CN111679017A - Method for measuring alkaloid content in gelsemium elegans - Google Patents
Method for measuring alkaloid content in gelsemium elegans 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
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- G—PHYSICS
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Abstract
The invention discloses a method for measuring alkaloid content in gelsemium elegans, and belongs to the field of organic analysis. Which comprises the following steps: adding a hydrochloric acid aqueous solution into a crushed gelsemium elegans sample for extraction, centrifuging, absorbing supernatant liquid to an activated MCX solid-phase extraction column to enrich a target substance, leaching, eluting, blow-drying, dissolving residues and filtering to obtain a test sample solution; preparing an alkaloid standard product into a proper standard solution, performing on-machine measurement on the proper standard solution and a sample solution, performing measurement by a liquid chromatograph through a gradient elution mass spectrometer, drawing a standard curve by utilizing the peak area and the concentration of the standard solution, and calculating through the standard curve to obtain the content of each alkaloid in the gelsemium elegans. The invention utilizes high performance liquid chromatography-triple quadrupole tandem mass spectrometry to carry out qualitative and quantitative determination on 11 kinds of gelsemium elegans alkaloids. And the alkaloid content of different parts in the gelsemium elegans plant is detected, so that data support is provided for researching safe and efficient application and toxic prevention and control of gelsemium elegans.
Description
Technical Field
The invention relates to the technical field of organic analysis, in particular to a method for measuring alkaloid content in gelsemium elegans.
Background
Gelsemium elegans (gelsemum elegans) is a traditional Chinese medicinal plant belonging to the family of loganaceae, the genus Gelsemium, also known as caulis sprangle and kudzu, and is widely distributed in the south to southwest regions of China. At present, the plants are discovered or planted in 9 provinces of China, such as Guangdong, Guangxi, Fujian, Guizhou, Yunnan and the like.
With the development of science and technology, people begin to research the chemical components, chemical structures and pharmacological toxicology of gelsemium elegans, and find that gelsemium elegans has quite wide clinical application, and has various efficacies of resisting tumors, easing pain, tranquilizing, treating cardiovascular diseases, regulating autoimmunity, treating skin diseases, being used for breeding livestock and poultry in animal husbandry and the like.
But its use is largely limited due to its high toxicity. The effective dose of the gelsemium elegans treatment dose is very close to the dose of the gelsemium elegans poisoning, and the gelsemium elegans treatment dose is often killed by accidental taking or suicide of the gelsemium elegans treatment dose from old to present and is one of well-known hypertoxic medicaments in the world.
Through searching the literature, the chemical components and pharmacological effects of gelsemium elegans are mainly researched in the literature at present, but the research on the activity of gelsemium elegans on the level of monomeric compounds and toxic action mechanisms of gelsemium elegans on a human body is relatively less, and the related action mechanisms are still unclear.
Although there are many studies on the alkaloid content measurement of gelsemium elegans plants, no study on 11 alkaloids of gelsemium elegans and simultaneous content measurement between different parts has been found so far. Therefore, a method capable of detecting the alkaloid content in the gelsemium elegans plants is needed, and data and theoretical support is made for the poisoning prevention and drug research of gelsemium elegans.
Disclosure of Invention
The invention aims at the problems in the prior art, and aims to provide a method for measuring the alkaloid content in gelsemium elegans.
In order to realize the purpose of the invention, the method is realized by the following technical scheme:
a method for measuring alkaloid content in gelsmium elegans comprises the following steps:
(1) preparing a test solution: adding hydrochloric acid aqueous solution into a crushed gelsemium elegans sample, carrying out ultrasonic extraction, centrifuging, absorbing supernatant liquid to an activated MCX solid-phase extraction column to enrich a target substance, leaching, eluting, blow-drying, adding methanol water to dissolve residues, and filtering with an organic filter membrane to obtain a test sample solution;
(2) drawing a standard curve: dissolving alkaloid standard substance with methanol to obtain standard solution; drawing a standard curve by using a standard solution;
(3) and (3) determination of a test solution: sucking the test solution to be tested for chromatographic gradient elution; then, determining a target substance parent ion according to chromatographic gradient elution and then performing mass spectrum scanning to obtain a measured value of the alkaloid content in gelsemium elegans;
the gelsemium elegans sample is flower, leaf, root or stem of gelsemium elegans plant; the target is an alkaloid to be measured.
