Method for detecting clenbuterol based on thin layer chromatography and surface enhanced resonance raman spectroscopy

Abstract

The application discloses a method for detecting clenbuterol based on thin layer chromatography and surface enhanced resonance Raman spectroscopy, and belongs to the technical field of food detection. The method comprises the following steps: (1) adopting thin layer chromatography to preliminarily separate the clenbuterol component from the matrix in the pig liver sample, and confirming the clenbuterol component in the pig liver sample under the ultraviolet lamp of 254 nm; (2) adding aromatic diazonium salt solution to the same Rf value of the clenbuterol component reference sample spot to generate azo compounds, and then adding a surface enhancer silver sol; (3) in-situ detecting the surface enhanced resonance Raman spectrum of the corresponding azo compound component spot, and if the surface enhanced resonance Raman spectrum of the pig liver sample solution at the same Rf value is the same as that of the reference sample solution, it is determined that the pig liver sample contains the clenbuterol component. The application has high specificity for detecting clenbuterol and salbutamol, high sensitivity, fast analysis speed, and is beneficial to fast detection of grass-roots units. The application is suitable for detecting clenbuterol.

Description

Technical Field

[0001] This invention belongs to the field of food testing technology, specifically relating to a method for detecting clenbuterol based on thin-layer chromatography and surface-enhanced resonance Raman spectroscopy. Background Technology

[0002] Clenbuterol is a specific, inexpensive hormone-like drug that reduces fat and increases lean meat ratio in farmed animals. Its main components include clenbuterol hydrochloride, ractopamine, salbutamol, and terbutaline. Clenbuterol was initially used to treat respiratory diseases such as bronchial asthma and chronic bronchitis in animals. Later, it was discovered that higher doses could achieve a redistribution of nutrients in animals. Adding clenbuterol to feed can accelerate animal growth and increase the lean meat percentage of pigs by nearly 10%. Clenbuterol is a non-protein hormone, heat-resistant, and accumulates in animal tissues after consumption, with the highest residue levels in internal organs. When humans consume it in large quantities or ingest high levels of residual internal organs, it can cause poisoning.

[0003] Currently, there are numerous methods for detecting ractopamine, including sensory evaluation, enzyme-linked immunosorbent assay (ELISA), and high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS). Sensory evaluation is the most direct method of assessment using the naked eye, observing the color of the meat. Pigs containing ractopamine generally have bright red meat after slaughter, with full and prominent hindquarter muscles and a thin layer of fat, typically less than 1 cm. Healthy meat is generally pale red and has good elasticity. ELISA is an immunological labeling technique that uses enzymes to label antigens or antibodies to detect the corresponding antigens or antibodies. Due to the specificity of ELISA principles, the antigens and antibodies used in establishing the method are highly active, making the results easily affected by enzymes. It also has high requirements for temperature and incubation conditions. Furthermore, ELISA uses pipettes throughout the process, requiring a high level of operator skill; slight inexperience or improper use can lead to inaccurate results and false positives. The most common rapid ractopamine detection method on the market is the rapid test strip. This method utilizes the immunological reaction between antigens and antibodies. Colloidal gold has a strong adsorption capacity for protein molecules. Furthermore, the negatively charged surface of colloidal gold allows it to bind to positively charged proteins (antigens, antibodies) via electrostatic interactions, forming stable gold-labeled antigens or antibodies. Colloidal gold immunochromatography offers convenient and low-cost results, with a detection time of 10–15 minutes, making it suitable for large-scale initial screening. However, commercially available cards have a sensitivity of 3–5 μg / L, while the industry standard NY / T 933−2005, "Determination of Clenbuterol Hydrochloride in Urine by Colloidal Gold Immunochromatography," sets the detection limit for clenbuterol by 3 μg / L. This slightly exceeds the industry standard, resulting in lower specificity and sensitivity, which can easily lead to inaccurate results and a higher likelihood of false positives. Additionally, visual interpretation can cause false positives or false negatives when the test line is blurry, and the results are difficult to preserve. High-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) is accurate and sensitive, but its higher operating cost makes it unsuitable for rapid testing at the grassroots level. Summary of the Invention

[0004] The purpose of this invention is to solve the problems existing in the above-mentioned technologies and to provide a method for detecting clenbuterol based on thin-layer chromatography and surface-enhanced resonance Raman spectroscopy.

