Method for detecting free steroid impurities in naturally-sourced conjugated estrogen drugs

Abstract

The invention provides a liquid chromatography-mass spectrometry (LC-MS) detection method for free steroidal impurities in a conjugated estrogen drug, which comprises the following steps: detecting and analyzing a starting material pregnant mare urine, a technical process sample, a conjugated estrogen drug and a preparation thereof of a conjugated estrogen drug to be detected by taking oestrone as a reference substance and adopting an LC-MS method in a selective ion monitoring (SIM) mode, so as to determine the content of free steroidal impurities in the conjugated estrogen drug. And calculating the content of oestrone in the conjugated estrogen related sample by using an external standard method according to an analysis result, and determining the content of free steroid impurities of maleestrone and 17 alpha-dihydromaleestrone in the sample by using a correction factor-added external standard method. The detection method is high in sensitivity, good in reproducibility and strong in specificity, and has a relatively good linear relation and a relatively high recovery rate in a common concentration range. Therefore, the content of the free steroid impurity components in the sample to be detected can be rapidly and accurately detected by utilizing the method, and the method has a good application prospect in the aspects of a technological process of a naturally-sourced conjugated estrogen drug and quality control of a finished product.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmaceutical analysis method, and particularly relates to a liquid chromatography-mass spectrometry detection method for free steroidal impurities in natural source conjugated estrogen drugs. BACKGROUND

[0002] Natural conjugated estrogen is a natural mixed conjugated estrogen extracted from animal urine. Natural conjugated estrogen drugs are mainly used for hormone replacement therapy (HRT) and are used for relieving clinical symptoms of any estrogen deficiency in the body, treating and preventing menopausal syndrome after female physiological or artificial menopause, and preventing and treating osteoporosis and coronary heart disease.

[0003] The chemical components of natural source conjugated estrogen bulk drugs are very complex, and the main active components are conjugated steroids, including conjugated estrogen, conjugated progestogen and conjugated androgen. In addition, the bulk drug also contains a small amount of free steroidal components, which have a low content but belong to the impurity components that need to be concerned.

[0004] In recent years, with the continuous deepening of pharmaceutical preparation research and the rapid development of pharmaceutical analysis technology, the International Conference on Harmonization (ICH) and the drug regulatory department have also put forward higher requirements for the quality control and safety of drug impurities, requiring comprehensive impurity analysis, clear impurity source and impurity change in the process. Especially, the quality control of raw materials and preparations with complex component systems is a common difficult problem in the industry, and the quality control of low-content, high-activity related impurities is the key.

[0005] USP conjugated estrogen bulk drug monograph controls estrone, equilin and 17a-dihydroequilin as impurities. According to the search of the European Chemicals Agency (ECHA) website, estrone has in vitro genetic toxicity evaluation. When other detection endpoints or less common cell types are used, more positive detection results will appear, and estrone may induce chromosomal aberrations. Such free hormone impurities often have a safety risk due to low content, so the accuracy of the measurement of very low concentration is very high.

[0006] As one of the few drugs containing complex ingredients approved by the US FDA, the natural conjugated estrogen raw material and its preparation are currently analyzed by GC-FID using derivatization treatment for free steroid impurities. The pre-treatment steps are more, the operation is more difficult, the time is longer, the detection fluctuation is large, and the sensitivity is low. A patent (CN 114184712A) discloses HPLC-UV for determining free steroids in conjugated estrogen tablets, which has relatively low sensitivity, uses three control samples for determination, and has high reagent cost. At the same time, because there is no strong conjugated system in the chemical structure of the three free steroids, there is no specific characteristic absorption wavelength, and the ultraviolet end absorption (205 nm) is used as the detection wavelength, which is easily affected by interference, affecting the repeatability and accuracy of the detection. The pre-treatment of the two methods is not suitable for the complex starting material of the natural source conjugated estrogen, the sample detection in the process, or the cream preparation, etc.

