CN107894475B - Liquid chromatography-tandem mass spectrometry quantitative method for simultaneously detecting multiple effective components in gelsmium elegans - Google Patents
Liquid chromatography-tandem mass spectrometry quantitative method for simultaneously detecting multiple effective components in gelsmium elegans Download PDFInfo
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Abstract
The invention discloses a liquid chromatography-tandem mass spectrometry quantitative method for simultaneously detecting multiple active ingredients in gelsemium, which takes gelsemium methanol solution, gelsemium methanol solution and gelsemine methanol solution as control sample solutions, takes gelsemium extract as a sample solution to be detected, adopts a high performance liquid chromatography triple quadrupole mass spectrometer for detection, and calculates the content of the multiple active ingredients in the sample solution to be detected according to the detection result. The quantitative analysis method provided by the invention is stable and reliable, simple and efficient, has high sensitivity and accuracy, has wide practical applicability, and can be applied to detection and quantitative analysis of any gelsemium elegans and derivative products thereof.
Description
Technical Field
The invention relates to a relative quantitative analysis method, in particular to a relative quantitative analysis method for simultaneously detecting a plurality of compounds in gelsemium elegans; belongs to the technical field of Chinese herbal medicine analysis.
Background
Gelsemium elegans is a whole plant of trigonella foenum-graecum belonging to the family of loguatae, and contains chemical components such as alkaloid, iridoid, phenolic acid, triterpene and grease, so far, the domestic and foreign literature reports report that more than 300 compounds are separated from gelsemium elegans. The gelsemium elegans has effects of resisting tumor, inflammation and neuropathic pain, and enhancing immunity, and is mainly used for treating rheumatoid arthritis pain and neuropathic pain, promoting animal growth, etc.
At present, more than 120 indole alkaloids have been reported to be separated from gelsemium, and are classified into six major classes of snakerootstalk (sarpagine), gelsemine (koumine), huclevidine (humantenine), gelsemine (gelsedine), gelsemine (gelsemine) and yohimbine (yohimbine) according to their structures. More than 100 kinds of non-alkaloid such as iridoid and phenolic acid are separated.
The literature reports about the content measurement of gelsemium components mainly include that plum crystal and the like adopt an acid dye colorimetric method to measure the content of total alkaloids of gelsemium and a preparation thereof, millet establishing and the like establish a single internal standard multi-control method to synchronously measure the content of gelsemine and gelsemine in gelsemium (traditional Chinese medicinal materials, 2013, 36 (10): 1626-.
Koumine has not been marketed as any product due to its toxicity limitation, but it is included in 2015 th veterinary pharmacopoeia due to its animal growth promoting effect, and thus has some wide clinical applications in veterinary medicine. At present, the method for extracting gelsemium elegans is various, such as alcohol extraction, water extraction, acid-alkali extraction and the like, the extraction process is different, the quality is different, and in order to prevent adverse events such as gelsemium elegans poisoning and the like and ensure the stable and controllable quality of related components taking gelsemium elegans as a raw material, multi-component analysis and relative quantitative analysis of effective components in gelsemium elegans are needed.
Chinese patent (Z L201510968431) discloses an analysis method for rapidly detecting and identifying chemical components in gelsemium elegans, and mass spectrum fragmentation rules and structural analysis are carried out on the components of the compound (Fitoterapia, 2017, 121: 94-105).
Disclosure of Invention
Aiming at the technical problems that the method for detecting the components of the gelsemium elegans compound in the prior art is difficult to realize the simultaneous quantitative analysis of a plurality of components in the gelsemium elegans compound, and the like, the invention aims to provide a relative quantitative analysis method for simultaneously detecting a plurality of compounds in the gelsemium elegans, which perfects a gelsemium elegans quality evaluation system, is stable, reliable, sensitive and accurate, has short detection period, is simple in sample pretreatment, and can solve the problem of quality monitoring of the gelsemium elegans under the condition of insufficient quantity of reference substances.
In order to achieve the technical purpose, the invention provides a liquid chromatogram-tandem mass spectrum quantitative method for simultaneously detecting a plurality of effective components in gelsemium elegans, which comprises the following steps: taking a gelsemine methanol solution, a gelsemium methanol solution, a gelsemine methanol solution and a gelsemine methanol solution as control sample solutions, taking a gelsemium extracting solution as a sample solution to be detected, detecting by adopting a high performance liquid chromatography triple quadrupole mass spectrometer, and calculating the content of various active ingredients in the sample solution to be detected according to a detection result.
