CN112649545A

CN112649545A - Method for quantitatively detecting saponin compounds

Info

Publication number: CN112649545A
Application number: CN202011490174.2A
Authority: CN
Inventors: 张峰; 冯峰
Original assignee: Chinese Academy of Inspection and Quarantine CAIQ
Current assignee: Chinese Academy of Inspection and Quarantine CAIQ
Priority date: 2020-12-15
Filing date: 2020-12-15
Publication date: 2021-04-13

Abstract

The invention discloses a method for quantitatively detecting saponin compounds. According to an embodiment of the invention, the method comprises: crushing the sample to obtain sample powder; subjecting the sample powder to ultrasonic extraction to obtain an extract; and detecting the extract by using a chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry system so as to obtain the content of the saponin compounds. The method adopts ultrasound for extraction, and effectively avoids saponin hydrolysis under heating. The chromatograph-quadrupole/electrostatic field orbit trap high-resolution mass spectrometry combined system is adopted for detection, and the detection accuracy and sensitivity are high.

Description

Method for quantitatively detecting saponin compounds

Technical Field

The invention relates to the field of analytical chemistry, in particular to a method for quantitatively detecting saponin compounds.

Background

The main active component of rhizoma Dioscoreae Septemlobae is steroid saponin compound, and has been mainly used as important raw material for synthesizing hormone medicine and contraceptive. In recent years, more and more researches show that the steroid saponin has wide pharmacological effects and important biological activities, such as anti-tumor effect, blood sugar reduction, anti-inflammation, prevention and treatment of cardiovascular diseases and the like. Because of its remarkable curative effect in treating cardiovascular and cerebrovascular diseases, it is used in many clinical applications, such as Diaoxinxuekang capsule containing 8 kinds of saponin extracted from Dioscorea panthaica Prain et Burkill and Dioscorea nipponica Makino as main components and peltate leaf perhexiline tablet containing water-soluble saponin extracted from rhizome of Dioscorea zingiberensis Wright as main component.

At present, there is no content determination item for the yam rhizome collected in the 'Chinese pharmacopoeia' 2015 edition, which brings certain difficulties for the quality control of yam rhizome. Therefore, the detection method of the saponin compound needs to be researched.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, one purpose of the invention is to provide a method for quantitatively detecting saponin compounds, which has the advantages of high detection accuracy and sensitivity, high extraction efficiency, simple and convenient operation, environmental protection and the like.

According to one aspect of the invention, the invention provides a method for quantitatively detecting saponin compounds. According to an embodiment of the invention, the method comprises: crushing the sample to obtain sample powder; subjecting the sample powder to ultrasonic extraction to obtain an extract; and detecting the extract by using a chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry system so as to obtain the content of the saponin compounds.

According to the method for quantitatively detecting the saponin compounds, provided by the embodiment of the invention, ultrasonic extraction is adopted, so that the saponin is effectively prevented from being hydrolyzed by heating. The chromatograph-quadrupole/electrostatic field orbit trap high-resolution mass spectrometry combined system is adopted for detection, and the detection accuracy and sensitivity are high.

In addition, the method for quantitatively detecting the saponin compounds according to the above embodiment of the present invention may further have the following additional technical features:

according to an embodiment of the present invention, the sample powder particle size is less than 60 mesh.

According to an embodiment of the invention, the ultrasound extraction comprises: mixing the sample powder with an ethanol solution to obtain a mixture; subjecting the mixture to ultrasound so as to obtain an ultrasound mixture; and subjecting the sonicated mixture to centrifugation to obtain the extract.

According to an embodiment of the present invention, the concentration of the ethanol solution is 33% ethanol solution.

According to the embodiment of the invention, the sample powder and the ethanol solution are mixed according to the liquid-material ratio of 120 mL: 1 g.

According to the embodiment of the invention, the ultrasonic time is 66min, and the power is 450W.

According to an embodiment of the invention, the chromatographic conditions of the detection are: a chromatographic column: ALORICH Ascentis C8, 10cm × 4.6mm, 3 μm; flow rate: 0.4 mL/min; sample chamber temperature: 10 ℃; column temperature: 25 ℃; sample introduction amount: 2 μ L.

