Method for determining content of ethyl hydroxide isomer in levonorgestrel starting material

Abstract

The invention discloses a method for determining the content of an ethyl hydroxide isomer in a levonorgestrel starting material, and belongs to the technical field of medicines. The method comprises the following steps: firstly, preparing a test solution and a contrast solution for later use; then setting high performance liquid detection conditions, taking amylose covalently bonded on the surface of silica gel as a filling agent, taking n-hexane as a mobile phase A, taking isopropanol as a mobile phase B, carrying out elution at the flow rate of 1.0 mL / min and the column temperature of 30 DEG C; the detection wavelength is 265 nm; taking 10 [mu] L of the test solution, injecting the test solution into a liquid chromatograph, precisely measuring 10 [mu] L of the reference solution, injecting the reference solution into the liquid chromatograph, and recording a chromatogram. According to the present invention, with the high performance liquid chromatography method, the three isomers of the ethyl hydroxylate as the levonorgestrel starting material can be rapidly, effectively, accurately and reliably separated and detected, such that the levonorgestrel product quality can be easily improved, and the medication safety of patients can be improved.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a method for determining the content of ethyl hydroxylate isomers of a levonorgestrel starting material. Background Art

[0002] Ethyl hydroxylate, chemically named (2R,3S)-2-ethyl-3-hydroxy-2(2-(6-methoxy-3,4-dihydro-1(2H)-naphthalenylidene)ethyl)cyclopentanone, is the starting material for the synthesis of levonorgestrel API. From its molecular structure, we can see the chiral carbons at positions 13 and 17. Therefore, establishing an analytical method for the isomers of ethyl hydroxylate for quality control is of great significance for the production of levonorgestrel API and preparations. Summary of the invention

[0003] The object of the present invention is to provide a method for determining the content of ethyl hydroxylate isomers of a levonorgestrel starting material, by adopting a high performance liquid chromatography method, the ethyl hydroxylate isomers of the levonorgestrel starting material can be separated and detected, which is beneficial to improving the product quality of levonorgestrel and improving the safety of medication for patients.

[0004] In order to solve the above problems, the technical solution adopted by the present invention is as follows:

[0005] A method for determining the content of ethyl hydroxylate isomers of levonorgestrel starting material, comprising the following steps:

[0006] 1) Prepare test solution, reference solution and mixed solution;

[0007] 2) Setting the HPLC detection conditions: the chromatographic column is filled with amylose covalently bonded to the surface of silica gel, the size is 250 mm × 4.6 mm, the filler particle size is 5 μm, the column temperature is 30-35°C, n-hexane is used as mobile phase A, isopropanol is used as mobile phase B, and gradient elution is performed;

[0008] 3) Take reference solution and mixed solution respectively, inject into liquid chromatograph, record chromatogram, and calculate the content of ethyl hydroxylate isomers.

[0009] Furthermore, the preparation method of the test solution is: take 10 mg of ethyl hydroxylate and place it in a 20 mL volumetric flask, add a solvent to dissolve it, dilute it to a fixed volume, and mix it to obtain the test solution; the preparation method of the reference solution is: measure 1 mL of the test solution and place it in a 100 mL volumetric flask, dilute it to the scale with a solvent, shake it well, and use it as a self-control solution; the preparation method of the mixed solution is: take ethyl hydroxylate and ethyl hydroxylate racemic body, dissolve and dilute it with a solvent to prepare a mixed solution containing 0.5 mg / mL of ethyl hydroxylate and 10 μg / mL of each isomer.

[0010] Furthermore, in the preparation process of the test solution, the reference solution and the mixed solution, the solvent used is ethanol.

[0011] Furthermore, in the step 2), the column temperature is 30° C. and the flow rate is 0.6 to 1.0 mL / min.

[0012] Furthermore, in the step 2), the flow rate is 1.0 mL / min.

[0013] Furthermore, in the HPLC detection conditions, the detection wavelength of the chromatograph detector is 265 nm.

[0014] Furthermore, the volume ratio of n-hexane to isopropanol in the mobile phase of the HPLC test is 70:30-95:5.

[0015] Furthermore, in the step 3), the reference solution measured is 10 μL; the mixed solution measured is 10 μL.

[0016] Furthermore, in the step 3), 10 μL of the reference solution is taken and injected into the liquid chromatograph, and the detection sensitivity is adjusted so that the peak height of the main component peak is about 10% to 20% of the full scale, and then 10 μL of the mixed solution is measured and injected into the liquid chromatograph to record the chromatogram.

