HPLC (High Performance Liquid Chromatography) analysis method capable of simultaneously and centrally controlling two different 7-dehydrocholesterol synthesis routes

Abstract

The invention discloses an HPLC (High Performance Liquid Chromatography) analysis method capable of simultaneously and centrally controlling two different 7-dehydrocholesterol synthesis routes, which comprises the following steps: adopting a chromatographic column taking octaalkylsilane bonded silica gel as a filler, taking water as a mobile phase A and taking an organic phase as a mobile phase B; carrying out gradient elution on reaction liquid samples on two different synthetic routes of cholesterol to 7-dehydrocholesterol; calculating a conversion rate according to an area normalization method containing a correction factor; the invention relates to an HPLC (High Performance Liquid Chromatography) analysis method for a 7-dehydrocholesterol synthesis route through According to the HPLC analysis method capable of simultaneously and centrally controlling the two different 7-dehydrocholesterol synthesis routes, all reaction solutions and intermediate samples on the two different 7-dehydrocholesterol synthesis routes can be effectively separated, reaction solvents are free of interference, accurate data are provided for selection and optimization of the 7-dehydrocholesterol process routes, and the method is suitable for industrial production. And a basis is provided for subsequent research on related substances of 7-dehydrocholesterol and transmission, transformation and removal of impurities.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical analytical chemistry, and particularly to an HPLC analytical method capable of simultaneously controlling two different synthetic routes of 7-dehydrocholesterol in the middle control process. Background Art

[0002] 7-dehydrocholesterol (7-DHC), with the chemical name 3β-hydroxy-5,7-cholestadiene, CAS number: 434-16-2, molecular formula: C27H44O, and molecular weight: 384.64, is a key intermediate for the preparation of vitamin D3 and its derivatives. Vitamin D3 is synthesized by a one-step photochemical reaction. Vitamin D3 is widely used in medicine, food and beverage, feed additives, and cosmetics. The annual demand is huge. Therefore, controlling the purity and yield of 7-dehydrocholesterol plays a key role in improving the total conversion rate of the vitamin D3 route, reducing costs and increasing efficiency, and enhancing competitiveness.

[0003] Patents such as CN102030794B, CN106397525A, CN112979738B, and CN117362376A have all reported the production and preparation methods of 7-dehydrocholesterol, but did not mention the in-process control detection methods; Patent WO2020104356A1 mentions gradient elution with acetonitrile and methyl tert-butyl ether as the mobile phase to detect 7-dehydrocholesterol; Patents CN105669813B and CN111138510B mention isocratic elution with acetonitrile-methanol as the mobile phase to detect the intermediate 7-ketocholesterol acetate of 7-dehydrocholesterol; Patent CN113621015B selects methanol as the mobile phase to control the aromatic impurities in the deazone reaction of 7-dehydrocholesterol. However, due to the large polarity differences of the intermediates in the reaction route, the above methods are not applicable to the detection of all reaction solutions and intermediate samples in the route.

[0004] Patents CN109761867B and CN112745253B respectively prepare 7-dehydrocholesterol from lanolin and stigmasterol through steps such as acetylation, oxidation, hydrazonation, deazonation, and hydrolysis, and finally produce vitamin D3 through light irradiation; Patent WO2024145583A1 provides two preparation methods for preparing 7-dehydrocholesterol from cholesterol and finally synthesizing vitamin D3. However, the entire reaction route of the above patents uses thin-layer chromatography for in-process control analysis, which cannot monitor the reaction process and accurately quantify.