Preferably, in the step (1), the mass concentration of the hydrochloric acid aqueous solution is 1% hydrochloric acid aqueous solution, and the mass ratio of the gelsemium elegans sample to the hydrochloric acid aqueous solution is 1: 50.
Preferably, in the step (1), the ultrasonic treatment is carried out for at least 30min under the condition of more than or equal to 100W and 40 kHz; the centrifugal treatment is carried out for at least 5min under the condition that the speed is more than or equal to 3000 r/min.
Preferably, in the step (1), the MCX solid phase extraction column needs to be activated by methanol and water which are larger than the supernatant before the supernatant is sucked into the MCX solid phase extraction column for enrichment; and discarding the effluent liquid and drying the MCX solid phase extraction column.
Preferably, in the step (1), the elution is carried out by using ammoniated methanol with the mass more than 5% of the supernatant, and the eluent is collected and dried by inert gas at the temperature of more than or equal to 40 ℃; then adding methanol water solution containing formic acid to dissolve the residue, and passing through an organic filter membrane with the aperture less than or equal to 0.22 μm to obtain a test solution.
Preferably, in the step (2), the alkaloid standard substance is dissolved by using the proportion of methanol to prepare a 1mg/ml standard stock solution, and then the standard stock solution is diluted by using methanol water containing formic acid to prepare a standard working solution of 4ng/ml, 10ng/ml, 20ng/ml, 40ng/ml, 100ng/ml and 200 ng/ml; and then drawing a standard curve by using the peak area obtained by measuring each alkaloid and the concentration of the standard working solution.
Preferably, in said step (3), the chromatographic gradient elution procedure is as follows:
gradient time is 0min, mobile phase A, mobile phase B85% and 15%
Gradient time is 1.50min, mobile phase A and mobile phase B are 55% and 45%
Gradient time is 2.00min, mobile phase A and mobile phase B are 10% and 90%
Gradient time is 2.50min, mobile phase A and mobile phase B are 0% and 100%
Gradient time is 3.50min, mobile phase A and mobile phase B are 0% and 100%
Gradient time is 3.51min, mobile phase A, mobile phase B85% and 15%
Gradient time is 5.50min, mobile phase A, mobile phase B85% and 15%
Wherein the mobile phase A is 0.1 percent of formic acid water solution by mass, and the mobile phase B is acetonitrile; the flow rate is 0.4 ml/min; the column temperature was 35 ℃.
Preferably, in the step (3), the conditions of the mass spectrum scan are as follows:
an ion source: ESI source
The detection mode is as follows: multiple reactive ion monitoring
Capillary voltage: positive ion mode, 3500V
Ion source temperature: 150 ℃ C
Flow rate of drying gas: 15L/min
Atomizing gas pressure: 25Psi
Temperature of sheath gas: 350 deg.C
The flow rate of the sheath gas: 12L/min
Sheath gas: nitrogen gas.
Preferably, in the step (3), the target substance parent ions are: 273.1m/z, 295.1m/z, 307.2m/z, 317.2m/z, 323.1m/z, 327.2m/z, 339.2m/z, 341.2m/z, 353.1m/z, 353.2m/z, 355.2 m/z.
Preferably, the alkaloids in gelsmium elegans comprise: gelsemine, gelsemide, (Z) -akumidine, gelsemine, Rankine gelsemide, speranskiane D, N-methoxy anhydrous senecine diol, and evergreen gelsemide.
The invention utilizes high performance liquid chromatography-triple quadrupole tandem mass spectrometry (HPLC-MS/MS) to carry out quantitative and qualitative determination analysis on 11 gelsemium target alkaloid, and explores the chromatographic conditions and mass spectrum conditions of each alkaloid in gelsemium to determine the optimal process parameters. The precision RSD of the 11 kinds of alkaloid instruments is 1.1-2.6%, the repeatability RSD is 2.1-3.3%, and the recovery rate is 93.2-98.8%; the measuring method has good precision, repeatability and stability. And the content of alkaloid at different parts in the gelsemium elegans plant is detected, so that reference is provided for researching safe and efficient application and toxic prevention and treatment of gelsemium elegans.
