CN112098551A - Method for detecting ginsenoside in health food - Google Patents
Method for detecting ginsenoside in health food Download PDFInfo
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- G01N30/02—Column chromatography
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- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
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Abstract
The invention relates to a method for detecting ginsenoside in health food, which comprises the following steps: (1) extraction: taking a sample to be detected, and extracting the sample by using a solvent to obtain a sample extraction solution; (2) purifying: purifying the sample extraction solution by using a solid phase extraction column, collecting the purified eluent, and performing constant volume by using a solvent to obtain a sample solution to be detected; the filler in the solid phase extraction column is a neutral alumina and XAD-2 macroporous resin composite filler; (3) and (3) detection: injecting the sample solution to be detected into a high performance liquid chromatography-tandem mass spectrometer for detection, wherein the conditions of the liquid chromatography comprise: a chromatographic column: c18 bonded silica gel chromatographic column; mobile phase: a is ammonium acetate solution containing formic acid, B is acetonitrile, and gradient elution is carried out. The method eliminates the influence of impurity peak on ginsenoside qualitative and quantitative determination under complex matrix, has high accuracy of ginsenoside qualitative and quantitative determination, and can simultaneously detect 9 most main ginsenosides in health food.
Description
Technical Field
The invention relates to the technical field of detection of traditional Chinese medicine health-care food, in particular to a quantitative detection method of ginsenoside in health-care food.
Background
Araliaceae (Araliaceae) plants such as ginseng, American ginseng, pseudo-ginseng and the like are not only traditional rare Chinese medicinal materials, but also are widely applied to health food as Chinese medicinal varieties capable of being used for health food management, and according to statistics, nearly 40% of health food contains ginseng, American ginseng or pseudo-ginseng (Li Qing, Jinrunhao, Jiang national taimen, and the like) in health food registered in China between 2006 2015 and 2015 [ J ] food science, 2017,38(3):292 298 ] through 298 ℃. Ginsenosides are the main active compounds of Ginseng, American Ginseng and pseudo-Ginseng and are classified into protopanaxadiol type saponin (PPD), protopanaxatriol type saponin (PPT) and oleanolic type saponin (Xia Y G, Song Y, Liang J, et al. Wherein, the content of 9 compounds such as ginsenoside Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1, Rg2 and the like exceeds 90 percent (the research on the chemical components of saponins in ginseng, Yangxibao, Yangxiwei, Liujian. J. modern Chinese medicine, 2013,15(5): 349) 358.; Fuzzatii N. analysis methods of ginsenosides. [ J ] Journal of Chromatography B,2004,812(1-2): 119) 133.).
Some methods for detecting ginsenosides are disclosed in the prior art, such as: the method comprises the steps of putting a prepared sample in a Soxhlet extractor, adding trichloromethane, heating and refluxing for 3 hours, discarding trichloromethane liquid, volatilizing solvent from dregs of a decoction, transferring the dregs of a decoction and a filter paper cylinder into a 100ml conical flask, precisely adding 50ml of water-saturated n-butyl alcohol, sealing, standing overnight, carrying out ultrasonic treatment for 30 minutes, filtering, discarding primary filtrate, precisely taking 25ml of subsequent filtrate, putting the subsequent filtrate in an evaporating dish, evaporating to dryness, dissolving residues by adding methanol, transferring to a constant volume. The method is complex to operate, and wastes time and labor; and a large amount of toxic reagent trichloromethane is used in the experiment, so that the harm to experiment operators and the environment is large.
Patent 200510013275.X discloses a method for measuring the content of ginsenoside Rb1 in a traditional Chinese medicine compound, and the method is based on the principle that C18 solid phase extraction filler adsorbs impurities of traditional Chinese medicine materials so as to eliminate interference on target component detection. The method is only suitable for detecting the ginsenoside Rb1, and the C18 is a universal filler because the filler is single, so that the method cannot be used for treating complex matrix samples.
Patent CN107064329A describes a method for measuring the content of five ginsenoside components in a Xueshuan xinmaining tablet, which comprises the steps of extracting ginsenoside in a sample by a direct extraction method, and simultaneously detecting the five ginsenoside components in the Xueshuan xinmaining tablet by a liquid chromatography-mass spectrometry combination method. The method is only suitable for products with single matrix components and cannot be suitable for processing complex matrix samples.
In the traditional SPE Extraction and purification method, Lou D W and the like adopt a C18 solid Phase Extraction method of a reverse Phase solid Phase Extraction filler to detect 6 Ginsenosides in a Pharmaceutical preparation (Lou D W, Saito Y, Jinno K.solid-Phase Extraction and High-Performance Liquid Chromatography for immunization biological derivatives in Pharmaceutical Preparations [ J ] Chromatography, 2005,62(7-8):349 and 354.); peng et al used an HLB solid phase extraction method suitable for extraction of hydrophilic and lipophilic compounds to detect 4 ginsenosides in various cosmetics (Peng Y, Wu Z J, Huo Y P, et al, Simultaneous determination of ginsenosides Rg1, Re, and Rb1 and nonoginsenosides R1 by soluble phase extraction found by UHPLC-MS/MS and excitation of the same associations in variations, of cosmetics, 2017,9(37): 5441-5448.). The two C18 and HLB solid phase extraction fillers are general fillers, and have poor effect on health food with complex components.
The existing detection research of ginsenoside mainly focuses on the content analysis of traditional Chinese medicine plants or traditional Chinese medicines, generally only aims at certain specific ginsenoside compounds, and lacks qualitative and quantitative research on various ginsenoside components in health-care food. In addition, the detection of ginsenoside is mainly performed by high performance liquid chromatography, but because ginsenoside has weak absorption in the ultraviolet region and has low response at the critical wavelength of ultraviolet absorption, a large amount of interference of miscellaneous peaks has a great influence on the detection result (Wumin, Zhaodan, Zuiliang, etc.. the research progress of ginsenoside analysis and determination method [ J ] Shanghai medical journal, 2018,52(5): 94-100.). The components of the health food are relatively complex, so the detection result is further influenced under the condition of not performing reasonable purification and enrichment (Xudunming, Laiyou silver, Chenyan, and the like, solid phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry is used for simultaneously determining 21 illegally added compounds [ J ] chromatography, 2019,37(7):778-785 ] in the health food). Solid Phase Extraction (SPE) is a physical extraction technology between a solid phase and a liquid phase, and the difference of the adsorption force of a target substance between the solid phase and the liquid phase enables a sample solution to be continuously enriched in a filler when passing through the solid phase extraction filler, so that an object to be detected is separated from other substances and further concentrated by changing an eluted solvent, and finally, a liquid component with higher purity is formed by elution. However, the solid phase extraction fillers adopted in the existing ginsenoside detection method are all universal fillers, and the effect on health-care food with complex components is poor. In a word, a method for accurately, qualitatively and quantitatively detecting various ginsenoside components in health-care food is lacked at present.
