CN114184712A - Method for detecting content of free steroid impurities in combined estrogen tablets - Google Patents
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Abstract
The invention provides a detection method for the content of free steroid impurities in a combined estrogen tablet, which comprises the following steps: performing high performance liquid chromatography analysis on a combined estrogen tablet solution to be detected and a steroid impurity standard reference solution, and calculating by using an external standard method according to an analysis result to obtain the content of free steroid impurities in the combined estrogen tablet; the free steroid impurities include any one or a combination of at least two of estrone, equilin or 17 alpha-dihydroequilin. The method provided by the invention has the advantages of good specificity, high precision, high accuracy, quick analysis, simple and convenient operation and important application value.
Description
Technical Field
The invention belongs to the field of pharmaceutical analysis, relates to a detection method for the content of free steroid impurities in a combined estrogen tablet, and particularly relates to a high performance liquid chromatography detection method for the content of free steroid impurities in the combined estrogen tablet.
Background
The combined estrogen tablet is an estrogen medicine and is mainly used for treating female climacteric syndrome. Women in menopause are easy to have vasomotor symptoms due to lack of estrogen, and when the vasomotor symptoms exist, the symptoms are manifested as hot flashes and hot flashes, and the symptoms can occur several times every day, thus seriously affecting the work and life of patients. Some estrogen can be supplemented properly by taking the combined estrogen tablets, which is beneficial for relieving the vasomotor symptoms.
The combined estrogen tablet contains estrogen mixture extracted from urine of pregnant mare, and is water soluble estrogen sodium sulfate mixture. Degradation of the sodium salt of estrogen sulphate produces a number of free steroidal impurities, mainly including estrone, equilenin and 17 α -dihydroequilenin. In the existing method for determining the content of the combined estrogen tablet, the impurities of non-free estrogen and free estrogen can not be distinguished, so that the content detection is inaccurate. Therefore, there is a need to develop a detection method for accurately determining the content of free steroid impurities in a combined estrogen tablet. The existing detection method for the content of free steroid impurities in the medicine is a gas chromatography method, is complex in operation, various in steps, not ideal in repeatability and precision, not suitable for being combined with estrogen tablets, and has certain application limitation.
Therefore, how to rapidly, efficiently, simply and economically measure the content of free steroid impurities in the combined estrogen tablets becomes a problem to be solved in the field.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for detecting the content of free steroid impurities in a combined estrogen tablet, and particularly provides a high performance liquid chromatography method for detecting the content of free steroid impurities in the combined estrogen tablet.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for detecting the content of free steroid impurities in a combined estrogen tablet, wherein the method comprises: performing high performance liquid chromatography analysis on a combined estrogen tablet solution to be detected and a steroid impurity standard reference solution, and calculating by using an external standard method according to an analysis result to obtain the content of free steroid impurities in the combined estrogen tablet;
the free steroid impurities include any one or a combination of at least two of estrone, equilin or 17 alpha-dihydroequilin.
The method provided by the invention is suitable for detecting any one single impurity component of estrone, equilin or 17 alpha-dihydroequilin in the combined estrone tablets, is also suitable for detecting the combination of estrone and equilin, the combination of equilin and 17 alpha-dihydroequilin, the combination of estrone and 17 alpha-dihydroequilin and is also suitable for simultaneously detecting estrone, equilin and 17 alpha-dihydroequilin.
The detection method provided by the invention has the advantages of good specificity, high precision, high accuracy, quick analysis, simple and convenient operation and important application value.
Preferably, the preparation method of the conjugated estrogen tablet solution to be detected comprises the following steps: and (3) dissolving the combined estrogen tablets to be detected by using the diluent, centrifuging, and collecting supernatant to obtain the compound estrogen tablets.
The preparation method of the steroid impurity standard control solution comprises the following steps: and (4) dissolving the steroid impurity standard substance by using the diluent, and diluting to obtain the steroid impurity standard substance.
Preferably, the rotation speed of the centrifugation is 2000-.
