A method for detecting propyl ketone impurities in nebivolol hydrochloride starting material

Abstract

The present invention belongs to the technical field of drug analysis, and in particular to a method for detecting propyl ketone impurities in a nebivolol hydrochloride starting material. The method of the present invention is to use high performance liquid chromatography, using octadecylsilane bonded silica gel as the filler of the chromatographic column, using trifluoroacetic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, and in the detection process, according to 0-30 min, 40% mobile phase A: 60% mobile phase B, the ratio of the test sample and the reference sample is eluted isocratic, and the detection of propyl ketone impurities is realized. The detection method successfully realizes the separation and detection of propyl ketone impurities and chloroketone intermediates, and has the advantages of strong specificity, high sensitivity, good precision and accuracy, and good durability.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for detecting propyl ketone impurities in a nebivolol hydrochloride starting material. Background Art

[0002] Nebivolol hydrochloride is a third-generation β-receptor blocker with strong selectivity. Its β1 receptor blocking strength is 290 times that of β2 receptor. It does not cause contraction of bronchial smooth muscle and vascular smooth muscle and has no endogenous sympathomimetic activity. It can be better used in the treatment of patients with mild to moderate hypertension, and can also be used for the treatment of angina pectoris and congestive heart failure.

[0003] 6-Fluoro-3,4-dihydro-2H-benzopyran-2-methanol-2-yl ethyl ketone chloride (hereinafter referred to as "chloroketone") is the key starting material for the production of nebivolol hydrochloride raw materials, and its structure is as follows:

[0004] .

[0005] In the above-mentioned synthesis process of chloroketone, it is necessary to go through the intermediate process of compound A, and then compound A is subjected to ring closure, chlorination, and diazomethane to increase the carbon chain to obtain the chloroketone product.

[0006] However, compound A needs to undergo Michael 1,4 addition to cyclize into a six-membered oxygen ring. In this process, compound A has two reaction sites (1 and 2), as shown below:

[0007] .

[0008] When compound A reacts at reaction site 1, the target product can be obtained. The reaction equation is as follows:

[0009] .

[0010] However, if compound A reacts at reaction site 2, 1-chloro-3-(5-fluoro-2,3-dihydro-benzofuran-3-1)-2-propyl ketone impurity (hereinafter referred to as "propyl ketone") will be obtained as a by-product. The reaction equation is as follows:

[0011] .

[0012] Although the electron-withdrawing ability of the carbonyl group in compound A is greater than that of the carboxylic acid group, the reaction at reaction site 1 is the main reaction to obtain the main product, and the reaction at reaction site 2 is a side reaction to obtain the by-product propyl ketone impurity, but this impurity can be substituted into the next reaction, resulting in difficulty in removing it from the final product and increasing the purification cost of the product. Therefore, the content of this impurity should be strictly controlled.

[0013] However, there is no method for detecting propyl ketone impurities in chloroketones in the prior art, and in the actual detection process, it is difficult to separate the propyl ketone impurities from the chloroketones, and the detection effect of the propyl ketone impurities is poor. Summary of the invention

[0014] In order to solve the above technical problems, the present invention provides a method for detecting propyl ketone impurities in nebivolol hydrochloride starting materials.

[0015] In a method for detecting propyl ketone impurities in a nebivolol hydrochloride starting material provided by the present invention, the nebivolol hydrochloride starting material is 6-fluoro-3,4-dihydro-2H-benzopyran-2-methanol-2-yl ethyl ketone chloride, and the nebivolol hydrochloride starting material contains propyl ketone impurities, and the propyl ketone impurity is 1-chloro-3-(5-fluoro-2,3-dihydro-benzofuran-3-1)-2-propyl ketone, and its structure is as follows:

[0016] ;

[0017] The detection method is to use high performance liquid chromatography, use 0.1%wt trifluoroacetic acid aqueous solution as mobile phase A, use acetonitrile as mobile phase B, and perform isocratic elution on the propyl ketone standard and the starting material sample respectively according to the following chromatographic conditions: 0-30 min, 40% mobile phase A: 60% mobile phase B (V: V); perform isocratic elution on the propyl ketone standard and the starting material sample respectively, obtain the standard curve and calculate the content of the propyl ketone impurity in the starting material sample.

