Extraction and separation method of five non-steroidal accompanying components in naturally-sourced conjugated estrogen drugs

Abstract

The invention discloses a method for extracting and separating five non-steroidal accompanying components in a natural-source conjugated estrogen drug, which is based on tracking and monitoring of HPLC-UV (High Performance Liquid Chromatography-Ultraviolet) and UPLC-Q-TOF MS (Ultra Performance Liquid Chromatography-Quantitative Time of Flight Mass Spectrometry) analysis technologies, and adopts a modernized separation technology to directionally separate and prepare the five non-steroidal components from an original research preparation, namely Baimeli tablets and a self-research raw material drug. According to the invention, chemical components of an originally developed preparation and a self-developed bulk drug are respectively researched, and five non-steroidal components are directionally separated by adopting separation technologies such as liquid-liquid extraction, macroporous resin chromatography, polyamide chromatography, silica gel chromatography and high performance liquid preparation; the method comprises the following steps: respectively identifying benzamido acetic acid (IMP-A), N-(2-phenylacetyl) glycine (IMP-B), indolyl sulfate (IMP-C), 2, 6-dihydroxy benzoic acid (IMP-D) and p-cresol sulfate (IMP-E) as the raw materials, namely, 2, 6-dihydroxy benzoic acid (IMP-C), 2, 6-dihydroxy benzoic acid (IMP-D) and 2, 6-dihydroxy benzoic acid (IMP-E). The five non-steroidal components prepared by the method provided by the invention are high in purity, and the definite chemical structures of the five non-steroidal components are beneficial to development, evaluation and quality control of conjugated estrogen imitation drugs and new products thereof.

Description

Technical Field

[0001] The invention relates to the technical field of natural drug chemistry, and in particular to a method for extracting and separating non-steroidal accompanying components in a natural source combined estrogen drug. Background Art

[0002] Conjugated estrogens (CE) are a natural conjugated steroidal mixed drug extracted and separated from the urine of pregnant mares from about 120 to 250 days. Its main components are sulfated estrogens produced by phase II metabolism of endogenous steroid components under the catalysis of sulfotransferase.

[0004] There has been no consensus on the composition of conjugated estrogen APIs, and the active ingredients have not been fully determined, which directly affects the development of generic drugs or their new products. With the continuous advancement of analytical technology, CE APIs contain a large number of unknown ingredients in addition to conjugated estrogen ingredients. Obviously, a single USP method cannot accurately reveal the material composition of CE APIs. In 2015, the FDA Center for Drug Evaluation and Research (CDER) published an article entitled "Marketplace Analysis of Conjugated Estrogens: Determining the Consistently Present Steroidal Content with LC-MS", developed an ultra-high performance liquid phase-high resolution mass spectrometry (UPLC-HRMS) analysis method, and made qualitative and semi-quantitative requirements for 60 major steroidal components. At the same time, the generic drug consistency evaluation guide "Draft Guidance on Conjugated Estrogens" was released. In addition to proposing relevant limiting requirements for conjugated steroid components, non-steroidal components were also defined as "Concomitant Components" and should be studied in accordance with the relevant guidelines for drug "impurities". Because the chemical structure and quantity of non-steroidal concomitant ingredients are not specified in the guidelines, in order to pass the FDA generic drug consistency evaluation, the non-steroidal concomitant ingredients in the original preparation must be separated and their structures identified. On the one hand, this meets the evaluation needs of conjugated estrogen generic drugs and related new products, and on the other hand, it allows process development to reasonably remove non-functional ingredients and ensure the safety of drug clinical applications to the greatest extent. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method for extracting and separating five non-steroidal accompanying components in a natural source combined estrogen drug, the method comprising the following steps:

[0006] (1) The preparation method of natural combined estrogen drug powder is as follows: take natural combined estrogen tablets, quickly and repeatedly rinse the surface of the tablets with a small amount of purified water, wash the pigment coating, and discard the rinsing solution; transfer the tablet core to a vacuum drying oven, and dry it at low temperature and reduced pressure to constant weight; then take the dried tablet core and crush it into coarse powder, or the natural combined estrogen drug powder is the natural combined estrogen raw material powder;

[0007] (2) taking the natural combined estrogen drug powder of step (1), adding an alcohol solvent (the amount of the alcohol solvent added is 25 to 100 times the mass of the natural combined estrogen drug powder), soaking for 1 hour, ultrasonically extracting twice, each time for 30 minutes, filtering with a 0.45 μm microporous filter membrane, combining the filtrate, concentrating to dryness under reduced pressure, and then adding an appropriate amount of purified water for re-dissolution;

[0008] (3) Add an alcoholic organic solvent in equal proportion to the aqueous solution of step (2), perform liquid-liquid extraction three times, discard the aqueous phase, separate and combine the clarified organic phases, and concentrate under reduced pressure until there is no alcohol taste (purified water is added to replace the organic phase during the concentration process), and finally add an appropriate amount of purified water to dilute and dissolve;

[0009] (4) The water dilution in step (3) is subjected to macroporous resin adsorption separation, using water as an eluent, collecting the water washing liquid in portions, and combining them according to the HPLC-UV detection results to obtain three components, namely component 1-1, component 1-2 and component 1-3;

[0010] (5) The component 1-1 described in step (4) is separated by polyamide adsorption, using acid water as the eluent, collecting the acid water-washed part of the 1st to 3rd column bed volume, adjusting the pH value to neutral, and concentrating under reduced pressure; the concentrate is used as C 18 Medium pressure column chromatography, using methanol-buffered saline solution (10:90) to methanol-buffered saline solution (20:90) as the mobile phase for gradient elution separation, collecting the target peak fraction, concentrating under reduced pressure, and freeze-drying to obtain compound IMP-A, wherein compound IMP-A is benzamidoacetic acid;

[0011] (6) The component 1-2 in step (4) is then separated by polyamide adsorption, and acid water, water, and alkaline alcohol solution are used as eluents in sequence, the acid water and water washing parts are discarded, and the 60% alkaline ethanol solution is collected in portions. According to the HPLC-UV detection results, three components are obtained, namely component 1-2-1, component 1-2-2, and component 1-2-3;