[0017] Furthermore, if there is an ethyl hydroxylate isomer peak in the chromatogram, the sum of the peak areas of the three isomers in the ethyl hydroxylate is no greater than half of the main peak area of ​​the control solution, i.e., 0.5%.

[0018] Compared with the prior art, the advantages of the present invention are as follows:

[0019] 1) The present invention adopts high performance liquid chromatography, which can quickly, effectively, accurately and reliably separate and detect the ethyl hydroxylate isomer of the levonorgestrel starting material, which is beneficial to improving the product quality of levonorgestrel and improving the safety of patient medication.

[0020] 2) Under the chromatographic conditions adopted by the present invention, the main component and the three isomers can be well separated, the ISO-1 separation degree is 3.2, the ISO-2 separation degree is 3.0, the main component separation degree is 2.3, the ISO-3 separation degree is 3.5, and the blank solvent has no interference.

[0021] 3) The sample injection precision of the present invention is good, and the peak area RSD of ethyl hydroxylate and ethyl hydroxylate isomers is ≤2.0%. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The chromatogram is tested under the chromatographic conditions of the present invention, wherein: Figure 1 a is the system suitability spectrum, Figure 1 b is the sample spectrum, Figure 1c is the control spectrum;

[0023] Figure 2 The chromatogram is tested in destructive testing, where: Figure 2 a is the undestructed spectrum of ethyl hydroxylate, Figure 2 b is the high temperature destruction spectrum of ethyl hydroxylate, Figure 2 c is the thermal decomposition spectrum of ethyl hydroxylate, Figure 2 d is the ethyl hydroxylate-light destruction spectrum;

[0024] Figure 3 is the standard curve, where Figure 3 a is the standard curve of ethyl hydroxylate, Figure 3 b is the standard curve of ethyl hydroxylate isomers. DETAILED DESCRIPTION

[0025] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0026] Example 1

[0027] A method for determining the content of ethyl hydroxylate isomers of levonorgestrel starting material, comprising the following steps:

[0028] (1) Preparation of test solution, control solution and mixed solution:

[0029] Preparation of the test solution: take 10 mg of ethyl hydroxylate and place it in a 20 mL volumetric flask, add ethanol to dissolve it, dilute it to the fixed volume, and mix it to obtain the test solution; Preparation of the control solution: measure 1 mL of the test solution and place it in a 100 mL volumetric flask, dilute it to the mark with ethanol, shake it well, and use it as the self-control solution; Preparation of the mixed solution: take appropriate amounts of ethyl hydroxylate and ethyl hydroxylate racemic body, dissolve and dilute them with ethanol to prepare a mixed solution containing 0.5 mg / mL of ethyl hydroxylate and about 10 μg / mL of each isomer; The solvent in the test solution, control solution and mixed solution is ethanol;

[0030] (2) Setting the HPLC detection conditions: the chromatographic column is filled with amylose covalently bonded to the surface of silica gel, with a size of 250 mm × 4.6 mm and 5 μm; the mobile phase is composed of n-hexane and isopropanol, with a volume ratio of n-hexane to isopropanol of 90:10-95:5; the column temperature is 30°C, and the flow rate is 1.0 mL / min; the detection wavelength of the HPLC detector is 265 nm;

[0031] (3) Take 10 μL of the self-control solution and inject it into the liquid chromatograph. Adjust the detection sensitivity so that the peak height of the main component peak is 10% to 20% of the full scale. Then take 10 μL of the mixed solution and inject it into the liquid chromatograph. Record the chromatogram. Figure 1 As shown. Figure 1 It can be seen that under this chromatographic condition, the main component and the three isomers can be well separated, the ISO-1 separation is 3.2, the ISO-2 separation is 3.0, the main component separation is 2.3, the ISO-3 separation is 3.5, and there is no interference from the blank solvent. This method is applicable.

[0032] Example 2

[0033] The detection method of Example 1 was verified from several aspects, including system applicability, destructive test, quantitative limit, detection limit, linear relationship, precision, accuracy, solution stability, etc., and is described in detail below.

[0034] 1) System suitability determination:

[0035] Take an appropriate amount of ethyl hydroxide racemic reference substance, accurately weigh it, dissolve it with a solvent and dilute it to make a stock solution containing 1 mg of ethyl hydroxide racemic compound per 1 mL. Take an appropriate amount of the stock solution and add a solvent to make a positioning solution containing 100 μg of each component per 1 mL.

