CN111103365A - Method for simultaneously, qualitatively and quantitatively analyzing 6 components in total flavonoids of wild jujube leaves based on MRM-IDA-EPI mode - Google Patents
Method for simultaneously, qualitatively and quantitatively analyzing 6 components in total flavonoids of wild jujube leaves based on MRM-IDA-EPI mode Download PDFInfo
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Abstract
The invention belongs to the technical field of traditional Chinese medicine active ingredient content determination, and provides a method for simultaneously, qualitatively and quantitatively analyzing 6 ingredients in total flavonoids in wild jujube leaves based on an MRM-IDA-EPI mode, wherein a wild jujube leaf total flavonoids sample is subjected to methanol ultrasonic extraction, diluted by 2 times and then filtered through a microporous filter membrane to obtain a test solution; using UPLC-QTRAP-MS technology to obtain MRM quantitative ion pair and EPI secondary full-scanning ion fragment qualitative data by MRM-IDA-EPI, and realizing simultaneous qualitative and quantitative analysis of one-time sample introduction; a standard curve for simultaneously measuring 6 components is established by using a series of concentration control solution, and the peak areas and the concentrations of the 6 chemical components have good linear relation in a measured concentration range. The RSD values of the repeatability results are less than 5 percent, and the requirements of pharmacopoeia regulations are met. The established method is stable and reliable, complete separation of two pairs of isomers is realized within 24 minutes, and the method is more accurate in quantification.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine active ingredient content determination, and particularly relates to a method for simultaneously, qualitatively and quantitatively analyzing 6 ingredients in total flavonoids in wild jujube leaves based on an MRM-IDA-EPI mode.
Background
The leaf of Zizyphus jujube is Zizyphus jujube of Zizyphus of RhamnaceaeZiziphusjujubaMill. Var.spinosaDried leaves of (Bunge) Hu ex H.F. Chou. It is recorded in Bencao gang mu, also known as Chin Ye, and has the actions of healing wound, removing toxicity and curing shank ecthyma. Modern pharmacological research shows that the wild jujube leaves have obvious health-care effects of tranquilizing and allaying excitement, resisting oxidation, protecting liver and the like. Chemical composition research shows that the wild jujube leaves contain rich nutrient components such as flavone, saponin, amino acid, nucleoside, trace elements and the like. In the early stage, the content of total flavonoids in wild jujube leaf samples of 9 different producing areas in Shanxi province is determined by an ultraviolet spectrophotometry, and the highest content of the total flavonoids in wild jujube leaf samples in Shanxi Jinzhong area is found to be 8 percent; UHPLC-Q-active orbital hydrazine high resolution mass spectrum and HPLC-CL technology are adopted to analyze and find that the wild jujube leaves mostly take quercetin and kaempferol as the main flavone, and H can be removed on line2O2And superoxide anion radicals. Therefore, the extraction and purification process of total flavonoids (ZSFF) of wild jujube leaves is optimized, and the total flavonoids with the purity of 41.5 percent are enriched. However, the quality control method of the total flavonoids in wild jujube leaves is still blank at present.
The ultra-high performance liquid chromatography-tandem quadrupole composite linear ion trap mass spectrometry (UPLC-QTRAP-MS) technology is an effective and feasible analysis method for researching complex components of traditional Chinese medicines. It is well known that traditional Multiple Reaction Monitoring Scans (MRMs) are the "gold standard" for quantitative analysis; and the Enhanced product ion scanning (EPI) mode of the linear ion trap can obtain the corresponding secondary fragment, and plays a qualitative role in compound quantification. When the method is based on data dependency acquisition (IDA), multi-reaction monitoring-triggered enhanced ion scanning (MRM-IDA-EPI) can be realized, and quantitative data of high-sensitivity MRM and a secondary full-scan mass spectrogram can be obtained at the same time for qualitative confirmation by one-time sample injection. The method can not only realize accurate quantification of a specific target compound, but also qualitatively identify the compound with high sensitivity in one-time operation without reducing qualitative and quantitative analysis performances.
