Determination of safflower and chrysoidine in traditional Chinese medicinal materials by in situ synthesized des-based dispersive liquid-liquid microextraction combined with high performance liquid chromatography

Abstract

The present application relates to the technical field of food safety detection, and particularly relates to a method for detecting saponin and quechidin in traditional Chinese medicinal materials based on in-situ synthesis of DES dispersion liquid-liquid microextraction combined with high performance liquid chromatography, comprising the following steps: adding thymol and methyl salicylate into a traditional Chinese medicinal material sample solution in sequence, then vortexing for 5 min, and placing into a centrifuge for centrifugal separation to obtain upper and lower layered liquid phases; wherein the molar ratio of thymol and methyl salicylate is 1:1; using a sample needle to suck the lower DES and adding the same volume of methanol, vortexing to mix, and detecting saponin and quechidin by using high performance liquid chromatography. The method is simple in operation, energy-saving, environmentally friendly, sensitive and accurate, and the detection of saponin and quechidin can reach the ng / mL level.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, and in particular to a method for detecting saponins and caustic soda in traditional Chinese medicines based on in-situ synthesized DES dispersion liquid-liquid microextraction combined with high performance liquid chromatography. Background Technology

[0002] Soap Yellow (also known as m-aniline yellow or Acid Yellow 36) and Ketilium (also known as Basic Orange 2) are two common azo basic industrial dyes. They are favored for their excellent dyeing properties, including high dyeing strength, low cost, and excellent lightfastness. These properties allow Soap Yellow and Ketilium to meet diverse color requirements and are widely used in dyeing and related fields. However, these two dyes cannot be used on traditional Chinese medicine materials and their products because they may pose a health hazard.

[0003] To enhance the appearance and color of their products, some merchants add inexpensive industrial dyes such as saponin and caustic soda to traditional Chinese medicinal materials (such as honey and kudzu root). These industrial dyes are not only highly toxic and persistent, but also have obvious or potential carcinogenic, teratogenic, and mutagenic effects. Long-term intake of these dyes may damage human cell structure and function, causing tissue lesions, increasing the risk of cancer, and potentially causing chronic toxic damage to multiple systems and organs, including the nervous, skeletal, reproductive, urinary, and hematopoietic systems. Therefore, it is necessary to test for the dyes saponin and caustic soda in traditional Chinese medicinal materials (such as honey and kudzu root).

[0004] Currently, the main methods for detecting illegally added dyes include ultraviolet-visible spectroscopy (UV-vis), fluorescence spectroscopy (FL), surface-enhanced Raman spectroscopy (SERS), thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). Each method has its advantages and limitations. UV-vis is simple to operate but lacks specificity; FL has high sensitivity, but requires high purity, concentration, and stability of the sample; SERS enables rapid detection, but the equipment is expensive and reproducibility needs improvement; TLC has limited separation efficiency; and while HPLC-MS / MS has high detection sensitivity, the analysis cost is considerable. Among these, HPLC, with its efficient separation capability and good quantitative analysis performance, is widely used in the detection of illegally added dyes.

[0005] However, since the content of illegally added dyes in honey and kudzu root is relatively low, sample pretreatment is usually required before detection to improve detection efficiency and avoid interference from the sample matrix on detection accuracy. Currently, commonly used pretreatment methods include traditional solvent extraction, liquid-liquid extraction (LLE), dispersive liquid-liquid microextraction (DLLME), solid-phase extraction (SPE), and molecularly imprinting technique (MIT).

[0006] DLLME has attracted widespread attention in the field of sample pretreatment due to its advantages such as high efficiency, speed, low cost, ease of operation, and high enrichment factor. However, conventional DLLME usually uses traditional organic solvents as extraction agents, which can easily cause environmental pollution. Therefore, it is particularly important to develop environmentally friendly extraction solvents to achieve a greener sample pretreatment technology, and thus realize the detection of saponins and caustic soda in traditional Chinese medicine.