Disclosure of Invention
Based on the above, the invention provides a method for detecting ginsenoside in health food, which eliminates the influence of impurity peaks under complex matrixes on the qualitative and quantitative determination of ginsenoside, has high accuracy of the qualitative and quantitative determination of ginsenoside, and can simultaneously detect 9 most main ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1 and Rg2 in the health food.
The specific technical scheme is as follows:
a method for detecting ginsenoside in health food comprises the following steps:
(1) extraction: taking a sample to be detected, and extracting the sample by using a solvent to obtain a sample extraction solution;
(2) purifying: purifying the sample extraction solution by using a solid phase extraction column, collecting the purified eluent, and performing constant volume by using a solvent to obtain a sample solution to be detected; the filler in the solid phase extraction column is a neutral alumina and XAD-2 macroporous resin composite filler;
(3) and (3) detection: injecting the sample solution to be detected into a high performance liquid chromatography-tandem mass spectrometer for detection, wherein the conditions of the liquid chromatography comprise:
a chromatographic column: c18 bonded silica gel chromatographic column;
mobile phase: a is ammonium acetate solution containing formic acid, B is acetonitrile, and gradient elution is carried out.
In some of these embodiments, the conditions of the liquid chromatography further comprise:
flow rate: 0.2-0.5 mL/min;
column temperature: 30 +/-10 ℃;
sample introduction amount: 2-10 μ L.
In some of these embodiments, the conditions of the liquid chromatography comprise:
a chromatographic column: c18 bonded silica gel chromatographic column with specification of 100 × 2.1mm and 1.9 μm;
mobile phase: a is ammonium acetate solution containing formic acid, B is acetonitrile, the concentration of ammonium acetate in the ammonium acetate solution is 5 +/-0.5 mmol/L, and the volume concentration of formic acid is 0.1 +/-0.05%;
conditions of gradient elution: 0-4.0 min, 81% A; 4.0-6.0 min, 81% -79% A; 6.0-8.0 min, 79-72% of A; 8.0-15.0 min, 72% -69% A; 15.0-20.0 min, 69% -54% A; 20-20.5 min, 54% -10% A; 20.5-22 min, 10% A; 22-22.5 min, 10% -81A%;
flow rate: 0.35-0.45 mL/min;
column temperature: 35-40 ℃;
sample introduction amount: 4-6 μ L.
In some of these embodiments, the conditions of mass spectrometry include:
an ion source: electrospray ion source, negative ion mode;
the scanning mode is as follows: monitoring multiple reactions;
ion transport capillary temperature: 250 ℃ and 400 ℃;
electrospray voltage: 2500-;
evaporation temperature: 300 ℃ and 450 ℃;
collision gas pressure: 1.5 +/-0.5 mTor;
auxiliary gas pressure: 12 +/-2 kPa;
sheath gas pressure: 35 +/-5 kPa.
In some of these embodiments, the conditions of mass spectrometry include:
ion transport capillary temperature: 340 ℃ and 360 ℃;
electrospray voltage: 2900-;
evaporation temperature: 390 ℃ and 410 ℃;
collision gas pressure: 1.5 +/-0.2 mTor;
auxiliary gas pressure: 12 +/-2 kPa;
sheath gas pressure: 35 +/-2 kPa.
In some of these embodiments, the conditions of mass spectrometry further comprise:
the monitoring ion pairs of the ginsenoside Rg1 are 799.5/637.5 and 799.5/475.5, the collision energy is 150 +/-10V, and the tube lens compensation voltage is 26 +/-2V and 33 +/-2V;
the ion pair for monitoring ginsenoside Re is 945.5/637.4, 945.5/475.5, the collision energy is 180 +/-10V, and the tube lens compensation voltage is 51 +/-2V and 36 +/-2V;
the monitoring ion pair of ginsenoside Rf is 799.5/475.4, 799.5/637.4, collision energy is 150 + -10V, and tube lens compensation voltage is 36 + -2V, 30 + -2V;
the monitoring ion pair of the ginsenoside Rg2 is 783.5/475.4 and 783.5/637.4, the collision energy is 150 +/-10V, and the tube lens compensation voltage is 36 +/-2V and 26 +/-2V;
the monitoring ion pair of ginsenoside Rb1 is 1153.6/1107.7, 1153.6/221.1, the collision energy is 180 +/-10V, and the tube lens compensation voltage is 28 +/-2V, 56 +/-2V;
the ion pair for monitoring ginsenoside Rc is 1077.6/783.5, 1077.6/945.5, collision energy is 180 + -10V, and tube lens compensation voltage is 41 + -2V, 40 + -2V;
the monitoring ion pair of ginsenoside Rb2 is 1077.6/783.5, 1077.6/945.5, the collision energy is 175 + -10V, and the tube lens compensation voltage is 46 + -2V, 38 + -2V;
the monitoring ion pair of ginsenoside Rb3 is 1077.6/783.5, 1077.6/945.5, collision energy is 180 + -10V, and tube lens compensation voltage is 42 + -2V, 41 + -2V;
the monitor ion pair of ginsenoside Rd is 945.5/783.5, 945.5/621.4, the collision energy is 164 +/-10V, and the tube lens compensation voltage is 36 +/-2V and 40 +/-2V.
In some of the embodiments, the packing in the solid phase extraction column is XAD-2 macroporous resin and neutral alumina from bottom to top respectively; the mass ratio of the XAD-2 macroporous resin to the neutral alumina is 3-4:1, and the ratio of the total weight of the filler to the column volume is 2.5-5g:6 mL.
In some of these embodiments, the XAD-2 macroporous resin and neutral alumina are present in a 4:1 mass ratio, and the packing has a total weight to column volume ratio of 2.5g:6 mL.
In some of these embodiments, the purging comprises the steps of: respectively activating the solid phase extraction column by using 3-3.5 column volumes of 20-90% ethanol aqueous solution and 3-3.5 column volumes of water, loading 0.3-0.35 column volumes of the sample extraction solution on the activated solid phase extraction column, leaching by using 3-3.5 column volumes of water, eluting by using 3-3.5 column volumes of 30-90% ethanol aqueous solution, and collecting eluent.
