Method for detecting ginsenoside components in shenlu tea

Abstract

The present application belongs to the technical field of analytical chemistry, and particularly relates to a detection method of ginsenoside components in Shenzhu tea. The graphene is covalently grafted and modified by 3-(4-ethoxyphenoxy)propyl trimethoxysilane, the prepared composite material is used as a solid-phase extraction filler, can effectively improve the removal rate of saccharides in the Shenzhu tea sample, the recovery rate of the standard addition and the detection precision, greatly prolongs the reusable number of the material, and can more accurately, stably and efficiently realize the enrichment and detection of ginsenosides in the Shenzhu tea.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically a method for detecting ginsenoside components in ginseng and deer antler tea. Background Technology

[0002] Ginsenosides are the main active ingredients of ginseng, American ginseng, Panax notoginseng, and other Araliaceae plants. They belong to the triterpenoid saponin class of compounds and are mainly classified into protopanaxadiol type (such as Rb1, Rb2, Rc, Rd), protopanatriol type (such as Rg1, Re, Rf), and oleanolic acid type (such as Ro) based on different aglycone structures. Modern pharmacological studies have shown that ginsenosides have various biological activities, including anti-tumor, immune enhancement, antioxidant, and anti-myocardial ischemia-reperfusion injury. Ginseng-deer antler tea, as a compound health food, is usually composed of deer antler, ginseng (or ginseng stems and leaves), and other Chinese medicinal materials, and has the effects of replenishing qi and blood, warming the kidneys and assisting yang. As an important functional component, the accurate determination of ginsenoside content is of great significance for the quality control, efficacy evaluation, and product stability investigation of ginseng-deer antler tea. However, the matrix of ginseng-deer antler tea is extremely complex. In addition to the target ginsenosides, it also contains a large number of sugars, proteins, amino acids, pigments, and interfering substances such as polypeptides and hormone analogs from deer antler.

[0003] Currently, the main method for determining ginsenosides is high-performance liquid chromatography (HPLC). HPLC typically uses octadecylsilane-bonded silica gel as the packing material and acetonitrile-water as the mobile phase for gradient elution (0–30 min, 19% acetonitrile; 30–55 min, 19%→29% acetonitrile). Detection is performed using an evaporative light scattering detector, and the contents of ginsenosides Rg1 and Re are calculated using an external standard two-point logarithmic equation. However, this method suffers from low detection sensitivity. Therefore, a more sensitive and selective high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS) method has been developed.

[0004] Sample pretreatment is a crucial step in determining the accuracy and reliability of the final analytical results. Ginsenosides are typically present in low concentrations in raw materials or formulations, requiring enrichment. Simultaneously, the sample matrix is ​​extremely complex, containing not only the target saponins but also a large number of interfering substances such as sugars, proteins, amino acids, and pigments. If these substances are not effectively removed, they will not only contaminate the analytical system but also produce severe matrix effects, leading to ion inhibition or enhancement, thereby significantly impacting the accuracy and reproducibility of quantitative analysis.

[0005] To address the aforementioned issues, researchers have developed various sample pretreatment techniques. Liquid-liquid extraction (LLE) is a traditional method, but it is cumbersome, time-consuming, requires large amounts of organic solvents, and is prone to emulsification. Solid-phase extraction (SPE), due to its advantages such as high recovery rate, high enrichment factor, and low organic solvent consumption, has become one of the most widely used pretreatment techniques. While commercially available SPE packing materials such as C18, HLB, and macroporous adsorption resins have some effectiveness, their selective adsorption capacity for ginsenosides is limited when processing complex samples. They often struggle to completely separate structurally similar saponins from a large number of interfering substances (especially sugars), resulting in unsatisfactory purification effects and low recovery rates.

[0006] To improve purification efficiency, researchers have begun exploring novel adsorption materials. Carbon nanomaterials, such as carbon nanotubes (CNTs) and graphene, have shown great potential in sample pretreatment due to their large specific surface area and unique π-π conjugated structure. Studies have shown that graphene has excellent adsorption performance for aromatic compounds containing benzene ring structures. However, directly using unmodified graphene also has some limitations: its sheet structure is prone to aggregation, leading to a reduction in effective adsorption area; its adsorption selectivity for target analytes is not strong enough, making it difficult to distinguish between different types of ginsenosides with similar structures, and the non-specific adsorption of interfering substances may also affect the purification effect; in addition, the elution of target analytes after adsorption may also be difficult. Although some studies have attempted to functionalize graphene, such as preparing magnetic graphene composites or silica-supported graphene, to improve its operational performance or increase adsorption capacity, there is still a lack of a solid-phase extraction adsorbent that can simultaneously achieve highly selective enrichment of ginsenosides, efficient removal of strongly polar interfering substances such as sugars, and has good material stability and reusability.

