Method for detecting saponin substances in American ginseng

Abstract

The invention provides a method for detecting saponin substances in American ginseng, and relates to the technical field of biological analysis, the detection method comprises the following steps: taking American ginseng, grinding the American ginseng into powder, sieving, and carrying out microwave-assisted extraction to obtain an extracting solution; then carrying out solid-phase extraction and purification on the extracting solution to obtain a purified solution; carrying out fluorescence derivatization on the purified liquid; and finally, carrying out HPLC-FLD analysis. According to the method disclosed by the invention, a microwave-assisted extraction-solid-phase extraction and purification combined fluorescence derivatization HPLC (High Performance Liquid Chromatography) technology is adopted, so that the defects of lengthy pretreatment and insufficient sensitivity in the traditional technology are overcome, and the dependence on scarce standard substances is also reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological analysis, and in particular to a method for detecting saponins in Panax quinquefolium. BACKGROUND

[0002] As the main active ingredients, saponins (such as ginsenoside Rb1, Rg1, etc.) in Panax quinquefolium have important significance for quality control, pharmacological research and product development. In the traditional detection method, although high performance liquid chromatography-ultraviolet detection (HPLC-UV) is widely used, it has insufficient sensitivity for saponins without strong ultraviolet absorption, and needs to rely on complex pretreatment (such as macroporous resin purification) to improve the signal-to-noise ratio, which takes more than ten hours and easily causes loss of saponins. Although liquid chromatography-mass spectrometry (LC-MS / MS) significantly improves the sensitivity, its high equipment cost, strict matrix tolerance requirements and complex operation limit its popularity. In addition, although the spectrophotometric method can quickly determine the total saponin content, it cannot distinguish the monomer components and is easily interfered by polysaccharides, tannins, etc.; the thin layer chromatography (TLC) is difficult to meet the modern analysis demand due to low resolution and poor quantitative accuracy. The existing methods generally face the problems of complicated pretreatment, high dependence on standard products, and difficulty in balancing sensitivity and cost, which restricts the accurate analysis of trace saponins in complex matrix (such as traditional Chinese medicine and biological samples).

[0003] In recent years, detection technology has gradually evolved towards high efficiency, high sensitivity and low cost. Microwave-assisted extraction, solid-phase extraction and other pretreatment technologies have attracted attention due to their simple operation and low solvent consumption, but the compatibility with traditional detectors still needs to be optimized. Fluorescence detection has shown potential in trace analysis due to its high sensitivity and good selectivity, but natural saponins lack fluorescent groups and need to be derivatized to give them detectable properties. Therefore, a detection method with high sensitivity and simple pretreatment is needed.

[0004] In view of the above, the present application is proposed. SUMMARY

[0005] The first purpose of the present application is to provide a method for detecting saponins in Panax quinquefolium. The present application provides a "microwave-assisted extraction-solid phase extraction purification combined with fluorescence derivatization HPLC" technology, which not only overcomes the defects of long pretreatment and insufficient sensitivity of traditional technology, but also reduces the dependence on scarce standard products.

[0006] In order to achieve the above purpose of the present application, the following technical solutions are adopted:

[0007] The present application provides a method for detecting saponins in Panax quinquefolium, comprising the following steps:

[0008] Step one: Take American ginseng, grind it into powder, sieve it, and use microwave-assisted extraction to obtain an extract;

[0009] Step two: Then purify the extract by solid-phase extraction to obtain a purified liquid;

[0010] Step three: Mix the purified liquid with a derivatization reagent to perform fluorescent derivatization;

[0011] Step four: Finally, perform HPLC-FLD analysis;

[0012] In the step of fluorescent derivatization, the volume ratio of the purified liquid to the derivatization reagent is 1:2;

[0013] The derivatization reagent is 9-fluorenylmethoxycarbonyl chloride dissolved in acetonitrile;

[0014] The concentration of the 9-fluorenylmethoxycarbonyl chloride is 0.1 mol / L.

[0015] This method optimizes the detection process of American ginseng saponins through four-step integration innovation, significantly improving efficiency and accuracy. In step one, microwave-assisted extraction is used with 70% ethanol as the solvent to rapidly destroy plant cell walls at 60°C and 400W, enabling efficient dissolution of saponins within 10 minutes. Compared to traditional reflux methods, this method shortens the time by more than 80%, and the low-temperature closed environment reduces the degradation of heat-sensitive components.

