Method for determining crystal form of conjugated estrogen of natural source mixture based on scanning electron microscope-confocal microscopy Rahmann coupling technology

Abstract

The invention relates to a rapid identification method for a crystal form of a complex mixture conjugated estrogen drug obtained by separation and purification of a natural pregnant mare urine source, and adopts a scanning electron microscope-confocal microscopy ramann coupling technology to determine a natural conjugated estrogen bulk drug and a preparation thereof. And carrying out difference analysis with conjugated estrogen from a synthesis source. According to the detection method, a microscopic area is positioned through a scanning electron microscope, fixed-point chemical analysis is performed through confocal microscopic Raman, the corresponding relation between morphology and crystal form is revealed, and the method is high in spatial resolution (micron level), high in specificity and good in reproducibility. Therefore, the method can be used for simply, quickly and accurately detecting the characteristic peak of the sample to be detected, reveals the special molecular arrangement mode and crystal structure characteristics of the conjugated estrogen drug produced by a specific process, and has a very good application prospect in the field of crystal form quality control of the drug.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for determining the crystal form of a natural source mixture combined estrogen medicine by using a scanning electron microscope and a confocal microscopic Raman spectrum combined technology, which is suitable for crystal form identification and source identification of raw medicine and preparations. BACKGROUND

[0002] Natural conjugated estrogen is a natural mixed conjugated estrogen extracted from pregnant mare urine, and mainly contains estrone sodium sulfate, mare estrone sodium sulfate and 17 alpha-dihydro-mare estrone sodium sulfate, and also contains other steroid compounds, and is a solid powder diluted with a diluent.

[0003] Natural conjugated estrogen drugs, such as Premarin ® Conjugated estrogen tablets are mainly used for hormone replacement therapy (HRT), for relieving clinical symptoms of any estrogen deficiency in the body, for treating and preventing menopausal syndrome after female physiology or artificial menopause, and for preventing and treating osteoporosis and coronary heart disease. At present, the FDA does not approve the Premarin ® conjugated estrogen tablets.

[0004] The United States Pharmacopoeia, the European Pharmacopoeia and the British Pharmacopoeia describe the natural source or synthetic source of the raw material medicine in the monograph of the conjugated estrogen raw material medicine, and the European Pharmacopoeia and the British Pharmacopoeia indicate that the conjugated estrogen is an amorphous powder, and other index items cannot distinguish the natural source or the synthetic source.

[0005] The crystal form of natural conjugated estrogen may affect the stability, solubility and bioavailability of the medicine, and since the components are complex and the content of the main components is low, the traditional analysis method (such as X-ray diffraction and infrared spectrum) is difficult to realize the synchronous correlation analysis of the microstructure and the chemical structure, and the detection is usually an amorphous powder, so it is difficult to correctly judge the crystal form.

[0006] Therefore, a high-resolution and high-specificity method is needed to distinguish the crystal form characteristics of natural and synthetic source conjugated estrogen from the microstructure level, and to improve the quality control means. SUMMARY

[0007] The application provides a conjugated estrogen medicine crystal form identification method based on a scanning electron microscope-confocal microscopic Raman combined technology, which realizes the crystal form identification and source determination of each component in the natural mixture by combining micro-morphology positioning and spot chemical analysis.

[0008] To achieve the object of the present application, the following technical solutions are adopted: Sample preparation: disperse the natural conjugated estrogen raw material or preparation uniformly on the conductive substrate, and synthesize conjugated estrogens (estrone sodium sulfate, equilin sodium sulfate, 17α-dihydroequilin sodium sulfate) to prepare samples.

[0009] Electron microscope morphology analysis: use the composition contrast mode (Det BSED-COMP) of the backscattered electron detector of the scanning electron microscope, voltage (HV) 15kV, and observe the microstructure of the sample to locate the micro area to be tested (the resolution reaches the micron level).

[0010] Raman spectrum collection: through the confocal microscopic Raman spectrometer, laser wavelength 785nm, power 60mW, integration time 0.5-1s, resolution 2cm -1 , and spectral range 100-4000cm -1 , the selected micro area is scanned, and the Raman characteristic peak (such as the sulfate ester bond in the range of 500-1800cm⁻¹ and the steroid skeleton vibration peak) is obtained.

[0011] Data comparison and analysis: compare the spectrum of the natural sample with the spectrum of the synthesis and the crystal form database, and determine the crystal form by the peak position, peak shape and relative intensity difference.