Drawings
FIG. 1 is a chromatogram of each alkaloid in a test solution prepared from a gelsemium elegans sample;
FIGS. 2-12 are HPLC-MS/MS spectrograms of gelsemine, (Z) -akuamidine, gelsemine, spergualine B, spergualine D, N-methoxy anhydrous lawsone diol, and evergreen gelsemine, respectively, in that order;
FIGS. 13-23 are respectively standard curves for gelsemine, (Z) -akmidine, gelsemine, speretine, N-methoxy anhydrous laevuline diol, and everglaucine at 3-200ng/ml in sequence.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. The drawings are for illustrative purposes only and are not intended to be limiting of the present invention. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Instrument and reagent
The Gelsemium elegans plant is purchased in Guangxi Yulin medicinal material market, the harvesting date is 2018 and 9 months, and the Gelsemium elegans plant is identified as Gelsemium elegans Benth (Gelsemii elegans Benth) of the genus humilis of the family Loganiaceae through the drug and food inspection of the Wuzhou city.
The apparatus used in the present invention comprises: a grinder DFT-100, an electronic balance Quintix224-1 CN, a vortex oscillator, an ultrasonic instrument, a centrifuge L009, an MCX solid phase extraction column (mixed type cation exchange solid phase extraction column) and a nitrogen blower; triple quadrupole liquid mass spectrometer Agilent1290-QQQ 6490.
The reagents used in the present invention are not particularly specified, and the% concentrations are mass percentage concentrations; the ratios referred to in the present invention are, for example, mass ratios unless otherwise specified.
The reagents used in the present invention include:
acetonitrile, methanol, formic acid (chromatographically pure, ohni, usa), MCX solid phase extraction column;
reagents required for pretreatment: methanol, 1% hydrochloric acid aqueous solution, 5% ammoniated methanol, 50% methanol water (containing 0.1% formic acid).
The extraction process requires reagents: 95% ethanol, sulfuric acid, dichloromethane and sodium hydroxide; the water is the Wahaha purified water. Information on 11 standard samples, such as gelsemine, evergeline, gelsemine, trigonelline B, gelsemine E, trigonelline, (Z) -akumidine, gelsemine, N-methoxy anhydrous palustrine diol D, gelsemine, and the like, is shown in Table 1.
TABLE 1 information of the standards
Preparation of Standard solutions
Standard stock solutions: weighing 11 standard substance of alkaloid components 10mg respectively, dissolving with methanol, diluting to 10mL, shaking, and making into standard stock solution with concentration of 1mg/mL respectively.
Mixing standard intermediate liquid: accurately sucking 0.10mL of each standard stock solution, diluting to 10mL with methanol, shaking up, and making into 10 μ g/mL mixed standard intermediate solution.
Standard curve solution: respectively and accurately sucking appropriate amount of mixed standard intermediate solution, diluting with 50% methanol water (containing 0.1% formic acid), shaking up to obtain series of mixed standard solutions, wherein the concentrations of the compounds are 4ng/mL, 10ng/mL, 20ng/mL, 40ng/mL, 100ng/mL and 200ng/mL in sequence (preparing mixed standard solutions with appropriate concentrations in a new preparation or according to the response condition of an instrument).
Preparation of test solution
Weighing 1g of crushed gelsemium elegans sample, placing the gelsemium elegans sample in a 100mL disposable centrifuge tube, adding 1% hydrochloric acid aqueous solution according to the proportion of 1:50, placing the gelsemium elegans sample on an oscillator for oscillation for 2min, then carrying out ultrasonic treatment (100W, 40kHz) for 30min, centrifuging the gelsemium elegans sample for 5min by using a centrifuge (3000r/min), sucking 2.00mL of supernate by using a disposable pipette to an activated MCX solid phase extraction column, activating the MCX solid phase extraction column by using 3mL of methanol and 3mL of water, leaching the MCX solid phase extraction column by using 3mL of water and 3mL of methanol in sequence, discarding all effluent liquid, pumping the MCX solid phase extraction column, eluting the column by using 5mL of 5% ammoniated methanol, collecting eluent, blowing the eluent to dry at 40 ℃ by nitrogen, adding 1.00mL of 50% methanol water (containing 0.1% formic acid). Or diluted properly according to the actual concentration for later use.
Preparation of blank solution
The method is the same as the method for preparing the test solution without adding a gelsemium elegans sample.