[0007] In summary, it is necessary to develop a suitable quality control method for free steroid impurities in natural source conjugated estrogen starting materials, raw materials and their preparations, to ensure the safety, stable and controllable quality of natural source conjugated estrogen drugs throughout the production process and the whole life cycle, and to provide new ideas and references for the quality control of drugs containing complex ingredients. SUMMARY

[0008] In view of the difficulty of the existing method in tracing the starting material and free steroid impurity content in complex matrix preparation, the present application provides a LC-MS method for determining the content of three free steroid impurities in natural conjugated estrogen drugs. The quantitative analysis method has good specificity and high accuracy, and can effectively realize the quality control of low content of related impurities in drugs.

[0009] To achieve the purpose of the present application, the following technical solutions are adopted:

[0010] A liquid chromatography-mass spectrometry method for detecting free steroid impurities in natural source conjugated estrogen drugs, wherein the natural source natural conjugated estrogen drug to be tested is detected and analyzed by LC-MS method in selective ion monitoring (SIM) mode, and the content of estrone in the related sample of the conjugated estrogen is calculated by external standard method according to the analysis result. The contents of equilin and 17α-dihydroequilin free steroid impurities in the sample are determined by external standard method with correction factor.

[0011] Specifically comprising the following steps:

[0012] (1) Preparation of control solution: weigh appropriate amounts of estrone, equilin and 17α-dihydroequilin control samples, and add ethanol solution to prepare mixed control solutions with different concentrations;

[0013] (2) Preparation of the pregnant mare's urine test sample solution: Take pregnant mare's urine, centrifuge, and accurately measure 10 ml of the supernatant. Add 10% to 15% sodium chloride, shake to dissolve, add 1,2-dichloroethane, extract, centrifuge, and blow dry. Dissolve the residue in 5 ml of an ethanol aqueous solution, shake well, centrifuge, and take the supernatant to obtain the solution.

[0014] (3) Preparation of the natural source conjugated estrogen raw material or its preparation test sample solution: Take natural source conjugated estrogen raw material or its preparation, accurately measure an appropriate amount (about 1.25 mg to 40 mg of conjugated estrogen), accurately measure the weight, ultrasonically treat, cool, re-measure the weight, make up the weight loss with 1,2-dichloroethane, centrifuge, accurately measure the supernatant, blow dry, dissolve the residue, shake well, centrifuge, and take the supernatant to obtain the solution.

[0015] (4) Standard curve: Inject the control sample solution of different concentrations prepared in step (1) into the liquid chromatograph-mass spectrometer to determine the peak position of the three components. Draw a standard curve with the concentration of the control sample solution as the abscissa and the peak area of the control sample quantitative ion extraction ion flow as the ordinate.

[0016] (5) Determination of the sample to be measured: Inject the test sample solution in steps (2) and (3) into the liquid chromatograph-mass spectrometer. According to the quantitative ion extraction ion flow peak area, calculate the content of the three free steroidal impurities in the sample to be measured according to the linear equation obtained in step (3).

[0017] Further, the liquid chromatography conditions in steps (4) and (5) are as follows: use a C 18 18 column as the chromatographic column, the column temperature is 38°C to 42°C, the flow rate is 0.28 ml / min ～ 0.32 ml / min, the injection volume is 1 μl, use 0.1% formic acid solution as the mobile phase A, 0.1% formic acid methanol solution as the mobile phase B, and elute with the mobile phase A-mobile phase B (45:55) isocratic elution for 50 min.

[0018] Further, the mass spectrometry conditions in steps (4) and (5) are as follows: electrospray ionization, positive ion scanning, selective ion monitoring mode; capillary voltage: 3500 V; drying gas temperature: 550°C; drying gas flow rate: 11.0 L / min; atomizing gas pressure: 55 psi; mass spectrometry collection time: 15 min to 30 min; quantitative ion m / z: 269, collision energy: 110; quantitative ion m / z: 271, collision energy: 90; gain factor: 1.

[0019] Further, in the (1), the ethanol aqueous solution is an 80% ethanol solution.

[0020] Further, in the sample (3), the ultrasonic treatment time is 10 to 60 minutes.