Preferably, the preparation method of the sample solution to be detected comprises the steps of carrying out ultrasonic treatment on the gelsemium elegans powder by using 75-85% ethanol in percentage by mass at the temperature of 55-65 ℃ for 20-40 min, filtering, collecting filtrate, repeating the operation once, combining the two filtrates, blowing 1m L filtrate by using nitrogen, adding the initial mobile phase with the same volume for redissolution, and filtering the mixture through a 0.22-micron microporous filter membrane to obtain the sample solution to be detected.
Preferably, the control sample solution comprises a series of gelsemine methanol solutions with different concentrations, a series of gelsemine methanol solutions with different concentrations and a series of gelsemine methanol solutions with different concentrations, which are obtained by respectively diluting the gelsemine solution, the gelsemine solution and the gelsemine solution with the concentrations of 100 mug/m L step by step according to multiple times.
In a more preferred embodiment, the gelsemium elegans powder comprises at least one of the powder of gelsemium elegans, root, stem and leaf.
Preferably, the high performance liquid chromatography conditions comprise that a chromatographic column is a Waters C18 chromatographic column, a mobile phase A is a 4-6 mM ammonium acetate solution, a mobile phase B is acetonitrile, the flow rate is 0.2-0.4 m L/min, the column temperature is 25-35 ℃, and the sample injection amount is 5-15 mu L;
a gradient elution procedure was used:
within 0-2min, the volume ratio of the mobile phase A to the mobile phase B is 90: 10;
within 2-7min, uniformly changing the volume ratio of the mobile phase A to the mobile phase B from 90:10 to 85: 15;
within 7-20min, the volume ratio of the mobile phase A to the mobile phase B is 85: 15;
the volume ratio of the mobile phase A to the mobile phase B is changed from 85:15 to 10:90 at a constant speed within 20-30 min;
the volume ratio of the mobile phase A to the mobile phase B is 10:90 within 30-33 min;
and the volume ratio of the mobile phase A to the mobile phase B is 90:10 within 33-40 min.
The preferred eluant and elution procedure of the present invention can achieve effective separation of multiple components in the gelsemium compound.
More preferably, the chromatographic column is a C18 chromatographic column, the caliber is 4.6mM, the length of the × chromatographic column is 150mM, the particle size is 5 μm, the mobile phase A is 5mM ammonium acetate solution, the mobile phase B is acetonitrile, the flow rate is 0.3m L/min, the temperature of the chromatographic column is 20 ℃, and the sample injection amount is 5 μ L.
In a further preferred embodiment, the ammonium acetate solution has a pH of 5.2.
Preferably, the mass spectrometry conditions are as follows: the ion source is an ESI ion source, the detection mode is positive ion detection, the scanning mode is multiple reaction monitoring, the desolvation temperature is 330-340 ℃, the capillary tube voltage is 2500-3500V, the atomizer pressure is 35-45 Psi, and the desolvation gas is N2，N2The flow rate of (A) is 10 to 15L/min.
In a more preferred embodiment, the mass spectrometry conditions are: the ion source is ESI ion source, the detection mode is positive ion detection, the scanning mode is multiple reaction monitoring, the desolvation temperature is 335 ℃, the capillary tube voltage is 3000V, the atomizer pressure is 40Psi, and the desolvation gas is N2，N2The flow rate was 12L/min.
Preferably, the method comprises detecting gelsemine methanol solution, gelsemine methanol solution and gelsemine methanol solution with different concentrations as control sample solutions, establishing the relationship between the concentration and peak area of each control sample solution, and adopting weighted 1/X2The least square method is used for carrying out regression calculation to obtain a linear regression equation and a correlation coefficient, then the sample solution to be detected is detected, the content of various effective components in the sample solution to be detected is calculated according to the detection result, and X is the concentration of the control sample solution and has the unit of mu g/m L.
Preferably, the effective components comprise alkaloids, iridoids, phenolic acids and the like.
