CN111707742A - Method for simultaneously detecting multiple components in rhodiola rosea by HPLC - Google Patents
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- CN111707742A CN111707742A CN202010507633.7A CN202010507633A CN111707742A CN 111707742 A CN111707742 A CN 111707742A CN 202010507633 A CN202010507633 A CN 202010507633A CN 111707742 A CN111707742 A CN 111707742A
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- 238000000034 method Methods 0.000 title claims abstract description 23
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 22
- 244000042430 Rhodiola rosea Species 0.000 title claims abstract description 19
- 235000003713 Rhodiola rosea Nutrition 0.000 title claims abstract description 19
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 78
- 239000000243 solution Substances 0.000 claims abstract description 15
- 239000000126 substance Substances 0.000 claims abstract description 15
- 238000001514 detection method Methods 0.000 claims abstract description 13
- RINHYCZCUGCZAJ-UHAHJPEESA-N (2s,3r,4s,5s,6r)-2-[(e)-3-phenylprop-2-enoxy]-6-[[(2s,3r,4s,5s)-3,4,5-trihydroxyoxan-2-yl]oxymethyl]oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)CO[C@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC\C=C\C=2C=CC=CC=2)O1 RINHYCZCUGCZAJ-UHAHJPEESA-N 0.000 claims abstract description 11
- JJYVNURTNGHITH-UHFFFAOYSA-N rosavin Natural products OC1COC(OCC2OC(OC(=O)C=Cc3ccccc3)C(O)C(O)C2O)C(O)C1O JJYVNURTNGHITH-UHFFFAOYSA-N 0.000 claims abstract description 11
- IEBFEMIXXHIISM-UHFFFAOYSA-N rozarin Natural products OC1C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OCC=CC=2C=CC=CC=2)O1 IEBFEMIXXHIISM-UHFFFAOYSA-N 0.000 claims abstract description 11
- RINHYCZCUGCZAJ-UHFFFAOYSA-N rozavin Natural products OC1C(O)C(O)COC1OCC1C(O)C(O)C(O)C(OCC=CC=2C=CC=CC=2)O1 RINHYCZCUGCZAJ-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000012085 test solution Substances 0.000 claims abstract description 11
- ILRCGYURZSFMEG-UHFFFAOYSA-N Salidroside Natural products OC1C(O)C(O)C(CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-UHFFFAOYSA-N 0.000 claims abstract description 8
- ILRCGYURZSFMEG-RQICVUQASA-N salidroside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-RQICVUQASA-N 0.000 claims abstract description 8
- 239000012528 membrane Substances 0.000 claims abstract description 6
- 239000002904 solvent Substances 0.000 claims abstract description 5
- 238000002156 mixing Methods 0.000 claims abstract description 4
- 238000004445 quantitative analysis Methods 0.000 claims abstract description 4
- 238000010835 comparative analysis Methods 0.000 claims abstract description 3
- 238000001914 filtration Methods 0.000 claims abstract description 3
- 239000012488 sample solution Substances 0.000 claims abstract 4
- 239000000523 sample Substances 0.000 claims abstract 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 24
- 230000003247 decreasing effect Effects 0.000 claims description 7
- 241001165494 Rhodiola Species 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 5
- 239000002994 raw material Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 238000002137 ultrasound extraction Methods 0.000 claims description 2
- 235000000640 Rosa roxburghii Nutrition 0.000 abstract description 2
- 240000002547 Rosa roxburghii Species 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract description 2
- 239000003814 drug Substances 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000010298 pulverizing process Methods 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- -1 rosalin Chemical compound 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a method for simultaneously detecting multiple components in rhodiola rosea by HPLC, which comprises the following steps: extracting radix Rhodiolae pulverized material with methanol as solvent to obtain sample solution; respectively preparing a salidroside standard substance solution, a rosavin standard substance solution and a rosavin standard substance solution, fixing the volume in a volumetric flask by using methanol, mixing the single standard substances in the same amount to prepare a mixed standard, and filtering the mixed standard through 0.4-0.5 ul of organic filter membrane; performing quantitative analysis on the mixed standard and the sample solution after the organic filter membrane is filtered by using HPLC at double wavelengths respectively; the column temperature is 26-30 ℃; the sample amount is 10 ul; detection wavelength: 0 → 8min set 276nm, 8 → 20min set 255 nm; and S4, obtaining a map of the test solution and a map of the mixed standard according to the HPLC analysis result, and performing comparative analysis. The invention provides a method for simultaneously detecting the content of salidroside, rosavin and rosa roxburghii in rhodiola rosea.
Description
Technical Field
The invention relates to the field of detection of traditional Chinese medicinal materials, in particular to a method for simultaneously detecting multiple components in rhodiola rosea by using HPLC (high performance liquid chromatography).
