CN116577436A - HPLC analysis method suitable for continuous measurement of hyperin and quercetin system - Google Patents
HPLC analysis method suitable for continuous measurement of hyperin and quercetin system Download PDFInfo
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- CN116577436A CN116577436A CN202310633893.2A CN202310633893A CN116577436A CN 116577436 A CN116577436 A CN 116577436A CN 202310633893 A CN202310633893 A CN 202310633893A CN 116577436 A CN116577436 A CN 116577436A
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- quercetin
- hyperin
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- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 title claims abstract description 264
- OVSQVDMCBVZWGM-SJWGPRHPSA-N Hyperin Natural products O[C@H]1[C@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OVSQVDMCBVZWGM-SJWGPRHPSA-N 0.000 title claims abstract description 134
- OVSQVDMCBVZWGM-DTGCRPNFSA-N quercetin 3-O-beta-D-galactopyranoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OVSQVDMCBVZWGM-DTGCRPNFSA-N 0.000 title claims abstract description 134
- FVQOMEDMFUMIMO-UHFFFAOYSA-N Hyperosid Natural products OC1C(O)C(O)C(CO)OC1OC1C(=O)C2=C(O)C=C(O)C=C2OC1C1=CC=C(O)C(O)=C1 FVQOMEDMFUMIMO-UHFFFAOYSA-N 0.000 title claims abstract description 133
- BBFYUPYFXSSMNV-UHFFFAOYSA-N quercetin-7-o-galactoside Natural products OC1C(O)C(O)C(CO)OC1OC1=CC(O)=C2C(=O)C(O)=C(C=3C=C(O)C(O)=CC=3)OC2=C1 BBFYUPYFXSSMNV-UHFFFAOYSA-N 0.000 title claims abstract description 133
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 title claims abstract description 132
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 title claims abstract description 132
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 title claims abstract description 132
- 235000005875 quercetin Nutrition 0.000 title claims abstract description 132
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- 238000000034 method Methods 0.000 title claims abstract description 49
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 46
- 238000005259 measurement Methods 0.000 title claims description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 72
- 238000004458 analytical method Methods 0.000 claims abstract description 51
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- 230000000052 comparative effect Effects 0.000 description 10
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 8
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- 239000012452 mother liquor Substances 0.000 description 7
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 206010011224 Cough Diseases 0.000 description 2
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- 235000006510 Nelumbo pentapetala Nutrition 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 230000036772 blood pressure Effects 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
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- XUDNWQSXPROHLK-OACYRQNASA-N 2-phenyl-3-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C(C=2C=CC=CC=2)OC2=CC=CC=C2C1=O XUDNWQSXPROHLK-OACYRQNASA-N 0.000 description 1
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- 239000008346 aqueous phase Substances 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
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- 239000011668 ascorbic acid Substances 0.000 description 1
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000002934 diuretic Substances 0.000 description 1
- 230000001882 diuretic effect Effects 0.000 description 1
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- 238000009776 industrial production Methods 0.000 description 1
- UWKQSNNFCGGAFS-XIFFEERXSA-N irinotecan Chemical compound C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 UWKQSNNFCGGAFS-XIFFEERXSA-N 0.000 description 1
- 229960004768 irinotecan Drugs 0.000 description 1
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- 230000014759 maintenance of location Effects 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 208000026435 phlegm Diseases 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000004801 process automation Methods 0.000 description 1
- 230000002633 protecting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- BPYKBJYZLASLKL-LKDYQKJYSA-N quercetin-3-O-beta-D-galactopyranoside Natural products CO[C@H]1O[C@@H](OC2=C(Oc3cc(O)cc(O)c3C2=O)c4ccc(O)c(O)c4)[C@H](O)[C@@H](O)[C@H]1O BPYKBJYZLASLKL-LKDYQKJYSA-N 0.000 description 1
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- 230000002048 spasmolytic effect Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
Classifications
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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
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- 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
- G01N30/60—Construction of the column
- G01N30/6052—Construction of the column body
-
- 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
- G01N30/86—Signal analysis
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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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The application relates to an HPLC analysis method suitable for continuously measuring a hyperin and quercetin system, which is characterized in that: the first step: setting liquid chromatography conditions; and a second step of: preparing a mobile phase for analysis, wherein; mobile phase A is ultrapure water; mobile phase B is methanol; and a third step of: setting the analysis program as isocratic elution and eluting concentration: mobile phase a: 20-40%, mobile phase B: 60-80%, flow rate: 0.5-2 mL/min; fourth step: preparing a plurality of solutions to be tested containing hyperin and quercetin samples; etc. The HPLC analysis method suitable for the system for continuously measuring the hyperin and the quercetin is simple to operate, saves a great deal of time cost and economic cost when continuously measuring the hyperin and the quercetin, greatly simplifies the complexity of operation and has good analysis effect. In addition, the sample containing the two substances can be measured, and the concentration of the two substances can be accurately calibrated at the same time.
