Method for detecting related impurities of acyl chloride compounds

Abstract

The invention provides a method for separating and detecting related impurities of acyl chloride compounds. The impurities comprise one or more of cyclopropanecarboxylic acid, gamma-butyrolactone, 2-butenyl chloride, 2-butenoic acid, butyryl chloride, butyric acid, cyclopropanecarboxylic acid methyl ester, 4-chlorobutyric acid methyl ester, 4-chlorobutyryl chloride and 4-chlorobutyric acid. According to the separation and detection method, substances obtained after hydrolysis treatment and esterification treatment of a cyclopropylformyl chloride substance to be detected are subjected to high performance liquid chromatography detection respectively, and effective separation and detection of 10 related impurities are achieved by combining formula calculation.

Description

Technical Field

[0001] The present application relates to the technical field of pharmaceutical analysis, and particularly to a method for detecting related impurities of acyl chloride compounds. Background Art

[0002] Acyl chloride compounds (such as cyclopropylcarbonyl chloride) are a very important class of compounds and are widely used as starting materials for drug substances in the pharmaceutical field. The synthetic by-products of cyclopropylcarbonyl chloride include structurally similar acyl chlorides, esters, and acids, specifically including 10 impurities such as cyclopropanecarboxylic acid, γ-butyrolactone, 2-butenoyl chloride, 2-butenoic acid, butyryl chloride, butyric acid, methyl cyclopropanecarboxylate, 4-chlorobutyryl chloride, methyl 4-chlorobutyrate, and 4-chlorobutyric acid. The impurity control of the starting material directly affects the product quality of the drug substance and further affects the quality of the pharmaceutical preparation. By quantitatively detecting the above 10 impurities in the starting material cyclopropylcarbonyl chloride, the safety and effectiveness of the corresponding drug substance and pharmaceutical preparation can be effectively controlled.

[0003] Due to the structural characteristics of acyl chloride compounds, they are unstable in water, acids, bases, or lower alcohols and are easily converted into substances such as hydrochloric acid, organic acids, and esters. For example, cyclopropylcarbonyl chloride, 2-butenoyl chloride, butyryl chloride, and 4-chlorobutyryl chloride are hydrolyzed to form cyclopropanecarboxylic acid, 2-butenoic acid, butyric acid, and 4-chlorobutyric acid, and are esterified with alcohols to form methyl cyclopropanecarboxylate, methyl 2-butenoate, methyl butyrate, and methyl 4-chlorobutyrate. Currently, the GC method is often used for direct injection to detect acyl chlorides and their impurities, but there is a problem that acyl chlorides react chemically with the packing material of the gas chromatography column, resulting in a sharp decrease in the column efficiency of the gas chromatography column. Moreover, there is no report in the prior art on the separation and detection method for 10 impurities such as cyclopropanecarboxylic acid, γ-butyrolactone, 2-butenoyl chloride, 2-butenoic acid, butyryl chloride, butyric acid, methyl cyclopropanecarboxylate, methyl 4-chlorobutyrate, 4-chlorobutyryl chloride, and 4-chlorobutyric acid in cyclopropylcarbonyl chloride compounds.

[0004] Therefore, it is of great practical significance to develop an analytical and detection method for cyclopropylcarbonyl chloride and its impurities with good separation effect, high sensitivity, reliable accuracy, and good reproducibility. Summary of the Invention

[0005] The present invention aims to provide a method for separating and detecting related impurities of acyl chloride compounds, which can quantitatively detect the following 10 different impurities in cyclopropylcarbonyl chloride compounds.

[0006]

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides a method for separating and detecting impurities related to acyl chloride compounds, and the impurities include one or more of cyclopropanecarboxylic acid, γ-butyrolactone, 2-butenoyl chloride, 2-butenoic acid, butyryl chloride, butyric acid, methyl cyclopropanecarboxylate, methyl 4-chlorobutyrate, 4-chlorobutyryl chloride, and 4-chlorobutyric acid.

[0009] In some embodiments of the present invention, the acyl chloride compound is a cycloalkyl acyl chloride or an alkyl acyl chloride; preferably, the acyl chloride compound is a C 3-8 cycloalkyl acyl chloride; more preferably, the acyl chloride compound is cyclopropylcarbonyl chloride.

[0010] In some embodiments of the present invention, the separation and detection method includes the following steps:

[0011] Step a: After the test substance is hydrolyzed, a test solution A is prepared, and the test solution A is detected by high performance liquid chromatography, and the content of one or more impurities in the test substance, such as methyl cyclopropanecarboxylate, methyl 4-chlorobutyrate, γ-butyrolactone, 2-butenoic acid, butyric acid, and 4-chlorobutyric acid, is calculated.

