A kind of detection method of teprenone

Abstract

The present invention relates to a detection method of teprenone, including a test solution: taking teprenone, accurately weighing, adding acetonitrile to dilute to make a solution with a concentration of 0.8-1.2mg / ml; a reference solution: taking a degradation impurity mixture, accurately weighing, adding acetonitrile to dilute to make a solution with a concentration of 0.8-1.2µg / ml; using pentafluorophenyl chemically bonded silica gel as a chromatographic column filler, using an ultraviolet detector, and a detection wavelength of 205nm-220nm; the mobile phase is acetonitrile-methanol-water; the flow rate is 0.8-1.2ml / min; the injection volume is 5-30µl; respectively measuring the reference solution and the test solution, injecting into a liquid chromatograph, injecting and recording a chromatogram, and calculating the content of each related substance. The present invention has strong specificity, can effectively separate the main degradation impurities from the main component, and has high sensitivity, good precision, stable solution, accurate and reliable, and can be applied to the impurity control of teprenone.

Description

Technical Field

[0001] The invention belongs to the field of medical technology and relates to a drug analysis and detection method, in particular to a teprenone detection method. Background Art

[0002] Teprenone, the English name is Teprenone, the chemical name is a mixture of mono-cis (5Z, 9E, 13E) and all-trans (5E, 9E, 13E) isomers of 6,10,14,18-tetramethyl-5,9,13,17-nonadecatetraen-2-one, with a molecular formula of C 23 H 38 O, molecular weight is 330.55, CAS number is 6809-52-5, chemical structure is as follows: .

[0003] It can be used to improve gastric mucosal lesions (erosion, bleeding, flushing, edema) in acute gastritis and acute exacerbation of chronic gastritis, and to treat gastric diseases such as gastric ulcer.

[0004] At present, teprenone is only included in the Japanese Pharmacopoeia. According to the literature report on page 5 of the IF file of Japan Eisai Teprenone Capsules, the main degradation impurities in teprenone are 6,10,14,18-tetramethyl-9,10-epoxy-5,13,17-nonadecatriene-2-one, 6,10,14,18-tetramethyl-13,14-epoxy-5,9,17-nonadecatriene-2-one, 6,10,14,18-tetramethyl-17,18-epoxy-5,9,13-nonadecatriene-2-one, and 6,10,14,18-tetramethyl-5,6-epoxy-9,13,17-nonadecatriene-2-one. The four impurities are not controlled in the pharmacopoeia standards.

[0005] Therefore, developing a detection method for teprenone with strong specificity and obvious advantages, which can effectively separate the main degradation impurities from the main components, and has high sensitivity, good precision, stable solution, accuracy and reliability, can be applied to the impurity control of teprenone, and provide a basis for teprenone quality research and impurity detection, has become a technical problem that urgently needs to be solved in this technical field. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for detecting teprenone, which has strong specificity, obvious advantages, can effectively separate the main degradation impurities from the main components, and has high sensitivity, good precision, stable solution, accuracy and reliability, and can be applied to the impurity control of teprenone.

[0007] The present invention solves the technical problem by adopting the following technical solutions:

[0008] A method for detecting teprenone comprises the following steps:

[0009] S1. Test solution: Take teprenone, weigh accurately, and dilute with acetonitrile to make a solution with a concentration of 0.8-1.2 mg / ml;

[0010] S2, reference solution: take the degradation impurity mixture, accurately weigh it, and dilute it with acetonitrile to make a solution with a concentration of 0.8-1.2µg / ml;

[0011] S3, using pentafluorophenyl chemically bonded silica gel as the chromatographic column filler, using an ultraviolet detector, the detection wavelength is 205nm~220nm; the mobile phase is acetonitrile-methanol-water; the flow rate is 0.8~1.2ml / min; the injection volume is 5~30μl;

[0012] S4. Take reference solution and test solution respectively, inject them into liquid chromatograph, inject and record the chromatogram, and calculate the content of each relevant substance.