In the preferred scheme, after the control sample solution and the sample solution to be detected are detected, the content of the control sample in the sample to be detected is calculated by adopting an external standard method according to a regression equation, and the content of 46 effective components in the semi-quantitative gelsemiquantitative with the sample to be detected is used for carrying out relative quantitative analysis.
The invention discloses a relative quantitative analysis method for simultaneously analyzing a plurality of effective components in gelsemium elegans, which combines the identification result of early Q-TOF on gelsemium elegans, specifically optimizes other compounds identified by Q-TOF through parent ions and child ions, determines ion pairs of multiple reaction detection (MRM), and finally establishes a standard curve of a compound response value and gelsemium mass.
The liquid chromatography-tandem mass spectrometry quantitative method for simultaneously detecting multiple effective components in gelsmium elegans comprises the following specific steps:
1) preparing a sample solution to be tested: pulverizing herba Gelsemii Elegantis sample, adding certain amount of extraction solvent, extracting, filtering, collecting filtrate, repeating the above operation once, mixing filtrates, volatilizing filtrate with certain volume, re-dissolving with initial mobile phase, passing through 0.22 μm microporous membrane to obtain sample solution to be measured, and analyzing with liquid chromatography-tandem mass spectrometer;
2) and (3) sample analysis: detecting by adopting a high performance liquid chromatography triple quadrupole mass spectrometer, wherein the high performance liquid chromatography conditions are as follows:
a gradient elution procedure was used:
within 0-2min, the volume ratio of the mobile phase A to the mobile phase B is 90: 10;
within 2-7min, uniformly changing the volume ratio of the mobile phase A to the mobile phase B from 90:10 to 85: 15;
within 7-20min, the volume ratio of the mobile phase A to the mobile phase B is 85: 15;
the volume ratio of the mobile phase A to the mobile phase B is changed from 85:15 to 10:90 at a constant speed within 20-30 min;
the volume ratio of the mobile phase A to the mobile phase B is 10:90 within 30-33 min;
within 33-40 min, the volume ratio of the mobile phase A to the mobile phase B is 90: 10;
the mass spectrum conditions are as follows: the ion source is an ESI ion source, the detection mode is positive ion detection, the scanning mode is multiple reaction monitoring, the desolvation temperature is 330-340 ℃, the capillary tube voltage is 2500-3500V, the atomizer pressure is 35-45 Psi, and the desolvation gas is N2，N2The flow rate of (A) is 10 to 15L/min.
3) Quantitative analysis of sample by detecting sample content with relative quantitative method, taking gelsemine, gelsemine and gelsemine of different concentrations as reference, taking concentration X (μ g/m L) of each reference as abscissa, taking peak area of each reference as ordinate, and weighting (1/X)2) And performing regression calculation by using a least square method, obtaining a linear regression equation and a correlation coefficient, and calculating the content of various effective components in the sample solution to be detected according to the detection result.
The technical scheme of the invention adopts a special eluent to realize the separation of effective components in gelsemium elegans, adopts conventional eluents such as water-methanol, water-acetonitrile and the like to difficultly realize the effective separation of the effective components in gelsemium elegans compounds, and adopts 5mM ammonium acetate-acetonitrile as a mobile phase system for comparative screening to find that the gelsemium elegans has better separation effect in the 5mM ammonium acetate-acetonitrile mobile phase system, proper elution capacity, better separation effect than water-methanol, water-acetonitrile and the like and higher response value of components to be detected. Preferably, the pH of the ammonium acetate solution is controlled to be weakly acidic (with the best effect of 5.2), so that the chromatographic peak pattern of the component to be detected can be improved, the separation effect between each chromatographic peak is good, and therefore, 5mM ammonium acetate-acetonitrile is selected as a mobile phase system.
According to the technical scheme, a control sample solution for quantification and a sample solution to be tested for quantification are prepared, the content of the control sample in the sample to be tested is calculated according to a regression equation by an external standard method, wherein the sample to be tested is gelsemium, the control sample is gelsemine, gelsemine and gelsemine, and the content of effective components in the gelsemium is relatively quantified by taking the sample to be tested as the relative control sample in multi-component analysis and relative quantification.
