A method for rapid detection of four kinds of stimulants based on surface-enhanced raman spectroscopy

Abstract

The application discloses a kind of based on surface-enhanced Raman spectrum rapid detection four kinds of stimulant method, specifically comprising the following steps: (1) the identification of characteristic peak and the screening of enhancement substrate, (2) standard SERS spectrum acquisition, (3) actual sample SERS spectrum acquisition.The stimulant in saliva and urine is extracted by liquid-liquid extraction, the organic layer is mixed with nanoparticles, then a coagulating agent is added and mixed again, and finally SERS detection is carried out.The SERS signal of the stimulant is obtained, realizing the ultra-sensitive detection of the stimulant commonly used in sports.The method provided by the application is simple in operation, low in cost and fast in detection speed, can be completed in about 60s, does not need large and precise instruments, the instrument is simple in later maintenance, does not need professional technical personnel to operate, greatly saves time and cost.

Description

Technical Field

[0001] This invention belongs to the field of rapid drug detection technology, specifically relating to a method for rapid detection of four stimulants based on surface-enhanced Raman spectroscopy. Background Technology

[0002] Today, sporting events have become an important means of national development and exchange. However, the abuse of doping has caused sports competitions to lose their original meaning. Doping not only disregards fairness but also causes physical and psychological harm to athletes. Furthermore, doping damages a country's image and can, to some extent, impact international exchange and development. Therefore, analyzing and detecting doping is of great significance to national development and the physical and mental health of athletes.

[0003] Currently, the main methods for doping detection and analysis are chromatography, mass spectrometry (MS), or a combination of both, along with ultraviolet-visible spectroscopy and chemiluminescence immunoassay. While each method has its advantages, they also have drawbacks such as bulky and expensive equipment, complex sample preparation, and the need for highly trained technicians. For example, MS-MS separates the components of the analyte before using the powerful component identification capabilities of mass spectrometry to determine its composition. Furthermore, doping detection primarily relies on blood and urine tests, which are complex procedures with long testing cycles, requiring sampling of athletes before and after competitions, making simple and rapid doping detection impossible. Therefore, to meet the needs of rapid detection and regulation, establishing a method that is fast, easy to sample, and low-cost is crucial.

[0004] Since the discovery of Raman scattering by Indian physicist Raman, nearly a century of development has seen the emergence of surface-enhanced Raman spectroscopy (SERS). With advancements in laser technology, computer science, and nanoscience, SERS has become increasingly popular due to its high surface sensitivity, lack of sample pretreatment requirements, ease of operation, rapid detection speed, high accuracy, and portable instrumentation. SERS detection has played a positive role in food safety, public safety, and national defense security. For example, SERS can qualitatively and quantitatively detect harmful and illegal additives (such as melamine and Sudan Red), excessive or out-of-range additives (such as synthetic pigments in food), pesticide residues in fruits and vegetables, and bacteria and viruses on food surfaces. Clearly, SERS holds promise as a commonly used rapid on-site analytical method for public safety. Currently, methods for detecting doping using SERS have also been reported, but these methods cannot provide rapid on-site detection and require complex sample pretreatment. While they overcome some shortcomings of methods like chromatography-mass spectrometry, a rapid, accurate, and low-cost detection method still needs to be developed for practical application. Summary of the Invention

[0005] The purpose of this invention is to overcome the defects of the existing technology and provide a method for rapid detection of four stimulants based on surface-enhanced Raman spectroscopy. The method involves liquid-liquid extraction of stimulants from saliva and urine, mixing the organic layer with nanosol, adding an agglomerating agent and mixing again, and finally performing SERS detection.

[0006] To achieve the above objectives, one of the technical solutions of the present invention is: a method for rapid detection of four stimulants based on surface-enhanced Raman spectroscopy, specifically comprising the following steps:

[0007] (1) Identification of characteristic peaks: Determine the location of the characteristic peaks of the stimulant;

[0008] (2) Collect SERS spectra of standard products: Sol and agglomerating agent were added to methanol aqueous solutions of stimulant standard products of different concentrations, and then SERS detection was performed to obtain SERS spectra of methanol aqueous solutions of stimulant standard products of different concentrations.

[0009] (3) Acquisition of SERS spectrum of actual sample: Mix the sample to be tested, buffer and extractant, and extract the organic phase for SERS detection to obtain the SERS spectrum of the actual sample of the stimulant.