[0007] Deep eutectic solvents (DES) are novel, environmentally friendly solvents prepared from hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs) in a specific molar ratio. They possess characteristics such as low melting point, low toxicity, biodegradability, good solubility, and high designability. In recent years, DES has gradually gained favor among analytical researchers in the field of sample pretreatment. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a method for detecting saponins and caustic soda in Chinese medicinal materials based on in-situ synthesized DES dispersion liquid-liquid microextraction combined with high performance liquid chromatography.

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

[0010] A method for detecting saponins and cauline in traditional Chinese medicinal materials based on in-situ synthesized DES dispersion-liquid microextraction combined with high performance liquid chromatography includes the following steps:

[0011] Step S1: Add thymol and methyl salicylate sequentially to the Chinese herbal sample solution, then vortex for 5 minutes, and centrifuge to separate the liquid phases into upper and lower layers; wherein, the molar ratio of thymol and methyl salicylate is 1:1.

[0012] Step S2: Use a syringe to draw up the lower layer of DES, add an equal volume of methanol, vortex mix, and use high performance liquid chromatography to detect saponin and caustic soda.

[0013] In one embodiment of the present invention, in step S1, the volume ratio of methyl salicylate to the traditional Chinese medicine sample solution is 1:160.

[0014] In one embodiment of the present invention, in step S1, the centrifugation speed is 1000 r / min.

[0015] In one embodiment of the present invention, the centrifugation time in step S1 is 3 minutes.

[0016] In one embodiment of the present invention, the vortex mixing time in step S2 is 3 to 5 seconds.

[0017] In one embodiment of the present invention, the concentration range of saponin in the Chinese medicinal material sample is 4-1000 ng / mL.

[0018] In one embodiment of the present invention, the concentration range of caustic soda in the Chinese medicinal material sample is 0.8-200 ng / mL.

[0019] In one embodiment of the present invention, in step S2, the high-performance liquid chromatography (HPLC) uses a WondaCract ODS-2 column with dimensions of 250 mm × 4.6 mm and 5 μm. The mobile phase includes mobile phase A and mobile phase B with a volume ratio of 75:25. Mobile phase A is methanol, and mobile phase B is 0.02 mol / L ammonium acetate. The detection wavelength is 430 nm, the injection volume is 20 μL, the flow rate is 1 mL / min, and the column temperature is 25 °C.

[0020] The beneficial effects of adopting the above technical solution are as follows:

[0021] This invention provides a method for detecting saponins and cauline in traditional Chinese medicinal materials using in-situ synthesized DES-based dispersion-liquid microextraction combined with high-performance liquid chromatography (HPLC). Thymol is used as the hydrogen bond acceptor (HBA), and methyl salicylate as the hydrogen bond donor (HBD). By forming DES in situ in the sample solution and using it as the extractant, a method for detecting saponins and cauline in traditional Chinese medicinal materials (such as honey and kudzu root) using dispersion-liquid microextraction combined with HPLC based on in-situ synthesized eutectic solvents is established. This method is simple to operate, energy-saving, environmentally friendly, sensitive, and accurate, achieving detection levels of ng / mL for saponins and cauline.

[0022] The establishment of this method not only provides an effective technical means for the detection of succinate and caustic soda, but also provides new ideas and references for the analysis of trace amounts of illegally added dyes in other Chinese medicinal materials. Attached Figure Description

[0023] Figure 1 This is a flowchart of a method for detecting saponins and caustic soda in traditional Chinese medicine based on in-situ synthesized DES using DLLME combined with high performance liquid chromatography, provided by the present invention.

[0024] Figure 2 Infrared spectra of thymol, methyl salicylate, and DES1.

[0025] Figure 3 The curves show the yellow color of soap in a series of mixed honey sample solutions of different concentrations.

[0026] Figure 4 The curves show the concentrations of caustic soda in a series of mixed honey sample solutions.

[0027] Figure 5 The curves show the yellow ochre content in mixed kudzu root sample solutions of various concentrations.