In some of these embodiments, the purging comprises the steps of: respectively activating the solid phase extraction column by using 3.2-3.4 column volumes of ethanol aqueous solution with volume fraction of 68-72% and 3.2-3.4 column volumes of water, loading 0.32-0.34 column volumes of the sample extraction solution on the activated solid phase extraction column, leaching by using 3.2-3.4 column volumes of water, eluting by using 3.2-3.4 column volumes of ethanol aqueous solution with volume fraction of 68-72%, and collecting eluent.
In some embodiments, the solvent in the purification step is 28-32% methanol aqueous solution by volume fraction.
In some of these embodiments, the solvent used for the extraction is water.
In some of these embodiments, the extraction is performed under ultrasonic conditions, wherein the power of the ultrasonic is 250-350W, and the frequency is 35-45 kHz.
In some embodiments, if the sample to be tested is a solid, the extracting includes the following steps: taking a sample to be detected, crushing, adding water according to the proportion of 28-32mL of water to the sample to be detected, and ultrasonically extracting for 1-3 times at the temperature of 45-55 ℃ for 20-40 minutes each time; if the sample to be detected is liquid, the extraction comprises the following steps: taking a sample to be detected, adding water according to the proportion of 18-22mL of water to the sample to be detected and 1g of water, and carrying out ultrasonic extraction for 20-40 minutes at the temperature of 45-55 ℃.
Compared with the prior art, the invention has the following beneficial effects:
(1) the detection method provided by the invention eliminates the influence of impurity peaks under complex matrixes on the qualitative and quantitative determination of the ginsenoside and the damage to large-scale expensive equipment. The detection method has the advantages of good linear range, low detection limit, high sensitivity, high recovery rate, good precision and stability, and small influence of matrix effect, and can be used for accurately detecting ginsenoside content in health food, controlling quality, and improving accuracy of qualitative and quantitative ginsenoside content in health food.
(2) The detection method provided by the invention adopts a mass spectrum combination technology, can simultaneously detect 9 most main ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1 and Rg2 in health-care food, and can completely separate three target substances of the ginsenosides Rb2, Rb3 and Rc which are isomeric structures mutually by optimizing chromatographic and mass spectrum conditions.
(3) The detection method provided by the invention does not need a process of heating reflux of trichloromethane and volatilizing a solvent, reduces the contact of detection personnel with toxic and harmful reagents, and reduces the environmental pollution caused by an experimental process.
(4) The detection method of the invention has the advantages that the pretreatment time of a single sample is about 1 hour, the detection time of an instrument is about 25 minutes, and the detection time is about one fifth of that of the method of Chinese pharmacopoeia.
(5) The detection method provided by the invention can meet the detection requirements of various different matrixes, the pretreatment process is simple and convenient, and the simultaneous detection of a large number of samples can be realized.
Drawings
Fig. 1 is a total ion flow diagram of a balsam pear and American ginseng soft capsule sample in example 1.
FIG. 2 is a total ion flow graph of the sample of American ginseng, hairy antler and rehmannia tablets in example 1.
Fig. 3 is a total ion flow diagram of a compound donkey-hide gelatin syrup oral liquid sample in example 1.
FIG. 4 is a graph showing the separation effect of 3 isomers of ginsenosides Rb2, Rb3 and Rc; the appearance sequence is ginsenoside Rc, Rb2, and Rb 3.
FIG. 5 is a total ion flow diagram of 9 kinds of ginsenosides, wherein the appearance order is ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, Rb3, Rd.
FIG. 6 is a graph showing the effect of different volumes of elution solvent on ginsenoside extraction rate.
In fig. 1 to 5, the abscissa represents time, and the ordinate represents relative abundance.
Detailed Description
The technical solution of the present invention is further illustrated by the following specific examples. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to only those steps or modules listed, but may alternatively include other steps not listed or inherent to such process, method, article, or device.
The "plurality" referred to in the present invention means two or more. "and/or" describes the association relationship of the associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.
The invention provides a method for detecting ginsenoside in health food, which comprises the following steps:
(1) extraction: taking a sample to be detected, and extracting the sample by using a solvent to obtain a sample extraction solution;
(2) purifying: purifying the sample extraction solution by using a solid phase extraction column, collecting the purified eluent, and performing constant volume by using a solvent to obtain a sample solution to be detected; the filler in the solid phase extraction column is a neutral alumina and XAD-2 macroporous resin composite filler;
(3) and (3) detection: injecting the sample solution to be detected into a high performance liquid chromatography-tandem mass spectrometer for detection, wherein the conditions of the liquid chromatography comprise:
a chromatographic column: c18 bonded silica gel chromatographic column;
mobile phase: a is ammonium acetate solution containing formic acid, B is acetonitrile, and gradient elution is carried out.
The method eliminates the influence of impurity peaks under complex matrixes on the qualitative and quantitative determination of the ginsenoside, has small influence of matrix effect, has a good linear range, low detection limit, high sensitivity, high recovery rate and good precision and stability, can be used for accurately detecting the content of the ginsenoside in health-care food, is used for quality control of the ginsenoside, can improve the qualitative and quantitative accuracy of the ginsenoside in the health-care food, and can simultaneously detect 9 most main ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1 and Rg2 in the health-care food.
Wherein, the structural formula of the 9 ginsenosides is as follows:
arap ═ arabinopyranosyl; araf ═ arabinofuranosyl; glc ═ glucosyl; xyl ═ xylosyl; rha-rhamnosyl radical
Further preference with respect to the purification conditions: the filler in the solid phase extraction column is selected from the composite of XAD-2 macroporous resin and neutral alumina, has strong adsorption capacity on saponin compounds, and can enrich target substances and separate the target substances from other interfering substances. In the experimental process, the selection and the dosage of the eluent are the key to clean or not, and the selection of the optimal volume of the eluent needs to take the elution rate of the ginsenoside, the elution rate of impurities and the dosage of the eluent into consideration. Deionized water or low-concentration ethanol aqueous solution can be used for leaching and removing impurities of the macroporous resin, ethanol aqueous solutions with different concentrations are commonly used for elution, and the concentration and the elution amount of the ethanol aqueous solution have great influence on the content of components to be separated. When water is used as an eluent, the ginsenoside can be retained and adsorbed in the solid-phase extraction column and cannot be eluted along with other water-soluble substances; purified water is preferred as the rinse solution for the solid phase extraction process. Further, the ginsenoside adsorbed in the filler can be gradually eluted by increasing the proportion of ethanol in the eluent, and experiments show that the ginsenoside can be basically and completely eluted when the proportion of ethanol in the eluent reaches 70 percent, and 70 percent (v/v) ethanol water solution is preferably used as the eluent for solid phase extraction. Further, taking the column volume of 6mL as an example, the effect of different volumes of elution solvent (5-30mL) on the extraction rate of ginsenosides was examined (see fig. 6). The result shows that when the elution solvent reaches 20mL, the ginsenoside can be basically and completely eluted, and the preferable dosage of the eluent is determined to be 20.0 mL.