The preparation method of the control sample solution for quantification in the technical scheme comprises the steps of respectively taking a proper amount of gelsemine, gelsemium, gelsemine and gelsemine reference substance powder into 7 10m L measuring flasks, dissolving the powder with methanol, diluting the powder to scale, shaking the solution evenly to prepare a stock solution of the control substance for quantification with a certain concentration, respectively precisely measuring a proper amount of the 7 stock solutions of the control substance for quantification into 10m L measuring flasks, diluting the stock solution to scale with methanol, shaking the solution evenly to prepare the control sample solution containing 100 mu g/m L of gelsemine, gelsemine and gelsemine per 1m L, and diluting the control sample solution step by step according to multiple.
The preparation method of the quantitative sample solution comprises precisely weighing 1g of kiss-gelsemium powder, adding 25m L-80% ethanol into a 50m L beaker, performing ultrasonic treatment at 60 ℃ for 0.5h, repeating the above operation once, combining the filtrates, blowing 1m L nitrogen, adding the initial mobile phase with the same volume for redissolution, and filtering with a 0.22 μm microporous membrane to obtain the quantitative sample solution.
The technical scheme of the invention optimizes the mass spectrum conditions of the high performance liquid chromatography-tandem triple quadrupole mass spectrum (HP L C-QQQ-MS/MS) coupling technology, and respectively inspects the scanning modes of positive ions and negative ions.
The technical scheme of the invention optimizes the declustering voltage (DP), the collision induced dissociation voltage (CE) and the outlet voltage (CXP) of an ion pair monitored by multiple ion reaction (MRM) of positive ion reaction of compounds in 4 reference samples of gelsemine, gelsemine and gelsemium, wherein the optimized relevant parameters of a reference product are shown in a table 1, and the optimized relevant parameters of a sample are shown in a table 2. The segmented compounds are illustrated: 1:1 min; 2:1.2 min; 3:10 min; 4:15 min; 5:17.8 min; 6:20 min; 7:22 min; 8:25.2 min; 9:27.8 min; 10:30.2 min; 11:32 min; 12:34.2 min.
TABLE 1 multiple ion reaction monitoring ion pairs and associated Voltage parameters for control
Table 2 multiple ion reaction monitoring of compounds in gelsmium elegans samples ion pairs and related voltage parameters
The technical scheme of the invention carries out methodology verification on the method for analyzing the multiple components of the effective components in the gelsmium elegans relatively quantitatively, and the method comprises the following specific steps:
specificity test: the reference sample solution for quantification and the sample solution to be measured for quantification are injected and the spectra are recorded, and the results are shown in fig. 2, and as can be seen from fig. 2, the retention times of the reference products gelsemine, gelsemine and gelsemine are 16.4, 19.1, 22.2 and 22.1 respectively, which shows that the selectivity of the quantitative analysis method of the invention is good.
Linear test, precisely measuring 100 μ g/m L quantitative control sample solution, gradually diluting to 1 μ g/m L, 0.2 μ g/m L, 0.1 μ g/m L, 0.02 μ g/m L, 0.01 μ g/m L, 0.005 μ g/m L, 0.002 μ g/m L and 0.001 μ g/m L standard solutions, sequentially injecting and recording the chromatogram, taking the concentration X (ng/m L) of each control as abscissa, taking the peak area of each control as ordinate, and weighting (1/X L)2) The least square method is used for regression calculation, and the obtained linear regression equation and the correlation coefficient are shown in the table 3.
Accurately weighing herba Gelsemii Elegantis sample 0.5g, 1g, 2g, 3g, 4g and 5g in three times in parallel, adding 25m L-80% ethanol in 50m L beaker, performing ultrasonic treatment at 60 deg.C for 0.5h, repeating the above steps once, mixing filtrates, blowing 1m L nitrogen, adding initial mobile phase with the same volume for redissolution, filtering with 0.22 μm microporous membrane to obtain sample solution to be measured, sequentially sampling and recording chromatogram, taking concentration X (μ g/m L) of each control as abscissa, taking peak area of each control as ordinate, and weighting (1/X L)2) The least square method is used for regression calculation, and the obtained linear regression equation and the correlation coefficient are shown in the table 4.
And (3) repeatability test: taking the same gelsemium sample, preparing 6 portions of sample solution to be tested for quantification respectively, injecting and recording the atlas, calculating the contents of gelsemine, gelsemine and gelsemine in gelsemium according to a regression equation, and statistically obtaining the relative standard deviation RSD of the compounds in the sample, wherein the RSD is shown in table 6 to be in accordance with the requirement of methodology verification, which shows that the quantitative analysis method of the invention has good repeatability.