Preferably, the concentration of conjugated estrogens in the conjugated estrogen tablet solution to be tested is 0.11-0.13mg/mL, for example, 0.11mg/mL, 0.112mg/mL, 0.114mg/mL, 0.116mg/mL, 0.118mg/mL, 0.12mg/mL, 0.122mg/mL, 0.124mg/mL, 0.126mg/mL, 0.128mg/mL, 0.13mg/mL, etc., the concentration of the steroid impurities in the standard control solution of the steroid impurities is 1.4-1.7 mug/mL, for example, 1.4. mu.g/mL, 1.42. mu.g/mL, 1.45. mu.g/mL, 1.47. mu.g/mL, 1.5. mu.g/mL, 1.52. mu.g/mL, 1.55. mu.g/mL, 1.57. mu.g/mL, 1.6. mu.g/mL, 1.62. mu.g/mL, 1.65. mu.g/mL, 1.67. mu.g/mL, 1.7. mu.g/mL, etc.
Preferably, the diluent comprises 80% to 95% aqueous methanol, e.g. 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% etc., said 80% to 95% being the volume percentage of methanol in solution.
Preferably, the chromatographic column used in the high performance liquid chromatography is an octaalkyl bonded silica gel chromatographic column.
Preferably, the chromatographic column has a size of 4.6mm x 250mm, 5 μm.
Preferably, the mobile phase used in the high performance liquid chromatography comprises a phase A and a phase B, wherein the phase A is a 30% acetonitrile aqueous solution, and the phase B is a 95% acetonitrile aqueous solution. The 30% and 95% refer to the volume percentage of acetonitrile in the solution.
Preferably, a gradient elution mode is adopted in the high performance liquid chromatography.
Preferably, the procedure of the gradient elution is:
in 0min, the volume of the phase A accounts for 100 percent, and the volume of the phase B accounts for 0 percent; then changing to 30min at constant speed, wherein the volume of the phase A accounts for 70-80%, and the volume of the phase B accounts for 20-30%; then changing to 33min at constant speed, wherein the volume of the phase A accounts for 5-15%, and the volume of the phase B accounts for 85-95%; in 33-37min, keeping the volume ratio of the phase A to be 5-15% and the volume ratio of the phase B to be 85-95%; then, changing the temperature to 40min at a constant speed, wherein the volume of the phase A accounts for 100 percent, and the volume of the phase B accounts for 0 percent; finally, within 40-45min, the volume ratio of the phase A is constant and 100 percent and the volume ratio of the phase B is constant and 0 percent.
Specific values of 70 to 80% are, for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, etc.
Specific values of 20 to 30% are, for example, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, and the like.
Specific values of 5 to 15% are, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, etc.
Specific values of 85 to 95% are, for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, and the like.
Preferably, the flow rate in the HPLC analysis is 1.4-1.6mL/min, such as 1.4mL/min, 1.42mL/min, 1.45mL/min, 1.47mL/min, 1.5mL/min, 1.52mL/min, 1.55mL/min, 1.57mL/min, 1.6mL/min, and the like.
Preferably, the sample volume in the HPLC analysis is 15-25. mu.L, such as 15. mu.L, 16. mu.L, 17. mu.L, 18. mu.L, 19. mu.L, 20. mu.L, 21. mu.L, 22. mu.L, 23. mu.L, 24. mu.L, 25. mu.L, etc.
Preferably, the column temperature in the HPLC analysis is set to 28-32 deg.C, such as 28 deg.C, 28.5 deg.C, 29 deg.C, 29.5 deg.C, 30 deg.C, 30.5 deg.C, 31 deg.C, 31.5 deg.C, 32 deg.C, etc.