[0005] In view of the existing mainstream processes for producing 7-dehydrocholesterol, namely the oxidation-reduction method and the bromination-debromination method, this invention designs a route and optimizes the analysis method. It can effectively separate all reaction solutions and intermediate samples in the two mainstream process routes of 7-dehydrocholesterol. The reaction solvent has no interference, providing accurate data for the selection and optimization of the 7-dehydrocholesterol process route. It is of great significance for the subsequent research on related substances of 7-dehydrocholesterol, as well as the transfer, transformation, and elimination of impurities. For this reason, we propose an HPLC analysis method that can simultaneously control two different 7-dehydrocholesterol synthesis routes. Summary of the Invention

[0006] Technical problems to be solved: In view of the deficiencies of the prior art, this invention provides an HPLC analysis method that can simultaneously control two different 7-dehydrocholesterol synthesis routes. It can effectively separate all reaction solutions and intermediate samples in the two different 7-dehydrocholesterol synthesis routes. The reaction solvent has no interference, providing accurate data for the selection and optimization of the 7-dehydrocholesterol process route, and providing a basis for the subsequent research on related substances of 7-dehydrocholesterol, as well as the transfer, transformation, and elimination of impurities. It can effectively solve the problems in the background technology.

[0007] Technical solution: To achieve the above object, the technical solution adopted by this invention is as follows: An HPLC analysis method that can simultaneously control two different 7-dehydrocholesterol synthesis routes, including the following operation steps: S1: Material preparation: Prepare the materials required for the analysis of the 7-dehydrocholesterol synthesis route, including a chromatographic column filled with octadecylsilane-bonded silica gel, water, an organic phase, and a reaction solution sample. S2: Mobile phase setting: Use a chromatographic column filled with octadecylsilane-bonded silica gel, with water as mobile phase A and the organic phase as mobile phase B. S3: Gradient elution: Perform gradient elution on the reaction solution samples in the two different synthesis routes from cholesterol to 7-dehydrocholesterol. S4: Conversion rate calculation: Calculate the conversion rate by the area normalization method with a correction factor. S5: HPLC analysis: Use the conversion rate to perform an HPLC analysis method on the 7-dehydrocholesterol synthesis route, where HPLC is high-performance liquid chromatography, used for separating multiple components in a mixture, identification, and quantitative analysis.

[0008] As a preferred technical solution of this application, the following operation steps are included in the S3 step for the synthesis route from cholesterol to 7-dehydrocholesterol: A1: Using cholesterol as the starting material (7-DHC-5), perform hydroxyl protection to obtain cholesterol ester (7-DHC-4). A2: Then, perform allylic oxidation to obtain 7-ketocholesterol ester (7-DHC-3). A3: Then, 7-aryl sulfonylhydrazone-cholesterol ester (7-DHC-2) is obtained through hydrazonation reaction; A4: Then, the hydrazone group is removed through de-hydrazonation reaction to obtain 7-dehydrocholesterol ester (7-DHC-1); A5: Finally, 7-dehydrocholesterol (7-DHC) is obtained through alkaline hydrolysis.

[0009] As a preferred technical solution of this application, the synthetic route II from cholesterol to 7-dehydrocholesterol in step S3 includes the following operating steps: B1: Using cholesterol as the starting material (7-DHC-5), hydroxyl protection is carried out to obtain cholesterol ester (7-DHC-4); B2: Then, bromination is carried out to obtain 7-bromo-cholesterol ester (7-DHC-3.2); B3: Then, debromination is carried out under alkaline conditions, and after purification and separation, 7-dehydrocholesterol ester (7-DHC-1) is obtained; B4: Finally, the acyl group is removed to obtain 7-dehydrocholesterol (7-DHC).

[0010] As a preferred technical solution of this application, in step S2, the chromatographic column model is a chromatographic column filled with octadecylsilane-bonded silica gel, the filler particle size is 1.5μm - 5.0μm, the chromatographic column diameter is 3.0mm - 5.0mm, and the chromatographic column length is 100mm - 250mm.

[0011] As a preferred technical solution of this application, in step S2, the organic phase solvent is one or more of acetonitrile, ethanol, methanol, isopropanol, and tetrahydrofuran.