Disclosure of Invention
The invention provides a method for simultaneously, qualitatively and quantitatively analyzing 6 components in total flavonoids in wild jujube leaves based on an MRM-IDA-EPI mode, wherein the 6 components in the total flavonoids in wild jujube leaves are subjected to multi-reaction monitoring-triggered enhanced ion scanning mode (MRM-IDA-EPI) detection, and the mode method can realize simultaneous acquisition of MRM quantitative data of 6 compounds in the total flavonoids in wild jujube leaves by one-time sample injection and simultaneous qualitative confirmation of a secondary full-scanning mass spectrogram.
The technical scheme adopted by the invention is as follows:
a method for simultaneously qualitatively and quantitatively analyzing 6 components in total flavone of folium Ziziphi Spinosae based on MRM-IDA-EPI mode comprises ultrasonically extracting total flavone of folium Ziziphi Spinosae with 70% methanol, diluting 2 times, and filtering with 0.45 μm microporous membrane to obtain test solution; using ultra-high performance liquid chromatography-tandem quadrupole composite linear ion trap mass spectrometry technology, obtaining MRM quantitative ion pair and EPI secondary full-scan ion fragment qualitative data by multi-reaction monitoring data dependent scanning-triggering enhanced ion scanning, and simultaneously performing qualitative and quantitative analysis by one-time sample introduction; adopts serial concentration reference substance solutions to establish and simultaneously measure rutin, hyperin, quercetin and quercetin-3-OGlucoside, Quercetin-3-O-standard curves of 6 ingredients of robinin and quercetin, and identifying the above compounds.
Equivalent to quercetin in the total flavonoids of the original medicinal materials3-O-Robinin, rutin, hyperoside, and quercetinMedicine prepared from Chinese medicinal herbs3-OGlucoside, Quercetin-3The contents of rhamnoside and quercetin are 1.04, 0.83, 0.01, 0.03, 0.02 and 0.0005%, respectively.
The method comprises the following specific steps:
(1) preparation of a test solution: precisely weighing 10 mg of total flavonoids powder of folium Ziziphi Spinosae, placing in a bottle with a stopper, precisely adding 25 mL of 70% methanol, weighing to a certain mass, and ultrasonically extracting for 20 min; standing at room temperature, weighing, and supplementing with 70% methanol to reduce loss; diluting with methanol by 2 times, and filtering with 0.45 μm microporous membrane to obtain filtrate as sample solution;
(2) preparation of mixed control solution: precisely weighing the reference substance, adding 70% methanol to prepare quercetin-3-OPreparing hyperin and quercetin-3-O-glucoside, quercitrin and quercetin single control solution; precisely sucking the single reference stock solutions respectively, mixing, adding methanol to obtain quercetin-containing solution with concentration of 50 μ g/mL3-O-robinin, rutin 52.8 μ g/mL, hyperin 0.544 μ g/mL, quercetin 1.034 μ g/mL-3-O-a mixed control solution of glucoside, 1.014 μ g/mL quercetin, 0.148 μ g/mL quercetin;
(3) and (3) detection: injecting the obtained test solution and the reference solution into an ultra-high performance liquid chromatography-tandem quadrupole composite linear ion trap mass spectrometer, and detecting according to the following chromatographic and mass spectrum conditions:
chromatographic conditions are as follows: the chromatographic column is ACQUITY UPLC HSST 3150 mm multiplied by 2.1 mm, 1.8 μm, and the mobile phase is as follows: a-B = acetonitrile-water containing 0.1% formic acid, gradient elution: 0-5 min, 15% A; 5-13 min, 15-16% A; 13-17 min, 16% A; 17-20 min, 16-16.5% A; 20-24 min, 16.5-100% A; the flow rate is 0.3 mL/min; the column temperature is 35 ℃; sampling amount is 2 muL;
mass spectrum conditions: an ion source: electrospray ion source ESI; scanning mode: MRM negative ion scanning; the source injection voltage IS IS-4500V; atomization temperature: 550 ℃; air Curtain gas, N2: 40.0 psi; atomizing gas GS1, N2：50psi; auxiliary gas GS2, N2：50 psi；
MS of 6 quantitative ion pairs1/MS2609.4/301.0, 609.4/301.1, 463.2/301.2, 447.2/301.2 and 301.1/151.2 respectively;
DP/CE is-80/-50, -100/-50, -60/-50, -100/-35, -50/-40, -50/-30, respectively;
MRM-IDA-EPI mode: IDA parameters: select 1 to 1 most intense peaks; selecting Dynamic Background Subtraction (Alter Dynamic Background Subtraction of Survey scan); threshold value Which exceeds: 500 cps; mass deviation Mass Tolerance: 250 mDa;
the EPI parameter is set to scan range: 50-650 Da; scan rate: 1000 Da/s; dynamically filling Dynamic fill time in the ion trap; DP: -80 eV; EP: -10 eV; and (5) CES: -45 ± 15 eV;
(4) the EPI data is compared with reference substances and reference documents through parent ion mass-to-charge ratio and daughter ion scanning map information, the structure of 6 chemical components in the total flavonoids of the wild jujube leaves is confirmed, and the MRM quantitative data respectively calculates the content of the 6 components in the total flavonoids of the wild jujube leaves by adopting a standard curve method.