[0012] Step b: After the test substance is esterified, a test solution B is prepared, and the test solution B is detected by high performance liquid chromatography, and the content of one or more impurities in the test substance, such as cyclopropanecarboxylic acid, 2-butenoic acid, butyric acid, and 4-chlorobutyric acid, is calculated.

[0013] Step c: According to the content of 2-butenoic acid, butyric acid, and / or 4-chlorobutyric acid obtained in step a and step b, the content of one or more impurities in the test substance, such as 2-butenoyl chloride, butyryl chloride, and 4-chlorobutyryl chloride, is calculated through a formula.

[0014] In some embodiments of the present invention, step c is to calculate the content of one or more impurities in the test substance, such as 2-butenoyl chloride, butyryl chloride, and 4-chlorobutyryl chloride, through the following formula:

[0015] C （2-丁烯酰氯） =[ C 总（2-丁烯酸） - C 0（2-丁烯酸） ×104.53 / 986.09, formula 1;

[0016] C （丁酰氯） =[ C 总（丁酸） - C 0（丁酸） ×106.55 / 88.11, formula 2;

[0017] C （4-氯丁酰氯） =[ C 总（4-氯丁酸） - C 0（4-氯丁酸） ×141.00 / 122.55, formula 3;

[0018] Among them, C 总（2-丁烯酸） , C 总（丁酸） , C 总（4-氯丁酸） are respectively the impurity contents of crotonic acid, butyric acid and 4-chlorobutyric acid measured in step a, and C 0（2-丁烯酸） , C 0（丁酸） , C 0（4-氯丁酸） are respectively the impurity contents of crotonic acid, butyric acid and 4-chlorobutyric acid measured in step b.

[0019] In some embodiments of the present invention, the hydrolysis treatment in step a includes: dissolving the cyclopropylcarbonyl chloride analyte in an organic solvent, adding water for hydrolysis reaction and making up the volume.

[0020] In some embodiments of the present invention, the mass-volume ratio of the analyte to the organic solvent in the hydrolysis treatment of step a, in mg / mL, is 100:(0.5 - 2), preferably 100:(0.8 - 1.5), for example 100:0.8, 100:0.9, 100:1, 100:1.1, 100:1.2, 100:1.3, 100:1.4, 100:1.5.

[0021] In some embodiments of the present invention, the organic solvent in the hydrolysis treatment of step a is selected from one of acetonitrile, acetone, dichloromethane, chloroform, and preferably acetonitrile.

[0022] In some embodiments of the present invention, the concentration of the analyte in the test solution A after adding water and making up the volume in the hydrolysis treatment of step a is 6 mg / mL - 15 mg / mL, preferably 8 mg / mL - 12 mg / mL, for example 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL.

[0023] In some embodiments of the present invention, the test solution A in step a is freshly prepared for immediate use.

[0024] In some embodiments of the present invention, the esterification treatment in step b includes: adding an alcohol esterification solvent to the cyclopropylcarbonyl chloride analyte for esterification reaction, standing until the reaction is complete, and diluting and making up the volume with a volume-making solvent.

[0025] In some embodiments of the present invention, the alcohol esterification solvent in the esterification treatment of step b is selected from one of methanol, ethanol, and propanol, and preferably methanol.

[0026] In some embodiments of the present invention, the volume-making solvent in the esterification treatment of step b is water.

[0027] In some embodiments of the present invention, in step b of the esterification treatment, the standing time until the reaction is completed is 30 minutes or more, preferably 30 minutes to 90 minutes, more preferably 40 minutes to 80 minutes, such as 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes.

[0028] In some embodiments of the present invention, in step b of the esterification treatment, the mass-to-volume ratio of the analyte to the alcohol esterification solvent, in mg / mL, is 100:(0.5 - 5), preferably 100:(1 - 4), such as 100:1, 100:1.5, 100:2, 100:2.5, 100:3, 100:3.5, 100:4.

[0029] In some embodiments of the present invention, in step b of the esterification treatment, after volume fixation with the volume-fixing solvent, the concentration of the analyte in the test sample solution B is 6 mg / mL - 15 mg / mL, preferably 8 mg / mL - 12 mg / mL, such as 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL.

[0030] In some embodiments of the present invention, the chromatographic column for the high-performance liquid chromatography is a C18 column, preferably a C18 hydrophilic column.

[0031] In some embodiments of the present invention, the column temperature for the high-performance liquid chromatography is 15°C to 25°C, preferably 18 - 23°C, such as 18°C, 19°C, 20°C, 21°C, 22°C, 23°C.

[0032] In some embodiments of the present invention, the flow rate for the high-performance liquid chromatography is 0.8 mL / min to 1.2 mL / min, preferably 0.9 mL / min - 1.1 mL / min, such as 0.9 mL / min, 1.0 mL / min, 1.1 mL / min.

