A method for preparing an olafluor analogue having an alkyl chain of C16:1

Abstract

The scheme provides a method for preparing an olafluor analogue with a C16:1 alkyl chain, comprising the following steps: S1: performing one chromatographic elution on olafluor raw materials; when mass spectrometry detects a mass spectrometry signal of a mass-to-charge ratio of the olafluor analogue with the C16:1 alkyl chain, collecting one eluate corresponding to the mass spectrometry signal; S2: performing secondary preparation chromatographic elution on the one eluate; when mass spectrometry detects a mass spectrometry signal of a mass-to-charge ratio of the olafluor analogue with the C16:1 alkyl chain, collecting a secondary eluate corresponding to the mass spectrometry signal; S3: concentrating the secondary eluate to obtain a concentrated component; dissolving the concentrated component in ammonia water, then extracting with n-hexane, separating the n-hexane layer, and blowing dry with nitrogen to obtain a purified component; and preparing the olafluor analogue with the C16:1 alkyl chain from the olafluor raw materials by mass spectrometry guidance.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of olaflur analogues, in particular to a method for preparing an olaflur analogue with a C16:1 alkyl chain. BACKGROUND

[0002] Olaflur, as an important fluorine-containing surfactant, has a wide range of applications in oral care (such as anti-caries agents) and material science, and its biological activity is closely related to the structure of the alkyl chain.

[0003] The olaflur raw material added in oral care products on the market is mainly prepared by special process from animal tallow. The tallow is rich in fatty acids with different alkyl chain lengths and saturations. The olaflur raw material prepared from the raw material is a mixture of structural analogues containing different alkyl chain lengths and saturations. Although the unique spatial conformation of various olaflur analogues can endow the molecules with better interfacial activity and biocompatibility, the effects of various components are different.

[0004] Since olaflur and its homologues and analogues do not have ultraviolet, visible and fluorescent absorption, and are difficult to volatilize, only evaporation light scattering or CAD, mass spectrometry methods are used for qualitative and quantitative analysis in chromatographic quantification. In synthesis, only the chemical synthesis of alkyl chains C18 (olaflur), C18:1 and C16 (olaflur homologues) has been realized on the market. The directed synthesis of olaflur analogues with a specific structure of a monounsaturated C16:1 chain (n=7) still faces key challenges. The synthesis of olaflur analogues with an unsaturated C16:1 chain (n=7) requires the preparation of the corresponding precursor raw material (Z)-1-bromo-7-hexadecene. However, the (Z)-1-bromo-7-hexadecene precursor raw material is not commercially available, which hinders the traditional synthesis path.

[0005] In summary, due to the commercial unavailability of the synthesis raw material for C16:1, there is currently no synthesis scheme for obtaining high-purity C16:1 olaflur analogue by chemical synthesis, and it is impossible to realize the quantitative analysis of C16:1 olaflur analogue. SUMMARY

[0006] The present application aims to provide a method for preparing an olaflur analogue with a C16:1 alkyl chain, which breaks through the constraints of key raw materials and uses mass spectrometry-guided preparative liquid chromatography separation to prepare high-purity C16:1 (n=7) olaflur analogue from olaflur raw material. This solves the problem that the specific configuration material cannot be obtained by conventional synthesis, and fills the market gap of long-chain fluorinated surfactants containing monounsaturated.

[0007] To achieve the above object, the technical scheme provides a method for preparing an olaparib analogue with an alkyl chain of C16:1, comprising the following steps:

[0008] S1: performing one chromatographic elution on olaparib raw material, and collecting one eluent corresponding to the mass spectrum signal when the mass spectrometer detects the mass spectrum signal of the mass-to-charge ratio of the olaparib analogue with an alkyl chain of C16:1, wherein the chromatographic conditions of the one chromatographic elution are as follows: the chromatographic column is Exmere Exsil Pure 120 Biphenyl, the column temperature is room temperature, the mobile phase A is 10 mM NH4AC and 0.1% formic acid water, the mobile phase B is methanol, and the elution mode is gradient elution;

[0009] S2: performing secondary chromatographic elution on the one eluent, and collecting secondary eluent corresponding to the mass spectrum signal when the mass spectrometer detects the mass spectrum signal of the mass-to-charge ratio of the olaparib analogue with an alkyl chain of C16:1, wherein the chromatographic conditions of the secondary chromatographic elution are as follows: the chromatographic column is Phenomenex Luna CN, the mobile phase A is 0.1% formic acid water, the mobile phase B is methanol, and the elution mode is gradient elution;

[0010] S3: concentrating the eluent to obtain a concentrated component, dissolving the concentrated component with ammonia water, extracting with n-hexane, separating the n-hexane layer, and blowing dry with nitrogen to obtain a purified component.

