Method for determining and analyzing content of beta-diketone ester compound

Abstract

The invention provides an analysis method of beta-diketone ester compounds in a sample to be detected and application of the beta-diketone ester compounds in product quality control. According to the analysis method disclosed by the invention, the ketone solvent is used as a diluent, the content of the beta-diketone ester compound is measured by directly using a gas chromatograph equipped with a hydrogen flame ionization detector, and the analysis method has the characteristics of simplicity in operation, high sensitivity, high precision, high accuracy, suitability for measuring the content of the trace beta-diketone ester compound and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and particularly relates to an analytical method for determining the content of beta-diketone ester compounds in a sample to be tested and application of the method in product quality control. Technical Background

[0002] The general structural formula of β-diketone ester compounds is as follows:

[0003]

[0004] For example, methyl acetoacetate is primarily used as an intermediate in the pharmaceutical industry, insecticides, and herbicides, and is widely used in pesticides, pharmaceuticals, and polymer stabilizers. Methyl acetoacetate is the starting material for benidipine hydrochloride and nicardipine hydrochloride, and is a known impurity in the starting material for lacidipine. If the starting material methyl acetoacetate contains ethyl acetoacetate as an impurity, impurities containing ethyl acetoacetate structures will be introduced during the synthesis of the API containing a methyl ester structure. Accurate quantitative detection of the content of β-diketone ester compounds, especially methyl acetoacetate and its impurity ethyl acetoacetate, is of great significance for the quality control of benidipine hydrochloride, nicardipine hydrochloride, and lacidipine.

[0005] The structure of methyl acetoacetate is as follows:

[0006]

[0007] The structure of ethyl acetoacetate is as follows:

[0008]

[0009] β-diketone compounds exhibit keto-enol tautomerism, potentially converting to the enol form during measurement, making accurate quantification difficult. Determination of the ethyl acetoacetate impurity in methyl acetoacetate is particularly challenging due to their structural similarity, resulting in difficult separation and overlapping peaks.

[0010] Existing methods use high-performance liquid chromatography to detect β-diketone compounds, but interconversion causes severe tailing of the target chromatographic peak and poor peak shape. Gas chromatography, however, suffers from low sensitivity and is unable to accurately determine the content. Furthermore, existing methods do not exist for analyzing the ethyl acetoacetate impurity content in methyl acetoacetate. Therefore, there is a need to develop a method for detecting β-diketone compounds that can accurately analyze the ethyl acetoacetate impurity in methyl acetoacetate, thereby providing a better approach for quality control of products using methyl acetoacetate as a raw material. Summary of the Invention

[0011] The first aspect of the present invention provides a method for analyzing β-diketone ester compounds in a sample to be tested, characterized in that a ketone solvent is used as a diluent and the content of the β-diketone ester compound is determined by gas chromatography.

[0012] The β-diketone ester compound is a compound represented by formula (II),

[0013]

[0014] Wherein, R1 and R2 are each independently -C 1-3 alkyl.

[0015] In some embodiments of the present invention, the ketone solvent is a ketone solvent containing α-hydrogen, preferably acetone, 2-butanone, cyclopentanone or 4-methyl-2-pentanone, more preferably, the ketone solvent is acetone.

[0016] In some embodiments of the present invention, R1 is methyl or ethyl, and R2 is methyl, ethyl, n-propyl or isopropyl. Preferably, R1 is methyl, and R2 is methyl or ethyl.

[0017] In some embodiments of the present invention, the sample to be tested contains a β-diketone ester compound. Preferably, the sample to be tested is a dipine drug starting material containing a β-diketone ester compound. More preferably, the dipine drug includes benidipine hydrochloride, nicardipine hydrochloride or lacidipine.

[0018] In some embodiments of the present invention, the sample to be tested is methyl acetoacetate, and the β-diketone ester compound to be measured is ethyl acetoacetate impurity in methyl acetoacetate.

[0019] In some embodiments of the present invention, the detector of the gas chromatography is a hydrogen flame ionization detector.