[0012] As a preferred technical solution of this application, in steps S3 - S5, the detection wavelength is 200 - 400nm, and the column temperature is 38 - 42°C.

[0013] As a preferred technical solution of this application, in steps S3 - S5, the flow rate is 0.2 - 2.0mL / min.

[0014] As a preferred technical solution of this application, in steps S3 - S5, the sample concentration is 0.2 - 2mg / mL, and the diluent is one or more of acetonitrile, ethanol, methanol, isopropanol, and tetrahydrofuran.

[0015] Beneficial effects: Compared with the prior art, the present invention provides an HPLC analysis method capable of simultaneously controlling two different 7-dehydrocholesterol synthesis routes, having the following beneficial effects: The HPLC analysis method capable of simultaneously controlling two different 7-dehydrocholesterol synthesis routes uses a chromatographic column filled with octadecylsilane-bonded silica gel, with water and an organic phase as the mobile phase, and performs gradient elution on the reaction solution samples on two different synthesis routes from cholesterol to 7-dehydrocholesterol. The conversion rate is calculated by the area normalization method with correction factors. For the existing mainstream processes for producing 7-dehydrocholesterol: the redox method and the bromination and debromination method, the routes are designed and the analysis method is optimized, which can effectively separate all reaction solution and intermediate samples on two different 7-dehydrocholesterol synthesis routes, and the reaction solvent has no interference, providing accurate data for the selection and optimization of the 7-dehydrocholesterol process route, providing a basis for the subsequent research on related substances of 7-dehydrocholesterol, and the transfer, transformation, and elimination of impurities. The HPLC analysis of the entire 7-dehydrocholesterol synthesis route is simpler, the operation is convenient, and the use effect is better than the traditional method. Brief Description of the Drawings

[0016] Figure 1 It is a schematic diagram of Synthesis Route 1 in the HPLC analysis method of the present invention capable of simultaneously controlling two different 7-dehydrocholesterol synthesis routes.

[0017] Figure 2 It is a schematic diagram of Synthesis Route 2 in the HPLC analysis method of the present invention capable of simultaneously controlling two different 7-dehydrocholesterol synthesis routes.

[0018] Figure 3 It is a schematic diagram of the applicability spectrum of Example 1 in the HPLC analysis method of the present invention capable of simultaneously controlling two different 7-dehydrocholesterol synthesis routes.

[0019] Figure 4 It is a schematic diagram of the applicability spectrum of Example 2 in the HPLC analysis method of the present invention capable of simultaneously controlling two different 7-dehydrocholesterol synthesis routes. Detailed Embodiments

[0020] The technical solutions of the present invention will be clearly and completely described below in conjunction with the drawings and specific embodiments. However, those skilled in the art will understand that the following described embodiments are some embodiments of the present invention, not all embodiments, and are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. Those not specified in the embodiments are carried out under conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified in the manufacturer are all conventional products that can be obtained through commercial purchase.

[0021] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention. In addition, the terms "first", "second", "third" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance.

[0022] In the description of the present invention, it should be noted that unless otherwise clearly specified and defined, the terms "installed", "connected", "connected" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0023] As Figures 1-4 shown, an HPLC analysis method that can simultaneously control two different 7-dehydrocholesterol synthesis routes includes the following operating steps: S1: Material preparation: Prepare the materials required for the analysis of the 7-dehydrocholesterol synthesis route, including a chromatographic column filled with octadecylsilane-bonded silica gel, water, an organic phase, and a reaction liquid sample; S2: Mobile phase setting: Use a chromatographic column filled with octadecylsilane-bonded silica gel, with water as mobile phase A and the organic phase as mobile phase B; S3: Gradient elution: Perform gradient elution on the reaction liquid samples on two different synthesis routes of cholesterol → 7-dehydrocholesterol; S4: Conversion rate calculation: Calculate the conversion rate according to the area normalization method with a correction factor; S5: HPLC analysis: Perform an HPLC analysis method on the 7-dehydrocholesterol synthesis route through the conversion rate, where HPLC is high performance liquid chromatography, which is used to separate multiple components in a mixture, identify, and perform quantitative analysis.