[0028] Figure 6 The curves show the concentrations of cauline in mixed kudzu root sample solutions of various concentrations.

[0029] Figure 7These are chromatograms of mixed reference standard solutions and various honey samples; a) mixed reference standard solution, b) blank honey sample, c) spiked honey sample, d) chromatogram of spiked honey sample after DLLME treatment.

[0030] Figure 8 These are chromatograms of mixed reference standard solutions and various types of kudzu root; a) mixed reference standard solution, b) blank kudzu root sample, c) spiked kudzu root sample, d) chromatogram of spiked kudzu root sample after DLLME treatment. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the present invention clearer, the invention will be described clearly and completely below in conjunction with specific embodiments.

[0032] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies, as detailed below:

[0033] Sodium thiocyanate, caustic soda, thymol, methyl salicylate, octanoic acid, decanoic acid, octanol, decyl alcohol, nonanol, and tetrabutylammonium chloride were all purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; heptanoic acid was purchased from Chengdu Xiya Chemical Co., Ltd.; phenol was purchased from Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd.; tetrabutylammonium bromide was purchased from Beijing Xingfu Fine Chemical Research Institute; acetic acid was purchased from Dima Technology (Tianjin) Co., Ltd.; sodium chloride was purchased from Tianjin Yongda Chemical Reagent Co., Ltd.; ammonium acetate was purchased from Tianjin Damao Chemical Reagent Factory; methanol, chromatographic grade, was purchased from Tedia Reagent Co., Ltd., USA; and 95% ethanol was purchased from Shijiazhuang Xinyu Sanyang Industrial Co., Ltd.

[0034] The honey and kudzu root were purchased from Hebei Lerentang Pharmaceutical Chain Co., Ltd.

[0035] Example 1

[0036] This embodiment provides a method for determining saponins and cauline in traditional Chinese medicinal materials based on in-situ synthesized DES using dispersion-liquid microextraction combined with high-performance liquid chromatography. Figure 1 As shown, it includes the following steps:

[0037] Step S1: Add 58.3 mg of thymol and 50 μL of methyl salicylate to 8 mL of Chinese herbal sample solution, then vortex for 5 min, and centrifuge at 3000 r / min for 3 min to obtain the upper and lower liquid phases.

[0038] Step S2: Use a syringe to draw 30 μL of DES into a liner tube, add an equal volume of methanol, vortex mix for 3-5 seconds, and use HPLC to detect the yellow iodine and caustic soda.

[0039] The chromatographic conditions are as follows:

[0040] Chromatographic column: Shimadzu WondaCract ODS-2 (250mm×4.6mm, 5μm), mobile phase: methanol: 0.02mol / L ammonium acetate = 75:25 (V / V), detection wavelength: 430nm, injection volume: 20μL, flow rate: 1mL / min, column temperature: 25℃.

[0041] Example 2: Optimization and Validation of the Method for Detecting Soap Yellow and Caclidin

[0042] 2.1 Solution preparation:

[0043] (1) Preparation of reference solution:

[0044] Accurately weigh appropriate amounts of yellow soap and coledin, place them in 10mL volumetric flasks respectively, dissolve and dilute with methanol to the mark, shake well, and prepare reference stock solutions containing the above reference solutions at a concentration of 1mg / mL.

[0045] Accurately measure specific volumes of yellow soap and caustic soda stock solutions into the same 10 mL volumetric flask, dilute to the mark with methanol, and mix well to prepare a mixed stock solution with a yellow soap concentration of 50 μg / mL and a caustic soda concentration of 10 μg / mL. Continuously dilute the mixed stock solution with ultrapure water to the desired concentration to obtain a series of mixed standard solutions (where the mixed standard solution with a yellow soap concentration of 50 ng / mL and a caustic soda concentration of 10 ng / mL was used to investigate influencing factors). Store at 4°C for later use.