[0018] Furthermore, the above detection method comprises the following steps:

[0019] (1) Establishment of the impurity standard curve of propyl ketone

[0020] Take the propyl ketone impurity reference substance and add it to the blank solution to prepare the reference solution;

[0021] Prepare a series of reference solution concentrations, inject them into a high performance liquid chromatograph, perform detection using the above chromatographic conditions, measure the chromatographic peak area, and obtain a standard curve of propyl ketone impurities;

[0022] (2) Determination of the content of propyl ketone impurity in the starting material of nebivolol hydrochloride

[0023] Take a sample of the nebivolol hydrochloride starting material to be tested, add blank solution to dissolve it, and obtain a test solution;

[0024] Take the test solution, inject it into the high performance liquid chromatograph, detect it under the same chromatographic conditions as in (1), and calculate it according to the standard curve obtained in (1) to obtain the content of the propyl ketone impurity in the nebivolol hydrochloride starting material sample to be tested;

[0025] Wherein, the blank solution is an acetonitrile aqueous solution with a volume fraction of 30% to 50%.

[0026] Preferably, the blank solution is an acetonitrile aqueous solution with a volume fraction of 40%.

[0027] Preferably, when the high performance liquid chromatography is used to detect the propyl ketone impurity in the nebivolol hydrochloride starting material, the chromatographic column used is octadecylsilane bonded silica gel, and its specifications are: 4.6 mm×150 mm, 5 μm.

[0028] As further preferred, the chromatographic column is selected from: any one of Thermo Hypersil GOLD C18, Thermo BDSC18, and Thermo Syncronis C18.

[0029] Preferably, the chromatographic conditions for the high performance liquid chromatography detection are:

[0030] During detection, the flow rate of mobile phase A and mobile phase B is: 0.8~1.5 mL / min; detection wavelength: 210 nm; column temperature: 25~35℃; injection volume: 10 μL.

[0031] The beneficial effects of the present invention are:

[0032] The present invention provides a detection method capable of accurately detecting 1-chloro-3-(5-fluoro-2,3-dihydro-benzofuran-3-1)-2-propyl ketone impurities in a nebivolol hydrochloride starting material, which has the advantages of good separation and high sensitivity and is of great significance for the quality control of the final product nebivolol hydrochloride. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is a high performance liquid chromatogram of the system suitability solution in Example 1 of the present invention;

[0034] Figure 2 is a high performance liquid chromatogram of the test solution in Example 1 of the present invention;

[0035] Figure 3 is the HPLC chromatogram of the blank solution in Example 1 of the present invention;

[0036] Figure 4 This is the regression curve of the propyl ketone impurity obtained in Example 1 of the present invention. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0038] Example 1

[0039] A method for detecting propyl ketone impurities in a nebivolol hydrochloride starting material, wherein the nebivolol hydrochloride starting material is 6-fluoro-3,4-dihydro-2H-benzopyran-2-methanol-2-yl ethyl ketone chloride, and the nebivolol hydrochloride starting material contains propyl ketone impurities, and the propyl ketone impurity is 1-chloro-3-(5-fluoro-2,3-dihydro-benzofuran-3-1)-2-propyl ketone, and its structure is as follows:

[0040] .

[0041] The content of the impurity was detected by high performance liquid chromatography, and the chromatographic conditions of high performance liquid chromatography detection were as follows:

[0042] Chromatographic column: octadecylsilane bonded silica gel (4.6 mm × 150 mm, 5 μm);

[0043] Mobile phase A: 0.1%wt trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile;

[0044] Flow rate: 1.0 mL / min; detection wavelength: 210 nm; column temperature: 30°C; run time: 30 min; injection volume: 10 μL;

[0045] Elution method: isocratic elution according to the following program: 0-30 min, 40% mobile phase A: 60% mobile phase B (V:V);

[0046] Blank solvent: 40% acetonitrile aqueous solution.

