A method for determining the content of dextrorotatory isomer in levonorgestrel by high performance liquid chromatography
Through the high -efficiency liquid phase method to detect the content of the right rotor in the left germination ketone, it solves the problem of difficult detection in existing technology, achieves fast and accurate detection effects, and improves the quality of drug products and drug use safety. sex.
Patent Information
- Application Number
- CN202211154388.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-09-19
- Publication Date
- 2025-01-28
- Estimated Expiration
- 2042-09-19
AI Technical Summary
The prior art is difficult to effectively detect the content of dexonorgestrel in levonorgestrel, which affects the product quality and drug safety of the drug.
The high -efficiency liquid phase method is adopted. By formulating the test solution and the control product solution, the proper liquid color spectrum testing conditions are set, and the Daicel IG color spectrum column and specific flow phase are used to record the chromatography and calculate the content of the right rotor.
A fast, effective, accurate, and reliable separation detection detection in the left rotor heterogeneity in the left progelive ketone, which improves the quality of the drug's product and the safety of medication.
Smart Images
Figure CN115598238B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of medical technology, and more specifically relates to a method for determining the content of dextrorotatory isomer in levonorgestrel by high performance liquid phase method. Background Art
[0002] Levonorgestrel, chemically known as (-)-13-ethyl-17-hydroxy-18,19-bis-demethyl-17α-pregn-4-ene-20-yn-3-one, is a widely used progestogen drug. From its molecular structure, it can be seen that the chiral carbon configuration of the steroid structure is stable, and only the chiral carbon atom at position 17 may produce a dextrorotatory isomer. The activity of levonorgestrel is twice that of the racemic isomer of norgestrel, from which it can be seen that the isomers have an effect on the activity. The 2020 edition of the Chinese Pharmacopoeia only has a limit requirement for the specific rotation, and there are many factors affecting the specific rotation. Therefore, the detection and separation of the dextrorotatory isomer of levonorgestrel is of great significance to the production and storage of raw materials and preparations. The structural formula of levonorgestrel is shown below:
[0003] Summary of the invention
[0004] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a method for determining the content of the dextrorotatory isomer in levonorgestrel by high performance liquid chromatography.
[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:
[0006] A method for determining the content of dextrorotatory isomer in levonorgestrel by high performance liquid chromatography, comprising:
[0007] (1) Prepare test sample solution and reference sample solution;
[0008] (2) Setting HPLC detection conditions;
[0009] (3) Take the test solution and the control solution separately, inject them into the liquid chromatograph, record the chromatogram, and calculate the content of the right-hand isomer.
[0010] Furthermore, in the method described above, the chromatographic column is DAICEL IG, with a length of 250 mm, an inner diameter of 4.6 mm, and a filler particle size of 5 μm.
[0011] Furthermore, the mobile phase consists of n-heptane, ethanol and trifluoroacetic acid in a volume ratio of 920-940:60-80:0.5.
[0012] Furthermore, the column temperature of the chromatographic column is 33°C to 37°C.
[0013] Furthermore, the flow rate of the mobile phase is 0.95-1.05 mL / min.
[0014] Furthermore, the detection wavelength of the liquid chromatograph is 237 nm.
[0015] Further, the preparation of the test solution: take about 10 mg of levonorgestrel and place it in a 20 mL volumetric flask, add 5 mL of ethanol to dissolve it, then add n-heptane to dilute to volume and mix well; the preparation of the reference solution: accurately measure 1 mL of the test solution and place it in a 100 mL volumetric flask, dilute it to the scale with solvent, shake well, and use it as the self-control solution.
[0016] In the method, the injection volume of the test solution and the control solution is 10 μL.
[0017] In the method described, the peak area of the right-handed isomer in the chromatogram of the test solution is not greater than the main peak area of the control solution, that is, the isomer content is not greater than 1.0%.