[0046] (2) Preparation of honey sample solution:

[0047] Weigh 1g of honey sample and place it in a 10mL centrifuge tube. Add 3mL of ultrapure water and vortex for 2 minutes until completely dissolved. Add 7mL of 95% ethanol and vortex for 5 minutes. Centrifuge at 3000r / min for 3 minutes. Collect the supernatant and filter it through a 0.45μm microporous membrane. Dilute 1mL of the filtered extract 10 times with ultrapure water to obtain the honey sample solution.

[0048] (3) Preparation of kudzu root sample solution:

[0049] Weigh 2g of kudzu root sample and place it in a 10mL centrifuge tube. Add 10mL of 95% ethanol and vortex extract for 5min. Centrifuge at 3000r / min for 3min. Collect the supernatant and filter it through a 0.45μm microporous membrane. Dilute 1mL of the filtered extract 10 times with ultrapure water to obtain the kudzu root sample solution.

[0050] 2.2 Chromatographic conditions:

[0051] Chromatographic column: Shimadzu WondaCract ODS-2 (250mm×4.6mm, 5μm), mobile phase: methanol: 0.02mol / L ammonium acetate = 75:25 (V / V), detection wavelength: 430nm, injection volume: 20μL, flow rate: 1mL / min, column temperature: 25℃.

[0052] 3 Results and Analysis

[0053] 3.1 Filtering of DES

[0054] Pipette 8 mL of the mixed reference standard solution into 10 mL centrifuge tubes, then add the 10 HBA and HBD solutions listed in Table 1 to each centrifuge tube in sequence. Vortex for 5 min, then centrifuge at 1000 r / min for 3 min to obtain the liquid phases with upper and lower layers; the upper layer is the sample solution and the lower layer is the DES phase.

[0055] 30 μL of DES was drawn into a liner using a microsyringe, and an equal volume of methanol was added. The mixture was vortexed for 3–5 seconds and then placed in a vial for HPLC analysis. The results are shown in Table 2.

[0056] Table 1 Preparation of different types of DES

[0057]

[0058] Table 2 DLLME results for different types of DES

[0059]

[0060] As shown in Table 2, the recovery rates of saponin and cauline were optimal when DES1 was selected as the extractant. Therefore, DES1 was chosen as the extractant for subsequent experiments.

[0061] In addition, to verify the formation of DES1, Fourier transform infrared spectroscopy (FT-IR) was used to characterize thymol, methyl salicylate, and DES. The results are as follows: Figure 2 As shown. In the spectrum of thymol, it is located at 3527 cm⁻¹. -1 The peak value characteristically indicates the stretching vibration of the hydroxyl group (OH). 1619 cm⁻¹ -1 1583cm -1 and 1517cm -1 The peak value at 2963 cm⁻¹ represents the stretching vibration of the carbon-carbon skeleton (C=C) of the benzene ring. Additionally, the peak value at 2963 cm⁻¹... -1 and 2871cm -1 The peak at 1290 cm⁻¹ corresponds to the stretching vibration of saturated carbon-hydrogen bonds (CH) in the molecule. -1 The peak value at that point corresponds to the stretching vibration of the carbon-oxygen single bond (CO).

[0062] In the spectrum of methyl salicylate, 3435 cm⁻¹ -1 The peak at 1615 cm⁻¹ represents the stretching vibration of the hydroxyl group (OH). -1 and 1583cm -1 The peak at 1306 cm⁻¹ represents the stretching vibration of the benzene ring skeleton (C=C). -1 The peak at 1680 cm⁻¹ is caused by the stretching vibration of the carbon-oxygen single bond (CO). -1 The peak value can be attributed to the carbonyl (C=O) stretching vibration.

[0063] In the DES1 infrared spectrum, it is located at 3527 cm⁻¹ -1 The stretching vibration of the hydroxyl group (OH) at that point shifted to a lower wavenumber of 3441 cm⁻¹. -1 At this location, the stretching vibrations of saturated carbon-hydrogen bonds (CH) also shifted to some extent; located at 1680 cm⁻¹ -1 The carbonyl (C=O) stretching vibration shifted to 1681 cm⁻¹. -1 At the same time, the characteristic peak of the stretching vibration of the benzene ring skeleton (C=C) appears at 1616 cm⁻¹. -1 and 1586cm -1 The shift in these peaks indicates a change in electron cloud density, which is usually caused by the formation of intermolecular hydrogen bonds.