[0033] In some embodiments of the present invention, the elution mode of the high-performance liquid chromatography is gradient elution. Mobile phase A is independently selected from phosphate buffer solutions, preferably potassium dihydrogen phosphate buffer solution, and mobile phase B is independently selected from one of acetonitrile, acetone, dichloromethane, and chloroform, preferably acetonitrile.

[0034] In some embodiments of the present invention, the method of gradient elution is as follows:

[0035] From 0 to 4 minutes, maintain the volume percentage of mobile phase A at 100% and the volume percentage of mobile phase B at 0%;

[0036] From 4 to 7 min, the volume percentage of mobile phase A decreases from 100% to 95%, and the volume percentage of mobile phase B increases from 0 to 5%.

[0037] From 7 to 10 min, maintain the volume percentage of mobile phase A at 95% and the volume percentage of mobile phase B at 5%.

[0038] From 10 to 45 min, the volume percentage of mobile phase A decreases from 95% to 45%, and the volume percentage of mobile phase B increases from 5% to 55%.

[0039] From 45 to 50 min, maintain the volume percentage of mobile phase A at 45% and the volume percentage of mobile phase B at 55%.

[0040] From 50 to 51 min, the volume percentage of mobile phase A increases from 45% to 100%, and the volume percentage of mobile phase B decreases from 55% to 0.

[0041] From 51 to 60 min, maintain the volume percentage of mobile phase A at 100% and the volume percentage of mobile phase B at 0.

[0042] In some embodiments of the present invention, the pH value of mobile phase A is 2.5 - 3.5, preferably 2.8 - 3.2, such as 2.8, 2.9, 3.0, 3.1, 3.2.

[0043] Compared with the prior art, the present application has at least the following beneficial effects:

[0044] The method for separating and detecting related impurities of acyl chloride compounds provided by the present application can effectively separate and detect one or more of 10 related impurities including methyl cyclopropanecarboxylate, butyric acid, 2-butenoic acid, methyl 4-chlorobutyrate, γ-butyrolactone, 4-chlorobutyric acid, cyclopropanecarboxylic acid, 2-butenoyl chloride, butyryl chloride, and 4-chlorobutyryl chloride in cyclopropylcarbonyl chloride. This separation and detection method has the advantages of good separation effect, high sensitivity, reliable accuracy, and good reproducibility. Description of the Drawings

[0045] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0046] Figure 1 Chromatogram of the specific impurity mixed reference solution in Example 2;

[0047] Figure 2Chromatogram of Example 5 with column temperature at 20 °C;

[0048] Figure 3 Chromatogram of Comparative Example 2 with methanol as the constant volume solvent for the test solution B;

[0049] Figure 4 Chromatogram of Comparative Example 3 with column temperature at 30 °C;

[0050] Figure 5 Chromatogram of Comparative Example 4 under the isocratic elution program in Table 5;

[0051] Figure 6 Chromatogram of Comparative Example 4 under the gradient elution program in Table 6;

[0052] Explanation of the reference numerals in the attached drawings:

[0053] In the above drawings, impurity A is cyclopropanecarboxylic acid, impurity B is γ-butyrolactone, impurity D is 2-butenoic acid, impurity F is butyric acid, impurity H is 4-chlorobutyric acid, impurity I is methyl cyclopropanecarboxylate, and impurity J is methyl 4-chlorobutyrate. Detailed implementation manners

[0054] To facilitate the understanding of this application, the following will provide a more comprehensive description of this application with reference to the relevant drawings. The preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of this application more thorough and comprehensive.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the description of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0056] In this application, "the first aspect", "the second aspect", "the third aspect", etc. are only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features.

[0057] In this application, among the technical features described in an open-ended manner, there are included closed technical solutions composed of the listed features, as well as open technical solutions including the listed features.

[0058] In this application, "one or more" means any one, any two, or any two or more of the listed items.

[0059] In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are considered continuous and include the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when the range refers to integers, it includes each integer between the minimum and maximum values of the range. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0060] In this application, the percentage concentration involved, unless otherwise specified, refers to the final concentration. The final concentration refers to the proportion of the added component in the system after adding the component.

[0061] In this application, for the temperature parameters, unless otherwise specified, both constant temperature treatment and treatment within a certain temperature range are allowed. The constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument.

[0062] In this application, the method steps that do not emphasize temperature usually refer to the method steps carried out under normal temperature or room temperature conditions. In this article, normal temperature and room temperature are interchangeable, and the specific temperature refers to 22°C to 25°C.

[0063] If there is no special instruction, all embodiments and optional embodiments of this application can be combined with each other to form a new technical solution.

[0064] If there is no special instruction, all technical features and optional technical features of this application can be combined with each other to form a new technical solution.

