Method for detecting 16-dehydroprogesterone in progesterone by high performance liquid chromatography
By using an Agilent InfinityLab Poroshell SB-C8 column and gradient elution technology, 16-dehydroprogesterone in progesterone was successfully separated and detected, solving the problem of difficult separation in existing technologies, improving the accuracy and sensitivity of detection, and ensuring drug quality.
Patent Information
- Application Number
- CN202511163316.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-19
- Publication Date
- 2025-11-07
AI Technical Summary
Current technology cannot effectively separate and detect 16-dehydroprogesterone in progesterone, which affects drug quality control.
An Agilent InfinityLab Poroshell SB-C8 column was used with acetonitrile and water as the mobile phase for gradient elution. The detection wavelength was 241 nm, the flow rate was 0.8–1.2 mL/min, and the column temperature was 30–40 °C, which achieved effective separation of 16-dehydroprogesterone and progesterone.
The method effectively separates 16-dehydroprogesterone and progesterone, and its detection method is highly specific, linear, sensitive, accurate, and reproducible, thus improving drug safety and quality control.
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Figure CN120908346A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of analytical chemistry, and particularly relates to a method for detecting 16-dehydropregnenolone in progesterone by high performance liquid chromatography. BACKGROUND
[0002] Progesterone, also known as pregnenolone, has very important uses in clinical practice as a drug, and is mainly used for treating threatened abortion, habitual abortion, menstrual disorders, functional uterine bleeding and the like caused by luteal insufficiency.
[0003] At present, the preparation method of progesterone mainly takes dienol ketone acetate as a starting material, and is prepared by three steps of hydrogenation, hydrolysis and WO's oxidation. If the hydrogenation of dienol ketone acetate is not complete, the residual dienol ketone acetate will generate impurity 16-dehydropregnenolone through hydrolysis and WO's oxidation.
[0004] However, neither the People's Republic of China Pharmacopoeia (2020 Edition) nor foreign common pharmacopoeias have added the inspection item of impurity 16-dehydropregnenolone under the inspection item of progesterone related substances, and the detection method referring to the above-mentioned existing pharmacopoeias cannot effectively separate 16-dehydropregnenolone, progesterone impurity C
(20R)-20-hydroxypregn-4-en-3-one
[0005] The present application aims to solve the above-mentioned problems, and provides a method for detecting 16-dehydropregnenolone in progesterone by high performance liquid chromatography, so as to make up for the blank in the field.
[0006] The technical scheme for realizing the present application is as follows: a method for detecting 16-dehydropregnenolone in progesterone by high performance liquid chromatography, characterized in that: an Agilent InfinityLab Poroshell SB-C8 column is used; a mixed mobile phase of mobile phase A and mobile phase B is used for gradient elution; the mobile phase A is acetonitrile; and the mobile phase B is water.
[0007] The gradient elution program is as follows: at 0-20 min, the volume ratio of the mobile phase A and the mobile phase B is balanced at 50%:50%; at 20-27 min, the volume ratio of the mobile phase A and the mobile phase B is gradiently increased to 80%:20%; at 27-40 min, the volume ratio of the mobile phase A and the mobile phase B is balanced at 80%:20%; at 40-42 min, the volume ratio of the mobile phase A and the mobile phase B is gradiently decreased to 50%:50%; and at 42-50 min, the volume ratio of the mobile phase A and the mobile phase B is balanced at 50%:50%.
[0008] Other conditions of the high performance liquid chromatography are as follows: The detection wavelength is 241 nm.
[0009] The column temperature is 30-40℃.
[0010] The injection volume is 10 μL.
[0011] The flow rate of the mobile phase is 0.8-1.2 mL / min.
[0012] The method of the present application can effectively separate 16-dehydroprogesterone and progesterone and impurities thereof, and can accurately detect the content of 16-dehydroprogesterone, and the method has strong specificity, good linearity, high sensitivity, good accuracy, good precision and good repeatability, thereby improving the safety and effectiveness of progesterone, and has very important significance for quality control of progesterone raw material medicine. BRIEF DESCRIPTION OF DRAWINGS
[0013] Figure 1 High performance liquid chromatogram of the system suitability solution in Example 1.
[0014] Figure 2 High performance liquid chromatogram of the test solution in Example 2.
[0015] Figure 3 High performance liquid chromatogram of the blank solution in Test Example 1.