Conditions of the apparatus
Chromatographic conditions
A chromatographic column: thermo Accucore aQ 2.1x100 mm, 2.6 μm or equivalent performance. Mobile phase: a was 0.1% formic acid water and B was acetonitrile, and the gradient elution procedure is shown in Table 2. Flow rate: 0.40 mL/min. Column temperature: 35 ℃ is carried out. Sample introduction amount: 1 μ L.
TABLE 2 gradient elution schedule
Conditions of Mass Spectrometry
An ion source: an ESI source.
The detection mode is as follows: multiple reactive ion monitoring (MRM). The collision gas is high-purity nitrogen, the dry gas, the atomization gas, the sheath gas and the like are nitrogen or other suitable gases, and corresponding parameters are adjusted before the collision gas is used, so that the sensitivity of the mass spectrum meets the detection requirement.
Capillary voltage: positive ion mode: 3500V.
Ion source temperature: at 150 ℃.
Flow rate of drying gas: 15L/min.
Atomizing gas pressure: 25 Psi.
Temperature of sheath gas: 350 ℃; sheath gas (N)2) Flow rate: 12L/min.
Parameters such as collision energy, fragmentation voltage, etc. should be optimized for optimal sensitivity, and information on monitored ion pairs and quantitative ion pairs is detailed in table 3.
TABLE 3 Compound name, formula, parent ion, daughter ion, fragmentation voltage, collision voltage and major fragment ion
Note:afor quantifying ions
Qualitative determination
The detection of the corresponding compound in the sample can be determined by measuring the sample and the standard solution under the conditions of high performance liquid chromatography-triple quadrupole tandem mass spectrometry, recording the chromatographic retention time of each compound in the sample and the standard solution, and when a chromatographic peak (within a variation range of + -2.5%) corresponding to the chromatographic peak retention time of a certain standard substance is detected in the sample, and the deviation of the relative abundance ratio of the selected monitoring ion pair in the chromatogram of the sample and the ion relative abundance ratio (k) of the standard solution of the corresponding concentration does not exceed the range specified in table 4.
TABLE 4 maximum permissible deviation of relative ion abundance in qualitative confirmation
Quantitative determination
Standard curve and linear range
Respectively and accurately sucking appropriate amount of mixed standard intermediate solution, diluting with 50% methanol water (containing 0.1% formic acid), shaking up to obtain series of mixed standard solutions, wherein the concentrations of the compounds are 4ng/mL, 10ng/mL, 20ng/mL, 40ng/mL, 100ng/mL and 200ng/mL in sequence, and preparing the mixed standard solution with appropriate concentration according to the response condition of the instrument or the new preparation. Analyzing according to the instrument condition under 2.5 items; under the condition of mass spectrum, the peak area of the corresponding mass number of each compound is measured in a multi-ion scanning mode. And respectively drawing standard curves by taking the peak area of each component as a vertical coordinate (Y) and the mass concentration as a horizontal coordinate (X), and performing linear regression to obtain a regression equation and a correlation coefficient.
Measurement of test solution
And (3) determining the sample solution according to the reference conditions of the instrument to obtain the chromatographic peak area of the corresponding component in the sample solution. And (5) obtaining the concentration of the components in the liquid to be measured according to the standard curve, and measuring in parallel for no less than two times.
Determination of blank solution
The blank solution and the test sample solution are measured.
2 calculation of results
Substituting the concentration measured by the liquid chromatography-mass spectrometry into the following formula to calculate the content:
in the formula: x-the content of each analyte in milligrams per kilogram (mg/kg);
c-reading the concentration of each substance to be detected in the test solution from the standard curve, wherein the unit is microgram per liter (mu g/L);
v-the final volumetric volume of the sample solution, in milliliters (mL);
m-mass represented by the sample solution in grams (g);
f-dilution factor.
The calculation results are expressed as the arithmetic mean of two independent measurements obtained under repetitive conditions, with the results remaining in three significant digits.
Methodology investigation
Precision test of instrument
A10 ng/ml control sample was injected, and the precision RSD (%) was calculated by performing parallel measurement 6 times under the instrument conditions of item 2.5.
Repeatability test
The same sample solution was subjected to parallel measurement 6 times under the instrument conditions of item 2.5, and the repetitive RSD (%) was calculated.
Sample application recoverability test
The same sample was taken and added with control substances at concentrations of 200. mu.g/kg and 1000. mu.g/kg, and the samples were subjected to parallel measurement 6 times under the instrument conditions of 2.5 items to calculate the recovery (%).
