Method for separating and preparing cis-trans isomers in vitamin K1

Abstract

The invention belongs to the technical field of separation and purification of organic compounds, and particularly relates to a method for separating and preparing cis-trans isomers in vitamin K1. The separation and preparation method of cis-trans isomers in vitamin K1 provided by the invention comprises the following steps: dissolving a vitamin K1 crude product in a first organic solvent to obtain a vitamin K1 solution; carrying out supercritical fluid chromatography separation on the vitamin K1 solution, and collecting corresponding eluents according to retention time in a spectrogram obtained by supercritical fluid chromatography separation to respectively obtain cis-isomer of vitamin K1 and trans-isomer of vitamin K1; a mobile phase for supercritical fluid chromatography separation comprises a mixture of supercritical carbon dioxide and a second organic solvent, and the second organic solvent is n-hexane or n-heptane; the filler in the chromatographic column for supercritical fluid chromatographic separation is silica gel. VK1 cis-trans isomers are well separated under the combination of a specific stationary phase and a mobile phase, and the purity of the obtained trans isomers is greater than 99%.

Description

Technical Field

[0001] The invention belongs to the technical field of separation and purification of organic compounds, and specifically relates to a method for separating and preparing cis-trans isomers in vitamin K1. Background Art

[0002] The chemical name of vitamin K1 is 2-methyl-3-phytol-1,4-naphthoquinone. It is a fat-soluble vitamin that is stable to air and moisture, but will be decomposed in sunlight. It is widely present in natural green plants and is a necessary substance for the liver to synthesize factors II, VII, IX, and X. Vitamin K1 injection is mainly used to treat various hemorrhagic diseases caused by vitamin K deficiency. Vitamin K1 exists in cis and trans isomers, of which the natural form is a 2,3-trans configuration. Although the cis isomer may account for a certain proportion in chemical synthetic products, the cis isomer has almost no physiological activity. The pharmacopoeia stipulates that the content of the cis isomer of vitamin K1 needs to be ≤21%.

[0003] Currently, vitamin K1 is mainly prepared by chemical synthesis, and it is difficult to control the proportion of cis isomers in vitamin K1 below 21%. Currently, HPLC is mainly used to separate cis and trans isomers in vitamin K1, but the purity of trans isomers separated by HPLC still needs to be improved. Summary of the invention

[0004] In view of this, the present invention provides a method for separating and preparing cis- and trans-isomers in vitamin K1. The purity of the trans-isomer of vitamin K1 obtained according to the method provided by the present invention is above 99%.

[0005] In order to solve the above technical problems, the present invention provides a method for separating and preparing cis-trans isomers in vitamin K1, comprising the following steps:

[0006] dissolving crude vitamin K1 in a first organic solvent to obtain a vitamin K1 solution;

[0007] The vitamin K1 solution is subjected to supercritical fluid chromatography separation, and corresponding eluents are collected according to the retention time in the spectrum obtained by the supercritical fluid chromatography separation to obtain a cis isomer of vitamin K1 and a trans isomer of vitamin K1, respectively;

[0008] The mobile phase of the supercritical fluid chromatographic separation comprises a mixture of supercritical carbon dioxide and a second organic solvent, wherein the second organic solvent is n-hexane or n-heptane; and the filler in the chromatographic column for supercritical fluid chromatographic separation is silica gel.

[0009] Preferably, the volume percentage of supercritical carbon dioxide in the mobile phase is 70-98%.

[0010] Preferably, the elution mode of the supercritical fluid chromatography separation includes isocratic elution or gradient elution.

[0011] Preferably, the isocratic elution time is 15 to 30 minutes, and the volume percentage of supercritical carbon dioxide in the mobile phase for the isocratic elution is 80 to 83%.

[0012] Preferably, the gradient elution procedure includes: from 0 to 15 min, the volume fraction of supercritical carbon dioxide in the mobile phase is reduced from 90% to 70%.