[0010] In a preferred embodiment of the present invention, the stimulant is at least one of clenbuterol, methadone, oxycodone, and chlordiazepoxide.

[0011] In a preferred embodiment of the present invention, the reinforcing substrate in step (2) is one of gold nanosol with a particle size of 30-60 nm or silver nanosol with a particle size of 80-100 nm.

[0012] In a preferred embodiment of the present invention, the agglomerating agent in step (2) is one or more of inorganic acids, inorganic bases, and inorganic salts, preferably one or more of NaOH, HCl, and NaCl, and the concentration of the agglomerating agent is 1.5-2.5M.

[0013] In a preferred embodiment of the present invention, the volume ratio of the stimulant standard methanol aqueous solution, gold sol, and agglomerating agent in step (2) is in the range of 2:(1-1.5):(1-2).

[0014] In a preferred embodiment of the present invention, the extractant in step (3) is one or a mixture of several organic reagents selected from cyclohexane, petroleum ether, n-hexane, dichloromethane, trichloromethane, and ethyl acetate.

[0015] In a preferred embodiment of the present invention, the buffer solution in step (3) is one of boric acid-borax buffer solution and citrate-disodium hydrogen phosphate buffer solution; the boric acid-borax buffer solution has a borax concentration of 0.03-0.07M, a boric acid concentration of 0.1-0.3M, a pH setting range of 7.4-9.0, and a corresponding volume ratio of borax to boric acid of (1-4):(9-1); the citrate-disodium hydrogen phosphate buffer solution has a disodium hydrogen phosphate concentration of 0.1-0.3M, a citric acid concentration of 0.05-0.15M, a pH setting range of 3.4-7.0, and a corresponding volume ratio of disodium hydrogen phosphate to citric acid of (1-5):(2.5-1).

[0016] Furthermore, the pH range of the buffer solution in step (3) is as follows: the buffer solution for clenbuterol is boric acid-borax buffer solution with a pH of 7.6 to 9.0; the buffer solution for methadone is boric acid-borax buffer solution with a pH of 7.0 to 9.0; the buffer solution for oxycodone is boric acid-borax buffer solution with a pH of 7.6 to 9.0; and the buffer solution for chlordiazepoxide is citrate-disodium hydrogen phosphate buffer solution with a pH of 6.0 to 7.0.

[0017] In a preferred embodiment of the present invention, the volume ratio of the sample to be tested, the buffer solution, and the extractant in step (3) is 2:(1-1.5):(1-2).

[0018] In a preferred embodiment of the present invention, in step (3), the extractant is ethyl acetate, the buffer is boric acid-borax buffer, the borax concentration is 0.05M, the boric acid concentration is 0.2M, the corresponding volume ratio of borax to boric acid is 1:4, and the buffer pH is 7.8; the volume ratio of the test sample containing the stimulant, the buffer, and the extractant is 2:1:2.

[0019] In a preferred embodiment of the present invention, the Raman spectrometer used for SERS detection in steps (2) and (3) is a portable Raman spectrometer with a wavelength of 785 nm and a power of 500 mW.

[0020] To achieve the above objectives, the second technical solution of the present invention is: the application of a method for rapid detection of four stimulants based on surface-enhanced Raman spectroscopy in the detection of stimulants in saliva and urine.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1. The method of the present invention can rapidly detect four illegal stimulants in saliva and urine in about one minute;

[0023] 2. The method of the present invention avoids complex and difficult detection conditions, such as bulky instruments and long detection time;

[0024] 3. The method of the present invention avoids the risks of disease transmission that may arise from collecting blood samples;

[0025] 4. The method of the present invention is low in cost, simple to operate, and requires no professional personnel. Attached Figure Description

[0026] Figure 1 These are the SERS spectra of the detection limits of the four doping standards in Example 1;

[0027] Figure 2 This is the SERS spectrum of the detection limit of actual saliva samples of four stimulants at different concentrations in Example 3;

[0028] Figure 3 This is the SERS spectrum of the detection limit of urine samples of four different concentrations of stimulants in Example 3;

[0029] Figure 4 This is the standard fitting curve of actual saliva samples of four different concentrations of stimulants from Example 4;

[0030] Figure 5 This is the standard fitting curve of actual urine samples of four stimulants at different concentrations in Example 4;

[0031] Figure 6 The SERS spectra of actual saliva and urine samples of unknown stimulant species in Example 9 are shown in (a) actual saliva sample and (b) actual urine sample. Detailed Implementation

[0032] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in more detail below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0033] The present invention will be further explained below with reference to the accompanying drawings.