[0011] The scheme captures the olaparib analogue with an alkyl chain of C16:1 from the olaparib raw material in a way of high-performance liquid chromatography and mass spectrometry, optimizes the one chromatographic elution and secondary chromatographic elution conditions to improve the separation efficiency of the olaparib analogue with an alkyl chain of C16:1, and realizes accurate identification and collection of the olaparib analogue with an alkyl chain of C16:1, thereby providing a high-purity sample for subsequent application.

[0012] In step S1, the olaparib raw material of the scheme is obtained by fluorination of C4-C18 mixed fatty acids in beef tallow.

[0013] It should be noted that the beef tallow is a renewable resource, and the C16-C18 mixed fatty acid is extracted and purified from the beef tallow by saponification and acidification, then the fluorine-containing group is introduced by activation, amidation and fluorination reaction, and the olaflur raw material is obtained by post-processing. The raw material is a mixture of olaflur analogs containing different carbon chains (C8-C18) and degrees of saturation (saturated, monounsaturated, such as C16:1, C18:1, etc., and the double bond is mainly cis), with less impurities, synergistic effect of each component, good interfacial activity and biocompatibility, and low cost. The olaflur analogs with C16:1 alkyl chain are separated from the olaflur raw material synthesized from beef tallow by mass spectrometry in the present scheme, so this way avoids the complex asymmetric olefin construction step compared to the total synthesis route, and conforms to the principle of green chemistry. Although the beef tallow composition is complex, but from the technical strategy, the fractionation preparation technology can be used to provide an economically viable alternative for the preparation of special structure fluorinated surfactants.

[0014] In some embodiments, the chromatographic column for one-time chromatographic elution is Exmere Exsil Pure 120 Biphenyl with a specification of 5um, 20*250mm.

[0015] In some embodiments, the flow rate of one-time chromatographic elution is 12mL / min.

[0016] In some embodiments, the gradient elution condition of one-time chromatographic elution is as follows:

[0017] 0~2min: 43% mobile phase A and 57% mobile phase B are used;

[0018] 2~20min: mobile phase A from 43% to 28% and mobile phase B from 57% to 72% are used;

[0019] 20~24min: mobile phase A from 28% to 15% and mobile phase B from 72% to 85% are used;

[0020] 25~32min: 0% mobile phase A and 100% mobile phase B are used;

[0021] 32.2~35min: 43% mobile phase A and 57% mobile phase B are used again.

[0022] Specifically, the one-time chromatographic elution of the present scheme is balanced at 0~2min, the polar and medium polar components are gradually separated at 2~20min, the mixed components containing C16:1 olaflur analogs are eluted at 20~24min, the residual strong hydrophobic impurities are eluted at 25~32min, and the chromatographic column is balanced again at 32.2~35min.

[0023] Specifically, the gradient elution condition of one-time chromatographic elution is shown in Table 1.

[0024] Table 1 Chromatography elution condition of primary chromatography elution

[0025] .

[0026] In some embodiments, the mass-to-charge ratio of the olaparib analogue with a C16:1 alkyl chain is 429.4, wherein the chemical formula of the olaparib analogue with a C16:1 alkyl chain is C 25 H 52 N2O3, wherein the alkyl chain is C16:1, and n = 7.

[0027] In other words, when the mass-to-charge ratio of the olaparib analogue with a C16:1 alkyl chain is detected by mass spectrometry to be 429.4, the eluate of the primary chromatography elution in the time period is collected.

[0028] In addition, since the olaparib analogue with a C16:1 alkyl chain is easily co-eluted with homologues of similar carbon chain length (such as C16:0) in conventional chromatography separation, affecting the purity of the product, the eluate obtained by primary elution contains various impurities and interfering tissues. In order to obtain the olaparib analogue with a C16:1 alkyl chain with high purity, the eluate needs to be further eluted.