The invention establishes a method for simultaneously qualitatively and quantitatively determining 6 chemical components in the total flavonoids in the wild jujube leaves in an MRM-IDA-EPI mode, and the method is used for evaluating the process stability of the total flavonoids in the wild jujube leaves prepared by extraction and purification.
The invention has the beneficial effects that: the invention adopts LC-MS/MS technology based on MRM-IDA-EPI mode to treat rutin, hyperoside, quercetin and quercetin-3-OGlucoside, Quercetin-3-OMeasuring the content of 6 chemical components of the locust glucoside and the quercetin, and identifying the compounds.
The total flavone of wild jujube leaves has more isomerides, and the method of the invention divides quercetin-3-O-robinin, rutin and hyperin, quercetin-3-OThe two pairs of isomers of the glucoside have more accurate quantification. Establishing a standard curve for simultaneously measuring 6 components by using serial concentration reference substance solutions, and obtaining peak areas and peak areas of 6 chemical components in total flavonoids of wild jujube leavesThe concentrations have good linear relation in the measured concentration range, and r is more than 0.9995. The RSD values of the repeatability results are less than 5 percent, and the requirements of pharmacopoeia regulations are met. The sample recovery rate is 99-108%, and the precision RSD is 1.83-2.95%. Quercetin in total flavonoids (equivalent to raw medicinal materials)3-O-Acacia glucoside, rutin, hyperin, quercetin-3-OGlucoside, Quercetin-3The contents of rhamnoside and quercetin are 1.04, 0.83, 0.01, 0.03, 0.02 and 0.0005%, respectively.
The established method is stable and reliable, and realizes quercetin-3-O-robinin, rutin and hyperin, quercetin-3-OThe complete separation of two pairs of isomers of glucoside can be used for simultaneously determining the content of 6 components in the total flavone of wild jujube leaves. The method has accurate quantification.
The method can obtain an EPI map and qualitatively analyze 6 compounds in the total flavonoids of the wild jujube leaves. The method can realize one-time sample injection and simultaneously obtain the MRM quantitative data and the second-level full-scan mass spectrogram (EPI) of 6 compounds in the total flavonoids of the leaves of the wild jujube, and is accurate and rapid.
Drawings
FIG. 1 is a chemical structural diagram of 6 components, wherein 1: quercetin-3-O-robinin; 2: rutin; 3: hyperin; 4: quercetin-3-O-a glucoside; 5: quercetin; 6: quercetin.
FIG. 2 is the MRM chromatogram of the control (A) and the total flavonoids from wild jujube leaves (B), wherein 1: quercetin-3-O-robinin; 2: rutin; 3: hyperin; 4: quercetin-3-O-a glucoside; 5: quercetin; 6: quercetin.
FIG. 3 is EPI profile of total flavonoids from leaves of Ziziphi Spinosae in MRM-IDA-EPI mode for 6 compounds, wherein 1: quercetin-3- O-robinin; 2: rutin; 3: hyperin; 4: quercetin-3-O-a glucoside; 5: quercetin; 6: quercetin.