[0013] Moreover, the degradation impurity mixture is a mixture of four degradation impurities: 6,10,14,18-tetramethyl-9,10-epoxy-5,13,17-nonadecatriene-2-one, 6,10,14,18-tetramethyl-13,14-epoxy-5,9,17-nonadecatriene-2-one, 6,10,14,18-tetramethyl-17,18-epoxy-5,9,13-nonadecatriene-2-one and 6,10,14,18-tetramethyl-5,6-epoxy-9,13,17-nonadecatriene-2-one.

[0014] Moreover, the molar ratio of the 6,10,14,18-tetramethyl-9,10-epoxy-5,13,17-nonadecatrien-2-one, 6,10,14,18-tetramethyl-13,14-epoxy-5,9,17-nonadecatrien-2-one, 6,10,14,18-tetramethyl-17,18-epoxy-5,9,13-nonadecatrien-2-one and 6,10,14,18-tetramethyl-5,6-epoxy-9,13,17-nonadecatrien-2-one is 30-45:20-35:15-35:5-15.

[0015] Moreover, the detection wavelength is 210 nm.

[0016] Moreover, the ratio of acetonitrile, methanol and water in the mobile phase is 450-500:450-500:45-55.

[0017] Moreover, the ratio of acetonitrile, methanol and water in the mobile phase is 475:475:50.

[0018] Furthermore, the flow rate was 1.0 ml / min.

[0019] Furthermore, the injection volume was 20 µl.

[0020] Moreover, the chromatographic column uses pentafluorophenyl chemically bonded silica gel as chromatographic column filler.

[0021] A method for preparing pentafluorophenyl chemically bonded silica gel, characterized in that it comprises the following steps:

[0022] (1) Soak the silica gel in 0.1-1 mol / L dilute hydrochloric acid for 20-24 hours, then wash with deionized water until neutral, and dry at 100-200°C for 8-12 hours to complete the pretreatment of the silica gel;

[0023] (2) dissolving pentafluorophenyltrimethoxysilane in toluene, mixing the pretreated silica gel and pentafluorophenyltrimethoxysilane solution in a mass ratio of 1:5-1:20, adding catalyst triethylamine, and reacting at 60-150°C for 8-12 hours;

[0024] (3) After the reaction is completed, the product is washed with methanol and dried at low temperature to obtain a pentafluorophenyl chemically bonded silica gel filler.

[0025] The advantages and positive effects of the present invention are:

[0026] 1. The present invention identifies four impurities in view of the degradation of teprenone, and analyzes them as 6,10,14,18-tetramethyl-9,10-epoxy-5,13,17-nonadecatriene-2-one, 6,10,14,18-tetramethyl-13,14-epoxy-5,9,17-nonadecatriene-2-one, 6,10,14,18-tetramethyl-17,18-epoxy-5,9,13-nonadecatriene-2-one, and 6,10,14,18-tetramethyl-5,6-epoxy-9,13,17-nonadecatriene-2-one; by screening the chromatographic conditions, the four impurities are merged into two chromatographic peaks for control, and finally a method for analyzing related substances is established, the analysis cycle is controlled at 20 minutes, and the main component and the degradation impurities can be effectively separated.

[0027] 2. The detection method of teprenone of the present invention has strong specificity and obvious advantages, can effectively separate the main degradation impurities from the main components, and has high sensitivity, good precision, stable solution, accuracy and reliability, and can be applied to the impurity control of teprenone.

[0028] 3. The detection method of teprenone of the present invention can accurately determine the impurities of 6,10,14,18-tetramethyl-9,10-epoxy-5,13,17-nonadecatriene-2-one, 6,10,14,18-tetramethyl-13,14-epoxy-5,9,17-nonadecatriene-2-one, 6,10,14,18-tetramethyl-17,18-epoxy-5,9,13-nonadecatriene-2-one and 6,10,14,18-tetramethyl-5,6-epoxy-9,13,17-nonadecatriene-2-one in teprenone, providing a basis for the quality research and impurity detection of teprenone.

