CN112461976A - Method for detecting multiple components in ginseng and aconite injection - Google Patents
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Abstract
The invention relates to the technical field of drug analysis and detection, in particular to a method for detecting multiple components in ginseng and aconite injection. The detection method comprises the steps of detecting saponin components and alkaloid components in the ginseng and aconite injection by adopting high performance liquid chromatography-mass spectrometry, wherein the scanning mode of the mass spectrometry is positive and negative ion scanning. The method provided by the embodiment of the invention can be used for simultaneously, rapidly and accurately detecting the content of the saponin component and the alkaloid component in the ginseng and aconite injection, can realize the detection of multiple components in the ginseng and aconite injection and simplifies the detection method of the ginseng and aconite injection.
Description
Technical Field
The invention relates to the technical field of drug analysis and detection, in particular to a method for detecting multiple components in ginseng and aconite injection.
Background
The injection is prepared from Ginseng radix Rubri and radix Aconiti lateralis Preparata, and contains ginsenoside and alkaloid as main effective components. At present, only alkaloid identification and limited inspection are available in national standards of ginseng and aconite injection; the ginsenoside content comprises Rg1, Re and total ginsenoside content detection, and the four quality control items adopt various detection methods. Specifically, the identification of alkaloid adopts thin-layer chromatography color development detection, the limitation of alkaloid and the content detection of ginsenoside Rg1 and Re adopt liquid phase analysis, and the content detection of total ginsenoside adopts ultraviolet visible light detection. The adoption of a plurality of different detection methods consumes a large amount of detection time and reagent, which indicates that the existing detection method is not efficient and environment-friendly and cannot accurately and quantitatively detect. In the prior art, the detection of alkaloid and ginsenoside components is single-component detection, so that the requirement of simultaneously and rapidly determining two components by one method cannot be met, and the content of more specific components in the two components cannot be detected.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for detecting multiple components in ginseng and aconite injection. The method provided by the embodiment of the invention can be used for simultaneously, rapidly and accurately detecting the content of the saponin component and the alkaloid component in the ginseng and aconite injection, can realize the detection of multiple components in the ginseng and aconite injection and simplifies the detection method of the ginseng and aconite injection.
The invention is realized by the following steps:
in a first aspect, the invention provides a method for detecting multiple components in ginseng and aconite injection, which comprises the step of detecting saponin components and alkaloid components in ginseng and aconite injection by adopting high performance liquid chromatography-mass spectrometry, wherein the scanning mode of the mass spectrometry is positive and negative ion scanning.
In alternative embodiments, the conditions for high performance liquid chromatography are: the chromatographic columns are SPE columns and C18 columns; the mobile phase A is 8-12mmol/L ammonium formate solution, the mobile phase B is 0.08-0.12% acetonitrile formic acid solution, and the mobile phase C is 18-25% mixed solution formed by 8-12mmol/L ammonium formate solution and acetonitrile; the flow rate is 0.5-1.0mL/min, and the column temperature is 30-50 ℃.
The mobile phase C is a mixed solution of analytically pure acetonitrile or acetonitrile having a purity of 99.9% and an ammonium formate solution having a concentration of 8 to 12mmol/L, and the mass concentration of acetonitrile in the mixed solution is 18 to 25%.
In an alternative embodiment, the high performance liquid chromatography comprises: sampling the SPE column, and then removing impurities from the SPE column;
preferably, the removing impurities comprises: removing impurities from the SPE column with the mobile phase C in 0-1 min;
the operation process of the high performance liquid chromatography comprises the following steps: after removing impurities from the SPE column, eluting the SPE column and the C18 column;
the elution process of the high performance liquid chromatography comprises the following steps: 1-4min, mobile phase A80%, mobile phase B20%; 4-7min, mobile phase A60% → 80%, mobile phase B40% → 20%; 7-11 min: mobile phase a 60%, mobile phase B40%; 11-16min, mobile phase A60% → 40%, mobile phase B40% → 60%; 16-19 min: mobile phase A40%, mobile phase B60%, 19-21 min: mobile phase a 40% → 80%, mobile phase B60% → 20%.
