Magnetic solid-phase extraction method for indole compounds in Baijiu and application of magnetic solid-phase extraction method

Abstract

The invention provides a magnetic solid-phase extraction method for indole compounds in Baijiu and application of the magnetic solid-phase extraction method, and belongs to the technical field of food detection. The magnetic solid-phase extraction method comprises the following steps: adsorbing indole substances (IAA and IPA) in Baijiu by using a GO-PAA-Fe3O4 composite material as an adsorbent, and then desorbing in an acidic acetone desorption solvent to complete the magnetic solid-phase extraction of the indole compounds. The method provided by the invention can effectively overcome the defects of low concentration of indole substances in Baijiu, complex matrix interference, poor enrichment effect, complex detection, consumption of a large amount of organic reagents and the like in the prior art.

Description

Technical Field

[0001] The invention relates to the technical field of food detection, and in particular to a magnetic solid phase extraction method for indole compounds in liquor and application thereof. Background Art

[0002] As a flavored alcoholic beverage, liquor research has gradually formed a development strategy with a dual orientation of "health & flavor" in order to meet consumers' pursuit of alcoholic beverage flavor and consumers' concern for their own health. Among them, 3-methylindole has attracted much attention as the key flavor substance of the "cellar aroma" flavor of Luzhou-flavor liquor. However, at present, many consumers have reported that some Luzhou-flavor liquors have a weak cellar aroma and when the cellar aroma in Luzhou-flavor liquor is too strong, it will highlight the "mud smell". Therefore, we believe that the systematic analysis of the metabolic pathway of 3-methylindole in the brewing process of Luzhou-flavor liquor can provide important theoretical guidance for the effective regulation of its aroma and style shaping. In recent years, many zoological studies have shown that indole-3-acetic acid and indole-3-propionic acid are important intermediates in the pathway of tryptophan metabolism to produce 3-methylindole. In addition, in the brewing process of Luzhou-flavor liquor, experts have deciphered that indole-3-acetic acid (IAA) and indole-3-propionic acid (IPA) are precursors for the production of 3-methylindole. Therefore, analyzing indole-3-acetic acid (IAA) and indole-3-propionic acid (IPA) is a key step in regulating the "cellar aroma" flavor of Luzhou-flavor liquor.

[0003] However, the detection of indole-3-acetic acid (IAA) and indole-3-propionic acid (IPA) faces many challenges. First, the concentrations of these two compounds in liquor are usually extremely low, and they are trace compounds, which are difficult to directly identify and quantify by conventional detection methods. Secondly, the matrix of liquor is extremely complex and contains a large number of volatile and non-volatile substances. These components will interfere with the detection of IAA and IPA, making the signal easily masked. In addition, the high alcohol content in liquor may also have a negative impact on the sensitivity of the detection. Due to the weak polarity of IAA and IPA, their extraction and enrichment processes are also very difficult. Conventional liquid-liquid extraction or solid phase extraction methods are difficult to effectively separate these compounds, and selective enrichment must be performed without destroying other flavor substances. In addition, the above-mentioned traditional methods generally have problems such as high consumption of organic solvents, cumbersome and time-consuming extraction methods, and poor targeting. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a magnetic solid phase extraction method for indole compounds in liquor and its application, so as to solve the defects of the prior art such as low concentration of indole substances in liquor, complex matrix interference, poor enrichment effect, complex detection and consumption of large amounts of organic reagents.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] One of the technical solutions of the present invention is a magnetic solid phase extraction method for indole compounds in liquor, using GO@PAA@Fe3O4 composite material as an adsorbent to adsorb indole compounds in liquor, and then desorbing them in an acidic acetone solution;

[0007] The indole compound is indole-3-acetic acid and / or indole-3-propionic acid.

[0008] The second technical solution of the present invention is a method for evaluating the cellar aroma flavor of Luzhou-flavor liquor. After indole compounds are extracted using the above-mentioned magnetic solid phase extraction method, HPLC-MS / MS analysis is performed, and qualitative testing is performed through the retention index and mass spectrometry fragmentation law of the analyte; the standard curve method is used, and the ion mode in the HPLC-MS / MS analysis is combined to perform quantitative testing on the analyte.