[0005] The method for detecting clenbuterol based on thin-layer chromatography and surface-enhanced resonance Raman spectroscopy is implemented according to the following steps:

[0006] I. Thin-layer chromatography (TLC) was used to initially separate the clenbuterol component in the pig liver sample from the matrix. Then, the clenbuterol component in the pig liver sample was confirmed by spot identification under a UV lamp at 254 nm based on the clenbuterol component reference spot.

[0007] 2. Add an aromatic diazonium salt solution to the spot with the same shift value as the clenbuterol component reference standard spot to generate an azo compound, and then add the surface enhancer silver sol.

[0008] 3. After adding the surface enhancer silver sol as described above, the surface enhanced resonance Raman spectrum (SERRS) of the corresponding azo compound component spots is detected in situ under a 532 nm laser light source. If the SERRS spectrum of the pig liver sample solution at the same ratio shift value is the same as that of the control solution, it can be confirmed that the pig liver sample contains clenbuterol, thus completing the detection method.

[0009] Furthermore, in step one, thin-layer chromatography is used to initially separate the clenbuterol component in the pig liver sample from the matrix. Then, under a UV lamp at 254 nm, the clenbuterol component in the pig liver sample is confirmed by spot analysis based on the clenbuterol component reference standard spots. The specific process is as follows:

[0010] a. Preparation of reference solutions: Prepare reference solutions with a concentration of 10 μg·mL using methanol as the solvent. -1 Salbutamol solution with a concentration of 10 μg·mL -1 All terbutaline solutions were stored in sealed brown bottles for later use.

[0011] b. Using silicone 60F 254 Aluminum alloy thin-layer plate was used as the stationary phase, and a mixture of ethyl acetate, isopropanol, water, and glacial acetic acid was used as the developing solvent. The reference solution and the pig liver sample solution were then spotted onto the same stationary phase at a volume of 4 μL. After saturation for 10 min, the sample was developed at 20 °C, removed, and air-dried to complete the initial separation of the clenbuterol component from the matrix. The clenbuterol component spots in the pig liver sample were then examined under a UV lamp at 254 nm based on the clenbuterol component reference spot.

[0012] Further, the preparation of the pig liver sample solution: commercially available pig liver samples are washed, air-dried, and crushed. Then, 10.00g is placed in a 50mL stoppered conical flask, methanol is added, and the mixture is sonicated at a frequency of 40kHz for 5min. After cooling to room temperature, the mixture is centrifuged at a speed of 4000r / min for 5min, filtered, and the filtrate is transferred to a rotary evaporator and heated to concentrate to 2mL to obtain the pig liver sample solution. The solution is then sealed and stored in a dark place for later use.

[0013] Furthermore, the volume ratio of ethyl acetate, isopropanol, water, and glacial acetic acid in the developing solvent is 5:2:1:2.

[0014] Further, in step two, the aromatic diazonium salt is prepared by mixing a 0.7 mg / mL dilute hydrochloric acid solution of p-aminothiophenol and a 0.03 g / mL sodium nitrite solution at a volume ratio of 1:1. The mixture is then reacted in an ice-salt bath at 0°C to 5°C until a yellow aromatic diazonium salt is formed. The pH is then adjusted to 8 to 10 with a 50% NaOH solution and stirred for 5 minutes.

[0015] Furthermore, in step two, the amount of aromatic diazonium salt solution used is 4 μL; and the amount of surface-enhancing silver sol used is 8 μL.

[0016] Further, in step two, the surface-enhancing silver sol is prepared as follows: 100 mL of silver nitrate solution with a concentration of 0.37 mg / mL is heated in a microwave oven until it just boils, then removed and 2.6 mL of sodium citrate solution with a mass-volume concentration of 1% is added. After mixing, the mixture is heated for another 2 min and cooled to obtain a gray-green sol, which is the surface-enhancing silver sol.