[0007] Therefore, developing a detection method that is simple to operate, highly efficient in purification, selective, has a short analysis time, and can simultaneously determine multiple ginsenosides is of great significance for the quality control of ginseng and deer antler tea and related ginseng health foods. Summary of the Invention

[0008] To address the above problems, this invention provides a method for detecting ginsenoside components in ginseng and deer antler tea, which solves the problems of low purification efficiency and poor selectivity in the current separation and detection of multiple ginsenosides in ginseng and deer antler tea.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A method for detecting ginsenoside components in ginseng and deer antler tea includes the following steps: solid-phase extraction (SPE) is used to purify the ginseng and deer antler tea extract using a solid-phase extraction packing material to obtain a test solution; then, the test solution is analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS), and the ginsenoside content in the test solution is calculated based on a standard curve. The solid-phase extraction packing material is prepared as follows: graphene oxide is dispersed in a solvent, and the pH of the system is adjusted to 4-5 to obtain a graphene oxide reaction solution; 3-(4-ethoxyphenoxy)propyltrimethoxysilane is dissolved in a solvent, water is added and stirred to obtain a silane coupling agent solution; the silane coupling agent solution is added dropwise to the graphene oxide reaction solution, heated to react, and then purified to obtain the solid-phase extraction packing material; the chemical structure of the 3-(4-ethoxyphenoxy)propyltrimethoxysilane is as follows:

[0011] .

[0012] Preferably, the graphene oxide has a sheet diameter of 1~1.5μm and a sheet thickness of 0.5~1nm.

[0013] Preferably, the mass ratio of the graphene oxide to the 3-(4-ethoxyphenoxy)propyltrimethoxysilane is 1:3~5.

[0014] Preferably, the heating reaction is carried out at a temperature of 50-70°C for 4-8 hours.

[0015] Preferably, the purification method is as follows: after heating the reaction, the system after the reaction is centrifuged to separate the precipitate, and then the precipitate is washed with ethanol and water in sequence. The washed solid precipitate is then dried to obtain solid phase extraction packing.

[0016] Preferably, the 3-(4-ethoxyphenoxy)propyltrimethoxysilane is prepared by the following method: 4-ethoxyphenol, sodium hydroxide and solvent are mixed and dissolved at 40~50°C, and then 3-chloropropyltrimethoxysilane is added dropwise. After the addition is complete, the temperature is raised to 80~85°C and the reaction is maintained at this temperature for 8~10 hours. After the reaction is completed, the mixture is washed, dried, purified by vacuum distillation and recrystallization to obtain 3-(4-ethoxyphenoxy)propyltrimethoxysilane.

[0017] Preferably, the recrystallization purification method is as follows: the crude product obtained by vacuum distillation and the mixed solvent are heated to 60-65°C, stirred and refluxed for 30-45 min, filtered, the filtrate is naturally cooled to 25-30°C, and then allowed to stand and cool at 5-8°C for 8-10 h, filtered again, and the solid obtained by the second filtration is dried to obtain 3-(4-ethoxyphenoxy)propyltrimethoxysilane; the mixed solvent is composed of n-hexane and ethyl acetate in a volume ratio of 6-8:1, and the mass ratio of the crude product to the mixed solvent is 1:7-8.

[0018] Preferably, the preparation method of the ginseng and deer antler tea extract is as follows: take ginseng and deer antler tea sample powder, add methanol aqueous solution with a mass fraction of 70~75%, vortex mix and then extract by ultrasonication, collect the supernatant by centrifugation, concentrate to remove methanol and then reconstitute to obtain the extract.

[0019] Preferably, the method for solid-phase extraction purification of ginseng and deer antler tea extract using solid-phase extraction packing material is as follows: solid-phase extraction packing material is packed into a solid-phase extraction column, ginseng and deer antler tea extract is loaded onto the column, followed by rinsing and elution. The eluent is collected, concentrated, and reconstituted to obtain the test solution. The loading flow rate is 0.5~0.8 mL / min. The rinsing is performed with water. The elution is performed with a 70~75% (w / w) ethanol aqueous solution at a flow rate of 0.8~1 mL / min.

