographic columns. Except for the chromatographic column, other chromatographic conditions are the same as in Example 1. The specifications of the chromatographic column are shown in Table 6.

[0069] Table 6

[0070] Using XBridge ® Phenyl column chromatography results are as follows Figure 6 As shown, the results using the ZORBAX SB-CN column are as follows: Figure 7 As shown, peaks 6 and 7 are both unknown impurities. Comparison revealed that the XBridge column... ® Phenyl detects a greater number of impurities, therefore the XBridge column is preferred. ® Phenyl.

[0071] Example 9 This embodiment is a comparative experiment on the selection of mobile phase buffer salt system for the detection method.

[0072] JKN2405 has a conjugate acid dissociation constant pKa of 3.81, is a weakly basic compound, and the initial selection of the mobile phase buffer salt is pH < 1.8 or pH > 5.8; Refer to the general principles for selecting buffer salts: pH 2-3: Phosphate, formic acid / ammonium formate, trifluoroacetic acid (TFA).

[0073] pH 4-6: Acetic acid / ammonium acetate, citrate.

[0074] pH 7-8: Potassium dihydrogen phosphate-sodium hydroxide, Tris-HCl.

[0075] Therefore, the ammonium acetate system was chosen. Considering the structure of the JKN2405 compound and its impurities, a 0.01 mol / L ammonium acetate solution (pH 6-7) was preferred as it could effectively suppress -NH2 protonation and reduce tailing.

[0076] Example 10 This embodiment is a comparative experiment of the detection method before and after using the impurity trapping column.

[0077] Except for the impurity trapping column, the chromatographic conditions in this embodiment are the same as in Example 1.

[0078] The blank solution was tested before and after using a Ghost-Buster Column (4.6 × 50 mm) for impurity collection. The results are as follows: Figure 8 As shown, peaks 6 to 9 are all unknown impurities. A is before using the impurity trapping column, and B is after using the impurity trapping column. The results show that there is baseline interference when the trapping column is not used; after using the impurity trapping column, there is no baseline interference. Therefore, the impurity trapping column is selected.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting organic impurities in JKN2405, characterized in that, The detection method includes using high performance liquid chromatography to detect organic impurities in the JKN2405 test sample solution; The chromatographic column is a combination of a phenyl column and an impurity trapping column.

2. The detection method according to claim 1, characterized in that, Mobile phase A is phosphate buffer or ammonium acetate solution, and mobile phase B is acetonitrile.

3. The detection method according to claim 2, characterized in that, Gradient elution includes: 0~25min: 90%~70% mobile phase A, 10%~30% mobile phase B; 25~40min: 70%~45% mobile phase A, 30%~55% mobile phase B; 40~45min: 45%~30% mobile phase A, 55%~70% mobile phase B; 45~60min: 30% mobile phase A, 70% mobile phase B; 60~65min: 30%~90% mobile phase A, 70%~10% mobile phase B; 65~80 min: 90% mobile phase A, 10% mobile phase B.

4. The detection method according to claim 3, characterized in that, The mobile phase A is an ammonium acetate solution; Preferably, the concentration of the ammonium acetate solution is 0.01~0.05 mol / L, and more preferably 0.01 mol / L.

5. The detection method according to claim 1, characterized in that, The chromatographic column has a particle size of 3.5 μm; Preferably, the size of the impurity collecting column is 4.6 × 50 mm.

6. The detection method according to claim 3, characterized in that, The phenyl column is preferably XBridge® Phenyl.

7. The detection method according to claim 1, characterized in that, The detection wavelength is 270nm; Preferably, the column temperature is 30°C; Preferably, the flow rate is 0.7 ml / min; Preferably, the injection volume is 10 μl.

8. The detection method according to claim 1, characterized in that, The JKN2405 test solution was prepared by dissolving the JKN2405 sample in a diluent. Each 1 ml of JKN2405 test solution contains 2 mg of JKN2405 sample; Preferably, the diluent is a 60% v / v acetonitrile solution.

9. The detection method according to any one of claims 1 to 8, characterized in that, The impurity content was calculated using the principal component external standard method with a correction factor.

10. The detection method according to claim 9, characterized in that, The impurities include at least one of VIP3156, VIP3157, C18082796-JIP1 or C18082796-JIP2.

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