[0012] (7) The pH value of the component 1-2-1 in step (6) was adjusted to neutral, concentrated to dryness under reduced pressure, mixed with anhydrous silica gel, dry-loaded, and separated by isocratic elution on a silica gel column using an organic acid ester: low carbon number fatty alcohol (70:30) as the mobile phase, collecting the target peak fractions, and concentrating under reduced pressure; the concentrate was purified by C18 Medium pressure column chromatography, using methanol-buffered saline solution (10:90) to methanol-buffered saline solution (20:80) as the mobile phase for gradient elution separation, collecting the target peak fraction, and concentrating under reduced pressure; further using preparative liquid phase for purification, using a preparative liquid phase column, acetonitrile-buffered saline solution (15:85) for isocratic elution separation, collecting the target peak fraction, concentrating under reduced pressure, and freeze-drying to obtain compound IMP-B, wherein the compound IMP-B is N-(2-phenylacetyl)glycine;

[0013] (8) The pH value of the component 1-2-2 in step (6) is adjusted to neutral and concentrated under reduced pressure. 18 Medium-pressure column chromatography, using methanol-buffered saline solution (20:80) to methanol-buffered saline solution (30:70) as the mobile phase for gradient elution separation, collecting the target peak fraction, and concentrating under reduced pressure; further using preparative liquid phase for purification, using a preparative liquid phase column, acetonitrile-buffered saline solution (20:80) for isocratic elution separation, collecting the target peak fraction, concentrating under reduced pressure, and freeze-drying to obtain compound IMP-D, wherein the compound IMP-D is 2,6-dihydroxybenzoic acid;

[0014] (9) The pH value of the components 1-2-3 in step (6) is adjusted to neutral and concentrated under reduced pressure. 18 Medium-pressure column chromatography, using methanol-buffered saline solution (20:80) to methanol-buffered saline solution (30:70) as the mobile phase for gradient elution separation, collecting the target peak fraction, and concentrating under reduced pressure; further using preparative liquid phase for purification, using a preparative liquid phase column, acetonitrile-buffered saline solution (20:80) for isocratic elution separation, collecting the target peak fraction, concentrating under reduced pressure, and freeze-drying to obtain compound IMP-C, wherein the compound IMP-C is indoxyl sulfate;

[0015] (10) The components 1-3 described in step (4) are separated by polyamide adsorption, using water as the eluent, collecting the water-washed fractions of 1 to 4 times the volume of the column bed, and concentrating under reduced pressure; the concentrate is treated with C 18 Medium-pressure column chromatography, gradient elution separation with methanol-buffered saline solution (20:80) to methanol-buffered saline solution (40:60), collecting the target peak fractions, and concentrating under reduced pressure; further purification using preparative liquid phase, using a preparative liquid phase column, isocratic elution separation with acetonitrile-buffered saline solution (23:77), collecting the target peak fractions, concentrating under reduced pressure, and freeze-drying to obtain compound IMP-E, which is p-cresol sulfate.

[0016] The separation method of the present invention is also preferably as follows: the alcohol solvent in step (2) is an alcohol compound having 1 to 4 carbon atoms.

[0017] The separation method of the present invention is also preferably as follows: the alcohol solvent in step (3) is an alcohol compound having 4 to 7 carbon atoms.

[0018] The separation method of the present invention is also preferably: the macroporous resin model in the step (4) is: D101, AB-8, LX-16, LX-32, XAD-2, SP70, SP850, etc.

[0019] The separation method of the present invention is also preferably: the polyamide eluent acid water in the steps (5) and (6) is an aqueous solution of hydrochloric acid, formic acid, acetic acid, etc., and its concentration is: 0.01-10%.

[0020] The separation method of the present invention is also preferably: the polyamide eluent alkaline alcohol solution in step (6) comprises: the alkali is sodium hydroxide with a concentration of 0.01-5%; the alcohol is methanol or ethanol with a concentration of 20-80%.

[0021] The separation method of the present invention is also preferably: the organic acid ester as the eluent in the silica gel column chromatography in step (7) is methyl formate, ethyl acetate, butyl acetate, etc.; and the low-carbon fatty alcohol is methanol or ethanol.

[0022] The separation method of the present invention is also preferably characterized in that the specifications of the polyamide used in each step are: 14-30 mesh, 30-60 mesh, 80-100 mesh, 100-200 mesh, etc.

[0023] The separation method of the present invention is also preferably: the C used in each step 18 Medium pressure column specifications: 800g, 25-50μm, 6.5×50cm.

[0024] The separation method of the present invention is also preferably: the preparative liquid chromatography column used in each step is: Agilent Eclipse XDB C 18 (9.4mm×250mm, 5μm).

[0025] The separation method of the present invention is also preferably: the mobile phase aqueous phase system used in the semi-preparative and preparative liquid phases in each step is a buffer system such as ammonium formate and acetamine, with a concentration of 0.01-0.5 mol / L.

[0026] The beneficial effects of the present invention are as follows: (1) polyamide is used as the main means of crude separation in the present invention, which reduces the use of a large amount of organic solvents compared with traditional silica gel separation; it has a higher sample loading capacity compared with ODS medium-pressure preparation, and is particularly suitable for crude separation of this variety; (2) the mobile phase aqueous phase system used in the semi-preparative and preparative liquid phases of the present invention is preferably a buffer salt such as ammonium formate or ammonium acetate, and the peak shape is significantly better than that of traditional mobile phases such as acid water and water; (3) the five compounds obtained are of high purity, which is convenient for structural identification and subsequent use as quantitative reference substances to establish a suitable quality control method; (4) from the perspective of structure-activity relationship, the five compounds obtained are significantly different from the main active ingredient in this drug: conjugated estrogen (steroid ring mother nucleus), that is, they are determined to be non-steroidal accompanying substances of naturally derived conjugated estrogen drugs, which meet the qualitative requirements of the FDA guidelines for the study of non-steroidal accompanying substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The chemical structures of five non-steroidal accompanying components and key HMBC-related signals (H→C).

[0028] Figure 2 IMP-A 1 H NMR spectrum.

[0029] Figure 3 IMP-A 13 C NMR spectrum.

[0030] Figure 4 N-(2-phenylacetyl)glycine (IMP-B) 1 H NMR spectrum.

[0031] Figure 5 N-(2-phenylacetyl)glycine (IMP-B) 13 C NMR spectrum.

[0032] Figure 6 Indoxyl sulfate (IMP-C) 1 HNMR spectrum.

