A method for simultaneously detecting ketamine and fluanisone based on SERS

Abstract

The present invention discloses a method for simultaneously detecting ketamine and fluanisone based on SERS, belonging to the technical field of rapid food detection. The method for simultaneously detecting ketamine and fluanisone based on SERS of the present invention is to form a SERS detection system by mixing colloidal gold sol and aqueous solution of coagulant with a mixed matrix standard solution or a pretreated sample to be detected, and collect Raman spectral information of the system; taking the characteristic peaks at 453±5 cm‑1 and 813±5 cm‑1 or 1222±5 cm‑1 respectively, a quantitative relationship model of ketamine and fluanisone is constructed; and according to the SERS spectral intensity of the sample to be detected at the characteristic peaks, the contents of ketamine and fluanisone in the sample to be detected are calculated. This method has high sensitivity, and the recovery rate and RSD meet the national standards during the detection process of food and beverages, can realize the rapid detection of trace ketamine and fluanisone in food, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to a method for simultaneously detecting ketamine and fluanisone based on SERS, belonging to the technical field of rapid food detection. Background Art

[0002] In recent years, the abuse of new drugs such as ketamine (KET) has been serious, which has seriously endangered the global public health security. The effects of excitement, hallucination, etc. caused by the abuse of ketamine are stronger than those of traditional drugs. Ketamine was first included in the list of the first category of psychotropic drugs. In order to avoid police searches and strict legal inspections, criminals synthesized fluanisone (FET) with a molecular structure similar to that of ketamine, that is, replacing the chlorine atom in ketamine with a fluorine atom. Due to the similar structures of fluanisone and ketamine, it is often used as a substitute for ketamine or mixed into ketamine for illegal drug use. In order to ensure the food safety of the people and create a safe and drug-free food market, it is of great significance to study the detection of the two new drugs KET and FET and establish a method that can quickly, conveniently and effectively identify whether KET and FET exist in food.

[0003] There are also many detection methods for KET and FET in human metabolites such as blood and urine. Common detection methods include gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), near-infrared spectroscopy (NIR), ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), etc. However, most of the above methods are chromatography and its coupling technologies. These methods have expensive detection equipment, complex pretreatment steps, long detection time, and there is currently no systematic detection method for these two substances, and the simultaneous detection method for these two substances in food is also rarely seen.

[0004] Bao Shaokui et al. proposed a method for simultaneously analyzing ketamine and fluanisone in blood based on liquid chromatography-tandem mass spectrometry (LC-MS / MS). This method found that the acetonitrile precipitation protein method was the most specific pretreatment method for KET and FET. The ketamine and fluanisone in blood showed a linear correlation in the concentration range of 1.50 - 150 ng / mL, and the method detection limits of ketamine and fluanisone were 0.30 ng / mL. However, this method is time-consuming, has complex sample pretreatment, expensive equipment, and poor practicability.

[0005] Based on Raman spectroscopy analysis, information such as molecular structure, molecular vibration and rotation can be studied. This method has been widely studied and applied in the chemical field. Raman spectroscopy technology has "fingerprint" specificity, and the pretreatment is simple, convenient and fast. The enhancement effect of surface-enhanced Raman spectroscopy (SERS) can reach 10 times that of ordinary Raman signals. 14times, and its significant signal intensity has great advantages in reducing the detection limit of the analyte. High sensitivity, simple and convenient operation process, and fast analysis speed are all unique advantages of SERS spectroscopy technology. Therefore, SERS spectroscopy technology is suitable for on-site detection and is currently widely used in trace detection in specific application scenarios, and is applicable to the rapid detection of drugs in food. "Hot spot" is an important theory of the SERS enhancement effect. The more "hot spots", the better the SERS enhancement effect.

[0006] Therefore, it is of great significance to establish a rapid, sensitive, effective and real-time feedback simultaneous detection method for ketamine and fluanisone based on SERS. Summary of the Invention

[0007] To solve the defects and deficiencies of the existing technology, the present invention establishes a method for simultaneously detecting ketamine and fluanisone based on SERS. This method is simple, fast, highly sensitive, and can be used for the rapid detection of drugs in food.

[0008] The object of the present invention is to provide a method for simultaneously detecting ketamine and fluanisone based on SERS, and the method includes the following steps:

[0009] (1) Synthesize SERS-active gold nanoparticles sol; prepare an aqueous solution of a coagulant promoter;

[0010] (2) Pretreatment of the analyte sample and the blank matrix; prepare a series of concentration of the mixed matrix standard solutions of fluanisone and ketamine based on the blank matrix;

[0011] (3) Mix the gold nanoparticles sol and the aqueous solution of the coagulant promoter in step (1) with the mixed matrix standard solution or the pretreated analyte sample in step (2) to form a SERS detection system, and collect the spectral information of the system using a Raman spectrometer; then, take the spectral intensity at the characteristic peak of 453±5 cm -1 as the ordinate and the concentration of ketamine in the mixed solution as the abscissa to construct a quantitative relationship model for ketamine; take the spectral intensity at the characteristic peak of 813±5 cm -1 as the ordinate and the concentration of fluanisone in the mixed solution as the abscissa to construct a quantitative relationship model for fluanisone; calculate the contents of ketamine and fluanisone in the analyte sample according to the SERS spectral intensity of the analyte sample at the characteristic peak and based on the quantitative relationship models of ketamine and fluanisone.