[0013] Preferably, the conditions for supercritical fluid chromatography separation include: back pressure of 10-15 MPa, column temperature of 30-35° C., injection volume of 5-1500 μL, detection wavelength of 254 nm, and flow rate of 6-30 mL / min.

[0014] Preferably, the diameter of the chromatographic column for supercritical fluid chromatography separation is 10 mm or 20 mm, and the length is 250 mm.

[0015] Preferably, the first organic solvent includes one or more of n-hexane, n-heptane and ethanol.

[0016] Preferably, the mass concentration of vitamin K1 in the vitamin K1 solution is below 350 mg / mL.

[0017] Preferably, the mass percentage of the cis isomer in the crude vitamin K1 is less than 60%.

[0018] The present invention provides a method for separating and preparing cis-trans isomers in vitamin K1, comprising the following steps: dissolving a crude vitamin K1 product in a first organic solvent to obtain a vitamin K1 solution; subjecting the vitamin K1 solution to supercritical fluid chromatography separation, collecting corresponding eluents according to the retention time in the spectrum obtained by the supercritical fluid chromatography separation, and obtaining the cis-isomer of vitamin K1 and the trans-isomer of vitamin K1 respectively; the mobile phase of the supercritical fluid chromatography separation comprises a mixture of supercritical carbon dioxide and a second organic solvent, wherein the second organic solvent is n-hexane or n-heptane; and the filler in the chromatographic column for supercritical fluid chromatography separation is silica gel. The present invention uses supercritical carbon dioxide and a second organic solvent as mobile phases, and silica gel as a stationary phase to separate and purify crude vitamin K1 (VK1), which can achieve good separation of VK1 cis-trans isomers, and the separation time is short, and the purity of the separated VK1 trans isomer is more than 99%. The separation process provided by the present invention uses less organic reagents, which can effectively reduce the reagent cost, simplify the post-processing procedure, improve the green environmental protection of the separation method, and achieve cost reduction and efficiency improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the spectrum obtained by supercritical fluid chromatography separation in Example 1;

[0020] Figure 2 This is the spectrum obtained by supercritical fluid chromatography separation in Example 2;

[0021] Figure 3 This is the spectrum obtained by supercritical fluid chromatography separation in Example 3;

[0022] Figure 4 This is the spectrum obtained by supercritical fluid chromatography separation of Comparative Example 1;

[0023] Figure 5 This is the spectrum obtained by supercritical fluid chromatography separation of Comparative Example 2;

[0024] Figure 6 This is the spectrum obtained by supercritical fluid chromatography separation of Comparative Example 3;

[0025] Figure 7 This is the HPLC spectrum of the trans isomer of vitamin K1 separated in Example 2;

[0026] Figure 8 This is the HPLC spectrum of the trans isomer of vitamin K1 separated in Example 3. DETAILED DESCRIPTION

[0027] The present invention provides a method for separating and preparing cis-trans isomers in vitamin K1, comprising the following steps:

[0028] dissolving crude vitamin K1 in a first organic solvent to obtain a vitamin K1 solution;

[0029] The vitamin K1 solution is subjected to supercritical fluid chromatography separation, and corresponding eluents are collected according to the retention time in the spectrum obtained by the supercritical fluid chromatography separation to obtain a cis isomer of vitamin K1 and a trans isomer of vitamin K1, respectively;

[0030] The mobile phase of the supercritical fluid chromatographic separation comprises a mixture of supercritical carbon dioxide and a second organic solvent, wherein the second organic solvent is n-hexane or n-heptane; and the filler in the chromatographic column for supercritical fluid chromatographic separation is silica gel.