According to the embodiment of the invention, the SPE column and the C18 column are adopted at the same time, but only the SPE column contains a sample, namely, the sample loading is carried out in the SPE column; and the C18 column contains no sample to be detected; in addition to the small molecules such as ginsenoside and alkaloid to be detected, the sample also contains large molecules such as polysaccharide, and the detection of the small molecules is affected by the large molecules, so that the influence of the large molecules needs to be removed. Specifically, the mobile phase C is used to elute only the SPE column (in this case, within 1min from the beginning of elution), so that the molecules with high polarity, such as saccharides, are dissolved/replaced and removed by the mobile phase C, and the sample in the SPE column is small molecules, such as alkaloids to be analyzed and detected and ginsenosides, so that ginsenosides and more specific substances in the alkaloids can be detected more accurately.
After the SPE column is eluted and impurity-removed, gradient elution is carried out by using eluent formed by the mobile phase A and the mobile phase B, and the elution not only is carried out on the SPE column, but also comprises the step of pumping liquid obtained by elution into a C18 column for further separation. For example, 1-4min mobile phase A80%, mobile phase B20%; 4-7min, mobile phase A60% → 80%, mobile phase B40% → 20%; the elution process is as follows: firstly, a mixed solution formed by mixing the mobile phase A80% and the mobile phase B20% sequentially passes through an SPE column and a C18 column, and then a mixed solution formed by mixing the mobile phase A60% → 80% and the mobile phase B40% → 20% sequentially passes through the SPE column and the C18 column.
In an alternative embodiment, the conditions of mass spectrometry are: atomizing air pressure nitrogen gas 40-60Psi, auxiliary air pressure helium gas 40-60Psi, air curtain air pressure 30-40Psi, spraying voltage 5500V, -4500V, ionization temperature 600 ℃, declustering voltage 100V, -100V, injection voltage 10V, -10V, and collision chamber injection voltage 13V, -16V; the working mode is as follows: multiple reaction detection mode (MRM), positive and negative ions are monitored simultaneously.
In an alternative embodiment, the process of mass spectrometry is: the process of mass spectrometry is as follows: the positive ion mode is adopted within 0.0-5.70 minutes, the negative ion mode is switched to the negative ion mode within 5.70-5.87 minutes, the negative ion mode is switched to the positive ion mode within 5.87-6.80 minutes, the negative ion mode is switched to the negative ion mode within 6.80-8.11 minutes, the positive ion mode is switched to the positive ion mode again within 8.11-8.41 minutes, and the positive ion mode is switched to the negative ion mode again within 10 minutes.
In an alternative embodiment, the saponin component is a ginsenoside component;
preferably, the ginsenoside components comprise 20(S) -ginsenoside F1, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, 20(S) -ginsenoside Rg2, 20(S) -ginsenoside Rg3, 20(R) -ginsenoside Rh1, ginsenoside Ro and pseudo-ginsenoside F11.
In an alternative embodiment, the detection limits of 20(S) -ginsenoside F1, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, 20(S) -ginsenoside Rg2, 20(S) -ginsenoside 36rg 3, 20(R) -ginsenoside Rh1, ginsenoside Ro and pseudoginsenoside F11 are in order: 1.08pg/mL, 1.56pg/mL, 3.97pg/mL, 2.91pg/mL, 3.14pg/mL, 1.13pg/mL, 2.72pg/mL, 1.52pg/mL, 2.02pg/mL, 54.0pg/mL, 15.7pg/mL, 1.02pg/mL, and 1.08 pg/mL.
In alternative embodiments, the alkaloid components include: aconitine, hypaconitine, benzoylaconine, benzoylhypaconine, benzoylmesaconine, niazoline, taraxamin, bulleyaconitine A, yunaconitine and songgonine;
preferably, the detection limits of aconitine, hypaconitine, benzoylaconine, benzoylhypaconine, benzoylmesaconine, niacropin, talamamin, bulleyaconitine A, yunaconitine and songonine are 7.10pg/mL, 6.21pg/mL, 0.68pg/mL, 0.62pg/mL, 0.06pg/mL, 1.52pg/mL, 1.41pg/mL, 0.58pg/mL, 7.55pg/mL and 1.31pg/mL in turn.