According to an embodiment of the invention, the mass spectrometric conditions of the detection are: an ion source: HESI source, positive and negative ion detection mode; flow rate of sheath gas: 35L/min; flow rate of auxiliary gas: 10L/min; spraying voltage: the positive ion mode is 3.0kV, and the negative ion mode is-3.5 kV; temperature of ion transfer tube: the positive ion mode is 325 ℃, and the negative ion mode is 350 ℃; temperature of the auxiliary gas: the positive ion mode is 300 ℃, and the negative ion mode is 350 ℃; scanning mode: full MS/dd-MS 2; the primary mass spectrum scanning range is m/z 500-1300, the Full MS resolution is 70000, the secondary mass spectrum scanning is based on data dependent acquisition of primary parent ions, the dd-MS2 resolution is 17500, and the scanning range is m/z 80-1300.

According to an embodiment of the present invention, the saponin compound is at least one selected from the group consisting of dioscin, pseudofibrillar saponin, vincristin, procariside, fibrillar saponin, pseudoprotodioscin, trillin, protodioscin, trigonella saponin, tenninal saponin, and diosgenin a.

According to an embodiment of the invention, the sample is dioscorea septemloba.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows a graphical representation of the results of the effect of different flows on trillin retention behavior, according to one embodiment of the present invention, wherein A: acetonitrile-water; b: acetonitrile-10 mM ammonium acetate; c: acetonitrile-10 mM ammonium formate;

fig. 2 shows a schematic representation of the results of different chromatography columns on the retention behavior of orthotrigonoside and fibrillar saponins according to one embodiment of the present invention, wherein a: ALORICH Ascentis C8 chromatography column; b: an Agilent XDB-C18 chromatography column; c: XBridge BEH C18 chromatography column; d: thermo Hypersil GOLD aQ chromatography column;

fig. 3 shows a schematic representation of the results of the effect of different flow rates on the retention behavior of orthotrigonoside and fibrillar saponins according to one embodiment of the present invention, wherein a: 0.5 mL/min; b: 0.4 mL/min; c: 0.3 mL/min;

FIG. 4 shows a schematic of the results of 11 saponin extraction ion streams (500 μ g/L) according to one embodiment of the present invention, where chromatographic peak names: 1: dioscin; 2: pseudofibrillary saponins; 3: scutellarin; 4: orthotrigonoside; 5: fibrillar saponins; 6: pseudoprotodioscin; 7: trillin; 8: protodioscin; 9: trigonella leaf saponin; 10: gracillin; 11: dioscin A.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.

It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.

According to an embodiment of the present invention, the sample powder particle size is less than 60 mesh. Therefore, the method is favorable for fully extracting the saponin compounds in the sample.

According to an embodiment of the present invention, the concentration of the ethanol solution is 33% ethanol solution. According to the embodiment of the invention, the ultrasonic time is 66min, and the power is 450W. Therefore, the extraction rate of the saponin is high.

According to an embodiment of the invention, the chromatographic conditions of the detection are: a chromatographic column: ALORICH Ascentis C8, 10cm × 4.6mm, 3 μm; flow rate: 0.4 mL/min; sample chamber temperature: 10 ℃; column temperature: 25 ℃; sample introduction amount: 2 μ L. Therefore, each saponin compound has good separation effect and high detection accuracy and sensitivity.

According to an embodiment of the invention, the mass spectrometric conditions of the detection are: an ion source: HESI source, positive and negative ion detection mode; flow rate of sheath gas: 35L/min; flow rate of auxiliary gas: 10L/min; spraying voltage: the positive ion mode is 3.0kV, and the negative ion mode is-3.5 kV; temperature of ion transfer tube: the positive ion mode is 325 ℃, and the negative ion mode is 350 ℃; temperature of the auxiliary gas: the positive ion mode is 300 ℃, and the negative ion mode is 350 ℃; scanning mode: full MS/dd-MS 2; the primary mass spectrum scanning range is m/z 500-1300, the Full MS resolution is 70000, the secondary mass spectrum scanning is based on data dependent acquisition of primary parent ions, the dd-MS2 resolution is 17500, and the scanning range is m/z 80-1300. Therefore, the spectrogram obtained by detection has the primary accurate mass number of the parent ions and the fragment ion information of the secondary mass spectrum, and can meet qualitative and quantitative requirements.

The present invention is described below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or apparatus used are conventional products which are commercially available, e.g. from Sigma, without reference to the manufacturer.