[0065] If there is no special instruction, all steps of this application can be carried out sequentially or randomly. For example, the method includes steps (a) and (b), which means that the method can include steps (a) and (b) carried out sequentially, or can also include steps (b) and (a) carried out sequentially. For example, it is mentioned that the method may further include step (c), which means that step (c) can be added to the method in any order. For example, the method can include steps (a), (b) and (c), or can also include steps (a), (c) and (b), or can also include steps (c), (a) and (b), etc.

[0066] If there is no special instruction, the "including" and "comprising" mentioned in this application mean open-ended, and can also be closed-ended. For example, the "including" and "comprising" can mean that other components not listed can also be included or comprised, or can only include or comprise the listed components.

[0067] Unless otherwise specified, the term "or" is inclusive in this application. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, any of the following conditions satisfies the condition "A or B": A is true (or present) and B is false (or absent); A is false (or absent) while B is true (or present); or both A and B are true (or present).

[0068] The following are specific embodiments. The purpose is to further elaborate on this application to help those skilled in the art and researchers better understand this application. The relevant technical conditions etc. do not constitute any limitation to this application. Any form of modification within the scope of the claims of this application is within the protection scope of the claims of this application.

[0069] Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods. For the experimental methods where specific conditions are not indicated in the examples, they are carried out under conventional conditions, such as the conditions described in literature, books, or the methods recommended by the manufacturers.

[0070] Example 1

[0071] Method for detecting related impurities in cyclopropylcarbonyl chloride by high performance liquid chromatography.

[0072] (1) Preparation of solutions

[0073] Stock solution of reference substances: Weigh accurately an appropriate amount of the impurity reference substance cyclopropanecarboxylic acid, dissolve it with acetonitrile and quantitatively dilute to prepare a solution containing about 4 mg per 1 mL as the stock solution of impurity reference substance (1). Weigh accurately appropriate amounts of the impurity reference substances γ-butyrolactone, 2-butenoic acid, butyric acid, 4-chlorobutyric acid, methyl cyclopropanecarboxylate, and methyl 4-chlorobutyrate respectively, dissolve them with acetonitrile and quantitatively dilute to prepare a solution containing about 0.3 mg per 1 mL, and name them as the stock solutions of impurity reference substances (2), (3), (4), (5), (6), and (7) respectively. The corresponding numbers of the stock solutions of reference substances are shown in Table 1.

[0074] Table 1 Numbers of stock solutions of reference substances

[0075]

[0076] Reference substance solution: Accurately measure appropriate amounts of the stock solutions of impurity reference substances (1), (2), (3), (4), (5), (6), and (7), and quantitatively dilute with water to prepare a mixed solution containing about 0.2 mg of cyclopropanecarboxylic acid and 15 μg of each of γ-butyrolactone, 2-butenoic acid, butyric acid, 4-chlorobutyric acid, methyl cyclopropanecarboxylate, and methyl 4-chlorobutyrate per 1 mL.

[0077] Blank solution A: Take an appropriate amount of hydrochloric acid, dissolve and dilute it with a 10% acetonitrile aqueous solution to prepare a 0.01% hydrochloric acid solution.

[0078] Test solution A (hydrolysis treatment): Take an appropriate amount of the test substance of cyclopropylcarbonyl chloride, dissolve it in acetonitrile, and immediately dilute it quantitatively with water to prepare a solution containing about 10 mg per 1 mL. Prepare fresh before use.

[0079] System suitability solution A: Take an appropriate amount of the test substance of cyclopropylcarbonyl chloride, add appropriate amounts of impurity reference stock solutions (2), (3), (4), (5), (6), and (7), and dilute quantitatively with water to prepare a mixed solution containing about 10 mg of cyclopropylcarbonyl chloride and 15 μg of γ-butyrolactone, 2-butenoic acid, butyric acid, 4-chlorobutyric acid, methyl cyclopropanecarboxylate, and methyl 4-chlorobutyrate per 1 mL. Prepare fresh before use.

[0080] Blank solution B: 30% aqueous methanol solution.

[0081] Test solution B (esterification treatment): Take 100 mg of the test substance of cyclopropylcarbonyl chloride, dissolve it in 3 mL of anhydrous methanol, shake well, let stand for 60 min, and dilute quantitatively with water to prepare a solution containing about 10 mg per 1 mL. Prepare fresh before use.

[0082] System suitability solution B: Take 100 mg of the test substance of cyclopropylcarbonyl chloride, dissolve it in 3 mL of anhydrous methanol, shake well, let stand for 60 min, then add appropriate amounts of impurity reference stock solutions (1), (3), (4), and (5), and dilute quantitatively with water to prepare a mixed solution containing about 10 mg of cyclopropylcarbonyl chloride, 0.2 mg of cyclopropanecarboxylic acid, and 15 μg of butyric acid, 2-butenoic acid, and 4-chlorobutyric acid per 1 mL. Prepare fresh before use.