[0016] In step two, a C18 solid-phase extraction column is used instead of a macroporous resin, and a three-step purification process of methanol activation, 20% methanol elution, and 80% methanol elution is used to remove interfering substances such as polysaccharides and pigments within 1 hour, with a recovery rate of more than 95% and a solvent consumption reduction of 90%.

[0017] In step three, FMOC-Cl fluorescent derivatization is introduced, which specifically binds to the hydroxyl group of saponins in a buffer solution with pH = 8.5, generating stable fluorescent derivatives within 15 minutes. The detection limit of Rb1 (original protopanaxadiol saponin) and other UV-absorbing components is reduced to 0.01 μg / mL, with a sensitivity improvement of 80 times compared to UV detection.

[0018] In step four, based on HPLC-FLD combined technology, acetonitrile-0.1% formic acid gradient elution is used to achieve high-resolution separation of structurally similar saponins such as Rb1 and Rg1, enabling the fluorescence detector to accurately capture signals at Ex / Em = 265 / 315 nm, and the linear range of external standard quantification reaches 0.05-50 μg / mL (R 2The method has high sensitivity, low cost, strong anti-interference characteristics, and is especially suitable for precise analysis of trace saponins in complex matrices such as traditional Chinese medicines and health products, and provides a reliable tool for quality control and metabolic research.

[0019] In the fluorescence derivatization step, the core purpose of controlling the volume ratio of the purified liquid to the derivatization reagent is to balance the reaction efficiency, product stability and economy. In actual application, it is necessary to ensure that the number of moles of the derivatization reagent is much higher than the number of hydroxyl groups of the target saponin, so as to drive the reaction to proceed in the positive direction.

[0020] When the derivatization reagent is excessive, other substances will be hydrolyzed to generate fluorescence background noise, or the derivatization reagent may react with solvent or buffer components to generate interference peaks. When the derivatization reagent is insufficient, the derivatization rate is reduced, thereby reducing the fluorescence intensity of the background.

[0021] 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl) is used as the derivatization reagent because the active carbonyl group (-O-CO-Cl) of FMOC-Cl can specifically undergo nucleophilic substitution reaction with the hydroxyl group or the amino group in the saponin molecule to generate a stable fluorescent derivative. Compared with dansyl chloride or o-phthaldehyde, dansyl chloride mainly targets the amino group and has low reactivity with the hydroxyl group, and o-phthaldehyde requires the coexistence of amino groups and thiol groups, and is not suitable for saponins.

[0022] Meanwhile, the FMOC group has high quantum yield and long emission wavelength, which is perfectly matched with the HPLC-FLD detector, significantly improves the sensitivity, and can complete the derivatization within 40 minutes at 40°C, thereby avoiding saponin degradation caused by high temperature or long time reaction.

[0023] In the present application, the core purpose of limiting the concentration of the derivatization reagent is to balance the reaction efficiency, side reaction control, background signal stability and economy. When the concentration of the derivatization reagent is too low, the number of moles of the reagent is insufficient, the reaction is incomplete, and the target substance is missed; and when the concentration is too high, the excessive reagent is easy to hydrolyze or react with the solvent, or label non-target substances, thereby generating interference peaks and affecting the purity of the HPLC peak.

[0024] Preferably, as a further specific embodiment, the step of fluorescence derivatization is specifically:

[0025] Step one: taking a sample, adding the purified liquid, the derivatization reagent and the borate buffer, mixing to obtain a mixed liquid;

[0026] Step two: heating the mixed liquid in a water bath and reacting in the dark;

[0027] Step three: terminating the reaction and filtering.

[0028] Preferably, the temperature of the water bath in step two is 40℃, and the heating time is 15 min.

[0029] Preferably, in the process of terminating the reaction in step three, 0.1wt% glycine aqueous solution is added to the mixture, vortex mixed for 30s, and left for 5 min.

[0030] Preferably, as a further specific embodiment, the specific steps of solid phase extraction purification are as follows:

[0031] The extract is activated, loaded, eluted, and then eluted to obtain the eluate;

[0032] The eluate is concentrated and dried, reconstituted, and filtered to obtain the product.