[0031] The present invention dissolves a crude vitamin K1 product in a first organic solvent to obtain a vitamin K1 solution. As a specific embodiment of the present invention, the mass percentage of the cis isomer in the crude vitamin K1 product may be less than 60%, may be 30-60%, and may further be 40-50%. As a specific embodiment of the present invention, the first organic solvent may include one or more of n-hexane, n-heptane and ethanol, and may specifically be a mixed solution of n-hexane and n-heptane, n-hexane, n-heptane or ethanol; the mass concentration of vitamin K1 in the vitamin K1 solution may be less than 350 mg / mL, and may also be 10-350 mg / mL, and may specifically be 10 mg / mL, 50 mg / mL, 100 mg / mL, 200 mg / mL, 300 mg / mL, 340 mg / mL, 345 mg / mL or 350 mg / mL.

[0032] In the present invention, the structural formula of vitamin K1 is as shown in Formula 1:

[0033]

[0034] As a specific embodiment of the present invention, the dissolution can be carried out under ultrasonic conditions. The present invention has no special requirements for the ultrasound, as long as the dissolution is complete; the dissolution is carried out under light-proof conditions to avoid decomposition of vitamin K1.

[0035] After obtaining the vitamin K1 solution, the present invention performs supercritical fluid chromatography separation on the vitamin K1 solution, collects the corresponding eluent according to the retention time in the spectrum obtained by the supercritical fluid chromatography separation, and obtains the cis isomer of vitamin K1 and the trans isomer of vitamin K1 respectively. In the present invention, the mobile phase of the supercritical fluid chromatography separation includes a mixture of supercritical carbon dioxide and a second organic solvent, and the volume percentage of supercritical carbon dioxide in the mobile phase can be 70-98%, and can be specifically 70%, 75%, 80%, 83%, 90% or 98%. In the present invention, the second organic solvent is n-hexane or n-heptane; the filler in the chromatographic column for supercritical fluid chromatography separation is silica gel (SI); the average particle size of the filler can be 4-6μm, and can be specifically 4μm, 5μm or 6μm. As a specific embodiment of the present invention, the elution mode of the supercritical fluid chromatographic separation includes isocratic elution or gradient elution; the time of the isocratic elution can be 15 to 30 minutes, and the volume percentage of supercritical carbon dioxide in the mobile phase for isocratic elution can be 80 to 83%, which can be specifically 80% or 83%; the procedure of the gradient elution can include: 0 to 15 minutes, the volume fraction of supercritical carbon dioxide in the mobile phase is reduced from 90% to 70%. The present invention adopts the isocratic elution method to achieve continuous separation preparation and improve separation efficiency.

[0036] As a specific embodiment of the present invention, the conditions for supercritical fluid chromatography separation may include: a back pressure of 10 to 15 MPa, a column temperature of 30 to 35°C, an injection volume of 5 to 1500 μL, a detection wavelength of 254 nm, and a flow rate of 6 to 30 mL / min; the back pressure may be specifically 10 MPa, 11 MPa, 12 MPa, 13 MPa, 14 MPa or 15 MPa; the column temperature may be specifically 30°C, 31°C, 32°C, 33°C, 34°C or 35°C; the The injection volume may be specifically 5 μL, 100 μL, 500 μL, 1000 μL or 1500 μL; the flow rate may be specifically 8 mL / min, 9 mL / min, 10 mL / min, 11 mL / min, 12 mL / min, 13 mL / min, 14 mL / min or 15 mL / min; the diameter of the chromatographic column for supercritical fluid chromatography separation may be 10 mm or 20 mm, and the length of the chromatographic column for supercritical fluid chromatography separation may be 250 mm. As a specific embodiment of the present invention, the conditions for supercritical fluid chromatography separation and preparation can be specifically as follows: back pressure of 10MPa, column temperature of 35°C, injection volume of 10μL, detection wavelength of 254nm, flow rate of 10mL / min, and chromatographic column specifications of 10×250mm; can also be specifically as follows: back pressure of 10MPa, column temperature of 35°C, injection volume of 40μL, detection wavelength of 254nm, flow rate of 10mL / min, and chromatographic column specifications of 10×250mm; can also be specifically as follows: back pressure of 10MPa, column temperature of 35°C, injection volume of 100μL, detection wavelength of 254nm, flow rate of 30mL / min, and chromatographic column specifications of 20×250mm.