[0021] Further, the method is used for quantitative analysis of free steroid impurities in natural source conjugated estrogen raw drug starting material, raw drug and its preparation.

[0022] The method has the advantages that: the method for detecting free steroid impurities in natural source conjugated estrogen raw drug starting material, raw drug and its preparation by using liquid chromatography-mass spectrometry, extracts free steroid components in the pregnant mare urine sample by using liquid-liquid extraction method, extracts free steroid components in the raw drug and its preparation by using ultrasonic method, the extraction operation is simple, efficient, time-saving, and the analysis is carried out by using liquid chromatography-mass spectrometry, the detection sensitivity, reproducibility and specificity are good, and there is a good linear relationship and a high recovery rate in a common concentration range. The content of equilin and 17α-dihydroequilin can be calculated by using the estrone control product and the correction factor, which can reduce the detection cost. Therefore, the method can be used to simply, quickly, accurately and low-cost detect the content of free steroid components in the related sample of conjugated estrogen, and has a good application prospect in the quality control of natural source conjugated estrogen drugs. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Extraction ion chromatogram of mixed control solution.

[0024] Figure 2 Extraction ion chromatogram of conjugated estrogen starting material pregnant mare urine.

[0025] Figure 3 Extraction ion chromatogram of conjugated estrogen raw drug.

[0026] Figure 4 Extraction ion chromatogram of conjugated estrogen tablets (self-made).

[0027] Figure 5 Extraction ion chromatogram of conjugated estrogen cream (self-made).

[0028] Figure 6 Extraction ion chromatogram of conjugated estrogen for injection (reference preparation).

[0029] Figure 7 Extraction ion chromatogram of conjugated estrogen tablets (reference preparation).

[0030] Figure 8 Extraction ion chromatogram of conjugated estrogen cream (reference preparation). DETAILED DESCRIPTION

[0031] The content of the application will be further described below in combination with the following examples and drawings of the specification, but is not limited to the protection scope of the application.

[0032] The technical solutions of the present application are further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the present application, and should not be regarded as specific limitations on the present application.

[0033] The instruments used in the embodiments of the present application are: 1260 HPLC chromatograph (Agilent, USA); 6120B mass spectrometer (Agilent, USA);

[0034] The reagents used in the embodiments of the present application are: acetonitrile, methanol and formic acid are mass spectrometry grade;

[0035] The control used in the embodiments of the present application is:

[0036] Name Purity Source Estrone 99.5％ China Institute for Drug Control Equilenin 99.9％ USP 17a-Dihydroequilenin 99.2％ USP Sodium estrone sulfate 98.0％ TRC

[0037] The test sample used in the embodiments of the present application is:

[0038] Example 1 (mass spectrometry parameter electrode mode investigation)

[0039] In this embodiment, LC-MS detection of free steroid impurities in natural source conjugated estrogen samples is developed, and the method is as follows:

[0040] (1) Analysis method

[0041]

[0042]

[0043] (2) Results

[0044] The mixed control solution is measured according to the method, and the results show that the positive mode response of the three free steroid target components is greater than the negative mode, the positive mode response of the main component estrone sodium sulfate and other adjacent interference peaks is lower than the negative mode, the positive mode is selected, and the results are as follows.

[0045] Table 2 response of three free steroids and estrone sodium sulfate in positive and negative modes

[0046] Name Response in negative mode Response in positive mode 17a-Dihydroequilenin 14.21 159.48 Equilenin 131.73 2555.53 Estrone 28.51 1654.03 Sodium estrone sulfate 11649.82 139.99

[0047] Example 2 (diluent investigation)

[0048] In this embodiment, LC-MS detection of free steroid impurities in natural source conjugated estrogen samples is developed:

[0049] Methanol is used as the diluent of the estrone control sample stock solution, and is refrigerated for about 24 h, and estrone is precipitated; the diluent is changed to ethanol, and no precipitation occurs, so ethanol is used as the diluent of the control sample stock solution.

[0050] The residue after nitrogen blowing was dissolved in 50% methanol solution by ultrasonic. To ensure complete dissolution, 80% ethanol solution was used as diluent.