[0016] Figure 4 High performance liquid chromatogram of the positioning solution in Test Example 1.
[0017] Figure 5 High performance liquid chromatogram of the test solution in Test Example 1.
[0018] Figure 6 Standard curve graph plotted in the linearity and range test of Test Example 3.
[0019] Figure 7 High performance liquid chromatogram of Comparative Example 1.
[0020] Figure 8 High performance liquid chromatogram of Comparative Example 2. DETAILED DESCRIPTION
[0021] (Example 1) I. Instruments and raw materials
[0022] High performance liquid chromatograph: Agilent 1260 liquid chromatograph.
[0023] Chromatographic column: Agilent InfinitrLab Poroshell 120 SB-C8 (4.6 mm x 150 mm, 2.7 μm).
[0024] Progesterone (PG) control; 16-dehydroprogesterone (16-dehydro PG or 16tuoqing-PG) control; Progesterone impurity C (imp C) control; Progesterone (PG) test sample.
[0025] II. Chromatographic conditions.
[0026] Detection wavelength: 241 nm.
[0027] Chromatographic column temperature: 35 °C.
[0028] Injection volume: 10 μL.
[0029] Mobile phase A: acetonitrile.
[0030] Mobile phase B: water.
[0031] Flow rate of mobile phase: 1.0 mL / min.
[0032] Diluent: acetonitrile.
[0033] The gradient elution program is as follows: at 0-20 min, the volume ratio of mobile phase A and mobile phase B is balanced at 50%:50%; at 20-27 min, the volume ratio of mobile phase A and mobile phase B is gradiently increased to 80%:20%; at 27-40 min, the volume ratio of mobile phase A and mobile phase B is balanced at 80%:20%; at 40-42 min, the volume ratio of mobile phase A and mobile phase B is gradiently decreased to 50%:50%; at 42-50 min, the volume ratio of mobile phase A and mobile phase B is balanced at 50%:50%.
[0034] See Table 1 for details.
[0035] Table 1 Elution time (min) Mobile phase A (%) Mobile phase B (%) 0 50 50 20 50 50 27 80 20 40 80 20 42 50 50 50 50 50
[0036] III. Experimental procedure.
[0037] 16-Dehydroprogesterone stock solution: precisely weigh 20 mg of 16-dehydroprogesterone control, place in a 100 mL volumetric flask, dissolve and dilute to the mark with diluent, mix well; precisely transfer 5 mL to a 50 mL volumetric flask, dilute to the mark with diluent, mix well, and obtain 16-dehydroprogesterone stock solution.
[0038] Progesterone impurity C stock solution: precisely weigh progesterone impurity C reference substance 20 mg, put into a 100 mL volumetric flask, dissolve and dilute to the mark with diluent, mix well; precisely transfer 5 mL into a 50 mL volumetric flask, dilute to the mark with diluent, mix well, and the progesterone impurity C stock solution is obtained.
[0039] System suitability solution: precisely weigh progesterone reference substance 20 mg into a 50 mL volumetric flask, dissolve with appropriate amount of diluent, and then precisely transfer 1.0 mL of the above-mentioned 16-dehydroprogesterone stock solution and 1.0 mL of the progesterone impurity C stock solution into the 50 mL volumetric flask, dilute to the mark with diluent, mix well, and the system suitability solution is obtained.
[0040] Precisely take 10 μL of the above-mentioned system suitability solution, inject into a high performance liquid chromatograph, record the chromatogram, and the results are shown in Figure 1 .
[0041] From Figure 1 It can be seen that the peak time of progesterone impurity C is 11.557 min, the peak time of 16-dehydroprogesterone is 12.747 min, the peak time of progesterone is 13.230 min, and the separation degree of 16-dehydroprogesterone and progesterone is 1.43, which basically meets the effective separation.
[0042] (Example 2) Test sample solution: precisely weigh progesterone test sample 15 mg into a 50 mL volumetric flask, dissolve and dilute to the mark with diluent, shake well, and the test sample solution is obtained.
[0043] Precisely take 10 μL of the above-mentioned test sample solution, inject into a high performance liquid chromatograph, and perform high performance liquid chromatography analysis according to the chromatographic conditions of Example 1, record the chromatogram, and the results are shown in Figure 2 .