[0012] In one embodiment, the synthesis of the gold nanoparticles sol in step (1) is as follows: add an aqueous solution of chloroauric acid with a mass fraction of 1% to the boiling aqueous solution, then add an aqueous solution of trisodium citrate with a mass fraction of 1%, continuously heat it to boiling and stir, cool, centrifuge, remove part of the supernatant, and concentrate it to one-sixth of the original volume.

[0013] In one embodiment, the particle size of the nano-gold in the nano-gold sol in step (1) is 50 - 75 nm.

[0014] In one embodiment, the volume ratio of the chloroauric acid aqueous solution to the trisodium citrate aqueous solution is 1:0.4 - 1.

[0015] In one embodiment, the centrifugation is carried out at 4000 - 5000 r / min for 15 - 20 min.

[0016] In one embodiment, the cooling refers to cooling to room temperature.

[0017] In one embodiment, the coagulant in step (1) includes one or more of NaCl, NaBr, NaF, and KCl.

[0018] In one embodiment, the concentration of the coagulant aqueous solution in step (1) is 0.4 - 1.0 mol / L.

[0019] In one embodiment, the coagulant aqueous solution is an NaBr aqueous solution.

[0020] In one embodiment, the sample to be measured in step (2) includes common foods such as beverages, sugars, biscuits, and chocolates; preferably cola, Gatorade, or white sugar.

[0021] In one embodiment, the blank matrix in step (2) is a sample of the same type as the corresponding sample to be detected, which does not contain fluanisone and ketamine; the pretreatment method of the blank matrix is the same as that of the corresponding sample to be measured.

[0022] In one embodiment, when the sample to be measured and the blank matrix are cola or Gatorade, the specific pretreatment is as follows: Take the blank cola / Gatorade sample and the sample to be measured of cola / Gatorade respectively, add NaOH solution to neutralize and remove carbonic acid, then extract pigments with ethyl acetate, shake and then centrifuge, and take the supernatant to obtain the extraction solution of the blank cola / Gatorade sample and the extraction solution of the sample to be measured of cola / Gatorade.

[0023] In one embodiment, the concentration of the NaOH solution is 1 - 2 mol / L, and the volume-mass ratio of the NaOH solution to the blank cola / Gatorade sample and the sample to be measured of cola / Gatorade is 0.1 - 0.2:1, ml:g.

[0024] In one embodiment, the centrifugation after shaking is carried out at 10000 - 15000 r / min for 10 - 15 s.

[0025] In one embodiment, the volume-mass ratio of ethyl acetate to the blank cola / Gatorade sample and the cola / Gatorade sample to be tested is 0.5 to 1:1, ml:g.

[0026] In one embodiment, the specific pretreatment when the sample to be tested and the blank matrix are white sugar is as follows: Take the blank white sugar sample and the white sugar sample to be tested and dissolve them in water respectively to obtain the blank white sugar sample extract and the white sugar sample extract to be tested.

[0027] In one embodiment, the mass-volume ratio of the blank white sugar sample and the white sugar sample to be tested to water is 1:2 to 5, g:ml.

[0028] In one embodiment, the concentration of ketamine in the ketamine and norketamine mixed standard solution in step (2) is 0 to 200 μg / g; the concentration of norketamine is 0 to 50 μg / g.

[0029] In one embodiment, the volume ratio of the gold nanosol, the coagulant aqueous solution and the mixed standard solution in step (2) or the sample to be tested after pretreatment in the SERS detection system in step (3) is 3 to 4.5:0.2 to 1.5:1.

[0030] In one embodiment, the Raman spectrometer in step (3) is Megrez 10, and the parameter settings are: laser power 785 nm; scanning power 400 mW; integration time 10 s; number of scans is 2 times.

[0031] Another object of the present invention is to provide an application of the above detection method in the detection of food and beverages.

[0032] The beneficial effects of the present invention:

[0033] (1) By combining theoretical calculation attribution with actual detection identification, the SERS characteristic peaks of ketamine and norketamine in the mixed solution are obtained in the present invention. The characteristic peaks of ketamine (KET) and norketamine (FET) are distinguished from the molecular perspective, and the accuracy of the quantitative relationship model constructed between the Raman intensities at each characteristic peak in the ketamine and norketamine mixed solution and the corresponding concentrations of ketamine and norketamine is combined. Finally, the SERS technology selected by the present invention is more scientific and reliable, and the method for simultaneously detecting ketamine and norketamine established by SERS is also more accurate and effective;

[0034] (2) On the basis of the existing technology, the present invention improves the enhancement substrate, enhances the signal by concentration, and the concentrated enhancement substrate can make more hot spots and reveal the signal;

[0035] (3) The present invention investigated the influence of the coagulant aqueous solution on the SERS enhancement effect. Different types of coagulants can affect the ionic strength and distribution in the mixed system; coagulants induce the aggregation of metal nanoparticles to obtain more "hot spots", and the more "hot spots", the stronger the SERS signal. Therefore, the stronger the ability of the coagulant to induce the aggregation of metal nanoparticles, the better the signal enhancement effect; under the same cation, among the halogen elements, the electron mobility and polarizability of Br are the strongest, followed by chlorine and fluorine in turn, and at the same time, Br - also has high adsorption, which all contribute to the aggregation of metal nanoparticles. Therefore, the enhancement effect is NaBr > NaCl > NaF in solution;