In an optional embodiment, the detection method further comprises: preparing a reference substance solution, an internal standard solution and a test solution before detection;
preferably, the control solution comprises a control prepared by respectively using standard substances of saponin components and alkaloid components;
preferably, the internal standard solution comprises a prepared internal standard solution prepared by respectively utilizing the linalool and the tenuifolin.
In an optional embodiment, the detection method further comprises: analyzing the reference substance solution and the internal standard solution with different concentrations by using a high performance liquid chromatography-mass spectrum, taking the peak area ratio (Y) of the reference substance and the internal standard as a longitudinal coordinate, the actual concentration ratio (X) of the reference substance and the internal standard as a horizontal coordinate, selecting 1/Y for alkaloid by weight and 1/X for ginsenoside by weight, and performing regression calculation to obtain a regression equation; when alkaloid is taken as a reference substance, the internal standard solution is a solution prepared from the camphoradin, when ginsenoside is taken as a reference substance, the internal standard solution is a solution prepared from the tenuifolin, and then a corresponding regression equation is obtained.
And analyzing the test solution by using high performance liquid chromatography-mass spectrometry, and substituting the integral value of each peak area into a regression equation to obtain the content of the saponin components and the alkaloid components.
The invention has the following beneficial effects: according to the embodiment of the invention, the high performance liquid chromatography and the mass spectrum are combined, and the mass spectrum is scanned in a scanning mode of switching positive ions and negative ions, so that the content of the saponin components and the alkaloid components in the ginseng and aconite injection can be efficiently and accurately detected.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic view of the connection of a mobile phase C elution apparatus for impurity removal according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of the connection of the apparatus for elution of mobile phase A and mobile phase B provided in the embodiment of the present invention;
fig. 3 is an ion flow diagram of a mixed standard in a positive-negative switching MRM mode according to an embodiment of the present invention;
fig. 4 is an ion flow diagram of a reference injection with lot number 18070101003 in the positive-negative switching MRM mode according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The embodiment of the invention provides a method for detecting multiple components in a ginseng and aconite injection, which comprises the following steps:
preparing a reference stock solution and an internal standard solution: respectively weighing 13 ginsenoside components such as 20(S) -ginsenoside F1, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, 20(S) -ginsenoside Rg2, 20(S) -ginsenoside Rg3, 20(R) -ginsenoside Rh1, ginsenoside Ro, pseudoginsenoside F11 and the like, and 10 alkaloid components such as aconitine, hypaconitine, benzoylaconitine, benzoylhypaconine, benzoylmesaconine, nicotianamine, talamcino, bulleyaconitine, sinomenine and the like, and respectively dissolving the components in a 20% methanol-acetonitrile solution (the mass concentration of methanol in the methanol-acetonitrile solution is 20%) to prepare a control stock solution.
Meanwhile, the camphor tree alkali (double internal standard substance 1) and the tenuifolin (double internal standard substance 2) are respectively dissolved with the methanol-acetonitrile solution to obtain internal standard solutions.
Preparing a test solution: diluting the ginseng and aconite injection with methanol by 10 times, then diluting with double internal standard solutions by 100 times, mixing uniformly, and filtering with a 0.22 mu m microporous filter membrane to obtain the ginseng and aconite injection. Wherein the double internal standard solution is a mixed solution formed by mixing the camphor-xanthine internal standard solution and the tenuifolin internal standard solution in equal volume.
Loading: the sample was added to the SPE column. The sample is the sample solution, the reference solution and the internal standard solution obtained by diluting the reference stock solution, and the like.
Meanwhile, the high liquid detection device and the mass spectrum detection device are connected in series. And mobile phases A, B and C were formulated separately.