Description
Technical Field
The application belongs to the field of analytical chemistry, and particularly relates to a High Performance Liquid Chromatography (HPLC) analysis method suitable for a hyperin and quercetin system.
Background
Hyperin, also known as quercetin-3-O-beta-D-galactopyranoside. Belongs to flavonol glycoside compounds, and has the property of being easily dissolved in ethanol, methanol, acetone and pyridine and being stable under normal conditions. Hyperin is widely distributed in various plants, such as fruits and whole plants of Hypericaceae, rosaceae, campanulaceae, labiatae, ericaceae, convolvulaceae, guttiferae, leguminosae, and Celastraceae. Has various physiological activities such as anti-inflammatory, spasmolytic, diuretic, cough relieving, blood pressure reducing, cholesterol reducing, protein assimilation, local and central pain relieving, heart and cerebral vessel protecting effects, etc., and is an important natural product. In recent years, the development of hyperin for treating depression, hepatitis B and other diseases has become a hotspot of research at home and abroad.
Quercetin, also known as quercetin, is dissolved in glacial acetic acid, and the alkaline aqueous solution is yellow and is almost insoluble in water. Can be used as medicine, has good effects of eliminating phlegm, relieving cough, and relieving asthma. In addition, it has effects of lowering blood pressure, enhancing capillary resistance, reducing capillary fragility, reducing blood lipid, dilating coronary artery, and increasing coronary blood flow. Can be used for treating chronic bronchitis. It also has adjuvant therapeutic effect on coronary heart disease and hypertension. The metal chelating agent can be used as an antioxidant for grease and ascorbic acid. Because the quercetin widely exists in plants, has wide sources and low cost, has the effects, can be used as a medicament and can also be used as a green feed additive for replacing antibiotics to be applied to livestock production.
In the biosynthesis process of hyperin, quercetin is a precursor substance thereof, so that the presence of quercetin is simultaneously caused during the detection of hyperin, and the detection of hyperin is affected by quercetin. In the prior art, a plurality of reports about a single substance analysis method exist, but few reports about a method for simultaneously analyzing two substances exist. In the prior art document HPLC method for simultaneously measuring the content of hyperin and quercetin in lotus plumule, 80% methanol is adopted for dissolution and ultrasonic wave is matched for dissolving a sample, and then methanol is adopted: acetonitrile: 0.01mol/L phosphoric acid = 35:10: 55. The method is used for eluting hyperin for about 6min and quercetin for about 15 min. The whole analysis procedure was 30min. The hyperin is linear at 0.05028-0.5531 mug and quercetin is linear at 0.04244-0.4668 mug. Therefore, the HPLC analysis method in the prior art has the defects of complicated analysis process, overlong analysis program time and difficulty in subsequent environment-friendly treatment due to the use of various organic solvents.
Therefore, research and search for a more efficient HPLC analysis method, which has universality on hyperoside and quercetin systems, can simplify the analysis process, shorten the analysis time, improve the analysis efficiency, provide a more accurate standard curve with wider measurement range for the content measurement of hyperoside and quercetin in samples with unknown concentrations, and further realize the continuous quantification of the content of hyperoside and quercetin in a plurality of samples with unknown concentrations.