The recitation of numerical ranges herein includes not only the above-recited values, but also any values between any of the above-recited numerical ranges not recited, and for brevity and clarity, is not intended to be exhaustive of the specific values encompassed within the range.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a detection method for the content of free steroid impurities in a combined estrogen tablet, which comprises the following steps: performing high performance liquid chromatography analysis on a combined estrogen tablet solution to be detected and a steroid impurity standard reference solution, and calculating by using an external standard method according to an analysis result to obtain the content of free steroid impurities in the combined estrogen tablet; the free steroid impurities include any one or a combination of at least two of estrone, equilin or 17 alpha-dihydroequilin. The method provided by the invention has the advantages of good specificity, high precision (RSD% is below 3.2), high accuracy (recovery rate is 87.4% -96.3%), quick analysis, simple operation and important application value.
Drawings
FIG. 1 is a chromatogram of a test drug solution of example 1, in which the 1-17. alpha. -dihydroequilenin peak, the 2-equilenin peak, and the 3-estrone peak are shown.
FIG. 2 is a chromatogram of a standard control solution of steroidal impurities of example 1, in which the peak of 1-17 α -dihydroequilenin, the peak of 2-equilenin, and the peak of 3-estrone are shown.
FIG. 3 is a chromatogram of a blank solution.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Specific information on the conjugated estrogen tablets related to the following examples is as follows:
specification: 0.3mg, manufacturer: south Tongli sub-pharmaceutical industry, product batch number: 0253007.
example 1
The embodiment provides a method for detecting the content of free steroid impurities in a combined estrogen tablet, which comprises the following specific steps:
(1) preparing a to-be-detected drug solution:
taking not less than ten combined estrogen tablets, and grinding into fine powder. Approximately 1.2mg of conjugated estrogens in the powder was placed in a 50mL centrifuge tube, 10.0mL 95% (v/v) methanol in water was added, the tube was closed, vortexed for 5min and sonicated for 5 min. Centrifuge at 4000rpm for 10 min. The supernatant was centrifuged again at 15000rpm for 5min until the solution was clear. And (4) loading the supernatant into a liquid phase sample injection vial to obtain the drug solution to be detected.
(2) Preparing a steroid impurity standard control solution:
weighing estrone, equilenin and 17 alpha-dihydroequilenin standard substances, 2.6mg respectively, placing in a 50mL measuring flask, dissolving with 95% (v/v) methanol water solution, diluting to scale, shaking up, and making into control stock solutions with each impurity concentration of 52 mu g/mL. Precisely measuring 3.0mL, placing in a 100mL measuring flask, adding 95% (v/v) methanol water solution to constant volume to scale, shaking up, and preparing into steroid impurity standard reference solution with each impurity concentration of 1.56 μ g/mL.
(3) High performance liquid chromatography
Instruments and conditions:
american waters E2690 chromatography system and Empower workstation, chromatography column: vortight Zorbax RX-C8, inner diameter 4.6mm, column length 250mm, filler particle size 5 μm; flow rate: 1.5 mL/min; sample introduction volume: 20 mu L of the solution; column temperature: 30 ℃; operating time: 45 min; detection wavelength: 205nm (ultraviolet detector);
mobile phase A: acetonitrile: water 3:7 (v/v);
mobile phase B: acetonitrile: water 95:5 (v/v);
gradient elution procedure:
and respectively carrying out high performance liquid chromatography analysis on the steroid impurity standard reference solution and the drug solution to be detected, recording a chromatogram, and calculating the content of free steroid impurities.
Example 2
The embodiment provides a method for detecting the content of free steroid impurities in a combined estrogen tablet, which comprises the following specific steps:
(1) preparing a to-be-detected drug solution:
taking not less than ten combined estrogen tablets, and grinding into fine powder. Approximately 1.1mg of conjugated estrogens in the powder is placed in a 50mL centrifuge tube, 10.0mL 80% (v/v) methanol in water is added, the tube is closed, vortexed for 5min and then sonicated for 5 min. Centrifuge at 5000rpm for 10 min. The supernatant was centrifuged again at 18000rpm for 5min until the solution was clear. And (4) loading the supernatant into a liquid phase sample injection vial to obtain the drug solution to be detected.