[0083] (2) Chromatographic conditions

[0084] Chromatographic column: Boston Μni C18-AQ, 4.6 mm × 250 mm, 3 µm

[0085] Column temperature: 20 °C

[0086] Flow rate: 1.0 mL / min

[0087] Wavelength: 200 nm

[0088] Injection volume: 100 μL

[0089] Mobile phase A: 0.02 moL / L potassium dihydrogen phosphate buffer solution (adjust the pH value to 3.0 with phosphoric acid)

[0090] Mobile phase B: Acetonitrile

[0091] Gradient elution program is shown in Table 2.

[0092] Table 2 Gradient elution program

[0093]

[0094] (3)Sample treatment, detection and analysis

[0095] Step a. Hydrolyze the analyte to be tested and inject the sample to detect the content of one or more impurities in γ-butyrolactone, crotonic acid, butyric acid, 4-chlorobutyric acid, methyl cyclopropanecarboxylate, and methyl 4-chlorobutyrate. The detected contents of crotonic acid, butyric acid, and 4-chlorobutyric acid include the impurity content of the analyte itself and the content of the converted acids formed by hydrolysis of acyl chloride impurities such as crotonyl chloride, butyryl chloride, and 4-chlorobutyryl chloride in the analyte, denoted as C 总（2-丁烯酸） , C 总（丁酸） and C 总（4-氯丁酸） . The operation is as follows:

[0096] Inject the prepared blank solution A, system suitability solution A, reference solution, and test solution A for detection respectively. The chromatogram of system suitability solution A shows that γ-butyrolactone, cyclopropanecarboxylic acid, crotonic acid, butyric acid, 4-chlorobutyric acid, methyl cyclopropanecarboxylate, and methyl 4-chlorobutyrate can be effectively separated and detected.

[0097] Step b. Esterify the analyte to be tested and inject the sample to detect the content of one or more impurities in cyclopropanecarboxylic acid, crotonic acid, butyric acid, and 4-chlorobutyric acid. The detected contents of crotonic acid, butyric acid, and 4-chlorobutyric acid are the impurity contents of the analyte itself, denoted as C 0（2-丁烯酸） , C 0（丁酸） and C 0（4-氯丁酸） . The operation is as follows:

[0098] Inject the prepared blank solution B, system suitability solution B, and test solution B for detection respectively. The chromatogram of system suitability solution B shows that cyclopropanecarboxylic acid, crotonic acid, butyric acid, and 4-chlorobutyric acid can be effectively separated and detected.

[0099] Step c. Calculation formula

[0100] The external standard method is used to calculate the content of each above-mentioned impurity by peak area. The calculation formula is:

[0101]

[0102] Where: At represents the peak area of the related substance in the chromatogram of the test solution;

[0103] Vt represents the dilution factor of the test solution, mL;

[0104] As represents the peak area of the related substance in the chromatogram of the reference solution;

[0105] Vs represents the dilution factor of the reference solution, mL;

[0106] Ws represents the weighed amount of the related substance reference substance, in mg;

[0107] 1000 represents the mass conversion factor, 1000 μg / mg.

[0108] P represents the purity of the related substance reference substance.

[0109] 1) In step a, according to the external standard method, the content of one or more impurities such as methyl cyclopropanecarboxylate, methyl 4-chlorobutyrate, γ-butyrolactone, crotonic acid, butyric acid, and 4-chlorobutyric acid in the analyte cyclopropylcarbonyl chloride is calculated based on the peak area, where crotonic acid, butyric acid, and 4-chlorobutyric acid are respectively denoted as C 总（2-丁烯酸） , C 总（丁酸） and C 总（4-氯丁酸） ;

[0110] 2) In step b, according to the external standard method, the content of one or more impurities such as cyclopropanecarboxylic acid, 2-butenoic acid, butyric acid, and 4-chlorobutyric acid in the analyte cyclopropylcarbonyl chloride is calculated based on the peak area, where 2-butenoic acid, butyric acid, and 4-chlorobutyric acid are respectively denoted as C 0（2-丁烯酸） , C 0（丁酸） and C 0（4-氯丁酸） ;

[0111] 3) The content of one or more impurities such as 2-butenoyl chloride, butyryl chloride, and 4-chlorobutyryl chloride in the analyte cyclopropylcarbonyl chloride is calculated by the following formula:

[0112] C （2-丁烯酰氯） = [C 总（2-丁烯酸） - C 0（2-丁烯酸） × 104.53 / 986.09, formula 1;

[0113] C （丁酰氯） = [C 总（丁酸） - C 0（丁酸） × 106.55 / 88.11, formula 2;

[0114] C （4-氯丁酰氯） = [C 总（4-氯丁酸） - C 0（4-氯丁酸） × 141.00 / 122.55, formula 3.

[0115] (4) Result analysis

[0116] The blank baseline is stable and does not interfere with the detection of related substances; the chromatograms of the system suitability solutions A and B show that all components can be effectively separated and the peak shapes are symmetrical, indicating that the system suitability of the detection method in this example is good and meets the detection requirements.