[0029] 4. The detection method of teprenone of the present invention uses pentafluorophenyl chemically bonded silica gel as a chromatographic column filler, and the chromatographic column filler has a strong retention and separation ability for oxidative degradation impurities in the product. At the same time, pentafluorophenyl has a strong hydrophobicity, which makes the chromatographic column have a good retention effect on hydrophobic compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a solution chromatogram of the separation degree of impurities of teprenone degradation in Example 1 of the present invention.

[0031] Figure 2 The chromatogram of the sample of teprenone-related substances in Example 1 of the present invention.

[0032] Figure 3 This is a linear result diagram of degradation of impurities in Example 1 of the present invention.

[0033] Figure 4 This is a chromatogram of the sample of teprenone-related substances in Example 4 of the present invention. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below through specific examples. The following examples are only illustrative and not restrictive, and the protection scope of the present invention cannot be limited thereto. Example 1

[0035] A method for detecting teprenone comprises the following steps:

[0036] S1. Test solution: Take teprenone, the API produced by Lianlong (Tianjin) Pharmaceutical Co., Ltd., and quantitatively dilute it with acetonitrile to prepare a solution containing 1 mg of teprenone per 1 ml;

[0037] S2. Reference solution: Take the degradation impurities, weigh accurately, dilute with acetonitrile to make a solution containing 1µg of degradation impurities per 1ml (for calculating the content of degradation impurities in teprenone);

[0038] S3, pentafluorophenyl chemically bonded silica gel was used as the chromatographic column filler, an ultraviolet detector was used, and the detection wavelength was 210nm; the mobile phase was acetonitrile-methanol-water (475:475:50); the flow rate was 1.0ml / min; the injection volume was 20µl;

[0039] S4. Take 20µl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, inject and record the chromatogram, and calculate the content of each degradation impurity.

[0040] The results are shown in Table 1.

[0041] Table 1 Determination results of teprenone degradation impurities

[0042]

[0043] 1. Tests on specificity of relevant substances

[0044] Blank solution, teprenone test solution and separation test solution (mixed solution of degradation impurities and teprenone) were prepared respectively, injected into liquid chromatograph, and chromatograms were recorded. The results are shown in Table 2.

[0045] Table 2 Separation results of teprenone and degradation impurities

[0046]

[0047] The results show: See Figure 1-Figure 2 , Figure 1 The chromatographic peak results are shown in Table 2-1. Figure 2 The chromatographic peak results are shown in Table 2-2. It can be seen that teprenone and degradation impurities, and degradation impurities and adjacent impurities can be effectively separated.

[0048] Table 2-1 Chromatographic peak results

[0049]

[0050] Table 2-2 Chromatographic peak results

[0051]

[0052] 2. Detection limit and quantification limit test

[0053] The signal-to-noise ratio method was used to determine the detection limit and quantification limit of degradation impurities. An appropriate amount of degradation impurity reference substance was taken and diluted to a certain concentration with acetonitrile. When the signal-to-noise ratio was ≥10, it was the quantification limit, and when the signal-to-noise ratio was ≥3, it was the detection limit. The results are shown in Table 3.

[0054] Table 3 Results of quantitation limit and detection limit tests

[0055]

[0056] The results showed that the quantitative limit and detection limit of degradation impurities met the determination requirements and the method had high sensitivity.

[0057] 3. Linearity test

[0058] Weigh an appropriate amount of degradation impurity reference solution, dilute it with acetonitrile, prepare solutions of different concentrations, inject them respectively and record the chromatograms, with concentration as the abscissa and peak area as the ordinate, to obtain the linear regression equation of the degradation impurities. The results are shown in Table 4.

[0059] Table 4 Degradation impurity linearity test results

[0060]

[0061] The results showed that the degradation impurities were in the range of 0.19μg / ml to 9.56μg / ml, indicating a good linear relationship.

[0062] 4. Accuracy test

[0063] Reference solution: Take an appropriate amount of degradation impurity reference, dissolve it in acetonitrile and dilute it to make solutions with concentrations of approximately 0.8 μg / ml, 1 μg / ml and 1.2 μg / ml respectively.