[0047] Preparation of each test solution:

[0048] Reference solution: Take 1 mg of propyl ketone impurity reference substance, add 40% by volume acetonitrile aqueous solution to dissolve and dilute to make a 0.2 μg / mL solution as the reference solution;

[0049] Test solution: Take 10 mg of the starting material chloroketone test sample to be tested, add 40% acetonitrile aqueous solution to dissolve and dilute to make a 0.10 mg / mL solution as the test solution;

[0050] System suitability solution: Weigh 0.1 mg of propyl ketone impurity reference and 10 mg of chloroketone test solution into a 50 mL volumetric flask, add 40% by volume acetonitrile aqueous solution to dissolve and dilute to the scale.

[0051] 1.1 Specificity test

[0052] Take the blank solution, system suitability solution and test solution prepared above and test them according to the above liquid chromatography conditions, record the chromatogram, and the test results are shown in Table 1 below and the chromatogram is shown in the attached Figure 1 To Attachment Figure 3 ,in, Figure 1 is the HPLC chromatogram of the system suitability solution. Figure 2 is the HPLC chromatogram of the test solution. Figure 3 HPLC chromatogram of blank solution.

[0053] Table 1 Specificity test results

[0054] name Blank solution retention time / min Retention time of test solution / min System suitability solution retention time / min Propyl Ketone Impurity Not yet peaked 5.022 5.039 Chloroketone test sample Not yet peaked 11.578 11.509

[0055] The results show that in the system suitability solution, the elution time of propyl ketone impurity is 5.039 min, and the elution time of chloroketone is 11.509 min. The separation degree between the two is 25.64, which is good. The tailing factors are both around 1.0, and the peak shape is good.

[0056] In addition, the blank solution and the test solution had no interference with the detection of propyl ketone impurities, and the method had good specificity.

[0057] 1.2 Solution stability test

[0058] Take 10 mg of the chloroketone test sample, add blank solvent to dissolve and quantitatively dilute to 100 mL to prepare a 0.10 mg / mL solution as the test solution. Place the test solution at room temperature, then take 5 μL of the test solution and inject it into the liquid chromatograph at 0, 1, 2, 4, 6, and 8 h, respectively, and record the chromatogram. The impurity content in the test sample was detected by HPLC at different times, as shown in Table 2 below.

[0059] Table 2 Impurity content of the test samples at different storage times

[0060] Time (h) 0 1 2 4 6 8 RSD(%) Propyl Ketone Impurity 2.807 2.853 2.976 2.953 2.967 2.818 2.70

[0061] The results in Table 2 show that the test sample solution after being dissolved in the blank solvent of the present invention still has good stability after being placed at room temperature for 8 hours.

[0062] 1.3 Precision test

[0063] (1) Instrument precision test

[0064] Accurately weigh 10 mg of the chloroketone test sample, dissolve and dilute it with 40% by volume acetonitrile aqueous solution to prepare a 0.10 mg / mL test sample solution.

[0065] Accurately measure 5 μL of the above solution, inject it into the high performance liquid chromatograph, inject it 6 times continuously, and record the chromatogram. The results of the instrument precision test are shown in Table 3 below.

[0066] Table 3 Precision test results

[0067] name 1 2 3 4 5 6 average RSD% Single impurity (%) 0.069 0.072 0.072 0.068 0.069 0.072 0.070 2.6

[0068] (2) Intermediate precision test

[0069] By changing the analysts and selecting different equipment, the influence of random variation factors on the detection precision was investigated. The operation method was the same as the repeatability test. The results are shown in Table 4 below.

[0070] Table 4 Precision test results

[0071] name 1 2 3 4 5 6 average RSD% Single impurity (%) 0.076 0.073 0.075 0.074 0.073 0.075 0.074 1.6

[0072] The results in Table 3-4 above show that the propyl ketone detection method of this product has good intermediate precision and good repeatability.