Detailed Description
The present invention will be described more fully with reference to the following examples, but it should not be construed that the scope of the above subject matter is limited to the following examples, and that all the technologies that can be realized based on the above contents of the present invention are within the scope of the present invention.
The reagents and equipment used in the examples were as follows:
1. the instrument comprises the following steps: an Agilent1290 ii hplc equipped with a G7120A quaternary pump, a G7116B column oven, a G7167B autosampler (Agilent, usa); 3200 QTRAP triple quadrupole linear ion trap mass spectrometer equipped with an ESI ion source, Analyst 1.5.2 software data System (AB SCIEX, USA); neoferrite 13R high speed refrigerated centrifuge (Likang, China); one hundred thousand analytical balances of the CPA225D type (Sartorius, germany); Milli-Q ultrapure water system (Millipore, USA).
2. Drugs and reagents: reference quercetin-3-ORobinin (batch No. 20161227), rutin (batch No. C1424058), hyperoside (batch No. 20161022), quercetin-3-OGlucoside (batch number HI 105405198) and quercetin (batch number 20160830) were purchased from chenguan biotechnology limited. Quercetin (batch No. Q111274) was purchased from Aladdin reagent (Shanghai) Co., Ltd. The mass fraction of the reference substance is more than 98% by HPLC normalization method. Methanol, acetonitrile and formic acid were purchased chromatographically pure from Thermo Fisher, usa. Ultrapure water was prepared from Milli-Q, and the remaining reagents were analytical grade. Folium Ziziphi Spinosae was collected from Shanxi province, Shanzhong city (sample number: 20170627004) 10 months in 2017, and was identified as Rhamnaceae plant fructus Ziziphi Spinosae by professor of Kagaku, Leishu-Leohui, Kagaku, KagaZiziphus jujubeMill. var.spinosa(Bunge) dried leaves of Huex H.F.Chou. Collected and naturally dried in the shade, and is reserved in the research room of the modern research center of traditional Chinese medicine of Shanxi university. The wild jujube leaf total flavonoids are obtained by purifying in 2018, 11 months and 17 days, the yield is 10 percent, and the purity is 41 percent by an ultraviolet spectrophotometry.
Example 1: solution preparation and chromatographic Mass Spectrometry conditions
Preparation of a test solution: weighing 10 mg of total flavonoids powder of wild jujube leaves, precisely weighing, placing in a conical flask with a plug, precisely adding 25 mL of 70% methanol, weighing, and ultrasonically extracting for 20 min. Standing at room temperature, weighing, and supplementing with 70% methanol to reduce loss. Filtering with 0.45 μm microporous membrane, and collecting the filtrate.
Preparation of control solutions: taking a proper amount of reference substance, precisely weighing, adding 70% methanol to prepare quercetin-3-OPreparing hyperin and quercetin-3-OSingle control solutions with glucoside, quercitrin and quercetin concentrations all 0.02 mg/mL. The control stock solutions were precisely pipetted with an appropriate amount of methanol to give mixed control solutions with concentrations of 50. mu.g/mL, 52.8. mu.g/mL, 0.544. mu.g/mL, 1.034. mu.g/mL, 1.014. mu.g/mL, and 0.148. mu.g/mL, respectively.
Chromatographic conditions are as follows: the chromatographic column is ACQUITY UPLC HSST3A (150 mm × 2.1 mm, 1.8 μm) column, a mobile phase of acetonitrile (A) -water (B) (containing 0.1% formic acid), and gradient elution for 0-5 min, 15% A; 5-13 min, 15-16% A; 13-17 min, 16% A; 17-20 min, 16-16.5A; 20-24 min, 16.5-100A; the flow rate is 0.3 mL/min; the column temperature is 35 ℃; and the sample size is 2 muL.
