CN108181400B - Method for simultaneously measuring contents of 6 chemical components in spina date seed water decoction by UHP L C-QQQ-MS/MS - Google Patents
Method for simultaneously measuring contents of 6 chemical components in spina date seed water decoction by UHP L C-QQQ-MS/MS Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Steroid Compounds (AREA)
Abstract
The invention relates to a method for simultaneously determining the contents of 6 chemical components in a spina date seed water decoction by adopting UHP L C-QQQ-MS/MS technology, belonging to the field of analysis of traditional Chinese medicine components.
Description
Technical Field
The invention relates to a method for simultaneously determining the contents of 6 chemical components in a spina date seed water decoction by adopting UHP L C-QQQ-MS/MS technology, belonging to the field of analysis of traditional Chinese medicine components.
Background
Although researchers have studied the content determination of chemical components in the spina date seeds at present, the spina date seeds contain numerous components such as flavonoids, saponins, alkaloids, triterpenes and the like and are limited by the performance of a detector of the traditional detection technology HP L C-DAD, and the content determination of various types of chemical components in the spina date seeds cannot be simultaneously performed rapidly, accurately and sensitively.
Disclosure of Invention
In order to solve the existing problems, the invention adopts UHP L C-QQQ-MS/MS technology to establish a method for simultaneously measuring the content of 6 different chemical components in the spina date seed water decoction, and the 6 chemical components structurally cover flavonoids, saponins and triterpenes, so that the established content measuring method can comprehensively, accurately and deeply control the quality of the spina date seed.
The invention adopts the following technical scheme:
1. preparation of a test solution:
according to the clinical application experience of the spina date seeds, precisely weighing 10g of the spina date seeds, adding 10 times of water into a decoction pot, soaking for 30min, boiling with strong fire, keeping a slightly boiling state, decocting for 1h, and filtering out liquid medicine by using 4 layers of gauze; adding 8 times of water into the residue, boiling with strong fire, decocting for 1 hr, filtering with 4 layers of gauze, mixing the decoctions, adding appropriate amount of water, and diluting to 200ml to obtain semen Ziziphi Spinosae water decoction sample.
Precisely transferring 0.2ml of semen Ziziphi Spinosae water decoction sample into 5ml measuring flask, adding methanol to constant volume to 5ml, ultrasonically treating for 10min, cooling, collecting appropriate amount of upper layer solution, centrifuging at 12000r/min for 10min, and collecting supernatant as test solution;
2. preparing a mixed reference substance solution:
accurately weighing appropriate amount of spinosin, 6' -feruloyl spinosin, spina date seed saponin A, spina date seed saponin B, camellianin B and betulinic acid reference substances respectively, adding methanol for dissolving and diluting, and shaking up to obtain mixed reference substance solutions with concentrations of 6.312 μ g/ml, 5.885 μ g/ml, 0.991 μ g/ml, 0.668 μ g/ml, 0.640 μ g/ml and 4.504 μ g/ml respectively;
3. the determination method comprises the following steps:
the concentration of 6 chemical components in the mixed reference substance and the test solution is measured by adopting a UHP L C-QQQ-MS/MS method.
Chromatographic conditions are as follows: the chromatographic column is Agilent C18(2.1mm × 100mm,1.8 μm) reverse phase chromatography column, gradient elution with A (water) -B (acetonitrile) as mobile phase, elution procedure:0-1.2min, the volume fraction of acetonitrile linearly increases from 5% to 31%, 1.2-2.5min, the volume fraction of acetonitrile linearly increases from 31% to 95%, 2.5-4.5min, the volume fraction of acetonitrile linearly increases from 95% to 100%, 4.5-5min, the volume fraction of acetonitrile remains at 100%. Flow rate: 0.3 ml/min; column temperature 40 ℃, sample introduction: 1 μ l.
Mass spectrum conditions: using ESI-The mode is detected, and the scanning mode adopts a multi-reaction monitoring mode (MRM). The parameters are as follows: the ion source temperature is 550 ℃; atomizing air 55 psi; auxiliary heating gas 55 psi; air curtain air 35 psi; nitrogen was used as the spray assist gas. The mass spectrum parameters Q1/Q3(m/z) of each component (the spinosad, 6' -feruloyl spinosad, spina date seed saponin A, spina date seed saponin B, camellin B and betulinic acid) are respectively as follows: 607.5/427.1, 783.2/427.4, 1205.9/1073.9, 1043.6/911.5, 725.2/284.0, 455.3/455.3; the declustering voltage (DP) is-26.46V, -25.83V, -60.00V, -27.20V, -23.72V and-97.00V respectively; the Collision Energy (CE) is: 41.60V, -50.92V, -60.00V, -49.77V, -53.07V, -20.00V.