[0017] Furthermore, the detection conditions for surface-enhanced resonance Raman spectroscopy described in step three are: microscope magnification of 10x, image pixel size of 540 μm, and scanning range of 100–3300 cm⁻¹. -1 The laser power was 10.0 mW, the exposure time was 0.05000 s, the number of scans was 30, the data acquisition and analysis software was OMNICxi, automatic baseline correction was performed, and the mapping software was Origin 6.1.

[0018] Furthermore, the surface-enhanced resonance Raman spectrum of the pig liver sample solution at the same ratio shift value in step three is the same as that of the reference solution. That is, the peak shape and position of the azo compound component spot are consistent with the peak shape and position of the azo compound component spot generated by the clenbuterol component reference, indicating that the pig liver sample contains salbutamol and terbutaline components.

[0019] Due to the complex composition of pig liver, this invention employs thin-layer chromatography (TLC) for rapid initial screening, followed by surface-enhanced resonance Raman spectroscopy (SERRS) for further confirmation, establishing a novel method for detecting clenbuterol in commercially available pig liver using TLC-SERRS. This invention has low requirements for pig liver sample preparation and is less affected by water, which is beneficial for rapid on-site determination and suitable for widespread use.

[0020] The existing TLC-SERRS method involves first mixing the sample solution with an aromatic diazonium salt. The target component, an aromatic secondary amine, reacts with the aromatic diazonium salt to form an azo compound. This mixture is then extracted to obtain an organic layer. The organic layer is then developed on a TLC plate to identify the azo compound component before silver sol is added for Raman spectroscopy. As can be seen, the existing method targets the aromatic secondary amine and is relatively cumbersome. In contrast, this invention first develops the sample solution on a TLC plate to identify the target component, then directly reacts it with the aromatic diazonium salt to form an azo compound. Silver sol is then added to the azo compound spot for surface-enhanced resonance Raman spectroscopy. Compared to the existing TLC-SERRS method, this method measures a different component structure and is more convenient and faster. It also requires less technical expertise from the testing personnel, making it suitable for rapid testing at the grassroots level.

[0021] This invention provides a novel method for detecting clenbuterol based on thin-layer chromatography and surface-enhanced resonance Raman spectroscopy. After separation of clenbuterol and terbutaline by thin-layer chromatography (TLC), the target spots are prepared into azo compounds, and then silver sol, a surface enhancer, is added. The surface-enhanced resonance Raman spectrum (SERRS) is then measured, establishing a new method for detecting clenbuterol in pig liver using a combined TLC and SERRS technique.

[0022] In this invention, salbutamol and terbutaline contain phenolic hydroxyl groups in their molecular structures. Under weakly alkaline conditions, they can undergo a diazo coupling reaction with aromatic diazonium salts to generate azo compounds, which lengthen the conjugated chains. Further interaction with surface enhancers causes a further redshift of the maximum absorption wavelength. When the wavelength of the excitation light coincides with or is close to the maximum absorption wavelength of the azo compound analyte, the resonance effect of the surface plasmons greatly enhances the detection signal, achieving highly sensitive and specific SERRS detection of these substances. This invention features convenient operation, simple equipment, high separation efficiency, good specificity, fast analysis speed, easily adjustable spectral parameters, and low cost.

[0023] The present invention has high specificity and high sensitivity for the detection of terbutaline and salbutamol. The detection limit (LOD) is the sample amount corresponding to the signal-to-noise ratio (S / N) of 3. The LOD of terbutaline is 2 ng and the LOD of salbutamol is 5 ng.

[0024] This invention is applicable to the detection of clenbuterol and terbutaline. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of TLC for salbutamol, terbutaline reference standards, and porcine liver samples in the examples, where 1: baseline; 2: silica gel 60F. 2543: Salbutamol reference standard; 4: Terbutaline reference standard; 5: Solvent front;

[0026] Figure 2 The TLC-SERRS image of the azo compound of terbutaline in the examples is shown, where: a: background of the surface enhancer silver sol on the silica gel plate; b: terbutaline solution spotting deposition amount of 6 μg; c: azo compound of terbutaline; d: azo compound of terbutaline with silver sol.