[0044] From Figure 2 It can be seen that the peak time of progesterone impurity C is 11.340 min, the peak time of 16-dehydroprogesterone is 12.597 min, the peak time of progesterone is 13.108 min, and the separation degree of 16-dehydroprogesterone and progesterone is 1.76, which is good.
[0045] (Test Example 1) This test example is a specificity experiment of the detection method of the present application.
[0046] Blank solution: diluent.
[0047] Positioning solution: precisely weigh 16-dehydroprogesterone reference substance 4 mg into a 10 mL volumetric flask, dissolve and dilute to the mark with diluent, shake well, and the positioning solution is obtained.
[0048] Test solution: Accurately weigh 20 mg of progesterone test sample and place it in a 50 mL volumetric flask. Add diluent to dissolve and dilute to the mark. Shake well to obtain the solution.
[0049] Accurately measure 10 μL each of the blank solution, positioning solution, and test solution, and inject them into the high-performance liquid chromatograph (HPLC). Perform HPLC analysis according to the chromatographic conditions of Example 1, and record the chromatograms. (See attached figures.) Figures 3-5 .
[0050] Depend on Figures 3-5 It can be seen that at a detection wavelength of 241 nm, the blank solution did not interfere with the sample determination; the retention time of 16-dehydroprogesterone in the positioning solution was 12.572 min, which was the same as the retention time of the corresponding substance in the test solution (12.606 min). The results were largely consistent, with a separation degree of 1.54 between 16-dehydroprogesterone and progesterone, indicating good separation.
[0051] (Experimental Example 2) This test example demonstrates the linearity and range of the detection method of the present invention.
[0052] 16-Dehydroprogesterone stock solution: Accurately weigh 20 mg of 16-dehydroprogesterone reference standard, place it in a 100 mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well to obtain 16-dehydroprogesterone stock solution.
[0053] Progesterone stock solution: Accurately weigh 20 mg of progesterone reference standard, place it in a 100 mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well to obtain progesterone stock solution.
[0054] Linear stock solution: Accurately transfer 1.0 mL each of 16-dehydroprogesterone stock solution and progesterone stock solution into the same 50 mL volumetric flask, and dilute to the mark with diluent to obtain the linear stock solution.
[0055] 5% linear solution (limit of quantitation solution): Take 0.05 mL of linear stock solution, place it in a 10 mL volumetric flask, add diluent to the mark, and shake well.
[0056] 10% linear solution: Take 0.1 mL of the linear stock solution, place it in a 10 mL volumetric flask, add diluent to the mark, and shake well to obtain the solution.
[0057] 50% linear solution: Take 0.5 mL of the linear stock solution, place it in a 10 mL volumetric flask, add diluent to the mark, and shake well to obtain the solution.
[0058] 100% linear solution: Take 1.0 mL of linear stock solution, place it in a 10 mL volumetric flask, add diluent to the mark, and shake well to obtain the solution.
[0059] 150% linear solution: take 1.5 mL of the linear stock solution, add to a 10 mL volumetric flask, dilute to the mark with diluent, shake well, and you get it.
[0060] 200% linear solution: take 2.0 mL of the linear stock solution, add to a 10 mL volumetric flask, dilute to the mark with diluent, shake well, and you get it.
[0061] 250% linear solution: take 2.5 mL of the linear stock solution, add to a 10 mL volumetric flask, dilute to the mark with diluent, shake well, and you get it.
[0062] Precisely take 10 μL of each linear solution above, inject into the chromatograph respectively, and perform high performance liquid chromatography analysis according to the chromatographic conditions of Example 1, record the chromatogram, calculate the peak area, and the results are shown in Table 2.
[0063] Take the concentration (μg / mL) as the abscissa and the peak area as the ordinate, draw the standard curve and prepare the linear regression equation, and the results are shown in Figure 6 .
[0064] From Figure 6 It can be seen that: 16-dehydroprogesterone is linear in the concentration range of 0.01971-0.98569 μg / mL (linear range 5%-250%), and the correlation coefficient is 0.9999; progesterone is linear in the concentration range of 0.02009-1.0047 μg / mL (linear range 5%-250%), and the correlation coefficient is 1.