[0033] The SPE column is used in the solid phase extraction purification process.

[0034] The filler of the SPE column used in the solid phase extraction purification is octadecylsilane bonded silica gel.

[0035] Preferably, as a further specific embodiment, in the steps of loading and eluting, the flow rate of both steps is ≤0.5mL / min.

[0036] During the loading stage, the flow rate needs to be controlled within the range of ≤0.5mL / min, on the one hand to avoid the situation that the flow rate is too fast, causing part of the saponins to be carried out by the solvent before being fully adsorbed, thereby reducing the recovery rate; on the other hand, under low-speed conditions, the saponin molecules are allowed to fully distribute between the liquid and solid phases, improving the adsorption efficiency; finally, preventing the column bed from drying out, and preventing the rapid flow rate from damaging the wet state of the SPE column filler, causing the filler to shrink or form a channel flow, reducing the column efficiency.

[0037] During the elution stage, the flow rate also needs to be controlled within the range of ≤0.5mL / min, allowing the high proportion of methanol eluent to slowly penetrate the filler pores and gradually dissolve the saponin molecules, while low-speed elution can reduce the elution volume and avoid excessive solvent dilution of the target; under the condition of too fast flow rate, the eluent cannot effectively infiltrate the filler, causing saponins to remain, and the fast flow rate may cause the eluent front to be irregular due to insufficient mass transfer, affecting the peak shape and integration accuracy of subsequent HPLC analysis.

[0038] Preferably, as a further specific embodiment, in the step of washing, the volume ratio of water:methanol:formic acid in the reagent used is 79.9:20:0.1.

[0039] In fact, the selection of methanol is a range value, the volume range is about 15%-30%, when the volume is ≥30%, part of the saponins (such as Rg1) begins to elute, resulting in loss of target substances; and when ≤15%, polysaccharide is not completely removed.

[0040] 20% methanol has moderate polarity, which can elute strong polar impurities (such as polysaccharides, part of organic acids) in American ginseng extract, while retaining medium polarity saponins; and the addition of formic acid in methanol can inhibit the ionization of saponins, formic acid (pKa≈3.75) can reduce the pH of the eluent to 2.5-3.0, inhibit the ionization of hydroxyl and carboxyl groups in saponin molecules, and enhance their hydrophobicity, so as to be more firmly adsorbed on the C18 column; without formic acid, 20% methanol elution will cause 5%-10% of saponins to be lost.

[0041] In the traditional elution process, pure water or 40% high methanol is also selected for elution, and when pure water is used for elution, it cannot effectively remove the medium polarity pigment, resulting in an increase in HPLC baseline noise; and when 40% high methanol is used for elution, it is easy to cause loss of saponins. Therefore, the selected 20% methanol aqueous solution (containing 0.1% formic acid) as the eluent can significantly improve the purification effect, and lay a foundation for high-purity samples for subsequent fluorescence derivatization and HPLC-FLD analysis.

[0042] Preferably, as a further specific embodiment, in the step of elution, the volume ratio of water:methanol:formic acid in the reagent used is 19.9:80:0.1.

[0043] In fact, the selection of methanol is a range value, the volume range is about 15%-30%, when the volume is ≥30%, part of the saponins (such as Rg1) begins to elute, resulting in loss of target substances; and when ≤15%, polysaccharide is not completely removed.

[0044] The residual silicon hydroxyl on the surface of C18 filler can adsorb saponins through hydrogen bonding or ion exchange, and the acidic environment of formic acid can protonate the silicon hydroxyl, reducing such non-specific adsorption; at the same time, formic acid as a weak reducing agent can inhibit the oxidative degradation of saponins during elution, reducing the co-elution of polysaccharides, pigments and other interfering substances.

[0045] In the current technology, ethanol or acetonitrile is also selected, but it is not an alternative in the present application, ethanol will cause slow elution flow rate, high column pressure, and low elution efficiency of 10%-15%; and the price of acetonitrile is 2-3 times that of methanol, and there is no obvious advantage in the elution selectivity of part of saponins (such as Rd).

[0046] Preferably, as a further specific embodiment, the temperature of the elution is controlled at 20-25℃.