And (3) stability test: preparing a quantitative sample solution to be detected, injecting samples for 0, 2, 4, 8, 12 and 24 hours after preparation, recording a map, and calculating and counting to obtain the relative standard deviation RSD of the compound in the sample, wherein the result is shown in Table 4, and the result shows that the sample solution to be detected is stable within 24 hours.
TABLE 3 Linear regression equation and correlation coefficient for each control sample
Table 4 linear regression equation, correlation coefficient, repeatability, stability and compound content of gelsmium elegans samples
As can be seen from tables 3 and 4, the compounds in each of the control samples and the samples exhibited a good linear relationship under the conditions of the quantitative analysis method of the present invention.
Precision test, 100 μ g/m L was precisely measured and quantified, diluted to 0.1 μ g/m L and 0.01 μ g/m L with a control sample solution, six injections were repeated for three consecutive days, and the intra-day and inter-day relative standard deviations RSD of the gelsemine, and gelsemine peak areas were counted, as shown in table 5.
TABLE 5 relative standard deviation in day and day for each control RSD
As can be seen from Table 5, the standard deviation RSD of each control sample under the condition of the quantitative analysis method of the present invention meets the requirement of methodology verification, indicating that the precision of the instrument is good.
TABLE 6 content of each control Compound
Measuring the content of the compound in the gelsmium elegans: the content of the compound in gelsemium elegans in table 4 is the content measured by a linear method established by a relative quantitative analysis method, and mg/g is the relative content of the compound in every 1g of gelsemium elegans sample. Compound content as determined by the linear equation established for the 4 controls in table 6.
m＝C*V；
V: the dilution ratio is 50, and the dilution ratio is,
the peak area average value of three samples, x and b are linear equation coefficients.
It can be seen from both tables 4 and 6 that the sequence of the 4 alkaloids in gelsemium is gelseminal > gelseminal.
Compared with the prior art, the technical scheme of the invention has the beneficial technical effects that:
the technical scheme of the invention realizes the multi-component analysis relative quantitative analysis of the effective components in gelsemium under the condition of no enough standard sample, the method combines the identification result of early Q-TOF on the compound in gelsemium, the basic instrument parameters of a high performance liquid chromatography triple quadrupole mass spectrometer are optimized by using the reference substances of gelsemine, gelsemine and gelsemine, the collision energy of each compound is changed under the condition of fixing the instrument parameters, the compound specificity of other compounds identified by Q-TOF is optimized through parent ions and daughter ions with high abundance ratio, the ion pair of the compound in 46 in multiple reaction detection (MRM) is determined, the methodology is examined and verified, and the standard curve of the response value and the gelsemium mass of each compound is established. Under the condition, the compound in the gelsemium elegans 46 can be simultaneously detected, and the content of the compound can be relatively quantitatively analyzed.
The invention has the advantages that the HP L C-QQQ-MS/MS (high performance liquid chromatography-triple quadrupole mass spectrometry) coupling technology is adopted, the sensitivity and the specificity are high, the complete separation of chromatographic peaks is not required, and a plurality of compounds in the gelsemium elegans can be accurately and quickly quantified.
Drawings
FIG. 1 is a structural formula of a reference, wherein a is gelsemine, b is gelsemine, c is gelsemine, and d is gelsemine.
FIG. 2 is a multiple ion reaction monitoring chromatogram for quantitative analysis specificity test, in which the corresponding compounds are 1-46.
Detailed Description
The following examples are intended to further illustrate the present disclosure, but not to limit the scope of the invention as claimed.