Example 1

In this embodiment, the method for quantitatively detecting saponins in the embodiment of the present invention is used for detecting saponins in dioscorea spongiosa, and specifically includes the following steps:

1. experimental part

1.1 instruments and reagents

The types and manufacturers of the instruments and reagents are shown in Table 1. \ u

Meter instrument and reagent information summary table

1.2 preparation of Standard solution

Dioscin, protodioscin and pseudoprotodioscin are purchased from ChromaDex corporation, tenninal and fibrillar dioscin are purchased from J & K corporation, diosgenin A, pseudofibrillar dioscin and trillin are purchased from Shanghai Mingsheng corporation, peltate leaf saponin, trigonella leaf saponin and prototrigonella leaf saponin are purchased from Nanjing-Senega corporation, and the mass fraction of 11 standard products is more than or equal to 98%.

Standard stock solutions of 11 saponins: accurately weighing each saponin standard substance 0.01g (to 0.0001g), diluting to 10mL with acetonitrile/water 5: 5(v/v) solution, and making into 1000mg/L standard stock solution, and storing at-40 deg.C.

11 kinds of saponin mixed standard working solution: transferring 0.2mL of each standard substance solution into a volumetric flask, diluting to a constant volume of 10mL to prepare a mixed standard substance stock solution of 20mg/L, diluting step by step to form a series of standard working solutions with mass concentration, and storing at 4 ℃.

1.3 sample pretreatment

Accurately weighing 0.2g of sample powder, sieving (sieving with a 60-mesh sieve) in a 50mL centrifuge tube, adding 24mL of 33% ethanol according to the liquid-material ratio of 120: 1(mL/g), carrying out ultrasonic extraction for 66min, carrying out ultrasonic power of 450W, centrifuging after extraction, 8000r/min, carrying out 5min, taking 1mL of supernatant, adding 9mL of 25% acetonitrile for dilution, and filtering with a 0.22-micron microporous filter membrane to be tested.

1.4 analysis conditions \ u

Chromatographic conditions are as follows: the column was ALORICH Ascentis C8(10 cm. times.4.6 mm, 3 μm) and the mobile phase was water (A) -acetonitrile (B). Gradient elution: 0-2min, 30% B; 2-10min, 30-45% B; 10-25min, 45-60% B; 25-27min, 60-95% B; 27-32min, 95-95% B; 32-32.1min, 95-30% B; 32.1-35min, 30% B. Wherein the flow rate is 0.4mL/min, the sampling chamber temperature is 10 ℃, the column temperature is 25 ℃, and the sampling amount is 2 muL.

Mass spectrum conditions: the ion source is an HESI source, a positive and negative ion detection mode, a sheath gas flow rate: 35L/min; flow rate of auxiliary gas: 10L/min; spraying voltage: the positive ion mode is 3.0kV, and the negative ion mode is-3.5 kV; temperature of ion transfer tube: the positive ion mode is 325 ℃, and the negative ion mode is 350 ℃; temperature of the auxiliary gas: the positive ion mode is 300 ℃, and the negative ion mode is 350 ℃; scanning mode: full MS/dd-MS 2; the primary mass spectrum scanning range is m/z 500-1300, the Full MS resolution is 70000, the secondary mass spectrum scanning is based on data dependent acquisition of primary parent ions, the dd-MS2 resolution is 17500, and the scanning range is m/z 80-1300. Separation of 11 analytes was completed within 35min, resulting in accurate mass numbers and accurate fragment ion information. The mass spectrometric information and the collision energies used in the positive and negative ion modes for the 11 compounds are shown in table 2.

Mass Spectrometry information for target Compounds in Table 211

2 results and discussion of the experiments

2.1 optimization of Mass Spectrometry conditions

The voltage and collision energy of the mass spectrum have important influence on the cracking of the target. The 11 saponin mixed standard samples are diluted to 1 mu g/mL by 30% acetonitrile, then direct mass spectrum sample injection is carried out through a peristaltic pump, and the mass spectrum response of the 11 saponin compounds is respectively examined under a positive ion mode and a negative ion mode. And selecting the excimer ion peak with the highest response as a parent ion, selecting different collision energies to break up the parent ion, and determining the optimal collision energy by observing the relative response intensity of the primary parent ion and the fragment ions thereof. The result shows that the response of each compound in the negative ion mode is obviously higher than that in the positive ion mode because the saponin substance structurally contains glycosyl which has a plurality of hydroxyl groups and is easier to lose hydrogen ions, and in the positive ion mode, a plurality of saponins with similar structures, such as dioscin, protodioscin and pseudo-protodioscin, form the same parent ion and are difficult to distinguish, while in the negative ion mode, each saponin can form stable parent ion [ M-H ] -, so that the [ M-H ] -of each target in the negative ion mode is selected as quantitative ions.