[0064] 80% accuracy solution: Take about 20 mg of teprenone, accurately weigh it, put it in a 20 ml volumetric flask, add 0.8 μg / ml reference solution and dilute to the scale, shake well, and prepare 3 solutions in the same way.

[0065] 100% accuracy solution: Take about 20 mg of teprenone, weigh accurately, place in a 20 ml volumetric flask, add 1 μg / ml reference solution and dilute to the scale, shake well, and prepare 3 solutions in the same way.

[0066] 120% accuracy solution: Take about 20 mg of teprenone, accurately weigh it, put it in a 20 ml volumetric flask, add 1.2 μg / ml reference solution and dilute to the scale, shake well, and prepare 3 solutions in the same way.

[0067] 20 μl of each recovery solution of each concentration was measured and injected into the liquid chromatograph, and the chromatogram was recorded. The recovery rate was calculated by dividing the measured amount of impurities by the added amount. The results are shown in Table 5;

[0068] Table 5 Accuracy test results

[0069]

[0070] The results showed that the average recovery rate of degradation impurities was between 90% and 110%, and the average RSD was less than 10%, indicating that the method had high accuracy. Example 2

[0071] A method for detecting teprenone comprises the following steps:

[0072] S1. Test solution: Take teprenone, the API produced by Lianlong (Tianjin) Pharmaceutical Co., Ltd., and quantitatively dilute it with acetonitrile to prepare a solution containing 1 mg of teprenone per 1 ml;

[0073] S2, reference solution: take the degradation impurity, weigh it accurately, dilute it with acetonitrile to make a solution containing 1µg of degradation impurity per 1ml;

[0074] S3, pentafluorophenyl chemically bonded silica gel was used as the chromatographic column filler, an ultraviolet detector was used, and the detection wavelength was 205nm; the mobile phase was acetonitrile-methanol-water (450:450:100); the flow rate was 0.8ml / min; the injection volume was 5µl;

[0075] S4. Take 5µl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, inject and record the chromatogram, and calculate the content of each degradation impurity.

[0076] The results are shown in Table 6.

[0077] Table 6 Determination results of teprenone degradation impurities

[0078] Example 3

[0079] A method for detecting teprenone comprises the following steps:

[0080] S1. Test solution: Take teprenone, the API produced by Lianlong (Tianjin) Pharmaceutical Co., Ltd., and quantitatively dilute it with acetonitrile to prepare a solution containing 1 mg of teprenone per 1 ml;

[0081] S2, reference solution: take the degradation impurity, weigh it accurately, dilute it with acetonitrile to make a solution containing 1µg of degradation impurity per 1ml;

[0082] S3, pentafluorophenyl chemically bonded silica gel was used as the chromatographic column filler, an ultraviolet detector was used, and the detection wavelength was 220nm; the mobile phase was acetonitrile-methanol-water (490:490:10); the flow rate was 1.2ml / min; the injection volume was 30µl;

[0083] S4. Take 30µl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, inject and record the chromatogram, and calculate the content of each degradation impurity.

[0084] The results are shown in Table 7.

[0085] Table 7 Determination results of teprenone degradation impurities

[0086]

[0087] The results show that: referring to Examples 1-3, if the chromatographic conditions are adjusted, there is basically no effect on the measurement results. Example 4

[0088] A method for detecting teprenone comprises the following steps:

[0089] S1. Test solution: Take teprenone, the API produced by Lianlong (Tianjin) Pharmaceutical Co., Ltd., and quantitatively dilute it with acetonitrile to prepare a solution containing 1 mg of teprenone per 1 ml;

[0090] S2, reference solution: take the degradation impurity, weigh it accurately, dilute it with acetonitrile to make a solution containing 1µg of degradation impurity per 1ml;

[0091] S3, octadecylsilane bonded silica gel was used as the column filler, UV detector was used, detection wavelength was 210nm; mobile phase was acetonitrile-methanol-water (475:475:50); flow rate was 1.0ml / min; injection volume was 20µl;

[0092] S4. Take 20µl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, inject and record the chromatogram. The unknown impurities in the test solution interfere with the determination of degradation impurities.