[0034] (1) Identification of characteristic peaks: Determine the positions of characteristic peaks for the four stimulants clenbuterol, methadone, oxycodone and chlordiazepoxide;

[0035] (2) Collect SERS spectra of standard products: Sol and agglomerating agent were added to methanol aqueous solutions of different concentrations of stimulant standard products, and then SERS detection was performed to obtain SERS spectra of methanol aqueous solutions of four different concentrations of stimulant standard products.

[0036] (3) Acquisition of SERS spectrum of actual sample: Mix the sample to be tested, buffer and extractant, and extract the organic phase for SERS detection to obtain the SERS spectrum of the actual sample of the stimulant.

[0037] The gold sol used in the following examples was prepared by the following method: This invention uses a 30nm gold sol to enhance the Raman detection signals of four stimulants. First, 2.424 mL of 0.825% chloroauric acid was added to 200 mL of already boiled ultrapure water, and boiling was continued for 10 min. Then, 4 mL of 1% sodium citrate solution was added. After the solution turned wine-red, boiling was maintained for 20 min to synthesize a gold sol of approximately 30nm. The sol was then allowed to cool naturally before use.

[0038] Example 1

[0039] (1) Determining the characteristic peak positions of the doping agents: By testing the SERS spectra of methanol solutions of four doping agent standards at a concentration of 100 μg / mL and comparing them with Raman databases and consulting literature, the characteristic peaks of the four doping agents were confirmed as follows: Clenbuterol's surface-enhanced Raman characteristic peak is at 382 cm⁻¹. -1 and 1599cm -1 The surface-enhanced Raman spectroscopy (SEM) characteristic peaks of methadone are at 678 cm⁻¹ and 764 cm⁻¹. -1 and 1000cm -1 The surface-enhanced Raman characteristic peak of oxycodone is at 575 cm⁻¹. -1 and 1275cm -1 The surface-enhanced Raman characteristic peak of chlorodiazepoxide is 682 cm⁻¹. -1 and 999cm -1 .

[0040] (2) Collect SERS spectra of standard samples:

[0041] Prepare standard aqueous solutions: Take 1000 μg / mL chlordiazepoxide methanol solution and oxycodone methanol solution, 100 μg / mL methadone and clenbuterol standards, respectively, and dilute with water to prepare a standard methanol aqueous solution with a concentration of 1 μg / mL.

[0042] Using a 30 nm gold sol as the reinforcing substrate, 100 μL each of methadone, oxycodone, and chlordiazepoxide standard aqueous solutions (1 μg / mL) were taken as the test samples, and 2 M KBr aqueous solution was used as the flocculant. The volume ratio of test sample, gold sol, and flocculant was 10:15:1. Similarly, 100 μL of clenbuterol standard test sample (1 μg / mL) was taken as the test sample, and 2 M KCl aqueous solution was used as the flocculant. The volume ratio of test sample, gold sol, and flocculant was 20:30:1. After adding the gold sol, the test samples were mixed appropriately using a pipette before adding the flocculant, and the mixture was mixed again before SERS detection. SERS detection was performed on methanol aqueous solutions of four stimulant standards at different concentrations (1000, 500, 250, 100, 50, 25, 10, 5, 2.5, and 1 ng / mL) using the same method. The results are as follows: Figure 1 The SERS spectra of standard aqueous solutions of the four stimulants are shown, and the limits of detection for the stimulants are as follows: clenbuterol 5 ng / mL, methadone 5 ng / mL, chlordiazepoxide 5 ng / mL, and oxycodone 25 ng / mL.

[0043] Blank control sample setup: When performing SERS tests on doping standards, the blank control sample is a methanol aqueous solution with a concentration of 1 μg / mL.