[0064] 3.2 Screening of the dosage of thymol and methyl salicylate

[0065] In the DLLME process, the amounts of thymol and methyl salicylate play a decisive role in the extraction efficiency. Insufficient amounts may lead to incomplete extraction, while excessive amounts, although ensuring complete extraction of the analyte, will reduce the enrichment factor and thus affect the detection sensitivity.

[0066] Pipette 8 mL of the mixed reference standard solution into a 10 mL centrifuge tube, and then add different amounts of thymol and methyl salicylate to each centrifuge tube according to Table 3. Vortex for 5 min, and centrifuge at 1000 r / min for 3 min to obtain the liquid phases with upper and lower layers; the upper layer is the sample solution and the lower layer is the DES phase.

[0067] 30 μL of DES was drawn into a liner using a microsyringe, and an equal volume of methanol was added. The mixture was vortexed for 3–5 seconds and then placed in a vial for HPLC analysis. The results are shown in Table 3.

[0068] Table 3. DLLME results for different amounts of thymol and methyl salicylate.

[0069]

[0070] Table 3 shows that the recovery rate gradually increased with increasing dosages of thymol and methyl salicylate. This is because the dosage was too low to achieve complete extraction of the target component. Simultaneously, the lower dosage also increased the sampling difficulty, leading to reduced reproducibility. The peak recovery rate was reached at 58.3 mg thymol and 50 μL methyl salicylate. However, further increasing the dosages of thymol and methyl salicylate led to a stable recovery rate, but resulted in a decrease in the enrichment fold, thus affecting the detection sensitivity. Therefore, this experiment selected 58.3 mg thymol and 50 μL methyl salicylate.

[0071] 3.3 Screening of pH values ​​for test solutions

[0072] Eight mL of the mixed standard solution was pipetted into a 10 mL centrifuge tube. The initial pH of the standard stock solution was measured without pH adjustment, and the result was 6. NaOH and acetic acid were then added to the centrifuge tubes to adjust the pH of the standard solution, resulting in multiple standard solutions with pH values ​​ranging from 3 to 9.

[0073] Add 58.3 mg of thymol and 50 μL of methyl salicylate to each centrifuge tube sequentially, vortex for 5 min, and centrifuge at 1000 r / min for 3 min to obtain a liquid phase with upper and lower layers; the upper layer is the sample solution and the lower layer is DES.

[0074] 30 μL of LDES was drawn into a liner using a microsyringe, and an equal volume of methanol was added. The mixture was vortexed for 3–5 seconds and then placed in a vial for HPLC analysis. The results are shown in Table 4.

[0075] Table 4. DLLME results for different pH values ​​of the test solutions

[0076]

[0077]

[0078] Table 4 shows that changes in pH value had no significant impact on the recovery rate of caustic soda, and all recovered samples reached ideal levels. In contrast, the recovery rate of yellow soap was better at pH values ​​of 3–6, but decreased with increasing pH values ​​from 7 to 9. The experimental results indicate that the recovery rates of both yellow soap and caustic soda can reach high levels without pH adjustment; therefore, this invention does not adjust the pH value.

[0079] 3.4 Screening of Salt Addition Amount

[0080] Pipette 8 mL of the mixed standard solution into a 10 mL centrifuge tube, and add different concentrations of NaCl (0, 0.01 g / mL, 0.02 g / mL, 0.04 g / mL, 0.08 g / mL, and 0.10 g / mL). Then, add 58.3 mg of thymol and 50 μL of methyl salicylate to each centrifuge tube in sequence. Vortex for 5 min, and centrifuge at 1000 r / min for 3 min to obtain a liquid phase with upper and lower layers. The upper layer is the sample solution, and the lower layer is the DES phase.