[0009] The present invention discloses the following technical effects:

[0010] The present invention uses the GO@PAA@Fe3O4 composite material as an adsorbent for magnetic solid phase extraction. The adsorbent can be quickly separated from the matrix only by applying an external magnetic field, providing a basis for more accurate qualitative and quantitative tests.

[0011] The GO@PAA@Fe3O4 composite material in the present invention has a higher targeting ability for indole substances in liquor, thereby achieving highly selective (targeted) extraction of indole substances in liquor, with better separation and enrichment effects, providing a basis for more accurate qualitative and quantitative testing.

[0012] The present invention uses GO@PAA@Fe3O4 composite material as an adsorbent to adsorb indole substances in liquor, and then desorbs them in a desorption solvent containing acetone to complete magnetic solid phase extraction of the indole substances, with high extraction efficiency, small sample amount required, and fast adsorption speed. The extraction method of the present invention can achieve micro-extraction with high efficiency, simple and fast operation, and has the advantages of accuracy, high efficiency, high sensitivity, and small amount of solvent used. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0014] Figure 1 To verify the optimization of the addition amount of GO@PAA@Fe3O4 in Example 1;

[0015] Figure 2 To verify the optimization of the pH value of the wine sample during the extraction process in Example 1;

[0016] Figure 3 To verify the zeta potential of GO@PAA@Fe3O4 in Example 1;

[0017] Figure 4 To verify the optimization of ionic strength during the extraction process in Example 1;

[0018] Figure 5 To verify the optimization process of adsorption time during the extraction process in Example 1;

[0019] Figure 6 To verify the optimization of the desorption solvent type in the desorption process in Example 1;

[0020] Figure 7 To verify the optimization of the pH value of the desorption solvent during the desorption process in Example 1;

[0021] Figure 8 To verify the optimization of desorption temperature during the desorption process in Example 1;

[0022] Fig. 9 To verify the optimization of desorption time during the desorption process in Example 1;

[0023] Fig.10 The figure is a schematic flow chart of the magnetic solid phase extraction method for indole compounds in liquor of the present invention. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] In recent years, with the development of analytical detection technology, magnetic solid-phase extraction (MSPE) has attracted much attention due to its advantages of high extraction efficiency and large contact area. Magnetic solid-phase extraction is a dispersed solid-phase extraction technology that uses magnetic or magnetizable materials as adsorbent matrices. This method can achieve rapid separation of the target analyte from the sample matrix within seconds by directly dispersing the magnetic adsorbent in the test solution under the action of an external magnetic field. It has the characteristics of easy separation, high efficiency, rapidity, simplicity and environmental friendliness, and has very high extraction capacity and extraction efficiency. It can simultaneously achieve the extraction, purification, separation and concentration of the analyte. Compared with traditional solid phase extraction technology, MSPE has a large contact area for the target analyte, high extraction efficiency, and can effectively avoid the problems of easy clogging of solid phase columns and cumbersome operation. In view of this, the present invention proposes to use GO@PAA@Fe3O4 composite materials as adsorbents combined with HPLC-MS / MS to achieve rapid and accurate detection of indole-3-acetic acid (IAA) and indole-3-propionic acid (IPA) in liquor.

[0030] Based on the above content, the present invention provides a magnetic solid phase extraction method for indole compounds in white wine, using GO@PAA@Fe3O4 composite material as an adsorbent to adsorb indole compounds in white wine, and then desorbing them in an acidic acetone solution; the indole compounds are indole-3-acetic acid and / or indole-3-propionic acid.

[0031] In some embodiments of the present invention, the method for preparing the GO@PAA@Fe3O4 composite material comprises the following steps:

[0032] dissolving polyacrylic acid (PAA) in potassium phosphate buffer to obtain a polyacrylic acid solution;

[0033] EDC and NHS were sequentially added into MES buffer solution containing GO powder for activation to obtain GO-EDC-NHS;

[0034] The polyacrylic acid solution is mixed with the GO-EDC-NHS and stirred to react to obtain a GO@PAA composite material;

[0035] After dispersing the GO@PAA composite material in water, the GO@PAA composite material was added dropwise to an acidic solution containing iron salt to prepare a N-GO@PAA@Fe3O4 composite material by a coprecipitation method;

[0036] The N-GO@PAA@Fe3O4 composite material is placed in an acidic acetone solution for activation to obtain the GO@PAA@Fe3O4 composite material.

