Detection method of teprenone impurities

Abstract

The invention discloses a teprenone impurity detection method, which improves the separation capacity of various impurities by optimizing a chromatographic column and optimizing chromatographic conditions, has the advantages of strong specificity, high detection sensitivity, stability and reliability, and can be used for the production process of teprenone bulk drugs and related preparation products and the quality monitoring of finished products. High-sensitivity and high-accuracy qualitative and quantitative detection is carried out on various impurities.

Description

Technical Field

[0001] The invention relates to a method for controlling the quality of teprenone, and in particular to a method for detecting impurities of teprenone. Background Art

[0002] Teprenone is a mixture of mono-cis (5Z, 9E, 13E) and all-trans (5E, 9E, 13E) 6,10,14,18-tetramethylnonadecan-5,9,13,17-tetraen-2-one. It is a digestive system antacid and gastric mucosal protective drug. Clinically, it mainly improves gastric mucosal lesions such as erosion, bleeding, redness, edema and gastric ulcer.

[0003]

[0004] The preparation process of teprenone is prone to produce a variety of process by-products, degradation products, etc. These impurities can be transferred to the raw materials or preparations through the subsequent process flow. Therefore, they directly affect the safety and effectiveness of the drugs. At present, there is no effective quality control method for these impurities.

[0005] Summary of the invention

[0006] Purpose of the invention: The present invention aims to provide a method for simultaneously and effectively detecting multiple teprenone impurities.

[0007] Technical solution: The method for detecting teprenone impurities of the present invention comprises the following steps:

[0008] (1) Prepare samples;

[0009] (2) Detection: A polyethylene glycol capillary column modified with nitroterephthalic acid was used for detection. The heating program was as follows:

[0010] The initial temperature was 208-212°C, maintained for 33 minutes, then raised to 230°C at a rate of 2°C per minute and maintained for 37 minutes;

[0011] (3)Analysis results.

[0012] Wherein, the sample prepared in step (1) comprises a test solution, a control solution, and a separation solution.

[0013] The preparation method of the test solution is as follows:

[0014] Take an appropriate amount of the contents of the teprenone capsule (approximately equivalent to 50 mg of teprenone), accurately weigh, place in a 10 mL volumetric flask, add about 6 mL of solvent, ultrasonically shake for 5 minutes to dissolve the teprenone, cool, dilute to the scale with solvent, shake well, and filter to obtain (containing approximately 5 mg / mL of teprenone).

[0015] The preparation method of the separation solution is as follows:

[0016] Step 1: Take appropriate amounts of impurity A, impurity B, impurity C, impurity D, impurity E and impurity F reference substances, add solvent to dissolve and dilute to make a solution containing about 250 μg / mL of each impurity per 1 mL;

[0017] Step 2: Take about 25 mg of teprenone, weigh it accurately, put it in a 5 mL volumetric flask, add about 5 mL of solvent, shake ultrasonically for 5 minutes to dissolve teprenone, cool, accurately add 0.5 mL of the above impurity stock solution, dilute to the scale with solvent, shake well, filter, and obtain the separation solution (containing about 5 mg / mL of teprenone and 25 μg / mL of each impurity).

[0018] The solvent is n-hexane-tetrahydrofuran (7:3, V / V).

[0019] Preferably, the impurities of teprenone are as follows:

[0020]

[0021]

[0022] Preferably, step (2) is detected using an FID detector, the injection port temperature is 248-252° C., the detector temperature is 248-252° C., and the split ratio is 20:1.

[0023] More preferably, the starting temperature is 210°C, the injection port temperature is 250°C, and the detector temperature is 250°C.

[0024] Preferably, step (2) is detected in constant flow mode, the carrier gas is nitrogen, and the carrier gas flow rate is 0.7 to 1.1 mL / min.

[0025] More preferably, the carrier gas flow rate is 0.9 mL / min.

[0026] Preferably, the capillary column has a length of 60 m, a column diameter of 0.25 mm, and a filler particle size of 0.25 μm.

[0027] More preferably, the capillary column is NanoChrom BP-FFAP, 0.25 mm×60 m, 0.25 μm, or an equivalent chromatographic column.

[0028] Preferably, the detection injection volume of step (2) is 4 μL.

[0029] Preferably, the detection linear range of each detection component is as follows:

[0030]

[0031]

[0032] Further preferably, the detection linear equation of each detection component is as follows:

[0033]

[0034] Preferably, the correction factor of impurity D is 2.3, the correction factor of impurity E is 1.3, and the correction factor of the remaining detection components is 1.