The method comprises the steps of setting a target List (Inclusion List) by adopting a primary mass spectrum full scan of a high-resolution mass spectrum and a data-dependent secondary mass spectrum scan, and when a parent ion in the List is found during the primary mass spectrum scan and the response intensity reaches a set threshold value, smashing the parent ion and carrying out the secondary scan. The selection of the resolution ratio is the key for accurately determining the quality of the high-resolution mass spectrum, the selected resolution ratio ensures that the interference substance and the target object can realize complete baseline separation, the matrix interference effect is removed, the obtained ion flow diagram is clearer, and accurate qualitative and quantitative determination can be carried out by extracting the accurate mass number of the first-stage mass spectrum and the second-stage mass spectrum. And finally, selecting a primary mass spectrum resolution of 70000 and a secondary mass spectrum resolution of 17500, and performing data acquisition to obtain a spectrogram which has the primary accurate mass number of the parent ions and the fragment ion information of the secondary mass spectrum, so that qualitative and quantitative requirements can be met. The information of the parent ion and fragment ion of 11 saponins is shown in Table 3.

TABLE 311 parent and fragment ion information for the target Compounds

2.2 optimization of chromatographic conditions

Because protodioscin, fibril saponin and other compounds are unstable in methanol, the-OH on the C-22 position is easy to be methylated and converted into methyl protodioscin, fibril saponin and the like, and methanol is not considered as an extraction solvent and a mobile phase. The elution effect of a mobile phase system consisting of acetonitrile, water, acetonitrile-10 mmol ammonium formate and acetonitrile-10 mmol ammonium acetate is compared, and the effect is best when the acetonitrile and water are used as mobile phases for elution by comparing the separation effect, the peak shape and the response value of saponin. Taking trillin with the smallest response among 11 saponins as an example, as shown in fig. 1, when the mobile phase is acetonitrile-10 mmol ammonium formate and acetonitrile-10 mmol ammonium acetate, the peak shape is poor, and when the mobile phase is acetonitrile and water, the peak shape is sharp and the linear relation is good, and finally acetonitrile and water are selected as the mobile phase. And then, determining gradient elution conditions through optimization experiments, and completing the detection of 11 saponin compounds within 35 min.

Comparing the effect of ALORICH Ascentis C8(4.6mm × 100mm, 3 μm), Agilent XDB-C18(4.6mm × 150mm, 5 μm), Xbridge BEH C18(4.6mm × 150mm, 5 μm), Thermo Hypersil GOLD aQ (4.6mm × 150mm, 5 μm) on the separation effect of 11 saponin compounds, see FIG. 2, taking a pair of isomer protofibril saponin and prototrigonal saponin as an example, when the above three C18 chromatographic columns are used, two saponins cannot be separated; when a C8 chromatographic column is used, the two saponins are successfully separated, and the peak shapes are symmetrical. This is probably due to the fact that the saponin structure contains sugar chains with higher polarity, so that the saponin has higher polarity, and the C8 chromatographic column has higher polarity than the C18 chromatographic column, so that the method is more suitable for the separation of the saponin. The final selection column was ALORICH Ascentis C8(10 cm. times.4.6 mm, 3 μm).

The effects on the separation effect of the saponin compounds at three different flow rates of 0.3mL/min, 0.4mL/min and 0.5mL/min were compared. As can be seen from FIG. 3, when the flow rate was 0.5mL/min, the fibril saponin and the trigonella saponin were poorly separated and the peak shape was not good. When the flow rates are 0.3mL/min and 0.4mL/min, the two are better separated, and the response values are similar. To obtain a shorter analysis time, a flow rate of 0.4mL/min was finally selected. The ion flow diagram of eleven saponin extractions is shown in fig. 4.