And (3) detection: then, at 0-1min, the SPE column is eluted with mobile phase C, and then the liquid obtained by elution is discarded (see FIG. 1), wherein the pump C in FIG. 1 refers to a pump for adding the mobile phase C. At this point, the SPE column and C18 column, high liquid detection, and mass spectrometry detection equipment were not connected. Then, the connection mode is switched, (see fig. 2), wherein the pump a in fig. 2 refers to a pump for adding mobile phase a, and the pump B refers to a pump for adding mobile phase B. At this moment, SPE post and C18 post and high liquid check out test set and mass spectrum check out test set are connected, then elute, specifically, the eluant passes through SPE post and C18 post in proper order, then enters into high liquid check out test set and mass spectrum check out test set and carries out analysis and detection. The function of the C18 column is equivalent to further solid phase extraction of the liquid obtained by eluting the SPE column, thereby ensuring the accuracy of the subsequent detection result. If the C18 column or SPE column is not adopted, the subsequent detection result is inaccurate.
Specifically, the specific process is as follows: mobile phase C (at this point, mobile phase C only acts on the SPE column and the solution obtained after elution is discarded) 0-1 min; 1-4min, mobile phase A80%, mobile phase B20%; 4-7min, mobile phase A60% → 80%, mobile phase B40% → 20%; 7-11 min: mobile phase a 60%, mobile phase B40%; 11-16min, mobile phase A60% → 40%, mobile phase B40% → 60%; 16-19 min: mobile phase A40%, mobile phase B60%, 19-21 min: mobile phase a 40% → 80%, mobile phase B60% → 20%. After 1min the eluent formed by mixing mobile phase A and mobile phase B was applied to SPE column and C18 column in turn.
Wherein the mobile phase A is 8-12mmol/L ammonium formate solution, the mobile phase B is 0.08-0.12% acetonitrile formate solution, and the mobile phase C is 18-25% mixed solution formed by 8-12mmol/L ammonium formate solution and acetonitrile; the flow rate is 0.5-1.0mL/min, and the column temperature is 30-50 ℃.
Further, the mass spectrometry process adopts a scanning mode of switching positive and negative ions for scanning, and specifically the mass spectrometry process comprises 6 stages: and detecting the songguoning, the Niaoling and the taraxamin in a positive ion mode for 0.0-5.70 minutes. And 5.70-5.87 minutes, switching to a negative ion mode, and detecting the ginsenoside Rg1 and the ginsenoside Re. 5.87-6.80 min, switching the negative ion mode to positive ion mode, and detecting benzoylaconine, benzoylhypaconine, benzoylmesaconine and bulleyaconitine A. Switching to negative ion mode at 6.80-8.11 min, and detecting ginsenoside F11, Rb1, Ro, Rf, Rc, Rb2, Rg2, and Rd in negative ion mode. And switching to positive ions again after 8.11-8.41 minutes, and detecting aconitine, aconitine and hypaconitine in a positive ion mode. Within the next 10 minutes, the positive ions are switched into negative ions again, and other ginsenosides such as the residual compound ginsenoside Rg3 and the like are detected in a negative ion mode.
Furthermore, the atomization pressure nitrogen is 40-60Psi, the auxiliary pressure helium is 40-60Psi, the air curtain pressure is 30-40Psi, the spraying voltage is 5500V, -4500V, the ionization temperature is 600 ℃, the declustering voltage is 100V, -100V, the injection voltage is 10V, -10V, and the injection voltage of the collision chamber is 13V, -16V; the working mode is as follows: multiple reaction detection mode (MRM), positive and negative ions are monitored simultaneously.
Then, taking the peak area ratio (Y) of the reference substance and the internal standard as a longitudinal coordinate, taking the actual concentration ratio (X) of the reference substance and the internal standard as a horizontal coordinate, selecting 1/Y for the weight of alkaloid and 1/X for the ginsenoside, and performing regression calculation to obtain a regression equation;
and analyzing the test solution by using high performance liquid chromatography-mass spectrometry, and substituting the integral value of each peak area into a regression equation to obtain the content of the saponin components and the alkaloid components.