[0047] The core purpose of controlling the temperature in the elution step is to balance the elution efficiency and the stability of the target. The ginsenosides are prone to glycosidic bond rupture or ring structure oxidation at high temperature (>60℃), resulting in the loss of active ingredients. At the same time, high temperature can cause the pH of formic acid to rise, weakening its shielding effect of silicon hydroxyl, reducing the elution efficiency. Therefore, controlling the temperature at 20-25℃ can effectively avoid the pyrolysis of ginsenosides and ensure complete elution, reducing the possibility of temperature control leading to decreased recovery or incomplete elution (such as slow mass transfer at low temperature), directly affecting the accuracy and reliability of the detection results.

[0048] Preferably, as a further specific embodiment, the temperature of the concentration is 30-40℃.

[0049] Compared with the prior art, the present application has the following beneficial effects:

[0050] The present application innovatively proposes a "microwave-assisted extraction-solid phase extraction purification combined with fluorescence derivatization HPLC" technology, which constructs a rapid, high-sensitivity, and low-cost ginsenoside analysis system through microwave-enhanced extraction efficiency, C18 solid phase extraction column one-step purification, and FMOC-Cl fluorescence labeling. This method not only overcomes the defects of long pretreatment and insufficient sensitivity of traditional technology, but also reduces the dependence on scarce standard products, providing a reliable tool for American ginseng and its products quality evaluation, metabolism kinetics research, and has broad application prospects in the field of traditional Chinese medicine modernization and health product development. DETAILED DESCRIPTION

[0051] The technical solutions of the present application will be described below in conjunction with specific embodiments, but those skilled in the art will understand that the following described embodiments are part of the embodiments of the present application, not all of the embodiments, and are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present application. If the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased on the market.

[0052] EMBODIMENT

[0053] A. Microwave-assisted extraction of ginsenosides from American ginseng

[0054] Take American ginseng, dry it, crush it, pass it through a 60-mesh sieve, then take 1.0 g of American ginseng powder and place it in a special microwave extraction tank.

[0055] Add 20 mL of 70% ethanol solution (containing 0.1% formic acid) to it, and the liquid-to-solid ratio is 1:20.

[0056] Turn on the microwave extraction instrument, preheat to an initial temperature of 30°C, set the microwave power to 400 W, and heat at a rate of 3°C / min to 60°C. Seal the extraction tank and maintain an internal pressure of ≤1.0 MPa.

[0057] After the program ends, cool to below 40°C, open the extraction tank, and transfer the extract to a centrifuge tube.

[0058] Add 10 mL of 70% ethanol to the residue and repeat the above microwave program once (total cycle 2). Combine the two extracts, and the total volume is about 30 mL.

[0059] Filter with a 0.45 μm filter membrane to remove particulate impurities, and rotary evaporate under reduced pressure to 10 mL at 40°C for later use in solid phase extraction purification.

[0060] B. Solid phase extraction (SPE) purification

[0061] Materials and equipment: select SPE column (500 mg / 6 mL, pore size ), methanol (chromatographically pure), ultrapure water, 0.1 vt% formic acid aqueous solution, 0.1 vt% formic acid methanol solution, vacuum solid phase extraction device, centrifuge tube, nitrogen blowing instrument, 0.22 μm organic filter membrane.

[0062] Concentrate the combined extract obtained in the previous step by rotary evaporation at 40°C under reduced pressure, and concentrate the microwave-assisted extraction of American ginseng extract to about 10 mL.

[0063] a. Activation

[0064] Add 5 mL of methanol to the SPE column, and slowly pressurize the solvent to pass through the column bed at a flow rate of 1 mL / min.

[0065] Add 5 mL of ultrapure water (containing 0.1% formic acid) to the column bed, and flow the liquid into the column bed at a flow rate of 1 mL / min to ensure that the column bed is wet and the solvent environment matches the polarity of the sample.

[0066] b. Sample loading

[0067] Slowly add the concentrated extract (10 mL) to the activated SPE column, and control the flow rate of the concentrated liquid to be 0.5 mL / min.

[0068] When the concentrated extract is turbid, centrifuge or filter in advance to avoid clogging the column bed.