Further preferences regarding chromatographic conditions: as can be seen from the above structural formula, the main molecular structures of the 9 ginsenosides are very similar. By comparison, the ginsenosides Rb2, Rb3 and Rc with protopanaxadiol type structures are isomers, the molecular structures are the same, only the difference exists in the spatial structures, the primary excimer ion peaks and ion fragments formed after the glycosyl is lost by cracking of the three substances are the same (m/z1077.6/783.5 and m/z 1077.6/945.5), and the retention time on a chromatographic column is close, so that the three substances are required to be completely separated during chromatographic separation so as to be accurately quantified. The invention preferably adopts a Hypersil Gold C18 chromatographic column (100 multiplied by 2.1mm,1.9 mu m), under the flow rate condition of 0.4mL/min, the three target substances of the ginsenoside Rb2, Rb3 and Rc are completely separated by adjusting and optimizing gradient elution conditions (see figure 4), and the better separation effect is realized on all 9 target substances (see figure 5). In addition, the invention inspects the separation effect of the mobile phase of the methanol-water and acetonitrile-water system relative to 9 ginsenosides under the same proportion, and the result shows that the elution capability of the mobile phase of the acetonitrile system is superior to that of the mobile phase of the methanol system, and the peak shape symmetry is better. The mass spectrum condition optimization process finds that the response signal of the saponin substance can be improved by adding formic acid into an acetonitrile-water mobile phase system, and in addition, the peak shape and the elution capacity of part of target substances can also be improved by adding a certain proportion of ammonium acetate. Further optimization of the amounts of ammonium acetate and formic acid added showed that better elution capacity and signal intensity were obtained with 0.1% formic acid and 5mmol/L ammonium acetate in the mobile phase of acetonitrile-water system, therefore 5mmol/L ammonium acetate solution (containing 0.1% formic acid) -acetonitrile mobile phase was preferably used as the final preferred conditions.
Further preferences regarding mass spectrometric conditions: the ionization and cracking process of ginsenoside is characterized in that saponin molecules [ M ] lose one proton to form [ M-H ] excimer ions, fragment ions formed by the collision of the excimer ions and collision gas molecules in a collision pool mainly form [ M-H-G ] after various glycosyl groups [ G ] are lost, the ion forming and cracking mechanisms are basically consistent, and the fragment ions have the typical characteristics of various types of saponin. Protopanaxadiol type saponin (PPD) is subjected to primary mass spectrum by taking a lost hydrogen atom as an excimer ion peak, and fragment ions of m/z783.5 and m/z945.5 are taken as main ions after fragmentation; the protopanaxatriol saponin (PPT) also has one missing hydrogen atom as an excimer ion peak in a primary mass spectrum, and fragment ions mainly comprising m/z 475.5 and m/z637.5 appear after fragmentation. After the primary mass spectrum excimer ion peak and the corresponding fragment ions are determined, mass spectrum conditions such as electrospray voltage, collision energy, tube lens compensation voltage and the like are further optimized through a large number of experiments, so that the detection sensitivity and accuracy of the detection method are further improved.
The optimal purification and detection conditions are finally obtained as follows:
and (3) purifying conditions: taking a column volume of 6mL as an example, respectively using 20mL of 70% (v/v) ethanol aqueous solution and 20mL of water to activate a neutral alumina/XAD-2 solid-phase extraction column, taking 2.0mL of sample extraction solution to load the activated solid-phase extraction column, respectively using 20mL of water to perform leaching, using 20mL of 70% (v/v) ethanol aqueous solution to perform elution, and collecting eluent.
Chromatographic conditions are as follows: a chromatographic column: hypersil Gold C18 column (100X 2.1mm,1.9 μm, Thermo Scientific Co., USA); mobile phase: a is 5mmol/L ammonium acetate solution (containing 0.1% formic acid, v/v), B is acetonitrile; gradient elution conditions: 0-4.0 min, 81% A; 4.0-6.0 min, 81% -79% A; 6.0-8.0 min, 79-72% of A; 8.0-15.0 min, 72% -69% A; 15.0-20.0 min, 69% -54% A; 20-20.5 min, 54% -10% A; 20.5-22 min, 10% A; 22-22.5 min, 10% -81A%. The flow rate is 0.4 mL/min; column temperature: 40 ℃; sample introduction amount: 5 μ L.
Mass spectrum conditions: mass spectrometry: a TSQ Quantum Access MAX triple quadrupole mass spectrometer; an ion source: electrospray ion source, negative ion mode (ESI-); the scanning mode is as follows: multiple Reaction Monitoring (MRM); ion transport capillary temperature: 350 ℃; electrospray voltage: 3000V; evaporation temperature: 400 ℃; collision gas pressure: 1.5 mTor; assist gas pressure (Arb): 12 kPa; sheath gas pressure: 35 kPa; the monitoring ion pair (Q1/Q3), Collision Energy (CE) and Tube Lens offset voltage (Tube Lens) for 9 analytes (ginsenosides Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, Rb3, Rd) are shown in table 1.
Table 19 Mass Spectrometry parameters of analytes
Quantitation of ion pairs.
The following is a further description with reference to specific examples.
Example 1
The embodiment provides a method for detecting ginsenoside in health food, and the specific detected health food is as follows: 1. balsam pear and American ginseng soft capsules; 2. american ginseng, hairy antler and rehmannia root tablets; 3. compound colla Corii Asini oral liquid. In the detection analysis of the common ginsenoside, 9 ginsenosides (Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, Rb3 and Rd) can not be effectively separated, and the 9 ginsenosides (Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, Rb3 and Rd) in the health food can not be analyzed and accurately quantified at the same time, so that most of interference substances influencing the measurement result can be effectively removed by adopting the method of the invention, the detection accuracy is improved, and the 9 ginsenosides (Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, Rb3 and Rd) can be accurately quantified. The specific method comprises the following steps:
1.1 laboratory instruments, reagents and materials
An experimental instrument: TSQ Quantum Access MAX triple quadrupole mass spectrometer (Thermo Scientific, USA); UltiMate 3000 ultra high performance liquid chromatography (Thermo Scientific, usa); sartorius BSA224S-CW electronic balance (Sartorius, germany); 5424R refrigerated centrifuge (Eppendorf, Germany); MIX-1 oscillatory vortex mixer (Shanghai Tomo scientific instruments Co., Ltd.); KQ-250E ultrasonic water bath (ultrasonic instruments, Inc. of Kunshan); Milli-QAdvantage A10 ultra pure water system (Millipore, USA); hypersil Gold C18 column (100X 2.1mm,1.9 μm, Thermo Scientific Co., USA)
Experimental reagents and materials: ammonium acetate (chromatographically pure, Shanghai Macklin Corp.); methanol, acetonitrile, formic acid (chromatographically pure, Merck, Germany); a solid phase extraction cartridge (neutral alumina/XAD-2, 500mg/2g,6 mL); the reference products ginsenoside Re (batch No. 110754-.