(2) Preparing a steroid impurity standard control solution:
weighing estrone, equilenin and 17 alpha-dihydroequilenin standard substances, 2.4mg respectively, placing in a 50mL measuring flask respectively, dissolving with 80% (v/v) methanol water solution, diluting to scale, shaking up, and making into control stock solutions with impurity concentrations of 48 mu g/mL. Precisely measuring 3.0mL, placing in a 100mL measuring flask, adding 95% (v/v) methanol water solution to constant volume to scale, shaking up, and preparing into steroid impurity standard reference solution with each impurity concentration of 1.44 μ g/mL.
(3) High performance liquid chromatography
Instruments and conditions:
american waters E2690 chromatography system and Empower workstation, chromatography column: vortight Zorbax RX-C8, inner diameter 4.6mm, column length 250mm, filler particle size 5 μm; flow rate: 1.4 mL/min; sample introduction volume: 18 mu L of the solution; column temperature: 28 ℃; operating time: 45 min; detection wavelength: 205nm (ultraviolet detector);
mobile phase A: acetonitrile: water 3:7 (v/v);
mobile phase B: acetonitrile: water 95:5 (v/v);
gradient elution procedure:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 30 | 70 | 30 |
| 33 | 15 | 85 |
| 37 | 15 | 85 |
| 40 | 100 | 0 |
| 45 | 100 | 0 |
And respectively carrying out high performance liquid chromatography analysis on the steroid impurity standard reference solution and the drug solution to be detected, recording a chromatogram, and calculating the content of free steroid impurities.
Example 3
The embodiment provides a method for detecting the content of free steroid impurities in a combined estrogen tablet, which comprises the following specific steps:
(1) preparing a to-be-detected drug solution:
taking not less than ten combined estrogen tablets, and grinding into fine powder. Approximately 1.3mg of conjugated estrogens in powder is placed in a 50mL centrifuge tube, 10.0mL 90% (v/v) aqueous methanol is added, the tube is closed, vortexed for 5min and sonicated for 5 min. Centrifuge at 3000rpm for 15 min. The supernatant was centrifuged again at 16000rpm for 5min until the solution was clear. And (4) loading the supernatant into a liquid phase sample injection vial to obtain the drug solution to be detected.
(2) Preparing a steroid impurity standard control solution:
weighing estrone, equilenin and 17 alpha-dihydroequilenin standard substances, 2.8mg respectively, placing in a 50mL measuring flask respectively, dissolving with 90% (v/v) methanol water solution, diluting to scale, shaking up, and making into control stock solutions with impurity concentrations of 56 μ g/mL. Precisely measuring 3.0mL, placing in a 100mL measuring flask, adding 95% (v/v) methanol water solution to constant volume to scale, shaking up, and preparing into steroid impurity standard reference solution with each impurity concentration of 1.68 μ g/mL.
(3) High performance liquid chromatography
Instruments and conditions:
american waters E2690 chromatography system and Empower workstation, chromatography column: vortight Zorbax RX-C8, inner diameter 4.6mm, column length 250mm, filler particle size 5 μm; flow rate: 1.6 mL/min; sample introduction volume: 22 mu L of the solution; column temperature: at 32 ℃; operating time: 45 min; detection wavelength: 205nm (ultraviolet detector);
mobile phase A: acetonitrile: water 3:7 (v/v);
mobile phase B: acetonitrile: water 95:5 (v/v);
gradient elution procedure:
and respectively carrying out high performance liquid chromatography analysis on the steroid impurity standard reference solution and the drug solution to be detected, recording a chromatogram, and calculating the content of free steroid impurities.
Example 4
This example provides a method of measuring the content of free steroidal impurities in a combined estrogen tablet which differs from example 1 only in that the column was replaced with Zorbax RX-C18, 4.6 x 250mm, 5 μm. All other conditions remained unchanged.
Example 5
This example provides a method for detecting the content of free steroidal impurities in a combined estrogen tablet, which differs from example 1 only in that mobile phase a is replaced by 30% (v/v) aqueous methanol and mobile phase B is replaced by 95% (v/v) aqueous methanol. Other conditions were unchanged.
