HPLC (High Performance Liquid Chromatography) analysis and detection method for 2, 4, 6-trifluorobenzoyl chloride and related substances thereof

Abstract

The invention discloses an HPLC (High Performance Liquid Chromatography) analysis and detection method for 2, 4, 6-trifluorobenzoyl chloride and related substances thereof, and in particular relates to an HPLC analysis method for deriving 2, 4, 6-trifluorobenzoyl chloride into methyl 2, 4, 6-trifluorobenzoate and four impurities in the methyl 2, 4, 6-trifluorobenzoate. According to the method, various impurities in 2, 4, 6-trifluorobenzoyl chloride can be rapidly, simply, accurately and efficiently eluted, separated and quantitatively detected according to the type and specification of the chromatographic column, the detection wavelength, the type of the mobile phase, the proportion of the mobile phase, the operation time and the like, and the method is beneficial to effectively detecting and controlling the product quality.

Description

Technical Field

[0001] The present invention belongs to the new technology field of drugs and relates to an analytical method for four impurities (process impurities) of 2,4,6-trifluorobenzoyl chloride. Specifically, it is a high performance liquid chromatography method using an ultraviolet detector, selecting appropriate mobile phase and chromatographic column to quantitatively analyze four impurities in 2,4,6-trifluorobenzoyl chloride. Background Art

[0002] 2,4,6-trifluorobenzoyl chloride is an important intermediate for synthesizing ramelteon hemisuccinate, which is prepared by acyl chlorination reaction of 2,4,6-trifluorobenzoic acid and mainly contains the following impurities shown in the structure:

[0003]

[0004] The presence of the above impurities will affect the purity of downstream products. In order to better control the quality of 2,4,6-trifluorobenzoyl chloride, reduce the impact of impurities on the production process and improve the safety of drugs, it is necessary to strictly monitor 2,4,6-trifluorobenzoyl chloride and its possible impurities. Summary of the Invention

[0005] The main object of the present invention is to overcome the deficiencies of the prior art and provide a high performance liquid chromatography detection method for 2,4,6-trifluorobenzoyl chloride and its related substances, which can quickly, sensitively, accurately and reliably detect 2,4,6-trifluorobenzoyl chloride and its related substances.

[0006] 2,4,6-trifluorobenzoyl chloride is a colorless liquid with a pungent odor. It reacts with water to form 2,4,6-trifluorobenzoic acid (impurity B) and hydrogen chloride; it reacts with alcohols to form 2,4,6-trifluorobenzoate and hydrogen chloride. For example, 2,4,6-trifluorobenzoyl chloride reacts with methanol to form a derivative E (methyl 2,4,6-trifluorobenzoate) with the following structure: Therefore, it is considered to derivatize it with alcohols to form esters under anhydrous conditions and then detect its related substances by high performance liquid chromatography. From the structural formula, it can be seen that the esters and impurities formed after derivatization of 2,4,6-trifluorobenzoyl chloride all have ultraviolet absorption and can be detected using an ultraviolet detector.

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

[0008] HPLC analysis and detection method for 2,4,6-trifluorobenzoyl chloride and its related substances, wherein the related substances are selected from one or more of four impurities A, B, C, and D in 2,4,6-trifluorobenzoyl chloride. Impurity A is 2,4,6-trifluorobenzamide; impurity B is 2,4,6-trifluorobenzoic acid; impurity C is 2,4,6-trifluorobenzonitrile; impurity D is methyl 2,4-difluorobenzoate; derivative E is 2,4,6-trifluorobenzoate, that is, the product converted after the derivatization reaction of 2,4,6-trifluorobenzoyl chloride, and the four impurities do not undergo derivatization reaction. This analysis method detects methyl 2,4,6-trifluorobenzoate after derivatization, so as to detect 2,4,6-trifluorobenzoyl chloride and the four impurities therein.

[0009] The method comprises the following steps:

[0010] 1) Preparation of test solution: Take an appropriate amount of 2,4,6-trifluorobenzoyl chloride, dissolve it in a derivatization solvent, shake it, and quantitatively dilute it with a dilution solvent to obtain a test solution;

[0011] 2) Preparation of system suitability solution:

[0012] 2-1) Stock solution of impurity reference substances: Take the reference substances of related substances A, B, C, D, and the reference substance of derivative E respectively, and prepare stock solutions of each impurity reference substance and derivative E with a dilution solvent; the derivative E is 2,4,6-trifluorobenzoic acid C1-4 alkyl alcohol ester, preferably methyl 2,4,6-trifluorobenzoate, ethyl 2,4,6-trifluorobenzoate, n-propyl 2,4,6-trifluorobenzoate, isopropyl 2,4,6-trifluorobenzoate or n-butyl 2,4,6-trifluorobenzoate;