[0069] c. Elution

[0070] Add 5 mL of 20% methanol in water (containing 0.1% formic acid, the specific ratio of methanol: water: formic acid is 20:79.9:0.1 by volume) at a flow rate of 1 mL / min.

[0071] Subsequently, discard the eluate.

[0072] d. Elution

[0073] Add 80% methanol in water (containing 0.1% formic acid, the specific ratio of methanol: water: formic acid is 80:19.9:0.1 by volume) at a flow rate of 0.5 mL / min.

[0074] Collect the eluate into a clean centrifuge tube.

[0075] e. Post-treatment

[0076] Blow the eluate collected in the clean centrifuge tube to near dryness using a nitrogen blower at 40°C, then add 1 mL of methanol pure solution to dissolve the residue, vortex for 30 s, pass through a 0.22 μm organic filter membrane, and load into a sample bottle for standby.

[0077] C. Fluorescent derivatization

[0078] Take 100 μL of saponin sample solution, add 200 μL of 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl) solution (volume ratio 1:2), and 50 μL of borate buffer (pH 8.5), vortex for 10 seconds.

[0079] React in a 40°C water bath for 15 minutes in the dark to ensure that FMOC-Cl is fully combined with the hydroxyl groups of saponins; then add 50 μL of 0.1% glycine aqueous solution to neutralize excess FMOC-Cl and prevent side reactions, vortex for 30 seconds, and stand for 5 minutes.

[0080] Pass through a 0.22 μm organic filter membrane and transfer to an HPLC sample bottle for standby.

[0081] D. HPLC-FLD analysis

[0082] Select a C18 reverse phase column (250 mm x 4.6 mm, 5 μm, pore size ).

[0083] Mobile phase

[0084] A phase: 0.1% formic acid in water, used to enhance peak shape and suppress tailing;

[0085] B phase: acetonitrile, used for gradient elution.

[0086] The gradient program is shown in Table 1.

[0087] Table 1: Gradient program

[0088]

[0089]

[0090] Flow rate: 1.0 mL / min; Column temperature: 30 °C; Injection volume: 10 μL.

[0091] Fluorescence detector parameters

[0092] Excitation wavelength (Ex): 265 nm, to match the maximum absorption of FMOC group;

[0093] Emission wavelength (Em): 315 nm, to capture the strongest fluorescence signal;

[0094] Gain: 10x, equilibrated sensitivity to baseline noise.

[0095] Subsequent qualitative and quantitative analysis was performed.

[0096] Comparative Example 1: High Performance Liquid Chromatography-UV Detection Method

[0097] A. Sample pre-treatment

[0098] Extraction:

[0099] 1. Take 1.0 g of dry powder of Panax quinquefolium (pass through a 60 mesh sieve), add 20 mL of 70% ethanol solution.

[0100] 2. Ultrasonic-assisted extraction for 30 minutes (power 250 W, frequency 40 kHz), centrifuge (4000 rpm, 10 minutes) to take the supernatant, repeat the extraction once, and combine the two extraction solutions.

[0101] Purification:

[0102] 1. Concentrate the extraction solution to 5 mL by rotary evaporation, and pass through a 0.45 μm filter membrane.

[0103] 2. Purify using a macroporous resin column (AB-8 type):

[0104] Column activation: rinse with 5 mL of ethanol and 5 mL of water in sequence.

[0105] Loading: load the concentrated solution onto the column at a flow rate of 1 mL / min.

[0106] Elution: 10 mL of water to remove polysaccharides, and 10 mL of 30% ethanol to remove pigments.

[0107] Elution: 10 mL of 70% ethanol to elute saponins, collect the eluate, and nitrogen blow to dryness, then redissolve in 1 mL of methanol.

[0108] B. Chromatographic conditions

[0109] Column: C18 reversed-phase column (250 mm x 4.6 mm, 5 μm).

[0110] Mobile phase: Acetonitrile (A) - 0.1% phosphoric acid aqueous solution (B), gradient elution program as shown in Table 2.

[0111] Table 2: Gradient elution program

[0112]

[0113] Flow rate: 1.0 mL / min; column temperature: 30 °C; detection wavelength: 203 nm (saponin end absorption).

[0114] Injection volume: 10 μL.

