Liquid chromatography-mass spectrometry detection method of phosphoramidite monomer

Abstract

The invention relates to the technical field of analysis, in particular to a liquid chromatography-mass spectrometry detection method of a phosphoramidite monomer. According to the liquid chromatography-mass spectrometry detection method for the phosphoramidite monomer, the molecular weight of the phosphoramidite monomer in a sample solution is detected by adopting reversed-phase high performance liquid chromatography-mass spectrometry; wherein the elution gradient of the liquid chromatography is as follows: the volume ratio of a mobile phase A to a mobile phase B is (29-31): (69-71) for 0 min; the volume ratio is 0: 100 after 20 min; the volume ratio is 0: 100; the volume ratio is (29 to 31): (69 to 71); the volume ratio is (29-31): (69-71); the mass spectrum conditions are as follows: the sheath gas flow velocity is 11-13; the auxiliary airflow speed is 0; the S-lens level is 34 to 36; the capillary temperature is 225-235 DEG C; and the spraying voltage is 2.7 to 2.9 kv. The method has the advantages of short analysis time, high sensitivity, high accuracy and high resolution.

Description

Technical Field

[0001] The invention relates to the field of analytical technology, in particular to a liquid chromatography-mass spectrometry (LC-MS) chromatography detection method for phosphoramidite monomers. Background Art

[0002] Nucleic acid drugs refer to drugs that can use small nucleic acid molecules such as siRNA, miRNA and antisense nucleic acids to specifically silence the expression of disease genes in order to cure specific diseases. Compared with other drugs targeting proteins, small nucleic acid drugs have the advantages of high specificity, high efficiency and long-term effectiveness. Small nucleic acid drugs are mainly divided into three types according to their mechanism of action: siRNA, miRNA and antisense nucleic acids. Among them, siRNA refers to double-stranded RNA or hairpin structure RNA that is cut by endonucleases to form siRNA, which generates sense strands and antisense strands under the action of helicases.

[0003] Nucleoside monomers are key materials for small nucleic acid drugs. The phosphoramidite monomers for synthesizing DNA are mainly composed of bases, deoxyribose, 5'-DMT, and 2-cyanoethyl and diisopropylamine groups at the 3' end. In the phosphoramidite monomers for RNA, 2'-OH needs to be protected to prevent cross-reaction between 2'-OH and 5'-OH during solid phase synthesis. The protecting group is generally TBDMS.

[0004] Since phosphoramidites form oligonucleotide chains by coupling with each other, each time a specific phosphoramidite is added, impurities specific to each phosphoramidite will be present throughout the synthesis process. Therefore, high-purity phosphoramidites are of great significance in the process of synthesizing oligonucleotides, and the purity of phosphoramidites needs to be tested.

[0005] In view of this, this invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide a liquid chromatography-mass spectrometry (LC-MS) chromatographic detection method for phosphoramidite monomers, which has the advantages of short analysis time, less sample required for circulation, high sensitivity, high accuracy, high resolution identification and quantification of target analytes, and can effectively control the quality of phosphoramidite.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0008] The present invention provides a liquid chromatography-mass spectrometry detection method for phosphoramidite monomers, comprising the following steps:

[0009] Reverse phase high performance liquid chromatography-mass spectrometry was used to detect the molecular weight of phosphoramidite monomers in the sample solution;

[0010] Wherein, the liquid chromatography conditions for the detection are as follows: the mobile phase includes mobile phase A and mobile phase B; elution gradient: 0min, the volume ratio of mobile phase A to mobile phase B is 29-31:69-71; 20min, the volume ratio of mobile phase A to mobile phase B is 0:100; 23min, the volume ratio of mobile phase A to mobile phase B is 0:100; 23.1min, the volume ratio of mobile phase A to mobile phase B is 29-31:69-71; 25min, the volume ratio of mobile phase A to mobile phase B is 29-31:69-71;

[0011] The mass spectrometry conditions for the detection are as follows: the sheath gas flow rate is 11-13; the auxiliary gas flow rate is 0; the S-lens level is 34-36; the capillary temperature is 225-235° C.; and the spray voltage is 2.7-2.9 kV.