Disclosure of Invention
Problems to be solved by the application
Aiming at the problems existing in the prior art, one of the purposes of the application is to unify the chromatographic column for analyzing hyperin and quercetin with the chromatographic column for analyzing peptides, and solve the problem that the time cost is wasted because the chromatographic column needs to be replaced when continuously analyzing and measuring the peptides, hyperin and quercetin samples.
It is still another object of the present application to solve the problem of achieving analysis of hyperin and quercetin without the addition of peak shape improving agents such as acids and salts.
The application further aims to solve the problem that the hyperoside and the quercetin sample can be accurately quantified simultaneously while being continuously analyzed by an HPLC method under the same system.
Still another object of the present application is to solve the problem of shortening the analysis time while achieving complete separation of hyperin and quercetin using conventionally used mobile phases and using a binary pump.
Still another object of the present application is to solve the problem of large errors caused by dilution of samples due to the narrow concentration ranges in which hyperin and quercetin are measurable in the prior art.
Furthermore, it is another object of the present application to solve the problem of low production efficiency due to long analysis time in the prior art.
Solution for solving the problem
The present application relates to:
1. an HPLC analysis method suitable for continuous measurement of hyperin and quercetin system, characterized in that:
the first step: setting liquid chromatography conditions;
and a second step of: preparing a mobile phase for analysis, wherein; mobile phase A is ultrapure water; mobile phase B is methanol;
and a third step of: setting the analysis program as isocratic elution and eluting concentration: mobile phase a: 20-40%, mobile phase B: 60-80%, flow rate: 0.5-2 mL/min;
fourth step: preparing a plurality of to-be-detected solutions containing hyperin and quercetin samples, wherein the hyperin and quercetin samples are samples containing one or two components of hyperin and quercetin;
fifth step: determining the solution of one of hyperin and quercetin sample to be detected by adopting the analysis program, and recording a chromatogram;
sixth step: optionally calculating the content of hyperin and quercetin according to the respective standard curves;
seventh step: adopting a mobile phase B to clean the chromatographic column for 1-5 min;
eighth step: repeating the fifth step to the seventh step, and sequentially measuring other unmeasured hyperin to be measured and quercetin samples.
2. The HPLC analysis method suitable for continuous measurement of hyperin and quercetin system according to item 1, characterized in that: the standard curve is established by preparing standard solutions of hyperin and quercetin in the same system and measuring the standard solutions by adopting the liquid chromatography condition.
3. The HPLC analysis method suitable for continuous measurement of hyperin and quercetin system according to item 1 or 2, characterized in that: the concentration of the hyperin and quercetin samples to be detected is 0.01-1.0 g/L.
4. The HPLC analysis method suitable for continuous measurement of hyperin and quercetin system according to item 1 or 2, characterized in that: the detection wavelength range is 300-400 nm.
5. The HPLC analysis method suitable for continuous measurement of hyperin and quercetin system according to item 1 or 2, characterized in that: the chromatographic column is a chromatographic column filled with C18.
6. The HPLC analysis method suitable for continuous measurement of hyperin and quercetin system according to item 1 or 2, characterized in that: the temperature of the chromatographic column is room temperature, the sample injection amount is 5-10 mu L, the temperature of the sample injector is the ambient temperature, and the sample collection time to be measured is 4-6 min.
7. The HPLC analysis method suitable for continuous measurement of hyperin and quercetin system according to item 3, characterized in that: the concentration of the hyperin and quercetin samples to be detected is 0.1-1.0 mg/mL.
ADVANTAGEOUS EFFECTS OF INVENTION
The application discovers a novel HPLC analysis method suitable for continuously measuring hyperin and quercetin, which not only can realize the continuous analysis and measurement of hyperin samples and quercetin samples, but also can realize the analysis and measurement of samples containing hyperin and quercetin simultaneously, and the chromatographic peak and peak shape are normally distributed, and the theoretical plate number is quite considerable; secondly, the HPLC analysis method suitable for continuously measuring the hyperin and the quercetin system can realize qualitative and quantitative analysis of the hyperin and the quercetin within 5 minutes. And the method is used for simultaneously analyzing the mixed samples of hyperin and quercetin with different concentrations, the two substances are not interfered with each other, and good linearity can be obtained between the two substances.