[0036] (4) The method established by the present invention can achieve the simultaneous detection of KET and FET, which is fast and convenient. It is not only applicable to the rapid detection in specific application scenarios, but can also be extended to the food system and is applicable to the rapid detection of drugs in food. Description of the Drawings

[0037] Figure 1 is the ultraviolet-visible spectral diagram of the gold nanoparticle solution in Example 1 of the present invention;

[0038] Figure 2 is the SERS spectral diagram of different concentration mixed standard solutions in Example 1 of the present invention;

[0039] Figure 3 is the SERS spectral diagram of different concentration mixed standard solutions in Example 2 of the present invention;

[0040] Figure 4 is the SERS spectral diagram of different concentration mixed standard solutions in Example 3 of the present invention;

[0041] Figure 5 is the Raman spectral diagram of the experimental solid Raman spectra of ketamine and fluanisone;

[0042] Figure 6 is the SERS spectral diagram of the mixed solution formed by different ratios of ketamine and fluanisone in Example 4 of the present invention (comparison of KET characteristic peaks);

[0043] Figure 7 is the SERS spectral diagram of the mixed solution formed by different ratios of ketamine and fluanisone in Example 4 of the present invention (comparison of FET characteristic peaks);

[0044] Figure 8 is the SERS spectral diagram of different concentration FET solutions in the mixed solution in Example 4 of the present invention and the linear relationship diagram between the concentration of the FET solution at the 813 cm -1 characteristic peak and the Raman intensity;

[0045] Figure 9SERS spectra of FET solutions with different concentrations in the mixed solution of Example 4 of the present invention and the linear relationship diagram between the concentration of the FET solution at the characteristic peak of 1222 cm -1 and the Raman intensity;

[0046] Figure 10 Comparison diagram of the SERS enhancement effects of different coagulants on KET and FET in Example 5 of the present invention;

[0047] Figure 11 SERS spectra of FET solutions with different concentrations and the linear relationship diagram in Comparative Example 1;

[0048] Figure 12 SERS spectra of KET solutions with different concentrations and the linear relationship diagram in Comparative Example 2. Detailed implementation manners

[0049] To better understand the present invention, the present invention will be further described below in conjunction with embodiments. The following embodiments are the applications of the method for simultaneous SERS detection of ketamine and fluanisone in food, and are respectively related to the applications in carbonated beverage Coke, non-carbonated beverage Gatorade electrolyte aqueous solution, and white sugar. However, the scope claimed by the present invention is not limited to the scope described in the embodiments.

[0050] The manufacturers of Coke and Gatorade beverages are both from PepsiCo Inc., USA; the white sugar is Baizuan premium white granulated sugar, and the manufacturer is Angel Yeast Co., Ltd.

[0051] The concentration of the ketamine stock solution is 1000 μg / mL; the concentration of the fluanisone stock solution is 1000 μg / mL.

[0052] Example 1

[0053] A method for simultaneous SERS detection of ketamine and fluanisone in Coke, the method comprising the following steps:

[0054] (1) Preparation of gold nanoparticles sol: Add 98.8 mL of ultrapure water into a 250 mL round-bottom flask, and perform magnetic stirring in an intelligent magnetic stirrer at 120 °C; then quickly add 1 mL of 1% chloroauric acid aqueous solution to the boiling aqueous solution, and quickly add 0.4 mL of 1% trisodium citrate aqueous solution after re-boiling. Keep heating to make it boil and stir for 15 min, cool to room temperature, then centrifuge the mixed solution at 5 °C and 4000 r / min for 15 min, remove part of the supernatant, and concentrate it to about one-sixth of the original to obtain gold nanoparticles with a particle size of 50 - 75 nm; the characterization of the gold nanoparticles solution is as Figure 1 shown;

[0055] (2) Preparation of coagulant promoter: Prepare an aqueous solution of NaBr with a concentration of 0.4 mol / L;

[0056] (3) Pretreatment of blank cola sample and cola sample to be tested: Take 1 g of blank cola sample and cola sample to be tested respectively, add 0.1 mL of NaOH solution with a concentration of 1 mol / L to neutralize and remove carbonic acid, then add 0.6 mL of ethyl acetate to extract pigments. After shaking, centrifuge at 10000 r / min for 10 s, and take the supernatant to obtain the blank cola sample extract and the cola sample extract to be tested;

[0057] (4) Preparation of matrix standard solutions of ketamine and fluanisone in cola

[0058] Mix the mother liquors of ketamine and fluanisone according to a volume ratio of 1:4 to form a mixed solution of ketamine and fluanisone. The concentration of ketamine in the mixed system is 200 μg / mL, and the concentration of fluanisone is 800 μg / mL; and add the mixed solution to the blank cola sample extract in step (3) with a volume of 500 μL according to volumes of 0, 12.5, 25, 50, 62.5, 100, 125, 250 μL respectively to prepare a series of matrix standard solutions with effective ketamine concentrations of 0, 2.5, 5, 10, 12.5, 20, 25, 50 μg / g and fluanisone concentrations of 0, 10, 20, 40, 50, 80, 100, 200 μg / g in cola;

[0059] (5) Determination of ketamine and fluanisone

[0060] Mix the nano-gold sol prepared in step (1), the solution to be tested in step (4), and the coagulant promoter prepared in step (2) according to a volume ratio of 4.5:1:0.5 to form a SERS detection system. Use a Raman spectrometer Megrez 10 to collect the spectral information of the system. The parameter settings of the Raman spectrometer are: laser power 785 nm; scanning power 400 mW; integration time 10 s; number of scans 2 times; respectively use the spectral intensity at the characteristic peak of 453 cm -1 as the ordinate and the ketamine concentration in the mixed solution as the abscissa to construct the quantitative relationship of ketamine; use the spectral intensity at the characteristic peak of 813 cm -1 as the ordinate and the fluanisone concentration in the mixed solution as the abscissa to construct the quantitative relationship model of fluanisone, and calculate the contents of ketamine and fluanisone in the cola sample to be tested according to the above quantitative relationship model.