[0020] Preferably, the ginsenoside is at least one of ginsenosides Rg1, Re, Rf, Rb1 and Rd.

[0021] The beneficial effects of the method for detecting ginsenoside components in ginseng and deer antler tea of ​​the present invention are as follows: The present invention uses 3-(4-ethoxyphenoxy)propyltrimethoxysilane to covalently graft graphene to modify it. The resulting composite material, as a solid-phase extraction filler, can effectively improve the sugar removal rate, spike recovery rate and detection precision of ginsenosides, while significantly extending the number of times the material can be reused. It can more accurately, stably and efficiently achieve the enrichment and detection of ginsenosides in ginseng and deer antler tea. Attached Figure Description

[0022] Figure 1 The image shows the 1H NMR spectrum of 3-(4-ethoxyphenoxy)propyltrimethoxysilane prepared in Example 1 of this invention. Detailed Implementation

[0023] To enable those skilled in the art to better understand the technical solution, the present invention will be described in detail below with reference to embodiments. The description in this part is only exemplary and explanatory, and should not be used to limit the scope of protection of the present invention in any way.

[0024] Example 1

[0025] The method for detecting ginsenoside components in ginseng and deer antler tea according to this embodiment includes the following steps:

[0026] (1) Add 152.18 g of 4-ethoxyphenol, 44.00 g of sodium hydroxide, and 400 mL of a mixed solvent consisting of toluene and methanol in a volume ratio of 9:1 to a three-necked flask. Then, install a spherical condenser and a constant-pressure dropping funnel on the three-necked flask, turn on the magnetic stirrer, heat to 50 °C, and stir until the sodium hydroxide is completely dissolved. Then, add 198.71 g of 3-chloropropyltrimethoxysilane dropwise to the three-necked flask at a dropping rate of 2-3 drops / s, and maintain the system temperature at 50-55 °C during the dropping process. After the dropping is completed, raise the temperature to 80 °C and maintain the temperature for the reaction. After 8 hours of reaction, heating was stopped, the system was cooled to room temperature, 300 mL of deionized water was added to the three-necked flask, stirred for 10 minutes, allowed to stand and separate into layers, and the lower aqueous phase (containing impurities such as sodium chloride) was discarded. The organic phase was washed three times with deionized water (200 mL of deionized water each time), and the pH of the final washing solution was tested and found to be neutral. The organic phase was transferred to a dry conical flask, 20 g of anhydrous sodium sulfate was added, and the flask was sealed and allowed to dry for 4 hours. The filtrate was filtered, and the filtrate was collected. Toluene was removed by vacuum distillation at 50 °C and -0.09 MPa to obtain a pale yellow crude product.

[0027] Anhydrous n-hexane and anhydrous ethyl acetate were then mixed at a volume ratio of 8:1 to obtain a mixed solvent. 20 g of the pale yellow crude product and 160 g of the mixed solvent were added to a 250 mL round-bottom flask. The flask was then placed in a constant-temperature water bath and heated to 60 °C. The mixture was stirred and refluxed for 30 min until the crude product was fully dissolved. The mixture was then filtered while hot to remove insoluble solid impurities. The filtrate was allowed to cool naturally to 25 °C at room temperature, then placed in a 5 °C refrigerator and allowed to stand for 8 h. The filtrate was then filtered using a Buchner funnel pre-cooled to 5 °C. The filter cake was washed twice with 10 mL of n-hexane pre-cooled to 5 °C. The washed solid was placed in a vacuum drying oven and dried at 30 °C and -0.09 MPa for 12 h to obtain 3-(4-ethoxyphenoxy)propyltrimethoxysilane. The 1H NMR spectrum of 3-(4-ethoxyphenoxy)propyltrimethoxysilane is shown below. Figure 1 As shown, the chemical structure is as follows:

[0028] .

