CN117310040A - Method for determining active ingredients in rhodiola rosea extract and application thereof - Google Patents
Method for determining active ingredients in rhodiola rosea extract and application thereof Download PDFInfo
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- CN117310040A CN117310040A CN202311316153.2A CN202311316153A CN117310040A CN 117310040 A CN117310040 A CN 117310040A CN 202311316153 A CN202311316153 A CN 202311316153A CN 117310040 A CN117310040 A CN 117310040A
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- salidroside
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- 239000000284 extract Substances 0.000 title claims abstract description 69
- 244000042430 Rhodiola rosea Species 0.000 title claims abstract description 57
- 235000003713 Rhodiola rosea Nutrition 0.000 title claims abstract description 57
- 238000000034 method Methods 0.000 title claims abstract description 39
- 239000004480 active ingredient Substances 0.000 title claims abstract description 23
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 87
- YCCILVSKPBXVIP-UHFFFAOYSA-N 2-(4-hydroxyphenyl)ethanol Chemical compound OCCC1=CC=C(O)C=C1 YCCILVSKPBXVIP-UHFFFAOYSA-N 0.000 claims abstract description 57
- 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 claims abstract description 54
- 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 52
- 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 52
- 239000012086 standard solution Substances 0.000 claims abstract description 31
- DBLDQZASZZMNSL-QMMMGPOBSA-N L-tyrosinol Natural products OC[C@@H](N)CC1=CC=C(O)C=C1 DBLDQZASZZMNSL-QMMMGPOBSA-N 0.000 claims abstract description 28
- 235000004330 tyrosol Nutrition 0.000 claims abstract description 28
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 claims abstract description 27
- 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 claims abstract description 26
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- 229960001285 quercetin Drugs 0.000 claims abstract description 26
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- 238000001514 detection method Methods 0.000 claims abstract description 20
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- 239000000126 substance Substances 0.000 claims abstract description 19
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- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 10
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- 235000019257 ammonium acetate Nutrition 0.000 claims description 10
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- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
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- 239000013076 target substance Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
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- IYRMWMYZSQPJKC-UHFFFAOYSA-N kaempferol Chemical compound C1=CC(O)=CC=C1C1=C(O)C(=O)C2=C(O)C=C(O)C=C2O1 IYRMWMYZSQPJKC-UHFFFAOYSA-N 0.000 description 2
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- MXYUKLILVYORSK-HBMCJLEFSA-N (-)-lobeline Chemical compound C1([C@@H](O)C[C@H]2N([C@H](CCC2)CC(=O)C=2C=CC=CC=2)C)=CC=CC=C1 MXYUKLILVYORSK-HBMCJLEFSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- UBSCDKPKWHYZNX-UHFFFAOYSA-N Demethoxycapillarisin Natural products C1=CC(O)=CC=C1OC1=CC(=O)C2=C(O)C=C(O)C=C2O1 UBSCDKPKWHYZNX-UHFFFAOYSA-N 0.000 description 1
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- DUEPRVBVGDRKAG-UHFFFAOYSA-N carbofuran Chemical compound CNC(=O)OC1=CC=CC2=C1OC(C)(C)C2 DUEPRVBVGDRKAG-UHFFFAOYSA-N 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
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- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
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- UXOUKMQIEVGVLY-UHFFFAOYSA-N morin Natural products OC1=CC(O)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UXOUKMQIEVGVLY-UHFFFAOYSA-N 0.000 description 1
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- 229930003231 vitamin Natural products 0.000 description 1
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Classifications
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- G—PHYSICS
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- 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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- 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
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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
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- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
- G01N30/724—Nebulising, aerosol formation or ionisation
- G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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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- 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
- G01N2030/042—Standards
- G01N2030/045—Standards internal
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/02—Column chromatography
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- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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- 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
- G01N2030/065—Preparation using different phases to separate parts of sample
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Abstract
The invention provides a method for measuring active ingredients in rhodiola rosea extract and application thereof, wherein the method comprises the following steps: s1, taking rhodiola rosea extract powder, adding an extract and an internal standard solution, performing ultrasonic extraction, cooling to room temperature, standing, sucking supernatant, and performing centrifugal purification and filtering by a microporous filter membrane to obtain a sample to-be-detected liquid; s2, dissolving salidroside, tyrosol, zoysin, salidroside, quercetin standard substances and an internal standard solution with methanol to prepare a series of standard working solutions; s3, analyzing the sample to-be-detected liquid and the series standard working solution by adopting an ultra-high performance liquid chromatography-tandem mass spectrometer, and quantitatively analyzing the content of salidroside, tyrosol, zoysin, salidroside and quercetin in the sample to-be-detected liquid by adopting an internal standard method. The method can efficiently detect the active ingredients such as salidroside, tyrosol, zoysin, salidroside, quercetin and the like in the rhodiola rosea extract, and has the advantages of low detection cost, high sensitivity and strong specificity.