[0037] In some embodiments of the present invention, the concentration of the polyacrylic acid solution is 25 mg / mL.

[0038] In some embodiments of the present invention, the mass ratio of EDC, NHS and GO powder is 16:24:5.

[0039] In some embodiments of the present invention, the activation condition is: shaking activation at 30-45°C.

[0040] In some embodiments of the present invention, the stirring reaction is carried out at a temperature of 15-35° C. and for a time of 4-8 h.

[0041] In some embodiments of the present invention, after the stirring reaction is completed, the step of centrifuging the reaction solution to collect precipitates, and washing and drying the collected precipitates is also included.

[0042] In some embodiments of the present invention, the iron salt is FeCl2·4H2O and FeCl3·6H2O; the molar ratio of FeCl2·4H2O and FeCl3·6H2O is 1:2; the acidic solution is 0.2-0.6 mol / L HCl solution; the molar volume ratio of FeCl2·4H2O, FeCl3·6H2O and HCl solution is 0.001 mol:0.002 mol:20 mL.

[0043] In some embodiments of the present invention, the co-precipitation method is specifically as follows: after dispersing the GO@PAA composite material in water, dropwise adding it into an acidic solution containing an iron salt, stirring evenly, and then dropwise adding ammonia water to the resulting mixed solution, so that the reaction system is in an alkaline condition of pH 10-12.0, and reacting at 85-95°C for 3.5-5.5h.

[0044] In some embodiments of the present invention, after the coprecipitation is completed, the steps of separating the product using a magnet, washing and drying the separated product are also included; the drying temperature is 80°C.

[0045] In some embodiments of the present invention, when the N-GO@PAA@Fe3O4 composite material is placed in an acidic acetone solution for activation, the acidic acetone solution is a mixed solution of formic acid and acetone in a volume ratio of (0.2-2):(98-99.8); the activation method is ultrasonic activation; after the activation is completed, the activated product is further washed and dried; the solution used for washing is acetonitrile and ultrapure water.

[0046] In some embodiments of the present invention, the amount of the GO@PAA@Fe3O4 composite material added is 1-5 mg per 1 ml of liquor, preferably 3 mg per 1 ml of liquor.

[0047] In some embodiments of the present invention, the adsorption time is 5-20 min, preferably 8-15 min, and more preferably 10 min.

[0048] In some embodiments of the present invention, the acidic acetone solution is a mixed solution of formic acid and acetone in a volume ratio of (0.2-2):(98-99.8).

[0049] In some embodiments of the present invention, before using the GO@PAA@Fe3O4 composite material as an adsorbent to adsorb indole compounds in white wine, the method further includes diluting the alcohol content of the white wine with water to 7-25° and adjusting the pH to 4-10 (preferably pH 4); the reagent used to adjust the pH is 0.1-0.3 mol / L dilute hydrochloric acid solution and / or 1-4 mol / L sodium hydroxide solution.

[0050] In some embodiments of the present invention, after the adsorption is completed, the step of collecting the adsorbed GO@PAA@Fe3O4 composite material using a magnet under the action of an external magnetic field is also included.

[0051] In some embodiments of the present invention, the desorption is specifically as follows: placing the adsorbed GO@PAA@Fe3O4 composite material in an acidic acetone solution and ultrasonically treating it for 10-20 minutes to desorb the indole substances; the temperature of the ultrasound is room temperature, and the ultrasonic power is 500-1000W.

[0052] In some embodiments of the present invention, after the desorption is completed, the desorption liquid is further dried with nitrogen, then re-dissolved with acetonitrile, and filtered.

[0053] The present invention also provides a method for evaluating the cellar aroma flavor of Luzhou-flavor liquor, wherein the indole compounds are extracted by the magnetic solid phase extraction method, and then HPLC-MS / MS analysis is performed, and a qualitative test is performed by using the retention index and mass spectrometry fragmentation law of the analyte, and a standard curve method is used, and the ion mode in the HPLC-MS / MS analysis is combined to perform a quantitative test on the analyte. The retention index is calculated by using an improved Kovats method.