[0029] (2) Take 1.0 g of graphene oxide (sheet diameter 1~1.5 μm, sheet thickness 0.5~1 nm, purity ≥99%) and add it to a mixed solvent consisting of 80 mL of anhydrous ethanol and 20 mL of deionized water. Disperse it ultrasonically for 30 min (40 kHz, 200 W) to obtain a uniform graphene oxide ethanol dispersion. Then add 0.1 mol / L hydrochloric acid to adjust the pH of the system to 4 to obtain the graphene oxide reaction solution. Take 3 g of 3-(4-ethoxyphenoxy)propyltrimethoxysilane and add it to 20 mL of anhydrous ethanol. Stir until uniform, then add 5 mL of deionized water and stir at room temperature for 30 min. The silane coupling agent solution was obtained by adding the silane coupling agent solution dropwise to the graphene oxide reaction solution. The mixture was heated to 60℃ under nitrogen protection and stirred for 6 hours (stirring rate 400 r / min). After the reaction was completed, the mixture was centrifuged (8000 r / min, 5 min) and the precipitate was collected. The precipitate was washed with anhydrous ethanol and deionized water three times each to remove ungrafted coupling agent and impurities. The washed solid precipitate was then vacuum dried at 50℃ and a vacuum degree of -0.09 MPa for 12 hours to obtain the modified graphene oxide composite material with a grafting rate of 8.7%.

[0030] The grafting rate was tested as follows: 10 mg each of graphene oxide and modified graphene oxide composite material were placed in a vacuum drying oven and dried at 50℃ and -0.09 MPa for 4 hours to remove surface adsorbed water and residual solvent. After drying, the powder was ground into powder using an agate mortar (to avoid uneven heat conduction due to excessively large particles). The powder was then sieved through a 100-mesh standard sieve (150 μm aperture) and the sieved powder was collected. The powder sample was sealed and stored for later use. 6 mg of the powder sample (dried and ground graphene oxide or modified graphene oxide composite material) was weighed and placed in an alumina crucible (the crucible needed to be calcined at 800℃ to constant weight beforehand to remove residual impurities). Then, under a nitrogen atmosphere, the temperature was increased from 30℃ to 300℃ at a rate of 10℃ / min. After reaching 300℃, the atmosphere was switched from nitrogen to air. After holding at this temperature for 1 min, the temperature was increased to 800℃ at a rate of 10℃ / min. The mass loss rate of graphene oxide and the modified graphene oxide composite material in the temperature range of 300~500℃ was recorded respectively, and the grafting rate was calculated as follows: Grafting rate G%=(W1−W0) / (1−W0)×100%, where W0 is the thermal weight loss value (%) of graphene oxide in the temperature range of 300~800℃, and W1 is the thermal weight loss value (%) of the modified graphene oxide composite material in the temperature range of 300~800℃.

[0031] (3) Weigh 2.0g of ginseng and deer antler tea sample powder, place it in a 50mL centrifuge tube, add 30mL of 70% methanol aqueous solution, vortex mix for 1min, ultrasonically extract for 30min (ultrasonic frequency 40kHz, power 200W), then centrifuge at 8000r / min for 10min, and collect the supernatant; the residue is extracted again with 20mL of 70% methanol, the two supernatants are combined, and the mixture is concentrated by rotary evaporation until no methanol residue is left. Add 2mL of methanol to the residue and vortex thoroughly, then add 8mL of deionized water, vortex mix, filter through a 0.22μm microporous membrane, and take the filtrate as the sample extract; take 300mg of modified graphene oxide composite material as solid phase extraction packing, uniformly fill it into a 25mL empty solid phase extraction column, and fix the upper and lower ends with degreased cotton to avoid material loss; rinse the solid phase extraction column with 10mL of anhydrous ethanol, and then rinse with 15mL of ethanol. The sample was balanced with deionized water at a flow rate of 1 mL / min to ensure adequate wetting of the material. Then, 10 mL of the sample extract was slowly injected into the solid-phase extraction column at a flow rate of 0.5 mL / min to allow the ginsenosides to fully interact with the composite material. The solid-phase extraction column was then rinsed with 20 mL of deionized water to remove water-soluble impurities such as sugars and amino acids, and the eluent was discarded. Next, 20 mL of 70% ethanol aqueous solution was used as the eluent at a flow rate of 0.8 mL / min. The eluent was collected, concentrated to dryness by rotary evaporation, and then reconstituted with 2 mL of 50% methanol aqueous solution. The solution was vortexed for 5 min and then filtered through a 0.22 μm organic filter membrane to obtain the test solution. Finally, the test solution was analyzed by HPLC-MS to obtain the characteristic peak areas of the five ginsenosides. The content of the five ginsenosides was calculated by substituting the characteristic peak areas into the standard curve.