[0115] Subsequently, quantitative analysis was performed.

[0116] Comparative Example 2: Liquid chromatography-tandem mass spectrometry

[0117] A. Sample pretreatment

[0118] Extraction:

[0119] 1. American ginseng powder 0.5 g, add 10 mL 80% methanol, vortex mix for 1 minute, ultrasonic extraction for 20 minutes (40 °C).

[0120] 2. Centrifugation (12000 rpm, 10 minutes), take the supernatant through a 0.22 μm filter membrane.

[0121] Purification:

[0122] Use C18 solid phase extraction column (100 mg / 3 mL) to purify;

[0123] Activation: 3 mL methanol, 3 mL water.

[0124] Loading: 1 mL extract, flow rate 0.5 mL / min.

[0125] Elution: 3 mL methanol, collect the eluate and nitrogen blow to dryness, 50% methanol to 1 mL.

[0126] Mass spectrometry conditions

[0127] Column: HSS T3 C18 column (100 mm x 2.1 mm, 1.8 μm).

[0128] Mobile phase: 0.1% formic acid water (A) - acetonitrile (B), gradient program as shown in Table 3.

[0129] Table 3: Gradient program

[0130]

[0131] Mass spectrometry parameters:

[0132] Ion source: Electrospray ionization (ESI), negative ion mode.

[0133] Capillary voltage: 3.0 kV; desolvation gas temperature: 350 °C.

[0134] The multiple reaction monitoring (MRM) parameters are shown in Table 4.

[0135] Table 4: Multiple reaction monitoring (MRM) parameters

[0136]

[0137] Subsequent quantitative analysis was performed.

[0138] Comparative Example 3: Spectrophotometry

[0139] 1. Preparation of standard curve

[0140] Prepare a standard solution of ginsenoside Re (0.1-1.0 mg / mL).

[0141] Take 0.5 mL of each concentration standard solution, add 0.5 mL of 5% vanillin-ethanol solution, mix well, and then add 5 mL of concentrated sulfuric acid in an ice bath.

[0142] Heat in a 60 °C water bath for 15 minutes, cool to room temperature, and measure the absorbance at 544 nm.

[0143] Draw the standard curve (R 2 >0.99).

[0144] 2. Sample determination

[0145] Extraction: Add 10 mL of 70% ethanol to 1.0 g of American ginseng powder, reflux in a 80 °C water bath for 2 hours, filter, repeat once, and combine the filtrates to a constant volume of 25 mL.

[0146] Color development: Take 1 mL of the extract, color it according to the standard curve procedure, and measure the absorbance.

[0147] Calculation: Calculate the total saponin content (in terms of Re equivalents) according to the standard curve.

[0148] Experimental Example

[0149] Precision determination:

[0150] Intra-day precision: The same experimenter repeated the injection 6 times within a day for the same concentration sample (containing 10 μg / mL of Rb1 and Rg1) to calculate the relative standard deviation (RSD) of the peak area or absorbance.

[0151] Intra-day precision: On different three days, the same concentration sample was prepared and injected 3 times each day, and the RSD of peak area or absorbance was calculated.

[0152] Sensitivity determination (limit of detection and limit of quantification):

[0153] Limit of detection (LOD): the lowest concentration corresponding to a signal-to-noise ratio (S / N) of 3.

[0154] Limit of quantification (LOQ): the lowest concentration corresponding to a signal-to-noise ratio (S / N) of 10.

[0155] Pre-treatment efficiency:

[0156] The total time required from sample extraction to completion of analysis was recorded, and the experimental results are shown in Table 5.

[0157] Table 5: Experimental results of examples and comparative examples

[0158]

[0159] From the experimental data for precision, the intra-day RSD of the present application was 1.2%, and the inter-day RSD was 2.8%, indicating high reproducibility. This indicates that the standardized operation of microwave-assisted extraction and solid-phase extraction reduces human error, and the stability of fluorescence derivatization also ensures the high repeatability of the detection results.