Background
Rhodiola rosea, another name: rhodiola rosea is a perennial herb with the height of 10-20cm, thick and strong root, conical shape, meat quality, brown yellow, most fibrous root at the root neck, short, thick and cylindrical rhizome, and most of scaly leaves arranged in an imbricate shape. The ecological floating bed grows in a high and cold non-pollution area with the altitude of 1800 and 2500 meters, and the growth environment is severe, so that the ecological floating bed has strong vitality and special adaptability. Can be used as a medicine, can tonify qi, clear away the lung-heat, benefit intelligence and nourish the heart, and is a traditional Chinese medicine with wide effect. It also has good skin caring effect, and can be used as skin care product or edible product.
Because the rhodiola plants in the market have wide sources, the varieties used in various places are different, the rhodiola components from different sources have larger differences, the existing method for detecting various components in the rhodiola has single detection component, the detection process is complex, and the reliability of the detection result is not high, so that a method capable of quickly and accurately detecting various components in the rhodiola is necessary to be developed.
Disclosure of Invention
In view of the above problems, the present invention aims to provide a method for simultaneously detecting multiple components in rhodiola rosea by HPLC, by which the detection of multiple components in rhodiola rosea can be efficiently and rapidly achieved.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for simultaneously detecting multiple components in rhodiola rosea by HPLC comprises the following steps:
s1, extracting the ground rhodiola root raw material with methanol as a solvent to obtain a test solution.
S2, preparing a salidroside standard substance solution, a rosavin standard substance solution and a rosavin standard substance solution respectively, fixing the volume in a volumetric flask by using methanol, mixing the single standard substances in the same amount to prepare a mixed standard, and filtering the mixed standard through 0.4-0.5 ul of organic filter membrane.
And S3, respectively carrying out quantitative analysis on the mixed standard and the test solution which pass through the organic filter membrane by using HPLC at double wavelengths.
The chromatographic conditions are as follows: the HPLC chromatographic column is a ZORBAX-SB-Aq column, and the mobile phase is as follows: methanol (A), 0.3% -0.5% acetic acid solution (B), the initial proportion A: b is 58-62: 38-42. The elution procedure was: 0 → 8min, A is increased from 58-62% to 82-88%, B is decreased from 38-42% to 12-18%; 8 → 20min, wherein A is increased from 82-88% to 92-98%, and B is decreased from 12-18% to 2-8%; 20 → 22min, A: b, keeping the ratio of 58-62: 38-42 unchanged. Column temperature: 26-30 ℃; the sample amount is 10 ul; detection wavelength: 0 → 8min set 276nm, 8 → 20min set 255 nm.
And S4, obtaining a map of the test solution and a map of the mixed standard according to the HPLC analysis result, and performing comparative analysis.
Preferably, the chromatographic conditions in step S2 are:
the HPLC column is a ZORBAX-SB-Aq column (5um, 4.6 mm. times.250 mm), and the mobile phase is: methanol (a), 0.4% acetic acid solution (B), starting ratio a: b is 60: 40; the elution procedure was: 0 → 8min, A rises from 60% to 85%, B falls from 40% to 15%; 8 → 20min, A rises from 85% to 95%, B drops from 15% to 5%; 20 → 22min, A: b, keeping the ratio of 60:40 unchanged.
Further, the methanol in the step S2 is a methanol solvent with a concentration of 60%.
Further, in step S1, the preparation of the test solution includes the following steps:
p1, pulverizing the rhodiola rosea raw material, and carrying out ultrasonic extraction for 25-40 min by using 50-70% methanol.
And P2, transferring the ultrasonic liquid into a volumetric flask, and performing constant volume with 50-70% methanol to obtain a test solution.
The invention has the following beneficial effects: the method for simultaneously detecting the content of salidroside, rosavin and rosa roxburghii in the rhodiola rosea is simple to operate, high in reliability of measurement results, few in experiment times, efficient, accurate and wide in applicability.
Drawings
FIG. 1 is a mixed standard map.
FIG. 2 is a test solution map.
Detailed Description
The invention is further described with reference to the following drawings and detailed description.
A method for simultaneously detecting multiple components in rhodiola rosea by HPLC comprises the following steps:
1. pulverizing radix Rhodiolae, accurately weighing, extracting with 60% methanol under ultrasound for 30min, transferring the ultrasound solution into a volumetric flask, and diluting to constant volume with 60% methanol to obtain the test solution.
2. Chromatographic conditions are as follows:
the chromatographic column is as follows: ZORBAX-SB-Aq 5um 4.6X 250 mm.
The mobile phase is as follows: methanol, 0.4% acetic acid, initial ratio 60:40, 0-8min methanol is increased from 60% to 85%, 0.4% acetic acid is decreased from 40% to 15%, 8-20min methanol is increased from 85% to 95%, 0.4% phosphoric acid is decreased from 15% to 5%, 20-22min methanol is decreased from 95% to 60%, 0.4% acetic acid is increased from 5% to 40%, 22-25min is maintained for 60: the 40 ratio was constant and the column was equilibrated with gradient changes as in table 1.
| Time/min | Methanol% | 0.4% acetic acid% |
| 0 | 60 | 40 |
| 8 | 85 | 15 |
| 20 | 95 | 5 |
| 22 | 60 | 40 |
| 25 | 60 | 40 |
Table 1: gradient change of mobile phase
Detection time: 25min, wherein the first 20min is gradient elution time, and the last 5min is post-operation, equilibrium chromatographic column time.