[0032] The standard curve was plotted as follows: Five ginsenoside single-standard stock solutions (1 mg / mL, dissolved and diluted with methanol) and a series of mixed standard working solutions (concentration gradients of 0.005, 0.01, 0.05, 0.1, and 0.5 μg / mL, diluted with methanol, freshly prepared and used immediately) were prepared, ensuring that each concentration of standard solution was free of turbidity and precipitation, and that the concentration was accurate (weighing error ≤ 0.01 mg, volume adjustment error ≤ 0.01 mL). Then, HPLC-MS detection was performed. During detection, the series of mixed standard working solutions were injected sequentially from low to high concentration, with each concentration injected three times, and the injection volume was 5 μL for each concentration, strictly controlling the injection rate to be consistent. (To avoid injection deviation); During the detection process, the characteristic peak areas of the five ginsenosides in each concentration standard solution are recorded simultaneously (based on the peak area of ​​the target ion pair monitored in MRM mode; abnormal peaks are recorded and evaluated (peak shape asymmetry factor 1.0~1.2, those outside the range are considered abnormal). The average peak area of ​​three parallel injections is taken as the response value corresponding to that concentration. If the RSD of the peak area of ​​a certain concentration is >5% after three injections, the sample needs to be re-injected to ensure data reliability. Linear regression analysis is performed with the mass concentration of the mixed standard working solution of each ginsenoside (X, unit μg / mL) as the abscissa and the average peak area (Y) as the ordinate, and a standard curve is plotted.

[0033] The chromatographic conditions for HPLC-MS detection are as follows: Column: BEH C 18 Chromatographic column (100 mm × 2.1 mm, 1.7 μm, Waters Corporation, USA); column temperature: 40 °C; mobile phase: Phase A was 5 mmol / L ammonium acetate solution (containing 0.1% formic acid), Phase B was acetonitrile; gradient elution program: 0–4.0 min, 81% A; 4.0–6.0 min, 81% A → 79% A; 6.0–8.0 min, 79% A → 72% A; 8.0–15.0 min, 72% A A→69%A; 15.0~20.0 min, 69%A→54%A; 20.0~20.5 min, 54%A→40%A; 20.5~21.0 min, 40%A→10%A; 21.0~22.0 min, 10%A; 22.0~22.1 min, 10%A→81%A; 22.1~24.0 min, 81%A; Flow rate: 0.4 mL / min; Injection volume: 5 μL. The mass spectrometry conditions for HPLC-MS detection are as follows: Ion source: Electrospray ionization source (ESI). - Scanning mode: Multiple reaction monitoring (MRM); Ion transport capillary temperature: 350℃; Electrospray voltage: 3000V; The MRM monitoring ion pairs and collision energies of the five ginsenosides are shown in Table 1.

[0034] Table 1. MRM monitoring of ion pairs and collision energies of five ginsenosides

[0035]

[0036] Comparative Example 1

[0037] The difference between the detection method of ginsenosides in this comparative example of ginseng and deer antler tea and the detection method of ginsenosides in ginseng and deer antler tea in Example 1 is that the modified graphene oxide composite material is replaced with the graphene oxide in step (2) of Example 1 in step (3) of the detection method of ginsenosides in ginseng and deer antler tea in this comparative example.

[0038] Comparative Example 2

[0039] The difference between the detection method of ginsenosides in this comparative example of ginseng and deer antler tea and the detection method of ginsenosides in ginseng and deer antler tea in Example 1 is that in step (2) of the detection method of ginsenosides in this comparative example, 3-(4-ethoxyphenoxy)propyltrimethoxysilane is replaced with aminopropyltrimethoxysilane.

[0040] Comparative Example 3

[0041] The difference between the detection method of ginsenosides in this comparative example of ginseng and deer antler tea and the detection method of ginsenosides in ginseng and deer antler tea in Example 1 is that in step (2) of the detection method of ginsenosides in this comparative example, 3-(4-ethoxyphenoxy)propyltrimethoxysilane is replaced with phenyltrimethoxysilane.