[0029] In step S2, the primary eluate is subjected to secondary chromatography elution, and when the mass-to-charge ratio of the olaparib analogue with a C16:1 alkyl chain is detected by mass spectrometry, the secondary eluate of the secondary chromatography elution in the corresponding time period is collected.

[0030] In some embodiments, the specifications of the chromatography column Phenomenex Luna CN for secondary chromatography elution are 5um, 21.2*250mm.

[0031] In some embodiments, the sample concentration for secondary chromatography elution is 10-50 mg / mL, the injection volume is 0.5-1 mL, and the flow rate is 12 mL / min.

[0032] In some embodiments, the gradient elution condition for secondary chromatography elution is:

[0033] 0~7 minutes: 80% mobile phase A and 20% mobile phase B are used;

[0034] 7~29 minutes: mobile phase A from 80% to 72%, and mobile phase B from 20% to 28% are used;

[0035] 29.2~32 minutes: 0% mobile phase A and 100% mobile phase B are used;

[0036] 32.2~35 minutes, return to the initial condition of 80% mobile phase A and 20% mobile phase B

[0037] In some embodiments, the concentrated purified component is weighed by the differential method.

[0038] Specifically, the gradient elution conditions of the secondary chromatography elution are shown in Table Two below:

[0039] Table Two: Chromatography elution conditions of the secondary chromatography elution

[0040] .

[0041] Due to the complexity of the composition of the tallow-derived olafluor raw material, and the lack of UV and fluorescence absorption of the olafluor raw material, it is also difficult to monitor by evaporation light scattering or CAD. Therefore, the mass spectrometry guided method is used to monitor the olafluor analog of C16:1.

[0042] Specifically, the mass spectrometry conditions of the mass spectrometry are: ion source ESI+, cone hole voltage 15.0 V, ion source temperature 120°C, probe temperature 600°C, ESI source inner capillary voltage 0.80 kV, monitoring mode full scan, mass range 100-1000 Da.

[0043] In step S3, the eluent obtained in step S1 is concentrated by vacuum rotary evaporation at 50°C to obtain a concentrated component. The concentrated component is dissolved in 2% ammonia water and extracted with n-hexane. After separating the n-hexane layer, the purified component is obtained by blowing dry with nitrogen.

[0044] In addition, in order to further detect the purity of the purified component, the present scheme further includes step S4: the purified component is prepared into a sample solution with a concentration of 20-50 ug / ml, and the component is detected by liquid chromatography-QDa mass spectrometry detector, wherein the chromatography conditions are: the chromatography column is Agilent HC-C8, the mobile phase A is 10 mM NH4AC and 0.1% formic acid, B is acetonitrile, the elution mode is gradient elution, and the flow rate is 1.0 mL / min.

[0045] In some embodiments, the gradient elution conditions of the chromatography elution for purity detection are:

[0046] 0~2 minutes, using 90% of mobile phase A and 10% of mobile phase B;

[0047] 2.1~5 minutes, using 60% of mobile phase A and 40% of mobile phase B;

[0048] 5.1~25 minutes, using 40% of mobile phase A and 40% of mobile phase B;

[0049] 25.1~30 minutes, return to the initial conditions of 90% of mobile phase A and 10% of mobile phase B.

[0050] Specifically, the purity test conditions are shown in Table Three below:

[0051] Table Three Purity Test Conditions

[0052]

[0053] Compared with the prior art, the technical scheme has the following characteristics and beneficial effects:

[0054] 1. Breakthrough in synthetic raw material limitation, first synthesized C16:1 olaflur analog. This scheme uses olaflur raw material obtained from beef tallow as raw material, and obtains olaflur analog with C16:1 alkyl chain through mass spectrometry guided preparation, to overcome the defect that traditional synthesis route is limited by the commercial unavailability of (Z)-1-bromo-7-hexadecene, and compared with the total synthesis route, this method avoids complex asymmetric olefin construction steps, in line with the principles of green chemistry. Although beef tallow components are complex, but from the technical strategy, fractionation preparation technology can be used to provide an economically viable alternative solution for the preparation of special structure fluorinated surfactants.