[0024] Furthermore, in step S3, the synthesis route of cholesterol → 7-dehydrocholesterol includes the following operating steps: A1: Using cholesterol as the starting material (7-DHC-5), perform hydroxyl protection to obtain cholesterol ester (7-DHC-4); A2: Then perform allylic oxidation to obtain 7-ketocholesterol ester (7-DHC-3); A3: Then, 7-aryl sulfonylhydrazone-cholesterol ester (7-DHC-2) is obtained through a hydrazonation reaction; A4: Then, a de-hydrazonation reaction is carried out to obtain 7-dehydrocholesterol ester (7-DHC-1); A5: Finally, 7-dehydrocholesterol (7-DHC) is obtained through alkaline hydrolysis.

[0025] Furthermore, the synthetic route II from cholesterol to 7-dehydrocholesterol in step S3 includes the following operating steps: B1: Using cholesterol as the starting material (7-DHC-5), hydroxyl protection is carried out to obtain cholesterol ester (7-DHC-4); B2: Then, bromination is carried out to obtain 7-bromo-cholesterol ester (7-DHC-3.2); B3: Then, debromination is carried out under an alkaline environment, and after refining and separation, 7-dehydrocholesterol ester (7-DHC-1) is obtained; B4: Finally, the acyl group is removed to obtain 7-dehydrocholesterol (7-DHC).

[0026] Furthermore, in step S2, the chromatographic column model is a chromatographic column filled with octadecylsilyl-bonded silica gel, the particle size of the packing is 1.5 μm to 5.0 μm, the diameter of the chromatographic column is 3.0 mm to 5.0 mm, and the length of the chromatographic column is 100 mm to 250 mm.

[0027] Furthermore, in step S2, the organic phase solvent is one or more of acetonitrile, ethanol, methanol, isopropanol, and tetrahydrofuran.

[0028] Furthermore, in steps S3 - S5, the detection wavelength is 200 - 400 nm, and the column temperature is 38 - 42 °C.

[0029] Furthermore, in steps S3 - S5, the flow rate is 0.2 - 2.0 mL / min.

[0030] Furthermore, in steps S3 - S5, the sample concentration is 0.2 - 2 mg / mL, and the diluent is one or more of acetonitrile, ethanol, methanol, isopropanol, and tetrahydrofuran.

[0031] Using a chromatographic column filled with octadecylsilyl-bonded silica gel, with water as mobile phase A and the organic phase as mobile phase B, gradient elution is carried out on the reaction solutions and intermediate samples on two different synthetic routes from cholesterol to 7-dehydrocholesterol, and the conversion rate is calculated by the area normalization method with a correction factor. The elution gradient is as follows: Preferably, the chromatographic column model is a chromatographic column filled with octadecylsilyl-bonded silica gel. A chromatographic column with a particle size of the packing of 1.5 μm to 5.0 μm, a diameter of the chromatographic column of 3.0 mm to 5.0 mm, and a length of the chromatographic column of 100 mm to 250 mm.

[0032] Preferably, the chromatographic column is an Agilentporoshell 120 SB-C8 column with a packing particle size of 2.7 μm, a column diameter of 4.6 mm, and a column length of 150 mm.

[0033] Preferably, the elution gradient is as follows: Preferably, elution gradient one is as follows: Preferably, elution gradient two is as follows: Preferably, the organic solvent is one or more of acetonitrile, ethanol, methanol, isopropanol, and tetrahydrofuran.

[0034] Preferably, the organic solvent is acetonitrile.

[0035] Preferably, the detection wavelength is 200 - 400 nm, the column temperature is 38 - 42 °C, and the flow rate is 0.5 - 2.0 mL / min.