[0020] In some embodiments of the present invention, the analysis method of the present invention comprises the following steps:

[0021] (1) taking appropriate amounts of a sample to be tested and a β-diketone ester compound, and using a ketone solvent as a diluent to prepare a sample solution and a standard solution;

[0022] (2) A glass capillary column with polyethylene glycol as the stationary phase;

[0023] (3) Set the nitrogen flow rate to 0.5-1.5 mL / min; split ratio 3:1-20:1;

[0024] (4) Set the detector temperature to 230°C and the injection port temperature to 190°C;

[0025] (5) Programmed heating: Initial temperature 100°C, maintain for 5 min; then heat to 130°C at a heating rate of 2°C / min; then heat to 170°C at a heating rate of 5°C / min; finally heat to 230°C at a heating rate of 20°C / min.

[0026] (6) Using a gas chromatograph equipped with a hydrogen flame ionization detector, inject 1 μL of the sample solution from step (1), record the chromatogram, and perform analysis.

[0027] Another aspect of the present invention provides a use of the analytical method according to the first aspect of the present invention in quality control of products using β-diketone ester compounds as materials.

[0028] In some embodiments of the present invention, the product using β-diketone ester compounds as raw materials includes dihydropyridine drugs.

[0029] The β-diketone ester compound presents a single chromatographic peak under the method of the present invention, and its content can be directly determined. The method has the characteristics of simple operation, high sensitivity, high precision, high accuracy, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Attachment Figure 1 : GC-MS ion current diagram of the methyl acetoacetate solution obtained according to Example 1 of the present invention Attachment Figure 2 : Blank solution spectrum obtained according to Example 2 of the present invention;

[0031] Attachment Figure 3 : The standard solution spectrum obtained according to Example 2 of the present invention;

[0032] Attachment Figure 4 : The methyl acetoacetate solution spectrum obtained according to Example 2 of the present invention;

[0033] Attachment Figure 5 : Selective solution spectrum obtained according to Example 2 of the present invention;

[0034] Attachment Figure 6 : Linear relationship diagram of ethyl acetoacetate obtained according to Example 5 of the present invention;

[0035] Attachment Figure 7 : Linear relationship diagram of methyl acetoacetate obtained according to Example 5 of the present invention.

[0036] Definition and Description

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0038] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0039] The terms "comprises," "comprising," "having," "containing," or "involving," and other variations thereof herein, are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

[0040] The term "alkyl" or "alkylene" refers to both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. 1-3 The term "alkyl" refers to a straight or branched chain saturated alkyl group containing 1 to 3 carbon atoms, for example, methyl, ethyl, n-propyl, isopropyl.

[0041] The term "detection limit" refers to the lowest concentration or amount of the analyte that can be detected by an analytical method under specified experimental conditions.

[0042] The term "limit of quantitation" refers to the lowest concentration or amount of an analyte in a sample that can be quantitatively determined by an analytical method. The lower the limit of quantitation, the higher the sensitivity.

[0043] The term "RRT" refers to relative retention time, which is the ratio of the retention time of an impurity to the retention time of the main component.

[0044] The term "FID detector" refers to a flame ionization detector.

[0045] The term "α-hydrogen" or "α-hydrogen" refers to a hydrogen atom directly attached to the carbon that is in the α-position relative to an electron withdrawing group.

[0046] The term "materials" is used to refer to raw materials (including starting materials, reagents, solvents), process aids, intermediates, APIs, labels and packaging materials.

[0047] The "drug" mentioned in the present invention is a substance used to prevent, treat and diagnose diseases. It can be a natural chemical substance and its preparation, a synthetic compound and its preparation, or a recombinant protein, antibody and its preparation formed by bioengineering.

[0048] The term "dihydropyridine" refers to a dihydropyridine calcium channel antagonist, including but not limited to benidipine hydrochloride, nicardipine hydrochloride, barnidipine, nimodipine, nifedipine, nisoldipine, amlodipine, nitrendipine or lacidipine.

[0049] In the present invention, the term "starting material" for drugs refers to the starting material of the active pharmaceutical ingredient.

[0050] The term "API starting material" refers to a raw material, intermediate, or API used in the production of an API that is incorporated into the API structure as a primary building block. API starting materials can be commercially available, purchased from one or more suppliers through contract or commercial agreement, or produced in-house. API starting materials generally have specific chemical properties and structures.

[0051] Throughout the present invention, all numerical values ​​disclosed herein are approximate, regardless of whether the word "about" or "approximately" is used. The numerical value of each numerical value may vary by less than ±10% based on the disclosed number, or by a reasonable variance recognized by one skilled in the art, such as ±1%, ±2%, ±3%, ±4%, or ±5%.