Further, the detection limits of 20(S) -ginsenoside F1, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, 20(S) -ginsenoside Rg2, 20(S) -ginsenoside Rg3, 20(R) -ginsenoside Rh1, ginsenoside Ro and pseudoginsenoside F11 are in turn: 1.08pg/mL, 1.56pg/mL, 3.97pg/mL, 2.91pg/mL, 3.14pg/mL, 1.13pg/mL, 2.72pg/mL, 1.52pg/mL, 2.02pg/mL, 54.0pg/mL, 15.7pg/mL, 1.02pg/mL, and 1.08 pg/mL.
The detection limits of aconitine, hypaconitine, benzoylaconine, benzoylhypaconine, benzoylmesaconine, niacinamide, taraxamine, bulleyaconitine A, yunaconitine and songchun are 7.10pg/mL, 6.21pg/mL, 0.68pg/mL, 0.62pg/mL, 0.06pg/mL, 1.52pg/mL, 1.41pg/mL, 0.58pg/mL, 7.55pg/mL and 1.31pg/mL in turn.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
Preparing a reference solution: respectively weighing 20(S) -ginsenoside F1, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, 20(S) -ginsenoside Rg2, 20(S) -ginsenoside Rg3, 20(R) -ginsenoside Rh1, ginsenoside Ro, pseudo-ginsenoside F11, aconitine, hypaconitine, benzoylaconine, benzoylhypaconine, benzoylmesaconine, nianine, Niaoling, talasamin, bulleyaconitine, yunaconitine and Songtongning 1mg, and respectively preparing stock solutions of different ginsenoside reference substances and different alkaloid reference substances with 100ml of methanol-acetonitrile solution (the mass concentration of methanol in the methanol-acetonitrile solution is 20%).
Weighing the xanthatin (internal substance 1) and the tenuifolin (double internal standard substance 2) to respectively prepare internal standard solutions of 1 ng/mL.
Test solution: the batch number is: 18070101003 diluting radix Ginseng and radix Aconiti lateralis Preparata injection with methanol 10 times, diluting with dual internal standard solution 100 times, mixing, and filtering with 0.22 μm microporous membrane to obtain test solution containing crude drug 0.3 mg/ml. Wherein the double internal standard solution is a mixed solution formed by mixing the camphor-xanthine internal standard solution and the tenuifolin internal standard solution in equal volume.
Standard curve: precisely sucking control stock solution, adding acetonitrile-water with mass concentration of 50% to 100ml, and shaking to obtain control solution with concentration of bornanine, Niaoling, taraxamine, Rg1, Re, benzoylmesaconine, bulleyaconitine, benzoylaconine, benzoylhypaconine, F11, Rb1, Ro, Rf, Rc, Rb2, 20(S) -Rg 2, Rd, 20(R) -Rh 1, 20(S) -F1, aconitine, sinomenine, hypaconine, and 20(S) -Rg3, wherein the concentrations of 77.87, 70.62, 68.34, 98.32, 102.60, 88.33, 78.79, 97.12, 93.88, 87.25, 112.62, 97.31, 89.59, 93.58, 90.34, 88.09, 98.38, 102.18, 87.72, 79.73, 77.83, 93.25, 97.89, 97.25, 97.89 ng/ml.
Respectively injecting 10 mu L of sample (both the reference solution and the corresponding internal standard solution) for determination, wherein; the chromatographic conditions are as follows: the chromatographic columns are SPE columns and C18 columns; the mobile phase A is 10mmol/L ammonium formate solution, the mobile phase B is 0.1% acetonitrile formate solution, and the mobile phase C is 20% acetonitrile and 10mmol/L ammonium formate solution; the flow rate was 0.8mL/min and the column temperature was 40 ℃.
And (3) an elution process: eluting SPE column with mobile phase C for 0-1min, mobile phase A80% and mobile phase B20% for 1-4 min; 4-7min, mobile phase A60% → 80%, mobile phase B40% → 20%; 7-11 min: mobile phase a 60%, mobile phase B40%; 11-16min, mobile phase A60% → 40%, mobile phase B40% → 60%; 16-19 min: mobile phase A40%, mobile phase B60%, 19-21 min: mobile phase a 40% → 80%, mobile phase B60% → 20%. And the eluent of 1-21min passes through SPE column and C18 column in turn.