Qualitative determination of results
As shown in fig. 1-12, chromatograms of gelsemine, (Z) -akimidine, gelsemine, calamine B, calamine D, N-methoxy anhydrous laodermamine diol, and everglamine in gelsemine samples were determined by HPLC-MS/MS.
As can be seen in figure 1, gelsemine and gelsemine, referenced 2, 3 therein; gelsemine, Aristolocrine, (Z) -akuamidine base, gelsemine, labeled 4, 5, 6; rankine gelsemide and kuwanin B with the labels of 7 and 8 have the phenomena of peak combination and insufficient separation degree; the reason is that all the measured substances are alkaloids, the structures of the alkaloids are very similar, and therefore the alkaloids cannot be completely separated, but because the method carries out quantitative measurement through mass spectrometry, only the molecular weight of each substance needs to be distinguished, and therefore the incomplete separation of each alkaloid in the chromatogram does not influence the result.
Results of sample content measurement
Standard curve and linear range results
As shown in fig. 13-23, respectively, the standard curves of gelsemine, (Z) -akimidine, gelsemine, rankine gelonine, calamine B, calamine D, N-methoxy anhydrous lawsone diol, and everglaucine at 3-200ng/ml are sequentially given. The results of the standard curve and associated data for 11 gelsemium alkaloids are shown in table 5.
Results of 511 gelsemium alkaloid standard curves in table
Determination results of alkaloid content in different parts of gelsemium elegans sample
The invention measures the contents of 11 alkaloids in four parts of gelsemium elegans, leaves, roots and stems, and the results are shown in Table 6.
TABLE 611 mean assay of gelsemium alkaloids (mg/g)
Methodological validation results
The precision, repeatability and recovery rate test results of 11 gelsemium alkaloids are shown in Table 7. The precision RSD% of 11 gelsemium alkaloid is 1.1% -2.4%, which shows that the precision of the instrument is good; the repeatability RSD% is 2.1% -3.3%, which shows that the repeatability of the method is good; the recovery rate of the standard substance with 200 mug/kg is 93.2-98.8%, and the recovery rate of the standard substance with 1000 mug/kg is 94.4-97.6%, which meets the standard requirement.
Conclusion
The method uses the MCX cation stationary phase extraction column to separate the gelsemium elegans alkaloid components, and the MCX solid phase extraction column has good retention capacity on alkaline compounds and has the characteristics of small organic solvent dosage, convenience, safety, high efficiency and the like. The HPLC-MS/MS method is an analysis method integrating high-efficiency separation and multi-component qualitative and quantitative determination, can well confirm the qualitative and quantitative determination of an analyte, and has the characteristics of high speed, high resolution and high sensitivity.
Comparing alkaloid content in different parts of gelsemium elegans
The gelsemium elegans leaf has the highest gelsemium content (average detected amount is 2.1mg/g), the next gelsemium elegans leaf has the highest gelsemium elegans content (average detected amount is 1.3mg/g), and the lowest gelsemium elegans leaf has the lowest content (average detected amount is 0.00014 mg/g). 11 alkaloid components can be detected from gelsemium elegans leaves, and the average detected amount is gelsemine, N-methoxy anhydrous laojieguinediol, gelonine, gelsemine, and gelsemine from high to low in sequence.
The gelsemium elegans (average detected amount is 1.4mg/g) is the highest in the gelsemium elegans, gelsemine (average detected amount is 1.3mg/g) is the next highest in the gelsemium elegans, and everglaucine (average detected amount is 0.00005mg/g) is the lowest in the gelsemium elegans. 11 alkaloid components can be detected from gelsemium elegans, and the average detected amount is koumine > gelsemina > gelsemine > trigonelline D > gelsemine > trigonelline B > (Z) -akuamidine > gelsemine > N-methoxy anhydrous laojiesine diol > Rankine geline > evergreen gelsemine from high to low in sequence.
The detected amounts of 11 alkaloids in two parts of gelsemium root and stem are sequentially gelsemine, N-methoxy anhydrous laojieguinediol, gelonine and gelsemine from high to low.
Of the 11 alkaloids, gelsemine and gelsemine were reported most pharmacologically active, while gelsemine was most toxic. Therefore, the content of gelsemine and gelsemine of different parts can be analyzed to be used as an index for judging the pharmaceutical parts of gelsemium; the content of gelsemine can be used as a pre-judging index of gelsemium toxicity parts.