[0036] Preferably, the detection wavelength is 210 nm, the column temperature is 40 °C, and the flow rate is 0.6 mL / min.

[0037] Preferably, the sample concentration is 0.2 - 2 mg / mL, and the diluent is one or more of acetonitrile, ethanol, methanol, isopropanol, and tetrahydrofuran.

[0038] Preferably, the sample concentration is 1.0 mg / mL, and the diluent is acetonitrile. Example

[0039] Using octadecylsilyl silica gel as the filler (Agilentporoshell 120 SB-C8, 150 × 4.6 mm, 2.7 μm), with water as mobile phase A and acetonitrile as mobile phase B, perform gradient elution; the detection wavelength is 210 nm, the flow rate is 0.6 ml / min, the column temperature is 40 °C, and the diluent is acetonitrile. Precisely measure 10 μl of the test solution and inject it into the liquid chromatograph, and record the chromatogram.

[0040] The elution gradient is as follows: In-process reaction solution: Take an appropriate amount of the reaction solution, dilute it with the diluent to prepare a solution containing approximately 1 mg of the sample per 1 ml, and shake well.

[0041] Starting material and intermediate sample solutions: Respectively take about 10 mg of each starting material and intermediate, accurately weigh, place them in a 10 ml volumetric flask, dissolve with the diluent, dilute to the scale, and shake well.

[0042] System suitability solution: Precisely transfer 1.0 ml of each of the above starting material and intermediate sample solutions into a 10 ml volumetric flask, dissolve with the diluent, dilute to the scale, and shake well.

[0043] Test result: The system suitability chromatogram of the first synthetic route of 7-dehydrocholesterol is shown in the appendix Figure 3 , where 7-DHC-5 (RT = 22.219 min), 7-DHC-4 (RT = 33.330 min), 7-DHC-3 (RT = 17.022 min), 7-DHC-2 (RT = 19.475 min), 7-DHC-1 (RT = 30.361 min), 7-DHC (RT = 17.946 min). Example

[0044] Use octadecylsilane-bonded silica gel as the packing material (Agilent poroshell 120 SB-C8, 150×4.6 mm, 2.7 μm), use water as mobile phase A and acetonitrile as mobile phase B for gradient elution; the detection wavelength is 210 nm, the flow rate is 0.6 ml / min, the column temperature is 40 °C, and the diluent: acetonitrile. Accurately measure 10 μl of the test solution, inject it into the liquid chromatograph, and record the chromatogram.

[0045] The elution gradient is as follows: In-process reaction solution: Take an appropriate amount of the reaction solution, dilute it with the diluent to make a solution containing about 1 mg of the sample per 1 ml, and shake well to obtain.

[0046] Starting material and intermediate sample solutions: Take about 10 mg of each starting material and intermediate respectively, accurately weigh, place in a 10 ml volumetric flask, dissolve with the diluent, dilute to the mark, and shake well to obtain.

[0047] System suitability solution: Accurately transfer 1.0 ml of each of the above starting material and intermediate sample solutions into a 10 ml volumetric flask, dissolve with the diluent, dilute to the mark, and shake well to obtain.

[0048] Test result: The system suitability chromatogram of the second synthetic route of 7-dehydrocholesterol is shown in the appendix Figure 4 , where 7-DHC-5 (RT = 14.799 min), 7-DHC-4 (RT = 19.289 min), 7-DHC-3.2 (RT = 11.731 min), 7-DHC-1 (RT = 17.256 min), 7-DHC (RT = 12.753 min).

[0049] It should be noted that in this text, relational terms such as first and second (No. 1, No. 2, etc.) are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprising", "including" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising an..." does not exclude the presence of additional identical elements in the process, method, article or device comprising said element.

[0050] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and what is described in the above embodiments and the specification is only to illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and all these changes and improvements fall within the scope of the present invention claimed.