[0117] Example 2 Specificity

[0118] (1) After the esterification treatment of the analyte in step b, the impurities 2-butenoyl chloride and butyryl chloride will be converted into methyl 2-butenoate and methyl butyrate. The preparation method of the specific mixed reference solution is as follows: Accurately measure appropriate amounts of the impurity reference stock solutions (1), (2), (3), (4), (5), (6), and (7), and appropriate amounts of the impurity references methyl 2-butenoate and methyl butyrate. Quantitatively dilute with water to prepare a mixed solution containing approximately 0.2 mg of cyclopropanecarboxylic acid and 15 μg of each of γ-butyrolactone, 2-butenoic acid, butyric acid, 4-chlorobutyric acid, methyl cyclopropanecarboxylate, methyl 4-chlorobutyrate, methyl 2-butenoate, and methyl butyrate per 1 mL.

[0119] (2) Inject the mixed reference solution of this example for detection, and the chromatographic conditions are the same as those in Example 1.

[0120] (3) The chromatogram of the mixed reference solution is as Figure 1 shown. It can be seen from Figure 1 that the impurities cyclopropanecarboxylic acid, γ-butyrolactone, 2-butenoic acid, butyric acid, 4-chlorobutyric acid, methyl cyclopropanecarboxylate, methyl 4-chlorobutyrate, methyl 2-butenoate, and methyl butyrate can all be effectively separated, and the peak shapes are symmetrical, indicating that the detection method of this example has good specificity.

[0121] Example 3 Limit of Quantitation and Limit of Detection

[0122] Take the impurity reference stock solutions (1) (2) (3) (4) (5) (6) (7), and dilute them step by step with water. The signal-to-noise ratio of about 3:1 is used as the limit of detection, and the signal-to-noise ratio of about 10:1 is used as the limit of quantitation. The inspection results are shown in Table 3 below. The detection method of this example has a stable baseline for consecutive injections, the sensitivity meets the detection requirements, and the precision is good, enabling accurate detection of a large number of samples.

[0123] Table 3 Limit of Quantitation and Limit of Detection

[0124]

[0125] Example 4 Accuracy and Repeatability

[0126] Prepare spiked recovery solutions of 50%, 100%, and 400% respectively.

[0127] Recovery sample solution 1: Take an appropriate amount of the analyte cyclopropylcarbonyl chloride, and respectively add the impurity reference stock solutions (1), (2), (3), (4), (5), (6), and (7). Dilute with water to prepare a solution containing 10 mg of cyclopropylcarbonyl chloride and 7.5 μg of each impurity per 1 mL. Prepare 3 parallel samples as the 50% recovery sample solution;

[0128] Recovery sample solution 2: Take an appropriate amount of the test substance of ciprofloxacin acyl chloride, and add the impurity reference stock solutions (1), (2), (3), (4), (5), (6), and (7) respectively. Dilute with water to prepare a solution containing 10 mg of ciprofloxacin acyl chloride and 15 μg of each impurity per 1 mL. Prepare 3 portions in parallel as the 100% recovery sample solution;

[0129] Recovery sample solution 3: Take an appropriate amount of the test substance of ciprofloxacin acyl chloride, and add the impurity reference stock solutions (1), (2), (3), (4), (5), (6), and (7) respectively. Dilute with water to prepare a solution containing 10 mg of ciprofloxacin acyl chloride and 60 μg of each impurity per 1 mL. Prepare 3 portions in parallel as the 400% recovery sample solution.

[0130] Inject the above solutions for analysis and calculate the recovery rate; according to the above 100% spiked recovery solution, prepare 6 portions in parallel, inject for analysis, and investigate the repeatability.

[0131] Recovery calculation formula:

[0132]

[0133] The results of the accuracy and repeatability investigations are shown in Table 4 below.

[0134] Table 4 Accuracy and repeatability investigations

[0135]

[0136] As can be seen from Table 4 above, the detection accuracy and repeatability of the detection method in this example for each impurity are both good, meeting the detection requirements.

[0137] Example 5 Durability investigation

[0138] (1) Solution preparation: Inject the system suitability solution A in Example 1 for detection.

[0139] (2) Chromatographic conditions:

[0140]

[0141] For conditions 1 - 3, the other chromatographic conditions are the same as those in Example 1 except for the column temperature; for conditions 4 - 6, the other chromatographic conditions are the same as those in Example 1 except for the flow rate.

[0142] (3) Result analysis

[0143] Under the conditions of different column temperatures of 18°C, 20°C, and 22°C in this example for conditions 1 - 3, each component in the system suitability solution A can be effectively separated and detected. The chromatogram at a column temperature of 20°C is as Figure 2 shown.