[0044] Example 2

[0045] Optimize the pH of the extractant and buffer:

[0046] In this embodiment, gold nanosols with a particle size of 30 nm were used as the reinforcing substrate. The standard aqueous solutions of methadone, oxycodone, chlordiazepoxide, and clenbuterol were extracted and detected. The extractant was optimized according to a volume ratio of 2:1:2 for the test sample, buffer solution, and extractant. Buffer solutions (chlordiazepoxide in a boric acid-borax buffer solution at pH 9.0, methadone in a boric acid-borax buffer solution at pH 8.7, oxycodone in a boric acid-borax buffer solution at pH 9.0, and chlordiazepoxide in a citrate-disodium hydrogen phosphate buffer solution at pH 6.2) and extractants (cyclohexane, petroleum ether, n-hexane, dichloromethane, chloroform, ethyl acetate, and a mixture of cyclohexane and ethyl acetate in a 5:1 volume ratio) were added sequentially to the standard aqueous solutions. The mixture was thoroughly vortexed and stabilized. After stabilization, the organic phase containing the standards was aspirated and tested according to the SERS detection procedure in Example 1. Based on the peak intensity and peak area at the position of the spectral characteristic peak, the extractant with the larger peak intensity and peak area is selected as the optimal extractant. The optimal extractants for clenbuterol and oxycodone are chloroform, the optimal extractant for methadone is cyclohexane, and the optimal extractant for chlordiazepoxide is a mixture of cyclohexane and ethyl acetate at a volume ratio of 5:1.

[0047] The pH of the buffer solution was optimized according to a 2:1:2 ratio of test sample, buffer solution, and extractant. For the test samples containing methadone, oxycodone, and clenbuterol standards, different pH values ​​(7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.7, 9.0) of boric acid-borax buffer solution and the corresponding optimal extractant were added sequentially, and the mixture was thoroughly vortexed. For the test samples containing chlordiazepoxide standards, different pH values ​​(3.4, 3.8, 4.2, 4.6, 5.0, 5.4, 5.0) were added sequentially. 8, 6.2, 6.6, 7.0) citrate-disodium hydrogen phosphate buffer solution and the corresponding final extractant were mixed thoroughly by vortexing. After stabilization, the organic phase was aspirated and tested according to the SERS detection procedure in Example 1. The pH value with the largest peak intensity and peak area at the position of the spectral characteristic peak was selected as the optimal buffer pH value. The optimal buffer pH values ​​for the four stimulants in this example are as follows: clenbuterol 8.7, methadone 7.8, chlordiazepoxide 6.0, and oxycodone 7.0.

[0048] The optimal ratio of test sample, buffer solution, and extractant volume is as follows: clenbuterol: 2:1:2, methadone: 2:1:1, oxycodone: 2:1:1.5, chlordiazepoxide: 2:1:1.

[0049] Blank control sample setup: When optimizing extraction conditions, the blank control sample is the corresponding organic reagent.

[0050] Example 3

[0051] Extraction of four stimulants from saliva and urine: 1000 μg / mL standard chlordiazepoxide methanol solution and oxycodone methanol solution, 100 μg / mL standard methadone methanol solution and clenbuterol methanol solution were diluted with ultrapure water to prepare standard methanol aqueous solutions with a concentration of 10 μg / mL. Then, the standard methanol aqueous solutions were further diluted with saliva and urine to obtain actual spiked samples of 1 μg / mL. The spiked samples were then serially diluted with saliva and urine to concentrations of 500, 250, 100, 50, 25, 10, 5, 2.5, and 1 ng / mL. Considering that large quantities of saliva samples are generally not possible, 400 μL of saliva and 400 μL of urine samples were taken each time. According to the optimized conditions in Example 2, the corresponding volumes of buffer and extraction solvent were added sequentially. Samples containing clenbuterol and oxycodone were extracted with chloroform, requiring appropriate force with a pipette for mixing to avoid emulsification; otherwise, centrifugation was necessary to separate the organic and aqueous layers. Samples containing methadone and chlordiazepoxide were first vortexed, then 30 μL of ethanol was added to break the emulsion. When adding chlordiazepoxide to ethanol, the pipette should be placed at the separation point of the organic and aqueous layers while rotating. After stabilization, the organic phase containing the above-mentioned stimulants was aspirated and detected according to the SERS detection procedure in Example 1. The SERS spectra of the four stimulants at different concentrations in saliva and urine in this example were obtained, as shown below. Figure 2 and Figure 3 As shown, the detection limits for the four stimulants in actual saliva samples in this embodiment were as follows: clenbuterol: 25 ng / mL, methadone: 10 ng / mL, oxycodone: 100 ng / mL, and chlordiazepoxide: 50 ng / mL. The detection limits for actual urine samples were as follows: clenbuterol: 25 ng / mL, methadone: 5 ng / mL, oxycodone: 50 ng / mL, and chlordiazepoxide: 50 ng / mL.

[0052] Blank control sample setup: During actual sample testing, the blank control is the organic phase extracted from unspecified saliva and urine.