The step 3 assay includes the determination of the linear relationship: precisely sucking the mixed reference substance solution, diluting according to a multiple dilution method to obtain 6 series reference substance solutions with different mass concentrations and dilution multiples of 1, 2, 4, 8, 16 and 32, respectively measuring under a test condition, recording chromatographic peak areas of 6 chemical components, drawing a standard curve by taking the reference mass concentration as a horizontal coordinate (X) and the peak area as a vertical coordinate (Y), and performing linear regression to obtain a standard curve equation.
The determination method in the step 3 comprises the following steps of: precisely sucking the mixed reference substance solution, continuously injecting samples for 6 times under the test condition, and recording peak areas of 6 chemical components.
The step 3 assay described above includes a reproducible assay: taking the same batch of wild jujube seed powder samples, preparing 6 parts of test solution in parallel according to the preparation method of the test solution, respectively measuring under the test condition, and recording peak areas of 6 chemical components.
The step 3 assay described above includes stability determination: taking the test solution, measuring under the test condition for 0, 2, 4, 8, 12, 24 and 48h respectively, and recording the peak areas of the 6 chemical components.
The step 3 comprises a sample recovery test: precisely sucking 0.1ml of spina date seed water decoction sample with known content under the item of preparation of test solution, placing the sample in a 5ml measuring flask, respectively and precisely adding 0.1ml of reference substance solution (the concentrations of the spinosad, 6' -feruloylsilonin, the spina date seed saponin A, the spina date seed saponin B, the camellianin B and the betulinic acid are 157.564, 73.535, 12.522, 8.042, 8.076 and 28.029 mu g/ml respectively) with certain concentration, adding a proper amount of methanol to fix the volume to 5ml, carrying out ultrasonic treatment for 10min, cooling, taking supernatant liquid to centrifuge at 12000r/min for 10min, sucking the supernatant liquid as the test solution, paralleling 6 parts, measuring under the test condition, recording peak areas of 6 chemical components, and calculating the sample adding recovery rate of the method.
The invention has the advantages that: 1. the experimental result shows that under the optimized test condition, the 6 chemical components tested in the spina date seed water decoction can be effectively separated within 5min under the gradient elution condition of a water-acetonitrile system, and the testing method has short period and high separation efficiency; 2. the mass spectrum has high sensitivity and strong selectivity, and can accurately give mass number information of components while accurately measuring the content of the components, thereby achieving the effect of identifying compounds at the same time; 3. the method simultaneously and quantitatively analyzes 6 different chemical components in the spina date seed water decoction, has high specificity, simple operation, good repeatability and accurate and reliable result, and can provide technical support and theoretical reference for clinical application and quality control of the spina date seeds.
Drawings
FIG. 1 is a mass chromatogram of extracted ion streams of 6 chemical components in a mixed control solution according to the present invention, wherein
A: a spinosin; b: 6' -feruloylscinolone acetonide; c: jujuboside A; d: jujuboside B; e: camellin B; f: betulinic acid.
FIG. 2 is a chemical structural diagram of 6 components to be tested in the test solution of the present invention, wherein
A: a spinosin; b: 6' -feruloylscinolone acetonide; c: jujuboside A; d: jujuboside B; e: camellin B; f: betulinic acid.
Detailed description of the invention
The foregoing aspects of the present invention are described in further detail by way of examples, but it should not be construed that the scope of the above-described subject matter is limited to the following examples, and that all the technologies implemented based on the above-described aspects of the present invention are within the scope of the present invention.
Example 1A method for simultaneously measuring the contents of 6 chemical components in spina date seed water decoction by UHP L C-QQQ-MS/MS comprises the following steps:
1. decoction pieces, instruments and reagents
1.1 sources of decoction pieces
Semen Ziziphi Spinosae is purchased from Holly field decoction pieces of Anhui (Holy, China); the products are all certified by professor Chenjianwei of the college of medicine of Nanjing university of traditional Chinese medicine.