[0037] As a specific embodiment of the present invention, the collection of the corresponding eluate needs to be carried out under light-proof conditions.

[0038] As a specific embodiment of the present invention, after receiving the corresponding eluate, the method may further include: performing post-treatment on the eluate to obtain the cis isomer of vitamin K1 and the trans isomer of vitamin K1 respectively; the post-treatment may be rotary evaporation. The present invention has no special limitation on the method of rotary evaporation as long as the second organic solvent in the eluate can be removed; the post-treatment may be performed under light-proof conditions.

[0039] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] The supercritical fluid chromatograph used for supercritical fluid chromatographic separation in the examples was purchased from Jiangsu Hanbang Technology Co., Ltd.

[0041] Example 1

[0042] The crude VK1 with a cis-isomer content of 30% was dissolved in n-hexane under ultrasonic conditions to obtain a VK1 solution with a mass concentration of 10 mg / mL;

[0043] The VK1 solution was subjected to supercritical fluid chromatography under the conditions of using a mixture of supercritical carbon dioxide and n-hexane as the mobile phase and silica gel as the chromatographic column filler (the temperature of carbon dioxide entering the pump was 8° C., and the elution method was gradient elution). The obtained chromatogram is shown in FIG. Figure 1 The conditions for supercritical fluid chromatography separation are shown in Table 1;

[0044] according to Figure 1 The corresponding eluate was collected at the medium retention time, and the eluate was rotary evaporated to obtain the cis isomer of vitamin K1 and the trans isomer of vitamin K1 respectively.

[0045] Table 1 Condition parameters for supercritical fluid chromatography separation in Example 1

[0046]

[0047] Example 2

[0048] The crude VK1 with a cis-isomer content of 30% was dissolved in n-hexane under ultrasonic conditions to obtain a VK1 solution with a mass concentration of 100 mg / mL;

[0049] The VK1 solution was subjected to supercritical fluid chromatography under the conditions of using a mixture of supercritical carbon dioxide with a supercritical carbon dioxide volume content of 83% and n-hexane as the mobile phase and silica gel as the chromatographic column filler (the temperature of carbon dioxide entering the pump was 8° C., and the elution method was isocratic elution). The obtained chromatogram is shown in FIG. Figure 2 The conditions for supercritical fluid chromatography separation are shown in Table 2;

[0050] according to Figure 2 The corresponding eluate was collected at the medium retention time, and the eluate was rotary evaporated to obtain the cis isomer of vitamin K1 and the trans isomer of vitamin K1 respectively.

[0051] Table 2 Condition parameters for supercritical fluid chromatography separation in Example 2

[0052]

[0053] Example 3

[0054] The crude VK1 with a cis-isomer content of 60% was dissolved in n-heptane under ultrasonic conditions to obtain a VK1 solution with a mass concentration of 150 mg / mL;

[0055] The VK1 solution was subjected to continuous (single needle sample load of 15 mg, single needle separation time of 10 min) supercritical fluid chromatography separation (the temperature of carbon dioxide entering the pump was 8° C., and the elution method was isocratic elution) under the conditions of using a mixture of supercritical carbon dioxide with a supercritical carbon dioxide volume content of 80% and n-heptane as the mobile phase and silica gel as the chromatographic column filler (the temperature of carbon dioxide entering the pump was 8° C., and the elution method was isocratic elution). The chromatogram obtained is as shown in Figure 3 The conditions for supercritical fluid chromatography separation are shown in Table 3;

[0056] according to Figure 3 The corresponding eluate was collected at the medium retention time, and the eluate was rotary evaporated to obtain the cis isomer of vitamin K1 and the trans isomer of vitamin K1 respectively.