Description
Technical Field
The invention relates to the technical field of detection of traditional Chinese medicinal materials, in particular to a method for measuring active ingredients in rhodiola rosea extract and application thereof.
Background
Rhodiola rosea (Rhodiola rosea) is a perennial herb of Rhodiola genus, and has the effects of resisting fatigue, depression, aging, oxidation, radiation, altitude stress, liver and heart protection, immunity regulation, etc. The composition is complex, and the separated chemical components are more than 40, including flavonoids, glycosides, coumarin, amino acids, polysaccharides, volatile oil, minerals and other substances, wherein the flavonoids and the glycosides are main effective components. The glycoside compounds mainly comprise salidroside, tyrosol and phenylpropanoids, the salidroside is considered as the functional component with the most medicinal value in the rhodiola, and has various functions of resisting oxidation, inflammation, liver fibrosis and the like, and the content of the salidroside is generally used as an important factor for evaluating the efficacy of the rhodiola. The phenylpropanoid compound mainly comprises lobeline, zoysin and zoysin, and the research shows that the compound has stronger capability of scavenging free radicals. The flavonoid compounds mainly comprise quercetin, kaempferol and the like, and are found to have the effects of resisting oxidization, regulating cardiovascular and cerebrovascular diseases and the like. The research on rhodiola rosea at home and abroad is more focused on various biological functions and pharmacological activities, the quantitative analysis research method for various effective components in rhodiola rosea is relatively few, and a High Performance Liquid Chromatography (HPLC) method, a pre-column derivatization combined HPLC-MS method and the like are mostly adopted. The invention takes salidroside, tyrosol, zosev-vitamin, salidroside and quercetin (each structural formula is shown as follows) in the rhodiola rosea extract as determination targets, establishes a high-efficiency, high-sensitivity and high-selectivity quantitative analysis method, provides a basis for quality evaluation of the rhodiola rosea extract, and provides a methodology foundation for research and detection of related active ingredients of the rhodiola rosea.
Disclosure of Invention
The invention aims to provide a method for measuring active ingredients in rhodiola rosea extract and application thereof, and the method can be used for efficiently detecting the active ingredients such as salidroside, tyrosol, zoysin, salidroside and quercetin in the rhodiola rosea extract, has low detection cost, high sensitivity and strong specificity, and can be used for quantitative analysis and quality evaluation of characteristic ingredients of the commercial rhodiola rosea extract and preliminary identification of rhodiola rosea medicinal material varieties.
In a first aspect of the present invention, there is provided a method for determining an active ingredient in rhodiola rosea extract, comprising the steps of:
s1, taking rhodiola rosea extract powder, adding an extract and an internal standard solution, performing ultrasonic extraction, cooling to room temperature, standing, sucking supernatant, and performing centrifugal purification and filtering by a microporous filter membrane to obtain a sample to-be-detected liquid;
s2, dissolving salidroside, tyrosol, zoysin, salidroside, quercetin standard substances and an internal standard solution with methanol to prepare a series of standard working solutions;
s3, analyzing the sample to-be-detected liquid and the series standard working solution by adopting an ultra-high performance liquid chromatography-tandem mass spectrometer, and quantitatively analyzing the content of salidroside, tyrosol, zoysin, salidroside and quercetin in the sample to-be-detected liquid by adopting an internal standard method.