[0065] Table 2 16-Dehydroprogesterone concentration (pg / mL) 16-Dehydroprogesterone peak area Progesterone concentration (pg / mL) Progesterone peak area 5% linear solution 0.01971 0.981 0.02009 0.612 10% linear solution 0.03943 2.238 0.04019 1.234 50% linear solution 0.19714 9.366 0.2009 6.156 100% linear solution 0.39428 18.580 0.4019 12.323 150% linear solution 0.59141 27.523 0.6028 18.193 200% linear solution 0.78855 37.339 0.8038 24.245 250% linear solution 0.98569 46.158 1.0047 30.315
[0066] (Test Example 3) This test example is the limit of quantification and limit of detection experiment of the detection method of the application.
[0067] Take the 5% linear solution in Test Example 2 as the limit of quantification solution, and dilute the limit of quantification solution 5 times as the limit of detection solution.
[0068] Precisely take 10 μL of each of the limit of quantification solution and the limit of detection solution, inject into the chromatograph respectively, and perform high performance liquid chromatography analysis according to the chromatographic conditions of Example 1, and the results are shown in Table 3.
[0069] Table 3
[0070] From Table 3, it can be seen that the limit of quantification is 0.005%, the limit of quantification signal-to-noise ratio is greater than 10, the limit of detection is 0.001%, and the limit of detection signal-to-noise ratio is greater than 3, indicating that the method of the application has high sensitivity.
[0071] (Test Example 4) This test example is the accuracy test of the detection method of the present application.
[0072] 80% impurity solution: 4.0 mL of the 16-dehydroprogesterone stock solution in test example 2 was taken precisely and placed in a 50 mL volumetric flask, which was diluted to the calibration mark with the diluent and shaken well; 1.0 mL of the solution was taken precisely and placed in a 50 mL volumetric flask in which 20 mg of progesterone was previously dissolved, which was diluted to the calibration mark with the diluent and shaken well. Three portions were prepared in parallel.
[0073] 100% impurity solution: 5.0 mL of the 16-dehydroprogesterone stock solution in test example 2 was taken precisely and placed in a 50 mL volumetric flask, which was diluted to the calibration mark with the diluent and shaken well; 1.0 mL of the solution was taken precisely and placed in a 50 mL volumetric flask in which 20 mg of progesterone was previously dissolved, which was diluted to the calibration mark with the diluent and shaken well. Three portions were prepared in parallel.
[0074] 120% impurity solution: 6.0 mL of the 16-dehydroprogesterone stock solution in test example 2 was taken precisely and placed in a 50 mL volumetric flask, which was diluted to the calibration mark with the diluent and shaken well; 1.0 mL of the solution was taken precisely and placed in a 50 mL volumetric flask in which 20 mg of progesterone was previously dissolved, which was diluted to the calibration mark with the diluent and shaken well. Three portions were prepared in parallel.
[0075] 10 μL of each of the three 80% impurity solutions, the three 100% impurity solutions and the three 120% impurity solutions was injected into the chromatograph, and high performance liquid chromatography analysis was performed according to the chromatographic conditions in example 1, the chromatogram was recorded, the recovery rate was calculated, and the results are shown in table 4.
[0076] Table 4
[0077] As can be seen from table 4, the recovery rate is in the range of 90% to 110%, and the RSD is less than 3%, indicating that the detection method of the present application has good accuracy.
[0078] (Test example 5) This test example is the precision test of the detection method of the present application.
[0079] 10 μL of the 100% impurity solution in test example 4 was injected into the chromatograph, and 6 injections were continuously performed, and high performance liquid chromatography analysis was performed according to the chromatographic conditions in example 1, the chromatogram was recorded, the peak area and content of 16-dehydroprogesterone were calculated, and the average value and relative standard deviation were calculated, and the results are shown in table 5.
[0080] Table 5 Peak area Content 1 needle 17.716 0.091% 2 needles 17.835 0.092% 3 needles 17.723 0.091% 4 needles 17.793 0.092% 5 needles 17.773 0.092% 6 needles 17.759 0.091% Average 17.767 0.092% RSD 0.25% 0.58%
[0081] As can be seen from table 5, the detection method of the present application has good precision.
[0082] (Test example 6) The present test example is a repeatability experiment of the detection method of the present application.
[0083] Precisely take 6 portions of 10 μL of the 100% impurity solution in Test Example 4, respectively inject into the chromatograph, perform high performance liquid chromatography analysis according to the chromatography conditions of Example 1, record the chromatogram, calculate the 16-dehydroprogesterone peak area and content, and calculate the relative standard deviation, and the results are shown in Table 6.