1.2 preparation of Standard solution
Standard stock solutions: accurately weighing 0.01g of each target standard substance, dissolving with methanol, preparing into 1mg/mL standard stock solution, and storing at-18 deg.C. Mixing standard solutions: respectively sucking a certain amount of standard stock solution, diluting with methanol, vortex mixing uniformly, preparing into a mixed standard solution with the mass concentration of 1.0 mug/mL, and storing at-18 ℃. Mixing standard working solution: transferring a proper amount of the mixed standard solution, and preparing a series of mixed standard working solutions of 0.005-0.5 mu g/mL by using methanol-water (30:70, v/v), wherein the working solutions are prepared on site.
1.3 conditions of analysis
1.3.1 chromatographic conditions
A chromatographic column: hypersil Gold C18 column (100X 2.1mm,1.9 μm, Thermo Scientific Co., USA); mobile phase: a is 5mmol/L ammonium acetate solution (containing 0.1% formic acid, v/v), B is acetonitrile; gradient elution conditions: 0-4.0 min, 81% A; 4.0-6.0 min, 81% -79% A; 6.0-8.0 min, 79-72% of A; 8.0-15.0 min, 72% -69% A; 15.0-20.0 min, 69% -54% A; 20-20.5 min, 54% -10% A; 20.5-22 min, 10% A; 22-22.5 min, 10% -81A%. The flow rate is 0.4 mL/min; column temperature: 40 ℃; sample introduction amount: 5 μ L.
1.3.2 Mass Spectrometry conditions
Mass spectrometry: a TSQ Quantum Access MAX triple quadrupole mass spectrometer; an ion source: electrospray ion source, negative ion mode (ESI-); the scanning mode is as follows: multiple Reaction Monitoring (MRM); ion transport capillary temperature: 350 ℃; electrospray voltage: 3000V; evaporation temperature: 400 ℃; collision gas pressure: 1.5 mTor; assist gas pressure (Arb): 12 kPa; sheath gas pressure: 35 kPa; the monitoring ion pair (Q1/Q3), Collision Energy (CE) and Tube Lens offset voltage (Tube Lens) for 9 analytes (ginsenosides Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, Rb3, Rd) are shown in table 1.
Table 19 Mass Spectrometry parameters of analytes
Quantitation of ion pairs.
1.4 sample pretreatment
1.4.1 extraction
Grinding the content of the tablet or capsule into powder, weighing 0.5g of the sample into a 50mL plastic centrifuge tube, adding about 15mL of purified water, performing vortex shaking and uniform mixing, performing ultrasonic extraction (power 300W and frequency 40kHz) at 50 ℃ for 30 minutes, centrifuging at 5000r/min for 5 minutes, transferring the supernatant into a 50mL measuring flask, re-extracting the residue with 30mL of purified water twice according to the conditions, merging the extracting solution into the measuring flask, performing constant volume to scale with the purified water, mixing uniformly to obtain a sample extracting solution, and purifying.
Liquid sample: weighing 2g of oral liquid into a 50mL plastic centrifuge tube, adding about 40mL of purified water, uniformly mixing by vortex shaking, performing ultrasonic extraction (power 300W and frequency 40kHz) at 50 ℃ for 30 minutes, fixing the volume to 50mL by using the purified water, and uniformly mixing to obtain a sample extraction solution to be purified.
1.4.2 sample extraction solution purification
Respectively activating a neutral alumina/XAD-2 solid-phase extraction column by using 20mL of 70% (v/v) ethanol aqueous solution and 20mL of water, loading 2.0mL of 1.4.1 obtained sample extraction solution on the activated solid-phase extraction column, respectively leaching by using 20mL of water, eluting by using 20mL of 70% (v/v) ethanol aqueous solution, and collecting eluent; the eluent is added with methanol-water (30:70, v/v) to reach 25.0mL, and after being mixed evenly, the eluent is filtered by a 0.22 mu m mixed filter membrane to obtain a sample solution to be detected.
1.5, detection on a computer: respectively and precisely absorbing 5 mu L of standard working solution and sample solution to be detected after sample purification, injecting into a high performance liquid chromatography-tandem mass spectrometer, measuring according to 1.3 analysis conditions, and calculating the content by a standard curve method.
1.6 results and analysis
The results are shown in FIGS. 1 to 3.
The results of measuring the contents of 9 ginsenosides (Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, Rb3, and Rd) in the balsam pear and American ginseng soft capsules, the American ginseng and hairy antler and rehmannia tablets and the compound donkey-hide gelatin oral liquid of the embodiment are shown in table 2.
TABLE 23 ginsenoside content in the samples
Example 2
The embodiment of the invention carries out methodology verification on the linear range, detection limit, recovery rate, precision, stability and matrix effect of the method for detecting the ginsenoside in the health food, and the result shows that the method can be used for accurately detecting the content of the ginsenoside in the health food and controlling the quality of the health food.
1.1 laboratory instruments, reagents and materials
The same as in example 1.
1.2 preparation of Standard solution
The same as in example 1.
1.3 conditions of analysis
1.3.1 chromatographic conditions
The same as in example 1.
1.3.2 Mass Spectrometry conditions
The same as in example 1.
1.4 sample pretreatment
1.4.1 extraction
Grinding the content of the tablet or capsule into powder, weighing 0.5g of the sample into a 50mL plastic centrifuge tube, adding about 15mL of purified water, performing vortex shaking and uniform mixing, performing ultrasonic extraction (power 300W and frequency 40kHz) at 50 ℃ for 30 minutes, centrifuging at 5000r/min for 5 minutes, transferring the supernatant into a 50mL measuring flask, re-extracting the residue with 30mL of purified water twice according to the conditions, merging the extracting solution into the measuring flask, performing constant volume to scale with the purified water, mixing uniformly to obtain a sample extracting solution, and purifying.