[0073] 1.4 Linearity and range tests

[0074] Accurately weigh an appropriate amount of propyl ketone reference substance, and use 40% acetonitrile aqueous solution to prepare a series of linear solutions containing 0.012 μg, 0.042 μg, 0.102 μg, 0.204 μg, 0.305 μg, and 0.408 μg of propyl ketone per 1 mL. Detection was carried out according to the above chromatographic conditions, and the chromatogram was recorded. A linear regression curve was drawn with propyl ketone concentration μg / mL as the horizontal and vertical scales and peak area as the vertical coordinate. The results are shown in Table 5 below. The obtained regression curve is shown in the attached figure. Figure 4 shown.

[0075] Table 5 Propyl ketone linear test results

[0076]

[0077] As can be seen from the table, propyl ketone has a good linear relationship in the concentration range of 0.012~0.408 μg / mL.

[0078] 1.5 Limit of Quantitation and Limit of Detection Test

[0079] Limit of quantitation: Take chloroketone reference substance and propyl ketone impurity reference substance respectively, add 40% acetonitrile aqueous solution with a volume fraction to prepare 0.1 mg / mL chloroketone test solution and 0.2 μg / mL propyl ketone impurity solution, dilute them step by step with methanol, detect according to the above-mentioned chromatographic conditions, record the chromatogram, and dilute to a concentration with a signal-to-noise ratio of 10:1, which is the quantitation limit solution.

[0080] Detection limit: Take the above quantitative limit solution, dilute it with 40% by volume acetonitrile aqueous solution to a concentration with a signal-to-noise ratio of 3, and perform detection according to the above chromatographic conditions. The results are shown in Table 6 below.

[0081] Table 6 Detection limit and quantification limit test results

[0082] type Limit of quantitation (ng / µL) Detection limit (ng / µL) Chloroketone concentration 1.206 0.404 Propyl ketone concentration 1.987 0.663

[0083] It can be seen from the above table that both the detection limit and the quantification limit meet the requirements for impurity detection.

[0084] 1.6 Accuracy Test

[0085] Propyl ketone impurity stock solution: Accurately weigh 1 mg of propyl ketone reference substance, dissolve and dilute it in 40% by volume acetonitrile aqueous solution, shake well to prepare a 0.10 mg / mL reference substance solution.

[0086] Test solution: Take 10 mg of the chloroketone test sample, dissolve and dilute it with 40% acetonitrile water to make a 0.10 mg / mL test solution.

[0087] Recovery solution: Take about 10 mg of the chloroketone test sample, accurately weigh 9 portions, and put them in a 100 mL volumetric flask. Accurately add 1.6 mL of propyl ketone impurity stock solution to 3 portions, dissolve and dilute to the scale with 40% acetonitrile water by volume to make an 80% recovery solution; accurately add 2 mL of propyl ketone impurity stock solution to 3 portions, dissolve and dilute to the scale with 40% acetonitrile water by volume to make a 100% recovery solution; accurately add 2.4 mL of propyl ketone impurity stock solution to 3 portions, dissolve and dilute to the scale with 40% acetonitrile water by volume to make a 120% recovery solution;

[0088] Accurately measure 5 μL of the above solution and inject it into the high performance liquid chromatograph respectively. Record the chromatogram and calculate the impurity content and recovery rate of propyl ketone by the external standard method. The results are shown in Table 7 below.

[0089] Table 7 Recovery test results

[0090]

[0091] The results in Table 7 show that the average recovery rate of propyl ketone impurity is 100.4%, the RSD is 1.48%, and the method has good accuracy.

[0092] Example 2 Durability Test

[0093] Minor adjustments were made to the chromatographic conditions for impurity detection to examine the durability of the chromatographic conditions. The specific plan is shown in Table 8 below.

[0094] Table 8 Durability test condition selection

[0095] Test Project Standard conditions Scope of verification Column temperature 30℃ ①25℃；②30℃；③35℃ Chromatographic columns <![CDATA[C 18 (4.6×250mm,5μm)]]> <![CDATA[①Thermo Hypersil GOLD C 18 ;②Thermo BDS C 18 ;③Thermo Syncronis C 18 ]]> Flow rate 1.0mL / min ①0.8mL / min; ②1.0mL / min; ③1.2mL / min

[0096] The specific operations are:

[0097] The preparation methods of the test solution and the reference solution are exactly the same as those in Example 1.