The column temperature was: 28 +/-0.2 degrees.
Sample introduction: 10 ul.
Wavelength: setting 276nm in 0-8min and 255nm in 8-20 min.
Preparing a standard substance solution, accurately weighing salidroside, rosalin, rosavin and rosavin respectively to 0.1mg, and dissolving in 60% methanol to a brown volumetric flask with a constant volume of 100 ml. Taking 1ml of each single standard substance, and mixing together to obtain four substance mixed standards. The mixed sample was passed through 0.45ul of organic filter, and 6 needles were continuously injected under the above chromatographic conditions, and the peak area results are shown in Table 2.
| Salidroside | Luosulin (Louiselin) | Luoxinwei medicine | Collateral flowing | |
| Area of a needle peak | 68.6 | 610.4 | 601.3 | 796.3 |
| Area of two needles | 68.5 | 611.6 | 607 | 800.9 |
| Area of three needles | 69.7 | 615.1 | 602.7 | 798.3 |
| Area of four needles | 70 | 613.8 | 603 | 801 |
| Area of five needles | 70.1 | 610.8 | 604.8 | 795.9 |
| Peak area of six needles | 70.5 | 611 | 606 | 799.5 |
| RSD％ | 1.1912 | 0.3094 | 0.359 | 0.2773 |
Table 2: peak area data results
The individual component retention time results are shown in table 3:
table 3: retention time results of ingredients
The final mixed standard map is shown in figure 1, and the test sample map is shown in figure 2. The detection result shows that the peak area of each component detected by the method is stable, the retention time is stable, the method has good reproducibility, the obtained chromatogram base line is stable, the chromatographic peak has good shape, the base line separation is achieved, and the method is suitable for simultaneous quantitative analysis of rhodiola rosea effective components, namely salidroside, rosavin, rosalin and rosalin.
While the invention has been particularly shown and described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (4)
1. A method for simultaneously detecting multiple components in rhodiola rosea by HPLC is characterized by comprising the following steps:
s1, extracting the ground rhodiola root raw material by using methanol as a solvent to prepare a test solution;
s2, preparing a salidroside standard substance solution, a rosavin standard substance solution and a rosavin standard substance solution respectively, fixing the volume in a volumetric flask by using methanol, mixing the single standard substances in the same amount to prepare a mixed standard, and filtering the mixed standard through 0.4-0.5 ul of an organic filter membrane;
s3, carrying out quantitative analysis on the mixed standard and the sample solution after the organic filter membrane is filtered by using HPLC at double wavelengths respectively;
the chromatographic conditions are as follows: the HPLC chromatographic column is a ZORBAX-SB-Aq column, and the mobile phase is as follows: methanol (A), 0.3% -0.5% acetic acid solution (B), the initial proportion A: b is 58-62: 38-42; the elution procedure was: 0 → 8min, A is increased from 58-62% to 82-88%, B is decreased from 38-42% to 12-18%; 8 → 20min, wherein A is increased from 82-88% to 92-98%, and B is decreased from 12-18% to 2-8%; 20 → 22min, A: b, keeping the ratio of 58-62: 38-42 unchanged; column temperature: 26-30 ℃; the sample amount is 10 ul; detection wavelength: 0 → 8min set 276nm, 8 → 20min set 255 nm;
and S4, obtaining a map of the test solution and a map of the mixed standard according to the HPLC analysis result, and performing comparative analysis.
2. The method for simultaneous HPLC detection of multiple components in rhodiola rosea according to claim 1, wherein: the chromatographic conditions in step S2 are:
the HPLC column is a ZORBAX-SB-Aq column (5um, 4.6 mm. times.250 mm), and the mobile phase is: methanol (a), 0.4% acetic acid solution (B), starting ratio a: b is 60: 40; the elution procedure was: 0 → 8min, A rises from 60% to 85%, B falls from 40% to 15%; 8 → 20min, A rises from 85% to 95%, B drops from 15% to 5%; 20 → 22min, A: b, keeping the ratio of 60:40 unchanged.
3. The method for simultaneous HPLC detection of multiple components in rhodiola rosea according to claim 1, wherein: the methanol in step S2 is a methanol solvent with a concentration of 60%.
4. The method for simultaneous HPLC detection of multiple components in rhodiola rosea according to claim 1, wherein: in step S1, the preparation of the sample solution includes the following steps:
p1, crushing the rhodiola rosea raw material, and carrying out ultrasonic extraction for 25-40 min by using 50-70% methanol;
and P2, transferring the ultrasonic liquid into a volumetric flask, and performing constant volume with 50-70% methanol to obtain a test solution.
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