Preferably, step S1 comprises the steps of:
s11, taking 0.1g of rhodiola rosea extract powder;
s12, adding 100ml of extract, adding 0.5ml of internal standard solution, weighing, performing ultrasonic extraction for 30min, cooling to room temperature, and supplementing the quality loss with the extract;
s13, standing for 5min, taking 2mL of supernatant into a centrifuge tube, and carrying out vortex centrifugation for 5min to obtain the supernatant;
s14, taking 1mL of supernatant, placing the supernatant into a brown volumetric flask, fixing the volume of the extract to 10mL, and filtering the extract by a microporous membrane with the size of 0.22 mu m to obtain a sample to-be-detected liquid.
Preferably, in step S1, the extract is an aqueous methanol solution, and the internal standard solution is a theophylline solution.
Preferably, the extract is an 80% aqueous methanol solution; the internal standard solution is 500ng/mL theophylline solution prepared by methanol.
Preferably, step S2 includes the steps of:
s21, taking a standard substance of salidroside, tyrosol, zoysin, salidroside and quercetin, and preparing a single standard substance stock solution with the concentration of 1mg/mL by using methanol;
s22, respectively taking equal volumes of single standard stock solution, and preparing 100 mug/ml mixed standard solution by using methanol;
s23, respectively taking 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 and 1.0ml of mixed standard substance solution, adding into a volumetric flask, respectively adding 50 mu L of internal standard solution, and adding methanol to a volume of 10ml to prepare a series of standard working solutions.
Preferably, in step S3, the eluent used in the liquid chromatography is an aqueous ammonium acetate solution and a formic acid-methanol solution.
Preferably, in step S3, the eluent used in the liquid chromatography is 2mmol/L ammonium acetate aqueous solution and 0.05% formic acid-methanol solution.
Preferably, in step S3, the sample to be measured is measured, and the content of the active component of rhodiola rosea is calculated according to each component and the area of the ion peak of the internal calibration quantity.
Preferably, the liquid to be detected of the sample is measured, quality control products are added before and after each batch of samples, and the stability of the system is monitored.
In a second aspect of the invention, there is provided the use of a method for determining the active ingredient in rhodiola rosea extract, said use being in the preparation of a kit for the detection of the active ingredient in rhodiola rosea extract.
Preferably, the kit comprises: eluent, internal standard solution, standard solution and extract.
Preferably, the eluent is an ammonium acetate aqueous solution and a formic acid-methanol solution; the extracting solution is methanol aqueous solution, and the internal standard solution is theophylline solution; the standard solution comprises: salidroside, tyrosol, zoysin, rhodiola rosea and quercetin.
Preferably, the kit further comprises: quality control.
Preferably, the preparation steps of the quality control product are as follows: the mixed standard stock solution was taken at 5. Mu.L and 100. Mu.L and each was fixed to a volume of 10ml with methanol.
Compared with the prior art, the invention has the beneficial effects that:
(1) Commercial rhodiola rosea extract manufacturers have a plurality of industry standards, and most of the commercial rhodiola rosea extract manufacturers produce the rhodiola rosea according to demands, and the quality of the rhodiola rosea extract cannot be ensured according to the detection of the enterprise standards. The invention can rapidly, efficiently and accurately detect the content of salidroside and tyrosol, can provide basis for quality evaluation of the rhodiola rosea extract, can determine the content of key characteristic components such as salidroside, zoysia (higher content in rhodiola rosea) and the like, and performs primary screening of raw materials.
(2) The extraction solvent used in the invention is 80% methanol aqueous solution, has good secondary extraction effect on characteristic components in the rhodiola rosea extract, has less environmental pollution, accords with the green development concept, and can reduce the influence of the solvent effect on the chromatographic peak shape.
(3) The eluent used in the invention is ammonium acetate aqueous solution and formic acid-methanol solution, a reasonable gradient elution program is arranged to improve the separation degree, ammonium acetate is added in the mobile phase under the mass spectrum anion scanning mode to facilitate ionization of the compound, and formic acid is added to facilitate improvement of the chromatographic peak-to-peak shape.
(4) The pretreatment method is simple, vortex centrifugation is carried out during sample purification, the filtering membrane is used for filtering, the operation is simple, convenient and efficient, and the cost is saved.