Liquid sample: weighing 2g of sample into a 50mL plastic centrifuge tube, adding about 40mL of purified water, uniformly mixing by vortex shaking, performing ultrasonic extraction (power 300W and frequency 40kHz) at 50 ℃ for 30 minutes, fixing the volume to 50mL by using the purified water, and uniformly mixing to obtain a sample extraction solution to be purified.
1.4.2 sample extraction solution purification
Respectively activating a neutral alumina/XAD-2 solid-phase extraction column by using 20mL of 70% (v/v) ethanol aqueous solution and 20mL of water, loading 2.0mL of 1.4.1 obtained sample extraction solution on the activated solid-phase extraction column, respectively leaching by using 20mL of water, eluting by using 20mL of 70% (v/v) ethanol aqueous solution, and collecting eluent; the eluent is added with methanol-water (30:70, v/v) to reach 25.0mL, and after being mixed evenly, the eluent is filtered by a 0.22 mu m mixed filter membrane to obtain a sample solution to be detected.
1.5, detection on a computer: respectively and precisely absorbing 5 mu L of standard working solution and sample solution to be detected after sample purification, injecting into a high performance liquid chromatography-tandem mass spectrometer, measuring according to 1.3 analysis conditions, and calculating the content by a standard curve method.
2.4 methodological considerations
2.4.1 Linear Range and detection Limit
Under the optimized chromatographic conditions (chromatographic conditions in 1.3.1), linear relations of 9 ginsenosides in the mass concentration range of 0.005-0.5 mug/mL are considered, detection is carried out under the optimized chromatographic and mass spectrometric conditions, the mass concentration of each analyte (ginsenosides Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, Rb3 and Rd) is taken as a horizontal coordinate, and the corresponding mass spectrum peak area is taken as a vertical coordinate, so that respective linear equations are obtained. The result shows that 9 ginsenosides have good linear relationship in a linear range, and the correlation coefficient is 0.9963-0.9994. The quantitative Limit (LOQ) of the method was determined with a signal-to-noise ratio (S/N) of 10, and the results are shown in Table 3. Therefore, the method for detecting the ginsenoside in the health food has high sensitivity, and can be better applied to the detection of 9 ginsenosides in the health food.
TABLE 39 Linear ranges, linear equations, correlation coefficients and quantitative limits for ginsenosides
Y is mass spectrum peak area; x is mass concentration, mu g/mL.
2.4.2 recovery and precision
According to the pretreatment method determined by the pretreatment of the 1.4 sample, 3 level standard addition recovery tests are carried out on the health food substrate (the radix puerariae soft capsule sample without ginsenoside detection), the addition levels are 1 time, 6 times and 12 times of the quantification limit, each addition level is repeated for 6 times, and the recovery rate and the precision are calculated. The results are shown in table 4, the standard recovery rate of the 9 ginsenosides in the health food is between 81.4% and 114.2%, and the precision is between 0.4% and 8.0%, and the results show that the detection method of the ginsenosides in the health food provided by the invention can meet the requirements of daily detection in a laboratory.
Table 49 ginsenoside recovery and precision (n ═ 6)
2.4.3 stability
Experiments investigate the stability of 9 ginsenosides after extraction and purification by a method of 1.4 sample pretreatment, and the obtained sample solution to be tested is placed in a sample bottle at room temperature for 1, 4,8, 12, 24 and 48 hours. After the corresponding standing time, the response values of the 9 ginsenosides are respectively measured, and the corresponding relative standard deviation is calculated, the result shows that (see table 5), the RSD value of the 9 ginsenosides is less than or equal to 8.66%, which shows that the sample solution to be measured obtained by the pretreatment and extraction method of the invention has good stability under the condition and can meet the measurement requirement.
Stability of the Table 59 ginsenosides
2.4.4 matrix Effect
Three representative matrix health-care foods of maca oyster peptide tablets, kudzu root soft capsules and compound donkey-hide gelatin oral liquid are respectively selected, the 3 products are not added with Araliaceae plant components such as ginseng, American ginseng and the like, the samples do not contain ginsenoside components, and the detection results of 9 ginsenosides are not detected, so the maca oyster peptide tablets, the kudzu root soft capsules and the compound donkey-hide gelatin oral liquid can be used as typical blank matrix samples. Processing the maca oyster peptide tablets, the kudzu root soft capsules and the compound donkey-hide gelatin oral liquid by a sample pretreatment method of 1.4, preparing a corresponding matrix standard curve by using the obtained sample matrix extraction solution to replace methanol-water (30:70, v/v) according to a preparation method of a 1.2 standard solution, and calculating a matrix effect factor (MF) of an analyte by comparing a response value of the matrix standard curve with a response value of a corresponding concentration curve. MF < 0.8 indicates significant matrix inhibition, and MF > 1.2 indicates significant matrix enhancement. The results show (see table 6) that the matrix effect factor of 9 ginsenosides in the tablet samples is between 0.922 and 1.023; the matrix effect factor in the capsule sample is between 0.896 and 0.953; the matrix effect factor in the oral liquid sample is between 0.933 and 1.051. The method for detecting the ginsenoside in the health food has the advantages that after the sample matrix is purified in the pretreatment process and diluted in a large volume, the influence of the matrix effect on the detection result of the ginsenoside is small, and the standard solution prepared by adopting a methanol-water (30:70, v/v) solvent can meet the measurement requirement.
TABLE 6 matrix Effect of different matrix samples
The results show that the detection method has the advantages of good linear range, low detection limit, high sensitivity, high recovery rate, good precision and stability and small influence of matrix effect, can be used for accurately detecting the content of the ginsenoside in the health food, and can be used for quality control of the health food.
Example 3
This example compares the effect of different purification regimes on the matrix effect factor and recovery of 9 ginsenosides.
1.1 laboratory instruments, reagents and materials
The same as in example 1.
1.2 preparation of Standard solution
The same as in example 1.
1.3 conditions of analysis
1.3.1 chromatographic conditions
The same as in example 1.
1.3.2 Mass Spectrometry conditions
The same as in example 1.
1.4 sample pretreatment
1.4.1 extraction
Grinding the contents of the soft capsule of the kudzuvine root into powder, weighing 0.5g of a sample, putting the sample into a 50mL plastic centrifuge tube, adding 1.00mL of a mixed standard solution of ginsenoside (Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, Rb3 and Rd) with the concentration of 0.1mg/mL, adding about 15mL of purified water, carrying out vortex shaking and uniform mixing, carrying out ultrasonic extraction (power 300W and frequency 40kHz) at 50 ℃ for 30 minutes, centrifuging at 5000r/min for 5min, transferring the supernatant into a 50mL measuring flask, Re-extracting residues with 30mL of purified water twice according to the conditions, merging the extracting solution into the measuring flask, carrying out constant volume calibration with the purified water, and mixing uniformly to obtain a sample extracting solution for purification.