[0036] Accurately weigh an appropriate amount of the test sample into a volumetric flask, add an appropriate amount of the racemic stock solution into the same volumetric flask, dissolve and dilute with solvent to make a mixed solution containing approximately 0.5 mg of ethyl hydroxylate and approximately 10 μg of each isomer per 1 mL, which serves as the system suitability solution.

[0037] 10 μL of blank solvent, system suitability solution and impurity location solution were injected into the HPLC, and the chromatograms were recorded to examine the separation of each component. The results are shown in Table 1. As shown in Table 1, at a detection wavelength of 265 nm, the peak order of the mixed solution is isomer 1, isomer 2, ethyl hydroxylate, isomer 3, and ethyl hydroxylate, isomers and other impurities can be effectively separated, and the solvent peak does not interfere with the determination of ethyl hydroxylate and isomers.

[0038] Table 1 Impurity location and separation

[0039]

[0040] 2) Destructive testing

[0041] Undestroyed solution: Take about 10 mg of this product, place it in a 20 mL volumetric flask, dissolve it with solvent and dilute it to the scale, shake well, and use it as the solution before destruction.

[0042] High temperature destruction solution: Take about 10 mg of this product, place it in a 20 mL volumetric flask, place it in a 105°C oven for destruction for 6 hours, cool to room temperature, then dissolve and dilute to the scale with a solvent, shake well, and use it as the high temperature destruction solution.

[0043] Pyrolysis destruction solution: Take about 10 mg of this product, place it in a 20 mL volumetric flask, add an appropriate amount of solvent to dissolve it, then heat it at 105°C for 6 hours, cool it to room temperature, then dissolve it with solvent and dilute it to the scale, shake it well, and use it as the pyrolysis destruction solution.

[0044] Light-damaged solution: Take the undamaged solution and place it under ultraviolet light for 2 hours to use as the light-damaged solution.

[0045] Take 10 μL of each of the above test solutions and inject them into the chromatograph, and record the chromatogram. Figure 2 As shown, the changes of each isomer were analyzed. The peak purity or spectral similarity was determined by diode array measurement for the test solution under various destruction conditions. The results are shown in Table 2. It can be seen from Table 2 that under various destruction conditions, isomer 1 and isomer 3 were not detected. After light and high temperature destruction, isomer 2 increased from 0.014% to 0.052% and 0.030%. The degradation products produced by the test sample can be effectively separated from the main peak, and the degradation products do not interfere with the determination of isomers. The purity of the main peak is not less than 990.

[0046] Table 2 Investigation results under various damage conditions

[0047]

[0048] 3) Limit of quantitation and limit of detection test

[0049] Accurately weigh an appropriate amount of ethyl hydroxylate racemic reference substance, dissolve it in a solvent, and then dilute it to make a mixed stock solution containing about 2 μg of ethyl hydroxylate and each isomer per 1 mL, and use the stepwise dilution method to measure its quantitative limit (S / N≥10) and detection limit (S / N≥3). The results are shown in Table 3. It can be seen from Table 3 that under the chromatographic conditions, the quantitative limit and detection limit of ethyl hydroxylate and its isomers meet the isomer inspection requirements.

[0050] Table 3 Results of determination of limit of quantitation and limit of detection

[0051]

[0052] 4) Linear relationship

[0053] Take an appropriate amount of ethyl hydroxylate and ethyl hydroxylate racemic reference substance, weigh accurately, dissolve in solvent and dilute to make a solution containing 20 μg of ethyl hydroxylate and 10 μg of ethyl hydroxylate isomer per 1 mL as linear stock solution. Prepare linear solution according to Table 4.

[0054] Table 4 Linear solution preparation

[0055]

[0056]

[0057] Take 10 μL of each of the above solutions and inject them into the HPLC. Each sample is injected twice, record the chromatogram, determine the peak area, and make a linear regression with the peak area A as the ordinate and the concentration C as the abscissa. Figure 3 The results are shown in Table 5 below.

[0058] Table 5 Linear relationship test results

[0059]

[0060] Note: The limit requirement is that the sum of the isomer contents shall not exceed 0.5%. The ethyl hydroxylate isomer reference substance is a mixed selection, and the linear relationship is calculated based on the sum of the isomer peak areas and the concentration.