Example 6
This example provides a method for detecting the content of free steroidal impurities in a combined estrogen tablet, which differs from example 1 only in that mobile phase a is replaced by 30% (v/v) aqueous ethanol and mobile phase B is replaced by 95% (v/v) aqueous ethanol. Other conditions were unchanged.
Example 7
This example provides a method for detecting the content of free steroidal impurities in a combined estrogen tablet, which differs from example 1 only in that the ratio of "mobile phase a: acetonitrile: water 3:7 (v/v); mobile phase B: acetonitrile: water 95:5(v/v) "was replaced with" mobile phase a: acetonitrile: water 35:65 (v/v); mobile phase B: acetonitrile: water 98:2(v/v) ". Other conditions were unchanged.
Example 8
This example provides a method for detecting the content of free steroidal impurities in a combined estrogen tablet, which differs from example 1 only in that the ratio of "mobile phase a: acetonitrile: water 3:7 (v/v); mobile phase B: acetonitrile: water 95:5(v/v) "was replaced with" mobile phase a: acetonitrile: 25:75(v/v) water; mobile phase B: acetonitrile: water 90:10(v/v) ". Other conditions were unchanged.
Example 9
This example provides a method for detecting the content of free steroidal impurities in a combined estrogen tablet, which differs from example 1 only in that the gradient elution procedure of liquid chromatography is changed to:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 30 | 65 | 35 |
| 33 | 5 | 95 |
| 37 | 5 | 95 |
| 40 | 100 | 0 |
| 45 | 100 | 0 |
Other conditions remained unchanged.
Example 10
This example provides a method for detecting the content of free steroidal impurities in a combined estrogen tablet, which differs from example 1 only in that the gradient elution procedure of liquid chromatography is changed to:
other conditions remained unchanged.
Example 11
This example provides a method for detecting the content of free steroidal impurities in a combined estrogen tablet, which differs from example 1 only in that the elution procedure of liquid chromatography is changed to isocratic elution: mobile phase A: 75%, mobile phase B: 25 percent. All other conditions remained unchanged.
Test example
The analysis conditions in the above examples were examined methodically, including feasibility, specificity, accuracy (recovery), and precision, as follows:
chromatograms obtained by analyzing the drug solution to be detected and the standard control solution of steroid impurities by high performance liquid chromatography in example 1 are respectively shown in fig. 1 and fig. 2. The 95% (v/v) methanol aqueous solution is vortexed for 5min, sonicated for 5min, centrifuged at 4000rpm for 10min, centrifuged at 15000rpm for 5min, and then loaded into a liquid phase sample injection vial as a blank solution, and the chromatogram obtained by high performance liquid chromatography analysis is shown in FIG. 3. The ordinate in the figure represents the time (min), in which the 1-17 α -dihydroequilenin peak, the 2-equilenin peak and the 3-estrone peak. The results show that: the retention time of the 17 alpha-dihydroequilenin peak is about 11.5min, the retention time of the equilenin peak is about 13.9min, the retention time of the equilenin peak is about 14.9min, the peaks do not interfere with each other, the peak shape is good, the separation effect is good, and the blank solution has no interference near the retention time of each peak, which shows that the method is fully feasible and has excellent specificity.
Accuracy (recovery)
Three recovery rate solutions of each impurity at a concentration of 0.3%, three recovery rate solutions of each impurity at a concentration of 3.4%, and six recovery rate solutions of each impurity at a concentration of 1.4% (12 recovery rate solutions for each impurity) were prepared, respectively, and the contents thereof were measured, and the measured values were compared with theoretical values to calculate the recovery rates, and the results are shown in tables 1-1, tables 1-2, and tables 1-3.