[0052] For numerical ranges herein, it is understood that each intervening value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated value or intervening value in that stated range is also encompassed within the present invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

[0053] In the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0054] If no specific experimental steps or conditions are specified in the examples herein, the conventional experimental steps or conditions described in the literature in this field can be followed. If no manufacturer is specified for the reagents or instruments used, all are commercially available conventional reagents. DETAILED DESCRIPTION

[0055] In order to better understand the technical solution of the present invention, the following is further described in conjunction with the specific embodiments of the present invention, but it is not limited to the present invention. Any changes or modifications that are easily realized by those skilled in the art will fall within the scope of the present invention.

[0056] Example 1: Method development

[0057] Using acetonitrile, ethyl acetate, dichloromethane, chloroform, methanol, ethanol, isopropanol and the like as diluents, gas chromatography analysis of methyl acetoacetate and ethyl acetoacetate was performed under the instrument and conventional measurement conditions of Example 2. The main peaks of methyl acetoacetate and ethyl acetoacetate did not show a single chromatographic peak. Representative typical chromatograms are shown in the attached Figure 1 .

[0058] Example 2: Specificity

[0059] Instruments and conditions:

[0060] Gas chromatograph detector: Agilent 7820A, FID detector.

[0061] Chromatographic column: glass capillary column with polyethylene glycol as stationary phase

[0062] Diluent: Acetone

[0063] Carrier gas: N2

[0064] Flow rate: 1.0 mL / min

[0065] Detector temperature: 230°C

[0066] Inlet temperature: 190°C

[0067] Column temperature: initial temperature 100°C, maintain for 5 min; then increase the temperature to 130°C at a heating rate of 2°C / min; then increase the temperature to 170°C at a heating rate of 5°C / min; finally increase the temperature to 230°C at a heating rate of 20°C / min.

[0068] Split ratio: 10:1

[0069] Injection volume: 1.0 μL

[0070] Solution preparation:

[0071] Diluent: Acetone

[0072] Blank solution: diluent

[0073] Standard stock solution: Weigh 25 mg of methyl acetoacetate and ethyl acetoacetate, accurately weigh them in a 25 mL volumetric flask, dissolve them in the diluent and dilute to the scale, and mix well.

[0074] Standard solution: Accurately transfer 1 mL of standard stock solution into a 100 mL volumetric flask, dilute to the mark with diluent, and mix well.

[0075] Methyl acetoacetate solution: Accurately weigh about 100 mg of methyl acetoacetate sample and place it in a 10 mL volumetric flask. Dissolve it in diluent and dilute to the mark, then mix well.

[0076] Selective solution: Accurately weigh about 100 mg of methyl acetoacetate sample and place it in a 10 mL volumetric flask. Dissolve it in the standard solution and dilute to the mark, then mix thoroughly.

[0077] Experimental steps:

[0078] Under the above conditions, accurately measure the blank solution, inject the sample, and record the chromatogram. Figure 2; Accurately measure the standard solution, inject the sample, and record the chromatogram, see attached Figure 3 ; Accurately measure the methyl acetoacetate solution, inject the sample, and record the chromatogram. Figure 4 ; Accurately measure the selective solution, inject the sample, and record the chromatogram, see attached Figure 5 The retention time and separation of the target peak were recorded. The results are shown in Table 1.

[0079] Table 1 Specificity results

[0080] Component name Retention time (min) Relative retention time Separation Methyl acetoacetate 7.67 1.0 -- Ethyl acetoacetate 8.55 1.12 3.29

[0081] The results showed that the blank solution had no interference with the detection of the main peak and known impurities, and the minimum separation between adjacent peaks was 3.29, as can be seen from the blank solution spectrum, standard solution spectrum, methyl acetoacetate solution spectrum and selective solution spectrum.

[0082] Example 3: Limit of Detection and Limit of Quantitation

[0083] Solution preparation:

[0084] Detection limit solution: Accurately pipette 1.5 mL of standard solution into a 10 mL volumetric flask, dilute to the mark with diluent, and mix well.

[0085] Quantitation limit solution: Accurately transfer 5 mL of the standard solution into a 10 mL volumetric flask, dilute to the mark with the diluent, and mix thoroughly.