[0054] In order to better understand the present invention, the content of the present invention is further explained in conjunction with the following examples, but the content of the present invention is not limited to the following examples. The flow diagram of the magnetic solid phase extraction method of indole compounds in liquor of the present invention is shown in Fig.10 .

[0055] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0056] The instrument conditions for HPLC-MS / MS analysis of the test substance in the embodiment are:

[0057] Liquid chromatography conditions:

[0058] The chromatographic column used was Agilent ZORBAX Eclipse Plus C18 (5 μm, 4.6 mm×250 mm); the column temperature was 30° C.; the flow rate was 0.5 mL / min; the injection volume was 10 μL; the mobile phase contained 2% formic acid solution (phase A) and acetonitrile (phase B); the gradient elution program was: 0-5 min, 20% B; 5-10 min, 20-70% B; 10-20 min, 70-80% B; 20-21 min, 80-20% B, 21-25 min, 20% B. The detection wavelength was 280 nm, and the absorption wavelength of the chromatographic peak was within the range of 400-600 nm.

[0059] Mass spectrometry conditions:

[0060] The ESI ionization source was used to perform precise mass measurement in the positive ion mode. The full mass spectrum data were obtained in the range of m / z 100-500. Among them, the optimal mass spectrometry parameters were as follows: the capillary voltage was set to 3500V; high-purity nitrogen (99.99%) was used as the nebulizing and drying gas. The nebulizing gas pressure was 45psi; at 300°C, the flow rate of the drying gas was 5.0L / min, and the flow rate of the sheath gas was 11.0L / min (250°C). In addition, the multiple reaction detection mode (MRM) was used for quantification.

[0061] Construction of standard curve:

[0062] The external standard method was used to quantitatively analyze the test compounds: the mass concentration of the test compound in each gradient standard solution was used as the horizontal coordinate (x), and the peak area of ​​each quantitative ion was used as the vertical coordinate (y), and the standard curve linear regression equation y=ax+b was established. Finally, the concentrations of IAA and IPA in each wine sample were calculated using the standard curve constructed above. The above experiments were performed in parallel 3 times.

[0063] Example 1

[0064] The preparation steps of GO@PAA@Fe3O4 composite material are as follows:

[0065] Step 1. Ultrasonic dispersion of PAA in 30.0 mL of 0.1 M potassium phosphate buffer (pH=7.4) to obtain a PAA solution having a concentration of 25 mg / mL.

[0066] Step 2. According to the EDC-NHS coupling reaction, the -COOH group on GO is activated and coupled with the -NH2 of PAA to form an amide bond. The specific steps are as follows: 50 mg of GO powder is fully dissolved in 10 mL of MES buffer solution (50.0 mM, pH = 6.0), and EDC and NHS are added in sequence to obtain a mixed solution (the concentration of EDC in the mixed solution is 16 mg / mL, and the concentration of NHS is 24 mg / mL). The mixed solution is slowly shaken in a shaker at 37°C for 15 minutes for activation, and then centrifuged at 3778.8×g for 10 minutes, and repeatedly rinsed with MES solution 3 times to remove the unreacted EDC and NHS solution to obtain GO-EDC-NHS.

[0067] Step 3. Slowly add all the GO-EDC-NHS prepared in step 2 to 30 mL of PAA solution, and mechanically stir the reaction at room temperature for 4 hours. Then centrifuge at 3778.8×g, remove the supernatant, and wash the reaction product repeatedly with ultrapure water until the washing liquid is neutral. Dry the product in a freeze dryer at -50°C for 24 hours to obtain a GO@PAA composite material.