The HPLC analysis method suitable for the continuous measurement of the hyperin and the quercetin system is simple to operate, can realize the continuous analysis of the hyperin and the quercetin, saves a great amount of time cost and economic cost, and greatly simplifies the complexity of the operation. The technology can save time, analyze the mobile phase used for the common components in the industry, unify the chromatographic column used by the application with the chromatographic column used by other peptide analysis of the company, so that the chromatographic column does not need to be manually replaced when different substance analysis is switched, and only the mobile phase needs to be replaced and then cleaned. Therefore, the method is more beneficial to the realization of full-process automation.
Drawings
FIG. 1 is an HPLC chart of standard solutions of the same concentrations of hyperin and quercetin in example 1 (concentrations of 0.01g/L, 0.025g/L, 0.05g/L, 0.1g/L, 0.2g/L, 0.4g/L, 0.6g/L, 0.8g/L, 1.0g/L, respectively).
FIG. 2 is a graph showing the peak area-concentration linear relationship between the concentrations of hyperin and quercetin (concentrations of 0.01, 0.025, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0g/L, respectively) in example 1.
FIG. 3 is a graph showing the peak area-concentration linear relationship between the concentrations of hyperin and quercetin (concentrations of 0.1, 0.2, 0.4, 0.8, and 1.0g/L, respectively) in example 1.
FIG. 4 is an HPLC chart of a mixture of hyperin (0.5 g/L) and quercetin (0.5 g/L) in example 2.
FIG. 5 is an HPLC chart of 0.5g/L hyperin in example 3.
FIG. 6 is an HPLC chart of 0.5g/L quercetin in example 4.
FIG. 7 is an HPLC chart of a mixture of hyperin (0.5 g/L) and quercetin (0.5 g/L) in example 5.
FIG. 8 is an HPLC chart of a mixture of hyperin (0.5 g/L) and quercetin (0.5 g/L) in comparative example 1.
FIG. 9 is an HPLC chart of a mixture of hyperin (0.5 g/L) and quercetin (0.5 g/L) in comparative example 2.
FIG. 10 is an HPLC chart of a mixture of hyperin (0.5 g/L) and quercetin (0.5 g/L) in comparative example 3.
FIG. 11 is an HPLC chart of a mixture of hyperin (0.5 g/L) and quercetin (0.5 g/L) in comparative example 4.
FIG. 12 is an HPLC chart of a mixture of hyperin (0.5 g/L) and quercetin (0.5 g/L) in comparative example 5.
Detailed Description
The HPLC analysis method suitable for continuously measuring the hyperin and quercetin system comprises the following steps:
the first step: setting liquid chromatography conditions;
and a second step of: preparing a mobile phase for analysis, wherein; mobile phase A is ultrapure water; mobile phase B is methanol;
and a third step of: setting the analysis program as isocratic elution and eluting concentration: mobile phase a: 20-40%, mobile phase B: 60-80%, flow rate: 0.5-2 mL/min;
fourth step: preparing a plurality of to-be-detected solutions containing hyperin and quercetin samples, wherein the hyperin and quercetin samples are samples containing one or two components of hyperin and quercetin;
fifth step: determining the solution of one of hyperin and quercetin sample to be detected by adopting the analysis program, and recording a chromatogram;
sixth step: optionally calculating the content of hyperin and quercetin according to the respective standard curves;
seventh step: adopting a mobile phase B to clean the chromatographic column for 1-5 min;
eighth step: repeating the fifth step to the seventh step, and sequentially measuring other unmeasured hyperin to be measured and quercetin samples.
The standard curve is established by preparing mixed standard solutions of hyperin and quercetin with different concentrations, and measuring the mixed standard solutions by adopting the HPLC chromatographic conditions, and is established according to the linear relation between the concentration and the peak area.