[0027] Figure 3 The TLC-SERRS diagrams of salbutamol and terbutaline in the examples are shown, where a: salbutamol, b: terbutaline;

[0028] Figure 4 The above are TLC images of pig liver samples in the examples, where: a': salbutamol reference standard; a: terbutaline reference standard; b-e: pig liver samples;

[0029] Figure 5 The above is a SERRS image of salbutamol in a pig liver sample from the examples, where: a': SERRS image of salbutamol reference standard; b'-e': spots in the pig liver sample that are identical to the salbutamol reference standard. f SERRS plot at the location;

[0030] Figure 6 The above is a SERRS diagram of terbutaline in a pig liver sample from the examples, where: a: SERRS diagram of terbutaline reference standard; b-e: spots in the pig liver sample that are identical to the terbutaline reference standard. f SERRS plot at the location;

[0031] Figure 7 The TLC-SERRS detection limit chromatogram for salbutamol in the examples is shown, where a: 1 ng; b: 2 ng; c: 5 ng; d: 10 ng; e: 5 μg;

[0032] Figure 8 The TLC-SERRS detection limit diagram for terbutaline in the examples is shown, where a: 1 ng; b: 2 ng; c: 5 ng; d: 10 ng; e: 5 μg. Detailed Implementation

[0033] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0034] Specific Implementation Method 1: This implementation method is based on thin-layer chromatography and surface-enhanced resonance Raman spectroscopy for the detection of clenbuterol, and it is implemented according to the following steps:

[0035] I. Thin-layer chromatography (TLC) was used to initially separate the clenbuterol component in the pig liver sample from the matrix. Then, the clenbuterol component in the pig liver sample was confirmed by spot identification under a UV lamp at 254 nm based on the clenbuterol component reference spot.

[0036] 2. Add an aromatic diazonium salt solution to the spot with the same shift value as the clenbuterol component reference standard spot to generate an azo compound, and then add the surface enhancer silver sol.

[0037] 3. After adding the surface enhancer silver sol as described above, the surface enhanced resonance Raman spectrum (SERRS) of the corresponding azo compound component spots is detected in situ under a 532 nm laser light source. If the SERRS spectrum of the pig liver sample solution at the same ratio shift value is the same as that of the control solution, it can be confirmed that the pig liver sample contains clenbuterol, thus completing the detection method.

[0038] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that, in step one, thin-layer chromatography is used to initially separate the clenbuterol component in the pig liver sample from the matrix. Then, under a UV lamp at 254 nm, the clenbuterol component in the pig liver sample is confirmed by spot identification based on the clenbuterol component reference standard spots. The specific process is as follows:

[0039] a. Preparation of reference solutions: Prepare reference solutions with a concentration of 10 μg·mL using methanol as the solvent. -1 Salbutamol solution with a concentration of 10 μg·mL -1 All terbutaline solutions were stored in sealed brown bottles for later use.

[0040] b. Using silicone 60F 254 An aluminum alloy thin-layer chromatography plate was used as the stationary phase, and a mixture of ethyl acetate, isopropanol, water, and glacial acetic acid was used as the developing solvent. The reference solution and the pig liver sample solution were spotted onto the same stationary phase at a volume of 4 μL. After saturation for 10 min, development was performed at 20°C. The sample was then removed and air-dried, completing the initial separation of the clenbuterol component from the matrix. The clenbuterol component spots in the pig liver sample were then examined under a UV lamp at 254 nm, based on the clenbuterol component reference spot. Other steps and parameters were the same as in Specific Implementation Method 1.