Example 1:
a relative quantitative analysis method for simultaneously detecting 46 compounds in gelsemium adopts a high performance liquid chromatography triple quadrupole mass spectrometer for detection, wherein the conditions of the high performance liquid chromatography are that a chromatographic column is a Waters C18 chromatographic column, a mobile phase A is 5mM ammonium acetate (PH is 5.2), a mobile phase B is acetonitrile, the flow rate is 0.3m L/min, the column temperature is 30 ℃, and the sample injection amount is 5 mu L for gradient elution, the gradient elution procedure is that the volume ratio of the mobile phase A to the mobile phase B is 90:10 and 2-7min within 0-2min, the volume ratio of the mobile phase A to the mobile phase B is 90:10 and is uniformly changed to 85:15 within 7-20min, the volume ratio of the mobile phase A to the mobile phase B is 85:15 and is 20-30min, the volume ratio of the mobile phase A to the mobile phase B is uniformly changed from 85:15 to 10:90 and 30-33min, the volume ratio of the mobile phase A to the mobile phase B is 10:90 and 3640: 35-40 min, and the volume ratio of the mobile phase A to the mobile phase B is 3610: 10: 15;
the mass spectrum conditions are as follows: the ion source is ESI ion source, the detection mode is positive ion detection, and the scanning mode is
Multiple reaction monitoring, desolvation temperature of 335 ℃, capillary voltage of 3000V, atomizer pressure of 40Psi, desolvation gas of N2, and flow rate of N2 of 12L/min;
solution preparation:
a quantitative reference substance solution is prepared by respectively taking a proper amount of gelsemine, gelsemine and gelsemine reference substance powder into 7 10m L measuring bottles, dissolving the powder with methanol, diluting the powder to a scale, shaking the solution evenly to prepare a quantitative reference substance stock solution with a certain concentration, respectively precisely measuring a proper amount of the 7 quantitative reference substance stock solutions into 10m L measuring bottles, diluting the stock solution to a scale with methanol, shaking the solution evenly to prepare a reference substance solution containing 100 mu g/m L of gelsemine per 1m L, and then diluting the reference substance solution step by step according to multiple.
Precisely weighing 1g of kiss-wire powder to be tested, adding 25m of L-80% of ethanol into a 50m L beaker, carrying out ultrasonic treatment at 60 ℃ for 0.5h, repeating the operation once, combining two filtrates, blowing 1m of L with nitrogen, adding the initial mobile phase with the same volume for redissolution, filtering with a 0.22 mu m microporous filter membrane, and taking the subsequent filtrate to obtain the sample solution to be tested.
The method comprises the following specific steps:
respectively preparing a quantitative reference substance solution and a quantitative sample solution to be detected, injecting the solutions, and calculating the content of the effective components in the sample to be detected according to a regression equation, wherein the sample to be detected is gelsemium elegans, and the reference substances are gelsemine, gelsemine and gelsemine. The results are shown in Table 5, where the contents of gelsemine, gelsemine and gelsemine were 0.23, 0.24, 0.15 and 0.06mg/g, respectively.
Example 2
A relative quantitative analysis method for simultaneously detecting 46 compounds in gelsemium adopts a high performance liquid chromatography triple quadrupole mass spectrometer for detection, wherein the conditions of the high performance liquid chromatography are that a chromatographic column is a Waters C18 chromatographic column, a mobile phase A is 5mM ammonium acetate (PH is 5.2), a mobile phase B is acetonitrile, the flow rate is 0.4m L/min, the column temperature is 25 ℃, the sample injection amount is 7 mu L, and gradient elution is carried out, and the gradient elution procedure is the same as that of case 1;
the mass spectrometry conditions were the same as in example 1.
The solution was prepared as in example 1.
The specific procedure was the same as in example 1.
Example 3
A relative quantitative analysis method for simultaneously detecting 46 compounds in gelsemium adopts a high performance liquid chromatography triple quadrupole mass spectrometer for detection, wherein the conditions of the high performance liquid chromatography are that a chromatographic column is a Waters C18 chromatographic column, a mobile phase A is 5mM ammonium acetate (PH is 5.2), a mobile phase B is acetonitrile, the flow rate is 0.5m L/min, the column temperature is 30 ℃, the sample injection amount is 10 mu L, and gradient elution is carried out, and the gradient elution procedure is the same as that of case 2;
the mass spectrometry conditions were the same as in example 1.
The solution was prepared as in example 2.
The specific procedure is the same as in example 2.