[0033] Figure 7 Indoxyl sulfate (IMP-C) 13 C NMR spectrum.

[0034] Figure 8 2,6-dihydroxybenzoic acid (IMP-D) 1 H NMR spectrum.

[0035] Fig. 9 2,6-dihydroxybenzoic acid (IMP-D) 13 C NMR spectrum.

[0036] Fig.10 For p-cresol sulfate (IMP-E) 1 HNMR spectrum.

[0037] Fig.11 For p-cresol sulfate (IMP-E) 13 C NMR spectrum.

[0038] Fig.12 It is a chemical structure of 10 main components that bind estrogen. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with the following examples and the accompanying drawings of the specification, but is not limited to the scope of protection of the present invention. The present invention discloses a method for extracting and separating five non-steroidal accompanying components in a natural source conjugated estrogen drug. Those skilled in the art can refer to the content of the present invention and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

[0040] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0041] Instruments used in the examples of the present invention: LC-20A ultra-high performance liquid chromatograph (Shimadzu Corporation, Japan); ABSCIEX Triple TOF TM 5600 high-resolution mass spectrometer (AB Sciex, USA); Agilent 1100 high-performance liquid chromatograph, VWD detector (Agilent, USA); AL204 electronic analytical balance (1 / 10,000, Mettler-Toledo Instrument Co., Ltd.); BT125D electronic analytical balance (1 / 100,000, Sartorius Scientific Instruments Co., Ltd.); BrukerASR-500 nuclear magnetic resonance instrument (Bruker, Germany); BUCHI Chromatography Pump B-688 medium-pressure column chromatography separation system (BUCHI, Switzerland);

[0042] Materials used in the embodiments of the present invention: Conjugated estrogen tablets (batch number: FP8635, Wyeth Pharmaceuticals Inc); self-developed raw materials (batch number: ZM20230301); unless otherwise specified, the reagents used in the present invention can be purchased from conventional channels or the market, the separation reagents are chemically pure, and the detection reagents are chromatographic or mass spectrometry grade;

[0043] Chromatographic and mass spectrometric conditions in the embodiments of the present invention are:

[0044] Chromatographic conditions: Hedera ODS-2C 18 (250mm×4.6mm,5μm) chromatographic column, with acetonitrile (A)-0.02mol / L ammonium formate water (B) as the mobile phase, gradient elution (0-20min, 8%A→28%A; 20-40min, 28%A→35%A, 40-50min, 35%A→50%A), detection wavelength 215nm, column temperature 30℃, flow rate 1.0ml / min, injection volume 5μL.

[0045] Mass spectrometry conditions: ion source: electrospray ion source (ESI); acquisition mode: negative ion detection mode; primary mass spectrometry conditions: scanning range m / z is 100-1000, capillary temperature is 550°C, ion spray no-load voltage is 5500V; declustering voltage is 100V, collision energy is 15V; secondary mass spectrometry conditions: scanning range m / z is 50-500, declustering voltage is 100V; collision energy is 35V; nebulizer is 60psi; auxiliary heater is 60psi; curtain gas is 40psi.

[0046] Example 1

[0047] 1. Pigment coating cleaning: Take 800 Premarin tablets, use 400 ml of purified water to quickly and repeatedly rinse the surface of the tablets, wash the pigment coating, and discard the eluent. Transfer the tablet core to a vacuum drying oven and dry it at low temperature and reduced pressure to constant weight. Then take the dried tablet core and crush it into coarse powder;

[0048] 2. Extraction of crude powder: Add 100 times the amount of methanol to the above crude powder, soak for 1 hour, perform ultrasonic extraction twice, 30 minutes each time, filter with a 0.45 μm microporous membrane, combine the filtrates, concentrate to dryness under reduced pressure, add 250 ml of purified water, and re-dissolve;

[0049] 3. Liquid-liquid extraction: Add 250 ml of n-butanol to the above aqueous solution, perform liquid-liquid extraction three times, discard the aqueous phase, separate and combine the clarified organic phases, and concentrate under reduced pressure until there is no alcohol taste (first concentrate to 5-10 ml, add 50 ml of purified water, continue to concentrate under reduced pressure to 5-10 ml, and repeat the operation twice), and finally add appropriate amount of purified water to dilute and dissolve to 1500 ml;

[0050] 4. Macroporous resin crude separation: The above water dilution was subjected to adsorption separation by pretreated LX-32 macroporous resin (column bed volume: 2 liters), 4 times column volume of water was used as eluent, and the water washing liquid was collected in portions (a total of 12 fractions). According to the HPLC-UV detection results, three components were obtained, namely component 1-1 (fraction 3), component 1-2 (fractions 4 to 6) and component 1-3 (fractions 7 to 11);

[0051] 5. Preparation of compound IMP-A: The above component 1-1 was separated by polyamide adsorption (column volume: 500 ml, 100-200 mesh), 0.1% formic acid water was used as eluent, and the acid-washed parts of the 1st to 3rd column volumes were collected, the pH value was adjusted to neutral, and the concentrated solution was concentrated under reduced pressure; 18 Medium pressure column chromatography (800g, 25-50μm, 6.5×50cm), with methanol-0.02mol / L ammonium formate aqueous solution (10:90) to methanol-0.02mol / L ammonium formate aqueous solution (20:90) as mobile phase for gradient elution separation, collecting the target peak fraction, concentrating under reduced pressure, and freeze drying to obtain compound IMP-A: benzamidoacetic acid. The purity of the compound was 98.80% by diameter detection;

[0052] 6. Polyamide crude separation: The above-mentioned component 1-2 was separated by polyamide adsorption (column bed volume: 2000 ml, 100-200 mesh), and 0.1% formic acid water 0.5 BV, purified water 3 BV, 0.1% NaOH 60% ethanol aqueous solution 4 BV were used as eluents in sequence, the acid water and water washing parts were discarded, and 0.1% NaOH 60% ethanol aqueous solution was collected in portions (a total of 8 fractions). According to the HPLC-UV detection results, 3 components were obtained, namely component 1-2-1 (fractions 1-3), component 1-2-2 (fractions 4 and 5) and component 1-2-3 (fraction 6);