[0012] Furthermore, the solvent of the sample solution includes acetonitrile.

[0013] Furthermore, the concentration of the sample solution is 0.1-0.5 mg / mL.

[0014] Furthermore, the mobile phase A comprises an aqueous solution of ammonium acetate with a concentration of 9 to 11 mmol / L; and the mobile phase B comprises mass spectrometry grade acetonitrile.

[0015] Furthermore, the liquid chromatography includes using a Thermo Vanquish high performance liquid chromatograph.

[0016] Furthermore, the chromatographic column of the liquid chromatography comprises a Water Acquily BEH C18 chromatographic column, the length of the chromatographic column is 95 to 105 mm, the inner diameter is 2.0 to 2.2 mm, and the filler particle size is 1.6 to 1.8 μm.

[0017] Furthermore, the mass spectrometry includes using a Q Exactive Plus mass spectrometer.

[0018] Furthermore, the mass spectrometry conditions for the detection are: negative ion full scan mode; mass scan range is 500-2000.

[0019] Furthermore, the mobile phase A and the mobile phase B are filtered through a microporous membrane and ultrasonically degassed in sequence, and then the sample solution is detected by reversed-phase high performance liquid chromatography-mass spectrometry.

[0020] Furthermore, the pore size of the microporous membrane is 0.20 to 0.25 μm;

[0021] And / or, the ultrasonic degassing time is 10 to 20 minutes.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The liquid chromatography-mass spectrometry (LC-MS) chromatography detection method for phosphoramidite monomers of the present invention has the advantages of short analysis time, high resolution, high precision, high sensitivity and good specificity, good reproducibility, small error and stable analysis result, good detection effect and simple operation, and can achieve the purpose of effectively controlling the quality of phosphoramidite. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 The method of Example 1 was used in the present invention to detect the liquid phase spectrum of 5-Me-DMT-2'-O-MOE-C(BZ)-CEPhosphoramidite.

[0026] Figure 2 The liquid phase spectrum of 5-Me-DMT-2'-O-MOE-C(BZ)-CEPhosphoramidite was detected by the method of Comparative Example 1 in the present invention.

[0027] Figure 3 The present invention adopts the method of Comparative Example 2 to detect the liquid phase spectrum of 5-Me-DMT-2'-O-MOE-C(BZ)-CEPhosphoramidite.

[0028] Figure 4 This is a graph showing the results of detecting the DMT-2'-O-MOE-A(BZ)-CE Phosphoramidite sample using the method of Example 1 of the present invention.

[0029] Figure 5 This is a graph showing the results of testing the DMT-2'-O-MOE-A(BZ)-CE Phosphoramidite sample using the method of Comparative Example 3 of the present invention.

[0030] Figure 6 This is a graph showing the results of the DMT-2'-O-MOE-A(BZ)-CE Phosphoramidite sample produced by the method of Comparative Example 4 of the present invention.

[0031] Figure 7 This is another result diagram of the detection of DMT-2'-O-MOE-A(BZ)-CE Phosphoramidite sample using the method of Example 1 of the present invention.

[0032] Figure 8 This is a graph showing the results of detecting a DMT-2'-O-MOE-T-CE Phosphoramidite sample using the method of Example 1 of the present invention.

[0033] Fig. 9 This is a graph showing the results of testing a 5-Me-DMT-2'-O-MOE-C(BZ)-CEPhosphoramidite sample using the method of Example 1 of the present invention.