[0051] Example 3 (Correction factor determination)

[0052] In this example, the analysis method was the same as that in Example 4.

[0053] Three component reference substances were precisely weighed, and three free steroid standard curve series solutions were respectively taken. After injection, the amount injected was linearly regressed against the response value (peak area, etc.) by least squares method to obtain three standard curves. Taking estrone as the main component, the ratio of the curve slopes of 17a-dihydroequilenin and estrone was the correction factor of 17a-dihydroequilenin, and the ratio of the curve slopes of equilenin and estrone was the correction factor of equilenin.

[0054] The determination was performed for 3 times, and the average value was calculated, and the results were as follows.

[0055] Table 3 17a-dihydroequilenin and equilenin correction factor determination

[0056]

[0057] Example 4 (Methodology verification)

[0058] (1) Analysis method

[0059]

[0060]

[0061] (2) System suitability

[0062] System suitability solution 1 μl was precisely measured and injected into liquid chromatography-mass spectrometry instrument, and 5 injections were continuously performed, and the chromatogram (extracted ion flow chart is shown in the attached Figure 1 ) was recorded. The results showed that 17a-dihydroequilenin: retention time RSD was 0.2%, and peak area RSD was 0.3%, which met the requirements; equilenin: retention time RSD was 0.2%, and peak area RSD was 0.8%, which met the requirements; estrone: retention time RSD was 0.2%, and peak area RSD was 0.7%, which met the requirements.

[0063] (3) Linear relationship investigation

[0064] Different concentrations of linear solutions were precisely measured, each 1 μl, and were respectively injected into liquid chromatography-mass spectrometry instrument, and the chromatogram was recorded. The peak area was linearly regressed against the concentration to draw a standard curve.

[0065] The results show that the 17a-dihydroequilin concentration is 0.300 μg / ml-19.996 μg / ml, the linear regression equation is y=11670x-3, the correlation coefficient R 2 The value is 1.000, the intercept of the Y-axis peak area is 0.1%, which meets the requirements;

[0066] The equilin concentration is 0.049 μg / ml-19.748 μg / ml, the linear regression equation is y=59067x+19911, the correlation coefficient R 2 The value is 0.998; the intercept of the Y-axis peak area is 3.4%, which meets the requirements.

[0067] The equilin concentration is 0.049 μg / ml-19.748 μg / ml, the linear regression equation is y=59067x+19911, the correlation coefficient R 2 The value is 0.998; the intercept of the Y-axis peak area is 3.4%, which meets the requirements.

[0068] (4) Detection limit and quantification limit

[0069] The control solution is diluted step by step, and when the signal-to-noise ratio S / N is 10, it is the quantification limit, and when the signal-to-noise ratio S / N is 3, it is the detection limit. The precision is investigated at the quantification limit concentration, and the results are as follows:

[0070] 1) Detection limit solution:

[0071] a. The 17a-dihydroequilin concentration is 0.045 μg / ml (equivalent to the concentration of pregnanuline of 0.0225 μg / ml), S / N is 5, which meets the requirements;

[0072] b. The equilin concentration is 0.006 μg / ml (equivalent to the concentration of pregnanuline of 0.003 μg / ml), S / N is 7, which meets the requirements;

[0073] c. The equilin concentration is 0.007 μg / ml (equivalent to the concentration of pregnanuline of 0.0035 μg / ml), S / N is 6, which meets the requirements;

[0074] 2) Quantification limit solution:

[0075] a. The 17a-dihydroequilin concentration is 0.150 μg / ml (equivalent to the concentration of pregnanuline of 0.075 μg / ml), S / N is between 11 and 20, the peak area RSD of 6 needles is 11.0%, which meets the requirements;

[0076] b. The equilin concentration is 0.020 μg / ml (equivalent to the concentration of pregnanuline of 0.010 μg / ml), S / N is between 14 and 22, the peak area RSD of 6 needles is 4.9%, which meets the requirements;

[0077] c. The estrone concentration was 0.025 μg / ml (equivalent to 0.013 μg / ml in the concentration of pregnant mare urine), S / N was between 11 and 20, and the RSD of peak area of 6 needles was 9.4%, which met the requirements.