[0053] 7. Preparation of compound IMP-B: The pH value of the above component 1-2-1 was adjusted to neutral, concentrated to dryness under reduced pressure, mixed with anhydrous silica gel, dry-loaded, and separated by isocratic elution on a silica gel column using ethyl acetate:methanol (70:30) as the mobile phase. The target peak fractions were collected and concentrated under reduced pressure. The concentrate was purified by C 18 Medium pressure column chromatography (800 g, 25-50 μm, 6.5 × 50 cm) was used to perform gradient elution separation using methanol-0.02 mol / L ammonium formate aqueous solution (10:90) to methanol-0.02 mol / L ammonium formate aqueous solution (20:80) as the mobile phase, and the target peak fractions were collected and concentrated under reduced pressure; further purification was performed using preparative liquid phase using Agilent Eclipse XDB C 18 (9.4mm×250mm, 5μm) preparative liquid column, acetonitrile-0.02mol / L ammonium formate aqueous solution (15:85) was used for isocratic elution and separation, and the target peak fraction was collected, concentrated under reduced pressure, and freeze-dried to obtain compound IMP-B: N-(2-phenylacetyl)glycine. The purity of the compound was 98.47% by diameter detection;

[0054] 8. Preparation of compound IMP-D: The pH value of the above component 1-2-2 was adjusted to neutral and concentrated under reduced pressure. 18Medium pressure column chromatography (800 g, 25-50 μm, 6.5 × 50 cm) was used to perform gradient elution separation using methanol-0.02 mol / L ammonium formate aqueous solution (20:80) to methanol-0.02 mol / L ammonium formate aqueous solution (30:70) as the mobile phase, and the target peak fractions were collected and concentrated under reduced pressure; further purification was performed using preparative liquid phase using Agilent Eclipse XDB C 18 (9.4mm×250mm, 5μm) preparative liquid column, acetonitrile-0.02mol / L ammonium formate aqueous solution (20:80) was used for isocratic elution and separation, and the target peak fraction was collected, concentrated under reduced pressure, and freeze-dried to obtain compound IMP-D: 2,6-dihydroxybenzoic acid. The purity of the compound was 95.90% by diameter detection;

[0055] 9. Preparation of compound IMP-C: The pH value of the above components 1-2-3 was adjusted to neutral and concentrated under reduced pressure. 18 Medium pressure column chromatography (800 g, 25-50 μm, 6.5 × 50 cm) was used to perform gradient elution separation using methanol-0.02 mol / L ammonium formate aqueous solution (20:80) to methanol-0.02 mol / L ammonium formate aqueous solution (30:70) as the mobile phase, and the target peak fractions were collected and concentrated under reduced pressure; further purification was performed using preparative liquid phase using Agilent Eclipse XDB C 18 (9.4mm×250mm, 5μm) preparative liquid column, acetonitrile-0.02mol / L ammonium formate aqueous solution (20:80) was used for isocratic elution and separation, and the target peak fraction was collected, concentrated under reduced pressure, and freeze-dried to obtain compound IMP-C: indoxyl sulfate. The purity of the compound was 96.61% by diameter detection;

[0056] 10. Preparation of compound IMP-E: The above components 1-3 were separated by polyamide adsorption (column volume: 500 ml, 100-200 mesh), water was used as eluent, and the water-washed parts of 1 to 4 times the column volume were collected and concentrated under reduced pressure; the concentrate was purified by C 18 Medium pressure column chromatography (800 g, 25-50 μm, 6.5 × 50 cm) was used for gradient elution separation with methanol-0.02 mol / L ammonium formate aqueous solution (20:80) to methanol-0.02 mol / L ammonium formate aqueous solution (40:60), and the target peak fractions were collected and concentrated under reduced pressure; further purification was performed using preparative liquid phase, using Agilent Eclipse XDB C 18(9.4mm×250mm, 5μm) preparative liquid column, acetonitrile-0.02mol / L ammonium formate aqueous solution (23:77) was used for isocratic elution and separation, and the target peak fraction was collected, concentrated under reduced pressure, and freeze-dried to obtain compound IMP-E: p-cresol sulfate. The purity of the compound was 100.00% by diameter detection;

[0057] 11. High-resolution mass spectrometry analysis: The structural characteristics of the five non-steroidal components were analyzed by combining chromatographic retention behavior, accurate relative molecular mass, secondary mass spectrometry fragment ions and literature information. In the high-resolution electrospray ionization mass spectrometry ESI-Q-TOF / MS(-) graph, obvious [MH] - The quasi-molecular ions are m / z 178.0525, m / z 192.0681, m / z 212.0035, m / z 153.0190, and m / z 187.0083, respectively. Their molecular formulas are determined to be C9H9NO3, C 10 H 11 NO3, C8H7NO4S, C7H6O4, C7H8O4S. Considering that the structure of the bound steroid compounds contains cyclopentane pyrophenanthrene nucleus and substituents such as sulfate or glucuronic acid ester, the number of carbon atoms is ≥18 and the molecular weight is ≥350, it can be determined that these five compounds are non-steroidal components; further based on the ultraviolet absorption spectrum characteristics and unsaturation of the compounds, it is speculated that the above compounds all contain benzene rings.

[0058] In the IMP-A secondary mass spectrum, it can be seen that the quasi-molecular ion [MH] - The loss of 44 (CO2) and 46 (HCOOH) formed fragment ions m / z 134.0610 and m / z 132.0451, respectively. It is speculated that there is a carboxyl group in the structure. In addition, a characteristic fragment ion of m / z 77.0406 of a benzene ring can be seen. In the secondary mass spectrum of IMP-B, a main characteristic fragment ion m / z 74.0257 was seen, and its corresponding molecular structure was C2H4O2N. It was speculated to be a carboxymethylamide structure, belonging to the acylglycine class of compounds. In the secondary mass spectra of IMP-C and IMP-E, fragment ions m / z 132.0457 and m / z 107.0501 were seen, respectively, which were the corresponding quasi-molecular ions [MH] - m / z212.0035 and m / z 187.0083 lose 80 (SO3) to form characteristic ions, and it is speculated that these two components are sulfate compounds. In the secondary mass spectrum of IMP-D, the fragment ion m / z 109.0293 [M-CO2-H] can be seen - , and m / z 135.0083 [M-H2O-H] - , and m / z 91.0194 [M-CO2-H2O-H] -, it is speculated that the structure should contain carboxyl and hydroxyl groups. See Table 1 for details.