[0018] Compared with the prior art, the present invention has the following beneficial effects:
[0019] The high performance liquid chromatography of the present invention can quickly, effectively, accurately and reliably separate and detect the dextrorotatory isomer in the levonorgestrel bulk drug. Under the chromatographic conditions of the present invention, the main component and the unknown impurities thereafter can be well separated, and the separation degree is 4.3, and there is no small inverted peak interference in the blank solvent, and the solvent peak does not interfere with the determination of the main component; and the sample destroyed under each condition is tested by peak purity, and the spectral purity is greater than 990, and the impurities produced by the destruction under each condition can be well separated from the main peak, and the detection specificity of related substances is good; at the same time, when the chromatographic condition parameters are slightly changed, the separation and inspection results of related substance impurities in levonorgestrel are not affected, and the durability is good. The present application can quickly, effectively, accurately and reliably separate and detect the dextrorotatory isomer in levonorgestrel, which is beneficial to improving the product quality of levonorgestrel and improving the safety of patient medication. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 The chromatogram is tested under the chromatographic conditions of the present invention;
[0021] Figure 2 It is the undestructed spectrum of levonorgestrel;
[0022] Figure 3 It is the levonorgestrel-photodestruction spectrum;
[0023] Figure 4 It is the high temperature destruction spectrum of levonorgestrel;
[0024] Figure 5 This is the thermal destruction spectrum of levonorgestrel;
[0025] Figure 6 is the standard curve, where Figure 6 a is the standard curve of the dextrorotatory isomer of levonorgestrel, Figure 6 b is the standard curve of levonorgestrel. DETAILED DESCRIPTION
[0026] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.
[0027] Example 1
[0028] A method for determining the content of dextrorotatory isomer in levonorgestrel by high performance liquid chromatography, comprising the following steps:
[0029] 1) Prepare a reference solution, a test solution and a mixed solution. The preparation of the test solution is as follows: take about 10 mg of levonorgestrel and place it in a 20 mL volumetric flask, add 5 mL of ethanol to dissolve it, and then add n-heptane to dilute it to the volume and mix it evenly; the preparation of the reference solution is as follows: accurately measure 1 mL of the test solution and place it in a 100 mL volumetric flask, dilute it to the mark with a solvent, and shake it evenly to serve as a self-reference solution; prepare a mixed solution and inject it to test the test effect of the method of the present invention. The preparation of the mixed solution is as follows: take appropriate amounts of levonorgestrel and norgestrel racemate, dissolve and dilute them with a solvent to prepare a mixed solution containing 0.5 mg / mL of levonorgestrel and 5 μg / mL of the dextrorotatory isomer of levonorgestrel; the solvent in the reference solution and the mixed solution consists of n-heptane-ethanol, and the ratio is 3:1.
[0030] 2) Setting HPLC detection conditions; Detection conditions: the chromatographic column is DAICEL IG, with a specification of 25 cm×0.46 cm, 5 μm, the mobile phase is composed of n-heptane-ethanol-trifluoroacetic acid, and the volume ratio of n-heptane to ethanol to trifluoroacetic acid is 930:70:0.5; the column temperature is 35°C, and the flow rate is 1.0 mL / min; the detection wavelength of the HPLC detector is 237 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 about 10% to 20% of the full scale. Then accurately measure 10 μL of the mixed solution and inject it into the liquid chromatograph. Record the chromatogram ( Figure 1 ).Depend on Figure 1 It can be seen that under this chromatographic condition, the main component and the subsequent unknown impurities can be well separated, with a separation degree of 4.3, and there is no tiny inverted peak interference in the blank solvent. This method is applicable.
[0032] Example 2
[0033] The detection method of Example 1 was validated from several aspects, including system applicability, destructive testing, limit of quantification, limit of detection, linear relationship, precision, accuracy, solution stability, etc., as described in detail below.
[0034] 1) System suitability determination:
[0035] Take appropriate amount of norgestrel and levonorgestrel reference substances, weigh accurately, dissolve and dilute with ethanol to prepare stock solutions containing about 1 mg / mL of norgestrel (containing about 0.5 mg / mL of levonorgestrel and dextro isomer) and 0.5 mg / mL of levonorgestrel. Take appropriate amount of norgestrel and levonorgestrel stock solutions, add solvent to prepare positioning solutions containing 20 μg / mL of norgestrel (containing about 10 μg / mL of levonorgestrel and dextro isomer) and 10 μg of levonorgestrel.