[0078] (5) Solution stability

[0079] The control solution and the test solution were respectively placed at room temperature for 0 h, 10 h, 15 h, 27 h, 32 h, 38 h, 42 h, 47 h and 52 h, the control stock solution was placed in the refrigerator for 19 days, the control solution (linear solution 4) was prepared, 1 μl was injected into the liquid chromatograph, and the stability was investigated.

[0080] The results showed that the RSD of the content of 17α-dihydroequilin within 52 h at room temperature was 1.0%, the RSD of the content of the test solution within 45 h at room temperature was 1.0%, the recovery rate of the control stock solution placed in the refrigerator for 19 days was 102.6%, and all met the requirements;

[0081] The RSD of the content of equilin within 52 h at room temperature was 1.1%, the RSD of the content of the test solution within 45 h at room temperature was 1.1%, the recovery rate of the control stock solution placed in the refrigerator for 19 days was 102.5%, and all met the requirements;

[0082] The RSD of the content of estrone within 52 h at room temperature was 1.0%, the RSD of the content of the test solution within 45 h at room temperature was 0.9%, and the recovery rate of the control stock solution placed in the refrigerator for 19 days was 103.7%, all of which met the requirements.

[0083] (6) Reproducibility test

[0084] Six test solutions of pregnant mare urine were prepared in parallel, the content of free steroidal impurities and the RSD value were calculated. The results are shown in Table 4. The RSD of the content of 17α-dihydroequilin in the six reproducible solutions was 1.6%, the RSD of the content of equilin was 0.7%, and the RSD of the content of estrone sodium sulfate was 1.1%, which met the requirements, and the method was good in reproducibility.

[0085] Table 4 Reproducibility results

[0086]

[0087] (7) Precision test

[0088] Six samples of pregnant mare urine were prepared in parallel on different dates and by different analysts to calculate the content of free steroid impurities and RSD values. The results are shown in Table 5. The RSD values for 17a-dihydroequilenin, equilenin and estrone in the six intermediate precision solutions were 1.5%, 0.7% and 1.1%, respectively. The RSD values for 17a-dihydroequilenin, equilenin and estrone in the 12 precision solutions were 1.5%, 0.7% and 2.7%, respectively, all of which met the requirements and the method had good precision.

[0089] Table 5. Intermediate precision results

[0090]

[0091]

[0092] (8) Accuracy test

[0093] 1) Standard addition stock solution

[0094] Accurately pipette 17a-dihydroequilenin control stock solution-15 ml, equilenin control stock solution-15 ml and estrone control stock solution-15 ml into a 50 ml volumetric flask, dilute to the mark with 80% ethanol solution, shake well to obtain the solution (total concentration of about 30 μg / ml, single concentration of about 10 μg / ml).

[0095] 2) Test sample solution (without standard addition)

[0096] Two samples of pregnant mare urine were prepared in parallel.

[0097] 3) 80% standard addition test sample solution

[0098] Accurately pipette 5 ml of pregnant mare urine and 5 ml of water, add 1.5 g of sodium chloride, dissolve, then add 2 ml of standard addition stock solution and 3 ml of 1,2-dichloroethane, shake to extract, centrifuge at 6000 rpm for 10 min, collect the organic phase A-1 and the aqueous phase A-2. Add 5 ml of 1,2-dichloroethane to the aqueous phase A-2, shake to extract, centrifuge at 6000 rpm for 10 min, collect the organic phase B-1 and combine with A-1. Take the combined organic phase, dry under nitrogen at room temperature, accurately pipette 5 ml of 80% ethanol solution to redissolve until completely dissolved, shake well, centrifuge at 13000 rpm for 10 min, take the supernatant for sampling.

[0099] Three samples were prepared in parallel.