[0059] Table 1 Mass spectrometry information of compounds IMP-A, B, C, D, E

[0060]

[0061] 12. Structural identification:

[0062] (1) The high-resolution electrospray ionization mass spectrum ESI-Q-TOF / MS(-) of IMP-A showed a quasi-molecular ion peak of m / z 178.0504 (theoretical value is 178.0510), and its molecular formula was determined to be C9H9NO3 with a degree of unsaturation of 6. 1 In the H-NMR spectrum (DMSO-d5, 500 MHz), a methylene signal (δ H 3.75, 2H, s), three groups of olefin hydrogen signals (δ H 7.46,2H,m)、(δ H 7.84,2H,m) and (δ H 7.52,1H,m), an imine hydrogen signal (δ H 8.34,1H,s). HSQC spectrum combined 13 C-NMR spectrum shows a methylene carbon signal δ C 42.8 (C-1'), four groups of olefin carbon signals δ C 127.4(C-2,6),δ C 128.4(C-3,5),δ C 131.4(C-4),δ C 134.3 (C-1), an amide carbon signal δ C 166.0 (C-1') and a carboxyl carbon signal δ C 171.3 (C-4'), indicating the presence of a benzene ring, an amide group, and a carboxyl group in the structure of the compound. Further, δ H 7.84(H-2,6) and δ C 131.4(C-4),δ C 166.0(C-1') related signal; δ H 3.75(H-3') and δ C 166.0(C-1'),δ C 171.3 (C-4') is significantly correlated, indicating that the amide substituent is connected to the benzene ring and the acetic acid group is connected to the nitrogen atom. In summary, compound IMP-A is determined to be benzoylaminoacetic acid. The NMR data are shown in Table 2 and the structural formula is shown in Figure 1 , NMR data see Figure 2, Figure 3 ;

[0063] (2) IMP-B quasi-molecular ion peak [MH] - The m / z is 192.0555 (theoretical value is 178.0666), and its molecular formula is determined to be C 10 H 11 NO3. Connect IMP-B and IMP-A 1 H-NMR spectrum and 13 After comparing the C-NMR spectra, it was found that the signals of the two components were similar, and the main difference was that there was an additional methylene signal δ in the structure of compound IMP-B. H 3.28 (2H, s) and δ C 42.2, which is consistent with the mass spectrometry information. Furthermore, in the HMBC spectrum, it can be observed that H-3(5) is significantly correlated with C-1, and H-1' is significantly correlated with C-2(6), suggesting that the substituent is connected to the C-1 position of the benzene ring; in addition, the correlation signal between H-4' and C-2' can be seen. Combined with the mass spectrometry information, it can be determined that the substituent is acetamidoacetic acid, and finally the compound IMP-B is determined to be N-(2-phenylacetyl)glycine. The NMR data are shown in Table 2, and the structural formula is shown in Figure 1 , NMR data see Figure 4 , Figure 5 ;

[0064] (3) In the ESI-Q-TOF / MS(-) graph, the quasi-molecular ion m / z 212.0027 (theoretical value is 212.0048) of IMP-C was observed, and its molecular formula was determined to be C8H7NO4S with a degree of unsaturation of 4. 1 In the H-NMR spectrum, five olefinic hydrogen signals (δ H 6.92,1H,t,J=7.2)、(δ H 7.021H,t,J=7.2)、(δ H 7.05,1H,d,J=2.5)、(δ H 7.26,1H,d,J=8.0)、(δ H 7.49, 1H, d, J = 8.0); 1 hydroxyl hydrogen signal (δ H 8.35,1H,s); 1 amino hydrogen signal (δ H 10.55, 1H, br). HSQC spectrum combined 13 In the C-NMR spectrum (DMSO-d5, 125 MHz), an oxygen-linked unsaturated quaternary carbon signal δ C 121.2 (C-3); 5 unsaturated tertiary carbon signals δ C 113.9(C-2),δ C121.2(C-3),δ C 117.9(C-5),δ C 120.8(C-6),δ C 111.1 (C-7); 2 unsaturated quaternary carbon signals δ C 133.1(C-8),δ C 131.9 (C-9). This indicates that the compound has a benzene ring and a sulfate group in its structure. Combined with the mass spectrometry information, it can be basically determined that there is an indole structure, so it is speculated that the structure of the compound is indole sulfate. The HMBC spectrum shows δ H 10.55(H-1) and δ C 131.9(C-9) has a significant correlation, δ H 7.05(H-2) and δ C 133.1(C-8),δ C 121.2(C-3) has a significant correlation, δ H 10.55(H-1) and δ H 7.49(H-4) and δ C 121.2 (C-3) has a significant correlation, suggesting that the sulfate group is connected to C-3 of the indole structure. Finally, compound IMP-C was determined to be indole sulfate. The NMR data are shown in Table 2 and the structural formula is shown in Figure 1 , NMR data see Figure 6 , Figure 7 ;

[0065] (4) In the ESI-Q-TOF / MS(-) spectrum of IMP-D, an obvious quasi-molecular ion m / z 153.0190 (theoretical value is 153.0193) was observed, and its molecular formula was determined to be C7H6O4 with a degree of unsaturation of 5. 1 In the H-NMR spectrum, two groups of olefinic hydrogen signals (δ H 6.03,2H,d,J=8.1)(δ H 6.92,1H,t,J=8.1), a group of phenolic hydroxyl hydrogen signals (δ H 14.68,2H,s), it is speculated that the compound has a symmetrical structure of benzene ring. 13 The C-NMR spectrum shows three groups of double bond carbon signals δ C 104.5(C-1),δ C 104.6(C-3,5),δ C 131.4 (C-4), 2 symmetrical oxygen-linked unsaturated carbon signals δ C 162.3 (C-2,6), 1 carboxyl carbon signal δ C 175.2 (C-1'). Combined with the mass spectrum information, it is speculated that the structure of the compound is 2,6-dihydroxybenzoic acid. In the HMBC spectrum, δH 6.03(H-3,5) and δ C 104.5(C-1) is significantly correlated, δ H 6.92(H-4) and δ C 162.3 (C-2,6) has a related signal, which further verifies the above structure. In summary, the compound IMP-D is determined to be 2,6-dihydroxybenzoic acid. The NMR data is shown in Table 2 and the structural formula is shown in Figure 1 , NMR data see Figure 8 , Fig. 9 ;