[0034] Fig.10 This is a graph showing the results of detecting a DMT-2'-O-MOE-G(iBu)-CEPhosphoramidite sample using the method of Example 1 of the present invention. DETAILED DESCRIPTION

[0035] The technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments, but it will be understood by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0036] In some embodiments of the present invention, a liquid chromatography-mass spectrometry detection method for phosphoramidite monomers is provided, comprising the following steps:

[0037] Reverse phase high performance liquid chromatography-mass spectrometry was used to detect the molecular weight of phosphoramidite monomers in the sample solution;

[0038] The liquid chromatography conditions for the test are as follows: the mobile phase includes mobile phase A and mobile phase B; the elution gradient is as follows: 0 min, the volume ratio of mobile phase A to mobile phase B is 29-31:69-71; 20 min, the volume ratio of mobile phase A to mobile phase B is 0:100; 23 min, the volume ratio of mobile phase A to mobile phase B is 0:100; 23.1 min, the volume ratio of mobile phase A to mobile phase B is 29-31:69-71; 25 min, the volume ratio of mobile phase A and The volume ratio of mobile phase B is 29-31:69-71; preferably, the elution gradient is: 0min, the volume ratio of mobile phase A and mobile phase B is 30:70; 20min, the volume ratio of mobile phase A and mobile phase B is 0:100; 23min, the volume ratio of mobile phase A and mobile phase B is 0:100; 23.1min, the volume ratio of mobile phase A and mobile phase B is 30:70; 25min, the volume ratio of mobile phase A and mobile phase B is 30:70;

[0039] The mass spectrometry conditions for detection are: sheath gas flow rate of 11-13; auxiliary gas flow rate of 0; S-lens level of 34-36; capillary temperature of 225-235°C; spray voltage of 2.7-2.9 kv; preferably, the sheath gas flow rate is 12; the auxiliary gas flow rate is 0; the S-lens level is 35; the capillary temperature is 230°C; and the spray voltage is 2.8 kv.

[0040] The detection method of the present invention has a short analysis time, and only takes about 30 minutes from sample pretreatment to completion of result analysis.

[0041] The detection method of the present invention has high resolution and can achieve better separation effect by selecting the type and ratio of the fixed mobile phase.

[0042] The detection method of the invention has high analytical precision, high sensitivity and good specificity.

[0043] The detection method of the present invention has good reproducibility and small error. The molecular weight detection error can be controlled within 10 ppm, and the analysis result is stable.

[0044] The detection method of the invention has good detection effect on nucleic acid monomers (phosphoramidite monomers) and related impurities.

[0045] The detection method of the present invention is simple to operate, the analysis is fully automated, and the detection is completed by an instrument. No matter whether one has an experimental basis or not, the operation can be performed as long as one has undergone operation training.

[0046] The present invention adopts reverse phase liquid chromatography-mass spectrometry to analyze and identify the phosphoramidite monomer, thereby achieving the purpose of effectively controlling the quality of the phosphoramidite.

[0047] In some embodiments of the present invention, the solvent of the sample solution comprises acetonitrile.

[0048] In some embodiments of the present invention, the concentration of the sample solution is 0.1-0.5 mg / mL; typically but not limiting, for example, the concentration of the sample solution can be 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL or a range consisting of any two thereof.

[0049] In some embodiments of the present invention, a sample is weighed and placed in a volumetric flask, and a solvent is added to dissolve the sample and then the volume is fixed to obtain a sample solution.

[0050] In some embodiments of the present invention, mobile phase A comprises an aqueous solution of ammonium acetate at a concentration of 9 to 11 mmol / L; mobile phase B comprises mass spectrometry grade acetonitrile; preferably, mobile phase A comprises an aqueous solution of ammonium acetate at a concentration of 10 mmol / L.

[0051] In some embodiments of the invention, liquid chromatography comprises using a Thermo Vanquish high performance liquid chromatograph.

[0052] In some embodiments of the present invention, the chromatographic column of the liquid chromatography comprises a Water Acquily BEH C18 chromatographic column having a length of 95 to 105 mm, an inner diameter of 2.0 to 2.2 mm, and a filler particle size of 1.6 to 1.8 μm; preferably, the length of the chromatographic column is 100 mm, the inner diameter is 2.1 mm, and the filler particle size is 1.7 μm.

[0053] In some embodiments of the invention, mass spectrometry comprises using a Q Exactive Plus mass spectrometer.