[0098] Accurately measure 5 μL of the test solution and the reference solution, perform the test according to the chromatographic detection conditions in Example 1, and record the chromatogram. The specific experimental results are shown in Table 9 below.

[0099] Table 9 Durability verification results

[0100]

[0101] It can be seen from the experimental results in Table 9 that fine-tuning of the detection conditions such as column temperature, flow rate, and chromatographic column model has little effect on the detection of propyl ketone impurities, and the separation between the peaks of each component is greater than 1.5, which shows that the detection method of the present invention has good durability.

[0102] Comparative Example 1

[0103] This comparative example provides a method for detecting propyl ketone impurities in nebivolol hydrochloride starting materials. The difference from Example 1 is that the chromatographic conditions are as follows:

[0104] Chromatographic column: octadecylsilane bonded silica gel (4.6 mm × 150 mm, 5 μm);

[0105] Mobile phase A: water, mobile phase B: methanol;

[0106] Flow rate: 1.0 mL / min; detection wavelength: 254 nm; column temperature: 25°C; run time: 30 min; injection volume: 10 μL;

[0107] Elution method: Isocratic elution according to the following program: 0-30 min, 80% mobile phase A: 20% mobile phase B (V:V);

[0108] Blank solvent: methanol.

[0109] The preparation methods of the test solution, the reference solution and the system suitability solution are the same as those of Example 1, except that the blank solution is methanol.

[0110] The test was carried out according to the chromatographic conditions of this comparative example, and the chromatogram was recorded. The test results showed that the elution time of the propyl ketone impurity in the system solution was 2.6 min, and the elution time of the chloroketone was 3.7 min. The separation degree between the two was 1.73, the impurity separation was incomplete, and there was an inverted peak after the blank solvent peak, so the propyl ketone impurity could not be accurately detected, and the tailing factor was about 0.8, and the tailing was serious.

[0111] Comparative Example 2

[0112] This comparative example provides a method for detecting propyl ketone impurities in nebivolol hydrochloride starting materials. The difference from Example 1 is that the chromatographic conditions are as follows:

[0113] Chromatographic column: octadecylsilane bonded silica gel (4.6 mm × 150 mm, 5 μm);

[0114] Mobile phase A: water, mobile phase B: acetonitrile;

[0115] Flow rate: 1.0 mL / min; detection wavelength: 254 nm; column temperature: 25°C; run time: 30 min; injection volume: 10 μL;

[0116] Blank solvent: acetonitrile; Elution method: gradient elution according to the program in Table 10 below.

[0117] Table 10 Gradient elution program

[0118] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 5 80 20 10 70 30 20 50 50 30 100 0

[0119] The preparation methods of the test solution, the reference solution and the system suitability solution are the same as those of Example 1, except that the blank solution is acetonitrile.

[0120] The detection was carried out according to the chromatographic conditions in this comparative example, and the chromatogram was recorded. The detection results showed that the elution time of the propyl ketone impurity in the system solution was 4.3 min, and the elution time of the chloroketone was 9.6 min. The separation degree between the two was 10.356, which was a good separation degree. However, the peak shapes of the two were poor and the tailing factors were about 0.5, and the peak tip of the main peak was forked.

[0121] Comparative Example 3

[0122] This comparative example provides a method for detecting propyl ketone impurities in nebivolol hydrochloride starting materials. The difference from Example 1 is that the chromatographic conditions are as follows:

[0123] Chromatographic column: octadecylsilane bonded silica gel (4.6 mm × 150 mm, 5 μm);

[0124] Mobile phase A: 0.1%wt phosphoric acid aqueous solution, mobile phase B: acetonitrile;

[0125] Flow rate: 1.0 mL / min; detection wavelength: 210 nm; column temperature: 25°C; run time: 30 min; injection volume: 10 μL; blank solvent: acetonitrile;

[0126] Elution method: Gradient elution was performed according to the procedure in Table 11 below.