[0061] The calculation results of the isomer correction factor are shown in Table 6 below:

[0062]

[0063] Where: K Main component: Slope of the standard curve of ethyl hydroxylate

[0064] K impurity: isomer standard curve slope

[0065] Table 6 Isomer Correction Factors

[0066] Element Slope Correction Factor Ethyl Hydroxide 37.2881 - Ethyl Hydroxide Isomers 34.7551 1.07

[0067] It can be seen from Tables 5 and 6 that ethyl hydroxylate and isomers have good linearity with their peak areas within their respective concentration ranges (r>0.999), and the correction factor of ethyl hydroxylate isomers is in the range of 0.9 to 1.1, and its content can be calculated by the self-control method.

[0068] 5) Injection precision

[0069] Accurately weigh an appropriate amount of ethyl hydroxylate and ethyl hydroxylate racemic reference substance, dissolve and dilute with a solvent to prepare a mixed solution containing about 5 μg of ethyl hydroxylate and about 3 μg of each isomer per 1 mL, take 10 μl and inject it into the liquid chromatograph, inject 6 times continuously, and record the peak area. The results are shown in Table 7. It can be seen from Table 7 that after 6 consecutive injections, the peak area RSD of ethyl hydroxylate and ethyl hydroxylate isomers is ≤2.0%, and the injection precision is good.

[0070] Table 7 Injection precision results

[0071]

[0072] 6) Solution stability test

[0073] The stability tests of the reference solution and the test solution were performed separately.

[0074] Stability of reference solution

[0075] Take an appropriate amount of ethyl hydroxylate and ethyl hydroxylate racemic reference substance, dissolve and dilute with a solvent to prepare a mixed solution containing about 5 μg of ethyl hydroxylate and about 3 μg of each isomer per 1 mL, and inject the sample at different times to investigate its intraday stability. The results are shown in Table 8. It can be seen from Table 8 that ethyl hydroxylate and ethyl hydroxylate isomers have good solution stability within 54 hours (RSD≤2.0%).

[0076] Table 8 Impurity reference solution stability results

[0077]

[0078]

[0079] Stability of test solution

[0080] The product was prepared into a test solution containing 0.5 mg / mL of ethyl hydroxylate according to the proposed method, and the samples were injected at different times. The impurity content was calculated by the area normalization method to investigate the intra-day stability of the impurities. The results are shown in Table 9. It can be seen from Table 9 that the absolute deviation of the isomers from the mean of the test solution within 54 hours was ≤0.025%, with no obvious change and good stability.

[0081] Table 9 Stability results of test solution

[0082]

[0083] 7) Repeatability test

[0084] Accurately weigh an appropriate amount of ethyl hydroxide test sample into a volumetric flask, dissolve it with ethanol, and dilute to the scale to prepare a solution containing about 0.5 mg of ethyl hydroxide per 1 mL as the test solution; pipette 1 mL of the test solution to 100 mL and dilute to the scale with ethanol as the self-control solution.

[0085] The test was repeated for 6 times. The results are shown in Table 10. As can be seen from Table 10, the absolute deviation between the sum of isomers and the mean of the 6 repeated sample test results is ≤0.05%, and the repeatability is good.

[0086] Table 10 Repeatability test results

[0087]

[0088]

[0089] 8) Accuracy test (sample recovery rate)

[0090] Accurately weigh an appropriate amount of the ethyl hydroxylate racemic body, add a solvent to dissolve and dilute it to prepare a mixed solution containing about 25 μg of the ethyl hydroxylate racemic body per 1 mL, which is used as the recovery rate stock solution.

[0091] Accurately weigh an appropriate amount of ethyl hydroxylate, then accurately pipette an appropriate amount of the above recovery rate stock solution, add solvent to dilute to prepare a mixed solution containing about 0.5 mg / mL of ethyl hydroxylate and 50%, 100%, 200% and 400% of the limit concentration of isomers, respectively. Prepare 3 portions for each concentration.

[0092] 10 μL of each of the above samples was injected, the peak area of ​​each isomer was recorded, and the recovery rate and RSD were calculated. The results are shown in Table 11. As can be seen from Table 11, the isomer recovery rate and the average recovery rate were both within the range of 90% to 108%, RSD ≤ 10%, and the recovery rate was good.

[0093] Table 11 Recovery rate and RSD results

[0094]

[0095] 9) Intermediate precision

[0096] Different operators and the same operator respectively tested the isomer content of the same batch of samples using different instruments and at different times according to the repeatability test method. The results are shown in Table 12. As can be seen from Table 12, there is no significant change in the isomer detection results of this product, and the intermediate precision of this method is good.