[0086] Experimental steps:

[0087] Under the conditions of Example 2, the detection limit solution and the quantification limit solution were accurately measured, sampled, the chromatogram was recorded, and the signal-to-noise ratio of the methyl acetoacetate peak and the ethyl acetoacetate peak was calculated. The signal-to-noise ratio of the quantification limit was not less than 10:1, and the signal-to-noise ratio of the detection limit was not less than 3:1. The results are shown in Table 2.

[0088] Table 2 Results of detection limit and quantification limit

[0089]

[0090] Example 4: Precision

[0091] Solution preparation:

[0092] Mixed solution: Accurately weigh about 100 mg of methyl acetoacetate sample and place it in a 10 mL volumetric flask. Dissolve it in the standard solution and dilute to the mark, then mix thoroughly.

[0093] Experimental steps:

[0094] Under the conditions of Example 2, 1.0 μL of each of the standard solution and the mixed solution was accurately measured and injected six times continuously. The peak area of ​​ethyl acetoacetate in the chromatogram was recorded, and the RSD% was calculated. The results are shown in Tables 3 and 4.

[0095] Table 3 Precision results of standard solutions

[0096] Number of injections 1 2 3 4 5 6 RSD% Ethyl acetoacetate peak area 7.267 7.165 7.386 7.295 7.115 7.144 1.4

[0097] Table 4 Precision results of mixed solutions

[0098]

[0099]

[0100] The results showed that the RSD of the peak area of ​​ethyl acetoacetate was less than 1.5% and that of methyl acetoacetate was less than 0.15% when the standard solution and the mixed solution were injected and measured six times continuously. The injection precision of this method was good.

[0101] Example 5: Linear range

[0102] Solution preparation:

[0103] Linear stock solution: Weigh 25 mg of methyl acetoacetate and ethyl acetoacetate, accurately weigh them into a 25 mL volumetric flask, dissolve them with diluent and dilute to the mark, mix well; accurately transfer 1 mL into a 10 mL volumetric flask, dilute to the mark with diluent, and mix well.

[0104] Linearity solution 1: Accurately pipette 0.5 mL of the stock solution into a 10 mL volumetric flask, dilute to the mark with the diluent, and mix well.

[0105] Linearity solution 2: Accurately pipette 1.0 mL of the stock solution into a 10 mL volumetric flask, dilute to the mark with the diluent, and mix well.

[0106] Linearity solution 3: Accurately transfer 2 mL of the stock solution into a 10 mL volumetric flask, dilute to the mark with the diluent, and mix thoroughly.

[0107] Linearity solution 4: Accurately pipette 5 mL of stock solution into a 10 mL volumetric flask, dilute to the mark with diluent, and mix well.

[0108] Linearity solution 5: Synlinear stock solution.

[0109] Experimental steps:

[0110] Under the conditions of Example 2, accurately measure 1.0 μL of each linear solution, inject the sample, record the peak areas of methyl acetoacetate and ethyl acetoacetate in the chromatogram, and plot the concentration and peak area. See the attached figure. Figure 6 and attached Figure 7 , calculate the regression equation and correlation coefficient.

[0111] Table 5 Linear concentration and peak area of ​​ethyl acetoacetate

[0112]

[0113] The above results show that ethyl acetoacetate has a good linear relationship in the range of 5.0395-100.79 μg / mL, and R 2 =0.9999.

[0114] Table 6 Linear concentration and peak area of ​​methyl acetoacetate

[0115]

[0116] The above results show that methyl acetoacetate has a good linear relationship in the range of 5.006-100.12 μg / mL, and R 2 =0.9999.

[0117] Example 6: Solution stability

[0118] Solution preparation:

[0119] Stability solution: Accurately weigh about 100 mg of methyl acetoacetate sample and place it in a 10 mL volumetric flask. Dissolve and dilute to the mark with the standard solution and mix well.

[0120] Experimental steps:

[0121] Under the conditions of Example 2, the stability solution was accurately measured and injected at 0 h, 12 h, and 48 h, and the peak areas of the target peak and other impurity peaks in the chromatogram were recorded, and the contents were calculated. The results are shown in Table 7.

[0122] Table 7 Solution stability results

[0123]

[0124]

[0125] The results showed that the peak area of ​​the stability solution remained basically unchanged after being placed at room temperature for 48 hours, indicating that the solution had good stability.

[0126] Example 7: Accuracy

[0127] Solution preparation:

[0128] Ethyl acetoacetate standard solution: Weigh 25 mg of ethyl acetoacetate into a 25 mL volumetric flask, dissolve it with diluent and dilute to the mark, mix well; then accurately transfer 1 mL of the above solution into a 100 mL volumetric flask, dilute to the mark with diluent, and mix well.