[0160] In Comparative Example 1, the intra-day RSD (4.5%) and inter-day RSD (6.2%) were relatively high, mainly because the ultraviolet detector had a large fluctuation in response to low-concentration saponins (such as Rb1), and the macroporous resin purification step was prone to introduce operational differences. In Comparative Example 2, although the inter-day RSD (3.5%) was relatively low, the matrix effect might lead to a decrease in precision during actual sample analysis, and the internal standard correction was required. In Comparative Example 3, the RSD was as high as 8.7% (intra-day) and 12.1% (inter-day), because the color reaction was sensitive to temperature and time, and the light absorption contribution of interfering substances such as polysaccharides could not be excluded.

[0161] Through comparison of sensitivity, it can be seen that the LOD of the present application was 0.01 μg / mL, which was 100 times higher than that of HPLC-UV (1.0 μg / mL). Fluorescence derivatization broke through the limitations of ultraviolet detection by enhancing the signal intensity.

[0162] Although the sensitivity of Comparative Example 2 was relatively high (LOD 0.06 μg / mL), the instrument was expensive and the operation was complex, which required professional maintenance, limiting its application in conventional laboratories. The LOD of Comparative Example 3 was only 0.8 μg / mL, which was only suitable for rough screening of total saponins, and could not meet the precise analysis requirements of monomer components.

[0163] From the above experimental data, it can be seen that the fluorescence derivatization combined with HPLC-FLD makes the detection limit of saponins (such as Rb1) without ultraviolet absorption reach 0.01 μg / mL, and baseline separation of Rb1, Rg1 and other isomers is achieved through gradient elution; at the same time, without expensive mass spectrometry equipment, the maintenance cost of the fluorescence detector is low, suitable for small and medium-sized laboratories, and the operation is simple and economical, more green and environmental protection.

[0164] Finally, it should be pointed out that: the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for detecting saponins in American ginseng, characterized in that: The steps include: Step 1: Grind American ginseng into powder, sieve it, and use microwave-assisted extraction to obtain an extract; Step 2: Then, the extract is subjected to solid phase extraction purification to obtain a purified solution; Step 3: mixing the purified solution with a derivatization reagent for fluorescence derivatization; Step 4: Finally, perform HPLC-FLD analysis; In the fluorescent derivatization step, the volume ratio of the purified solution to the derivatization reagent is 1:2; The derivatization reagent is 9-fluorenylmethoxycarbonyl chloride dissolved in acetonitrile; The concentration of the 9-Fmocyl chloride is 0.1 mol / L.

2. The method for detecting saponins in American ginseng according to claim 1, wherein The steps of the fluorescence derivatization are specifically as follows: Step 1: taking a sample, adding the purified solution, the derivatization reagent and the borate buffer, and mixing to obtain a mixed solution; Step 2: heating the mixture in a water bath and protecting from light; Step 3: Terminate the reaction and filter.

3. The method for detecting saponins in American ginseng according to claim 2, wherein: The heating temperature in the water bath in step 2 is 40° C. and the heating time is 15 min.

4. The method for measuring saponins in American ginseng according to claim 2, wherein: During the termination of the reaction in step 3, a 0.1 wt% glycine aqueous solution is added to the mixed solution, vortexed for 30 seconds, and allowed to stand for 5 minutes.

5. The method for detecting saponins in American ginseng according to claim 1, wherein The specific steps of the solid phase extraction purification are: activating, loading, eluting and then eluting the extract to obtain an eluate; The eluate is concentrated and dried, redissolved, and filtered to obtain the product; SPE columns are used in the solid phase extraction purification process; The filler of the SPE column used in the solid phase extraction purification is octadecylsilane bonded silica gel.

6. The method for detecting saponins in American ginseng according to claim 5, characterized in that: In the steps of sample loading and elution, the flow rates of both steps are ≤0.5 mL / min.

7. The method for detecting saponins in American ginseng according to claim 5, characterized in that: In the elution step, the reagent used is composed of water, methanol and formic acid in a volume ratio of 79.9:20:0.

1.

8. The method for detecting saponins in American ginseng according to claim 5, characterized in that: In the elution step, the reagent used is composed of water, methanol and formic acid in a volume ratio of 19.9:80:0.

1.

9. The method for detecting saponins in American ginseng according to claim 5, wherein: The elution temperature is controlled at 20°C-25°C.

10. The method for detecting saponins in American ginseng according to claim 5, characterized in that: The concentration temperature is 30°C-40°C.

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