[0068] Step 4. The preparation of magnetic GO@PAA mainly adopts the coprecipitation method of iron salt and GO@PAA. Specifically, 0.1988g FeCl2·4H2O and 0.5406g FeCl3·6H2O were dissolved in 20mL, 0.4mol / L HCl solution to obtain FeCl3 / FeCl2 / HCl solution. In addition, 0.06g of the GO@PAA composite material obtained in step 3 was ultrasonically dispersed in 20mL of ultrapure water at 35°C for 15min. The uniformly dispersed GO@PAA solution was added dropwise to the FeCl3 / FeCl2 / HCl solution and magnetically stirred for 1h at room temperature. After stirring, 160mL, 1.25M ammonia solution was added dropwise at a rate of 10 drops / min (so that the reaction system was in an alkaline condition of pH 10-12.0) and reacted at 90°C for 4h. Finally, the reaction product was adsorbed by a magnet and repeatedly washed with ultrapure water until the washing liquid was neutral. The washed product was dried in an oven at 60 °C overnight to obtain N-GO@PAA@Fe3O4 composite material (denoted as N-GPF).

[0069] Step 5. Take 15 mg of the N-GO@PAA@Fe3O4 composite material obtained in step 4 in a 10 mL centrifuge tube, add 5 mL of 0.5% acidic acetone solution (a mixed solution of formic acid and acetone with a volume ratio of 0.5:99.5) and ultrasonically activate it at 35°C for 15 minutes. Then, the above reaction product is adsorbed by a magnet and repeatedly washed with acetonitrile and ultrapure water until the washing liquid is neutral. The washed product is placed in a 60°C oven and dried for 6 hours to remove all solvents. Finally, the GO@PAA@Fe3O4 composite material is obtained.

[0070] Example 2

[0071] The magnetic solid phase extraction of indole substances in liquor is as follows:

[0072] Step 1. Preparation of liquor sample: Take 4 mL of liquor sample in a 25 mL centrifuge tube, add ultrapure water to dilute the alcohol content to 15°, and adjust the pH to 4 with 1 mol / L sodium hydroxide solution using 0.2 mol / L dilute hydrochloric acid solution.

[0073] Step 2. Adsorption process of the analyte in the liquor sample: Take 5 mL of the liquor sample prepared in step 1, add 15 mg of the GO@PAA@Fe3O4 composite material prepared in Example 1 to the liquor sample, and vortex extract for 10 minutes at a speed of 701.2×g to achieve adsorption equilibrium so that the GO@PAA@Fe3O4 composite material can be fully adsorbed. After the extraction is complete, use a magnet to separate the GO@PAA@Fe3O4 composite material from the supernatant under the action of an external magnetic field, and remove the supernatant.

[0074] Step 3. Desorption process of the analyte in the liquor sample: The GO@PAA@Fe3O4 composite material treated in step 2 was redispersed and dissolved in 0.5mL of 0.5% acidic acetone solution (a mixed solution of formic acid and acetone with a volume ratio of 0.5:99.5), and GO@PAA@ (a mixed solution of formic acid and acetone with a volume ratio of 0.5:99.5) was ultrasonically desorbed at 35°C for 15min (the elution process was repeated 3 times); the 3 eluates were combined, dried with N2 at 25°C, and then re-dissolved with 0.5mL of acetonitrile solution; finally, after filtering with a 0.22μm organic filter membrane, 10μL of the sample was taken for HPLC analysis.

[0075] Test Example 1

[0076] Optimization of magnetic solid phase extraction method for indole compounds in liquor (5 mL of liquor sample was selected in the experiment)

[0077] 1.1 Enrichment rate evaluation

[0078] The present invention adopts the enrichment ratio (EF) to evaluate the effectiveness of the extraction efficiency.

[0079] EF(%)=C d / C0×100

[0080] In the formula, C d is the concentration of the compound to be tested in the desorption solution after extraction; C0 is the initial added concentration of the compound to be tested measured in the simulated liquor sample.

[0081] 1.2 Optimization of GO@PAA@Fe3O4 addition

[0082] The amount of adsorbents such as graphene can affect the extraction rate of target analytes to a certain extent. Therefore, in order to clarify the effect of the amount of GO@PAA@Fe3O4 on the extraction rate of indole-3-acetic acid (IAA) and indole-3-propionic acid (IPA), we investigated the addition of 10 mg, 15 mg, 20 mg, 25 mg and 30 mg of GO@PAA@Fe3O4. The results are shown in Figure 2. Figure 1As shown. The results show that when only 15 mg of GO@PAA@Fe3O4 adsorbent is used, the extraction rates of IAA and IPA are the highest, and the extraction rates are 427.9% and 442.2%, respectively. However, as the addition amount of GO@PAA@Fe3O4 adsorbent is further increased from 15 mg to 30 mg, the extraction rates of IAA and IPA both decrease significantly, and are less than 272.9% and 235.1%, respectively. The reason may be that GO@PAA@Fe3O4 agglomerates due to the increase in the addition amount, which leads to a reduction in the area of ​​IAA and IPA adsorbed on GO@PAA@Fe3O4. In summary, 15 mg of GO@PAA@Fe3O4 is selected as the optimal adsorbent dosage.