[0129] Sample solution: Weigh 25 mg of ethyl acetoacetate into a 25 mL volumetric flask. Dissolve and dilute to volume with ethyl acetoacetate standard solution. Mix thoroughly. Prepare 6 replicates.

[0130] Experimental steps:

[0131] Under the conditions of Example 2, 6 portions of the sample solution were accurately measured and injected, the chromatograms were recorded, and the recovery rate of ethyl acetoacetate was calculated. The results are shown in Table 8.

[0132] Table 8 Recovery results

[0133] Adding solution 1 2 3 4 5 6 RSD% Recovery rate 102.1 102.5 102.3 100.8 102.5 102.5 0.64

[0134] The results showed that the recovery rate was between 100.8% and 102.5% with an RSD of 0.64% at the concentration of ethyl acetoacetate standard solution, indicating that the method had good accuracy.

[0135] Example 8: Test sample detection

[0136] Solution preparation:

[0137] Test solution: Weigh 100 mg of the test sample into a 10 mL volumetric flask, dissolve it in the diluent and dilute to the mark, then mix thoroughly.

[0138] Experimental steps:

[0139] Take different batches of test samples and prepare test sample solutions according to the above steps. Under the conditions of Example 2, inject the samples, record the chromatograms, and calculate the content of ethyl acetoacetate. The results are shown in Table 9.

[0140] Table 9 Content results of ethyl acetoacetate in different batches of methyl acetoacetate

[0141]

[0142]

[0143] The results show that the analytical method of the present invention has good sensitivity and a wide linear range, which is beneficial for effectively controlling the quality of related β-diketone ester compounds in the production quality management of products using β-diketone ester compounds as raw materials.

Claims

1. A method for analyzing β-diketone ester compounds in a sample to be tested, characterized in that: The content of β-diketone ester compounds was determined by gas chromatography using ketone solvents as diluents. The β-diketone ester compound is a compound represented by formula (II), Wherein, R1 and R2 are each independently -C 1-3 alkyl.

2. The analysis method according to claim 1, characterized in that The ketone solvent is a ketone solvent containing α-hydrogen, preferably acetone, 2-butanone, cyclopentanone or 4-methyl-2-pentanone, more preferably, the ketone solvent is acetone.

3. The analysis method according to claim 1 or 2, characterized in that R1 is methyl or ethyl, R2 is methyl, ethyl or propyl, preferably, R1 is methyl, R2 is methyl or ethyl.

4. The analysis method according to any one of claims 1 to 3, characterized in that The sample to be tested contains a β-diketone ester compound. Preferably, the sample to be tested is a dipine drug starting material containing a β-diketone ester compound. More preferably, the dipine drug includes benidipine hydrochloride, nicardipine hydrochloride or lacidipine.

5. The analysis method according to any one of claims 1 to 4, characterized in that The sample to be tested is methyl acetoacetate, and the β-diketone ester compound to be measured is the ethyl acetoacetate impurity in methyl acetoacetate.

6. The analysis method according to any one of claims 1 to 5, characterized in that The detector of the gas chromatography is a hydrogen flame ionization detector.

7. The analytical method according to any one of claims 1 to 6, characterized in that The method includes the following steps: (1) taking appropriate amounts of a sample to be tested and a β-diketone ester compound, and using a ketone solvent as a diluent to prepare a sample solution and a standard solution; (2) A glass capillary column with polyethylene glycol as the stationary phase; (3) Set the nitrogen flow rate to 0.5-1.5 mL / min; split ratio 3:1-20:1; (4) Set the detector temperature to 230°C and the injection port temperature to 190°C; (5) Programmed heating: Initial temperature 100°C, maintain for 5 min; then heat to 130°C at a heating rate of 2°C / min; then heat to 170°C at a heating rate of 5°C / min; finally heat to 230°C at a heating rate of 20°C / min. (6) Using a gas chromatograph equipped with an FID detector, inject 1 μL of the sample solution from step (1), record the chromatogram, and perform analysis.

8. Use of the analytical method according to any one of claims 1 to 7 in quality control of products using β-diketone ester compounds as raw materials.

9. The use according to claim 8, characterized in that The products using β-diketone ester compounds as materials include dihydropyridine drugs.

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