[0054] In some embodiments of the present invention, the mass spectrometry conditions for detection are: negative ion full scan mode; mass scan range is 500-2000.

[0055] In some embodiments of the present invention, after the mobile phase A and the mobile phase B are filtered through a microporous membrane and ultrasonically degassed in sequence, the sample solution is detected by reversed-phase high performance liquid chromatography-mass spectrometry.

[0056] In some embodiments of the present invention, the pore size of the microporous membrane is 0.20-0.25 μm; the pore size of the microporous membrane may be 0.20 μm, 0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm or a range of any two thereof.

[0057] In some embodiments of the present invention, the ultrasonic degassing time is 10 to 20 minutes; typically but not limiting, for example, the ultrasonic degassing time can be 10 minutes, 15 minutes, 20 minutes or a range of any two thereof.

[0058] In some embodiments of the present invention, the LC-MS detection method of phosphoramidite monomers specifically comprises the following steps:

[0059] Weigh the sample and place it in a volumetric flask, add solvent to dissolve it and then make up the volume to obtain a sample solution;

[0060] After the mobile phase A and the mobile phase B were filtered through a microporous membrane with a pore size of 0.20 to 0.25 μm and ultrasonically degassed for 10 to 20 min, the sample solution was detected by reversed-phase high performance liquid chromatography-mass spectrometry;

[0061] Among them, the liquid chromatography conditions for detection are as follows:

[0062] The mobile phase includes mobile phase A and mobile phase B;

[0063] Mobile phase A was an aqueous solution of ammonium acetate with a concentration of 10 mmol / L; mobile phase B was mass spectrometry grade acetonitrile; elution gradient: 0 min, the volume ratio of mobile phase A to mobile phase B was 30:70; 20 min, the volume ratio of mobile phase A to mobile phase B was 0:100; 23 min, the volume ratio of mobile phase A to mobile phase B was 0:100; 23.1 min, the volume ratio of mobile phase A to mobile phase B was 30:70; 25 min, the volume ratio of mobile phase A to mobile phase B was 30:70;

[0064] Thermo Vanquish high performance liquid chromatograph;

[0065] Water Acquily BEH C18 column, column length 100 mm, inner diameter 2.1 mm, filler particle size 1.7 μm;

[0066] The mass spectrometry conditions for detection are as follows:

[0067] The sheath gas flow rate was 12;

[0068] The auxiliary gas flow rate is 0;

[0069] S-lens level is 35;

[0070] The capillary temperature is 230°C;

[0071] The spray voltage was 2.8 kV;

[0072] Q Exactive Plus mass spectrometer;

[0073] Negative ion full scan mode;

[0074] The mass scan range is 500-2000.

[0075] Example 1

[0076] The LC-MS detection method of phosphoramidite monomers provided in this embodiment comprises the following steps:

[0077] instrument:

[0078] Thermo Vanquish high-performance liquid chromatograph with UV detector and full-wavelength detection;

[0079] Water Acquily BEH C18 column (100 × 2.1 mm, 1.7 μm);

[0080] Q Exactive Plus mass spectrometer;

[0081] Ultrasonic degassing device.

[0082] Sample solution preparation:

[0083] Accurately weigh 1 mg of sample and place it in a 10 mL volumetric flask. Add acetonitrile to dissolve it and then make up the volume to obtain a sample solution.

[0084] Detection:

[0085] After mobile phase A and mobile phase B were filtered through a microporous membrane with a pore size of 0.22 μm, ultrasonic degassing was performed for 15 min, and then the sample solution was detected by reversed-phase high performance liquid chromatography-mass spectrometry; the injection volume of the sample solution was 0.2 μg, and after the peak appeared, the experimental data was compared with the calculated reference mass number;

[0086] Among them, diluent / blank: acetonitrile;

[0087] Liquid chromatography conditions:

[0088] Mobile phase: 10 mmol / L ammonium acetate aqueous solution, mass spectrometry grade acetonitrile;