[0127] Table 11 Gradient elution program

[0128] Time (min) Mobile phase A (%) Mobile phase B (%) 0 60 40 10 50 50 20 40 60 30 20 80

[0129] The preparation methods of the test solution, reference solution and system suitability solution are the same as those of Example 1, except that the blank solvent is acetonitrile.

[0130] The detection was carried out according to the chromatographic conditions in this comparative example, and the chromatogram was recorded. The detection results showed that the separation degree between the propyl ketone impurity and the chloroketone in the system solution was 15.273, and the separation degree was good, but the retention time of the main peak was 26 min, which was too late and affected the detection efficiency; the peak shapes of the two were good, and the tailing factor was about 0.9.

Claims

1. A method for detecting propyl ketone impurities in nebivolol hydrochloride starting materials, characterized in that: The nebivolol hydrochloride starting material is 6-fluoro-3,4-dihydro-2H-benzopyran-2-methanol-2-yl ethyl ketone chloride, and the nebivolol hydrochloride starting material contains propyl ketone impurity, and the propyl ketone impurity is 1-chloro-3-(5-fluoro-2,3-dihydro-benzofuran-3-1)-2-propyl ketone, and its structure is as follows: ； The detection method is to use high performance liquid chromatography, with 0.1%wt trifluoroacetic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, and perform isocratic elution on the propyl ketone standard and the starting material sample respectively according to the following chromatographic conditions: based on the volume of mobile phase A and mobile phase B, 0-30 min, 40% mobile phase A: 60% mobile phase B; obtain a standard curve and calculate the content of propyl ketone impurities in the starting material sample.

2. The method for detecting propyl ketone impurities in a nebivolol hydrochloride starting material according to claim 1, characterized in that: The steps include: (1) Establishment of the standard curve of propyl ketone impurity: Take the propyl ketone impurity reference substance and add it to the blank solution to prepare the reference solution; Prepare a series of concentrations of reference solution, inject them into a high performance liquid chromatograph respectively, perform detection according to the chromatographic conditions of claim 1, measure the chromatographic peak area, and obtain a standard curve of propyl ketone impurity; (2) Determination of the content of propyl ketone impurity in the starting material of nebivolol hydrochloride: Take a sample of the nebivolol hydrochloride starting material to be tested, add blank solution to dissolve it, and obtain a test solution; Take the test solution, inject it into the high performance liquid chromatograph, detect it under the same chromatographic conditions as in (1), and calculate it according to the standard curve obtained in (1) to obtain the content of the propyl ketone impurity in the nebivolol hydrochloride starting material sample to be tested; Wherein, the blank solution is an acetonitrile aqueous solution with a volume fraction of 30% to 50%.

3. The method for detecting propyl ketone impurities in a nebivolol hydrochloride starting material according to claim 2, characterized in that: The blank solution is an acetonitrile aqueous solution with a volume fraction of 40%.

4. The method for detecting propyl ketone impurities in a nebivolol hydrochloride starting material according to claim 2, characterized in that: When the high performance liquid chromatography method is used to detect the propyl ketone impurity in the nebivolol hydrochloride starting material, the chromatographic column used is octadecylsilane bonded silica gel, and its specifications are: 4.6 mm×150 mm, 5 μm.

5. The method for detecting propyl ketone impurities in a nebivolol hydrochloride starting material according to claim 4, characterized in that: The chromatographic column is selected from: any one of Thermo Hypersil GOLD C18, Thermo BDS C18, and ThermoSyncronis C18.

6. The method for detecting propyl ketone impurities in a nebivolol hydrochloride starting material according to claim 2, characterized in that: The chromatographic conditions for the HPLC detection are: During detection, the flow rate of mobile phase A and mobile phase B is: 0.8~1.5 mL / min; detection wavelength: 210 nm; column temperature: 25~35℃; injection volume: 10 μL.

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