[0055] 2. Realize accurate identification and collection of high-purity C16:1 olaflur analog. In view of the technical bottleneck of co-flow of homologues in beef tallow derivatives, through optimization of LC-MS separation conditions (mobile phase gradient, stationary phase selection) and mass spectrometry monitoring mode, high-efficiency enrichment and impurity control of target components are realized, and the product purity can meet the detection requirements (>90%), providing reliable commercial reference material for subsequent application research.

[0056] 3. Has academic value and application premise, assists in the research of olaflur. Through LC-MS technology, the key reaction nodes such as fluorination can be monitored in real time, guiding the adjustment of process parameters and improving the yield and consistency. The obtained C16:1 olaflur analog can be used to study the influence of unsaturated chain configuration on the performance of fluorinated surfactants. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 is a mass spectrum obtained by chromatographic elution condition 1.

[0058] Figure 2 is a mass spectrum obtained by chromatographic elution condition 2.

[0059] Figure 3 is a mass spectrum obtained by chromatographic elution condition 3.

[0060] Figure 4 is a mass spectrum obtained by chromatographic elution condition 4.

[0061] Figure 5 is a mass spectrum obtained by chromatographic elution condition 5.

[0062] Figure 6is the mass spectrum obtained under chromatographic elution condition 6.

[0063] Figure 7 is the mass spectrum obtained under chromatographic elution condition 7.

[0064] Figure 8 is the mass spectrum obtained under chromatographic elution condition 7. DETAILED DESCRIPTION

[0065] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art belong to the scope of protection of the present application.

[0066] Those skilled in the art should understand that in the disclosure of the present application, the terms "longitudinal", "transverse", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like indicate the orientation or positional relationship shown in the drawings, which are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, therefore the above terms cannot be understood as a limitation of the present application.

[0067] The mass spectrometry conditions mentioned in the following examples are as follows:

[0068] Waters QDa single quadrupole mass spectrometer detector is used for orafenacin raw material detection, and the specific parameters are as follows: ion source ESI+, cone hole voltage 15.0V, ion source temperature (Source Temperature) 120℃, probe temperature (Probe Temperature) 600℃.

[0069] Example 1, component analysis of orafenacin raw material:

[0070] The SI source inner capillary voltage is 0.80 kV, the monitoring mode is full scan, and the mass range is 100-1000 Da.

[0071] Take orafenacin raw material and place it under chromatographic condition 1 for elution separation and mass spectrometry analysis to obtain the mass spectrum as shown in Figure 1shown, wherein the chromatographic condition 1 is chromatographic column Unitary C18 4.6*250mm, 5um; column temperature: room temperature; sample concentration 1 mg / mL, injection volume: 40uL; mobile phase: A: 10mM NH4AC, 0.1% formic acid water, B: 10mM NH4AC, 0.1% formic acid methanol, gradient elution conditions are shown in Table Four as follows:

[0072] Table Four Gradient elution conditions of chromatographic condition 1

[0073] .

[0074] The gradient conditions are continued to be optimized using the above mobile phase and chromatographic column to obtain chromatographic condition 2, and the olafluor raw material is placed under chromatographic condition 2 for elution separation and mass spectrometry analysis to obtain a mass spectrum as shown in Figure 2 shown, wherein the chromatographic condition 1 is chromatographic column Unitary C18 4.6*250mm, 5um; column temperature: room temperature; sample concentration 1 mg / mL, injection volume: 40uL; mobile phase: A: 10mM NH4AC, 0.1% formic acid water, B: 10mM NH4AC, 0.1% formic acid methanol, gradient elution conditions are shown in Table Five as follows:

[0075] Table Five Gradient elution conditions of chromatographic condition 2

[0076]

[0077] Example Two, Preparation Method Development and Optimization of C16:1:

[0078] Referring to the analysis method of Example One and directly scaling up the chromatographic column to a preparative chromatographic column of the same type number to obtain elution condition 3, and placing the olafluor raw material under chromatographic condition 3 for elution separation and mass spectrometry analysis to obtain a mass spectrum as shown in Figure 3 shown, wherein the chromatographic condition 3 is chromatographic column Unitary C18 20*250mm, 5um; column temperature: room temperature; mobile phase: A: 10mM ammonium acetate, 0.1% formic acid water, B: methanol, gradient elution conditions are shown in Table Six as follows:

[0079] Table Six Gradient elution conditions of chromatographic condition 3

[0080] .