[0057] Table 3 Condition parameters for supercritical fluid chromatography separation in Example 3

[0058]

[0059] Comparative Example 1

[0060] The crude VK1 with a cis-isomer content of 30% was dissolved in ethanol under ultrasonic conditions to obtain a VK1 solution with a mass concentration of 50 mg / mL;

[0061] The VK1 solution was subjected to supercritical fluid chromatography under the conditions of using a mixture of supercritical carbon dioxide with a supercritical carbon dioxide volume content of 98% and ethanol as the mobile phase and silica gel (MSU) bonded with imidazole groups on the surface as the chromatographic column filler (the temperature of carbon dioxide entering the pump was 8° C., and the elution method was isocratic elution). The obtained chromatogram is shown in FIG. Figure 4 The conditions for supercritical fluid chromatography separation are shown in Table 4.

[0062] Table 4 Condition parameters for supercritical fluid chromatography separation of comparative example 1

[0063]

[0064]

[0065] Comparative Example 2

[0066] The crude VK1 with a cis-isomer content of 30% was dissolved in n-hexane under ultrasonic conditions to obtain a VK1 solution with a mass concentration of 10 mg / mL;

[0067] The VK1 solution was subjected to supercritical fluid chromatography under the conditions of using a mixture of supercritical carbon dioxide with a supercritical carbon dioxide volume content of 98% and ethanol as the mobile phase and silica gel as the chromatographic column filler (the temperature of carbon dioxide entering the pump was 8° C., and the elution method was isocratic elution). The obtained chromatogram is shown in FIG. Figure 5 The conditions for supercritical fluid chromatography separation are shown in Table 5.

[0068] Table 5 Condition parameters for supercritical fluid chromatography separation of comparative example 2

[0069]

[0070] Comparative Example 3

[0071] The crude VK1 with a cis-isomer content of 30% was dissolved in n-hexane under ultrasonic conditions to obtain a VK1 solution with a mass concentration of 10 mg / mL;

[0072] The VK1 solution was subjected to supercritical fluid chromatography under the conditions of using a mixture of supercritical carbon dioxide and n-hexane as the mobile phase and silica gel (MSU) bonded with imidazole groups on the surface as the chromatographic column filler (the temperature of carbon dioxide entering the pump was 8° C., and the elution method was gradient elution). The obtained chromatogram is shown in FIG. Figure 6 The conditions for supercritical fluid chromatography separation are shown in Table 6.

[0073] Table 6 Condition parameters for supercritical fluid chromatography separation of comparative example 3

[0074]

[0075]

[0076] Depend on Figures 1 to 6 It can be seen that the method according to the embodiment can well separate the trans isomer from the crude VK1; the method according to the comparative example cannot well separate the trans isomer from the crude VK1. Figure 1 and Figure 6 It can be seen that the use of silica gel chromatography column for supercritical fluid chromatography separation can well separate the trans isomer from the crude VK1. Figure 2 and Figure 5 It can be seen that when the chromatographic column is a silica gel column and the mobile phase is supercritical fluid CO2 and ethanol, the cis and trans isomers of VK1 cannot be effectively separated, indicating that the selection of a suitable mobile phase for supercritical fluid chromatography can well separate the trans isomer from the crude VK1. Figure 3 and Figure 4 It can be seen that when the mobile phase is carbon dioxide and ethanol, the separation effect is poor, and the cis and trans isomers of VK1 cannot be effectively separated. When carbon dioxide and n-heptane are used as the mobile phase, the separation effect is good, the sample loading capacity is larger, and it is more conducive to industrial-grade production and preparation.