Mass spectrum conditions: an ion source: electrospray ion source (ESI); scanning mode: MRM negative ion scanning; the source injection voltage (IS) IS-4500V; atomization temperature: the temperature is 550 ℃; air Curtain gas (N, Curtain gas)2): 40.0 psi; atomizing gas (GS 1, N)2): 50 psi; auxiliary gas (GS 2, N)2): 50 psi; . The EPI qualitative parameters are as follows, dynamic collision energy CEs: 40 +/-15 eV; the scanning speed is 1000 Da/s; the scanning threshold is 500 cps. The detailed mass spectral parameters of the 6 quantitative ion pairs are shown in table 1.
Table 16 mass spectral data of the components
Example 2 qualitative analysis: the EPI parameters were optimized for 6 compounds according to the above chromatographic and mass spectrometric conditions, respectively, to obtain high quality secondary fragment information, see table 2. Taking rutin as an example, reference documents (plum self-red, Weiyue, Van-Ying, etc.. rutin's ElectricitySpray ion trap mass spectrometry) found that in negative ion mode, rutinose was lost to form characteristic ion fragments 301, and fragments 301 continued to fragment to form fragments 273.0, 255.1, 179.0, 151.0, etc. Other 5 compounds were identified by comparison with controls and literature (Yan, Du-Chenghui, Von-Hongyao, etc.. integrate UHPLC-QOxctive orbital hydrazine high resolution mass spectrometry with HPLC-CL techniques for analysis of antioxidant active ingredients in Ziziphus jujube leaves; Phyllosa, Yanming, Yanxiangyu, etc.. UPLC-QTOF-MS analysis of chemical ingredients of Ziziphus jujube leaves in Taihang mountainous area) as follows: quercetin-3-O-robinin, hyperoside, quercetin-3-OGlucoside, Quercetin-3-rhamnoside, quercetin.
TABLE 2 ion fragment of 6 components injected by needle pump in full scan mode
Example 3 methodology review:
1. linear range investigation: precisely measuring a proper amount of the mixed reference substance solution, and gradually diluting the mixed reference substance solution with 7 concentration gradients by using 70% methanol. According to the above-mentioned chromatographic and mass spectrometric analysis, the peak area of the index component is used as ordinate (C: (Y) Mass concentration is abscissa (X) And performing linear regression to obtain a regression equation, a correlation coefficient (r) and a linear range, wherein the results show that the correlation coefficients (r) of the mass concentration and the peak area of the 6 index components are both greater than 0.9995, which indicates that the method has good linear relation. The mixed control solutions were diluted from high to low according to the concentration gradient of mass, and the amount of each control at the signal-to-noise ratio S/N =10 and S/N =3 was used as the limit of quantitation (LOD) and the limit of detection (LOD), respectively, and the results are shown in table 3.
TABLE 36 regression equation, correlation coefficient, Linear Range, LOQ and LOD for the components
2. And (3) precision test: precisely sucking the same mixed reference solution, and performing chromatography and mass spectrometryContinuously feeding samples for 6 times in the same day, recording peak areas of 6 index components, and calculating RSD values of the peak areas. The results are shown in Table 4, quercetin-3- O-robinin, rutin, hyperoside, quercetin-3-O-The RSD values of the peak areas of the glucoside, the quercitrin and the quercetin are respectively 2.82%, 2.44%, 1.99%, 2.91%, 2.65% and 2.56%, which indicates that the precision of the instrument is good. And (3) continuously taking 3 days, precisely sucking the mixed reference substance solution every day, continuously feeding samples for 3 times in the same day according to the chromatographic and mass spectrum conditions, recording the peak areas of the 6 index components for 3 days, and calculating the RSD value of the peak areas. The results are shown in Table 4, quercetin-3-O-robinin, rutin, hyperoside, quercetin-3-O-The RSD values of the peak areas of the glucoside, the quercitrin and the quercitrin are respectively 2.90%, 2.36%, 2.74%, 2.95%, 2.88% and 1.83%, which indicates that the daytime precision of the instrument is good.