[0081] As shown in Figure 3 , it is found that after scaling up to a preparative LC, the peak shape is obviously broadened, causing serious cross between adjacent components, and the preparation conditions need to be further optimized to obtain chromatographic condition 4, and the olafluor raw material is placed under chromatographic condition 4 for elution separation and mass spectrometry analysis to obtain a mass spectrum as shown in Figure 4shown, wherein the chromatographic condition 4 is Exmere Exsil Pure 120 Biphenyl, 5um, 20*250mm; column temperature: room temperature; mobile phase: A: 10mM ammonium acetate, 0.1% formic acid water, B: methanol; flow rate: 12 mL / min, gradient elution conditions are shown in Table Seven as follows:

[0082] Table Seven Gradient elution conditions of chromatographic condition 4

[0083] .

[0084] Further purification study of the olaparib analog of C16:1:

[0085] For the components obtained in Example Two, the components were vacuum concentrated and then subjected to secondary HPLC optimization:

[0086] Firstly, based on Exmere Exsil Pure 120 Biphenyl, 5um, 20*250mm column, the mobile phase system was replaced to optimize the separation design chromatographic condition 5, and the concentrated components were subjected to purification separation under chromatographic condition 5 to obtain the mass spectrum as shown in Figure 5 , wherein the chromatographic condition 5 is as follows: column: Exmere Exsil Pure 120 Biphenyl, 5um, 20*250mm, column temperature: room temperature; mobile phase: A: 0.1% formic acid water, B: acetonitrile, gradient elution conditions are shown in Table Eight as follows:

[0087] Table Eight Gradient elution conditions of chromatographic condition 5

[0088] .

[0089] As shown in Figure 5 , after analysis, the main component MS 429.3 contains MS 417.3 component, which does not meet the expected purification target.

[0090] This scheme needs to be further optimized, and the column is replaced and chromatographic condition 6 is designed, and the concentrated components are subjected to purification separation under chromatographic condition 6 to obtain the mass spectrum as shown in Figure 6 , wherein the chromatographic condition 6 is as follows: column: Phenomenex Luna CN, 5um, 21.2*250mm, column temperature: room temperature; mobile phase: A: 0.1% FA water, B: ACN, gradient elution conditions are shown in Table Nine as follows:

[0091] Table Nine Gradient elution conditions of chromatographic condition 6

[0092] .

[0093] As Figure 6 shown, MS: 417.3 was found to still co-elute with the target peak MS: 429.3 upon analysis, so further optimization of separation conditions was required.

[0094] This solution continues to use Phenomenex Luna CN, 5um, 21.2*250mm, changes the mobile phase system and designs the chromatographic condition 7, and places the olafluor raw material under the chromatographic condition 7 for purification and separation to obtain a mass spectrum as shown in Figure 7 , wherein the chromatographic condition 7 is as follows: Phenomenex Luna CN, 5um, 21.2*250mm, column temperature: room temperature; mobile phase: A: 0.1% formic acid water, B: methanol, gradient elution conditions are as shown in the following table ten:

[0095] Table ten Gradient elution conditions of chromatographic condition 7

[0096] .

[0097] As Figure 7 can be seen, this solution collects by time condition segmentation, and then obtains some high-purity MS 429.3 components. Further, the purity of the component with mass-to-charge ratio 429.4 of the C16:1 olafluor analog collected is detected by mass spectrometry, and the test conditions are shown in Table three, and the mass spectrum of the C16:1 olafluor analog is shown in Figure 8 .

[0098] Those skilled in the art should understand that each technical feature of the above embodiments can be combined arbitrarily, and in order to make the description concise, each technical feature in the above embodiments has not been described all possible combinations, however, as long as the combination of these technical features does not exist contradictory, it should be considered as the scope of the present application.

[0099] The above embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the present application. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, some modifications and improvements can be made, which are all within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.