[0061] The test results are as follows:

[0062] 1. The SERS spectrograms of different concentration mixed standard solutions in cola are as Figure 2 shown; The concentration of ketamine (KET) in the cola system and 453 cm -1The characteristic peak intensity at [location] shows a logarithmic linear correlation, and the linear equation is y = 1271.6ln(x) + 2377, R 2 = 0.9755, and the linear range is 2.5 - 50 μg / g, with good results.

[0063] For ketamine (KET), there is also a good linear relationship between the characteristic peak intensity at 813 cm -1 and the concentration. The linear equation is y = 752.27ln(x) - 506.02, R 2 = 0.9601 for the standard curve, and the linear range is 10 - 100 μg / g.

[0064] 2. To evaluate the accuracy of the established detection method, two technical indicators, namely the recovery rate and the within-laboratory coefficient of variation (RSD), were analyzed.

[0065] For the recovery rate, three levels of one-fold, two-fold, and ten-fold of the detection limit or the lowest detectable concentration of this experimental method should be selected for repeated tests (n ≥ 6). The reference range of the recovery rate refers to Appendix F in GB / T 27404 - 2008 "Laboratory Quality Control Specification for Physical and Chemical Testing of Foods". The within-laboratory coefficient of variation (RSD) is based on the data of the recovery rate experiment, and the reference range refers to Table F.2 in Appendix F.

[0066] In the present invention, the lowest detectable concentration of ketamine is 2.5 μg / g, and six repeated tests were carried out at three levels of ketamine concentrations of 2.5, 5, and 25 μg / g; the lowest detectable concentration of fluoramine ketone is 10 μg / g, and six repeated tests were carried out at three levels of fluoramine ketone concentrations of 10, 20, and 100 μg / g. The recovery rates and RSDs of KET and FET in cola are shown in Table 1; the recovery rates in Table 1 are 95.8% - 108.9%, and the RSDs are 5.2% - 7.5%, which can meet the detection requirements of food physical and chemical indicators.

[0067] Table 1 Recovery Rates and RSDs of KET and FET in Cola

[0068]

[0069] Example 2

[0070] A method for simultaneously detecting ketamine and fluoramine ketone in Gatorade based on SERS, the method comprising the following steps:

[0071] (1) Preparation of gold nanoparticles sol: Add 98.8 mL of ultrapure water into a 250 mL round-bottom flask, and perform magnetic stirring in an intelligent magnetic stirrer at 120 °C. Then, quickly add 1 mL of 1% aqueous chloroauric acid solution to the boiling aqueous solution. After re-boiling, rapidly add 0.4 mL of 1% aqueous sodium citrate solution. Keep heating to make it boil and stir for 15 min, then cool to room temperature. Then, centrifuge the mixed solution at 5 °C and 4000 r / min for 15 min, remove part of the supernatant, and concentrate it to about one-sixth of the original volume. The particle size of the gold nanoparticles is 50 - 75 nm;

[0072] (2) Preparation of coagulant: Prepare 0.4 mol / L aqueous NaBr solution;

[0073] (3) Pretreatment of blank Gatorade sample and Gatorade sample to be measured: Take 1 g of blank Gatorade sample and Gatorade sample to be measured respectively, add 0.1 mL of 1 mol / L NaOH solution to neutralize and remove carbonic acid, then add 0.6 mL of ethyl acetate to extract pigments. After oscillation, centrifuge at 10000 r / min for 10 s, and take the upper clear liquid to obtain the blank Gatorade sample extract and the Gatorade sample extract to be measured;

[0074] (4) Establishment of standard curves of ketamine and fluanisone in Gatorade

[0075] Mix the stock solutions of ketamine and fluanisone according to a volume ratio of 1:4 to form a mixed solution of ketamine and fluanisone. The concentration of ketamine in the mixed system is 200 μg / mL, and the concentration of fluanisone is 800 μg / mL. Then, add the mixed solution to the blank Gatorade sample extract in step (3) with a volume of about 500 μL according to volumes of 0, 12.5, 25, 50, 62.5, 100, 125, 250 μL respectively. After preparation, the effective concentrations of ketamine in Gatorade are 0, 2.5, 5, 10, 12.5, 20, 25, 50 μg / g, and the concentrations of fluanisone are 0, 10, 20, 40, 50, 80, 100, 200 μg / g.