The process of mass spectrometry is as follows: the positive ion mode is adopted for 0-5.7 minutes, 5.7-5.87 is switched into negative ions, 5.87-6.80 is switched into positive ions, 6.80-8.11 is switched into negative ions again, 8.11-8.41 is converted into positive ions, and finally the negative ions are switched, and the reaction is kept for 10 minutes. And the mass spectrum conditions are as follows: atomization pressure nitrogen 55Psi, auxiliary pressure helium 55Psi, air curtain pressure 35Psi, spraying voltage 5500V, -4500V, ionization temperature 600 ℃, declustering voltage 100V, -100V, injection voltage 10V, -10V, and collision chamber injection voltage 13V, -16V. See fig. 3 and 4.
Taking the peak area ratio (Y) of the reference substance and the internal standard as a vertical coordinate, taking the actual concentration ratio (X) of the reference substance and the internal standard as a horizontal coordinate, selecting 1/Y for the weight of alkaloid and 1/X for the ginsenoside, and performing regression calculation to obtain a regression equation, wherein the result is shown in Table 1.
Precisely sucking 10 μ l of test solution, introducing sample according to the above chromatographic conditions, measuring, and introducing the integral value of each peak area into a regression equation to obtain the content of saponin components and alkaloid components, with the results shown in Table 1.
TABLE 1 detection results of ginseng and aconite injection (ug/mL)
Example 2
The same method as in example 1 was adopted to detect the batch numbers as follows: 18080601005, 18100301006, 18120105003, 18120205006, 19010201005, 19010301005, 19011001002, 19030101003 and 19030501004, and the test results are shown in tables 2 and 3.
TABLE 2 detection results of ginseng and aconite injection (ug/mL)
TABLE 3 detection results of radix Ginseng and radix Aconiti lateralis Preparata injection (μ g/mL)
According to tables 1-3, it can be seen that ginsenoside has a strong response value in an anion mode and alkaloid has a strong response value in an anion mode, 13 ginsenosides with a definite structure and 10 alkaloids as main components of radix aconiti lateralis preparata are simultaneously measured in a unified analysis period through a simultaneous monitoring mode of positive and negative ions, so that the purpose of monitoring multiple components of a Chinese patent medicine preparation is met, the quality condition of raw materials for radix aconiti lateralis preparata injection can be monitored, and whether traditional Chinese medicinal materials containing pseudo-ginsenosides are mixed or not can be judged. Greatly shortens the analysis time, improves the analysis efficiency and provides reference basis for the quality control method of the Chinese patent medicine.
Method verification
In general, the alkaloid component responds only in the positive ion mode to [ M + H]+ ion as base peakIons. The saponin components respond to positive and negative ions, but have different forms. Under the positive ion mode of the ginsenoside components, the ginsenoside components are cracked under the condition of low transmission voltage (120V), no specificity exists between fragments and parent ions, the fragments are easily interfered by background ions/other compound ions, and the corresponding relation between the parent ions and the parent ions is not easy to distinguish. In the negative ion mode, the saponin compounds form different charge adduction ions ([ M + HCOO)]-、[M-H]-、[M+Cl]-And/or [ M + CF3COO]-) Usually in the form of [ M + HCOO]-The response is highest. Thus, in MS analysis, alkaloids are selected [ M + H]+The ginsenoside is [ M + HCOO [ ] -ginsenoside]-The ions serve as precursor ions. Determining the DP value and the CE value by observing the maximum response value of mass spectrum fragment ions, and obtaining the table below;
the control stock solution was diluted 0, 10, and 20 times in order to prepare solutions with gradient concentrations from high to low, and the samples were injected under the chromatographic conditions of example 1, wherein the concentration with signal-to-noise ratio of 3(S/N ═ 3) was taken as the detection limit, the concentration with signal-to-noise ratio of 10(S/N ═ 10) was taken as the quantification limit, and the detection results are as follows:
precision: taking 3 parts of reference substance solution with 0-fold, 10-fold and 20-fold high, medium and low concentrations diluted by the reference substance stock solution, respectively carrying out HPLC-MS analysis, repeatedly carrying out sample introduction for 6 times, wherein the sample introduction amount is 10 mu L, and recording peak areas; measuring for 3 days by the same method, and calculating the precision within and during the day. The measured RSD for all compounds was less than 10%, see table 4.