[0041] Specific Implementation Method 3: This implementation method differs from Specific Implementation Method 2 in that the preparation of the pig liver sample solution is as follows: Commercially available pig liver samples are washed, air-dried, and crushed. 10.00g is placed in a 50mL stoppered conical flask, methanol is added, and the mixture is sonicated at 40kHz for 5 minutes. After cooling to room temperature, the mixture is centrifuged at 4000r / min for 5 minutes, filtered, and the filtrate is transferred to a rotary evaporator and concentrated to 2mL to obtain the pig liver sample solution. This solution is then sealed and stored in a dark place for later use. Other steps and parameters are the same as in Specific Implementation Method 2.

[0042] Specific Implementation Method Four: This implementation method differs from Specific Implementation Method Two in that the volume ratio of ethyl acetate, isopropanol, water, and glacial acetic acid in the developing solvent is 5:2:1:2. Other steps and parameters are the same as in Specific Implementation Method Two.

[0043] Specific Implementation Method Five: This implementation method differs from Specific Implementation Method Two in that the preparation of the aromatic diazonium salt in step two is as follows: A dilute hydrochloric acid solution of 0.7 mg / mL p-aminothiophenol and a sodium nitrite solution of 0.03 g / mL are mixed at a volume ratio of 1:1. The mixture is then reacted in an ice-salt bath at 0℃~5℃ until a yellow aromatic diazonium salt is formed. The pH is then adjusted to 8~10 with a 50% (w / v) NaOH solution, and the mixture is stirred for 5 minutes. The remaining steps and parameters are the same as in Specific Implementation Method Two.

[0044] Specific Implementation Method Six: This implementation method differs from Specific Implementation Method Two in that the amount of aromatic diazonium salt solution used in step two is 4 μL; and the amount of surface-enhancing agent silver sol used is 8 μL. Other steps and parameters are the same as in Specific Implementation Method Two.

[0045] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Method One in that the preparation of the surface-enhancing silver sol in step two is as follows: Take 100 mL of a 0.37 mg / mL silver nitrate solution, heat it in a microwave oven until it just begins to boil, then remove it and add 2.6 mL of a 1% (w / v) sodium citrate solution. Mix well and continue heating for 2 minutes. After cooling, a grayish-green sol is obtained, which is the surface-enhancing silver sol. Other steps and parameters are the same as in Specific Implementation Method One.

[0046] The preparation of the surface-reinforcing silver sol in this embodiment ensures that the surface-reinforcing silver sol has good particle size, particle shape and stability, and has a good reinforcing effect.

[0047] Specific Implementation Method Eight: This implementation method differs from Specific Implementation Method Seven in that the detection conditions for surface-enhanced resonance Raman spectroscopy in step three are: microscope magnification of 10x, image pixel size of 540μm, and scanning range of 100~3300cm. -1 A 5.0 μm confocal pinhole aperture was used, with area scanning as the scanning method, a laser power of 10.0 mW, an exposure time of 0.05000 s, 30 scans, and OMNICxi software for data acquisition and analysis, automatic baseline correction, and Origin 6.1 software for plotting. Other steps and parameters were the same as in Specific Implementation Method Seven.

[0048] Specific Implementation Method Nine: This implementation method differs from Specific Implementation Method One in that the surface-enhanced resonance Raman spectrum of the pig liver sample solution at the same ratio shift value in step three is the same as that of the reference solution. That is, the peak shape and position of the azo compound component spots are consistent with those of the azo compound component spots generated by the clenbuterol reference standard, indicating that the pig liver sample contains salbutamol and terbutaline components. Other steps and parameters are the same as in Specific Implementation Method One.

[0049] The technical solutions provided by the present invention will be described in detail through the following embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0050] Example:

[0051] The method for detecting clenbuterol based on thin-layer chromatography and surface-enhanced resonance Raman spectroscopy is implemented according to the following steps:

[0052] I. Thin-layer chromatography (TLC) was used to initially separate the clenbuterol component in the pig liver sample from the matrix. Then, the clenbuterol component in the pig liver sample was confirmed by spot identification under a UV lamp at 254 nm based on the clenbuterol component reference spot.

[0053] 2. Add an aromatic diazonium salt solution to the spot with the same shift value as the clenbuterol component reference standard spot to generate an azo compound, and then add the surface enhancer silver sol.