[0042] Comparative Example 4

[0043] The difference between the detection method of ginsenosides in this comparative example of ginseng and deer antler tea and the detection method of ginsenosides in ginseng and deer antler tea in Example 1 is that in step (2) of the detection method of ginsenosides in this comparative example, 3-(4-ethoxyphenoxy)propyltrimethoxysilane is replaced with (3-methoxypropyl)trimethoxysilane.

[0044] Comparative Example 5

[0045] The difference between the detection method of ginsenosides in this comparative example of ginseng and deer antler tea and the detection method of ginsenosides in ginseng and deer antler tea in Example 1 is that in step (2) of the detection method of ginsenosides in this comparative example, 3-(4-ethoxyphenoxy)propyltrimethoxysilane is replaced with trimethoxy(3-phenoxypropyl)silane.

[0046] Experimental Example 1

[0047] To evaluate the purification effect of the solid-phase extraction column packing material (i.e., graphene oxide adsorbent material) on ginsenosides in ginseng and deer antler tea samples in the detection methods of ginseng and deer antler tea in Example 1 and Comparative Examples 1-5, the same batch and the same mass (2.0g) of ginseng and deer antler tea samples were treated according to the steps (3) of Example 1 and Comparative Examples 1-5, respectively, to obtain 6 test solutions; the residual sugar content in each test solution was determined by the phenol-sulfuric acid colorimetric method (with glucose as standard and detection wavelength 490nm), and the sugar removal rate was calculated as follows: Sugar removal rate (%) = (initial sugar content in sample - residual sugar content in test solution) / initial sugar content in sample × 100%; 3 samples were tested in parallel, and the average value was taken. The results are shown in Table 2.

[0048] Table 2 Purification effect of ginsenosides on ginseng and deer antler tea samples

[0049]

[0050] As shown in Table 2, in Example 1, the use of 4-ethoxyphenoxypropyltrimethoxysilane-modified graphene oxide as a solid-phase extraction filler resulted in a higher sugar removal rate after purification of the ginseng and deer antler tea sample, which was superior to Comparative Examples 1 and 2-5. The effective improvement in impurity removal rate indicates that this invention can effectively remove interfering substances in the sample matrix, reduce baseline noise, and thus significantly improve detection sensitivity.

[0051] Comparative Example 1, using unmodified graphene oxide, showed a low sugar removal rate, indicating limited purification effect and sensitivity enhancement for ginsenosides. The reason may be as follows: unmodified graphene oxide, due to its layered stacking and polar surface, forms a complex porous structure, physically trapping some sugars; while the modified graphene oxide has better dispersibility, making sugars easier to elute. Comparative Examples 2-5, using aminopropyl, phenyl, methoxypropyl, and phenoxypropyl silanes respectively to modify graphene oxide, all showed significantly lower sugar removal rates than Example 1. This indicates that the ethoxy substituent on the benzene ring plays a crucial role in the purification process. Compared to other groups, the ethoxy group may provide more suitable steric hindrance and electronic effects, enhancing the specific interaction between the graphene surface and the ginsenoside sugar chains, thus more effectively repelling the non-specific adsorption of polar sugar impurities while retaining the target analyte.

[0052] Experimental Example 2

[0053] Take the same batch of ginseng and deer antler tea sample powder as in Example 1, add a mixed standard of five ginsenosides of known concentration (spiking level: medium concentration, 60 μg / g) to obtain spiked samples; according to the complete methods of Example 1 and Comparative Examples 1-5, detect the content of the five ginsenosides in the spiked samples, and calculate the average of the spiked recovery rate and relative standard deviation (RSD, n=3); the recovery rate is calculated as follows: recovery rate (%) = (detected content after spiking - background content of sample) / spiking amount × 100%; the experimental results are shown in Table 3.

[0054] Table 3. Spike recovery rate and relative standard deviation experimental results

[0055]

[0056] As shown in Table 3, the detection method of Example 1 showed a recovery rate of 95.3% to 98.5% for the five ginsenosides, with an average RSD of only 2.1%, indicating that the method of the present invention has high accuracy, good precision, and stable and reliable detection results.

[0057] The detection method in Comparative Example 1 showed a recovery rate of only 74.2%–81.2% for the five ginsenosides, with an RSD as high as 6.7%, indicating insufficient adsorption capacity, strong non-specific adsorption, and poor quantitative accuracy. The detection methods in Comparative Examples 2–5 showed a recovery rate of 85.6%–91.8% for the five ginsenosides, with an RSD of 3.1%–4.2%, both of which were significantly inferior in accuracy and precision to Example 1. Therefore, this invention uses 3-(4-ethoxyphenoxy)propyltrimethoxysilane-modified graphene oxide, which possesses specific recognition and efficient enrichment capabilities for ginsenosides, effectively improving the accuracy and stability of the detection method.