Therefore, the medicinal value of the four parts is root, leaf, flower and stem; while the toxicity is leaf > flower > root > stem. In conclusion, the root part of gelsemium elegans has the highest medicinal value and the leaf part of gelsemium elegans has the highest toxicity, and reference information is provided for research or prevention and treatment of poisoning.
Claims (10)
1. A method for measuring alkaloid content in gelsmium elegans is characterized by comprising the following steps:
(1) preparing a test solution: adding hydrochloric acid aqueous solution into a crushed gelsemium elegans sample, carrying out ultrasonic extraction, centrifuging, absorbing supernatant liquid to an activated MCX solid-phase extraction column to enrich a target substance, leaching, eluting, blow-drying, adding methanol water to dissolve residues, and filtering with an organic filter membrane to obtain a test sample solution;
(2) drawing a standard curve: dissolving alkaloid standard substance with methanol to obtain standard solution; drawing a standard curve by using a standard solution;
(3) and (3) determination of a test solution: sucking the test solution to be tested for chromatographic gradient elution; then, determining a target substance parent ion according to chromatographic gradient elution and then performing mass spectrum scanning to obtain a measured value of the alkaloid content in gelsemium elegans;
the gelsemium elegans sample is flower, leaf, root or stem of gelsemium elegans plant; the target is an alkaloid to be measured.
2. The method for measuring alkaloid content in gelsemium as claimed in claim 1, wherein: in the step (1), the mass concentration of the hydrochloric acid aqueous solution is 1% of the hydrochloric acid aqueous solution, and the mass ratio of the gelsemium elegans sample to the hydrochloric acid aqueous solution is 1: 50.
3. The method for measuring alkaloid content in gelsemium as claimed in claim 1, wherein: in the step (1), the ultrasonic treatment is carried out for at least 30min under the condition of more than or equal to 100W and 40 kHz; the centrifugal treatment is carried out for at least 5min under the condition that the speed is more than or equal to 3000 r/min.
4. The method for measuring alkaloid content in gelsemium as claimed in claim 1, wherein: in the step (1), the supernatant is sucked into the MCX solid-phase extraction column and needs to be activated by methanol and water which are larger than the supernatant before being enriched; and discarding the effluent liquid and drying the MCX solid phase extraction column.
5. The method for measuring alkaloid content in gelsemium as claimed in claim 1, wherein: in the step (1), the elution is carried out by using ammoniated methanol with the mass more than 5% of the supernatant, and the eluent is collected and dried by inert gas at the temperature of more than or equal to 40 ℃; then adding methanol water solution containing formic acid to dissolve the residue, and passing through an organic filter membrane with the aperture less than or equal to 0.22 μm to obtain a test solution.
6. The method for measuring alkaloid content in gelsemium as claimed in claim 1, wherein: in the step (2), the alkaloid standard substance is dissolved by the proportion of methanol to prepare 1mg/ml standard stock solution, and then the standard stock solution is diluted by methanol water containing formic acid to prepare standard working solutions of 4ng/ml, 10ng/ml, 20ng/ml, 40ng/ml, 100ng/ml and 200 ng/ml; and then drawing a standard curve by using the peak area obtained by measuring each alkaloid and the concentration of the standard working solution.
7. The method for measuring alkaloid content in gelsemium as claimed in claim 1, wherein: in said step (3), the chromatographic gradient elution procedure is as follows:
gradient time is 0min, mobile phase A, mobile phase B85% and 15%
Gradient time is 1.50min, mobile phase A and mobile phase B are 55% and 45%
Gradient time is 2.00min, mobile phase A and mobile phase B are 10% and 90%
Gradient time is 2.50min, mobile phase A and mobile phase B are 0% and 100%
Gradient time is 3.50min, mobile phase A and mobile phase B are 0% and 100%
Gradient time is 3.51min, mobile phase A, mobile phase B85% and 15%
Gradient time is 5.50min, mobile phase A, mobile phase B85% and 15%
Wherein the mobile phase A is 0.1 percent of formic acid water solution by mass, and the mobile phase B is acetonitrile; the flow rate is 0.4 ml/min; the column temperature was 35 ℃.