[0076] (5) Determination of ketamine and fluanisone

[0077] Mix the gold nanoparticles sol prepared in step (1), the solution to be measured in step (4), and the coagulant prepared in step (2) according to a volume ratio of 4.5:1:0.5 to form a SERS detection system. Use a Raman spectrometer Megrez 10 to collect the spectral information of the system. The parameter settings of the Raman spectrometer are: laser power 785 nm; scanning power 400 mW; integration time 10 s; number of scans 2 times; respectively with the characteristic peak 453 cm -1Using the spectral intensity at [specific wavenumber] as the ordinate and the concentration of ketamine in the mixed solution as the abscissa, a quantitative relationship for ketamine was constructed; using the spectral intensity at -1 as the ordinate and the concentration of fluanisone in the mixed solution as the abscissa, a quantitative relationship model for fluanisone was constructed, and based on the above quantitative relationship model, the contents of ketamine and fluanisone in the sample of Gatorade to be tested were calculated. -1 Using the spectral intensity at -1 as the ordinate and the concentration of fluanisone in the mixed solution as the abscissa, a quantitative relationship model for fluanisone was constructed, and based on the above quantitative relationship model, the contents of ketamine and fluanisone in the sample of Gatorade to be tested were calculated.

[0078] The test results are as follows:

[0079] 1. The SERS spectra of different concentration mixed standard solutions in Gatorade are as shown in Figure 3 ; The concentration of ketamine (KET) in the Gatorade system has a logarithmic linear correlation with the intensity of the characteristic peak at 453 cm -1 . The linear equation is y = 909ln(x) + 3650.7, R 2 = 0.9777, and the linear range is 2.5 - 50 μg / g, with good results.

[0080] There is also a good linear relationship between the intensity of the characteristic peak of fluanisone (FET) at 813 cm -1 and the concentration. The linear equation is y = 855.71ln(x) - 740.45, R 2 = 0.9199 for the standard curve, and the linear range is 10 - 100 μg / g.

[0081] 2. To evaluate the accuracy of the established detection method, two technical indicators, namely the recovery rate and the within-laboratory coefficient of variation (RSD), of the method were analyzed.

[0082] For the recovery rate, three levels that are one-fold, two-fold, and ten-fold of the detection limit or the lowest detectable concentration of this experimental method should be selected for repeated tests (n≥6). The reference range of the recovery rate refers to Appendix F in GB / T 27404 - 2008 "Laboratory Quality Control Specification for Food Physical and Chemical Analysis". The within-laboratory coefficient of variation (RSD) is based on the data of the recovery rate experiment, and the reference range refers to Table F.2 in Appendix F.

[0083] The lowest detectable concentration of ketamine in this method is 2.5 μg / g, and six repeated tests were carried out at three levels of ketamine concentrations of 2.5, 5, and 25 μg / g. The lowest detectable concentration of fluanisone is 10 μg / g, and six repeated tests were carried out at three levels of fluanisone concentrations of 10, 20, and 100 μg / g. The recovery rates and RSDs of KET and FET in Gatorade are shown in Table 2. The recovery rates in Table 2 are 90.1% - 107.8%, and the RSDs are 4.4% - 8.3%, which can meet the detection requirements of food physical and chemical indicators.

[0084] Table 2 Recovery rates and RSDs of KET and FET in Gatorade

[0085]

[0086] Example 3

[0087] A method for simultaneously detecting ketamine and fluanisone in white sugar based on SERS, the method comprising the following steps:

[0088] (1) Preparation of gold nanoparticles sol: Add 98.8 mL of ultrapure water into a 250 mL round-bottom flask, and perform magnetic stirring in an intelligent magnetic stirrer at 120 °C; then quickly add 1 mL of 1% aqueous chloroauric acid solution to the boiling aqueous solution, and quickly add 0.4 mL of 1% aqueous sodium citrate solution after re-boiling. Keep heating to make it boil and stir for 15 min, cool to room temperature, and then centrifuge the mixed solution at 5 °C and 4000 r / min for 15 min. Remove part of the supernatant and concentrate it to about one-sixth of the original. The particle size of the gold nanoparticles is 50 - 75 nm;

[0089] (2) Preparation of coagulant: Prepare 0.4 mol / L aqueous NaBr solution;

[0090] (3) Pretreatment of blank white sugar sample and white sugar sample to be tested: Respectively take 1 g of blank white sugar sample and white sugar sample to be tested, add 2 mL of deionized water to dissolve, and obtain blank white sugar sample extract and white sugar sample extract to be tested;

[0091] (4) Establishment of standard curves of ketamine and fluanisone in white sugar

[0092] Mix the mother solutions of ketamine and fluanisone according to a volume ratio of 1:4 to form a mixed solution of ketamine and fluanisone. The concentration of ketamine in the mixed system is 200 μg / mL, and the concentration of fluanisone is 800 μg / mL. Then add the mixed solution to the blank white sugar sample extract in step (3) with a volume of about 2 mL according to volumes of 0, 200, 300, 400, 500, 600, 850 μL. After configuration, the effective concentration of ketamine in white sugar is 0, 40, 60, 80, 100, 120, 170 μg / g, and the concentration of fluanisone is 0, 160, 240, 320, 400, 480, 680 μg / g.