[0054] 3. After adding the surface enhancer silver sol as described above, the surface enhanced resonance Raman spectrum (SERRS) of the corresponding azo compound component spots is detected in situ under a 532 nm laser light source. If the SERRS spectrum of the pig liver sample solution at the same ratio shift value is the same as that of the control solution, it can be confirmed that the pig liver sample contains clenbuterol, thus completing the detection method.

[0055] In step one of this embodiment, thin-layer chromatography is used to initially separate the clenbuterol component in the pig liver sample from the matrix. Then, under a UV lamp at 254 nm, the clenbuterol component in the pig liver sample is confirmed by spot identification based on the clenbuterol component reference standard spots. The specific process is as follows:

[0056] a. Preparation of reference solutions: Prepare reference solutions with a concentration of 10 μg·mL using methanol as the solvent. -1 Salbutamol solution with a concentration of 10 μg·mL -1 All terbutaline solutions were stored in sealed brown bottles for later use.

[0057] b. Using silicone 60F 254Aluminum alloy thin-layer plate was used as the stationary phase, and a mixture of ethyl acetate, isopropanol, water, and glacial acetic acid was used as the developing solvent. The reference solution and the pig liver sample solution were then spotted onto the same stationary phase at a volume of 4 μL. After saturation for 10 min, the sample was developed at 20 °C, removed, and air-dried to complete the initial separation of the clenbuterol component from the matrix. The clenbuterol component spots in the pig liver sample were then examined under a UV lamp at 254 nm based on the clenbuterol component reference spot.

[0058] Preparation of the pig liver sample solution described in this embodiment: Wash commercially available pig liver samples, air dry them naturally, and crush them. Then, take 10.00g and place them in a 50mL stoppered conical flask, add methanol, sonicate at a frequency of 40kHz for 5min, cool to room temperature, centrifuge at a speed of 4000r / min for 5min, filter, transfer the filtrate to a rotary evaporator and heat to concentrate to 2mL to obtain the pig liver sample solution, which should be sealed and stored in a dark place for later use.

[0059] In this embodiment, the volume ratio of ethyl acetate, isopropanol, water, and glacial acetic acid in the developing solvent is 5:2:1:2.

[0060] Preparation of the aromatic diazonium salt in step two of this embodiment: A dilute hydrochloric acid solution of 0.7 mg / mL p-aminothiophenol and a sodium nitrite solution of 0.03 g / mL are mixed at a volume ratio of 1:1. The mixture is then reacted in an ice-salt bath at 0℃~5℃ until a yellow aromatic diazonium salt is formed. The pH is then adjusted to 8~10 with a 50% NaOH solution and stirred for 5 min.

[0061] In step two of this embodiment, the amount of aromatic diazonium salt solution used is 4 μL; the amount of surface enhancer silver sol used is 8 μL.

[0062] Preparation of surface-enhancing silver sol in step two of this embodiment: Take 100 mL of silver nitrate solution with a concentration of 0.37 mg / mL, heat it in a microwave oven until it just boils, then take it out and add 2.6 mL of sodium citrate solution with a mass-volume concentration of 1%. After mixing, continue heating for 2 min and cool to obtain a gray-green sol, which is the surface-enhancing silver sol.

[0063] The detection conditions for surface-enhanced resonance Raman spectroscopy described in step three of this embodiment are: microscope magnification of 10x, image pixel size of 540μm, and scanning range of 100~3300 cm⁻¹. -1 The laser power was 10.0 mW, the exposure time was 0.05000 s, the number of scans was 30, the data acquisition and analysis software was OMNICxi, automatic baseline correction was performed, and the mapping software was Origin 6.1.

[0064] In step three of this embodiment, the surface-enhanced resonance Raman spectrum of the pig liver sample solution at the same ratio shift value is the same as that of the reference solution. That is, the peak shape and position of the azo compound component spot are consistent with the peak shape and position of the azo compound component spot generated by the clenbuterol component reference, indicating that the pig liver sample contains salbutamol and terbutaline components.

[0065] In this embodiment, Thermo Fisher Scientific, USA, DXRxi Raman Imaging Microscope was used.