1.2 instruments
Shimadzu high performance liquid chromatography system (Shimadzu L C30 AD pump, SI L30 AC autosampler, 30AC column oven), Triple quadrupolole 5500 Mass spectrometer (AB Sciex, USA) ESI ion source, Mass Spectroscopy workstation: AnalystTF1.6, an ultrasonic cleaner (KQ-500, ultrasound apparatus Co., Ltd., Kunshan), an AG 285 electronic balance (METT L ER TO L EDO, d is 0.01mg), a water purifier (Nanjing Yipudida development Co., Ltd., model: EPED-10TF, No. 0903090818), and a high-speed centrifuge (Changshan Xiangzhi centrifuge apparatus Co., Ltd., model: XZ-6G).
1.3 reagent
Spiromone (batch No. S-043-150626) (purity > 98%), 6' -feruloylspiromone (batch No. A-011-; theabroside B (lot number 170313-080) (purity > 98%), betulinic acid (lot number JYB 201701) (purity > 98%) control was purchased from Nanjing Jinyibai Biotech Co., Ltd.
Acetonitrile is mass spectrum pure (Merck company, USA), and ultrapure water is prepared by self.
2. Method and results
2.1 preparation of Mixed control solutions
Accurately weighing appropriate amount of spinosin, 6' -feruloyl spinosin, spina date seed saponin A, spina date seed saponin B, camellianin B and betulinic acid reference substances respectively, adding methanol for dissolving and diluting, and shaking up to obtain mixed reference substance solutions with concentrations of 6.312 μ g/ml, 5.885 μ g/ml, 0.991 μ g/ml, 0.668 μ g/ml, 0.640 μ g/ml and 4.504 μ g/ml respectively;
2.2 preparation of test solutions
According to the clinical application experience of the spina date seeds, precisely weighing 10g of the spina date seeds, adding 10 times of water into a decoction pot, soaking for 30min, boiling with strong fire, keeping a slightly boiling state, decocting for 1h, and filtering out liquid medicine by using 4 layers of gauze; adding 8 times of water into the residue, boiling with strong fire, decocting for 1 hr, filtering with 4 layers of gauze, mixing the decoctions, adding appropriate amount of water to obtain a volume of 200ml, and collecting the decoction as semen Ziziphi Spinosae water decoction sample. Precisely transferring 0.2ml of semen Ziziphi Spinosae water decoction sample into 5ml measuring flask, adding methanol to constant volume to 5ml, ultrasonically treating for 10min, cooling, collecting appropriate amount of upper layer solution, centrifuging at 12000r/min for 10min, and collecting supernatant as test solution;
2.3 chromatographic conditions and Mass Spectrometry parameter conditions
Chromatographic conditions are as follows: the chromatographic column is Agilent C18(2.1mm × 100mm,1.8 μm) reverse phase chromatography column, gradient elution is carried out with A (water) -B (acetonitrile) as mobile phase, the elution procedure is 0-1.2min, the volume fraction of acetonitrile is linearly increased from 5% to 31%, 1.2-2.5min, the volume fraction of acetonitrile is linearly increased from 31% to 95%, 2.5-4.5min, the volume fraction of acetonitrile is linearly increased from 95% to 100%, 4.5-5min, the volume fraction of acetonitrile is maintained at 100%, the flow rate is 0.3ml/min, the column temperature is 40 ℃, and the sample injection amount is 1 μ l.
Mass spectrum conditions: using ESI-The mode is detected, and the scanning mode adopts a multi-reaction monitoring mode (MRM). The parameters are as follows: the ion source temperature is 550 ℃; atomizing air 55 psi; auxiliary heating gas 55 psi; air curtain air 35 psi; nitrogen was used as the spray assist gas. Each component (spinosin, 6' -feruloylspirocin, spina date seed saponin A, and Ziziphi SpinosaeBenosaponin B, camellin B, betulinic acid) has mass spectrum parameters Q1/Q3(m/z) respectively: 607.5/427.1, 783.2/427.4, 1205.9/1073.9, 1043.6/911.5, 725.2/284.0, 455.3/455.3; the declustering voltage (DP) is-26.46V, -25.83V, -60.00V, -27.20V, -23.72V and-97.00V respectively; the Collision Energy (CE) is: 41.60V, -50.92V, -60.00V, -49.77V, -53.07V, -20.00V.