[0066] (5) In the IMP-E high-resolution electrospray ionization mass spectrometry ESI-Q-TOF / MS(-) spectrum, an obvious quasi-molecular ion m / z 187.0058 (theoretical value is 187.0071) was observed, and its molecular formula was determined to be C7H8O4S. 1 The H-NMR spectrum showed a methyl signal (δ H 2.24,3H,s) and two groups of olefinic hydrogen signals (δ H 7.03,2H,d,J=8.9),(δ H 7.05, 2H, d, J = 8.9), HSQC spectrum combined 13 C-NMR spectrum shows a methyl carbon signal δ C 20.3 (C-1'), 3 double bond carbon signals δ C 120.4 (C-2,6), 129.1 (C-3,5), 131.8 (C-4) and one oxygen-linked unsaturated quaternary carbon signal δ C 151.3 (C-1), indicating that there is a benzene ring in the structure of the compound and the two substituents are para-substituted. Combined with the mass spectrometry information, it can be basically determined that the substituents are methyl and sulfate. In the HMBC spectrum, δ H 7.0(H-2,6) and δ C 131.8(C-4) is related; δ H 7.1(H-3,5) and δ C 151.3(C-1),δ C 20.3(C-1') has a significant correlation; δ H 2.24(H-1') and δ C 129.1(C-3,5) is significantly correlated, indicating that the methyl group is connected to the C-4 of the benzene ring and the sulfate is connected to the C-1 of the benzene ring. Based on the above information, IMP-E was determined to be p-cresol sulfate. The NMR data are shown in Table 2 and the structural formula is shown in Figure 1 , NMR data see Fig.10 , Fig.11 .

[0067] Table 2 NMR data of compounds IMP-A, B, C, D, E (C5D5N-d5, 1 H-NMR:500MHz, 13 C-NMR: 125MHz)

[0068]

[0069] 13. Referring to the chemical structures of the above compounds IMP-A, B, C, D, and E, it is not difficult to find that they are significantly different from the main active ingredient of this drug: conjugated estrogen (steroid ring nucleus) (the chemical structure of conjugated estrogen is shown in Fig.12 ), which means it is determined to be a non-steroidal accompanying ingredient of a naturally derived combined estrogen drug, meeting the FDA's research requirements for non-steroidal accompanying ingredients in the consistency evaluation of generic drugs. Subsequently, these five high-purity samples will be used as reference substances to develop quantitative quality control methods to ensure drug safety.

[0070] Example 2

[0071] 1. Extraction: Take the self-developed raw material drug, add 100 times the amount of methanol, soak for 1 hour, ultrasonically extract twice, 30 minutes each time, filter with a 0.45μm microporous membrane, combine the filtrate, concentrate to dryness under reduced pressure, add 250ml of purified water, and re-dissolve;

[0072] 2. Liquid-liquid extraction: Add 250 ml of n-butanol to the above aqueous solution, perform liquid-liquid extraction three times, discard the aqueous phase, separate and combine the clarified organic phases, and concentrate under reduced pressure until there is no alcohol taste (first concentrate to 5-10 ml, add 50 ml of purified water, continue to concentrate under reduced pressure to 5-10 ml, and repeat the operation twice), and finally add appropriate amount of purified water to dilute and dissolve to 1500 ml;

[0073] 3. Macroporous resin crude separation: The above water dilution was subjected to adsorption separation by pretreated LX-32 macroporous resin (column bed volume: 2 liters), 4 times column volume of water was used as eluent, and the water washing liquid was collected in portions (a total of 12 fractions). According to the HPLC-UV detection results, three components were obtained, namely component 1-1 (fraction 3), component 1-2 (fractions 4 to 6) and component 1-3 (fractions 7 to 11);

[0074] 4. Preparation of compound IMP-A: The above component 1-1 was separated by polyamide adsorption (column volume: 500 ml, 100-200 mesh), 0.1% formic acid water was used as eluent, and the acid-water-washed part of the 1st to 3rd column volume was collected, the pH value was adjusted to neutral, and the concentrated solution was concentrated under reduced pressure; 18Medium pressure column chromatography (800 g, 25-50 μm, 6.5×50 cm) was used to perform gradient elution separation using methanol-0.02 mol / L ammonium formate aqueous solution (10:90) to methanol-0.02 mol / L ammonium formate aqueous solution (20:90) as the mobile phase, and the target peak fractions were collected, concentrated under reduced pressure, and freeze-dried to obtain compound IMP-A: benzoylaminoacetic acid;

[0075] 5. Polyamide crude separation: The above-mentioned component 1-2 was separated by polyamide adsorption (column bed volume: 2000 ml, 100-200 mesh), and 0.1% formic acid water 0.5 BV, purified water 3 BV, 0.1% NaOH 60% ethanol aqueous solution 4 BV were used as eluents in sequence, the acid water and water washing parts were discarded, and 0.1% NaOH 60% ethanol aqueous solution was collected in portions (a total of 8 fractions). According to the HPLC-UV detection results, 3 components were obtained, namely component 1-2-1 (fractions 1-3), component 1-2-2 (fractions 4 and 5) and component 1-2-3 (fraction 6);

[0076] 6. Preparation of compound IMP-B: The pH value of the above component 1-2-1 was adjusted to neutral, concentrated to dryness under reduced pressure, mixed with anhydrous silica gel, dry-loaded, and separated by isocratic elution on a silica gel column using ethyl acetate:methanol (70:30) as the mobile phase, collecting the target peak fractions and concentrating under reduced pressure; the concentrate was purified by C 18 Medium pressure column chromatography (800 g, 25-50 μm, 6.5 × 50 cm) was used to perform gradient elution separation using methanol-0.02 mol / L ammonium formate aqueous solution (10:90) to methanol-0.02 mol / L ammonium formate aqueous solution (20:80) as the mobile phase, and the target peak fractions were collected and concentrated under reduced pressure; further purification was performed using preparative liquid phase using Agilent Eclipse XDB C 18 (9.4 mm × 250 mm, 5 μm) preparative liquid column, acetonitrile-0.02 mol / L ammonium formate aqueous solution (15:85) was used for isocratic elution and separation, and the target peak fraction was collected, concentrated under reduced pressure, and freeze-dried to obtain compound IMP-B: N-(2-phenylacetyl)glycine;