The mobile phase is that the mobile phase A is ultrapure water; mobile phase B was methanol. The analytical procedure was isocratic elution, elution concentration: mobile phase a: 20-40%, mobile phase B:60 to 80%, preferably mobile phase a: 25-35%, mobile phase B:65 to 75%, more preferably mobile phase a:30%, mobile phase B:70%. The range is selected to obtain the best analysis results. The flow rate of the mobile phase is 0.5-2 mL/min, preferably 1mL/min. The flow rate is critical to select, the analysis time can be shortened by increasing the flow rate, however, the properties of the hyperin and the quercetin are very close, and if the flow rate is too fast, the two substances cannot be separated, so that the respective concentrations cannot be accurately calculated; too slow a flow rate can lead to an increase in chromatographic peak width, affecting analysis accuracy. Meanwhile, in order to enable the peaks of the two targets to be completely displayed, the concentration of the peaks is not too high, otherwise, the peaks of the targets are capped, and therefore, even if the peaks appear at the peaks, the peaks cannot be judged; but the concentration should not be too low, otherwise when a peak of impurities occurs, the peak is misjudged to be uneven due to the fact that the peak is too low. Therefore, when the concentrations of hyperoside to be detected and quercetin samples are in the linear range, namely between 0.01 and 1.0g/L, not only the qualitative property of each substance can be realized, but also the accurate quantification of each substance can be realized according to the respective standard curve.
The hyperin and quercetin samples to be tested are prepared according to the following method: for example, when preparing 1.0g/L sample, firstly accurately weighing 2.0mg hyperin and quercetin respectively, respectively placing the two into different centrifuge tubes, respectively adding methanol to dissolve and fix the volume to 1mL, thus obtaining mother liquor of hyperin and quercetin samples with the concentration of 2.0 g/L. Respectively sucking 500 mu L of each of the two mother solutions, and placing the two mother solutions in the same centrifuge tube to obtain a mixed solution with the concentration of both substances being 1.0g/L.
The detection wavelength is determined by the nature of the substance to be detected and is not affected by the analysis conditions. The detection wavelength is determined to be in the preferred range of 300 to 400nm, more preferably 340 to 380nm, particularly preferably 360nm, by full wavelength scanning of both substances. The reason is that: based on the full wavelength scan of the hyperoside and quercetin solutions, both showed strong light absorption at 360nm, so 360nm was most preferred as the detection wavelength for HPLC analysis.
The chromatographic column is a conventionally used chromatographic column, preferably a chromatographic column with octadecylsilane chemically bonded phase as filler, such as irinotecan Sin deg.C hrom ODS-AP
The flow rate is generally set to 0.5 to 2mL/min, preferably 1mL/min.
The column temperature was room temperature (typically 25 ℃).
The sample amount is 3 to 10. Mu.L, preferably 5. Mu.L.
The temperature of the sample injector is ambient temperature, and the sample collection time to be measured is 3-6 min, preferably 5min.
The technical scheme of the application is further described and illustrated by the following specific examples.
The application is further illustrated by the following examples, which are not to be construed as limiting the application, in conjunction with the accompanying drawings. It should be understood, however, that these examples are illustrative only and are not intended to limit the present application. Unless otherwise indicated, all starting materials used in the examples of the present application were those commonly used in the art, and all methods used in the examples were those commonly used in the art.
Examples
EXAMPLE 1 analysis of hyperin and quercetin
Instrument and conditions:
an Agilent1260InfinityII LC high performance liquid chromatograph and an OpenLabCDS2 software system are adopted; with an Eilit SinoChrom ODS-AP(250X 4.6 mm) is an analytical column, the column temperature is 25 ℃; the detection wavelength was 360nm.
The experimental steps are as follows:
accurately weighing 2mg of hyperin and 2mg of quercetin standard substances respectively, mixing, placing into a centrifuge tube, and quantifying to 1mL by using methanol to obtain mother solutions with the concentration of hyperin and quercetin of 2g/L respectively. Mobile phase: a: ultrapure water; and B, methanol.
Elution concentration: 30% of phase A; 70% of phase B.
Flow rate: 1.0mL/min
The temperature is 25 DEG C
Sample injection amount: 5 mu L
Analysis procedure: and (5) performing isocratic elution on the sample by adopting the elution concentration.
Based on 2g/L mother liquor prepared in the experimental step, accurately sucking 5 mu L, 12.5 mu L, 25 mu L, 50 mu L, 100 mu L, 200 mu L, 300 mu L, 400 mu L and 500 mu L respectively, placing the mother liquor into a clean centrifuge tube, and adding methanol correspondingly to supplement 1mL respectively. The concentration of hyperin and quercetin is 0.01g/L, 0.025g/L, 0.05g/L, 0.1g/L, 0.2g/L, 0.4g/L, 0.6g/L, 0.8g/L and 1.0g/L respectively.