[0013] 2-2) System suitability solution: Take the stock solutions of each impurity reference substance and derivative E, and prepare a system suitability solution with a dilution solvent;

[0014] 3) Chromatographic conditions: Use high performance liquid chromatography,

[0015] The injection volume is 5 - 50 μl,

[0016] The detection wavelength is 205 nm - 220 nm,

[0017] The flow rate of the mobile phase is 0.5 - 2.0 ml / min,

[0018] The chromatographic column is: a chromatographic column with octadecylsilane chemically bonded silica as the stationary phase,

[0019] The column temperature is 20 - 40 °C,

[0020] Mobile phase: A mixture of one or more of water, phosphate buffer solution, and phosphoric acid solution is used as mobile phase A, and a mixture of one or more of acetonitrile, methanol, or tetrahydrofuran solution is used as mobile phase B.

[0021] Gradient elution is performed, and the gradient elution program is as follows:

[0022] From 0 to 18 min, the proportion of mobile phase A is 80% - 20%, and the proportion of mobile phase B is 20% - 80%;

[0023] From 18 to 20 min, the proportion of mobile phase A is 20%, and the proportion of mobile phase B is 80%;

[0024] From 20 to 20.1 min, the proportion of mobile phase A is 20% - 80%, and the proportion of mobile phase B is 80% - 20%;

[0025] From 20.1 to 26 min, the proportion of mobile phase A is 80%, and the proportion of mobile phase B is 20%;

[0026] 4) Determination method: Take the test solution in step 1) and the system suitability solution in step 2), inject them into a high-performance liquid chromatograph, record the chromatogram, and perform high-performance liquid chromatography quantitative analysis on the 4 impurities in 2,4,6-trifluorobenzoyl chloride according to the peak area normalization method with correction factor or the external standard method.

[0027] Furthermore, in step 1), the derivatization solvent is selected from C1-4 alkyl alcohols, preferably a mixture of one or more of methanol, ethanol, n-propanol, isopropanol, and n-butanol, and preferably, the derivatization solvent is selected from methanol; the content of 2,4,6-trifluorobenzoyl chloride in the test solution is 0.5 - 5 mg / ml, preferably 0.8 - 4 mg / ml, and most preferably 1 mg / ml.

[0028] Furthermore, in steps 1) and 2), the dilution solvent is selected from a mixture of one or more of methanol, ethanol, water, and acetonitrile. Preferably, the dilution solvent is a mixed solvent of methanol and water, and preferably, the dilution solvent is a mixed solvent of water and methanol with a volume ratio of 90:10.

[0029] Furthermore, the concentration of the reference stock solution of derivative E in step 2) is 0.5 - 5 mg / ml, preferably 1 mg / ml;

[0030] The concentrations of the reference stock solutions of impurity A and impurity C are both 5 - 20 μg / ml, preferably 10 μg / ml;

[0031] The concentration of the reference stock solution of impurity B is 10 - 50 μg / ml, preferably 25 μg / ml;

[0032] The concentration of the reference stock solution of impurity D is 3 - 10 μg / ml, preferably 7.5 μg / ml.

[0033] Furthermore, in the system suitability solution in step 2), the concentration of the reference substance E derivative is 0.125 - 1.25 mg / ml, preferably 0.25 mg / ml;

[0034] The concentrations of impurity A and impurity C are 0.25 - 1 μg / ml, preferably 0.5 μg / ml;

[0035] The concentration of impurity B is 0.5 - 2.5 μg / ml, preferably 1.25 μg / ml;

[0036] The concentration of the reference stock solution of impurity D is 0.15 - 0.5 μg / ml, preferably 0.375 μg / ml.

[0037] Furthermore, in step 3), the injection volume is 10 μl, the detection wavelength is 210 nm, and the mobile phase flow rate is 1.0 ml / min.

[0038] Furthermore, in step 3), the column temperature is 25 - 35 °C, preferably, the column temperature is 30 °C.

[0039] Furthermore, in step 3), the mobile phase A is 0.1% phosphoric acid aqueous solution, and the mobile phase B is acetonitrile.