[0093] The results show: See Figure 4 As shown in the figure, 13.630 is an oxidative degradation impurity and 14.510 is an unknown impurity. This indicates that when octadecylsilane bonded silica gel is used as the chromatographic column filler, the separation degree between oxidative degradation impurities and unknown impurities is low.

[0094] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for detecting teprenone, characterized in that: The steps include: S1. Test solution: Take teprenone, accurately weigh it, and dilute it with acetonitrile to make a solution with a concentration of 0.8-1.2 mg / ml; S2, reference solution: take the degradation impurity mixture, accurately weigh it, and dilute it with acetonitrile to make a solution with a concentration of 0.8-1.2µg / ml; S3, using pentafluorophenyl chemically bonded silica gel as the chromatographic column filler, using an ultraviolet detector, the detection wavelength is 205nm~220nm; the mobile phase is acetonitrile-methanol-water; the flow rate is 0.8~1.2ml / min; the injection volume is 5~30μl; S4. Take the reference solution and the test solution respectively, inject them into the liquid chromatograph, inject and record the chromatogram, and calculate the content of degradation impurities; The degradation impurity mixture is a mixture of four degradation impurities: 6,10,14,18-tetramethyl-9,10-epoxy-5,13,17-nonadecatriene-2-one, 6,10,14,18-tetramethyl-13,14-epoxy-5,9,17-nonadecatriene-2-one, 6,10,14,18-tetramethyl-17,18-epoxy-5,9,13-nonadecatriene-2-one and 6,10,14,18-tetramethyl-5,6-epoxy-9,13,17-nonadecatriene-2-one.

2. The method for detecting teprenone according to claim 1, wherein: The molar ratio of the 6,10,14,18-tetramethyl-9,10-epoxy-5,13,17-nonadecatriene-2-one, 6,10,14,18-tetramethyl-13,14-epoxy-5,9,17-nonadecatriene-2-one, 6,10,14,18-tetramethyl-17,18-epoxy-5,9,13-nonadecatriene-2-one and 6,10,14,18-tetramethyl-5,6-epoxy-9,13,17-nonadecatriene-2-one is 30-45:20-35:15-35:5-15.

3. The method for detecting teprenone according to claim 1, characterized in that: The detection wavelength is 210 nm.

4. The method for detecting teprenone according to claim 1, wherein: The ratio of acetonitrile, methanol and water in the mobile phase is 450-500:450-500:45-55.

5. The method for detecting teprenone according to claim 4, characterized in that: The ratio of acetonitrile, methanol and water in the mobile phase is 475:475:

50.

6. The method for detecting teprenone according to claim 1, characterized in that: The flow rate is 1.0 ml / min.

7. The method for detecting teprenone according to claim 1, characterized in that: The injection volume was 20 µl.

8. The method for detecting teprenone according to claim 1, characterized in that: The chromatographic column uses pentafluorophenyl chemically bonded silica gel as chromatographic column filler.

9. The method for detecting teprenone according to claim 8, characterized in that: The preparation method of pentafluorophenyl chemically bonded silica gel filler comprises the following steps: (1) Soak the silica gel in 0.1-1 mol / L dilute hydrochloric acid for 20-24 hours, then wash with deionized water until neutral, and dry at 100-200°C for 8-12 hours to complete the pretreatment of the silica gel; (2) dissolving pentafluorophenyltrimethoxysilane in toluene, mixing the pretreated silica gel and pentafluorophenyltrimethoxysilane solution in a mass ratio of 1:5-1:20, adding catalyst triethylamine, and reacting at 60-150°C for 8-12 hours; (3) After the reaction is completed, the product is washed with methanol and dried at low temperature to obtain a pentafluorophenyl chemically bonded silica gel filler.

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