[0077] 7. Preparation of compound IMP-D: The pH value of the above component 1-2-2 was adjusted to neutral and concentrated under reduced pressure. 18 Medium pressure column chromatography (800 g, 25-50 μm, 6.5 × 50 cm) was used to perform gradient elution separation using methanol-0.02 mol / L ammonium formate aqueous solution (20:80) to methanol-0.02 mol / L ammonium formate aqueous solution (30:70) as the mobile phase, and the target peak fractions were collected and concentrated under reduced pressure; further purification was performed using preparative liquid phase using Agilent Eclipse XDB C18 (9.4 mm × 250 mm, 5 μm) preparative liquid column, acetonitrile-0.02 mol / L ammonium formate aqueous solution (20:80) was used for isocratic elution and separation, and the target peak fraction was collected, concentrated under reduced pressure, and freeze-dried to obtain compound IMP-D: 2,6-dihydroxybenzoic acid;

[0078] 8. Preparation of compound IMP-C: The pH value of the above components 1-2-3 was adjusted to neutral and concentrated under reduced pressure. 18 Medium pressure column chromatography (800 g, 25-50 μm, 6.5 × 50 cm) was used to perform gradient elution separation using methanol-0.02 mol / L ammonium formate aqueous solution (20:80) to methanol-0.02 mol / L ammonium formate aqueous solution (30:70) as the mobile phase, and the target peak fractions were collected and concentrated under reduced pressure; further purification was performed using preparative liquid phase using Agilent Eclipse XDB C 18 (9.4 mm × 250 mm, 5 μm) preparative liquid column, acetonitrile-0.02 mol / L ammonium formate aqueous solution (20:80) was used for isocratic elution and separation, and the target peak fraction was collected, concentrated under reduced pressure, and freeze-dried to obtain compound IMP-C: indoxyl sulfate;

[0079] 9. Preparation of compound IMP-E: The above components 1-3 were separated by polyamide adsorption (column volume: 500 ml, 100-200 mesh), water was used as eluent, and the water-washed parts of 1 to 4 times the column volume were collected and concentrated under reduced pressure; the concentrate was purified by C 18 Medium pressure column chromatography (800 g, 25-50 μm, 6.5 × 50 cm) was used for gradient elution separation with methanol-0.02 mol / L ammonium formate aqueous solution (20:80) to methanol-0.02 mol / L ammonium formate aqueous solution (40:60), and the target peak fractions were collected and concentrated under reduced pressure; further purification was performed using preparative liquid phase, using Agilent Eclipse XDB C 18 (9.4 mm × 250 mm, 5 μm) preparative liquid column, acetonitrile-0.02 mol / L ammonium formate aqueous solution (23:77) was used for isocratic elution and separation, and the target peak fraction was collected, concentrated under reduced pressure, and freeze-dried to obtain compound IMP-E: p-cresol sulfate;

[0080] 10. The above compounds IMP-A, B, C, D, and E were detected by high-resolution mass spectrometry and nuclear magnetic resonance and were consistent with the non-steroidal accompanying ingredients in the original Premarin tablets.

Claims

1. A method for extracting and separating five non-steroidal accompanying components in a natural conjugated estrogen drug, characterized in that: The following steps are involved: (1) Take the powder of natural conjugated estrogen drug, soak it in alcohol solvent, extract it by ultrasonic, filter it with filter membrane, concentrate it to dryness under reduced pressure, and then re-dissolve it in purified water; (2) Add an alcoholic organic solvent in equal proportions to the aqueous solution in step (1), perform liquid-liquid extraction, discard the aqueous phase, and concentrate the organic phase under reduced pressure until there is no alcohol smell, and finally add purified water to dilute and dissolve; (3) The water dilution in step (2) is subjected to macroporous resin adsorption separation, using water as the eluent, collecting the water washing liquid in portions, and combining them according to the HPLC-UV detection results to obtain three components, namely component 1-1, component 1-2 and component 1-3; (4) Separating, purifying, concentrating under reduced pressure, and freeze-drying the components 1-1, 1-2, and 1-3 obtained in step (3) to obtain compounds such as benzamidoacetic acid, N-(2-phenylacetyl)glycine, 2,6-dihydroxybenzoic acid, indoxyl sulfate, and p-cresol sulfate.

2. The method for extracting and separating five non-steroidal accompanying components in the natural conjugated estrogen drug according to claim 1, characterized in that: The specific steps of obtaining benzamidoacetic acid from component 1-1 in step (4) are as follows: component 1-1 is separated by polyamide adsorption, acid water is used as eluent, the acid water washing part of the first to third column bed volume is collected, the pH value is adjusted to neutral, and the concentrated solution is concentrated under reduced pressure; 18 Medium-pressure column chromatography was performed with methanol-buffered saline solution as the mobile phase for gradient elution separation, and the target peak fractions were collected, concentrated under reduced pressure, and freeze-dried to obtain compound IMP-A. Structural identification showed that the compound IMP-A was benzamidoacetic acid; wherein the volume ratio of methanol-buffered saline solution was 10:90~20:

90.