[0100] 4) 100% standard addition test sample solution

[0101] Accurately weigh 5 ml of pregnant mare's urine and 5 ml of water, add 1.5 g of sodium chloride, dissolve, then add 2.5 ml of the labeled stock solution and 2.5 ml of 1,2-dichloroethane, shake and extract, centrifuge at 6000 rpm for 10 min, collect the organic phase A-1, and the aqueous phase A-2. Add 5 ml of 1,2-dichloroethane to the aqueous phase A-2, shake and extract, centrifuge at 6000 rpm for 10 min, collect the organic phase B-1, and combine with A-1. Take the combined organic phase, dry under nitrogen at room temperature, accurately weigh 5 ml of 80% ethanol solution, redissolve until completely dissolved, shake well, centrifuge at 13000 rpm for 10 min, and take the supernatant for sampling.

[0102] Prepare 3 samples in parallel.

[0103] 5) 120% labeled sample solution

[0104] Accurately weigh 5 ml of pregnant mare's urine and 5 ml of water, add 1.5 g of sodium chloride, dissolve, then add 2.5 ml of the labeled stock solution and 2.5 ml of 1,2-dichloroethane, shake and extract, centrifuge at 6000 rpm for 10 min, collect the organic phase A-1, and the aqueous phase A-2. Add 5 ml of 1,2-dichloroethane to the aqueous phase A-2, shake and extract, centrifuge at 6000 rpm for 10 min, collect the organic phase B-1, and combine with A-1. Take the combined organic phase, dry under nitrogen at room temperature, accurately weigh 5 ml of 80% ethanol solution, redissolve until completely dissolved, shake well, centrifuge at 13000 rpm for 10 min, and take the supernatant for sampling.

[0105] Prepare 3 samples in parallel.

[0106] 6) Results

[0107] Accurately weigh 1 μl of each of the accuracy solutions, inject into the liquid chromatograph-mass spectrometer, record the chromatogram, calculate the recovery rates of the three main components according to the standard curve method, and the labeled recovery rate = (measured amount of labeled sample - known sample amount) / added amount x 100%, and the results are shown in Tables 6-8.

[0108] Results: The labeled recovery rates of the 9 accuracy solutions of 17α-dihydroequilin were between 96.2% and 104.6%, the labeled recovery rate RSD was 3.6%; the labeled recovery rates of the 9 accuracy solutions of equilin were between 99.3% and 112.0%, the labeled recovery rate RSD was 4.5%; the labeled recovery rates of the 9 accuracy solutions of estrone were between 98.9% and 111.4%, the labeled recovery rate RSD was 4.2%, all of which met the requirements, and the accuracy of the method was good.

[0109] Table 6 Accuracy results of 17α-dihydroequilin

[0110]

[0111] Table 7 Accuracy results for equilenin

[0112]

[0113]

[0114] Table 8 Accuracy results for estrone

[0115]

[0116] (9) Robustness

[0117] The degree of tolerance of the determination results was evaluated by changing the column temperature and flow rate with slight variations in the determination conditions. The factors changed were as follows:

[0118]

[0119] One μl of the standard curve solution and one μl of the sample solution were precisely measured and injected into the liquid chromatograph-mass spectrometer, respectively, and the chromatogram was recorded. The content of the three main components was calculated according to the standard curve method and the change in the correction factor was evaluated. The results are shown in Tables 9 to 11.

[0120] Under the condition of changing the flow rate (0.28 ml / min, 0.32 ml / min), the system suitability was in accordance with the requirements, the RSD of the content of 17a-dihydroequilenin was 1.0%, the RSD of the content of equilenin was 1.5%, and the RSD of the content of estrone was 0.9%, which met the requirements, and the method was robust. Under the condition of changing the column temperature (38°C, 42°C), the system suitability was in accordance with the requirements, the RSD of the content of 17a-dihydroequilenin was 3.6%, the RSD of the content of equilenin was 1.9%, and the RSD of the content of estrone was 1.8%, and the RSD of the correction factor of 17a-dihydroequilenin was 1.2%, and the RSD of the correction factor of equilenin was 0%. The method was robust.