[0089] Thermo Vanquish high performance liquid chromatograph;

[0090] Water Acquily BEH C18 column, column length 100 mm, inner diameter 2.1 mm, filler particle size 1.7 μm;

[0091] The elution gradient is shown in Table 1:

[0092] Table 1

[0093] time Ammonium acetate aqueous solution Mass spectrometry grade acetonitrile 0min 30％ 70％ 20min 0％ 100％ 23min 0％ 100％ 23.1min 30％ 70％ 25min 30％ 70％

[0094] Mass spectrometry conditions:

[0095] The sheath gas flow rate was 12;

[0096] The auxiliary gas flow rate is 0;

[0097] S-lens level is 35;

[0098] The capillary temperature is 230°C;

[0099] The spray voltage was 2.8 kV;

[0100] Q Exactive Plus mass spectrometer;

[0101] Negative ion full scan mode;

[0102] The mass scan range is 500-2000.

[0103] Comparative Example 1

[0104] The LC-MS detection method for phosphoramidite monomers provided in this comparative example refers to Example 1, except that the elution gradient is as shown in Table 2:

[0105] Table 2

[0106] time Ammonium acetate aqueous solution Mass spectrometry grade acetonitrile 0min 40％ 60％ 12min 10％ 90％ 12.1min 40％ 60％ 15min 40％ 60％

[0107] Comparative Example 2

[0108] The LC-MS detection method for phosphoramidite monomers provided in this comparative example refers to Example 1, except that the elution gradient is as shown in Table 3:

[0109] Table 3

[0110]

[0111]

[0112] Comparative Example 3

[0113] The LC-MS detection method of phosphoramidite monomer provided in this comparative example refers to Example 1, except that the mass spectrometry conditions are: sheath gas flow rate of 8; auxiliary gas flow rate of 0; S-lens level of 15; capillary temperature of 210°C; and spray voltage of 2.1 kV.

[0114] Comparative Example 4

[0115] The LC-MS detection method of phosphoramidite monomer provided in this comparative example refers to Example 1, except that the mass spectrometry conditions are: sheath gas flow rate is 20; auxiliary gas flow rate is 0; S-lens level is 40; capillary temperature is 300°C; and spray voltage is 3.0 kV.

[0116] Test example

[0117] The liquid phase spectra of 5-Me-DMT-2'-O-MOE-C(BZ)-CEPhosphoramidite were detected by the methods of Example 1, Comparative Example 1 and Comparative Example 2, respectively. Figure 1 , Figure 2 and Figure 3 shown.

[0118] from Figure 1 to Figure 3 It can be seen that the method of Comparative Example 1 does not produce a main peak in the elution gradient; the method of Comparative Example 2 does not have a good elution gradient for separating impurities.

[0119] The DMT-2'-O-MOE-A(BZ)-CEPhosphoramidite samples were tested using the methods of Example 1, Comparative Example 3 and Comparative Example 4, and the results are as follows: Figure 4 , Figure 5 and Figure 6 shown.

[0120] from Figures 4 to 6 It can be seen that the mass spectrometry conditions in the detection method of the present invention are better than the mass spectrometry conditions in Comparative Examples 3 and 4 in detecting nucleic acid monomers and related impurities.

[0121] The samples DMT-2'-O-MOE-A(BZ)-CE Phosphoramidite, DMT-2'-O-MOE-T-CE Phosphoramidite, 5-Me-DMT-2'-O-MOE-C(BZ)-CE Phosphoramidite and DMT-2'-O-MOE-G(iBu)-CE Phosphoramidite were tested by the method of Example 1. The results are as follows: Figure 7 , Figure 8 and Fig. 9 As shown; Figure 7 for Figure 4 A partial enlarged view of .