The standard solutions were measured by the above method, and a chromatogram was recorded as shown in fig. 1.
In addition, the spectrograms of hyperin and quercetin at each concentration were integrated to obtain peak areas at the corresponding concentrations, and standard curves of the hyperin and quercetin concentrations and absorption peak areas were drawn, respectively, as shown in fig. 2, so that the hyperin and quercetin concentrations were in a linear relationship with the corresponding peak areas at 0.01-1.0 g/L.
The linear regression equation for hyperin concentration-peak area is shown below:
y 1 ＝a x+b
wherein: y is 1 : peak area
Concentration of hyperin
a:15949.06611
b:-26.19737
R 2 :0.99693
The concentration-peak area linear regression equation for quercetin is shown below:
y α ＝a x+b
wherein: y is α : peak area
Concentration of quercetin
a:9230.44497
b:15.18095
R 2 :0.99352
The retention times and theoretical plate numbers for hyperin and quercetin at each concentration are shown in the following table:
as is clear from the above table, the peak area and concentration relationship is linear but the analysis error is large when the concentration of hyperin and quercetin is less than 0.05g/L, and the error includes the operation error generated during sample preparation and the measurement error generated during analysis. Obviously, the lower the concentration of the formulated sample, the greater the operating error.
The linearity is better when the concentration of hyperin and quercetin are both in the range of 0.1-1 g/L, and the error is less than 10%, as shown in figure 3. At this time, the linear regression equation of hyperin concentration-peak area is as follows:
y 2 ＝a x+b
wherein: y is 2 : peak area
Concentration of hyperin
a:5930.87223
b:-85.32494
R 2 :0.99982
The concentration-peak area linear regression equation for quercetin is shown below:
y β ＝a x+b
wherein: y is β : peak area
Concentration of quercetin
a:9064.11965
b:17.27113
R 2 :0.99412
The detected concentrations and error comparisons of hyperin and quercetin in the two concentration ranges are shown in the following table:
EXAMPLE 2 verification of the Linear relationship of hyperin and quercetin
After analysis of the sample of example 1, the column was washed with methanol for 5min.
HPLC analysis was performed on hyperoside and quercetin in the same manner as in example 1 except that a mixed solution of 0.5g/L hyperoside and 0.5g/L quercetin was used, and the chromatograms thereof are shown in FIG. 4. The peak areas of hyperin are respectively substituted into a standard curve y 1 Y 2 The calculated concentrations are respectively: 0.4948g/L and 0.5011g/L, with errors of 1.04% and 0.22%, respectively; substituting peak areas of quercetin into standard curve y α And y β The calculated concentrations are respectively: 0.4950g/L and 0.5041g/L, the errors are respectively: 1% and 0.82%. Therefore, the standard curve of each substance in different concentration ranges has higher accuracy, and is suitable for calculating the concentration of the sample with unknown concentration.
EXAMPLE 3.0.5g/L hyperin Linear relationship verification
After analysis of the sample of example 1, the column was washed with methanol for 5min.
250 mu L of hyperin mother liquor in the example 1 is accurately sucked, and methanol is used for supplementing the volume to 1mL to obtain the hyperin solution with the concentration of 0.5 g/L. HPLC analysis was performed on the hyperin solution in the same manner as in example 1, and the chromatogram thereof is shown in FIG. 5. The peak areas are respectively substituted into a standard curve y 1 Y 2 The calculated concentrations are respectively0.512 and 0.5235g/L, errors were 2.4% and 4.7%.
EXAMPLE 4.0.5g/L quercetin Linear relationship verification
After analysis of the sample of example 1, the column was washed with methanol for 5min.
250 mu L of the quercetin mother liquor in the example 1 is accurately sucked, and the volume is supplemented to 1mL by methanol to obtain the quercetin solution with the concentration of 0.5 g/L. HPLC analysis was performed on the quercetin solution in the same manner as in example 1, and the chromatogram thereof is shown in FIG. 6. The peak areas are respectively substituted into a standard curve y α And y β The calculated concentrations were 0.527g/L and 0.5367g/L, with errors of 5.4% and 7.3%, respectively.