[0035] Preferably, the step (3) uses the principal component self-control method with a correction factor to analyze the results.

[0036] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0037] The present invention improves the separation ability of various impurities by optimizing the chromatographic column and the chromatographic conditions. The method has strong specificity, high detection sensitivity, stability and reliability. It can be used for the production process of teprenone raw materials and related preparations and the quality monitoring of finished products, and can perform high-sensitivity and high-accuracy qualitative and quantitative analysis of various impurities therein. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Chromatographic analysis of the mixed solution in Example 1 Figure 1 ;

[0039] Figure 2 Chromatographic analysis of the mixed solution in Example 1 Figure 2 ;

[0040] Figure 3 The chromatogram of the separation solution in Example 2;

[0041] Figure 4 is the chromatogram of the blank solution in Example 3;

[0042] Figure 5 It is the chromatogram of the test solution in Example 3;

[0043] Figure 6 The chromatogram of the separation solution in Comparative Example 1;

[0044] Figure 7 This is the chromatogram of the separation solution in Comparative Example 2. DETAILED DESCRIPTION

[0045] The technical solution of the present invention is further described below in conjunction with embodiments.

[0046] Example 1: Determination of chromatographic column

[0047] The solvent for preparing the mixed solution was n-hexane-tetrahydrofuran (7:3, V / V).

[0048] Mixed solution: Take appropriate amounts of impurity A, impurity B, impurity C, impurity F reference substance and teprenone, add solvent to dissolve and dilute to prepare a mixed solution containing approximately 5 mg of teprenone and 400 μg of each impurity per 1 mL.

[0049] The chromatographic detection conditions are as follows:

[0050] The capillary column with nitroterephthalic acid-modified polyethylene glycol as the stationary phase was used as the chromatographic column; the detector was an FID detector; the injection port temperature was 250°C; the detector temperature was 250°C; the column temperature was 210°C; and the carrier gas was nitrogen. The effects of different specifications of chromatographic columns on the separation of various impurities were compared (chromatographic column 1: Agilent DB-FFAP, 0.53mm×30m, 1μm; chromatographic column 2: NanoChrom BP-FFAP, 0.25mm×60m, 0.25μm).

[0051] like Figure 1 As shown, when using chromatographic column 1, the separation degree of impurities A & C from impurity B is poor, and the separation degree is less than 1.0. Figure 2 As shown in the figure, when using chromatographic column 2, the separation between impurities A & C and impurity B is better, the separation is greater than 1.0, and the separation between the main peak and the adjacent impurities is greater than 1.0. Taking into account the separation between the main component and the adjacent impurity peaks and the separation between the impurities, chromatographic column 2, namely NanoChrom BP-FFAP chromatographic column, 60m×0.25mm, 0.25μm, was finally selected.

[0052] Example 2: Determination of detection method

[0053] The solvent for the preparation of the separation solution was n-hexane-tetrahydrofuran (7:3, V / V).

[0054] Separation solution: Take appropriate amounts of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and teprenone reference substance, add solvent to dissolve and dilute to prepare a mixed solution containing approximately teprenone: 5 mg, impurities: 25 μg each per 1 mL.

[0055] The chromatographic detection conditions are as follows:

[0056] The chromatographic column is a capillary column with polyethylene glycol modified with nitroterephthalic acid as the stationary liquid (NanoChromBP-FFAP, 60m×0.25mm, 0.25μm or similar polarity); the detector is an FID detector; the injection port temperature is 250℃; the detector temperature is 250℃; the split ratio is 20:1; the constant flow mode is adopted, the flow rate is 0.9mL / min, and the injection volume is 4μL; the carrier gas is nitrogen; the heating program: the initial column temperature is 210℃, maintained for 33min, and the temperature is increased to 230℃ at a heating rate of 2℃ / min and maintained for 37min.

[0057] like Figure 3 As shown, referring to the Japanese Pharmacopoeia standard, impurity A and impurity C overlap and are combined for control; the separation degrees of the remaining impurities are all >1.0, and the peak shapes are good.

[0058] Example 3: Determination of Related Substances in Teprenone Capsules

[0059] 1. Gas chromatography conditions

[0060] The chromatographic column is a capillary column with nitroterephthalic acid-modified polyethylene glycol as the stationary liquid (NanoChromBP-FFAP, 60m×0.25mm, 0.25μm or a chromatographic column with similar polarity); the detector is an FID detector; the injection port temperature is 250℃; the detector temperature is 250℃; the split ratio is 20:1; the constant flow mode is adopted, the flow rate is 0.9mL / min, and the injection volume is 4μL; the carrier gas is nitrogen; the heating program: the initial column temperature is 210℃, maintained for 33min, and the temperature is increased to 230℃ at a heating rate of 2℃ / min and maintained for 37min.