[0081] 30 μL of DES was drawn into a liner using a microsyringe, an equal volume of methanol was added, the mixture was vortexed for 3–5 s, and then placed in a vial for HPLC analysis. The results are shown in Table 5.

[0082] Table 5. DLLME Results with Different Salt Addition Amounts

[0083]

[0084] Table 5 shows that the recovery rates of the two target compounds differed with increasing salt concentration. The recovery rate of yellow soap reached its optimal level with no salt added and with a salt concentration of 0.02 g / mL. However, the recovery rate of yellow soap decreased with a salt concentration of 0.10 g / mL, possibly because the increased salt concentration led to increased solution viscosity, hindering the diffusion and migration of yellow soap to DES1, thus reducing extraction efficiency. In contrast, caustic soda maintained a high recovery rate under conditions of 0-0.10 g / mL salt addition. Based on the above experimental results and analysis, to ensure ideal recovery rates for both yellow soap and caustic soda, this invention ultimately selected the absence of NaCl as the optimal condition.

[0085] 3.5 Screening of Vortex Time

[0086] Vortexing time is a key parameter affecting DLLME, directly related to the degree of contact between the extractant and the target analyte. An appropriate vortexing time promotes thorough mixing between the extractant and the target substance, thereby improving extraction efficiency.

[0087] Pipette 8 mL of the mixed reference standard solution into a 10 mL centrifuge tube. Add 58.3 mg of thymol and 50 μL of methyl salicylate to each centrifuge tube in sequence. Vortex for 1 min, 2 min, 3 min, 4 min, 5 min, and 6 min, respectively, and centrifuge at 1000 r / min for 3 min to obtain the liquid phases with upper and lower layers. The upper layer is the sample solution and the lower layer is the DES phase.

[0088] 30 μL of DES was drawn into a liner using a microsyringe, an equal volume of methanol was added, the mixture was vortexed for 3–5 s, and then placed in a vial for HPLC analysis. The results are shown in Table 6.

[0089] Table 6. DLLME results for different vortex times.

[0090]

[0091] As shown in Table 6, with the extension of vortex time, yellow soap reached its optimal recovery rate at 4 minutes, while cauline reached its optimal recovery rate at 5 minutes. Further extension of the vortex time resulted in a stable recovery rate for both target components. To ensure that both target components achieve ideal recovery rates, this invention selected a vortex time of 5 minutes.

[0092] 3.6 Selection of centrifugation speed

[0093] Pipette 8 mL of the mixed reference standard solution into a 10 mL centrifuge tube. Add 58.3 mg of thymol and 50 μL of methyl salicylate to each centrifuge tube sequentially. Vortex for 5 min, then centrifuge at 1000 r / min, 2000 r / min, 3000 r / min, 4000 r / min, and 5000 r / min for 3 min respectively to obtain the upper and lower liquid phases; the upper layer is the sample solution and the lower layer is the DES phase.

[0094] 30 μL of DES was drawn into a liner using a microsyringe, an equal volume of methanol was added, the mixture was vortexed for 3–5 s, and then placed in a vial for HPLC analysis. The results are shown in Table 7.

[0095] Table 7 DLLME results at different centrifugation speeds

[0096]

[0097] As shown in Table 7, the recovery rate of the analyte did not change significantly with increasing centrifugation speed, and a high recovery rate was achieved at all speeds. However, a lower centrifugation speed can reduce energy consumption. Therefore, this invention selected a centrifugation speed of 1000 r / min.

[0098] 3.7 Selection of Centrifugation Time

[0099] Centrifugation time can affect the separation of the extractant and analyte, thus impacting the extraction recovery rate. If the centrifugation time is too short, the two phases may not separate sufficiently, failing to achieve the desired separation; conversely, if the centrifugation time is too long, energy consumption will increase.