[0083] 1.3 Optimization of pH value of wine samples during extraction

[0084] Adjusting the wine sample to a suitable pH value is crucial to improving the extraction efficiency of the target analyte. The effects of 2, 4, 6, 8, 10, and 12 pH conditions on the extraction efficiency of indole-3-acetic acid (IAA) and indole-3-propionic acid (IPA) are shown in the following table. Figure 2 As shown. The results show that when the pH increases from 2.0 to 4.0, the adsorption rate of IAA and IPA by GO@PAA@Fe3O4 increases rapidly, and at pH 4.0, the extraction rate is the highest, which is 520.3% and 643.9%, respectively. However, when the pH value continues to increase, the extraction rate of IAA and IPA decreases. Especially under strong alkaline conditions (i.e., pH = 12.0), the extraction rates of IAA and IPA are 31.4% and 22.7%, respectively. The reasons may be that: (a) The strength of the adsorption between IAA and IPA and GO@PAA@Fe3O4 is mainly affected by hydrogen bonding, π-π interaction and electrostatic interaction. In addition, (b) since IAA and IPA belong to indole acid compounds (R-COOH), the carboxyl groups they contain will dissociate in aqueous solution: R-COO- is produced, so IAA and IPA can exist in the sample solution in two forms, namely ions and molecules, respectively, but their concentrations are different. Literature research shows that the pKa values ​​of IAA and IPA are 4.75 and 4.77, respectively; according to pKa = pH-log [(R-COO-) / (R-COOH)], in the present invention, when the pH increases from 2.0 to 4.0, the above dissociation reaction proceeds in the reverse direction, resulting in an increase in the concentration of IAA and IPA in molecular form. Therefore, under the influence of π-π interaction and hydrogen bonding, the adsorption capacity of GO@PAA@Fe3O4 for IAA and IPA is significantly improved. In addition, as Figure 3As shown in the figure, the isoelectric point of GO@PAA@Fe3O4 is pH 5.35. Therefore, when the pH is in the range of 2.0-4.0, electrostatic adsorption can also occur between the positively charged surface of GO@PAA@Fe3O4 and the negatively charged IAA or IPA. However, when the pH is ≤ 2.0, due to the increase in the number of positive ions (H+) in the wine sample to be tested, it will compete with the positively charged surface of GO@PAA@Fe3O4 to adsorb IAA or IPA at the same time, thereby reducing the electrostatic adsorption between GO@PAA@Fe3O4 and IAA and IPA, and ultimately reducing the extraction rate of IAA and IPA. (c) When the pH is in the range of 4.0-5.35, the zeta potential of GO@PAA@Fe3O4 decreases with increasing pH (see Figure 3 ), thereby weakening the adsorption of IAA and IPA by GO@PAA@Fe3O4. In addition, as the pH continues to increase (pH>5.35), the above adsorbents and the target analytes are negatively charged, and electrostatic repulsion occurs between GO@PAA@Fe3O4 and IAA and IPA. Especially when pH=12.0, the above electrostatic repulsion is significant, which ultimately leads to the lowest extraction efficiency of IAA and IPA by GO@PAA@Fe3O4. In summary, adjusting the pH of the sample solution to 4.0 is the optimal pH value.