[0144] Under the conditions of 4 - 6 in this example, with different flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min, each component in the system suitability solution A can achieve effective separation and detection.

[0145] Comparative Example 1

[0146] Investigate the influence of the esterification solvent on the impurity detection when preparing the test solution B.

[0147] (1) Preparation of test solution B:

[0148] Sample 1: Take an appropriate amount of the test substance cyclopropylcarbonyl chloride, dissolve it in absolute ethanol, shake well, let it stand for 60 min, and dilute it with water to make a solution containing about 10 mg per 1 mL, freshly prepared before use.

[0149] Sample 2: Take an appropriate amount of the test substance cyclopropylcarbonyl chloride, dissolve it in absolute methanol, shake well, let it stand for 60 min, and quantitatively dilute it with water to make a solution containing about 10 mg per 1 mL, freshly prepared before use.

[0150] (2) Inject Sample 1 and Sample 2 of this example for detection respectively, and the chromatographic conditions are the same as in Example 1.

[0151] (3) Result analysis

[0152] If the esterification of the test substance cyclopropylcarbonyl chloride is incomplete, the residual cyclopropylcarbonyl chloride will hydrolyze to form cyclopropanecarboxylic acid when diluted with water. Therefore, it is confirmed whether the test substance is completely esterified by detecting the peak area of cyclopropanecarboxylic acid in Sample 1 and Sample 2. The detection and comparison results of the peak area of cyclopropanecarboxylic acid in Sample 1 and Sample 2 are shown in the following table.

[0153]

[0154] As can be seen from the above table, the peak area of cyclopropanecarboxylic acid detected in Sample 1 is much higher than that in Sample 2, which proves that when the esterification solvent is ethanol, the reaction with cyclopropylcarbonyl chloride is incomplete, and a large amount of residual cyclopropylcarbonyl chloride reacts with water to form cyclopropanecarboxylic acid; when the esterification solvent is methanol, the reaction is complete, and the measured result is the peak area of cyclopropanecarboxylic acid impurity in the test substance.

[0155] Comparative Example 2

[0156] Investigate the influence of the volume - fixing solvent on the impurity detection when preparing the test solution B (volume - fixed with methanol)

[0157] (1) Preparation of test solution B: Take an appropriate amount of the test substance cyclopropylcarbonyl chloride, dissolve it in absolute methanol and fix the volume to make a solution containing about 10 mg per 1 mL, shake well, let it stand for 60 min, and inject immediately.

[0158] (2) Inject the test solution B of this example for detection, and the chromatographic conditions are the same as in Example 1.

[0159] (3)Result analysis

[0160] The chromatogram of the test solution B in this example is as Figure 3 shown. The peak shape of ciprofloxacin carboxylic acid is severely fronting, and the integration is inaccurate.

[0161] Comparative Example 3

[0162] Investigate the influence of column temperature on the detection method

[0163] (1)Solution preparation: Inject the system suitability solution A in Example 1 for detection.

[0164] (2)Chromatographic conditions: Set the column temperature at 30 °C, and the other chromatographic conditions are the same as those in Example 1.

[0165] (3)Result analysis

[0166] Under the condition of a column temperature of 30 °C in this example, the chromatogram of the system suitability solution A is as Figure 4 shown. 4-Chlorobutyric acid completely coincides with the previous unknown impurity peak and cannot be effectively separated and detected.

[0167] Comparative Example 4

[0168] Investigate the influence of the elution program on the detection method

[0169] Set the isocratic elution program and gradient elution program of the mobile phase respectively, as shown in Table 5 - Table 6 below, and the other chromatographic conditions are the same as those in Example 1.

[0170] Inject the system suitability solution A in Example 1 for detection. The chromatogram of the isocratic elution program in Table 5 is as Figure 5 shown, and the chromatogram of the gradient elution program in Table 6 is as Figure 6 shown. It can be seen from Figure 5 that only two relatively small peaks are detected within 60 min, which does not meet the detection requirements. It can be seen from Figure 6 that the impurity 4-chlorobutyric acid coincides with the previous unknown impurity peak and fails to achieve effective separation and detection.

[0171] Table 5 Isocratic elution program

[0172]

[0173] Table 6 Gradient elution program

[0174]