Precision, reproducibility, stability of the ingredients of Table 46
3. And (3) stability test: taking the same batch of wild jujube leaf total flavone powder samples, about 10 mg, precisely weighing, preparing a test sample solution according to the method described in example 1, standing at room temperature for 0, 2, 4, 6, 12 and 24 hours, performing sample injection analysis according to the chromatographic and mass spectrum conditions described in example 1, performing analysis by taking peak areas as indexes, calculating RSD values of peak areas of 6 index components, and displaying the results in the form of quercetin-3-O-robinin, rutin, hyperoside, quercetin-3-O-The peak areas RSD of the glucoside, the quercitrin and the quercitrin in 24 h are respectively 2.59%, 2.50%, 2.53%, 2.95% and 1.32%, which indicates that the sample solution is stable in 24 h.
4. And (3) repeatability test: taking 6 parts of the same wild jujube leaf total flavone sample powder, each part of which is about 10 mg, precisely weighing, preparing a test solution according to the method in the embodiment 1, carrying out sample injection analysis according to the chromatographic and mass spectrum conditions in the embodiment 1, respectively recording peak areas of 6 index components, and calculating the mass fraction and the RSD value of the peak areas. Quercetin + in 6 samples3-O-robinin, reedD, hyperin and quercetin-3-O-The mass fractions of the glucoside, the quercitrin and the quercetin are 104, 83.78, 0.95, 2.93, 2.49 and 0.05 mg/g in sequence, and the RSDs are 0.73%, 2.12%, 4.10%, 2.79%, 2.39% and 2.22% respectively, which shows that the precision of the extraction method is good. The results are shown in Table 4.
5. Sample recovery rate test: taking 6 parts of the total flavonoids in the wild jujube leaves with known 6 component contents, each part is about 5 mg, precisely weighing, placing in a conical flask with a stopper, respectively and precisely adding a mixed reference substance solution which is 50%, 100% and 150% of the 6 component contents in the 5 mg sample, preparing a sample solution according to the method of example 1, carrying out sample injection analysis according to the chromatographic and mass spectrum conditions of example 1, recording the peak areas of index components, and calculating the average sample adding recovery rate and the corresponding RSD value. As can be seen from Table 5, the accuracy of the extraction method is good.
TABLE 56 sample recovery rates for the components
Example 4 determination of 6 ingredients of the total flavonoids from wild jujube leaves sample: 3 parts of the tested total flavonoids of the wild jujube leaves, each part of which is about 10 mg, are precisely weighed, a test solution is prepared according to the method described in the example 1, sample injection analysis is carried out according to the chromatographic and mass spectrum conditions described in the example 1, the peak areas of the index components are recorded, and the contents of 6 index components are calculated. Quercetin + in 3 samples3-O-robinin, rutin, hyperoside, quercetin-3-O-Glucoside and quercetin-3The contents of rhamnoside and quercetin are 10.410, 8.298, 0.096, 0.295, 0.246 and 0.005%, respectively, wherein quercetin is contained in the composition-3-OThe contents of robinin and rutin, both of which are 18.71% of the total flavone extract of wild jujube leaves, are high, as shown in table 6.
TABLE 66 Total Flavonoids content of the compounds in Zizyphus jujube leaves
Claims (4)
1. A method for simultaneously, qualitatively and quantitatively analyzing 6 components in total flavonoids of wild jujube leaves based on an MRM-IDA-EPI mode is characterized in that: ultrasonically extracting a wild jujube leaf total flavone sample by using 70 percent methanol, diluting by 2 times, and then passing through a 0.45 mu m microporous filter membrane to obtain a test solution; using ultra-high performance liquid chromatography-tandem quadrupole composite linear ion trap mass spectrometry technology, obtaining MRM quantitative ion pair and EPI secondary full-scan ion fragment qualitative data by multi-reaction monitoring data dependent scanning-triggering enhanced ion scanning, and simultaneously performing qualitative and quantitative analysis by one-time sample introduction; adopts serial concentration reference substance solutions to establish and simultaneously measure rutin, hyperin, quercetin and quercetin-3-OGlucoside, Quercetin-3-O-standard curves of 6 ingredients of robinin and quercetin, and identifying the above compounds.