[0093] (5) Determination of ketamine and fluanisone

[0094] Mix the nano-gold sol prepared in step (1), the test solution in step (4), and the coagulant promoter prepared in step (2) according to a volume ratio of 4.5:1:0.5 to form a SERS detection system. Use a Raman spectrometer Megrez 10 to collect the spectral information of the system. The parameters of the Raman spectrometer are set as follows: laser power 785 nm; scanning power 400 mW; integration time 10 s; number of scans 2 times; respectively, with the spectral intensity at the characteristic peak of 453 cm -1 as the ordinate and the concentration of ketamine in the mixed solution as the abscissa to construct the quantitative relationship of ketamine; with the spectral intensity at the characteristic peak of 813 cm -1 as the ordinate and the concentration of fluanisone in the mixed solution as the abscissa to construct the quantitative relationship model of fluanisone, and calculate the contents of ketamine and fluanisone in the test white sugar sample according to the above quantitative relationship model.

[0095] The test results are as follows:

[0096] 1. The SERS spectrograms of the mixed standard solutions with different concentrations in white sugar are as shown in Figure 4 ; The concentration of ketamine (KET) in the white sugar system has a linear correlation with the intensity of the characteristic peak at 453 cm -1 . The linear equation is y = 21.421x + 801.01, R 2 = 0.9626, and the linear range is 40 - 170 μg / g, with good results.

[0097] There is also a good linear relationship between the intensity of the characteristic peak of fluanisone (FET) at 813 cm -1 and the concentration. The linear equation is y = 2.6508x + 624.34, R 2 = 0.9649 standard curve, and the linear range is 160 - 680 μg / g.

[0098] 2. To evaluate the detection method established in the experiment, two technical indicators, namely the recovery rate and the within-laboratory coefficient of variation (RSD), of the method were analyzed.

[0099] For the recovery rate, three levels that are one-fold, two-fold, and ten-fold of the detection limit or the lowest detection concentration of this experimental method should be selected for repeated tests (n ≥ 6). The reference range of the recovery rate refers to Appendix F in GB / T 27404-2008 "Laboratory Quality Control Specification for Physical and Chemical Testing of Foods". The within-laboratory coefficient of variation (RSD) is based on the data of the recovery rate experiment, and the reference range refers to Table F.2 in Appendix F.

[0100] In this method, the lowest detectable concentration of ketamine is 20 μg / g. Six repeated tests were conducted on concentrations of 20, 40, and 200 μg / g. Since 200 μg / g is not within the linear range, it was diluted to 100 μg / g and then the repeated tests were carried out. The lowest detectable concentration of fluanisone is 160 μg / g. Six repeated tests were conducted on three levels of fluanisone concentrations at 160, 320, and 1600 μg / g. Since 1600 μg / g is not within the linear range, it was diluted to 400 μg / g and then the repeated tests were carried out. The recoveries and RSDs of ketamine and fluanisone in white sugar are shown in Table 3. The recoveries in Table 3 are 94.6%-114.6%, and the RSDs are 4.0%-7.0%, which can meet the detection requirements of food physical and chemical indicators.

[0101] Table 3 Recoveries and RSDs of ketamine and fluanisone in white sugar

[0102]

[0103] Example 4 Determination of characteristic peaks of fluanisone and ketamine in the mixed solution

[0104] The Raman spectral information of KET and FET solids was collected in the experiment, as shown in the appendix Figure 5 , and it can be seen from the figure that the characteristic peaks of KET are 454 cm -1 , 594 cm -1 , 651 cm -1 , 1041 cm -1 , 1448 cm -1 etc.; the characteristic peaks of FET are 540 cm -1 , 655 cm -1 , 776 cm -1 , 813 cm -1 , 1032 cm -1 , 1222 cm -1 , 1614 cm -1 , 1730 cm -1 etc.

[0105] However, in the case of the mixed solution formed by different ratios of KET and FET (1:99, 2:98, 10:90, 14:85, 17:83, 20:80, 50:50), only the specific peak of KET molecules appearing at 453 cm -1 has a good linear relationship with the KET concentration; as Figure 6 shown, so the present invention selects 453 cm -1 as the characteristic peak of KET.

[0106] In the case of mixed solutions formed with different ratios of KET and FET (1:99, 2:98, 10:90, 14:85, 17:83, 20:80, 50:50), obvious characteristic peaks of FET appear at 813 cm -1 and 1222 cm -1 as shown in Figure 7 ; The characteristic peak intensities at 813 cm -1 and 1222 cm -1 are respectively taken to construct the standard curve of FET, as shown in Figure 8 and Figure 9 . The concentration of FET has a logarithmic linear correlation with the Raman intensity at 813 cm -1 , and the linear equation is y = 663.49ln(x) - 438.71, with R 2 being 0.9445; The concentration of FET has a logarithmic linear correlation with the Raman intensity at 1222 cm -1 , and the linear equation is y = 1162.3ln(x) - 1288, with R 2 being 0.8962; It is found that the linear effect between the concentration of FET and the Raman intensity at 813 cm -1 is better and more suitable for the quantitative analysis of FET.

[0107] Example 5

[0108] Determination of Coagulant in SERS Detection System

[0109] Prepare four inorganic salt solutions of NaCl, NaBr, NaF, and KCl with a concentration of 1 mol / L each, and mix them according to the ratio of enhancement substrate: 100 μg / mL KET / FET solution: coagulant = 400:100:100 to form a SERS detection system.