2.3 optimization of pretreatment conditions

In the embodiment, not only the cracking rule of the saponin is researched, but also the content of the saponin component in the dioscorea spongiosa is measured. Due to the large structural difference of each saponin, the pretreatment conditions of the sample need to be optimized. The protodioscin, fibril saponin and other saponins belong to protosaponins, are easy to hydrolyze under the heating condition, remove glucose on C-26 position, and then perform F-ring cyclization to generate corresponding secondary saponin compounds. Therefore, the pretreatment method is not suitable for heating, and an ultrasonic-assisted extraction method is selected. The saponin substances can be effectively extracted by destroying the plant cell walls through ultrasonic-assisted extraction. It has become a common method for extracting natural active substances because of its simple and convenient operation and low energy consumption. In optimizing the parameters of ultrasound-assisted extraction, the interaction between the variables should be considered simultaneously. In order to optimize the optimal parameters and simultaneously study the interrelations between the key variables, Response Surface analysis (RSM) is used herein to process the data. The response surface analysis is a reasonable design experimental scheme, a multivariate quadratic regression equation is utilized to fit the functional relationship between the factors and the response values, and the regression equation is further analyzed to obtain the optimal process parameters, so that the multivariate problem is solved. The method is different from the most widely used orthogonal test design method, and has the characteristics of short test period, high accuracy of regression equation, capability of researching interaction among multiple variables and the like. In the embodiment, 5 factors of the extraction solvent, the solvent concentration, the material-liquid ratio, the ultrasonic power and the ultrasonic time, which mainly influence the extraction rate, are selected, the extraction amount of 11 saponins is used as an investigation index for evaluating the extraction efficiency, and single-factor investigation is firstly carried out. On the basis of a single-factor experiment result, a three-factor and three-level Box-Behnken test (BBD) design scheme is adopted, and 3 influencing factors for extracting the dioscorea spongiosa saponin by an ultrasonic-assisted method are deeply researched and condition optimized.

2.3.1 response surface test

According to the Design principle of a Box-Behnken test in Design Expert 8.5 software and the single-factor test result of extracting saponin, the test determines that an extracting solvent is ethanol, the extracting power is 450W, ethanol concentration (A), ultrasonic time (B) and liquid-material ratio (C) which have obvious influence on the extraction rate of saponin are selected as investigation factors, and as the test estimates the extraction rate by taking the extraction rate of various steroid saponins as an index, a 'total evaluation normalization value (OD)' is introduced as the index for estimating the extraction rate. "OD" is the average of the extraction yields of 11 saponins. The test factors and the horizontal design are shown in Table 4, and the response surface test results are shown in Table 5.

TABLE 4 response surface analysis factors and levels

TABLE 5 BBD design matrix and Experimental results

a OD overall desirability

a total score normalized to one

And (3) carrying out data analysis on the test result in the table 4 by using Design Expert 8.5 program software, taking the total evaluation normalization value of the yield of 11 saponins as a response value, and carrying out regression fitting on the response value and each factor to obtain a regression equation: y ═ 0.54+0.024A +0.014B-9.948E-003C-4.208E-003AB-5.309E-003AC-1.798E-003BC-0.066a2-0.024B2-0.052C2, and the results of analysis of variance and significance tests on the regression equation are shown in table 3.7. As can be seen from Table 3.5, the P-value of the model in the regression term is P < 0.0001, demonstrating that the model is significant and the method is reliable. And the mismatching item P is 0.1211 & gt 0.05, which shows that the mismatching item difference is not obvious, namely the quadratic regression model can successfully fit the influence of ethanol concentration, ultrasonic time and liquid material ratio on the extraction rate of the saponin, and can replace the real point of the test to analyze the yield of the saponin. The decision coefficient (R2Adj) of the regression model is 0.9746, which indicates that the model can explain 97.46% of response value changes, and only 2.54% of variation cannot be explained by the model, so that the established model is reliable, can well explain the change of saponin yield, and can effectively predict the saponin yield. Analysis of various factors can show that when P is less than 0.0001, the influence of the corresponding test conditions on the saponin yield is highly significant; when P is less than 0.05, the influence of the factor on the saponin extraction effect is considered to be significant. The significance of the regression equation coefficients can be obtained from table 6, the ethanol concentration (a), the ultrasonic time (B), the secondary term of the ethanol concentration (a2), the secondary term of the ultrasonic time (B2), and the secondary term of the liquid-to-material ratio (C2) have a highly significant effect on the saponin yield, and the p value of the variable interaction term in the model is observed, so that the interaction between variables is not significant. According to the obtained model, the optimal process conditions of 11 saponins in the yam rhizome can be predicted: the concentration of ethanol is 33.47%, the ultrasonic time is 65.84min, and the liquid-material ratio is 122.23mL/g, the OD theoretical value of saponin under the condition is 0.54%, for the convenience of practical operation, the concentration of ethanol is 33%, the ultrasonic time is 66min, and the liquid-material ratio is 120 mL/g.