1.4.2 sample extraction solution purification
The method of the example is as follows:
respectively activating a neutral alumina/XAD-2 solid-phase extraction column by using 20mL of 70% (v/v) ethanol aqueous solution and 20mL of water, loading 2.0mL of 1.4.1 obtained sample extraction solution on the activated solid-phase extraction column, respectively leaching by using 20mL of water, eluting by using 20mL of 70% (v/v) ethanol aqueous solution, and collecting eluent; the eluent is added with methanol-water (30:70, v/v) to reach 25.0mL, and after being mixed evenly, the eluent is filtered by a 0.22 mu m mixed filter membrane to obtain a sample solution to be detected.
The process of comparative example 1 is as follows:
respectively activating a C18 solid phase extraction column by using 20mL of 70% (v/v) ethanol aqueous solution and 20mL of water, loading a sample extraction solution obtained by 2.0mL of 1.4.1 on the activated solid phase extraction column, respectively leaching by using 20mL of water, eluting by using 20mL of 70% (v/v) ethanol aqueous solution, and collecting eluent; the eluent is added with methanol-water (30:70, v/v) to reach 25.0mL, and after being mixed evenly, the eluent is filtered by a 0.22 mu m mixed filter membrane to obtain a sample solution to be detected.
The procedure of comparative example 2 was as follows:
respectively activating an HLB solid-phase extraction column by using 20mL of 70% (v/v) ethanol aqueous solution and 20mL of water, loading a sample extraction solution obtained by 2.0mL of 1.4.1 on the activated solid-phase extraction column, respectively leaching by using 20mL of water, eluting by using 20mL of 70% (v/v) ethanol aqueous solution, and collecting an eluent; the eluent is added with methanol-water (30:70, v/v) to reach 25.0mL, and after being mixed evenly, the eluent is filtered by a 0.22 mu m mixed filter membrane to obtain a sample solution to be detected.
1.5, detection on a computer: respectively and precisely absorbing 5 mu L of standard working solution and sample solution to be detected after sample purification, injecting the solution into a high performance liquid chromatography-tandem mass spectrometer, measuring according to 1.3 analysis conditions, calculating the content by a standard curve method, and then calculating a matrix effect factor (the method is the same as the example 2) and the recovery rate.
1.6 results
The results show (as shown in table 7): after the sample extraction solution is purified by a neutral alumina/XAD-2 composite solid-phase extraction column, the matrix effect of 9 ginsenosides is obviously lower than that of the purification mode of C18 and HLB solid-phase extraction columns; and after the purification by a neutral alumina/XAD-2 composite solid phase extraction column, the recovery rate of 9 ginsenosides is also obviously superior to that of the other two purification modes.
TABLE 7 matrix effect factor and recovery of 9 ginsenosides for different purification modes
Example 4
This example examined the effect of different volumes of elution solvent on ginsenoside extraction rate in the sample extraction solution purification step.
1.1 laboratory instruments, reagents and materials
The same as in example 1.
1.2 preparation of Standard solution
The same as in example 1.
1.3 conditions of analysis
1.3.1 chromatographic conditions
The same as in example 1.
1.3.2 Mass Spectrometry conditions
The same as in example 1.
1.4 sample pretreatment
1.4.1 extraction
Grinding the contents of the soft capsule of the kudzuvine root into powder, weighing 0.5g of a sample, putting the sample into a 50mL plastic centrifuge tube, adding 1.00mL of a mixed standard solution of ginsenoside (Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, Rb3 and Rd) with the concentration of 0.1mg/mL, adding about 15mL of purified water, carrying out vortex shaking and uniform mixing, carrying out ultrasonic extraction (power 300W and frequency 40kHz) at 50 ℃ for 30 minutes, centrifuging at 5000r/min for 5min, transferring the supernatant into a 50mL measuring flask, Re-extracting residues with 30mL of purified water twice according to the conditions, merging the extracting solution into the measuring flask, carrying out constant volume calibration with the purified water, and mixing uniformly to obtain a sample extracting solution for purification.
1.4.2 sample extraction solution purification
The step is provided with 6 parallel treatments, and 6 sample extraction solutions are eluted by using elution solvents with different volumes, which specifically comprises the following steps:
respectively activating a neutral alumina/XAD-2 solid-phase extraction column by using 20mL of 70% (v/v) ethanol aqueous solution and 20mL of water, loading 2.0mL of 1.4.1 obtained sample extraction solution on the activated solid-phase extraction column, firstly leaching by using 20mL of water, then respectively eluting 6 parallel sample extraction solutions by using 5mL, 10mL, 15mL, 20mL, 25mL and 30mL of 70% (v/v) ethanol aqueous solution, and collecting eluent; the eluent is added with methanol-water (30:70, v/v) to reach 25.0mL, and after being mixed evenly, the eluent is filtered by a 0.22 mu m mixed filter membrane to obtain a sample solution to be detected.
1.5, detection on a computer: respectively and precisely absorbing 5 mu L of standard working solution and sample solution to be detected after sample purification, injecting into a high performance liquid chromatography-tandem mass spectrometer, measuring according to 1.3 analysis conditions, calculating the content by a standard curve method, and then calculating the recovery rate.
1.6 results
The results are shown in fig. 6 and show that: when the elution solvent reaches 20mL, the 9 ginsenosides can be basically and completely eluted, and the dosage of the eluent is preferably determined to be 20.0 mL.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A method for detecting ginsenoside in health food is characterized by comprising the following steps:
(1) extraction: taking a sample to be detected, and extracting the sample by using a solvent to obtain a sample extraction solution;
(2) purifying: purifying the sample extraction solution by using a solid phase extraction column, collecting the purified eluent, and performing constant volume by using a solvent to obtain a sample solution to be detected; the filler in the solid phase extraction column is a neutral alumina and XAD-2 macroporous resin composite filler;
(3) and (3) detection: injecting the sample solution to be detected into a high performance liquid chromatography-tandem mass spectrometer for detection, wherein the conditions of the liquid chromatography comprise:
a chromatographic column: c18 bonded silica gel chromatographic column;
mobile phase: a is ammonium acetate solution containing formic acid, B is acetonitrile, and gradient elution is carried out.
2. The method for detecting ginsenoside in health food as in claim 1, wherein the conditions of the liquid chromatography further comprise:
flow rate: 0.2-0.5 mL/min;
column temperature: 30 +/-10 ℃;
sample introduction amount: 2-10 μ L.