[0058] Experimental Example 3

[0059] Take 300 mg each of the modified graphene oxide composite materials prepared in Example 1 and Comparative Examples 2-5, and pack them into three identical solid-phase extraction columns (i.e., three parallel samples for each group of materials). Perform solid-phase extraction purification on the ginseng and deer antler tea samples according to step (3) in Example 1, and calculate the recovery rates of ginsenosides Rg1, Re, and Rb1. This is the first cycle. After each elution, immediately regenerate the solid-phase extraction column online: rinse the column bed with 5 mL of anhydrous ethanol (flow rate 1 mL / min) and 10 mL of deionized water (flow rate 1 mL / min) in sequence, and drain the residual liquid in the column for later use. Continue the next extraction-elution-detection cycle the next day. Record the spiked recovery rates of Rg1, Re, and Rb1 in each cycle. When the recovery rate of any ginsenoside is lower than 85% for the first time, and subsequent cycles cannot restore it to above 85%, stop using the column, and record the number of the previous cycle as the effective reuse number of the material. The final number of uses for each group of materials is represented by the average of the three parallel columns (results are rounded to the nearest integer). The experimental results are shown in Table 4.

[0060] Table 4. Number of reusable times of the modified graphene oxide composite materials prepared in Examples 1 and Comparative Examples 2-5

[0061]

[0062] As shown in Table 4, the modified graphene oxide composite material prepared in Example 1 can be reused up to 12 times. After multiple regenerations, it can still maintain a high ginsenoside recovery rate, demonstrating excellent structural stability and service life.

[0063] The modified graphene oxide composite materials prepared in Comparative Examples 2-5 can only be reused 4-7 times, with a service life significantly shorter than that of Example 1. The unmodified graphene oxide used in Comparative Example 1 could not meet the reuse requirements, and no relevant tests were conducted. Therefore, this invention uses 3-(4-ethoxyphenoxy)propyltrimethoxysilane modification, which makes the grafted layer on the graphene surface more stable and more resistant to leaching and regeneration, effectively improving the material's service life.

[0064] In summary, this invention utilizes 3-(4-ethoxyphenoxy)propyltrimethoxysilane to covalently graft graphene, and the resulting composite material, used as a solid-phase extraction filler, effectively improves the sugar removal rate, spike recovery rate, and detection precision of ginsenosides, while significantly extending the material's reusability. Compared to unmodified graphene oxide and other silane-modified graphene oxides, this invention significantly enhances separation efficiency, purification efficiency, detection accuracy, stability, and service life, enabling more accurate, stable, and efficient enrichment and detection of ginsenosides in ginseng and deer antler tea. The reasons are as follows: In the 3-(4-ethoxyphenoxy)propyltrimethoxysilane molecule, the propyl flexible chain acts as a connecting arm, effectively alleviating the shielding effect of the graphene substrate on the electron cloud of the benzene ring; at the same time, the large π bond of the benzene ring forms a stable π-π stacking interaction with the π electron system of the tetracyclic triterpenoid skeleton of ginsenosides, combined with the hydrogen bonding interaction between the ether bond oxygen atom and the hydroxyl group of the sugar chain, thereby achieving efficient and simultaneous adsorption of different types of ginsenosides.

[0065] It should be noted that, in this document, the terms "comprising," "including," and any other variations are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Specific examples have been used in this document to illustrate the principles and implementation methods of the present invention. These examples are merely for the purpose of helping to understand the method and core ideas of the present invention. The above descriptions are only preferred embodiments of the present invention. It should be pointed out that, due to the limitations of written expression and the objective existence of infinite specific structures, those skilled in the art can make several improvements, modifications, or variations without departing from the principles of the present invention, and can also combine the above technical features in an appropriate manner. These improvements, modifications, variations, or combinations, or the direct application of the concept and technical solution of the present invention to other situations without modification, should all be considered within the scope of protection of the present invention.