[0036] Accurately weigh an appropriate amount of levonorgestrel into a 20 mL volumetric flask, then add an appropriate amount of norgestrel stock solution into the same volumetric flask, dissolve it in 5 mL of ethanol, and then dilute it with n-heptane to prepare a mixed solution containing about 5 μg / mL of dextrorotatory isomer and about 0.5 mg / mL of levonorgestrel as the system suitability solution.
[0037] 10 μL of blank solvent, system suitability solution and each impurity location solution were respectively injected into the high performance liquid chromatograph, and the chromatogram was recorded to examine the separation of each component. The results are shown in Table 1. As can be seen from Table 1, at a detection wavelength of 237 nm, the peak order of the mixed solution is dextrorotatory isomer, levonorgestrel, levonorgestrel, dextrorotatory isomer and other impurities can be effectively separated, and the solvent peak does not interfere with the determination of levonorgestrel and dextrorotatory isomer.
[0038] Table 1 Impurity location and separation
[0039] Peak time Levonorgestrel dextroisomer Levonorgestrel Positioning solution (minutes) 11.712 15.881 SST(minutes) 11.713 15.538 Separation 7.6 4.3
[0040] 2) Destructive testing
[0041] Preparation of test solution: Since a chiral column (normal phase) was used to detect isomers, acid, alkali and oxidation destruction tests were not performed.
[0042] Solution before destruction: Take about 10 mg of levonorgestrel and place it in a 20 mL volumetric flask. Add 5 mL of ethanol to dissolve it, then add n-heptane to dilute to volume and mix well.
[0043] Light-damaged solution: Take about 10 mg of levonorgestrel and place it in a 20 mL volumetric flask, add 5 mL of ethanol to dissolve it, then add n-heptane to dilute and mix. After light-damaged at ultraviolet wavelengths of 254 nm and 365 nm for 20 minutes, use this as the light-damaged solution.
[0044] High temperature destruction solution: Take about 10 mg of levonorgestrel and place it in a 20 mL volumetric flask. Destroy it under high temperature conditions for 4 hours. Take it out and let it cool at room temperature. Add 5 mL of ethanol to dissolve it, then add n-heptane to make up the volume and mix well to prepare the high temperature destruction solution.
[0045] Pyrolysis destruction solution: Take about 10 mg of levonorgestrel and place it in a 20 mL volumetric flask, add 5 mL of ethanol to dissolve it, then add a small amount of n-heptane to destroy it under high temperature conditions for 4 hours, take it out and let it cool at room temperature, add 5 mL of ethanol to dissolve it, then add n-heptane to make up the volume, mix well, and use it as the pyrolysis destruction solution.
[0046] Take 10 μL of each of the above test solutions and inject them into the chromatograph, and record the chromatogram ( Figure 2-5 ), analyze the changes of each impurity. For the test solution under various destruction conditions, the diode array was used for peak purity or spectral similarity determination. The results are shown in Table 2. It can be seen from Table 2 that under various destruction conditions, the degradation products produced by the test sample can be effectively separated from the main peak, the degradation products do not interfere with the determination of the right-handed isomer, the reduction in the destruction of the main peak should be basically consistent with the decrease in its content, the purity of the main peak is not less than 990, and the material is conserved.
[0047] Table 2 Material balance investigation results
[0048]
[0049] 3) Limit of quantitation and limit of detection test
[0050] Take the stock solution of norgestrel reference substance, add solvent to dilute to make 1mL of solution containing 1μg norgestrel (including levonorgestrel and dextro isomer about 0.5μg each), and measure its limit of quantification (S / N≥10) and limit of detection (S / N≥3) by dilution method. The results are shown in Table 3. It can be seen from Table 3 that under the chromatographic conditions, the limit of quantification and limit of detection of levonorgestrel and its dextro isomer meet the requirements of isomer inspection.
[0051] Table 3 Limit of quantitation and detection limit
[0052]
[0053] 4) Linear relationship
[0054] Take a 1 mg / mL stock solution of norgestrel reference substance, add solvent to dilute it to make a solution containing 20 μg norgestrel (including about 10 μg of levonorgestrel and dextrorotatory isomer) per 1 mL as the linear stock solution. Prepare the linear solution according to Table 4.