(5) The invention reduces the interference of matrix effect by dilution method. In the extraction process, a target substance is extracted, and a plurality of substances other than the target exist in the extracting solution, so that the qualitative and quantitative analysis of the target substance on an analysis instrument is influenced, and the influence of matrix effect on detection needs to be considered. The matrix effect is generally not eliminated but its effect can be reduced by an effective route. Considering the characteristics of the sample and the target substance, the invention adopts a dilution mode to reduce the matrix effect. The method of the invention basically eliminates the influence of matrix effect on final detection data because the method has the advantages that the measurement result after dilution is more accurate and the matrix effect is obviously improved by diluting the supernatant obtained after ultrasonic extraction and centrifugal purification of the extract powder with known target component content by 10 times and then sampling the supernatant and comparing the response signals of each target component before and after dilution with the concentration calculated according to a standard curve.
(6) The method of the invention has lower detection Limit (LOD) and quantification Limit (LOQ), which shows that the method of the invention has high sensitivity.
(7) The method proves that the method is accurate in operation and good in repeatability through the investigation of precision, stability, reproducibility and standard adding recovery rate.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of a method for determining active ingredients in rhodiola rosea extract;
FIG. 2 is a total ion flow chromatogram of a standard substance provided by the invention, wherein 1 represents salidroside, 2 represents theophylline internal standard, 3 represents tyrosol, 4 represents zoysin, 5 represents rhodiola rosea, and 6 represents quercetin;
fig. 3 is a total ion flow chromatogram of a rhodiola rosea extract sample and a quantitative ion chromatogram of each component in a multi-reaction monitoring mode, wherein A is a total ion flow chromatogram (internal standard: theophylline) of the rhodiola rosea extract (sample 1#), B is salidroside, C is tyrosol, D is zoysin, E is salidroside, and F is quercetin.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms also include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
As shown in fig. 1, this embodiment provides a method for determining active ingredients in rhodiola rosea extract, comprising the following steps:
(1) Accurately weighing radix Rhodiolae extract powder 0.1g to 0.1mg.
(2) Extraction of samples: 100mL of extract (80% aqueous methanol solution) was accurately added, 0.5mL of internal standard solution (theophylline solution prepared by methanol, concentration of 500 ng/mL) was added, the mixture was weighed, extracted by ultrasonic for 30min (500W, 40 kHz), cooled to room temperature, and the quality of the extract was complemented by the quality of the loss.
(3) Sample purification: standing for 5min, sucking 2mL of supernatant into a 5mL centrifuge tube, and vortex centrifuging for 5min to obtain supernatant.
(4) 1mL of the filtrate is removed, the filtrate is placed in a 10mL brown volumetric flask, the volume of the extract is fixed to the scale, and the extract is filtered by a 0.22 mu m organic phase filter membrane to be used as a sample to be measured liquid for UPLC-MS/MS analysis.
(5) And (3) standard curve preparation: the ratio of the standard substance of each characteristic component to the concentration of the internal standard substance is taken as an abscissa, the ratio of the peak area of each characteristic component to the ion peak area of the internal standard quantity is taken as an ordinate, linear regression is carried out, a correction curve is established, and the correlation coefficient is more than or equal to 0.99. The mixed standard was diluted stepwise with the sample concentrations at signal to noise ratios (S/N) of 3 and 10 being the limit of detection (LOD) and limit of quantification (LOQ). Regression equations and linear ranges of the components are shown in Table 1.
TABLE 1 Standard working curves and Linear relations of five active ingredients
| Composition of the components | Linear range ng/mL | Regression equation | R 2 |
| Salidroside | 20～10000 | Y＝1.1319X+0.1563 | 0.9995 |
| Tyrosol | 50～10000 | Y＝21.1970X+2.2214 | 0.9994 |
| Trapeziwei (Chinese character) | 20～5000 | Y＝4.6234X+0.3310 | 0.9997 |
| Rhodiola rosea extract | 100～10000 | Y＝58.8420X+0.4712 | 0.9992 |
| Quercetin | 50～10000 | Y＝0.4443X+0.0620 | 0.9996 |
(6) Ultra-high performance liquid chromatography tandem mass spectrometry determination: the prepared series of standard working solutions and sample to-be-detected solutions are sucked and respectively injected into an ultra-high performance liquid chromatography-tandem mass spectrometer, wherein figure 2 shows a total ion flow chromatogram of the standard, 1 represents salidroside, 2 represents theophylline internal standard, 3 represents tyrosol, 4 represents zoysin, 5 represents rhodiola rosea element and 6 represents quercetin.