2.4 Linear relationship investigation
Precisely sucking the mixed reference substance solution, diluting according to a multiple dilution method to obtain 6 series reference substance solutions with different mass concentrations and dilution multiples of 1, 2, 4, 8, 16 and 32 times respectively, respectively measuring under a test condition, recording chromatographic peak areas of 6 chemical components, drawing a standard curve by taking the reference mass concentration as an abscissa (X) and the peak area as an ordinate (Y), and performing linear regression to obtain a standard curve equation, wherein the result is shown in table 1. The results showed that 6 chemical components were in good linear relationship within the specified range.
TABLE 1 Linear equation, correlation coefficient and linear range of 6 chemical components in semen Ziziphi Spinosae
2.5 precision test
Precisely sucking the reference substance solution, continuously injecting sample for 6 times under the condition of 'item 2.3', and recording the peak areas of 6 chemical components. The results show that the RSD (n ═ 6) of the peak areas of the spinosin, 6' -feruloyl spinosin, spina date seed saponin a, spina date seed saponin B, camellianin B and betulinic acid are 0.60%, 2.51%, 2.75%, 2.91%, 1.12% and 2.62% respectively, which indicates that the precision of the instrument is good.
2.6 repeatability test
Taking the same batch of semen Ziziphi Spinosae decoction pieces, preparing 6 parts of sample solution in parallel according to the method for preparing sample solution under item 2.2, measuring under item 2.3, and calculating the concentration of each chemical component. The results show that the average concentrations of the spinosad, the 6' -feruloyl spinosad, the spina date seed saponin A, the spina date seed saponin B, the camellin B and the betulinic acid are 3.807, 1.451, 0.225, 0.186, 0.101 and 0.863 mu g/ml respectively; RSD is 1.20%, 2.64%, 2.60%, 2.21%, 2.98% and 1.18%, respectively, which indicates that the repeatability of the method is good.
2.7 stability test
The test solution is taken and placed at room temperature, and is respectively measured under the conditions of 'item 2.3' for 0, 2, 4, 8, 12, 24 and 48h, and the peak areas of 6 chemical components are recorded. The results show that the RSD of the peak areas of the spinosad, 6' -feruloyl spinosad, spina date seed saponin A, spina date seed saponin B, camellin B and betulinic acid are respectively 2.96%, 2.53%, 2.82%, 2.10%, 2.31% and 2.72%, which indicates that the test sample is stable when placed at room temperature for 48 hours.
2.8 sample recovery test
Precisely sucking 0.1ml of spina date seed water decoction sample with known content under the item of preparation of test solution, placing the sample in a 5ml measuring flask, respectively and precisely adding 0.1ml of reference solution (spinosad, 6' -feruloylspirocin, spina date seed saponin A, spina date seed saponin B, camellin B and betulinic acid) with certain concentration, adding a proper amount of methanol to fix the volume to 5ml, carrying out ultrasonic treatment for 10min, cooling, taking supernatant, centrifuging at 12000r/min for 10min, sucking supernatant as test solution, parallelly preparing 6 parts, measuring under test conditions, recording peak areas of 6 chemical components, and calculating the sample adding recovery rate of the method. The results are shown in Table 2. The recovery rate of the 6 chemical components is in the range of 95.00-105.00%, and the RSD is less than or equal to 3.0%, which shows that the method is accurate and reliable.
TABLE 26 recovery of chemical components (n ═ 6)
2.9 measurement of sample content
Taking 10 batches of spina date seed medicinal material decoction pieces, preparing a sample solution according to a sample solution preparation method under '2.2' items, performing sample injection analysis on 3 batches in parallel under '2.3' conditions, recording peak areas of 6 chemical components and calculating the content of the chemical components, wherein the results are shown in Table 3.