[0100] Pipette 8 mL of the mixed reference stock solution into a 10 mL centrifuge tube. Add 58.3 mg of thymol and 50 μL of methyl salicylate to each centrifuge tube in sequence. Vortex for 5 min, and centrifuge at 1000 r / min for 1 min, 2 min, 3 min, 4 min, and 5 min respectively to obtain a sample solution with upper and lower layers. The upper layer is the sample solution, and the lower layer is the DES phase.

[0101] 30 μL of DES was drawn into a liner using a microsyringe, an equal volume of methanol was added, the mixture was vortexed for 3–5 s, and then placed in a vial for HPLC analysis. The results are shown in Table 8.

[0102] Table 8. DLLME results at different centrifugation times.

[0103]

[0104] As shown in Table 8, from 1 to 3 minutes, the recovery rates of both analytes gradually increased with increasing centrifugation time, reaching a maximum at 3 minutes. Subsequently, as the centrifugation time was gradually extended, the recovery rates tended to stabilize. Therefore, this invention selected a centrifugation time of 3 minutes.

[0105] 3.8 Methodological Examination

[0106] (1) Linear range and limit of quantitation

[0107] Take 1 mL of the mixed reference stock solution and place it in a 50 mL volumetric flask. Dilute to volume with honey sample solution and kudzu root sample solution, respectively, to obtain a mixed honey sample solution and a mixed kudzu root sample solution. Serially dilute the mixed honey sample solution and the mixed kudzu root sample solution to prepare a series of linear reference solutions of different concentrations. Perform HPLC analysis. Plot the peak area A of each analyte on the ordinate and the concentration c (this concentration excludes the saponins and caustic soda contained in the honey and kudzu root samples; for example, the saponin concentration in both the mixed honey and mixed kudzu root sample solutions is 1000 ng / mL, and the caustic soda concentration is 200 ng / mL) on the abscissa to obtain a standard curve. Figures 3-6 As shown in Table 9, the results were obtained.

[0108] Table 9. Linearity and detection limit of different sample detection methods

[0109]

[0110] (2) Recovery and precision of the method

[0111] Accurately measure 100 μL, 10 μL, and 2 μL of the mixed reference stock solution, respectively, and concentrate and evaporate to dryness under vacuum. Add 10 mL of honey sample solution and 10 mL of kudzu root sample solution to obtain three different concentrations of honey and kudzu root test solutions: low, medium, and high. The low concentration consisted of 2 ng / mL caustic soda and 10 ng / mL saponin; the medium concentration consisted of 10 ng / mL caustic soda and 50 ng / mL saponin; and the high concentration consisted of 100 ng / mL caustic soda and 500 ng / mL saponin.

[0112] For any concentration of the test solution, measure 8 mL of the test solution into a 10 mL centrifuge tube, add 58.3 mg of thymol and 50 μL of methyl salicylate, vortex for 5 min, and centrifuge at 1000 r / min for 3 min to obtain a liquid phase with upper and lower layers; the upper layer is the sample solution and the lower layer is the DES phase.

[0113] 30 μL of DES was drawn into a liner using a microsyringe, and an equal volume of methanol was added. The mixture was vortexed for 3–5 seconds and then placed in a vial for HPLC analysis. Six parallel treatments were performed for each concentration. The recovery rates of the detection method are shown in Tables 10 and 11.

[0114] Table 10 Recovery rates and RSD data of caudin and saponins in honey

[0115]

[0116] Table 11 Recovery rates and RSD data of cauline and saponin in kudzu root

[0117]

[0118] As shown in Tables 9 and 10, the recovery rates of saponin in honey samples were 91.8–109.8% with an RSD of 1.6–2.6%, and the recovery rates of cauline were 94.2–101.5% with an RSD of 1.1–2.8%. In kudzu root samples, the recovery rates of saponin were 90.9–103.2% with an RSD of 1.1–2.4%, and the recovery rates of cauline were 90.4–101.4% with an RSD of 2–2.6%, indicating that the method is accurate and reliable.