[0175] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0176] The above-described embodiments only express several implementation manners of the present application, which are convenient for understanding the technical solution of the present application specifically and in detail, but should not be construed as a limitation on the protection scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can be made, and these all belong to the protection scope 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 by the present application are all within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the content of the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A method for separating and detecting related impurities of acyl chloride compounds, characterized in that, the impurities include one or more of cyclopropanecarboxylic acid, γ-butyrolactone, 2-butenoyl chloride, 2-butenoic acid, butyryl chloride, butyric acid, methyl cyclopropanecarboxylate, methyl 4-chlorobutyrate, 4-chlorobutyryl chloride and 4-chlorobutyric acid; the separation and detection method includes the following steps: Step a: After the test substance is hydrolyzed, a test solution A is prepared, and the test solution A is detected by high performance liquid chromatography, and the content of one or more impurities in the test substance, such as methyl cyclopropanecarboxylate, methyl 4-chlorobutyrate, γ-butyrolactone, 2-butenoic acid, butyric acid and 4-chlorobutyric acid, is calculated; Step b: After the test substance is esterified, a test solution B is prepared, and the test solution B is detected by high performance liquid chromatography, and the content of one or more impurities in the test substance, such as cyclopropanecarboxylic acid, 2-butenoic acid, butyric acid and 4-chlorobutyric acid, is calculated; Step c: According to the content of 2-butenoic acid, butyric acid and / or 4-chlorobutyric acid obtained in Step a and Step b, the content of one or more impurities in the test substance, such as 2-butenoyl chloride, butyryl chloride and 4-chlorobutyryl chloride, is calculated by a formula.

2. The separation and detection method according to claim 1, characterized in that, The acyl chloride compound is cycloalkyl acyl chloride or alkyl acyl chloride; preferably, the acyl chloride compound is C 3-8 cycloalkyl acyl chloride; more preferably, the acyl chloride compound is cyclopropylcarbonyl chloride.

3. The separation and detection method according to claim 1 or 2, wherein Step c is to calculate the content of one or more impurities in the test substance, such as 2-butenoyl chloride, butyryl chloride and 4-chlorobutyryl chloride, by the following formula: C （2-丁烯酰氯） = [C 总（2-丁烯酸） - C 0（2-丁烯酸） × 104.53 / 986.09, Equation 1; C （丁酰氯） = [ C 总（丁酸） - C 0（丁酸） × 106.55 / 88.11, Equation 2; C （4-氯丁酰氯） = [C 总（4-氯丁酸） - C 0（4-氯丁酸） × 141.00 / 122.55, Equation 3; wherein, C 总（2-丁烯酸） and C 总（丁酸） and C 总（4-氯丁酸） are the impurity contents of crotonic acid, butyric acid and 4-chlorobutyric acid measured in step a, respectively, C 0（2-丁烯酸） and C 0（丁酸） and C 0（4-氯丁酸） are the impurity contents of crotonic acid, butyric acid and 4-chlorobutyric acid measured in step b, respectively.

4. The separation and detection method according to any one of claims 1-3, characterized in that, the hydrolysis treatment in Step a includes: dissolving the cyclopropylcarbonyl chloride test substance in an organic solvent, adding water for hydrolysis reaction and making up the volume.

5. The separation and detection method according to any one of claims 1-4, characterized in that, the esterification treatment in Step b includes: adding an alcohol esterification solvent to the cyclopropylcarbonyl chloride test substance for esterification reaction, standing until the reaction is complete, and adding a volume-making solvent for dilution and volume-making.

6. The separation and detection method according to claim 5, characterized in that, the alcohol esterification solvent in Step b is selected from one of methanol, ethanol and propanol, preferably methanol.

7. The separation and detection method according to claim 5, characterized in that, the volume-making solvent in Step b is water.

8. The separation and detection method according to claim 5, characterized in that, the mass-volume ratio of the test substance to methanol, in mg / mL, is 100:(0.5-5), preferably 100:(1-4).

9. The separation and detection method according to any one of claims 1-8, characterized in that, the elution mode of the high performance liquid chromatography used in Step a and Step b is gradient elution, the mobile phase A of the high performance liquid chromatography used in Step a and Step b is independently selected from phosphate buffer solutions, preferably potassium dihydrogen phosphate buffer solution, and the mobile phase B is independently selected from one of acetonitrile, acetone, dichloromethane and chloroform, preferably acetonitrile.

10. The separation and detection method according to any one of claims 1-9, characterized in that, The method of gradient elution is as follows: From 0 to 4 min, maintain the volume percentage of mobile phase A at 100% and the volume percentage of mobile phase B at 0; From 4 to 7 min, the volume percentage of mobile phase A decreases from 100% to 95%, and the volume percentage of mobile phase B increases from 0 to 5%; From 7 to 10 min, maintain the volume percentage of mobile phase A at 95% and the volume percentage of mobile phase B at 5%; From 10 to 45 min, the volume percentage of mobile phase A decreases from 95% to 45%, and the volume percentage of mobile phase B increases from 5% to 55%; From 45 to 50 min, maintain the volume percentage of mobile phase A at 45% and the volume percentage of mobile phase B at 55%; From 50 to 51 min, the volume percentage of mobile phase A increases from 45% to 100%, and the volume percentage of mobile phase B decreases from 55% to 0; From 51 to 60 min, maintain the volume percentage of mobile phase A at 100% and the volume percentage of mobile phase B at 0.

Images