Table 310 Subscription seed water decoction 6 chemical component content determination results (mug/g)
3. Discussion of the related Art
Analyzing the content of 6 chemical components in the spina date seed water decoction by adopting a UHP L C-QQQ-MS/MS method, comprehensively considering the structure and polarity characteristics of different types of components, and performing ESI (electronic signature analysis)-Detection is carried out in an ion mode, monitoring is carried out in an MRM mode, and all components have good response in different ion channels; the method optimizes the mobile phase to realize effective separation of each component, compares an organic phase (methanol/acetonitrile) with a water phase (water/0.1% formic acid water), and finally finds that when acetonitrile-water is used as a mobile phase system for gradient elution, 6 chemical components can realize effective separation and obtain good peak shapes, the retention time of each component is greatly shortened, and the mobile phase system improves the experimental efficiency, saves reagents and has practical economic significance. The selection of the solvent considers methanol, a 60% methanol solution and a 60% ethanol solution, and finally finds that the methanol can effectively dissolve 6 components in the spina date seed water decoction, so that the solvent adopts the methanol.
4. Conclusion
The method established by the invention is simple to operate, and the 6 chemical components obtain good separation effect and response under the conditions selected in the invention. The method can complete the determination and analysis of the component content within 5min, has accurate determination result and good stability, is a reliable method for determining various structural types of chemical components in the spina date seed water decoction, and can provide experimental basis for the comprehensive quality control and evaluation of the spina date seeds.
Claims (1)
1. A method for simultaneously measuring the contents of 6 chemical components in spina date seed water decoction by using UHP L C-QQQ-MS/MS is characterized by comprising the following steps:
(1) preparing a mixed reference substance solution:
accurately weighing appropriate amount of spinosin, 6' -feruloyl spinosin, spina date seed saponin A, spina date seed saponin B, camellianin B and betulinic acid reference substances respectively, adding methanol for dissolving and diluting, and shaking up to obtain mixed reference substance solutions with concentrations of 6.312 μ g/ml, 5.885 μ g/ml, 0.991 μ g/ml, 0.668 μ g/ml, 0.640 μ g/ml and 4.504 μ g/ml respectively;
(2) preparing a test solution:
precisely weighing 10g of semen Ziziphi Spinosae, adding 10 times of water, soaking in a decocting pot for 30min, boiling with strong fire, decocting for 1 hr, and filtering with 4 layers of gauze; adding 8 times of water into the residue, boiling with strong fire, decocting for 1 hr, filtering with 4 layers of gauze, mixing the decoctions, adding appropriate amount of water, and diluting to 200ml to obtain semen Ziziphi Spinosae water decoction sample;
precisely transferring 0.2ml of semen Ziziphi Spinosae water decoction sample into 5ml measuring flask, adding methanol to constant volume to 5ml, ultrasonically treating for 10min, cooling, collecting appropriate amount of upper layer solution, centrifuging at 12000r/min for 10min, and collecting supernatant as test solution;
(3) the determination method comprises the following steps:
the UHP L C-QQQ-MS/MS method is adopted to determine the concentration of 6 chemical components in the mixed reference substance and the test solution:
chromatographic conditions are as follows: the chromatographic column is Agilent C18The specification is 2.1mm × 100mm,1.8 mu m, the reversed phase chromatographic column, the fluidity A is water, the fluidity B is acetonitrile, gradient elution is carried out, the elution procedure is 0-1.2min, the volume fraction of the acetonitrile is linearly increased from 5% to 31%, 1.2-2.5min, the volume fraction of the acetonitrile is linearly increased from 31% to 95%, 2.5-4.5min, the volume fraction of the acetonitrile is linearly increased from 95% to 100%, 4.5-5min, the volume fraction of the acetonitrile is maintained at 100%, the flow rate is 0.3ml/min, the column temperature is 40 ℃, and the sample injection amount is 1 mu l;
mass spectrum conditions: using ESI-Detecting a mode, wherein a scanning mode adopts a multi-reaction monitoring mode (MRM), and parameters are as follows:the ion source temperature is 550 ℃; atomizing air 55 psi; auxiliary heating gas 55 psi; air curtain air 35 psi; the nitrogen is used as spray auxiliary gas, and mass spectrum parameters Q1/Q3(m/z) of the ingredients of the spinosad, 6' -feruloyl spinosad, spina date seed saponin A, spina date seed saponin B, camellin B and betulinic acid are respectively as follows: 607.5/427.1, 783.2/427.4, 1205.9/1073.9, 1043.6/911.5, 725.2/284.0, 455.3/455.3; the declustering voltage (DP) is-26.46V, -25.83V, -60.00V, -27.20V, -23.72V and-97.00V respectively; the Collision Energy (CE) is: 41.60V, -50.92V, -60.00V, -49.77V, -53.07V, -20.00V.
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