(6.1) liquid chromatography parameters
Chromatographic column: ZORBAX Eclipse Plus C18 chromatography column (3.0 mm. Times.50 mm,1.8 μm). Mobile phase: a is 2mmol/L ammonium acetate aqueous solution and B is 0.05% formic acid-methanol solution. Gradient elution procedure is as in table 1; flow rate: 0.3ml/min; column temperature: 30 ℃; sample injection amount: 1 μl, sample injection time 8min. The mobile phase gradient set up is shown in table 2.
TABLE 2 mobile phase gradient setup
(6.2) Mass Spectrometry parameters
Ion source: AJS ESI source (atmospheric electrospray ion source with agilent jet technology); the temperature of the drying gas is 250 ℃; the flow rate of the drying gas is 7.0L/min; the spray gas pressure was 30psi; the sheath gas temperature is 325 ℃; sheath gas (N) 2 ) The flow rate is 11.0L/min; the capillary voltage is 4000 (+) 3500 (-); the nozzle voltage was 0V. The scanning mode is MRM mode, the detection mode is anion detection, and the mass spectrum parameters are shown in Table 3.
Table 3 mass spectral parameters
(7) Calculation of determination result of active ingredient of rhodiola rosea extract
And (3) measuring the extracted sample to-be-measured liquid, and calculating the content of the active ingredients of the rhodiola rosea according to each ingredient in the sample and the area of the ion peak of the internal calibration quantity. And adding quality control samples before and after each batch of samples, and monitoring the stability of the system.
(8) The method has the advantages of detection limit, quantitative limit, precision, stability, repeatability and standard recovery rate:
(8.1) detection Limit and quantitative Limit
The limit of detection (LOD) was calculated with S/n=3, and the limit of quantification (LOQ) was calculated with S/n=10, as shown in table 4.
TABLE 4 method detection Limit (LOD) and quantification Limit (LOQ)
| Composition of the components | LOD(mg/g) | LOQ(mg/g) |
| Salidroside | 0.0058 | 0.0215 |
| Tyrosol | 0.0346 | 0.1205 |
| Trapeziwei (Chinese character) | 0.0065 | 0.0224 |
| Rhodiola rosea extract | 0.0456 | 0.1526 |
| Quercetin | 0.0050 | 0.0162 |
(8.2) precision
The mixed standard solution of salidroside, tyrosol, zosev-vitamin, salidroside and quercetin is respectively and precisely absorbed, 1 mu L of sample is injected each time, 6 times of continuous measurement are carried out, and the measured peak areas RSD are respectively 2.93%, 2.05%, 3.39%, 1.55% and 3.12%, which indicates that the instrument precision is good.
(8.3) stability
The salidroside, tyrosol, zoysin, salidroside and quercetin sample solutions are respectively and precisely absorbed, sample injection is carried out for 4, 8, 12, 16, 20 and 24 hours, peak areas of 5 active components are measured, and the RSD of the peak areas is calculated to be 2.88%, 3.47%, 2.55%, 2.16% and 1.32%, which shows that the stability is better.
(8.4) reproducibility
6 sample solutions were prepared in parallel, 1. Mu.L of the sample was taken, the peak areas of the five active ingredients were examined, and the RSDs of the peak areas were calculated to be 2.64%, 2.95%, 1.60%, 4.03% and 2.71%, respectively. The method has good reproducibility.
(8.5) labeling recovery rate
Accurately weighing radix Rhodiolae extract 0.1g to 0.1mg, adding control single stock solutions with different volumes according to 80%, 100% and 120% of salidroside, tyrosol, zosev-vitamin, salidroside and quercetin content, processing according to detection conditions, measuring each concentration level for 3 times, and calculating the standard recovery rate of 5 active ingredients.
The method of this example was used to determine the rhodiola rosea extract (sample 1 #) to obtain a chromatogram shown in fig. 3, wherein a is the total ion flow chromatogram (internal standard: theophylline) of the rhodiola rosea extract (sample 1 #), B is salidroside, C is tyrosol, D is zoysin, E is rhodiola rosea extract, and F is quercetin.