[0061] 2. Solution preparation

[0062] Solvent, blank solution: n-hexane-tetrahydrofuran (7:3, V / V).

[0063] Test solution: Take an appropriate amount of the contents of teprenone capsules (equivalent to about 50 mg of teprenone), place in a 10 mL volumetric flask, add about 6 mL of solvent, shake ultrasonically for 5 min to dissolve teprenone, cool, dilute to the scale with solvent, shake well, and filter to obtain.

[0064] Control solution: Accurately measure 1 mL of the test solution, place it in a 100 mL volumetric flask, dilute to the scale with solvent, shake well, and obtain.

[0065] Sensitivity solution: Accurately measure 1 mL of the control solution, place it in a 10 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0066] Accurately measure 4 μL of each of the above solutions, inject them into the gas chromatograph, record the chromatogram, and calculate according to the main component self-reference method.

[0067] 3. System suitability investigation

[0068] The blank solution, control solution and test solution were measured. Figure 4-5 As shown, using the chromatographic conditions of the present invention, the blank solution has no interference, and the main component and impurities as well as the impurities can be effectively separated, indicating that the method has good system applicability.

[0069] 4. Sensitivity test

[0070] The main component (mono-cis (5Z, 9E, 13E) and all-trans isomers (5E, 9E, 13E) of 6,10,14,18-tetramethylnonadecan-5,9,13,17-tetraen-2-one) reference substance and impurity reference substance stock solution were prepared with reference to Example 1. An appropriate amount of each impurity reference substance stock solution was accurately measured, and the solution was gradually diluted and determined with a solvent. The signal-to-noise ratio was about 10:1 as the quantitative limit, and the detection sensitivity of each impurity was investigated. As shown in Table 1, the sensitivity of this method is good.

[0071] Table 1 Sensitivity investigation results

[0072]

[0073] 5. Linear inspection

[0074] The main component (single cis (5Z, 9E, 13E) and all-trans isomers (5E, 9E, 13E) of 6,10,14,18-tetramethylnonadecan-5,9,13,17-tetraene-2-one) reference substance and impurity reference substance stock solution were prepared with reference to Example 1. An appropriate amount of each impurity reference substance stock solution was accurately measured and diluted with a solvent to prepare a series of concentrations as linear solutions of each component. As shown in Table 2, the chromatographic conditions of the present invention were used, and the linear relationship of each component to be measured was good.

[0075] Table 2 Linearity test results

[0076]

[0077]

[0078] 6. Correction Factor

[0079] The calibration factor was determined using the standard curve method and the results are shown in Table 3.

[0080] Table 3 Correction factors

[0081] / Slope Correction Factor Determine the correction factor Principal component 1 80.3819 / / Principal component 2 79.6745 1.009 1.0 Impurity A & Impurity C 71.2705 1.128 1.0 Impurity B 73.2285 1.098 1.0 Impurity D 34.3246 2.342 2.3 Impurity E 60.5841 1.327 1.3 Impurity F 72.7498 1.105 1.0

[0082] 7. Solution stability study

[0083] At different times, 4 μL of the separation solution, test solution and control solution were injected into the gas chromatograph and the chromatogram was recorded. The results are shown in Tables 4 to 5. The test solution was measured at room temperature at 0h, 45h and 68h, and the absolute difference between the impurity content at each time point and 0h was less than 0.03%, and no new impurities ≥ 0.03% appeared. It shows that the test solution is stable for at least 68h at room temperature. The separation solution was measured at room temperature at 0h, 45h and 68h, and at each time point, the ratio of the peak area of ​​each impurity peak in the separation solution to the peak area of ​​0h was between 80% and 120%. It shows that the separation solution is stable for at least 68h at room temperature. The control solution was measured at room temperature at 0h, 45h and 72h, and at each time point, the ratio of the peak area of ​​each impurity peak in the control solution to the peak area of ​​0h was between 80% and 120%. It shows that the control solution is stable for at least 72h at room temperature.

[0084] Table 4 Stability test of relevant substance solutions - test solution

[0085]

[0086] Table 5 Stability test of related substance solutions - separation solution

[0087]

[0088] 8. Accuracy test

[0089] Accurately weigh 3 portions of teprenone samples, add impurity reference solutions with impurity limits of 50%, 100%, and 150%, respectively, dissolve with solvent and dilute to the scale as test solutions. Accurately measure each test solution and dilute 1000 times as control solutions. Accurately measure 4 μL of the above test solution and control solution and inject them into the gas chromatograph, record the chromatogram, and calculate the recovery rate of each impurity [(measured amount-background amount) / added amount]. As shown in Table 6, the recovery rates of each impurity are between 80% and 120%, and the RSD is less than 10.0%, indicating that this method has good accuracy and is suitable for the detection of related substances of this product.