Claims

1. A method of preparing an olafluor analogue having an alkyl chain of C16:1, characterised in that, Comprising the following steps: S1: performing a first chromatographic elution on the olafluor raw material, and collecting the eluent corresponding to the mass spectrum signal when the mass spectrometer detects a mass-to-charge ratio mass spectrum signal of the C16:1 olafluor analog; wherein the chromatographic conditions of the first chromatographic elution are: the chromatographic column is Exmere Exsil Pure 120 Biphenyl, the column temperature is room temperature, the mobile phase A1 is 10 mM NH4AC and 0.1% formic acid water, the mobile phase B1 is methanol, and the elution mode is gradient elution; wherein the gradient elution conditions of the first chromatographic elution are: 0-2 minutes: 43% of the mobile phase A1 and 57% of the mobile phase B1; 2-20 minutes: the mobile phase A1 from 43% to 28% and the mobile phase B1 from 57% to 72%; 20-24 minutes: the mobile phase A1 from 28% to 15% and the mobile phase B1 from 72% to 85%; 25-32 minutes: 0% of the mobile phase A1 and 100% of the mobile phase B1; 32.2-35 minutes: return to 43% of the mobile phase A1 and 57% of the mobile phase B1; wherein the olafluor raw material is obtained by fluorination of C4-C18 mixed fatty acids in beef tallow, wherein the C16-C18 mixed fatty acids are first extracted and purified from beef tallow by saponification and acidification, and then an active amide is introduced by activation, amidation and fluorination, and the olafluor raw material is obtained by post-treatment, and the olafluor raw material is a mixture of olafluor analogs with different carbon chains and degrees of saturation; S2: performing a second preparative chromatographic elution on the first eluent, and collecting the second eluent corresponding to the mass spectrum signal when the mass spectrometer detects a mass-to-charge ratio mass spectrum signal of the C16:1 olafluor analog; wherein the chromatographic conditions of the second chromatographic elution are: the chromatographic column is Phenomenex Luna CN, the mobile phase A2 is 0.1% formic acid water, the mobile phase B2 is methanol, and the elution mode is gradient elution; wherein the gradient elution conditions of the second chromatographic elution are: 0-8 minutes: 77% of the mobile phase A2 and 23% of the mobile phase B2; 8-25 minutes: the mobile phase A2 from 77% to 63% and the mobile phase B2 from 23% to 27%; 25-29 minutes: the mobile phase A2 from 63% to 30% and the mobile phase B2 from 27% to 70%; 29-29.2 minutes: the mobile phase A2 from 30% to 0% and the mobile phase B2 from 70% to 100%; 29.2-32 minutes: 0% of the mobile phase A2 and 100% of the mobile phase B2; 32-32.2 minutes: the mobile phase A2 from 0% to 77% and the mobile phase B2 from 100% to 23%; 32.2-35 minutes: 77% of the mobile phase A2 and 23% of the mobile phase B2; S3: concentrating the second eluent to obtain a concentrated component, dissolving the concentrated component with ammonia water, and then extracting with n-hexane to obtain a purified component, The mass to charge ratio of the olafluor analogue with an alkyl chain of C16:1 is 429.4 and the chemical formula of the olafluor analogue with an alkyl chain of C16:1 is C 25 H 52 N2O3.

2. The process for preparing an olafluor analogue with an alkyl chain of C16:1 according to claim 1, characterized in that, Comprising: S4: The purified component was allocated into sample solution with a concentration of 20-50 ug / ml, and liquid chromatography-QDa mass spectrometry detector was used for purity detection, wherein the chromatographic conditions were as follows: the chromatographic column was Agilent HC-C8, the mobile phase A3 was 10 mM NH4AC and 0.1% formic acid, the mobile phase B3 was acetonitrile, and the elution mode was gradient elution.

3. The method of preparing an olafluor analogue with an alkyl chain of C16:1 according to claim 1, characterized in that, The specification of the chromatographic column for one-time chromatography elution was 5um, 20*250mm.

4. The process for preparing an olafluor analogue with an alkyl chain of C16:1 according to claim 1, characterized in that, The concentrated component was obtained by vacuum rotary evaporation concentration treatment at 50 DEG C, the concentrated component was dissolved by 2% ammonia water, then extracted by n-hexane, the n-hexane layer was separated, and then dried by nitrogen to obtain the purified component.

5. The process of claim 1 for preparing an olafluor analogue with an alkyl chain of C16:1, characterized in that, The specification of the chromatographic column for two-time chromatography elution was 21.2*250mm, 5um.

6. The method of preparing an olafluor analogue with an alkyl chain of C16:1 according to claim 1, characterized in that, The sample concentration for two-time chromatography elution was 10-50 mg / mL, the injection volume was 0.5-1mL, and the flow rate was 12 mL / min.

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