[0085] 1.4 Optimization of ionic strength during extraction

[0086] Adding an appropriate amount of NaCl to the wine sample can improve the extraction efficiency of the target analyte to a certain extent. In the present invention, we investigated the effects of adding 5 different salt amounts, namely 0 mol / L, 0.5 mol / L, 1.0 mol / L, 2.0 mol / L and 4.0 mol / L (saturated), on the extraction rate of IAA and IPA in the wine sample. Figure 4 As shown in the figure, when the added concentration of NaCl increases from 0 mol / L to 0.5 mol / L, the extraction rates of IAA and IPA are significantly reduced, reaching 258.8% and 397.3%, respectively; however, when it is further increased to the saturated salt addition (i.e., 4.0 mol / L), the extraction efficiency of GO@PAA@Fe3O4 for IAA and IPA does not change significantly. This may be because the viscosity of the wine sample solution increases with the increase in NaCl concentration; when the solution viscosity is high, the diffusion coefficients of IAA and IPA will be significantly reduced, thereby affecting the mass transfer rate when transferring from the sample solution to GO@PAA@Fe3O4 and being unfavorable to the extraction of IAA and IPA. In summary, not adding NaCl will be more conducive to the extraction effect of IAA and IPA.

[0087] 1.5 Optimization of adsorption time during extraction

[0088] Adsorption time is an important factor affecting the extraction efficiency of the target analyte. Therefore, the present invention investigates the effects of 6 different adsorption times, namely 30s, 2.5min, 5min, 10min, 20min and 10min, on the extraction rate of IAA and IPA in the wine sample. Figure 5 As shown in the figure, both IAA and IPA can quickly reach adsorption equilibrium within 10 minutes, and their extraction rates reach 680.7% and 634.6%, respectively. However, when the adsorption time continues to increase (10-30 minutes), the extraction rates of IAA and IPA remain unchanged. Therefore, 10 minutes of extraction adsorption is selected as the optimal adsorption time.

[0089] 1.6 Optimization of desorption solvent type during desorption

[0090] The desorption solvent is an important factor that determines the extraction effect. In the present invention, in order to investigate the effect of different extraction solvents on the extraction rate of IAA and IPA in the wine samples to be tested, we selected four solvents, namely methanol, acetonitrile, acetone and dichloromethane, to extract IAA and IPA in the wine samples. The extraction rate results after elution with each desorption solvent are shown in the figure below. Figure 6 As shown. The results show that when acetone is selected as the desorption solvent, the extraction efficiency of IAA and IPA is the best, reaching 408.5% and 556.1%, respectively; however, when dichloromethane is used, the extraction efficiency of IAA and IPA is the lowest. Due to the presence of a large number of polar functional groups on the surface of GO@PAA@Fe3O4, such as -COOH, -OH, -CO-NH-, it can not only be well dispersed in polar solvents, but also interact with the target analytes to promote adsorption; however, compared with acetone, methanol and acetonitrile, dichloromethane has the weakest polarity. According to the principle of like dissolves like, the above adsorbents are very easy to agglomerate in dichloromethane, thereby significantly reducing the adsorption efficiency of GO@PAA@Fe3O4 for IAA and IPA (see Figure 6 , illustration), in summary, acetone was selected as the optimal desorption solvent.

[0091] 1.7 Optimization of the pH of the desorption solvent during desorption

[0092] In addition to investigating the effect of the type of desorption solvent on the extraction rate of IAA and IPA in the wine samples, we also investigated the effect of adding 8 different acidic and alkaline desorption solvents, including acetone, 0.5% acidic acetone, 1.0% acidic acetone, 2.0% acidic acetone, 5% acidic acetone, 1.0% alkaline acetone, 2.0% alkaline acetone and 5% alkaline acetone, on the extraction rate of IAA and IPA in the wine samples. The results are as follows Figure 7As shown. When 0.5% acidic acetone was selected as the desorption solvent, the extraction rates of IAA and IPA were the highest. The reasons may be: (a) When GO@PAA@Fe3O4 is in an alkaline environment (pH ≥ 7), the oxygen-containing functional groups (such as -COOH and -OH) on its surface are easily ionized, making GO@PAA@Fe3O4 negatively charged ( Figure 3 ). Therefore, under the strong electrostatic repulsion, the target analytes (IAA and IPA) are favored to be eluted from the adsorbent. However, in an alkaline environment, the OH- in the sample solution will easily compete with IAA and IPA in the form of negative ions for H + , which will eventually reduce the generation of IAA and IPA, thereby affecting their extraction efficiency. Therefore, when alkaline acetone is selected as the extraction solvent, the desorption rates of IAA and IPA are both low. (b) When the pH value of the desorption solvent is in the range of 5.35-7.0 (i.e., 0.5% acidic acetone), IAA and IPA can be easily eluted from GO@PAA@Fe3O4 due to the existence of electrostatic repulsion; at the same time, under the action of H+, IAA and IPA are finally formed. However, (c) when the concentration of formic acid in acetone continues to increase, the surface of GO@PAA@Fe3O4 is positively charged, and electrostatic adsorption occurs between it and the negatively charged IAA or IPA, which eventually leads to a significant decrease in the desorption rate of IAA and IPA. Therefore, 0.5% acidic acetone is selected as the optimal desorption solvent, and the desorption rates of IAA and IPA are 686.3% and 626.5%, respectively.