Preparing a standard working curve, and calculating a measurement result: quantitative analysis is carried out by an internal standard method, and the content of characteristic components in a sample is converted into gram weight, and the formula is as follows:
M＝c×V/m；
wherein:
the gram weight (mg/g) of characteristic components in the M-rhodiola rosea extract;
c-instrument measurement of concentration value (ng/mL);
v-volume of extract;
m-quality of rhodiola rosea extract (g).
The average value of the two determinations was taken as the determination result, and the result was accurate to 0.01mg/g.
The results of testing five different batches of rhodiola rosea extract (sample 1# -5 #) are shown in Table 5: the average content of salidroside in 5 batches of samples is 13.13mg/g (mass fraction is 1.31%), which accords with the nominal value (the mass fraction of the salidroside is more than or equal to 1%).
TABLE 5 rhodiola rosea extract detection results
And (3) adding a mark and recovering rate: in order to judge the accuracy of the method, adding a salidroside standard solution into a sample 1# to ensure that the addition standard quantity is respectively 80%, 100% and 120% of background value, carrying out the same sample pretreatment, obtaining the content of the salidroside in the sample at the moment by using UPLC-MS/MS on-machine detection, obtaining the average addition standard recovery rate of the salidroside to be 101.6%, meeting the reasonable addition standard recovery rate range, and obtaining the results (see Table 6) of the addition standard recovery rate of the tyrosol, the zosev-ing, the salidroside and the quercetin to be 97.9-103.4% by using the same method, thus indicating that the method is accurate.
TABLE 6 recovery rate by adding standard
Example 2
This example provides a kit for detecting an active ingredient in rhodiola rosea extract based on the assay method of example 1, comprising the following reagents:
(1) Eluent: mobile phase: a is 2mmol/L ammonium acetate aqueous solution and B is 0.05% formic acid-methanol solution.
(2) Internal standard solution: preparing an internal standard stock solution with the theophylline concentration of 0.1mg/mL by using methanol, and storing in a refrigerator at the temperature of 4 ℃ in a dark place for 3 months; in the series of standard working solutions and the sample to-be-measured solutions, the concentration of each internal standard is kept at 500ng/mL.
(3) Standard solution: accurately weighing salidroside, tyrosol, zoysin, salidroside and quercetin standard substances to 0.1mg, and dissolving with methanol to obtain single reference stock solution with each substance concentration of 1 mg/mL. Equal volumes of stock solutions were aspirated separately with a pipette and 100. Mu.g/ml of mixed standard solution was prepared with methanol. Precisely measuring 5 active ingredient mixed standard substance solutions 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 and 1.0ml to 10ml volumetric flasks, respectively adding 50 mu L of internal standard solution, adding methanol to a constant volume to 10ml to prepare a series of standard working solutions: 10. 20, 50, 100, 200, 500, 1000, 2000, 5000, 10 000ng/ml.
(4) Extract liquid: 80% aqueous methanol (methanol: water=80:20, V/V);
(5) Quality control product: the stock solutions of the above mixed standards were taken at 5. Mu.L and 100. Mu.L and fixed to 10ml with methanol, respectively, to obtain QC (L) and QC (H), and the corresponding concentrations of quality control substances in QC (L) and QC (H) are shown in Table 7.