[0012] Morphology-crystal form correlation: combine the crystal habit (such as needle-shaped and flaky) in the electron microscope image with the Raman spectrum characteristics to establish the correspondence between the morphology and the crystal form.

[0013] The present application has the following beneficial effects: High resolution: the scanning electron microscope-Raman combined technology realizes the morphology and chemical analysis of the micron level area, and accurately locates the crystal form of each component in the mixture.

[0014] Strong specificity: the Raman spectrum can distinguish the configuration difference of the sulfate ester group and the lattice vibration mode, and effectively identify the crystal form characteristics of the natural and synthetic conjugated estrogens.

[0015] Fast and efficient: without complex pretreatment, multiple component crystal form identification can be completed in a single detection, which is suitable for the quality control of raw materials and preparations.

[0016] The scanning electron microscope-Raman combined technology detection method for the natural conjugated estrogen crystal form of the present application has the advantages of simple operation, high efficiency, short time consumption, and the use of scanning electron microscope-Raman combined technology for analysis, high detection sensitivity and strong specificity. Therefore, the method can be used to simply, quickly and accurately detect the crystal form of the conjugated estrogen raw material, and has good application prospect in the quality control of natural source conjugated estrogen drugs. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 This is a scanning electron microscope image of a natural conjugated estrogen raw material in a specific embodiment of the present invention.

[0018] Figure 2 The images shown are scanning electron microscope images and Raman spectra of the synthesized sodium estrone sulfate in a specific embodiment of the present invention.

[0019] Figure 3 The images shown are scanning electron microscope images and Raman spectra of sodium estradiol sulfate synthesized in a specific embodiment of the present invention.

[0020] Figure 4 The images shown are scanning electron microscope images and Raman spectra of sodium 17α-dihydromareneestrol sulfate synthesized in a specific embodiment of the present invention.

[0021] Figure 5 This represents the Raman spectral difference region between natural and synthetically sourced bound estrogens in a specific embodiment of the present invention.

[0022] Figure 6 This is to verify the specificity of the method in a specific embodiment of the present invention.

[0023] Figure 7 This is a comparison of the Raman spectra of natural conjugated estrogen with batch number ZM20240802 in a specific embodiment of the present invention.

[0024] Figure 8 This is a comparison of the Raman spectra of natural conjugated estrogen with batch number ZM20240803 in a specific embodiment of the present invention.

[0025] Figure 9 For comparison of the Raman spectra of natural conjugated estrogen with batch number ZM20240804 in a specific embodiment of the present invention.

[0026] Figure 10 For comparison of the Raman spectra of natural conjugated estrogen with batch number ZM20240807 in a specific embodiment of the present invention. Detailed Implementation

[0027] Taking natural conjugated estrogen raw materials extracted from pregnant mare urine as an example: Typical crystalline regions, including blocky and spherical shapes, were observed under a scanning electron microscope. Raman analysis showed that the spectra of the blocky regions matched those of the diluent, and the blocky / spherical regions were consistent with the undiluted bound estrogen.

[0028] The present invention will be further described below with reference to the following embodiments and accompanying drawings, but is not limited to the scope of protection of the present invention.

[0029] Example 1 In this embodiment, the powder is photographed by scanning electron microscope using microscopic imaging method, and the particle habitus in the synthetic conjugated estrogens [estrone sodium sulfate (batch number: 14-ATH-129-1) (a and b), equilin sodium sulfate (batch number: 12-MP-131-1) (a and b), 17a-dihydroequilin sodium sulfate (batch number: Y1-S-24-01) (a and b)] is determined, and then the characteristic peaks of Raman spectrum of specific regions are determined.

[0030] The particle habitus of the diluent (a) under scanning electron microscope Figure 1 a) The particle habitus is blocky, and the synthetic conjugated estrogens (b) The particle habitus is spherical or blocky porous, and the conjugated estrogens raw material (batch number: ZM20240802) (c and d) The inner blocky particles match the diluent, and the inner spherical or blocky porous particles match the synthetic conjugated estrogens. Figure 1 Figure 1 Figure 1 d) The inner blocky particles match the diluent, and the inner spherical or blocky porous particles match the synthetic conjugated estrogens. Raman analysis is performed on specific regions, and the characteristic peaks of the synthetic conjugated estrogens are 1309 cm-1, 1602 cm-1, and 1650 cm-1. -1 -1 Figure 7 Example