[0093] 1.8 Optimization of desorption temperature and time during desorption

[0094] Correctly selecting the desorption temperature can not only accelerate the desorption rate of the target analyte and improve the extraction rate; more importantly, it can effectively solve the problems of thermal decomposition of the target analyte and thermal volatilization of the desorption solvent to a certain extent. Therefore, in the present invention, we investigate the effects of 5 different desorption temperatures from 25-50°C on the extraction rates of IAA and IPA in the wine samples to be tested. Figure 8 As shown, when the desorption temperature is 35 °C, the extraction rate of IAA and IPA by GO@PAA@Fe3O4 is optimal, reaching 722.7% and 623.4%, respectively. Therefore, 35 °C is selected as the optimal desorption temperature.

[0095] In addition, we also investigated the extraction rates of IAA and IPA in the wine samples after ultrasonic desorption for 1-15 min. The results showed that (see Fig. 9), the desorption time had no significant effect on the extraction rate of IAA and IPA. When the ultrasonic desorption was 15 min, the extraction rates of IAA and IPA were the highest. In summary, 15 min of ultrasonic desorption was selected as the optimal desorption time, and the extraction rates of IAA and IPA could reach 867.1% and 717.5%, respectively.

[0096] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A magnetic solid phase extraction method for indole compounds in liquor, characterized in that: GO@PAA@Fe3O4 composite material was used as adsorbent to adsorb indole compounds in liquor, and then desorbed in acidic acetone solution. The indole compound is indole-3-acetic acid and / or indole-3-propionic acid.

2. The magnetic solid phase extraction method for indole compounds in liquor according to claim 1, characterized in that: The added amount of the GO@PAA@Fe3O4 composite material is 1-5 mg per 1 ml of liquor.

3. The magnetic solid phase extraction method for indole compounds in liquor according to claim 1, characterized in that: The adsorption time is 5-20 minutes.

4. The magnetic solid phase extraction method for indole compounds in liquor according to claim 1, characterized in that: The acidic acetone solution is a mixed solution of formic acid and acetone in a volume ratio of (0.2-2):(98-99.8).

5. The magnetic solid phase extraction method for indole compounds in liquor according to claim 1, characterized in that: Before using the GO@PAA@Fe3O4 composite material as an adsorbent to adsorb indole compounds in liquor, the method also includes the steps of diluting the alcohol content of the liquor to 7-25° with water and adjusting the pH to 4-10.

6. The magnetic solid phase extraction method for indole compounds in liquor according to claim 1, characterized in that: After the adsorption is completed, the method further includes collecting the adsorbed GO@PAA@Fe3O4 composite material using a magnet under the action of an external magnetic field.

7. The magnetic solid phase extraction method for indole compounds in liquor according to claim 1, characterized in that: After the desorption is completed, the desorption liquid is dried with nitrogen, then re-dissolved with acetonitrile, and filtered.

8. A method for evaluating the cellar aroma flavor of Luzhou-flavor liquor, characterized in that: After the indole compounds are extracted using the magnetic solid phase extraction method according to any one of claims 1 to 7, HPLC-MS / MS analysis is performed, and a qualitative test is performed using the retention index and mass spectrometry fragmentation law of the analyte; a standard curve method is used in combination with the ion mode in the HPLC-MS / MS analysis to perform a quantitative test on the analyte.

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