EXAMPLE 5.0.5g/L hyperin and 1g/L quercetin Mixed solution Linear relationship verification
After analysis of the sample of example 1, the column was washed with methanol for 5min.
250 mu L of hyperin mother liquor in the example 1 is accurately sucked, 500 mu L of quercetin mother liquor is added into the same centrifuge tube, and methanol is used for supplementing the volume to 1mL, so that a mixed solution of hyperin with the concentration of 0.5g/L and quercetin with the concentration of 1.0g/L is obtained. This mixed solution was subjected to HPLC analysis in the same manner as in example 1, and the chromatogram thereof is shown in FIG. 7. Integrating the two substances to obtain respective peak areas, and substituting the peak areas of hyperin into corresponding standard curve y 1 Y 2 The concentration of the hyperin obtained by calculation is 0.505g/L and 0.5172g/L respectively, and the error is 1.0% and 3.4%; substituting peak areas of quercetin into corresponding standard curves y α And y β The calculated quercetin concentrations were 1.046g/L and 1.0646g/L, with errors of 4.6% and 6.5%, respectively.
Comparative example 1.
The ratio of water in the mobile phase was reduced from 30% to 10%, and the ratio of methanol was correspondingly increased from 70% to 90%, and the mixed sample of hyperoside and quercetin in the concentration of 0.5g/L in example 2 was analyzed and measured, and as shown in FIG. 8, the chromatographic peaks of hyperoside and quercetin were connected together, and separation failed.
Comparative example 2.
The analytical measurement was carried out on a mixed sample of 0.5g/L hyperin and 0.5g/L quercetin in the same manner as in example 2 except that methanol in the mobile phase was changed to acetonitrile, and as a result, as shown in FIG. 9, the chromatographic peaks of both were still joined together and the respective peaks were deformed to a different extent than in example 1, so that the spectrogram analysis was impossible.
Comparative example 3.
As shown in FIG. 10, it is understood from a comparison of FIGS. 10 and 4 that the addition of phosphoric acid in the mobile phase can still achieve the analysis of the hyperoside and quercetin mixed system, but the analysis effect is comparable to that of the method of the present application, except that the water in the mobile phase is changed to 0.07% phosphoric acid aqueous solution, and the analysis of the 0.5g/L hyperoside and 0.5g/L quercetin mixed sample is performed in the same manner as in example 2.
Comparative example 4.
As shown in FIG. 11, it is understood from the comparison between FIG. 11 and FIG. 4 that the addition of salt in the mobile phase can still analyze the mixed system of hyperoside and quercetin, but the analysis effect is not significantly improved as compared with the method of the present application, except that the water in the mobile phase is changed to the 0.02M phosphate aqueous solution, and the mixed sample of hyperoside and quercetin of 0.5g/L is analyzed and measured in the same manner as in example 2.
Comparative example 5.
As shown in fig. 12, it is understood from fig. 12 that no substance was detected, except that the ratio of water in the mobile phase was increased to 80% and the ratio of methanol was decreased to 20%, and the mixed sample of hyperin and quercetin was analyzed and measured at 0.5g/L in the same manner as in example 2. The reason for this analysis is that both substances are water insoluble and cannot be eluted when eluting with a high proportion of aqueous phase and are therefore undetected.
Experiments prove that the application can completely dissolve the sample by adopting 100% methanol without helping dissolution by ultrasonic wave. The mobile phase is methanol: water = 70:30, and eluting the hyperin for about 3.4min and the quercetin for about 3.9min by using the method. The whole analysis procedure was 5min. By using the analysis method of the application, hyperin and quercetin are respectively linear in the range of 0.01-1 g/L, but the error is smaller in the linear range of 0.1-1 g/L, and the error is less than 10%. It can be seen that the present application is superior to the existing references both in terms of mobile phase ratio and in terms of analysis time. In addition, when the high performance liquid chromatography analysis is continuously carried out on the sample 1 according to the chromatographic conditions of the application, the mobile phase ratio can completely elute hyperin and quercetin without residue almost in the sample measurement process.