[0097] Table 12 Intermediate precision test results

[0098]

[0099]

[0100] 10) Durability test

[0101] The robustness of the method for the ethyl hydroxylate isomer of levonorgestrel starting material was verified mainly from four aspects: different flow rates, different column temperatures, different initial ratios and different chromatographic columns.

[0102] The test was carried out according to the conditions listed in Table 13 below. The test results are shown in Table 14 below.

[0103] Table 13 Durability test conditions

[0104] parameter Scope of change Mobile phase ratio ±1％ Column temperature Column temperature ±2℃ Flow rate Flow rate ±0.02ml / min Different columns The silica gel surface is covalently bonded with amylose as a filler

[0105] Table 14 Durability test results

[0106]

[0107] It can be seen from Table 14 that when the flow rate, column temperature, and mobile phase ratio are slightly changed or the same type of chromatographic column is replaced, the ethyl hydroxylate isomers and ethyl hydroxylate of the levonorgestrel starting material can be effectively separated, with no obvious effect on the test results, which meets the requirements, indicating that the chromatographic conditions are durable.

[0108] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for determining the content of ethyl hydroxylate isomers of levonorgestrel starting material, characterized in that: Here are the steps: 1) Prepare test solution, reference solution and mixed solution; 2) Setting the HPLC detection conditions: the chromatographic column is filled with amylose covalently bonded to the surface of silica gel, the size is 250 mm × 4.6 mm, the filler particle size is 5 μm, the column temperature is 30-35°C, n-hexane is used as mobile phase A, isopropanol is used as mobile phase B, and gradient elution is performed; 3) Separately measure the reference solution and the mixed solution, inject them into the liquid chromatograph, record the chromatogram, and calculate the content of the ethyl hydroxylate isomer.

2. The method for measuring the content of ethyl hydroxylate isomers of levonorgestrel starting material according to claim 1, characterized in that: The preparation method of the test solution is as follows: 10 mg of ethyl hydroxylate is placed in a 20 mL volumetric flask, a solvent is added to dissolve it, the volume is fixed, and the test solution is obtained; the preparation method of the reference solution is as follows: 1 mL of the test solution is measured and placed in a 100 mL volumetric flask, the volume is diluted with a solvent, and the volume is shaken to serve as a self-reference solution; the preparation method of the mixed solution is as follows: ethyl hydroxylate and ethyl hydroxylate racemic body are taken, and the ethyl hydroxylate racemic body is dissolved and diluted with a solvent to prepare a mixed solution containing 0.5 mg / mL of ethyl hydroxylate and 10 μg / mL of each isomer.

3. The method for measuring the content of ethyl hydroxylate isomers of levonorgestrel starting material according to claim 2, characterized in that: In the preparation process of the test solution, the reference solution and the mixed solution, the solvent used is ethanol.

4. The method for measuring the content of ethyl hydroxylate isomers of levonorgestrel starting material according to claim 1, characterized in that: In the step 2), the column temperature is 30° C. and the flow rate is 0.6 to 1.0 mL / min.

5. The method for measuring the content of ethyl hydroxylate isomers of levonorgestrel starting material according to claim 4, characterized in that: In the step 2), the flow rate is 1.0 mL / min.

6. The method for determining the content of ethyl hydroxylate isomers of levonorgestrel starting material according to claim 1, characterized in that: In the high performance liquid phase detection conditions, the detection wavelength of the chromatograph detector is 265 nm.

7. The method for determining the content of ethyl hydroxylate isomers of levonorgestrel starting material according to claim 1, characterized in that: The volume ratio of n-hexane to isopropanol in the mobile phase of the HPLC test is 70:30-95:

5.

8. The method for determining the content of ethyl hydroxylate isomers of levonorgestrel starting material according to claim 1, characterized in that: In the step 3), the reference solution measured is 10 μL; the mixed solution measured is 10 μL.

9. The method for determining the content of ethyl hydroxylate isomers of levonorgestrel starting material according to claim 1, characterized in that: In the step 3), 10 μL of the reference solution is taken and injected into the liquid chromatograph, and the detection sensitivity is adjusted so that the peak height of the main component peak is about 10% to 20% of the full scale. Then 10 μL of the mixed solution is measured and injected into the liquid chromatograph to record the chromatogram.

10. The method for determining the content of ethyl hydroxylate isomers of levonorgestrel starting material according to claim 1, characterized in that: If there are ethyl hydroxylate isomer peaks in the chromatogram, the sum of the peak areas of the three isomers in ethyl hydroxylate shall not be greater than half of the main peak area of ​​the control solution, i.e., 0.5%.

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