[0121] Table 9 Robustness results (change in flow rate)

[0122]

[0123] Table 10 Robustness results (change in column temperature)

[0124]

[0125] Table 11 Robustness results of the correction factor

[0126]

[0127]

[0128] The comprehensive methodology verified all the results of the investigated items, indicating that the method is sensitive, reproducible and accurate.

[0129] Example 5 (sample determination)

[0130] The analytical method was the same as in Example 4; the test sample solution was prepared as follows

[0131]

[0132]

[0133] 1 μl of the above test sample solution was injected into the liquid chromatograph, and the chromatogram was recorded, wherein the pregnenolone extraction ion flow chart is shown in Figure 1 Figure 2 , the conjugated estrogen raw material extraction ion flow chart is shown in Figure 2 Figure 3 , the conjugated estrogen tablet (self-made) extraction ion flow chart is shown in Figure 3 Figure 4 , the conjugated estrogen cream (self-made) extraction ion flow chart is shown in Figure 4 Figure 5 , the conjugated estrogen injection (reference preparation) extraction ion flow chart is shown in Figure 5 Figure 6 , the conjugated estrogen tablet (reference preparation) extraction ion flow chart is shown in Figure 6 Figure 7 , the conjugated estrogen cream (reference preparation) extraction ion flow chart is shown in Figure 7 Figure 8 .

[0134] The contents of the three free steroid impurities in the conjugated estrogen related samples were measured and calculated, the results of the pregnenolone are shown in Table 12, and the results of the raw material and preparation are shown in Table 13. The contents of the raw material and preparation were low, the equilin was 0.0081% to 0.1687%, 17α-dihydroequilin was not detected to 0.1417%, and the estrone was 0.0516% to 0.6148%.

[0135] Table 12: Content results of free steroid impurities in the raw material pregnenolone (μg / ml)

[0136] Type Batch No. Equilenin 17a-Dihydroequilenin Estrone Total content Pregnenolone YMN2405 3.76 3.81 4.83 12.40 Pregnenolone YMN0108 1.87 3.09 1.98 6.94

[0137] Table 13: Content results of free steroid impurities in the raw material and preparation as a percentage of conjugated estrogen

[0138] Type Batch No. Equilenin 17a-Dihydroequilenin Estrone Total content Conjugated estrogens drug substance (home-made) LL0801 0.0116％ 0.0097％ 0.0513％ 0.0725％ Conjugated estrogens drug substance (home-made) LL0802 0.0081％ 0.0092％ 0.0513％ 0.0686％ Conjugated estrogens drug substance (home-made) LL0803 0.0098％ 0.0113％ 0.0579％ 0.0790％ Conjugated estrogens tablets (home-made) LL0101 0.1384％ 0.1317％ 0.2538％ 0.5240％ Conjugated estrogens tablets (home-made) LL0502 0.1687％ 0.1417％ 0.3044％ 0.6148％ Conjugated estrogens tablets (home-made) LL0904 0.0842％ 0.0961％ 0.2235％ 0.4038％ Conjugated estrogens cream (home-made) LL1216 0.0928％ Not detected 0.2343％ 0.3270％ Conjugated estrogens cream (home-made) LL1217 0.1408％ 0.0688％ 0.4019％ 0.6115％ Conjugated estrogens cream (home-made) LL1219 0.0449％ Not detected 0.1282％ 0.1730％ Conjugated estrogens for injection (reference) 19P049A 0.0296％ 0.0137％ 0.0443％ 0.0876％ Conjugated estrogens tablets (reference) LD4346 0.0235％ 0.0180％ 0.0102％ 0.0516％ Conjugated estrogens cream (reference) 3X5W 0.0497％ Not detected 0.0853％ 0.1350％

[0139] It is to be understood, of course, that the foregoing describes and illustrates only the principles of the application and that this application is to be considered in its broadest and widest sense and that the appended claims are to be construed accordingly. Thus, although the present application has been described in detail with regard to implementations shown and described above, it should be apparent that various modifications can be made of the application without departing from its spirit and scope. Accordingly, it is intended that all such modifications fall within the scope of the application.