[0053] Example 4

[0054] Fitting the standard curve:

[0055] When fitting the standard curve, clenbuterol was taken at 382 cm. -1 Characteristic peak, methadone taken as 1000 cm⁻¹ -1 Characteristic peak, oxycodone taken at 1275 cm⁻¹ -1 Characteristic peak, chlorine nitrogen peak at 682 cm⁻¹ -1 Characteristic peaks.

[0056] like Figure 4 and Figure 5As shown, the peak areas of the characteristic peaks in the SERS spectra of different concentrations of clenbuterol and methadone in saliva and different concentrations of methadone in urine obtained in Example 3, and their corresponding sample concentrations, were fitted to the logarithm (base 10) to obtain a standard curve with good linearity. Similarly, the peak areas of the characteristic peaks in the SERS spectra of different concentrations of oxycodone and chlordiazepoxide in saliva and different concentrations of clenbuterol, oxycodone, and chlordiazepoxide in urine obtained in Example 3, and their corresponding sample concentrations, were fitted to the standard curve with good linearity.

[0057] Example 5

[0058] Five urine samples from healthy male individuals were tested. Each sample was spiked with 1 μg / mL of a methanol-water solution containing four different stimulant standards, diluted with urine to prepare a 1 μg / mL spiked urine solution. Further dilution with urine was performed, and the corresponding buffer and extraction solvent were added sequentially according to the optimized scheme in Example 2. After stabilization, 100 μL of the organic layer was taken and tested using the SERS detection procedure in Example 1. The detection limits of the four stimulants in the actual urine samples from the five healthy male individuals are shown in Table 1.

[0059] Table 1

[0060]

[0061] Example 6

[0062] Five saliva samples from healthy male individuals were tested. A 1 μg / mL spiked saliva solution was prepared by diluting four different stimulant standards (10 μg / mL) with methanol-water solution. The solution was then further diluted with saliva. Following the optimized procedure in Example 2, the corresponding buffer and extraction solvent were added sequentially according to the specified proportions. After stabilization, 100 μL of the organic layer was taken and tested using the SERS detection procedure described in Example 1. The detection limits of the four stimulants in the actual saliva samples from the five healthy male individuals are shown in Table 2.

[0063] Table 2

[0064]

[0065]

[0066] Example 7

[0067] Five urine samples from healthy female individuals were tested. A 1 μg / mL spiked urine solution was prepared by diluting the urine with a 10 μg / mL methanol-water solution of four different stimulant standards. The solution was then further diluted with urine, and the corresponding buffer and extraction solvent were added sequentially according to the optimized scheme in Example 2. After stabilization, 100 μL of the organic layer was taken and tested using the SERS detection procedure in Example 1. The detection limits of the four stimulants in the actual urine samples from the five healthy female individuals are shown in Table 3.

[0068] Table 3

[0069]

[0070] Example 8

[0071] Five saliva samples from healthy female individuals were tested. A 1 μg / mL spiked urine solution of four different stimulant standards was prepared by diluting the saliva with a 10 μg / mL methanol aqueous solution. This solution was then further diluted with saliva. Following the optimized procedure in Example 2, the corresponding buffer and extraction solvent were added sequentially according to the specified proportions. After stabilization, 100 μL of the organic layer was taken and tested using the SERS detection procedure described in Example 1. The detection limits of the four stimulants in the actual saliva samples from the five healthy female individuals are shown in Table 4.

[0072] Table 4

[0073]

[0074]

[0075] Example 9

[0076] For unknown samples, it is impossible to know whether they contain one or more stimulants. Therefore, to simultaneously detect four stimulants, a mixed test method was developed. A mixed standard solution of the four stimulants at a concentration of 1 μg / mL was prepared, and then serially diluted with saliva and urine to 500, 250, 100, 50, 25, 10, 5, 2.5, and 1 ng / mL, respectively. Ethyl acetate was used as the extraction solvent, and the buffer solution pH was 7.8 (the buffer solution was a boric acid-borax buffer, with 0.05 M borax and 0.2 M boric acid, corresponding to a borax to boric acid volume ratio of 1:4). The volume ratio of the test sample containing the four stimulants, the buffer solution, and the extraction solvent was 2:1:2. Based on the test process and steps in the above examples, the following results were obtained: Figure 6 The results are shown. The minimum detectable level of the four stimulants in saliva is 100 ng / mL, which is then determined by observing the characteristic peak at 380 cm⁻¹. -1 and 1275cm -1It was found that clenbuterol and oxycodone were undetectable, while methadone and chlordiazepoxide were still detectable at 25 ng / mL. At lower concentrations, all four stimulants were undetectable. In urine samples, the minimum level at which all four stimulants could be detected simultaneously was 100 ng / mL. Oxycodone was again undetectable at lower concentrations. Clenbuterol could still be detected at 50 ng / mL along with the other two stimulants, but it was undetectable at lower concentrations. Methadone and chlordiazepoxide were also detectable at 25 ng / mL, but all four stimulants were undetectable at lower concentrations.