[0122] from Figure 7 It can be seen that the results of the DMT-2'-O-MOE-A(BZ)-CE Phosphoramidite sample analysis showed that the main peak molecular weight was 930.40, and the molecular weight detection error was within 5ppm. Figure 8 It can be seen that the results of the DMT-2'-O-MOE-T-CEPhosphoramidite sample analysis and detection show that the main peak molecular weight is 817.36, and the molecular weight detection error is within 5ppm; Fig. 9It can be seen that the results of the analysis and detection of 5-Me-DMT-2'-O-MOE-C(BZ)-CE Phosphoramidite sample show that the main peak molecular weight is 920.40, and the molecular weight detection error is within 5ppm; Fig.10 It can be seen that the results of the DMT-2'-O-MOE-G(iBu)-CE Phosphoramidite sample analysis test show that the main peak molecular weight is 912.41, and the molecular weight detection error is within 5ppm; it can be seen that the molecular weight detection error of the present invention is small, the sample can be accurately and effectively analyzed and identified, and it has the advantages of high resolution, high sensitivity, and strong interference elimination ability.

[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A liquid chromatography-mass spectrometry detection method for phosphoramidite monomers, characterized in that: The steps include: Reverse phase high performance liquid chromatography-mass spectrometry was used to detect the molecular weight of phosphoramidite monomers in the sample solution; Wherein, the liquid chromatography conditions for the detection are as follows: the mobile phase includes mobile phase A and mobile phase B; elution gradient: 0min, the volume ratio of mobile phase A to mobile phase B is 29-31:69-71; 20min, the volume ratio of mobile phase A to mobile phase B is 0:100; 23min, the volume ratio of mobile phase A to mobile phase B is 0:100; 23.1min, the volume ratio of mobile phase A to mobile phase B is 29-31:69-71; 25min, the volume ratio of mobile phase A to mobile phase B is 29-31:69-71; The mass spectrometry conditions for the detection are as follows: the sheath gas flow rate is 11-13; the auxiliary gas flow rate is 0; the S-lens level is 34-36; the capillary temperature is 225-235° C.; and the spray voltage is 2.7-2.9 kV.

2. The liquid chromatography-mass spectrometry detection method of phosphoramidite monomers according to claim 1, characterized in that: The solvent of the sample solution includes acetonitrile.

3. The liquid chromatography-mass spectrometry detection method of phosphoramidite monomer according to claim 1, characterized in that: The concentration of the sample solution is 0.1-0.5 mg / mL.

4. The liquid chromatography-mass spectrometry detection method of phosphoramidite monomers according to claim 1, characterized in that: The mobile phase A comprises an aqueous solution of ammonium acetate with a concentration of 9 to 11 mmol / L; and the mobile phase B comprises mass spectrometry grade acetonitrile.

5. The liquid chromatography-mass spectrometry detection method of phosphoramidite monomers according to claim 1, characterized in that: The liquid chromatography includes using a Thermo Vanquish high performance liquid chromatograph.

6. The liquid chromatography-mass spectrometry detection method of phosphoramidite monomers according to claim 1, characterized in that: The chromatographic column of the liquid chromatography comprises a Water Acquily BEH C18 chromatographic column, the length of the chromatographic column is 95-105 mm, the inner diameter is 2.0-2.2 mm, and the filler particle size is 1.6-1.8 μm.

7. The LC-MS detection method of phosphoramidite monomers according to claim 1, characterized in that: The mass spectrometry includes the use of a Q Exactive Plus mass spectrometer.

8. The liquid chromatography-mass spectrometry detection method of phosphoramidite monomers according to claim 1, characterized in that: The mass spectrometry conditions for the detection are: negative ion full scan mode; mass scan range is 500-2000.

9. The liquid chromatography-mass spectrometry detection method of phosphoramidite monomers according to claim 1, characterized in that: After the mobile phase A and the mobile phase B are filtered through a microporous membrane and degassed by ultrasound in sequence, the sample solution is detected by reversed-phase high performance liquid chromatography-mass spectrometry.

10. The liquid chromatography-mass spectrometry detection method of phosphoramidite monomers according to claim 9, characterized in that: The pore size of the microporous membrane is 0.20 to 0.25 μm; And / or, the ultrasonic degassing time is 10 to 20 minutes.

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