[0090] Table 6 Accuracy calculation table

[0091]

[0092] In summary, the quality control method for the related substances of teprenone capsules of the present invention has strong specificity, high sensitivity, good linear relationship and high accuracy.

[0093] Comparative Example 1

[0094] Solvent and diluent for the preparation of separation solution: n-hexane-tetrahydrofuran (7:3, V / V).

[0095] Separation solution: Take appropriate amounts of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and teprenone reference substance, add solvent to dissolve and dilute to prepare a mixed solution containing approximately teprenone: 5 mg, impurities: 25 μg each per 1 mL.

[0096] The chromatographic detection conditions are as follows:

[0097] Chromatographic column: NanoChrom BP-FFAP, 0.25mm×60m, 0.25μm; detector: FID detector; injection port temperature: 250℃; detector temperature: 250℃; split ratio: 20:1; column temperature: 235℃, constant flow mode, flow rate: 1.0mL / min, injection volume: 4μL; carrier gas: nitrogen. Take the separation solution for injection.

[0098] like Figure 6 As shown, the separation between the main peak and the adjacent impurities is good, impurity A overlaps with impurity C, and the separation between it and impurity B is greater than 1.0. The separation between impurity D and impurity E is 0.95, which is less than 1.0, and the separation does not meet the requirements.

[0099] Comparative Example 2

[0100] Solvent and diluent for the preparation of separation solution: n-hexane-tetrahydrofuran (7:3, V / V).

[0101] Separation solution: Take appropriate amounts of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and teprenone reference substance, add solvent to dissolve and dilute to prepare a mixed solution containing approximately teprenone: 5 mg, impurities: 25 μg each per 1 mL.

[0102] The chromatographic detection conditions are as follows:

[0103] Chromatographic column: NanoChrom BP-FFAP, 0.25mm×60m, 0.25μm; detector: FID detector; injection port temperature: 250℃; detector temperature: 250℃; split ratio: 20:1; constant flow mode, flow rate: 1.3mL / min, injection volume: 4μL; carrier gas: nitrogen; heating program: starting column temperature: 210℃, maintained for 40min, heated to 230℃ at a heating rate of 10℃ / min, maintained for 30min. Take the separation solution for injection.

[0104] like Figure 7 As shown, the main peak has good separation from adjacent impurities. Impurity A overlaps with impurity C, and the separation between it and impurity B is greater than 1.0. The responses of impurity D and impurity E are increased, and the signal-to-noise ratios are 360 ​​and 315, respectively. However, the separation between the two peaks is only 0.68, which is less than 1.0.

Claims

1. A method for detecting impurities of teprenone, characterized in that: The following steps are involved: (1) Prepare samples; (2) Detection: A polyethylene glycol capillary column modified with nitroterephthalic acid was used for detection. The heating program was as follows: The initial temperature was 208-212°C, maintained for 33 minutes, then raised to 230°C at a rate of 2°C per minute and maintained for 37 minutes; (3)Analysis results.

2. The detection method according to claim 1, characterized in that: The impurities of teprenone are as follows:

3. The detection method according to claim 1, characterized in that: Step (2) is detected by using an FID detector, the injection port temperature is 248-252°C, the detector temperature is 248-252°C, and the split ratio is 20:

1.

4. The detection method according to claim 1 or 3, characterized in that: The starting temperature is 210°C, the injection port temperature is 250°C, and the detector temperature is 250°C.

5. The detection method according to claim 1, characterized in that: Step (2) is detected in constant flow mode, the carrier gas is nitrogen, and the carrier gas flow rate is 0.7 to 1.1 mL / min.

6. The detection method according to claim 5, characterized in that: The carrier gas flow rate is 0.9 mL / min.

7. The detection method according to claim 1, characterized in that: The capillary column has a length of 60 m, a column diameter of 0.25 mm, and a filler particle size of 0.25 μm.

8. The detection method according to claim 7, characterized in that: The capillary column is NanoChrom BP-FFAP, 0.25 mm×60 m, 0.25 μm or an equivalent chromatographic column.

9. The detection method according to claim 1, characterized in that: The detection injection volume of step (2) is 4 μL.

10. The detection method according to claim 1, characterized in that: The linear range of each test component is as follows:

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