[0066] The dynamic thin-layer chromatograph was used by Beijing Jinjianzhiguang Pharmaceutical Information Technology Center.

[0067] Made with 60F silicone from Merck, Germany. 254 Aluminum alloy thin sheet.

[0068] result:

[0069] The TLC diagrams of salbutamol and terbutaline reference standards and porcine liver samples are shown below. Figure 1 Where 1: baseline; 2: silicone 60F 254 Aluminum alloy thin-layer plate; 3: Salbutamol reference standard; 4: Terbutaline reference standard; 5: Solvent front; 6-7: Pig liver sample. This figure is a schematic diagram to illustrate the identification process of clenbuterol and terbutaline in this method using TLC. As shown in the figure, salbutamol and terbutaline reference standards each contain only one component, so only one component spot appears on the TLC plate: the salbutamol spot and the terbutaline spot. Pig liver sample, however, is a mixture containing several components, so several component spots will appear on the TLC plate. If it contains both salbutamol and terbutaline components, then at the same ratioshift value (R0) as the reference standard... f If similar spots appear at the location, namely salbutamol component spots and terbutaline component spots, it can be preliminarily determined that it may contain these two components.

[0070] TLC-SERRS plot of the azo compound of terbutaline, see Figure 2The figure shows the following: a: background of the surface-enhancing silver sol on the silica gel plate; b: terbutaline solution spotting deposition amount of 6 μg; c: terbutaline azo compound; d: terbutaline azo compound plus silver sol. As shown in the figure, when the terbutaline solution spotting deposition amount is 6 μg (b), there is no Raman spectral signal. Furthermore, the characteristic Raman spectral signal is also weak after terbutaline reacts with aromatic diazonium salts to form an azo compound (c). Therefore, simply using chemical derivatization to generate the azo compound is insufficient for the detection of terbutaline. Thus, silver sol is used as the active substrate added to the detection system of the generated azo molecules. Because the target spot is a colored compound with a long conjugated system and maximum absorption in the visible light region, when the micro Raman spectrometer uses a 532 nm laser as the light source, the Raman detection signal is greatly enhanced due to the resonance effect of the surface plasmons (d), achieving surface-enhanced resonance Raman spectroscopy (SERRS) detection of terbutaline and improving the sensitivity of terbutaline detection. Raman background signal of silver sol on thin film (a) at 3200 cm⁻¹ -1 There is an absorption peak with an intensity of around 2000 nearby, which is very different in shape and position from the SERRS peak of the terbutaline azo spot. Therefore, it has little impact on the SERRS detection of terbutaline and can be ignored.

[0071] TLC-SERRS chromatograms of salbutamol and terbutaline are shown in [reference needed]. Figure 3 Where a: salbutamol, b: terbutaline. As shown in the figure, the SERRS patterns of terbutaline and salbutamol are significantly different and clearly distinguishable, which can clearly reflect their structural fingerprint information, demonstrating the high selectivity of this method for the detection of terbutaline and salbutamol.

[0072] TLC image of pig liver sample, see Figure 4 Where: a': salbutamol reference standard; a: terbutaline reference standard; b-e: pig liver samples. As shown in the figure, the pig liver samples have the same R-value as the reference standards salbutamol and terbutaline. f Similar spots appeared in all areas, suggesting that the sample may contain these two components.

[0073] Serum SERRS plot of salbutamol in pig liver sample, see Figure 5 Where: a': SERRS plot of salbutamol reference standard; b'-e': spots in porcine liver samples that are identical to the salbutamol reference standard. f The SERRS plot at the location. To avoid false positive results from TLC, further confirmation using Raman spectroscopy is required. The spots in the porcine liver sample showed the same R value as the salbutamol component of the reference standard. fAfter adding diazonium salt and silver sol, the SERRS of the sample was measured. As shown in the figure, the peak shape and position of the SERRS of the pig liver sample were consistent with those of the azo compound component generated by the clenbuterol reference standard, indicating that the pig liver sample contained salbutamol.