[0084] Table 6 Peak area Content Solution 1 17.178 0.086% Solution 2 16.998 0.086% Solution 3 17.003 0.087% Solution 4 17.233 0.089% Solution 5 17.276 0.091% Solution 6 17.093 0.092% RSD 0.69% 2.92%
[0085] As can be seen from Table 6, the detection method of the present application has good repeatability.
[0086] (Comparative Example 1) The present comparative example is to detect 16-dehydroprogesterone by referring to the detection method of progesterone related substances in the prior art “Chinese Pharmacopoeia (2020 Edition)”.
[0087] Chromatographic column: Waters XBridge C8, 4.6 mm x 250 mm, 5 μm.
[0088] Detection wavelength: 241 nm.
[0089] Chromatographic column temperature: 30°C.
[0090] Injection volume: 10 μL.
[0091] Mobile phase: methanol / acetonitrile / water = 25% / 35% / 40%.
[0092] Flow rate of mobile phase: 1.0 mL / min.
[0093] Diluent: mobile phase.
[0094] Elution program: isocratic elution, time 25 min.
[0095] Precisely take 10 μL of the positioning solution and the test solution in Test Example 1, inject into the high performance liquid chromatograph, perform high performance liquid chromatography analysis according to the above chromatography conditions, record the chromatogram, and the results are shown in Figure 7 .
[0096] As can be seen from Table 6, the detection method of the present application has good repeatability. Figure 7 It can be seen that 16-dehydroprogesterone (16-dehydro PG) and progesterone (PG) overlap in peak position, and effective separation of the main peak and impurities cannot be achieved.
[0097] (Comparative Example 2) The present comparative example is to detect 16-dehydroprogesterone by referring to the detection method of progesterone related substances in the prior art “European Pharmacopoeia”.
[0098] Column: Agilent ZORBAX Eclipse XDB C18, 4.6 mm × 150 mm, 5 μm.
[0099] Detection wavelength: 241nm.
[0100] Column temperature: 35℃.
[0101] Injection volume: 10 μL.
[0102] Mobile phase A: Acetonitrile.
[0103] Mobile phase B: Water.
[0104] Mobile phase flow rate: 1.0 mL / min.
[0105] Diluent: Acetonitrile / Water = 80% / 20%.
[0106] Elution procedure: gradient elution, see Table 7 for details.
[0107] Table 7 Time (min) Mobile phase A (%) Mobile phase B (%) 0 50 50 20 50 50 27 80 20 45 80 20
[0108] Accurately measure 10 μL each of the positioning solution and the test solution from Example 1, inject them into the high-performance liquid chromatograph (HPLC), perform HPLC analysis under the chromatographic conditions described above, record the chromatograms, and see the results shown in the figure. Figure 8 .
[0109] Depend on Figure 8 It can be seen that the peak of 16-dehydroprogesterone (16-dehydroPG) is adjacent to the peak of progesterone (PG), and the peak of impurity C overlaps with the peak of 16-dehydroprogesterone (16-dehydroPG), so they cannot be effectively separated.
Claims
1. A method for detecting 16-dehydroprogesterone in progesterone by high performance liquid chromatography, characterized by: The adopted chromatographic column is Agilent InfinityLab Poroshell SB-C8; a mixed mobile phase of mobile phase A and mobile phase B is used for gradient elution; the mobile phase A is acetonitrile; and the mobile phase B is water.
2. The method for detecting 16-dehydroprogesterone in progesterone by high performance liquid chromatography according to claim 1, characterized in that The gradient elution procedure is as follows: the volume ratio of the mobile phase A and the mobile phase B is balanced at 50%:50% at 0-20 min; the volume ratio of the mobile phase A and the mobile phase B is gradiently increased to 80%:20% at 20-27 min; the volume ratio of the mobile phase A and the mobile phase B is balanced at 80%:20% at 27-40 min; the volume ratio of the mobile phase A and the mobile phase B is gradiently decreased to 50%:50% at 40-42 min; and the volume ratio of the mobile phase A and the mobile phase B is balanced at 50%:50% at 42-50 min.
3. The method for detecting 16-dehydroprogesterone in progesterone by high performance liquid chromatography according to claim 1 or 2, characterized in that Other conditions of the high performance liquid chromatography are as follows: the detection wavelength is 241 nm; the chromatographic column temperature is 30-40 ℃; the injection amount is 10 μL; and the flow rate of the mobile phase is 0.8-1.2 mL / min.