[0119] 3.9 Theoretical Verification

[0120] The chromatograms of the mixed reference standard solution and various honey samples are shown below. Figure 7 and Figure 8 As shown, by Figure 7 and Figure 8It can be seen that the chromatogram of the mixed reference standard solution showed corresponding peaks of thymol and kudzu root. Neither the honey blank sample nor the kudzu root blank sample showed corresponding peaks of thymol and kudzu root. However, when the honey sample or kudzu root sample contained thymol or kudzu root, corresponding peaks of thymol and kudzu root appeared (e.g., honey spiked sample or kudzu root spiked sample). Subsequently, using the method of this invention, 58.3 mg of thymol and 50 μL of methyl salicylate were added to obtain a mixed solution. After vortex extraction and centrifugation, 30 μL of the lower layer of DES was aspirated with a syringe, and an equal volume of methanol was added. The corresponding peaks of thymol and kudzu root in the chromatogram obtained by vortex mixing were enhanced, which further demonstrates the reliability of this method.

[0121] It should be noted that the aforementioned honey spiked sample refers to the medium-concentration honey test sample in step 2 of Example 8, and the kudzu root spiked sample refers to the medium-concentration kudzu root test sample in step 2 of Example 8.

[0122] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting saponins and caudin in traditional Chinese medicinal materials based on in-situ synthesized DES dispersion-liquid microextraction combined with high performance liquid chromatography, characterized in that, Includes the following steps: Step S1: Add thymol and methyl salicylate sequentially to the Chinese herbal sample solution, then vortex for 5 minutes, and centrifuge to separate the liquid phases into upper and lower layers; wherein, the molar ratio of thymol and methyl salicylate is 1:

1. Step S2: Use a syringe to draw up the lower layer of DES, add an equal volume of methanol, vortex mix, and use high performance liquid chromatography to detect saponin and caustic soda; In the high performance liquid chromatography method, a WondaCractODS-2 column is used, the specifications of which are 250mm×4.6mm and 5μm. The mobile phase consists of mobile phase A and mobile phase B with a volume ratio of 75:

25. Mobile phase A is methanol and mobile phase B is 0.02 mol / L ammonium acetate. The detection wavelength is 430 nm, the injection volume is 20 μL, the flow rate is 1 mL / min, and the column temperature is 25 °C.

2. The method for detecting saponins and cauline in traditional Chinese medicinal materials based on in-situ synthesized DES dispersion-liquid microextraction combined with high performance liquid chromatography, as described in claim 1, is characterized in that... In step S1, the volume ratio of methyl salicylate to the herbal sample solution is 1:

160.

3. The method for detecting saponins and cauline in traditional Chinese medicinal materials based on in-situ synthesized DES dispersion-liquid microextraction combined with high performance liquid chromatography, as described in claim 1, is characterized in that... In step S1, the centrifugation speed is 1000 r / min.

4. The method for detecting saponins and caudin in traditional Chinese medicinal materials based on in-situ synthesized DES dispersion-liquid microextraction combined with high performance liquid chromatography according to claim 3, characterized in that, In step S1, the centrifugation time is 3 minutes.

5. The method for detecting saponins and cauline in traditional Chinese medicinal materials based on in-situ synthesized DES dispersion-liquid microextraction combined with high performance liquid chromatography, as described in claim 1, is characterized in that... In step S2, the vortex mixing time is 3~5s.

6. The method for detecting saponins and cauline in traditional Chinese medicinal materials based on in-situ synthesized DES dispersion-liquid microextraction combined with high performance liquid chromatography, as described in claim 1, is characterized in that... The concentration range of saponin in the Chinese medicinal material samples was 4~1000 ng / mL.

7. The method for detecting saponins and cauline in traditional Chinese medicinal materials based on in-situ synthesized DES dispersion-liquid microextraction combined with high performance liquid chromatography, as described in claim 1, is characterized in that... The concentration range of caustic soda in the Chinese medicinal material samples was 0.8~200 ng / mL.

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