TABLE 1-1
Tables 1 to 2
Tables 1 to 3
Precision degree
6 parts of recovery rate sample drug solutions (combined estrogen tablets) are prepared in parallel respectively, the drug solutions are tested by the same analyst under the same condition as much as possible, and the relative standard deviation (RSD%) of the content of free steroid impurities in the 6 parts of the drug solutions to be tested is calculated. The results are shown in table 2:
TABLE 2
From the data in tables 1 and 2, it can be seen that: by adopting the method for detecting the content of the free steroid impurities in the combined estrogen tablets, the recovery rate result of each impurity at each concentration level is good (87.4% -96.3%), which indicates that the method has higher accuracy. In the precision test, 6 samples are prepared in parallel for detection, and the relative standard deviation of the obtained results is below 3.2 and less than the limit of 10.0, which indicates that the precision of the method is good.
The applicant states that the present invention is illustrated by the above examples of the method for detecting the content of free steroid impurities in a combined estrogen tablet according to the present invention, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be implemented by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Claims (10)
1. A method for detecting the content of free steroid impurities in a combined estrogen tablet is characterized by comprising the following steps: performing high performance liquid chromatography analysis on a combined estrogen tablet solution to be detected and a steroid impurity standard reference solution, and calculating by using an external standard method according to an analysis result to obtain the content of free steroid impurities in the combined estrogen tablet;
the free steroid impurities include any one or a combination of at least two of estrone, equilin or 17 alpha-dihydroequilin.
2. The assay of claim 1 wherein said test conjugated estrogen tablet solution is prepared by a method comprising: dissolving the combined estrogen tablet to be detected by using a diluent, centrifuging, and collecting supernatant to obtain the compound estrogen tablet;
the preparation method of the steroid impurity standard control solution comprises the following steps: dissolving the steroid impurity standard substance by using a diluent, and diluting to obtain the steroid impurity standard substance;
preferably, the rotation speed of the centrifugation is 2000-.
3. The assay of claim 1 or claim 2 wherein the conjugated estrogens in the conjugated estrogen tablet solution to be assayed is from 0.11 to 0.13mg/mL and the concentration of steroid impurities in the standard control solution of steroid impurities is from 1.4 to 1.7 μ g/mL.
4. The assay of claim 2 or claim 3 wherein the diluent comprises an 80% to 95% aqueous solution of methanol.
5. The detection method according to any one of claims 1 to 4, wherein the column used in the high performance liquid chromatography is an octaalkyl bonded silica gel column.
6. The detection method according to claim 5, wherein the chromatographic column has a size of 4.6mm x 250mm, 5 μm.
7. The detection method according to any one of claims 1 to 6, wherein the mobile phase used in the high performance liquid chromatography comprises a phase A and a phase B, wherein the phase A is a 30% acetonitrile aqueous solution, and the phase B is a 95% acetonitrile aqueous solution.
8. The detection method according to any one of claims 1 to 7, wherein a gradient elution method is used in the high performance liquid chromatography;
preferably, the procedure of the gradient elution is:
in 0min, the volume of the phase A accounts for 100 percent, and the volume of the phase B accounts for 0 percent; then changing to 30min at constant speed, wherein the volume of the phase A accounts for 70-80%, and the volume of the phase B accounts for 20-30%; then changing to 33min at constant speed, wherein the volume of the phase A accounts for 5-15%, and the volume of the phase B accounts for 85-95%; in 33-37min, keeping the volume ratio of the phase A to be 5-15% and the volume ratio of the phase B to be 85-95%; then, changing the temperature to 40min at a constant speed, wherein the volume of the phase A accounts for 100 percent, and the volume of the phase B accounts for 0 percent; finally, within 40-45min, the volume ratio of the phase A is constant and 100 percent and the volume ratio of the phase B is constant and 0 percent.
9. The assay of any one of claims 1-8, wherein the flow rate in the high performance liquid chromatography assay is 1.4-1.6 mL/min.
10. The detection method according to any one of claims 1 to 9, wherein the sample volume in the high performance liquid chromatography is 15 to 25 μ L;
preferably, the column temperature in the high performance liquid chromatography is set to 28-32 ℃.
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