Claims (6)
1. A liquid chromatogram-tandem mass spectrum quantitative method for simultaneously detecting a plurality of active ingredients in gelsmium elegans is characterized in that: taking a gelsemine methanol solution, a gelsemium methanol solution, a gelsemine methanol solution and a gelsemine methanol solution as control sample solutions, taking a gelsemium extracting solution as a sample solution to be detected, detecting by adopting a high performance liquid chromatography triple quadrupole mass spectrometer, and calculating the content of various active ingredients in the sample solution to be detected according to a detection result;
the high performance liquid chromatography conditions are as follows:
the chromatographic column is a Waters C18 chromatographic column;
the mobile phase A is 4-6 mM ammonium acetate solution, the mobile phase B is acetonitrile, the flow rate is 0.2-0.4 m L/min, the column temperature is 25-35 ℃, and the sample injection amount is 5-15 mu L;
a gradient elution procedure was used:
within 0-2min, the volume ratio of the mobile phase A to the mobile phase B is 90: 10;
within 2-7min, uniformly changing the volume ratio of the mobile phase A to the mobile phase B from 90:10 to 85: 15;
within 7-20min, the volume ratio of the mobile phase A to the mobile phase B is 85: 15;
the volume ratio of the mobile phase A to the mobile phase B is changed from 85:15 to 10:90 at a constant speed within 20-30 min;
the volume ratio of the mobile phase A to the mobile phase B is 10:90 within 30-33 min;
within 33-40 min, the volume ratio of the mobile phase A to the mobile phase B is 90: 10;
the mass spectrum conditions are as follows: the ion source is an ESI ion source, the detection mode is positive ion detection, the scanning mode is multiple reaction monitoring, the desolvation temperature is 330-340 ℃, the capillary tube voltage is 2500-3500V, the atomizer pressure is 35-45 Psi, and the desolvation gas is N2，N2The flow rate of (A) is 10-15L/min;
detecting gelsemine methanol solution, gelsemine methanol solution and gelsemine methanol solution with different concentrations as control sample solutions, establishing relationship between each control sample solution concentration and peak area, and adopting weighted 1/X2Performing regression calculation by using a least square method to obtain a linear regression equation and a correlation coefficient, detecting the sample solution to be detected, and calculating the content of various effective components in the sample solution to be detected according to a detection result, wherein X is the concentration of the control sample solution and the unit is mu g/m L;
the effective components include alkaloids, iridoids and phenolic acids.
2. The liquid chromatography-tandem mass spectrometry quantitative method for simultaneously detecting multiple active ingredients in gelsemium elegans as claimed in claim 1, wherein the sample solution to be detected is prepared by subjecting gelsemium elegans powder to ultrasonic treatment with 75-85% ethanol by mass at 55-65 ℃ for 20-40 min, filtering, collecting filtrate, repeating the above operation once, combining the two filtrates, blowing 1m L filtrate with nitrogen, adding initial mobile phase with the same volume for redissolution, and filtering with 0.22 μm microporous membrane to obtain the sample solution to be detected.
3. The method for quantifying multiple active ingredients in gelsmium elegans according to claim 2 by liquid chromatography-tandem mass spectrometry, wherein the method comprises: the herba Gelsemii Elegantis powder comprises at least one of herba Gelsemii Elegantis, root, stem and leaf.
4. The method for quantifying multiple active ingredients in gelsmium elegans according to claim 1 by liquid chromatography-tandem mass spectrometry, wherein the method comprises the steps of:
the chromatographic column is a C18 chromatographic column, the caliber of the chromatographic column is 4.6mm, the length of the chromatographic column is × mm, and the particle size is 5 mu m;
the mobile phase A was 5mM ammonium acetate solution, the mobile phase B was acetonitrile, the flow rate was 0.3m L/min, the column temperature was 20 ℃ and the amount of sample was 5. mu. L.
5. The method for quantifying multiple active ingredients in gelsmium elegans according to claim 4 by liquid chromatography-tandem mass spectrometry, wherein the method comprises: the pH of the ammonium acetate solution was 5.2.
6. The method for quantifying multiple active ingredients in gelsmium elegans according to claim 1 by liquid chromatography-tandem mass spectrometry, wherein the method comprises the steps of: the mass spectrum conditions are as follows: the ion source is ESI ion source, the detection mode is positive ion detection, the scanning mode is multiple reaction monitoring, the desolvation temperature is 335 ℃, the capillary tube voltage is 3000V, the atomizer pressure is 40Psi, and the desolvation gas is N2，N2The flow rate was 12L/min.
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