In addition, the mobile phase in the prior art needs to be proportioned in advance, and the mobile phase needs to be eluted by adopting high proportion of organic matching high proportion of phosphoric acid, and a ternary pump is needed to realize analysis. The application does not need to perform prior mobile phase preparation work, has simpler mobile phase composition, can realize analysis by only having a binary pump, and is easier to operate. Meanwhile, the chromatographic column for analysis is unified with most of chromatographic columns for substance analysis of the company, so that automatic analysis is more facilitated, and time cost and labor cost caused by replacing the chromatographic column are saved. Compared with the method for simultaneously measuring the content of hyperin and quercetin in lotus plumule by HPLC in the prior art, the method of the application shortens the analysis time to 1/3.5 of the prior art, and the method of the prior art requires at least 17.5min, but the method of the application requires only 5min, which has great significance in industrial production.
In conclusion, the HPLC analysis method suitable for hyperin and quercetin can successfully separate two substances in a system, can form a stable and linear standard curve, and can accurately calculate the concentration of hyperin in the system, so that the concentration calibration can be carried out on samples with unknown concentrations. The method is simple to operate, and can continuously analyze the independent hyperin and quercetin, so that not only can a great amount of time cost and economic cost be saved, but also the complexity of the operation is greatly simplified, and meanwhile, a good analysis effect is achieved.
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (7)
1. An HPLC analysis method suitable for continuous measurement of hyperin and quercetin system, characterized in that:
the first step: setting liquid chromatography conditions;
and a second step of: preparing a mobile phase for analysis, wherein; mobile phase A is ultrapure water; mobile phase B is methanol;
and a third step of: setting the analysis program as isocratic elution and eluting concentration: mobile phase a: 20-40%, mobile phase B: 60-80%, flow rate: 0.5-2 mL/min;
fourth step: preparing a plurality of to-be-detected solutions containing hyperin and quercetin samples, wherein the hyperin and quercetin samples are samples containing one or two of hyperin and quercetin;
fifth step: determining the solution of one of hyperin and quercetin sample to be detected by adopting the analysis program, and recording a chromatogram;
sixth step: optionally calculating the content of hyperin and quercetin according to a standard curve;
seventh step: adopting a mobile phase B to clean the chromatographic column for 1-5 min;
eighth step: repeating the fifth step to the seventh step, and sequentially measuring other unmeasured hyperin to be measured and quercetin samples.
2. The HPLC analysis method suitable for continuous determination of hyperin and quercetin system according to claim 1, characterized in that: the standard curve is established by preparing standard solutions of hyperin and quercetin separately and performing the assay using the liquid chromatography conditions.
3. The HPLC analysis method suitable for continuous determination of hyperin and quercetin system according to claim 1, characterized in that: the concentration of the hyperin and quercetin samples to be detected is 0.01-1.0 g/L.
4. HPLC analysis method suitable for the continuous determination of hyperin and quercetin systems according to claim 1 or 2, characterized in that: the detection wavelength range is 300-400 nm.
5. HPLC analysis method suitable for the continuous determination of hyperin and quercetin systems according to claim 1 or 2, characterized in that: the chromatographic column is a chromatographic column filled with C18.
6. HPLC analysis method suitable for the continuous determination of hyperin and quercetin systems according to claim 1 or 2, characterized in that: the temperature of the chromatographic column is room temperature, the sample injection amount is 5-10 mu L, the temperature of the sample injector is the ambient temperature, and the sample collection time to be measured is 4-6 min.
7. The HPLC analysis method suitable for continuous measurement of hyperin and quercetin system according to claim 3, wherein: the concentrations of the hyperin and the quercetin sample are respectively 0.1-1 g/L.
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| CN102827220A (en) * | 2012-08-24 | 2012-12-19 | 湖州市食品药品检验所 | Method for separating rutin, hyperoside, isoquercitrin and quercetin from lotus leaves |
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| CN102827220A (en) * | 2012-08-24 | 2012-12-19 | 湖州市食品药品检验所 | Method for separating rutin, hyperoside, isoquercitrin and quercetin from lotus leaves |
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