[0040] Furthermore, the gradient elution program in step 3) is as follows:

[0041]

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The present invention discloses an HPLC analysis and detection method for 2,4,6-trifluorobenzoyl chloride and its related substances. This method uses the convenient and fast high performance liquid chromatography method, and adopts the derivatization method to detect 2,4,6-trifluorobenzoyl chloride and its 4 related impurities, which is beneficial to effectively detect and control the product quality. Description of the Drawings

[0044] Figure 1 is the chromatogram of the blank solvent;

[0045] Figure 2 is the chromatogram of the mixed sample of derivative E (methyl 2,4,6-trifluorobenzoate) and 4 impurities;

[0046] Figure 3 is the chromatogram of the test sample of 2,4,6-trifluorobenzoyl chloride. Detailed Embodiments

[0047] The present invention will be further explained below in conjunction with embodiments, but the embodiments do not limit the present invention in any form.

[0048] The following embodiments facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all obtained from conventional biochemical reagent stores unless otherwise specified.

[0049] Instrument: High performance liquid chromatograph, including a liquid phase pump, an ultraviolet detector, and an injector.

[0050] Reagents: Phosphoric acid (analytical grade), acetonitrile, methanol.

[0051] Chromatographic column: Octadecylsilane-bonded silica gel chromatographic column.

[0052] Example 1:

[0053] Determination was carried out with reference to the high performance liquid chromatography method (General Principles 0512, Volume IV, Chinese Pharmacopoeia 2020 Edition).

[0054] Chromatographic conditions and system suitability test

[0055] Using octadecylsilane-bonded silica gel as the filler (Waters Xterra RP18, 4.6 mm × 150 mm, 5 μm);

[0056] Using 0.1% phosphoric acid as mobile phase A and acetonitrile as mobile phase B;

[0057] Perform linear gradient elution according to Table 1;

[0058] The flow rate is 1.0 ml per minute;

[0059] The column temperature is 30 °C;

[0060] The detection wavelength is 210 nm;

[0061] The injection volume is 10 μl.

[0062] Table 1 Gradient elution program

[0063]

[0064] Determination method

[0065] Take 2,4,6-trifluorobenzoyl chloride, dissolve it in methanol and dilute to make a solution containing about 1 mg per 1 ml, shake for 30 min, as the stock solution of the test sample; accurately measure 5 ml of the stock solution of the test sample, place it in a 20 ml volumetric flask, dilute to the mark with the solvent, and shake well to obtain the test sample solution;

[0066] Take the reference substance of derivative E, dissolve it with methanol and dilute to prepare a stock solution containing about 1 mg per 1 ml as the stock solution of derivative E;

[0067] Precisely measure appropriate amounts of the reference substances of impurity A, impurity B, impurity C and impurity D, dilute with methanol to prepare a solution containing about 10 μg of impurity A, 25 μg of impurity B, 10 μg of impurity C and 7.5 μg of impurity D per 1 ml as the mixed stock solution of impurities;

[0068] Take 5 ml of the stock solution of derivative E, place it in a 20-ml volumetric flask, dissolve it with the solvent (water - methanol (90:10)), precisely add 1 ml of the mixed stock solution of impurities, dilute to the mark with the solvent (water - methanol (90:10)), and shake well as the system suitability solution.

[0069] Precisely measure 10 μl each of the test solution and the system suitability solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0070] If there are impurity peaks in the chromatogram of the test solution, calculate by the peak area normalization method with correction factor, and the content of each impurity should meet the limit requirements in Table 2, and the peaks less than 0.03% are ignored.

[0071] In the chromatogram of the system suitability solution, the elution order of the peaks is impurity A, impurity B, impurity C, impurity D, and derivative E in turn.

[0072] Table 2 Provisions on the limits of known impurities

[0073]

[0074] In summary, the method of the present invention can effectively detect four extremely trace impurities in 2,4,6-trifluorobenzoyl chloride, which is beneficial to product quality control.