2. The method for simultaneously, qualitatively and quantitatively analyzing 6 components in total flavonoids of zizyphus jujube leaves based on MRM-IDA-EPI mode as claimed in claim 1, wherein: equivalent to quercetin in the total flavonoids of the original medicinal materials3-O-Acacia glucoside, rutin, hyperin, quercetin-3-OGlucoside, Quercetin-3The contents of rhamnoside and quercetin are 1.04, 0.83, 0.01, 0.03, 0.02 and 0.0005%, respectively.
3. The method for simultaneously, qualitatively and quantitatively analyzing 6 components in total flavonoids of zizyphus jujube leaves based on MRM-IDA-EPI mode as claimed in claim 1, wherein: the method comprises the following specific steps:
(1) preparation of a test solution: precisely weighing 10 mg of total flavonoids powder of folium Ziziphi Spinosae, placing in a bottle with a stopper, precisely adding 25 mL of 70% methanol, weighing to a certain mass, and ultrasonically extracting for 20 min; standing at room temperature, weighing, and supplementing with 70% methanol to reduce loss; diluting with methanol for 2 times, filtering with 0.45 μm microporous membrane, and collecting filtrate to obtain sample solution;
(2) preparation of mixed control solution: precisely weighing the reference substance, adding 70% methanol to prepare quercetin-3-O-robinin and rutin single reference stock solutions, prepared to concentrations of 0.02 each by adding 70% methanolHyperin and quercetin in mg/mL3-O-glucoside, quercitrin and quercetin single control solution; precisely sucking the single reference stock solutions respectively, mixing, adding methanol to obtain quercetin-containing solution with concentration of 50 μ g/mL3-O-robinin, rutin 52.8 μ g/mL, hyperin 0.544 μ g/mL, quercetin 1.034 μ g/mL-3-O-a mixed control solution of glucoside, 1.014 μ g/mL quercetin, 0.148 μ g/mL quercetin;
(3) and (3) detection: injecting the obtained test solution and the reference solution into an ultra-high performance liquid chromatography-tandem quadrupole composite linear ion trap mass spectrometer, and detecting according to the following chromatographic and mass spectrum conditions:
chromatographic conditions are as follows: the chromatographic column is ACQUITY UPLC HSST3150 mm multiplied by 2.1 mm, 1.8 μm, and the mobile phase is as follows: a-B = acetonitrile-water containing 0.1% formic acid, gradient elution: 0-5 min, 15% A; 5-13 min, 15-16% A; 13-17 min, 16% A; 17-20 min, 16-16.5% A; 20-24 min, 16.5-100% A; the flow rate is 0.3 mL/min; the column temperature is 35 ℃; sampling amount is 2 muL;
mass spectrum conditions: an ion source: electrospray ion source ESI; scanning mode: MRM negative ion scanning; the source injection voltage IS IS-4500V; atomization temperature: 550 ℃; air Curtain gas, N2: 40.0 psi; atomizing gas GS1, N2: 50 psi; auxiliary gas GS2, N2：50 psi；
MS of 6 quantitative ion pairs1/MS2609.4/301.0, 609.4/301.1, 463.2/301.2, 447.2/301.2 and 301.1/151.2 respectively;
DP/CE is-80/-50, -100/-50, -60/-50, -100/-35, -50/-40, -50/-30, respectively;
MRM-IDA-EPI mode: IDA parameters: select 1 to 1 most intense peaks; selecting Dynamic Background Subtraction (Alter Dynamic Background Subtraction of Survey scan); threshold value Which exceeds: 500 cps; mass deviation Mass Tolerance: 250 mDa;
the EPI parameter is set to scan range: 50-650 Da; scan rate: 1000 Da/s; dynamically filling Dynamic fill time in the ion trap; DP: -80 eV; EP: -10 eV; and (5) CES: -45 ± 15 eV;
(4) the EPI data is compared with reference substances and reference documents through the information of parent ion mass-to-charge ratio, daughter ion scanning maps and the like, the structure of 6 chemical components in the total flavonoids of the wild jujube leaves is confirmed, and the MRM quantitative data respectively calculates the content of the 6 components in the total flavonoids of the wild jujube leaves by adopting a standard curve method.
4. Use of the method of claim 3, wherein: the application in the process stability evaluation of the total flavonoids in the wild jujube leaves prepared by extraction and purification.
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