Claims

1. A method for detecting free steroidal impurities in natural source conjugated estrogen pharmaceuticals by liquid chromatography-mass spectrometry, characterized in that, The natural source natural conjugated estrogen drug to be tested is detected and analyzed by using LC-MS method in SIM mode, with estrone as a control sample, the content of estrone in the related sample of the conjugated estrogen is calculated by using external standard method according to the analysis result, and the contents of the free steroidal impurities of equilin and 17α-dihydroequilin in the sample are determined by using external standard method with a correction factor.

2. The method for detecting free steroidal impurities in natural origin conjugated estrogens drug substance by LC-MS as claimed in claim 1, wherein, Specifically, the following steps are included: (1) Preparation of control sample solution: estrone, equilin and 17α-dihydroequilin control samples are weighed, and ethanol aqueous solution is added to prepare mixed control sample solutions with different concentrations; (2) Preparation of pregnant mare's urine sample solution: pregnant mare's urine is taken, centrifuged, and the supernatant is precisely taken, 10ml of 10%-15% sodium chloride is added, shaken to dissolve, 1,2-dichloroethane is added for extraction, centrifuged, dried, and the residue is dissolved in 5ml of ethanol aqueous solution, shaken, centrifuged, and the supernatant is obtained; (3) Preparation of natural source conjugated estrogen raw material or preparation sample solution: natural source conjugated estrogen raw material or preparation is weighed (about 1.25mg-40mg of conjugated estrogen), precisely weighed, added with 1,2-dichloroethane, weighed, ultrasonically treated, cooled, weighed again, the weight loss is made up with 1,2-dichloroethane, centrifuged, the supernatant is precisely taken, dried, the residue is dissolved, shaken, centrifuged, and the supernatant is obtained; (4) Standard curve: the control sample solutions with different concentrations prepared in step (1) are injected into a liquid chromatograph-mass spectrometer, and the peak positions of the three components are determined; the concentration of the control sample solution is taken as the abscissa, and the peak area of the control sample quantitative ion extraction ion flow is taken as the ordinate to draw a standard curve; (5) Determination of the sample to be tested: the sample solutions in steps (2) and (3) are injected into a liquid chromatograph-mass spectrometer, the peak area of the quantitative ion extraction ion flow is determined, and the content of the three free steroidal impurities in the sample to be tested is calculated according to the linear equation obtained in step (3).

3. The method for detecting free steroidal impurities in natural origin conjugated estrogens drug substance by LC-MS as claimed in claim 2, wherein, The liquid chromatography conditions in steps (4) and (5) are as follows: using C 18 18 column as the chromatographic column, column temperature: 38-42°C; flow rate: 0.28-0.32 ml / min; injection volume: 1 μ l; using 0.1% formic acid solution as mobile phase A and 0.1% formic acid methanol solution as mobile phase B, eluting with mobile phase A-mobile phase B (45:55) isocratic, running for 50 min.

4. The method for detecting free steroidal impurities in natural origin conjugated estrogens drug substance by LC-MS as claimed in claim 2, wherein, In steps (4) and (5), the mass spectrometry conditions are as follows: electrospray ionization, positive ion scanning, selective ion monitoring mode; capillary voltage: 3500V; dry gas temperature: 550℃; dry gas flow rate: 11.0L / min; atomization gas pressure: 55psi; mass spectrometry collection time: 15min-30min; quantitative ion m / z: 269, collision energy: 110; quantitative ion m / z: 271, collision energy: 90; gain factor:

1.

5. The method for detecting free steroidal impurities in natural origin conjugated estrogens drug substance by LC-MS as claimed in claim 2, wherein, In the step (1), the ethanol aqueous solution is 80% ethanol solution.

6. The method for detecting free steroidal impurities in natural origin conjugated estrogens drug substance by LC-MS as claimed in claim 2, wherein, In the sample (3), the ultrasonic treatment time is 10-60 minutes.

7. The method for detecting free steroidal impurities in natural origin conjugated estrogens drug substance by LC-MS as claimed in claim 2, wherein, The method is used for quantitative analysis of free steroidal impurities in natural source conjugated estrogen raw material, raw material and preparation.

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