TABLE 6 ANOVA TABLE

2.4 method validation

2.4.1 Linear relationship, detection Limit, quantification Limit

Taking the original concentration of each saponin in rhizoma Dioscoreae Septemlobae as the central point of a standard curve, sucking a certain amount of standard stock working solution, diluting the solution with initial mobile phase step by step into standard working solution with serial concentrations, measuring under the condition of 1.4, taking the concentration of the standard working solution as the abscissa (X), and performing linear regression with the peak area corresponding to each saponin primary parent ion as the ordinate (Y) to obtain the standard curve. And (3) diluting the standard solution, wherein the concentration corresponding to the chromatographic peak S/N of 3 is taken as the detection limit of the method, and the concentration corresponding to the chromatographic peak S/N of 10 is taken as the quantification limit of the method. The results show that the concentrations of the components and the peak areas thereof have good linear relations, and the linear equation, the linear range, the correlation coefficient, the detection limit and the quantitative limit are shown in Table 7.

TABLE 7 Linear Range, Linear equation, correlation coefficient (r2), detection limits and quantitation limits (units: μ g/L) for the target Compound

2.4.2 precision test

Repeatedly sampling 11 saponin mixed standard samples with the concentration of 100 mug/L for 6 times in one day, and calculating relative standard deviation RSD (standard deviation), namely the precision in the day; the assay was repeated for 6 days and RSD, i.e. the inter-day precision, was calculated. The precision in the day and the precision in the day are respectively 1.78-6.87% and 2.32-7.22%, which shows that the precision of the instrument is good.

2.4.3 repeatability test

6 parts of the same sample powder are taken and prepared according to a 3.2.3 sample processing method, the analysis condition is 3.2.4, the RSD is between 2.25 and 6.98 percent by calculating the content of each saponin, and the method is proved to have good repeatability.

2.4.4 stability test

And (3) sampling the same test solution for 0, 2, 4, 6, 8, 10, 12 and 24 hours, respectively, recording the peak area of each saponin, and calculating that the RSD between 6 parallel samples is between 2.69 and 7.17 percent, which indicates that the test solution is stable within 24 hours.

2.4.5 recovery rate with standard addition

And verifying the accuracy of the method by adopting a labeling recovery method. Weighing rhizoma Dioscoreae Septemlobae sample 0.2g with known saponin content, and adding mixed standard solution of 11 kinds of saponin. The standard solution was added at 3 levels of 50%, 100%, 150% of the background content of each saponin in the sample. The sample was prepared according to 1.3 sample treatment method and measured to calculate the recovery rate of spiked samples. Under the condition of three concentration levels, the recovery rate of each saponin is 82.52-107.51%, and the RSD is 1.86-7.12%, which shows that the method has good accuracy and can be used for measuring the content of each saponin.

2.4.6 determination of saponin content in actual sample

The content of 11 saponins in 10 batches of rhizoma Dioscoreae Septemlobae collected from different regions was determined, and the data of the target substance content determination are shown in Table 8. In each sample of dioscorea spongiosa, the highest and second highest saponins were protodioscin and dioscin, respectively, with a relatively low content of diosmin a, while trillin, trigonella triterpenes and prototrigonella triterpenes were significantly lower than the other saponins, and were not detected in a few samples.