Claims

1. A method for detecting ginsenoside components in ginseng and deer antler tea, characterized in that, Includes the following steps: Solid-phase extraction (SPE) was used to purify the extract of ginseng and deer antler tea to obtain the test solution. The test solution was then analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS), and the content of ginsenosides in the test solution was calculated based on the standard curve. The preparation method of the SPE was as follows: graphene oxide was dispersed in a solvent, and the pH of the system was adjusted to 4-5 to obtain a graphene oxide reaction solution; 3-(4-ethoxyphenoxy)propyltrimethoxysilane was dissolved in a solvent, and water was added and stirred to obtain a silane coupling agent solution; the silane coupling agent solution was added dropwise to the graphene oxide reaction solution, and the mixture was heated and purified to obtain the SPE. The chemical structure of 3-(4-ethoxyphenoxy)propyltrimethoxysilane is as follows: 。 2. The method for detecting ginsenoside components in ginseng and deer antler tea according to claim 1, characterized in that, The graphene oxide has a sheet diameter of 1~1.5μm and a sheet thickness of 0.5~1nm.

3. The method for detecting ginsenoside components in ginseng and deer antler tea according to claim 2, characterized in that, The mass ratio of the graphene oxide to the 3-(4-ethoxyphenoxy)propyltrimethoxysilane is 1:3~5.

4. The method for detecting ginsenoside components in ginseng and deer antler tea according to claim 1, characterized in that, The heating reaction is carried out at a temperature of 50-70°C for 4-8 hours.

5. The method for detecting ginsenoside components in ginseng and deer antler tea according to claim 1, characterized in that, The purification method is as follows: the reaction system is centrifuged to separate the precipitate, and then the precipitate is washed with ethanol and water in sequence. The washed solid precipitate is then dried to obtain solid phase extraction packing.

6. The method for detecting ginsenoside components in ginseng and deer antler tea according to any one of claims 1-5, characterized in that, The 3-(4-ethoxyphenoxy)propyltrimethoxysilane was prepared by the following method: 4-ethoxyphenol, sodium hydroxide and solvent were mixed and dissolved at 40~50℃, and then 3-chloropropyltrimethoxysilane was added dropwise. After the addition was completed, the temperature was raised to 80~85℃ and the reaction was maintained for 8~10h. After the reaction was completed, the mixture was washed, dried, purified by vacuum distillation and recrystallization to obtain 3-(4-ethoxyphenoxy)propyltrimethoxysilane.

7. The method for detecting ginsenoside components in ginseng and deer antler tea according to claim 6, characterized in that, The recrystallization purification method is as follows: the crude product obtained by vacuum distillation and the mixed solvent are heated to 60-65°C, stirred and refluxed for 30-45 min, filtered, and the filtrate is naturally cooled to 25-30°C, then allowed to stand and cool at 5-8°C for 8-10 h, filtered again, and the solid obtained by the second filtration is dried to obtain 3-(4-ethoxyphenoxy)propyltrimethoxysilane; the mixed solvent is composed of n-hexane and ethyl acetate in a volume ratio of 6-8:1, and the mass ratio of the crude product to the mixed solvent is 1:7-8.

8. The method for detecting ginsenoside components in ginseng and deer antler tea according to any one of claims 1-5, characterized in that, The preparation method of the ginseng and deer antler tea extract is as follows: Take ginseng and deer antler tea sample powder, add methanol aqueous solution with a mass fraction of 70~75%, vortex mix and then extract by ultrasonication, collect the supernatant by centrifugation, concentrate to remove methanol and then redissolve to obtain the extract.

9. The method for detecting ginsenoside components in ginseng and deer antler tea according to any one of claims 1-5, characterized in that, The method for solid-phase extraction purification of ginseng and deer antler tea extract using solid-phase extraction packing material is as follows: Solid-phase extraction packing material is packed into a solid-phase extraction column, ginseng and deer antler tea extract is loaded onto the column, followed by rinsing and elution. The eluent is collected, concentrated, and reconstituted to obtain the test solution. The loading flow rate is 0.5–0.8 mL / min. The rinsing is performed with water. The elution is performed with a 70–75% (w / w) ethanol aqueous solution at a flow rate of 0.8–1 mL / min.

10. The method for detecting ginsenoside components in ginseng and deer antler tea according to any one of claims 1-5, characterized in that, The ginsenoside is at least one of ginsenosides Rg1, Re, Rf, Rb1 and Rd.

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