[0075] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. HPLC analysis and detection method for 2,4,6-trifluorobenzoyl chloride and its related substances Characterized in that The related substances are selected from one or more of 4 impurities in 2,4,6-trifluorobenzoyl chloride. Impurity A is 2,4,6-trifluorobenzamide; Impurity B is 2,4,6-trifluorobenzoic acid; Impurity C is 2,4,6-trifluorobenzonitrile; Impurity D is methyl 2,4-difluorobenzoate; The method includes the following steps: 1) Preparation of test solution: Take 2,4,6-trifluorobenzoyl chloride, dissolve it with a derivatization solvent, shake it, and quantitatively dilute it with a dilution solvent to obtain a test solution; 2) Preparation of system suitability solution: 2-1) Stock solution of impurity reference substance: Take the reference substance of related substance A, the reference substance of related substance B, the reference substance of related substance C Reference substance, reference substance of related substance D, reference substance of derivative E, and prepare stock solutions of each impurity reference substance with methanol; The derivative E is an alkyl alcohol ester of 2,4,6-trifluorobenzoic acid C1-4, preferably methyl 2,4,6-trifluorobenzoate, ethyl 2,4,6-trifluorobenzoate, n-propyl 2,4,6-trifluorobenzoate, isopropyl 2,4,6-trifluorobenzoate or n-butyl 2,4,6-trifluorobenzoate; 2-2) System suitability solution: Take the stock solutions of each impurity reference substance and prepare a system suitability solution with a dilution solvent; 3) Chromatographic conditions: Use high performance liquid chromatography The injection volume is 5 - 50 μl, The detection wavelength is 205 nm - 220 nm, The flow rate of the mobile phase is 0.5 - 2.0 ml / min, The chromatographic column is: a chromatographic column with octadecylsilyl-bonded silica gel as the stationary phase, The column temperature is 20 - 40 °C, Mobile phase: A mixture of one or more of water, phosphate buffer solution, and phosphoric acid solution is used as mobile phase A, and a mixture of one or more of acetonitrile, methanol, or tetrahydrofuran solution is used as mobile phase B, Gradient elution, and the gradient elution program is: 0 - 18 min, the proportion of mobile phase A is 80% - 20%, and the proportion of mobile phase B is 20% - 80%; 18 - 20 min, the proportion of mobile phase A is 20%, and the proportion of mobile phase B is 80%; 20 - 20.1 min, the proportion of mobile phase A is 20% - 80%, and the proportion of mobile phase B is 80% - 20%; 20.1 - 26 min, the proportion of mobile phase A is 80%, and the proportion of mobile phase B is 20%; 4) Determination method: Take the test solution in step 1) and the system suitability solution in step 2), inject them into a high performance liquid chromatograph, record the chromatogram, and perform high performance liquid chromatography quantitative analysis on 4 impurities in 2,4,6-trifluorobenzoyl chloride according to the peak area normalization method with correction factor or the external standard method.

2. The HPLC analysis and detection method according to claim 1 Characterized in that In step 1), the derivatization solvent is selected from C1-4 alkyl alcohols, preferably a mixture of one or more of methanol, ethanol, n-propanol, isopropanol, and n-butanol. Preferably, the derivatization solvent is selected from methanol; The content of 2,4,6-trifluorobenzoyl chloride in the test solution is 0.5 - 5 mg / ml.

3. The HPLC analysis and detection method according to claim 1, characterized in that, the dilution solvent described in steps 1) and 2) is selected from one or more of methanol, ethanol, water and acetonitrile, preferably, the dilution solvent is a mixed solvent of methanol and water, and preferably, the dilution solvent is a mixed solvent of water and methanol with a volume ratio of 90:

10.

4. The HPLC analysis and detection method according to claim 1, characterized in that, the concentration of the reference stock solution of derivative E in step 2) is 0.5 - 5 mg / ml, preferably 1 mg / ml; the concentrations of the reference stock solutions of impurity A and impurity C are both 5 - 20 μg / ml, preferably 10 μg / ml; the concentration of the reference stock solution of impurity B is 10 - 50 μg / ml, preferably 25 μg / ml; the concentration of the reference stock solution of impurity D is 3 - 10 μg / ml, preferably 7.5 μg / ml.

5. The HPLC analysis and detection method according to claim 1, characterized in that, the concentration of the reference substance of derivative E in the system suitability solution in step 2) is 0.125 - 1.25 mg / ml, preferably 0.25 mg / ml; the concentrations of impurity A and impurity C are 0.25 - 1 μg / ml, preferably 0.5 μg / ml; the concentration of impurity B is 0.5 - 2.5 μg / ml, preferably 1.25 μg / ml; the concentration of the reference stock solution of impurity D is 0.15 - 0.5 μg / ml, preferably 0.375 μg / ml.

6. The HPLC analysis and detection method according to claim 1, characterized in that, the injection volume in step 3) is 10 μl, the detection wavelength is 210 nm, and the flow rate of the mobile phase is 1.0 ml / min.

7. The HPLC analysis and detection method according to claim 1, characterized in that, the column temperature in step 3) is 25 - 35 °C, preferably, the column temperature is 30 °C.

8. The HPLC analysis and detection method according to claim 1, characterized in that, the mobile phase A in step 3) is 0.1% phosphoric acid aqueous solution, and the mobile phase B is acetonitrile.

9. The HPLC analysis and detection method according to claim 1, characterized in that, the gradient elution program in step 3) is:

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