[0031] In this embodiment, the powder is photographed by scanning electron microscope using microscopic imaging method, and the particle habitus in the synthetic conjugated estrogens [estrone sodium sulfate (batch number: 14-ATH-129-1) (a and b), equilin sodium sulfate (batch number: 12-MP-131-1) (a and b), 17a-dihydroequilin sodium sulfate (batch number: Y1-S-24-01) (a and b)] is determined, and then the characteristic peaks of Raman spectrum of specific regions are determined. Figure 2 Figure 2 Figure 3 Figure 3 Figure 4 Figure 4 The particle habitus of the diluent (a) under scanning electron microscope

[0032] The particle habitus of synthetic estrone sodium sulfate is rod-shaped, and there are secondary particles formed by rod-shaped particles; the particle habitus of synthetic equilin sodium sulfate is flaky, and there are secondary particles formed by flaky particles; the particle habitus of synthetic 17a-dihydroequilin sodium sulfate is needle-shaped and rod-shaped, and there are secondary particles formed by needle-shaped and rod-shaped particles. Raman analysis shows that the characteristic peaks (c, c, and c) of the synthetic products are quite different from those of natural conjugated estrogens (d), which can be used to determine the source of the drug and the consistency of the process. Figure 2 Figure 3 Figure 4 Figure 5 Example

[0033] ​​​​​​​​​​​​​​In this embodiment, the natural conjugated estrogen raw material (containing diluent, sodium citrate, conjugated estrogen, etc.) (batch number: ZM20240802), diluent (batch number: 0007773263), sodium citrate (batch number: 20230501), and synthetic conjugated estrogen (estrone sodium sulfate, sodium sulfate, 17α-dihydroequilin sulfate) are respectively photographed by scanning electron microscope using microscopic imaging method, and then specific regions are taken for Raman spectrum characteristic peak comparison. Figure 6 ).

[0034] The auxiliary materials and synthetic conjugated estrogen do not interfere with the determination of the crystal form of natural conjugated estrogen, and the specificity is good. Example

[0035] In this embodiment, four batches of natural conjugated estrogen raw materials (batch number: ZM20240802, ZM20240803, ZM20240804, ZM20240807) are respectively photographed by scanning electron microscope using microscopic imaging method, and then specific regions are taken for Raman spectrum characteristic peak comparison. The Raman spectrum characteristic peaks of ZM20240803, ZM20240804, and ZM20240807 are shown in Table 1. Figures 8-10 ).

[0036] The characteristic peaks of the four batches are good in reproducibility.

Claims

1. A method for determining a crystalline form of conjugated estrogens of natural origin mixture, characterized in that, The scanning electron microscope-confocal micro-Raman combined technology is used for analysis, and specifically includes the following steps: (1) sample preparation: the sample to be tested is uniformly dispersed on a conductive substrate, and is separately prepared and detected; (2) electron microscope morphology analysis: the scanning electron microscope is used to observe the micro-morphology of the sample, and the micro-area to be tested is positioned; (3) Raman spectrum acquisition: through the confocal micro-Raman spectrometer, the selected micro-area is scanned, and the Raman characteristic peak is obtained; (4) data comparison and analysis: the spectra of different detection samples are compared with the synthesized spectrum and the crystal form database, and the crystal form is determined through the peak position, peak shape and relative intensity difference; morphology-crystal form correlation: the crystal habit in the electron microscope image and the Raman spectrum characteristics are combined, and the corresponding relationship between the morphology and the crystal form is established.

2. The method of claim 1, wherein the natural source mixture is a mixture of natural plant extracts. In the (1), the sample is one or more of the following: natural conjugated estrogen raw material drug or preparation, synthetic estrone sodium sulfate, synthetic marenostrif sodium sulfate, and synthetic 17 alpha-dihydroequilin sulfate.

3. The method of claim 1, wherein the natural source mixture is a mixture of natural plant extracts. In the (2), the component contrast mode (Det BSED-COMP) of the backscattered electron detector of the scanning electron microscope is used, the voltage (HV) is 15kV, and the micro-morphology of the sample is observed.

4. The method of claim 1, wherein the natural source mixture is a mixture of natural plant extracts. In the (3) described, the laser wavelength of Raman spectrometer is 785nm, the power is 60mW, the integration time is 0.5~1s, the resolution is 2cm -1 , and the spectral range is 100~4000cm -1 .

5. The method of claim 1, wherein the natural source mixture is a mixture of natural plant extracts. In the (3), the sulfate ester bond and the steroid skeleton vibration peak are between 500-1800 cm⁻¹.

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