And (3) sample recovery rate: based on the sample recovery rate calculation formula (%) (measured value-amount of component to be measured contained in sample)/amount of added control × 100%, sample recovery rates at high, medium and low concentrations of 23 compounds were calculated, respectively, and the results were all greater than 85%, as shown in table 4.
TABLE 4 precision and recovery rate during and during day
According to the table, the method has good precision, accuracy and repeatability and can meet the requirement of quantitative detection.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for detecting multiple components in ginseng and aconite injection is characterized by adopting high performance liquid chromatography-mass spectrometry to detect saponin components and alkaloid components in ginseng and aconite injection, and the scanning mode of the mass spectrometry is positive and negative ion scanning.
2. The method for detecting the multiple components in the ginseng radix aconiti lateralis preparata injection as claimed in claim 1, wherein the conditions of the high performance liquid chromatography are as follows: the chromatographic columns are SPE columns and C18 columns;
the mobile phase A is 8-12mmol/L ammonium formate solution, the mobile phase B is 0.08-0.12% acetonitrile formic acid solution, and the mobile phase C is 18-25% mixed solution formed by 8-12mmol/L ammonium formate solution and acetonitrile; the flow rate is 0.5-1.0mL/min, and the column temperature is 30-50 ℃.
3. The method for detecting the multiple components in the ginseng radix aconiti lateralis preparata injection as claimed in claim 1, wherein the operation process of the high performance liquid chromatography comprises: sampling the SPE column, and then removing impurities from the SPE column;
preferably, the removing impurities comprises: removing impurities from the SPE column with the mobile phase C in 0-1 min;
the operation process of the high performance liquid chromatography comprises the following steps: removing impurities from the SPE column, and eluting;
the elution process of the high performance liquid chromatography comprises the following steps: 1-4min, mobile phase A80%, mobile phase B20%; 4-7min, mobile phase A60% → 80%, mobile phase B40% → 20%; 7-11 min: mobile phase a 60%, mobile phase B40%; 11-16min, mobile phase A60% → 40%, mobile phase B40% → 60%; 16-19 min: mobile phase A40%, mobile phase B60%, 19-21 min: mobile phase a 40% → 80%, mobile phase B60% → 20%.
4. The method for detecting the multiple components in the ginseng and aconite injection according to claim 1, wherein the conditions of mass spectrum are as follows: atomizing air pressure nitrogen gas 40-60Psi, auxiliary air pressure helium gas 40-60Psi, air curtain air pressure 30-40Psi, spraying voltage 5500V, -4500V, ionization temperature 600 ℃, declustering voltage 100V, -100V, injection voltage 10V, -10V, and collision chamber injection voltage 13V, -16V; the working mode is as follows: and in a multi-reaction detection mode, positive ions and negative ions are monitored simultaneously.
5. The method for detecting the multiple components in the ginseng and aconite injection according to claim 4, wherein the mass spectrometry process comprises: the positive ion mode is adopted within 0.0-5.70 minutes, the negative ion mode is switched to the negative ion mode within 5.70-5.87 minutes, the negative ion mode is switched to the positive ion mode within 5.87-6.80 minutes, the negative ion mode is switched to the negative ion mode within 6.80-8.11 minutes, the positive ion mode is switched to the positive ion mode again within 8.11-8.41 minutes, and the positive ion mode is switched to the negative ion mode again within 10 minutes.
6. The method for detecting the multiple components in the ginseng radix aconiti lateralis preparata injection as claimed in any one of claims 1 to 5, wherein the saponin components are ginseng saponin components;
preferably, the ginsenoside component comprises 20(S) -ginsenoside F1, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, 20(S) -ginsenoside Rg2, 20(S) -ginsenoside Rg3, 20(R) -ginsenoside Rh1, ginsenoside Ro and pseudoginsenoside F11.