Claims

1. An HPLC analysis method capable of simultaneously controlling two different 7-dehydrocholesterol synthesis routes, characterized in that: The steps include: S1: Material preparation: Prepare the materials needed for the analysis of the synthetic route of 7-dehydrocholesterol, including a chromatographic column filled with octaalkylsilane bonded silica gel, water, an organic phase, and a reaction solution sample; S2: Mobile phase setting: using octaalkylsilane bonded silica gel as a chromatographic column filler, water as mobile phase A, and organic phase as mobile phase B; S3: Gradient elution: gradient elution of the reaction solution samples from two different synthetic routes of cholesterol → 7-dehydrocholesterol; S4: Conversion rate calculation: The conversion rate is calculated by the area normalization method including the correction factor; S5: HPLC analysis: The HPLC analysis method of the 7-dehydrocholesterol synthesis route is performed by conversion rate, wherein HPLC is high performance liquid chromatography, which is used for separation, identification and quantitative analysis of multiple components in a mixture.

2. The HPLC analysis method capable of simultaneously controlling two different 7-dehydrocholesterol synthesis routes according to claim 1, characterized in that: The synthesis route of cholesterol → 7-dehydrocholesterol in step S3 includes the following steps: A1: Using cholesterol as the starting material (7-DHC-5), the hydroxyl group was protected to obtain cholesterol ester (7-DHC-4); A2: After allylic oxidation, 7-ketocholesterol ester (7-DHC-3) is obtained; A3: Then, 7-arylsulfonylhydrazone-cholesterol ester (7-DHC-2) is obtained through hydrazolation reaction; A4: Dehydrazone reaction to obtain 7-dehydrocholesterol ester (7-DHC-1); A5: Finally, 7-dehydrocholesterol (7-DHC) is obtained through alkaline hydrolysis.

3. The HPLC analysis method capable of simultaneously controlling two different 7-dehydrocholesterol synthesis routes according to claim 1, characterized in that: The second synthesis route of cholesterol→7-dehydrocholesterol in step S3 includes the following steps: B1: Using cholesterol as the starting material (7-DHC-5), the hydroxyl group was protected to obtain cholesterol ester (7-DHC-4); B2: After further bromination, 7-bromo-cholesterol ester (7-DHC-3.2) is obtained; B3: Then debromination under alkaline environment, purification and separation to obtain 7-dehydrocholesterol ester (7-DHC-1); B4: Finally, the acyl group is removed to obtain 7-dehydrocholesterol (7-DHC).

4. The HPLC analysis method capable of simultaneously controlling two different 7-dehydrocholesterol synthesis routes according to claim 1, characterized in that: The chromatographic column model in step S2 is a chromatographic column with octaalkylsilane bonded silica gel as a filler, the filler particle size is 1.5 μm~5.0 μm, the chromatographic column diameter is 3.0 mm~5.0 mm, and the chromatographic column length is 100 mm~250 mm.

5. The HPLC analysis method capable of simultaneously controlling two different 7-dehydrocholesterol synthesis routes according to claim 1, characterized in that: The organic phase solvent in step S2 is one or more of acetonitrile, ethanol, methanol, isopropanol, and tetrahydrofuran.

6. The HPLC analysis method capable of simultaneously controlling two different 7-dehydrocholesterol synthesis routes according to claim 1, characterized in that: In the steps S3-S5, the detection wavelength is 200-400 nm and the column temperature is 38-42° C.

7. The HPLC analysis method capable of simultaneously controlling two different 7-dehydrocholesterol synthesis routes according to claim 1, characterized in that: The flow rate in the steps S3-S5 is 0.2-2.0 mL / min.

8. The HPLC analysis method capable of simultaneously controlling two different 7-dehydrocholesterol synthesis routes according to claim 1, characterized in that: In the steps S3-S5, the sample concentration is 0.2-2 mg / mL, and the diluent is one or more of acetonitrile, ethanol, methanol, isopropanol, and tetrahydrofuran.

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