11 kinds of saponin content determination (unit: mg/kg) in 810 samples of rhizoma Dioscoreae Septemlobae

3. Small knot

The method for quantitatively detecting the saponin compounds provided by the embodiment of the invention is based on high performance liquid chromatography-quadrupole/electrostatic field orbit trap high-resolution mass spectrometry, establishes a high-sensitivity and high-accuracy analysis method for qualitatively and quantitatively researching 11 steroidal saponin compounds in dioscorea spongiosa, and performs content determination on saponins in 10 batches of dioscorea spongiosa from different production places. In addition, the mass spectrum cracking way and rule of the eleven saponins in the positive and negative ion mode are analyzed and summarized. The method has the following advantages:

(1) the chromatographic conditions are optimized, and the influence of different chromatographic columns, mobile phases and flow velocities on chromatographic peak shapes and separation degrees is examined, so that the separation degree of each compound is good, the peak shapes are sharp, and the response is high. The method comprises the following steps of extracting steroid saponin compounds in the rhizoma dioscoreae septemlobae by adopting a response surface method and an ultrasonic auxiliary extraction method, carrying out single-factor tests on an extraction solvent, ethanol concentration, a solid-liquid ratio, ultrasonic time and ultrasonic power, selecting the ethanol concentration, the ultrasonic time and a liquid-material ratio on the basis, carrying out a three-factor three-level test, and optimizing extraction parameters of 11 types of saponins by a Box-Behnken design method, wherein the extraction parameters are as follows: the extraction solvent is 33% ethanol, the liquid-material ratio is 120: 1(mL/g), the ultrasonic power is 450W, and the ultrasonic time is 66 min. The method has the advantages of high extraction efficiency, simple operation, environmental friendliness, etc.

(2) The methodological results show that 11 saponins have good linear relation in respective linear range (r2 is more than 0.9956), and the detection limit and the quantitative limit range are respectively 0.2-1 mug/L (S/N is 3) and 0.6-3 mug/L (S/N is 10); the daily precision (RSD) is less than 6.87% (n: 6), and the daytime precision is less than 7.22% (n: 6). Under the condition of adding the standard in three concentration levels, the recovery rate of each saponin is 82.52-107.51%, and the RSD is 1.86-7.12%.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A method for quantitatively detecting saponin compounds is characterized by comprising the following steps:

crushing the sample to obtain sample powder;

subjecting the sample powder to ultrasonic extraction to obtain an extract; and

and detecting the extract by using a chromatograph-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry system so as to obtain the content of the saponin compounds.

2. The method of claim 1, wherein the sample powder particle size is less than 60 mesh.

3. The method of claim 1, wherein the ultrasound extraction comprises:

mixing the sample powder with an ethanol solution to obtain a mixture;

subjecting the mixture to ultrasound so as to obtain an ultrasound mixture; and

subjecting the sonicated mixture to centrifugation to obtain the extract.

4. The method of claim 1, wherein the ethanol solution has a concentration of 33% ethanol solution.

5. The method of claim 1, wherein the sample powder is mixed with the ethanol solution at a liquid-to-material ratio of 120 mL: 1 g.

6. The method of claim 1, wherein the ultrasound is at 66min and 450W power.

7. The method of claim 1, wherein the detected chromatographic conditions are:

a chromatographic column: ALORICH Ascentis C8, 10cm × 4.6mm, 3 μm;

flow rate: 0.4 mL/min;

sample chamber temperature: 10 ℃;

column temperature: 25 ℃;

sample introduction amount: 2 μ L.

8. The method of claim 1, wherein the detected mass spectrometry conditions are:

an ion source: HESI source, positive and negative ion detection mode;

flow rate of sheath gas: 35L/min;

flow rate of auxiliary gas: 10L/min;

spraying voltage: the positive ion mode is 3.0kV, and the negative ion mode is-3.5 kV;

temperature of ion transfer tube: the positive ion mode is 325 ℃, and the negative ion mode is 350 ℃;

temperature of the auxiliary gas: the positive ion mode is 300 ℃, and the negative ion mode is 350 ℃;

scanning mode: FullMS/dd-MS 2;

the primary mass spectrum scanning range is m/z 500-1300, the FullMS resolution is 70000, the secondary mass spectrum scanning is based on data dependent acquisition of primary parent ions, the dd-MS2 resolution is 17500, and the scanning range is m/z 80-1300.

9. The method according to claim 1, wherein the saponin compound is at least one selected from the group consisting of dioscin, pseudofibrillar saponin, vincristoside, Prodelgenin, fibrillar saponin, pseudoprotodioscin, trillin, protodioscin, trigonella elegans saponin, tenninal saponin, and diosgenin A.

10. The method of claim 1, wherein the sample is Dioscorea tokoro.