TABLE 7 quality control product concentration (in ng/mL)
| Composition of the components | QC(L) | QC(H) |
| Salidroside | 50 | 1000 |
| Tyrosol | 50 | 1000 |
| Trapeziwei (Chinese character) | 50 | 1000 |
| Rhodiola rosea extract | 50 | 1000 |
| Quercetin | 50 | 1000 |
The instrument and reagent product information involved in the invention is as follows:
salidroside (CAS number: 10338-51-9, purity not less than 98%, lot number: S101157), shanghai Ala Ding Shiji; tyrosol (CAS number: 501-94-0, purity > 98%, batch number: 188255), sigma, USA; corp vitamin (CAS number: 84954-92-7, purity > 98%, lot number: R115719), shanghai Ala Ding Shiji; rhodiola rosea (CAS number: 86831-54-1, purity greater than or equal to 98%, lot number: R890362), shanghai Meilin reagent; quercetin (CAS number: 117-39-5, purity greater than or equal to 99.1%, batch number: Q111273), shanghai Ala Ding Shiji; commercial rhodiola rosea extract (the nominal rhodioside is more than or equal to 1 percent), and the biological technology of the western blue-green cereal Co., ltd; chromatographic grade methanol and acetonitrile, carbofuran corporation, usa; chromatographic grade formic acid, dikma Reagent company; the water is self-made primary water in a laboratory, and the requirements meet GB/T6682. Agilent 1290UPLC ultra performance liquid chromatography tandem ulvo triple quadrupole mass spectrometer, column: ZORBAX Eclipse Plus C18 chromatography column (3.0 mm. Times.50 mm,1.8 μm).
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. The method for measuring the active ingredients in the rhodiola rosea extract is characterized by comprising the following steps of:
s1, taking rhodiola rosea extract powder, adding an extract and an internal standard solution, performing ultrasonic extraction, cooling to room temperature, standing, sucking supernatant, and performing centrifugal purification and filtering by a microporous filter membrane to obtain a sample to-be-detected liquid;
s2, dissolving salidroside, tyrosol, zoysin, salidroside, quercetin standard substances and an internal standard solution with methanol to prepare a series of standard working solutions;
s3, analyzing the sample to-be-detected liquid and the series standard working solution by adopting an ultra-high performance liquid chromatography-tandem mass spectrometer, and quantitatively analyzing the content of salidroside, tyrosol, zoysin, salidroside and quercetin in the sample to-be-detected liquid by adopting an internal standard method.
2. The method according to claim 1, wherein step S1 comprises the steps of:
s11, weighing 0.1g of rhodiola rosea extract powder;
s12, adding 100ml of extract, adding 0.5ml of internal standard solution, weighing, performing ultrasonic extraction for 30min, cooling to room temperature, and supplementing the quality loss with the extract;
s13, standing for 5min, taking 2mL of supernatant into a centrifuge tube, and carrying out vortex centrifugation for 5min to obtain the supernatant;
s14, taking 1mL of supernatant, placing the supernatant into a brown volumetric flask, fixing the volume of the extract to 10mL, and filtering the extract by a microporous membrane with the size of 0.22 mu m to obtain a sample to-be-detected liquid.
3. The method according to claim 1, wherein in step S1, the extract is an aqueous methanol solution and the internal standard solution is a theophylline solution.
4. A method according to claim 3, wherein the extract is an 80% aqueous methanol solution; the internal standard solution is 500ng/mL theophylline solution prepared by methanol.
5. The method according to claim 1, wherein step S2 comprises the steps of:
s21, taking a standard substance of salidroside, tyrosol, zoysin, salidroside and quercetin, and preparing a single standard substance stock solution with the concentration of 1mg/mL by using methanol;
s22, respectively taking equal volumes of single standard stock solution, and preparing 100 mug/ml mixed standard solution by using methanol;
s23, respectively taking 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 and 1.0ml of mixed standard substance solution, adding into a volumetric flask, respectively adding 50 mu L of internal standard solution, and adding methanol to a volume of 10ml to prepare a series of standard working solutions.
6. The method according to claim 1, wherein in step S3, the eluent used in the liquid chromatography is an aqueous ammonium acetate solution and a formic acid-methanol solution.
7. The method according to claim 6, wherein in step S3, the eluent used in the liquid chromatography is 2mmol/L ammonium acetate aqueous solution and 0.05% formic acid-methanol solution.
8. The application of the method for measuring the active ingredients in the rhodiola rosea extract is characterized in that the application is a detection kit for preparing the active ingredients in the rhodiola rosea extract.
9. The use according to claim 8, wherein the kit comprises: eluent, internal standard solution, standard solution and extract.
10. The use according to claim 9, wherein the eluent is an aqueous ammonium acetate solution and a formic acid-methanol solution; the extracting solution is methanol aqueous solution, and the internal standard solution is theophylline solution; the standard solution comprises: salidroside, tyrosol, zoysin, rhodiola rosea and quercetin.
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