3. The method for detecting ginsenoside in health food as in claim 2, wherein the conditions of the liquid chromatography comprise:
a chromatographic column: c18 bonded silica gel chromatographic column with specification of 100 × 2.1mm and 1.9 μm;
mobile phase: a is ammonium acetate solution containing formic acid, B is acetonitrile, the concentration of ammonium acetate in the ammonium acetate solution is 5 +/-0.5 mmol/L, and the volume concentration of formic acid is 0.1 +/-0.05%;
conditions of gradient elution: 0-4.0 min, 81% A; 4.0-6.0 min, 81% -79% A; 6.0-8.0 min, 79-72% of A; 8.0-15.0 min, 72% -69% A; 15.0-20.0 min, 69% -54% A; 20-20.5 min, 54% -10% A; 20.5-22 min, 10% A; 22-22.5 min, 10% -81A%;
flow rate: 0.35-0.45 mL/min;
column temperature: 35-40 ℃;
sample introduction amount: 4-6 μ L.
4. The method for detecting ginsenoside in health food as in claim 1, wherein the conditions of mass spectrometry comprise:
an ion source: electrospray ion source, negative ion mode;
the scanning mode is as follows: monitoring multiple reactions;
ion transport capillary temperature: 250 ℃ and 400 ℃;
electrospray voltage: 2500-;
evaporation temperature: 300 ℃ and 450 ℃;
collision gas pressure: 1.5 +/-0.5 mTor;
auxiliary gas pressure: 12 +/-2 kPa;
sheath gas pressure: 35 +/-5 kPa.
5. The method for detecting ginsenoside in health food as in claim 4, wherein the conditions of mass spectrometry comprise: ion transport capillary temperature: 340 ℃ and 360 ℃;
electrospray voltage: 2900-;
evaporation temperature: 390 ℃ and 410 ℃;
collision gas pressure: 1.5 +/-0.2 mTor;
auxiliary gas pressure: 12 +/-2 kPa;
sheath gas pressure: 35 +/-2 kPa.
6. The method for detecting ginsenoside in health food as in claim 1, wherein the conditions of mass spectrometry comprise:
the monitoring ion pairs of the ginsenoside Rg1 are 799.5/637.5 and 799.5/475.5, the collision energy is 150 +/-10V, and the tube lens compensation voltage is 26 +/-2V and 33 +/-2V;
the ion pair for monitoring ginsenoside Re is 945.5/637.4, 945.5/475.5, the collision energy is 180 +/-10V, and the tube lens compensation voltage is 51 +/-2V and 36 +/-2V;
the monitoring ion pair of ginsenoside Rf is 799.5/475.4, 799.5/637.4, collision energy is 150 + -10V, and tube lens compensation voltage is 36 + -2V, 30 + -2V;
the monitoring ion pair of the ginsenoside Rg2 is 783.5/475.4 and 783.5/637.4, the collision energy is 150 +/-10V, and the tube lens compensation voltage is 36 +/-2V and 26 +/-2V;
the monitoring ion pair of ginsenoside Rb1 is 1153.6/1107.7, 1153.6/221.1, the collision energy is 180 +/-10V, and the tube lens compensation voltage is 28 +/-2V, 56 +/-2V;
the ion pair for monitoring ginsenoside Rc is 1077.6/783.5, 1077.6/945.5, collision energy is 180 + -10V, and tube lens compensation voltage is 41 + -2V, 40 + -2V;
the monitoring ion pair of ginsenoside Rb2 is 1077.6/783.5, 1077.6/945.5, the collision energy is 175 + -10V, and the tube lens compensation voltage is 46 + -2V, 38 + -2V;
the monitoring ion pair of ginsenoside Rb3 is 1077.6/783.5, 1077.6/945.5, collision energy is 180 + -10V, and tube lens compensation voltage is 42 + -2V, 41 + -2V;
the monitor ion pair of ginsenoside Rd is 945.5/783.5, 945.5/621.4, the collision energy is 164 +/-10V, and the tube lens compensation voltage is 36 +/-2V and 40 +/-2V.
7. A method for detecting ginsenoside in health food according to any one of claims 1-6, wherein the filler in the solid phase extraction column is XAD-2 macroporous resin and neutral alumina respectively from bottom to top; the mass ratio of the XAD-2 macroporous resin to the neutral alumina is 3-4:1, and the ratio of the total weight of the filler to the column volume is 2.5-5g:6 mL; preferably, the mass ratio of the XAD-2 macroporous resin to the neutral alumina is 4:1, and the ratio of the total weight of the filler to the column volume is 2.5g:6 mL.
8. A method for detecting ginsenosides in a health food according to any one of claims 1 to 6, wherein the purification comprises the steps of: respectively activating the solid phase extraction column by using 3-3.5 column volumes of 20-90% ethanol aqueous solution and 3-3.5 column volumes of water, loading 0.3-0.35 column volumes of the sample extraction solution on the activated solid phase extraction column, leaching by using 3-3.5 column volumes of water, eluting by using 3-3.5 column volumes of 30-90% ethanol aqueous solution, and collecting eluent; preferably, the first and second electrodes are formed of a metal,
the purification comprises the following steps: respectively activating the solid phase extraction column by using 3.2-3.4 column volumes of ethanol aqueous solution with volume fraction of 68-72% and 3.2-3.4 column volumes of water, loading 0.32-0.34 column volumes of the sample extraction solution on the activated solid phase extraction column, leaching by using 3.2-3.4 column volumes of water, eluting by using 3.2-3.4 column volumes of ethanol aqueous solution with volume fraction of 68-72%, and collecting eluent.
9. A method for detecting ginsenoside in health food according to any one of claims 1-6, wherein the solvent for volume determination by the solvent in the purification step is a methanol aqueous solution with volume fraction of 28-32%; and/or the solvent for extraction is water; and/or the presence of a gas in the gas,
the extraction is carried out under the ultrasonic condition, the power of the ultrasonic is 250-350W, and the frequency is 35-45 kHz.
10. The method for detecting ginsenoside in health food according to any one of claims 1-6, wherein if the sample to be detected is a solid, the extraction comprises the following steps: taking a sample to be detected, crushing, adding water according to the proportion of 28-32mL of water to the sample to be detected, and ultrasonically extracting for 1-3 times at the temperature of 45-55 ℃ for 20-40 minutes each time; if the sample to be detected is liquid, the extraction comprises the following steps: taking a sample to be detected, adding water according to the proportion of 18-22mL of water to the sample to be detected and 1g of water, and carrying out ultrasonic extraction for 20-40 minutes at the temperature of 45-55 ℃.
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