8. The method for measuring alkaloid content in gelsemium as claimed in claim 1, wherein: in the step (3), the conditions of the mass spectrum scanning are as follows:
an ion source: ESI source
The detection mode is as follows: multiple reactive ion monitoring
Capillary voltage: positive ion mode, 3500V
Ion source temperature: 150 ℃ C
Flow rate of drying gas: 15L/min
Atomizing gas pressure: 25Psi
Temperature of sheath gas: 350 deg.C
The flow rate of the sheath gas: 12L/min
Sheath gas: nitrogen gas.
9. The method for measuring alkaloid content in gelsemium as claimed in claim 1, wherein: in the step (3), the target substance parent ions are respectively: 273.1m/z, 295.1m/z, 307.2m/z, 317.2m/z, 323.1m/z, 327.2m/z, 339.2m/z, 341.2m/z, 353.1m/z, 353.2m/z, 355.2 m/z.
10. The method for measuring alkaloid content in gelsemium according to any of claims 1-9, wherein: the alkaloids in gelsmium elegans comprise: gelsemine, gelsemide, (Z) -akumidine, gelsemine, Rankine gelsemide, speranskiane D, N-methoxy anhydrous senecine diol, and evergreen gelsemide.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104807908A (en) * | 2015-05-12 | 2015-07-29 | 广西壮族自治区梧州食品药品检验所 | Analytical method of gelsemine and koumine in gelsmium elegans |
| CN104833752A (en) * | 2015-05-12 | 2015-08-12 | 广西壮族自治区梧州食品药品检验所 | Method for measuring gelsemine and koumine in gelsmium elegans |
| CN104991019A (en) * | 2015-06-29 | 2015-10-21 | 公安部物证鉴定中心 | Liquid chromatography-tandem mass spectrometry detection method for Geliemine and Koumine in biological sample |
| CN105572284A (en) * | 2015-12-22 | 2016-05-11 | 湖南农业大学 | Analysis method for quickly detecting and identifying chemical components in gelsemium elegans |
| CN107894475A (en) * | 2017-11-20 | 2018-04-10 | 湖南农业大学 | The Liquid Chromatography-Tandem Mass Spectrometry quantitative approach that plurality of active ingredients detects simultaneously in elegant jessamine |
| CN108008056A (en) * | 2017-10-24 | 2018-05-08 | 中国农业科学院蜜蜂研究所 | Application of the elegant jessamine alkaloid as the biomarker of toxic honey |
| CN108414643A (en) * | 2018-04-11 | 2018-08-17 | 江苏省家禽科学研究所 | The triple level four bars Mass Spectrometry detection methods of liquid chromatogram-of biogenic amine in a kind of cold freshly-slaughtered poultry |
-
2020
- 2020-07-29 CN CN202010742641.XA patent/CN111679017A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104807908A (en) * | 2015-05-12 | 2015-07-29 | 广西壮族自治区梧州食品药品检验所 | Analytical method of gelsemine and koumine in gelsmium elegans |
| CN104833752A (en) * | 2015-05-12 | 2015-08-12 | 广西壮族自治区梧州食品药品检验所 | Method for measuring gelsemine and koumine in gelsmium elegans |
| CN104991019A (en) * | 2015-06-29 | 2015-10-21 | 公安部物证鉴定中心 | Liquid chromatography-tandem mass spectrometry detection method for Geliemine and Koumine in biological sample |
| CN105572284A (en) * | 2015-12-22 | 2016-05-11 | 湖南农业大学 | Analysis method for quickly detecting and identifying chemical components in gelsemium elegans |
| CN108008056A (en) * | 2017-10-24 | 2018-05-08 | 中国农业科学院蜜蜂研究所 | Application of the elegant jessamine alkaloid as the biomarker of toxic honey |
| CN107894475A (en) * | 2017-11-20 | 2018-04-10 | 湖南农业大学 | The Liquid Chromatography-Tandem Mass Spectrometry quantitative approach that plurality of active ingredients detects simultaneously in elegant jessamine |
| CN108414643A (en) * | 2018-04-11 | 2018-08-17 | 江苏省家禽科学研究所 | The triple level four bars Mass Spectrometry detection methods of liquid chromatogram-of biogenic amine in a kind of cold freshly-slaughtered poultry |
Non-Patent Citations (1)
| Title |
|---|
| 王杰 等: "钩吻老茎与嫩茎生物碱成分的差异分析", 《中国中药杂志》 * |
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