[0110] Among them, the NaCl solution is the control group. NaF and NaBr solutions with different anions and KCl solution with different cations are selected to explore the influence of different cations and anions on the SERS enhancement effect of KET and FET. The results are shown in Figure 10 ;

[0111] As shown by Figure 10 ; When using NaCl and NaBr as coagulants, the characteristic peaks of KET at 453 cm -1 and 651 cm -1 and the characteristic peaks of FET at 660 cm -1 and 813 cm -1Enhancement can be obtained everywhere, and there is no interference from the solvent background peak of methanol. The enhancement effect of the NaBr solution as a coagulant promoter is significantly better than that of the NaCl solution. Therefore, it is best to choose the NaBr solution as the coagulant promoter in the SERS detection system. The enhancement effect of the NaF solution as a coagulant promoter on the characteristic signals of KET and FET is weak. The background signal value of the methanol solvent of KCl has low interference, and the enhancement effect on KET and FET is also not good.

[0112] It can be found that among the cations, sodium ions have the best enhancement effect on the SERS detection system, and among the anions, bromide ions have the greatest impact on the SERS enhancement effect. The enhancement effect is NaBr > NaCl > NaF ≈ KCl. Choose the NaBr solution as the coagulant promoter in the SERS detection system.

[0113] Comparative Example 1

[0114] A method for detecting ketamine in cola based on SERS, the method comprising the following steps:

[0115] (1) Preparation of gold nanoparticle sol: Add 98.8 mL of ultrapure water to a 250 mL round-bottom flask and perform magnetic stirring in an intelligent magnetic stirrer at 120 °C. Then quickly add 1 mL of 1% aqueous chloroauric acid solution to the boiling aqueous solution, and immediately add 0.4 mL of 1% aqueous sodium citrate solution after re-boiling. Keep heating to make it boil and stir for 15 min, cool to room temperature, and then centrifuge the mixed solution at 5 °C and 4000 r / min for 15 min. Remove part of the supernatant and concentrate it to about one-sixth of the original volume to obtain gold nanoparticles with a particle size of 50 - 75 nm.

[0116] (2) Preparation of coagulant promoter: Prepare 0.4 mol / L aqueous NaBr solution.

[0117] (3) Pretreatment of blank cola sample and test cola sample: Take 1 g of blank cola sample and test cola sample respectively, add 0.1 mL of 1 mol / L NaOH solution to neutralize and remove carbonic acid, then add 0.6 mL of ethyl acetate to extract pigments. After shaking, centrifuge at 10000 r / min for 10 s, and take the upper clear liquid to obtain the blank cola sample extract and the test cola sample extract.

[0118] (4) Preparation of matrix standard solution of ketamine in cola

[0119] Add ketamine to the 500 μL blank cola sample extract in step (3) respectively to prepare a series of matrix standard solutions of ketamine in cola with concentrations of 0, 1, 5, 10, 25, 50, and 100 μg / g.

[0120] (5) Determination of ketamine

[0121] Mix the nano-gold sol prepared in step (1), the solution to be tested in step (4), and the coagulant promoter prepared in step (2) in a volume ratio of 4.5:1:0.5 to form a SERS detection system. Use a Raman spectrometer Megrez 10 to collect the spectral information of the system. The parameters of the Raman spectrometer are set as follows: laser power 785 nm; scanning power 400 mW; integration time 10 s; number of scans 2 times; with the spectral intensity at the characteristic peak of 660 cm -1 as the ordinate and the fluanisone concentration as the abscissa to construct a quantitative relationship model of fluanisone, and calculate the content of fluanisone in the to-be-tested cola sample according to the above quantitative relationship model.

[0122] The test results are as follows:

[0123] 1. The SERS spectrograms of the matrix standard solutions of fluanisone with different concentrations in cola are as Figure 11 shown; the concentration of fluanisone (FET) in the cola system has a linear correlation with the intensity of the characteristic peak at 660 cm -1 . The linear equation is y = 2408.6ln(x) - 3199.8, and R 2 = 0.9564. The linear range is 5 - 100 μg / g, and the results are good.

[0124] 2. To evaluate the detection method established in the experiment, two technical indicators, namely the recovery rate and the coefficient of variation within the laboratory (RSD), of the method were analyzed.

[0125] For the addition and recovery of fluanisone at concentrations of 5, 10, and 50 μg / g, six repeated experiments were carried out; the average recovery rate of fluanisone in cola was 91.2% - 100.2%, and the RSD was 5.4% - 10.4%.

[0126] Comparative Example 2

[0127] A method for detecting ketamine in cola based on SERS, the method comprising the following steps:

[0128] (1) Preparation of nano-gold sol: Add 98.8 mL of ultrapure water to a 250 mL round-bottom flask and perform magnetic stirring in an intelligent magnetic stirrer at 120 °C; then quickly add 1 mL of 1% chloroauric acid aqueous solution to the boiling aqueous solution, and quickly add 0.4 mL of 1% trisodium citrate aqueous solution after re-boiling. Keep heating to make it boil and stir for 15 min, cool to room temperature, and then centrifuge the mixed solution at 5 °C and 4000 r / min for 15 min. Remove part of the supernatant and concentrate it to about one-sixth of the original to obtain nano-gold particles with a particle size of 50 - 75 nm;

[0129] (2) Preparation of coagulant: Prepare an aqueous solution of NaBr with a concentration of 0.4 mol / L;

[0130] (3) Pretreatment of blank cola sample: Take 1 g of blank cola sample and the cola sample to be tested respectively, add 0.1 mL of NaOH solution with a concentration of 1 mol / L to neutralize and remove carbonic acid, then add 0.6 mL of ethyl acetate to extract pigments. After shaking, centrifuge at 10000 r / min for 10 s, and take the supernatant to obtain the extract of the blank cola sample;