3. The method for extracting and separating five non-steroidal accompanying components in the natural conjugated estrogen drug according to claim 1, characterized in that: The specific extraction steps of the component 1-2 in step (4) are as follows: component 1-2 is then separated by polyamide adsorption, acid water, water, and alkaline alcohol solution are used as eluents in sequence, the acid water and water washing parts are discarded, and 60% alkaline ethanol solution is collected in portions, and three components are obtained by merging according to the HPLC-UV detection results, namely component 1-2-1, component 1-2-2, and component 1-2-3; The steps of obtaining N-(2-phenylacetyl)glycine from component 1-2-1 are as follows: adjusting the pH value of component 1-2-1 to neutral, concentrating to dryness under reduced pressure, mixing with anhydrous silica gel, loading by dry method, performing isocratic elution separation by silica gel column chromatography with organic acid ester-low carbon number fatty alcohol as mobile phase, collecting target peak fractions, and concentrating under reduced pressure; the concentrate is subjected to C 18 Medium-pressure column chromatography, using methanol-buffered saline solution as the mobile phase for gradient elution separation, collecting the target peak fraction, and concentrating under reduced pressure; further using preparative liquid phase for purification, using a preparative liquid phase column, using acetonitrile-buffered saline solution for isocratic elution separation, collecting the target peak fraction, concentrating under reduced pressure, and freeze-drying to obtain compound IMP-B. After structural identification, the compound IMP-B is N-(2-phenylacetyl)glycine; wherein the volume ratio of organic acid ester to low-carbon fatty alcohol is 70:30; the volume ratio of methanol to buffered saline solution is 10:90~20:80; the volume ratio of acetonitrile to buffered saline solution is 15:85; The steps of obtaining 2,6-dihydroxybenzoic acid from component 1-2-2 are as follows: adjusting the pH value of component 1-2-1 to neutral, concentrating under reduced pressure; 18 Medium-pressure column chromatography, using methanol-buffered saline solution as the mobile phase for gradient elution separation, collecting the target peak fraction, and concentrating under reduced pressure; further using preparative liquid phase for purification, using a preparative liquid phase column, using acetonitrile-buffered saline solution for isocratic elution separation, collecting the target peak fraction, concentrating under reduced pressure, and freeze-drying to obtain compound IMP-D. After structural identification, the compound IMP-D is 2,6-dihydroxybenzoic acid; wherein the volume ratio of methanol to buffered saline solution is 20:80-30:70, and the volume ratio of acetonitrile to buffered saline solution is 20:80; The steps of obtaining indoxyl sulfate from component 1-2-3 are as follows: adjusting the pH value of component 1-2-3 to neutral, concentrating under reduced pressure; 18 Medium-pressure column chromatography, using methanol-buffered saline solution as the mobile phase for gradient elution separation, collecting the target peak fractions, and concentrating under reduced pressure; further purification using preparative liquid phase, using a preparative liquid phase column, isocratic elution separation with acetonitrile-buffered saline solution, collecting the target peak fractions, concentrating under reduced pressure, and freeze-drying to obtain compound IMP-C. After structural identification, the compound IMP-C is indoxyl sulfate; wherein the volume ratio of methanol to buffered saline solution is 20:80~30:70, and the volume ratio of acetonitrile to buffered saline solution is 20:

80.

4. The method for extracting and separating five non-steroidal accompanying components in the natural conjugated estrogen drug according to claim 1, characterized in that: The specific separation steps of the components 1-3 in step (4) are as follows: the components 1-3 are separated by polyamide adsorption, water is used as the eluent, the water-washed parts of the 1st to 4th column bed volumes are collected, and the concentrated liquid is concentrated under reduced pressure; 18 Medium-pressure column chromatography, gradient elution and separation with methanol-buffered saline solution, collecting target peak fractions, and concentrating under reduced pressure; further purification using preparative liquid phase, using a preparative liquid phase column, isocratic elution and separation with acetonitrile-buffered saline solution, collecting target peak fractions, concentrating under reduced pressure, and freeze-drying to obtain compound IMP-E. Structural identification shows that the compound IMP-E is p-cresol sulfate; wherein the volume ratio of methanol to buffered saline solution is 20:80~40:60, and the volume ratio of acetonitrile-buffered saline solution is 23:

77.

5. The extraction and separation method according to claim 1, characterized in that: The alcohol solvent in step (1) is one or more alcohol compounds having 1 to 4 carbon atoms; The alcohol organic solvent in step (2) is one or more alcohol compounds having 4 to 7 carbon atoms; The macroporous resin model in the step (3) is one or more of D101, AB-8, LX-16, LX-32, XAD-2, SP70, and SP850.

6. The method for extracting and separating five non-steroidal accompanying components in the natural conjugated estrogen drug of claim 1 according to claim 2 or 3, characterized in that: The acid water is one or more of hydrochloric acid aqueous solution, formic acid aqueous solution and acetic acid aqueous solution, and the concentration of the acid water is 0.01-10%.

7. The method for extracting and separating five non-steroidal accompanying components in the natural conjugated estrogen drug according to claim 3, characterized in that: The alkaline alcohol solution comprises: the alkali is sodium hydroxide with a concentration of 0.01-5%; the alcohol is methanol or ethanol with an alcohol concentration of 20-80%; and the rest is pure water.

8. The method for extracting and separating five non-steroidal accompanying components in the natural conjugated estrogen drug according to claim 3, characterized in that: The organic acid ester is one or more of methyl formate, ethyl acetate and butyl acetate; the low-carbon fatty alcohol is methanol or ethanol.

9. The method for extracting and separating five non-steroidal accompanying components in the natural conjugated estrogen drug according to any one of claims 2 to 5, characterized in that: The polyamide specification is one or more of 14-30 mesh, 30-60 mesh, 80-100 mesh, and 100-200 mesh; The C 18 Medium pressure column specifications: 800g, 25-50μm, 6.5×50cm; The preparative HPLC column: Agilent Eclipse XDB C 18 , specifications are 9.4mm×250mm, 5μm; The buffered salt solution is one or more of ammonium formate solution, ammonium acetate solution or ammonium carbonate solution, and the concentration of the buffered salt solution is 0.01-0.5 mol / L.

10. The method for extracting and separating five non-steroidal accompanying components in the natural conjugated estrogen drug according to claim 1, characterized in that: The preparation method of natural combined estrogen drug powder is as follows: take natural combined estrogen tablets, quickly and repeatedly rinse the tablet surface with a small amount of purified water, wash the pigment coating, and discard the elution solution; transfer the tablet core to a vacuum drying oven, and dry it at low temperature and reduced pressure to constant weight; then take the dried tablet core and crush it into coarse powder, or the natural combined estrogen drug powder is the natural combined estrogen raw material powder; In the step (1), the natural combined estrogen drug powder is dissolved in an alcohol solvent, the mass of the alcohol solvent is 25 to 100 times the mass of the natural combined estrogen drug powder, soaked for 1 hour, ultrasonically extracted twice, each time for 30 minutes, filtered through a 0.45 μm microporous filter membrane, the filtrate is combined, concentrated to dryness under reduced pressure, and then re-dissolved with an appropriate amount of purified water; In the step (2), the aqueous solution in step (2) is added with an alcohol organic solvent in equal proportions, and liquid-liquid extraction is performed three times. The aqueous phase is discarded, and the clarified organic phases are separated and combined; vacuum concentration is performed: purified water is added to replace the organic phase during the vacuum concentration process until there is no alcohol taste; and finally, an appropriate amount of purified water is added to dilute and dissolve.

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