[0055] Table 4 Linear solution preparation
[0056]
[0057] Accurately measure 10 μL of the above solution and inject it into the HPLC instrument to record the chromatogram ( Figure 6 ), the peak area was determined, and a linear regression was performed with the peak area A as the ordinate and the concentration C as the abscissa. The results are shown in Table 5 below.
[0058] Table 5 Linear relationship test results
[0059]
[0060]
[0061] The calculation results of the known impurity correction factors are shown in Table 6:
[0062]
[0063] K 左炔诺孕酮 :Levonorgestrel standard curve slope
[0064] K 左炔诺孕酮右旋异构体 : Slope of the dextrorotatory isomer of levonorgestrel
[0065] Table 6 Correction factors for known impurities
[0066] Element Slope Correction Factor Levonorgestrel 29.3311 - Levonorgestrel dextroisomer 29.3369 1.00
[0067] It can be seen from Tables 5 and 6 that each known impurity has a good linear relationship with its peak area within its corresponding concentration range (r>0.999), and the correction factor for the dextrorotatory isomer of levonorgestrel is 1.00, and its content can be calculated by the self-control method.
[0068] 5) Injection precision
[0069] Take the stock solution of norgestrel reference substance with a concentration of 1 mg / mL, add solvent to dilute to make 1mL of solution containing 10μg norgestrel (containing about 5μg of levonorgestrel and dextro isomer), take 10μL and inject into 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 injections, the peak area RSD% of levonorgestrel and dextro isomer is ≤2.0%, and the injection precision is good.
[0070] Table 7 Injection precision results
[0071] Injection precision A Levonorgestrel dextroisomer ALevonorgestrel 1 157.104 157.032 2 157.032 156.538 3 156.881 156.825 4 156.892 156.631 5 157.240 157.024 6 157.136 156.788 RSD(%) 0.09％ 0.13％
[0072] 6) Solution stability test
[0073] The stability tests of the reference solution and the test solution were performed separately.
[0074] ①Stability of reference solution:
[0075] Take an appropriate amount of norgestrel reference substance stock solution with a concentration of 1 mg / mL, add solvent to dilute to make 1 mL of solution containing 10 μg norgestrel (containing about 5 μg of levonorgestrel and dextro isomer), and inject at different times to investigate its intraday stability. The results are shown in Table 8. It can be seen from Table 8 that the levonorgestrel and dextro isomer reference substances have good solution stability within 24 hours (RSD≤2.0%).
[0076] Table 8 Impurity reference solution stability results
[0077] ②Stability of test solution:
[0078] Time (hours) <![CDATA[Peak area 右旋异构体 > <![CDATA[Peak area 左炔诺孕酮 > 0 157.104 157.032 4 156.395 156.164 7 156.100 155.746 9 156.053 155.623 11 156.239 156.415 14 156.035 155.724 16 156.056 155.625 21 158.047 157.477 24 160.095 159.886 RSD% 0.87％ 0.88％
[0079] The product was prepared into a test solution containing 0.5 mg / mL of levonorgestrel 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 isomer content of the test product was 0.016% to 0.017% within 24 hours, with no obvious change, and good stability.
[0080] Table 9 Stability results of test solution
[0081] Time (hours) Dextrorotatory isomer content / % 0 0.017 4 0.016 7 0.016 9 0.016 11 0.017 14 0.016 16 0.017 21 0.017 24 0.017
[0082] 7) Repeatability test
[0083] Place the levonorgestrel test sample in a 20 mL volumetric flask, add 5 mL of ethanol to dissolve it, and then add n-heptane to dilute to the scale to prepare a solution containing approximately 0.5 mg per mL as the test solution; transfer 1 mL of the test solution to 100 mL and dilute to the scale with solvent as the self-control solution.
[0084] The determination was repeated for 6 times. The results are shown in Table 10. As can be seen from Table 10, the absolute value of the difference between the dextrorotatory isomer content and the mean value of the 6 repeated sample test results is ≤ 10.0% of the limit, and the repeatability is good.