[0131] (4) Preparation of ketamine matrix standard solution in cola

[0132] Add ketamine to the extract of the blank cola sample in step (3) with a volume of 500 μL respectively to prepare a series of ketamine matrix standard solutions with ketamine concentrations of 0, 1, 5, 10, 25, 50, and 100 μg / g in cola;

[0133] (5) Determination of ketamine

[0134] Mix the nanogold sol prepared in step (1), the solution to be tested in step (4), and the coagulant prepared in step (2) according to a volume ratio of 4:1:1 to form a SERS detection system. Use a Raman spectrometer Megrez 10 to collect the spectral information of the system. The parameters of the Raman spectrometer are set as follows: laser power 785 nm; scanning power 400 mW; integration time 10 s; number of scans 2 times; with the spectral intensity at the characteristic peak of 651 cm -1 as the ordinate and the ketamine concentration as the abscissa to construct a quantitative relationship model of ketamine, and calculate the content of ketamine in the cola sample to be tested according to the above quantitative relationship model.

[0135] The test results are as follows:

[0136] 1. The SERS spectra of ketamine standard solutions with different concentrations in cola are as Figure 12 shown; The concentration of ketamine (KET) in the cola system has a linear correlation with the intensity of the characteristic peak at 651 cm -1 . The linear equation is y = 3764ln(x) + 1169, R2 = 0.9775, and the linear range is 1 - 100 μg / g, and the results are good.

[0137] 2. To evaluate the detection method established in the experiment, two technical indicators, the recovery rate and the coefficient of variation (RSD) in the laboratory, of the method were analyzed.

[0138] Add and recover ketamine at concentrations of 1, 2, and 10 μg / g, and conduct six repeated experiments; The average recovery rate of ketamine in cola is 101.1% - 109.6%, and the RSD is 6.5% - 10.6%.

Claims

1. A method for simultaneously detecting ketamine and fluanisone based on SERS, characterized in that, The method includes the following steps: (1) Synthesize nano-gold sol with SERS activity; prepare an aqueous solution of a coagulant; the coagulant is NaBr; the concentration of the aqueous solution of the coagulant is 0.4 - 1.0 mol / L; (2) Pretreat the sample to be tested and the blank matrix; prepare a series of concentration mixed matrix standard solutions of fluanisone and ketamine based on the blank matrix; (3) Mix the nano-gold sol and the coagulant aqueous solution in step (1) with the mixed matrix standard solution or the pre-treated sample to be measured in step (2) to form a SERS detection system, and collect the spectral information of the system using a Raman spectrometer; then, with the spectral intensity at the characteristic peak of 453 ± 5 cm -1 as the ordinate and the ketamine concentration in the mixed solution as the abscissa, construct a quantitative relationship model of ketamine; with the spectral intensity at the characteristic peak of 813 ± 5 cm -1 or 1222 ± 5 cm -1 as the ordinate and the fluanisone concentration in the mixed solution as the abscissa, construct a quantitative relationship model of fluanisone; calculate the contents of ketamine and fluanisone in the sample to be measured according to the SERS spectral intensity of the sample to be measured at the characteristic peak and based on the quantitative relationship models of ketamine and fluanisone; In the SERS detection system, the volume ratio of the nano-gold sol, the aqueous solution of the coagulant, and the mixed standard solution in step (2) or the sample to be tested after pretreatment is 3 - 4.5:0.2 - 1.5:

1.

2. The method according to claim 1, wherein The synthesis of the nano-gold sol in step (1) is as follows: Add an aqueous solution of chloroauric acid with a mass fraction of 1% to the boiling aqueous solution, then add an aqueous solution of trisodium citrate with a mass fraction of 1%, continuously heat it to boiling and stir, cool, centrifuge, remove part of the supernatant, and concentrate it to one-sixth of the original volume.

3. The method according to claim 2, characterized in that, The volume ratio of the aqueous solution of chloroauric acid to the aqueous solution of trisodium citrate is 1:0.4 - 1.

4. The method according to claim 1, characterized in that, The sample to be tested in step (2) includes one or more of beverages, sugars, biscuits, and chocolates.

5. The method according to claim 1, characterized in that, When the sample to be tested and the blank matrix are cola or Gatorade, the pretreatment in step (2) is specifically as follows: Take the blank cola / Gatorade sample and the sample to be tested of cola / Gatorade respectively, add NaOH solution to neutralize and remove carbonic acid, then extract pigments with ethyl acetate, shake and centrifuge, and take the upper clear liquid to obtain the extraction solution of the blank cola / Gatorade sample and the extraction solution of the sample to be tested of cola / Gatorade; when the sample to be tested and the blank matrix are white sugar, the pretreatment is specifically as follows: Take the blank white sugar sample and the sample to be tested of white sugar respectively, add water to dissolve them to obtain the extraction solution of the blank white sugar sample and the extraction solution of the sample to be tested of white sugar.

6. The method according to claim 1, wherein The Raman spectrometer in step (3) is Megrez 10, and the parameter settings are: laser power 785 nm; scanning power 400 mW; integration time 10 s; the number of scanning times is 2 times.

7. Application of the method according to any one of claims 1 - 6 in the detection of beverages and foods.

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