7. The method for detecting the multiple components in the radix aconiti lateralis preparata injection as claimed in claim 6, wherein the detection limits of 20(S) -ginsenoside F1, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, 20(S) -ginsenoside Rg2, 20(S) -ginsenoside Rg3, 20(R) -ginsenoside Rh1, ginsenoside Ro and pseudo-ginsenoside F11 are as follows in sequence: 1.08pg/mL, 1.56pg/mL, 3.97pg/mL, 2.91pg/mL, 3.14pg/mL, 1.13pg/mL, 2.72pg/mL, 1.52pg/mL, 2.02pg/mL, 54.0pg/mL, 15.7pg/mL, 1.02pg/mL, and 1.08 pg/mL.
8. The method for detecting the multiple components in the ginseng and aconite injection according to claim 1, wherein the alkaloid components comprise: aconitine, hypaconitine, benzoylaconine, benzoylhypaconine, benzoylmesaconine, niazoline, taraxamin, bulleyaconitine A, yunaconitine and songgonine;
preferably, the detection limits of aconitine, hypaconitine, benzoylaconine, benzoylhypaconine, benzoylmesaconine, niacropin, talamamin, bulleyaconitine A, yunaconitine and songonine are 7.10pg/mL, 6.21pg/mL, 0.68pg/mL, 0.62pg/mL, 0.06pg/mL, 1.52pg/mL, 1.41pg/mL, 0.58pg/mL, 7.55pg/mL and 1.31pg/mL in turn.
9. The method for detecting the multiple components in the ginseng and aconite injection according to claim 1, wherein the method further comprises: preparing a reference substance solution, an internal standard solution and a test solution before detection;
preferably, the control solution comprises a control prepared by respectively using standard substances of saponin components and alkaloid components;
preferably, the internal standard solution comprises a prepared internal standard solution prepared by respectively utilizing the linalool and the tenuifolin.
10. The method for detecting the multiple components in the ginseng and aconite injection according to claim 9, wherein the method further comprises: analyzing the reference substance solution and the internal standard solution with different concentrations by using a high performance liquid chromatography-mass spectrum, taking the peak area ratio (Y) of the reference substance and the internal standard as a longitudinal coordinate, the actual concentration ratio (X) of the reference substance and the internal standard as a horizontal coordinate, selecting 1/Y for alkaloid by weight and 1/X for ginsenoside by weight, and performing regression calculation to obtain a regression equation;
and analyzing the test solution by using high performance liquid chromatography-mass spectrometry, and substituting the integral value of each peak area into a regression equation to obtain the content of the saponin components and the alkaloid components.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115494185A (en) * | 2022-08-08 | 2022-12-20 | 威海海洋生物医药产业技术研究院有限公司 | Method for analyzing PPD, PPT and OA type saponin distribution of American ginseng root, stem and leaf |
| CN118010898A (en) * | 2024-04-09 | 2024-05-10 | 成都大学 | Method for detecting effective components of aconite |
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| CN1501079A (en) * | 2002-11-13 | 2004-06-02 | 雅安三九药业有限公司 | Quality controlling method for Shenfu injection preparation |
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| CN1501079A (en) * | 2002-11-13 | 2004-06-02 | 雅安三九药业有限公司 | Quality controlling method for Shenfu injection preparation |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115494185A (en) * | 2022-08-08 | 2022-12-20 | 威海海洋生物医药产业技术研究院有限公司 | Method for analyzing PPD, PPT and OA type saponin distribution of American ginseng root, stem and leaf |
| CN115494185B (en) * | 2022-08-08 | 2024-04-26 | 威海海洋生物医药产业技术研究院有限公司 | Analysis method for distribution of American ginseng rootstock leaf PPD, PPT and OA type saponins |
| CN118010898A (en) * | 2024-04-09 | 2024-05-10 | 成都大学 | Method for detecting effective components of aconite |
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