[0077] The trans isomers separated in Example 2 and Example 3 were subjected to high performance liquid chromatography according to the conditions in Table 7 (using silica gel as the chromatographic column filler, a mixture of n-hexane and n-pentanol in a volume ratio of 2000:1 as the mobile phase for isocratic elution, the column temperature was 30° C., and the flow rate was 1 mL / min), and a liquid chromatogram was obtained, as shown in FIG. Figure 7 , 8 As shown, Figure 7 is the liquid chromatography spectrum of the trans isomer obtained in Example 2, Figure 8 This is the liquid chromatography spectrum of the trans isomer obtained in Example 3.

[0078] Table 7 HPLC detection conditions

[0079]

[0080]

[0081] Depend on Figure 7 It can be seen that the purity of the trans isomer separated in Example 2 is 99.44%, and the content of the cis isomer is less than 1%.

[0082] Depend on Figure 8 It can be seen that the purity of the trans isomer separated in Example 3 is 100%.

[0083] It can be seen from the above test results that the supercritical fluid chromatography separation performed by the present invention under a specific combination of stationary phase and mobile phase can well separate the cis and trans isomers in the crude VK1 product, and the separation time is short; the purity of the separated trans isomer is above 99%. The separation and preparation method provided by the present invention has broad application prospects in the field of preparation of high-purity vitamin K1 trans isomers.

[0084] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for separating and preparing cis-trans isomers in vitamin K1, comprising the following steps: dissolving crude vitamin K1 in a first organic solvent to obtain a vitamin K1 solution; The vitamin K1 solution is subjected to supercritical fluid chromatography separation, and corresponding eluents are collected according to the retention time in the spectrum obtained by the supercritical fluid chromatography separation to obtain a cis isomer of vitamin K1 and a trans isomer of vitamin K1, respectively; The mobile phase of the supercritical fluid chromatographic separation comprises a mixture of supercritical carbon dioxide and a second organic solvent, wherein the second organic solvent is n-hexane or n-heptane; and the filler in the chromatographic column for supercritical fluid chromatographic separation is silica gel.

2. The method for separating and preparing cis-trans isomers in vitamin K1 according to claim 1, characterized in that: The volume percentage of supercritical carbon dioxide in the mobile phase is 70-98%.

3. The method for separating and preparing cis-trans isomers in vitamin K1 according to claim 2, characterized in that: The elution mode of the supercritical fluid chromatography separation includes isocratic elution or gradient elution.

4. The method for separating and preparing cis-trans isomers in vitamin K1 according to claim 3, characterized in that: The isocratic elution time is 15 to 30 minutes, and the volume percentage of supercritical carbon dioxide in the mobile phase for the isocratic elution is 80 to 83%.

5. The method for separating and preparing cis-trans isomers in vitamin K1 according to claim 3, characterized in that: The gradient elution procedure includes: from 0 to 15 minutes, the volume fraction of supercritical carbon dioxide in the mobile phase is reduced from 90% to 70%.

6. The method for separating and preparing cis-trans isomers in vitamin K1 according to claim 1, characterized in that: The conditions for supercritical fluid chromatography separation include: back pressure of 10-15 MPa, column temperature of 30-35° C., injection volume of 5-1500 μL, detection wavelength of 254 nm, and flow rate of 6-30 mL / min.

7. The method for separating and preparing cis-trans isomers in vitamin K1 according to claim 1 or 6, characterized in that: The diameter of the chromatographic column for supercritical fluid chromatographic separation is 10 mm or 20 mm, and the length is 250 mm.

8. The method for separating and preparing cis-trans isomers in vitamin K1 according to claim 1, characterized in that: The first organic solvent includes one or more of n-hexane, n-heptane and ethanol.

9. The method for separating and preparing cis-trans isomers in vitamin K1 according to claim 1 or 8, characterized in that: The mass concentration of vitamin K1 in the vitamin K1 solution is below 350 mg / mL.

10. The method for separating and preparing cis-trans isomers in vitamin K1 according to claim 1, characterized in that: The mass percentage of the cis isomer in the crude vitamin K1 is less than 60%.

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