[0077] The above embodiments are merely optimized implementations of the present invention, used to illustrate the principles and effects of the present invention, and are not intended to limit the present invention. It should be noted that any modifications made to the above embodiments by those skilled in the art without departing from the spirit and scope of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for rapid detection of four stimulants based on surface-enhanced Raman spectroscopy, characterized in that, Includes the following steps: (1) Identification of doping characteristic peaks: Determine the location of the characteristic peaks of the doping agents; wherein the doping agents are at least one of clenbuterol, methadone, oxycodone, and chlordiazepoxide; the surface-enhanced Raman characteristic peak of clenbuterol is at 382 cm⁻¹. -1 and 1599 cm -1 The surface-enhanced Raman characteristic peak of methadone is 678 cm⁻¹. -1 764 cm -1 and 1000 cm -1 The surface-enhanced Raman characteristic peak of oxycodone is at 575 cm⁻¹. -1 and 1275cm -1 The surface-enhanced Raman characteristic peak of chlorodiazepoxide is 682 cm⁻¹. -1 and 999cm -1 The minimum detection limits for doping are as follows: clenbuterol 5 ng / mL, methadone 5 ng / mL, chlordiazepoxide 5 ng / mL, oxycodone 25 ng / mL; (2) Collect SERS spectra of standard products: Nano sol and agglomerating agent were added to methanol aqueous solutions of stimulant standard products of different concentrations, and then SERS detection was performed to obtain SERS spectra of methanol aqueous solutions of stimulant standard products of different concentrations. (3) Acquisition of SERS spectrum of actual sample: The test sample, buffer solution, and extractant were mixed, and the organic phase was aspirated for SERS detection to obtain the SERS spectrum of the actual doping sample; the volume ratio of the doping standard methanol aqueous solution, nano-sol, and agglomerating agent was 2:(1-1.5):(1-2), and the volume ratio of the test sample, buffer solution, and extractant was 2:(1-1.5):(1-2). The extractant was one or more of the organic reagents selected from cyclohexane, petroleum ether, n-hexane, dichloromethane, trichloromethane, and ethyl acetate. The buffer solution was one of boric acid-borax buffer solution and citrate-disodium hydrogen phosphate buffer solution, and the borax concentration in the boric acid-borax buffer solution was 0.03-0.07%. M, boric acid concentration is 0.1-0.3M, buffer pH is 7.4-9.0, borax to boric acid volume ratio is (1-4):(9-1), citrate-disodium hydrogen phosphate buffer concentration is 0.1-0.3M, citric acid concentration is 0.05-0.15M, buffer pH is 3.4-7.0, disodium hydrogen phosphate to citric acid volume ratio is (1-5):(2.5-1), when the extractant is ethyl acetate, the buffer is boric acid-borax buffer, borax concentration is 0.05M, boric acid concentration is 0.2M, corresponding borax to boric acid volume ratio is 1:4, buffer pH is 7.8, the volume ratio of the test sample containing stimulants, buffer, and extractant is 2:1:

2.

2. The method for rapid detection of four stimulants based on surface-enhanced Raman spectroscopy as described in claim 1, characterized in that, In step (2), the nanosol is a gold nanosol with a particle size of 30-60 nm or a silver nanosol with a particle size of 80-100 nm.

3. The method for rapid detection of four stimulants based on surface-enhanced Raman spectroscopy as described in claim 1, characterized in that, The concentration of the agglomerating agent in step (2) is 1.5-2.5M, and the agglomerating agent is one or more of inorganic acids, inorganic bases, and inorganic salts.

4. The method for rapid detection of four stimulants based on surface-enhanced Raman spectroscopy as described in claim 3, characterized in that, The agglomerating agent is one or more of NaOH, HCl, and NaCl.

5. The application of the rapid detection method for four stimulants as described in any one of claims 1-4 in the detection of stimulants in saliva and urine.

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