[0085] Table 10 Repeatability results
[0086]
[0087] 8) Accuracy test (sample recovery rate)
[0088] Take a 1 mg / mL stock solution of norgestrel reference substance, add solvent to dilute it to make a solution containing 200 μg of norgestrel (including about 100 μg of levonorgestrel and dextrorotatory isomer) per 1 mL, which is used as the recovery rate stock solution.
[0089] Accurately draw the above recovery rate stock solution and dilute it according to Table 11 to prepare 40%, 100% and 200% solutions, and prepare 3 copies for each concentration. Draw 10 μL of each of the above samples for injection, record the peak area of the right-hand isomer, and calculate its recovery rate and RSD. The results are shown in Table 11. It can be seen from Table 12 that the recovery rate and average recovery rate of the right-hand isomer are in the range of 101.0% to 104.3%, and the RSD% of each recovery rate is 1.1%, which is a good recovery rate.
[0090] Table 11 Recovery solution preparation
[0091]
[0092] Table 12 Recovery results
[0093]
[0094]
[0095] 9) Intermediate precision
[0096] The same batch of samples were tested for isomer content by the same operator at different times, by different operators, and by the same operator using different instruments according to the repeatability test method. The results are shown in Table 13. It can be seen from Table 13 that the intermediate precision of the isomer test method of this product is good.
[0097] Table 13 Intermediate precision test results
[0098] Experiment Name Experimenter Instrument No. Isomer content (%) Repeatability Experimenter A CA15A23008 0.018％ Different time Experimenter A CA15A23008 0.017％ Different instruments Experimenter A CA15A23008 0.017％ Different people Experimenter B CA15A23003 0.018％
[0099] 10) Durability test
[0100] The robustness of the levonorgestrel isomer method was verified mainly from three aspects: different proportions of mobile phase, different flow rates and different column temperatures.
[0101] The experiment was carried out according to the conditions in Table 14, and the results are shown in Table 15. It can be seen from Table 15 that when the mobile phase ratio, flow rate, and column temperature are slightly changed or the same type of chromatographic column is replaced, the dextrorotatory isomer of levonorgestrel and levonorgestrel can be effectively separated, and there is no obvious effect on the test results, which meets the requirements, indicating that the chromatographic conditions have good durability.
[0102] Table 14 Durability conditions
[0103] parameter Specified parameters Scope of change Mobile phase ratio n-Heptane-Ethanol-Trifluoroacetic Acid (930:70:0.5) ±1％ Column temperature 35℃ Column temperature ±2℃ Flow rate 1.0mL / min Flow rate ±0.05mL / min
[0104] Table 15 Durability results
[0105] .
Claims
1. A method for determining the content of the dextrorotatory isomer in levonorgestrel by high performance liquid chromatography, characterized in that: include: (1) Prepare test sample solution and reference sample solution; (2) Setting the HPLC detection conditions; the chromatographic column is DAICEL IG, with a length of 250 nm, an inner diameter of 4.6 nm, and a filler particle size of 5 μm; the mobile phase is composed of n-heptane, ethanol, and trifluoroacetic acid, with a volume ratio of 920-940:60-80:0.5; the column temperature of the chromatographic column is 33°C-37°C; the flow rate of the mobile phase is 0.95-1.05 mL / min; the detection wavelength of the liquid chromatograph is 237 nm; (3) Take the test solution and the reference solution respectively, inject them into the liquid chromatograph, record the chromatogram, and calculate the content of the right-hand isomer; preparation of the test solution: take about 10 mg of levonorgestrel and place it in a 20 mL volumetric flask, add 5 mL of ethanol to dissolve it, then add n-heptane to dilute to volume and mix well; preparation of the reference solution: accurately measure 1 mL of the test solution and place it in a 100 mL volumetric flask, dilute to the mark with solvent, shake well, and use it as the self-reference solution.
2. The method for determining the content of the dextrorotatory isomer in levonorgestrel by high performance liquid chromatography according to claim 1, characterized in that: The injection volume of the test solution and the control solution was 10 μL.
3. The method for determining the content of the dextrorotatory isomer in levonorgestrel by high performance liquid chromatography according to claim 1, characterized in that: The peak area of the right-hand isomer in the chromatogram of the test solution is not greater than the main peak area of the control solution.