[0074] See terbutaline SERRS image of pig liver sample. Figure 6 Where: a: SERRS plot of terbutaline reference standard; b-e: spots in porcine liver samples that are identical to the terbutaline reference standard. f The SERRS plot is shown. To avoid false positive results from TLC, further confirmation using Raman spectroscopy is needed. The spots in the porcine liver sample showed the same R value as the terbutaline control. f After adding aromatic diazonium salt and silver sol, the SERRS of the sample was measured. As shown in the figure, the peak shape and position of the SERRS of the pig liver sample were consistent with those of the azo compound component generated by the terbutaline reference standard, indicating that the pig liver sample contained terbutaline.

[0075] The TLC-SERRS detection limit chromatogram for salbutamol is shown in [link to chromatogram]. Figure 7 Where a: 1 ng; b: 2 ng; c: 5 ng; d: 10 ng; e: 5 μg; the detection limit chromatogram for terbutaline TLC-SERRS is shown in [reference needed]. Figure 8 Where a: 1 ng; b: 2 ng; c: 5 ng; d: 10 ng; e: 5 μg; it can be seen that the sample amount corresponding to a signal-to-noise ratio (S / N) of 3 is used as the limit of detection (LOD). The LOD of salbutamol is 5 ng, and the LOD of terbutaline is 2 ng.

Claims

1. A method for detecting clenbuterol based on thin layer chromatography and surface enhanced resonance Raman spectroscopy, characterized in that It is achieved by the following steps: I. The thin layer chromatography is used to preliminarily separate the clenbuterol components from the matrix in the pig liver sample, and then the spot confirmation of the clenbuterol components in the pig liver sample is carried out under the ultraviolet lamp 254 nm according to the spot of the clenbuterol component reference substance; II. The aromatic diazonium salt solution is added at the same Rf value of the spot of the clenbuterol component reference substance to generate azo compounds, and then the surface enhancer silver sol is added; III. After the surface enhancer silver sol is added in the above step, the surface enhanced resonance Raman spectrum of the corresponding azo compound component spot is detected in situ under the 532 nm laser light source. If the surface enhanced resonance Raman spectrum at the same Rf value of the pig liver sample solution is the same as that of the reference substance solution, it is confirmed that the pig liver sample contains the clenbuterol component, and the detection method is completed; The specific process of the spot confirmation of the clenbuterol components in the pig liver sample under the ultraviolet lamp 254 nm according to the spot of the clenbuterol component reference substance after the thin layer chromatography is preliminarily used to separate the clenbuterol components from the matrix in the pig liver sample is as follows: a. Preparation of the control solution: 10 μg·mL -1 of salbutamol solution and 10 μg·mL -1 of terbutaline solution were respectively prepared with methanol as the solvent, and were stored in a brown bottle for later use; b, Silica gel 60F 254 The aluminum alloy thin layer plate was used as the stationary phase, and a mixture of ethyl acetate, isopropyl alcohol, water and glacial acetic acid was used as the developing agent. Then, the control solution and the pig liver sample solution were spotted on the same stationary phase, with a sample size of 4 μL. After being saturated for 10 min, the sample was developed at 20°C. After being taken out and dried, the preliminary separation of the clenbuterol components and the matrix was completed. Then, under the ultraviolet lamp at 254 nm, the spots of the clenbuterol components in the pig liver sample were observed according to the spots of the clenbuterol component control. The preparation of the pig liver sample solution: The commercially available pig liver sample is washed, naturally dried and crushed, then 10.00 g is taken and placed in a 50 mL conical flask with a plug, methanol is added, ultrasonic is performed at an ultrasonic frequency of 40 kHz for 5 min, cooled to room temperature, centrifuged at a speed of 4000 r / min for 5 min, filtered, the filtrate is transferred to a rotary evaporator and concentrated to 2 mL, the pig liver sample solution is obtained, and stored in a dark place after sealing. The volume ratio of ethyl acetate, isopropyl alcohol, water and glacial acetic acid in the developing agent is 5:2:1:

2.

2. The method for detecting clenbuterol based on thin layer chromatography and surface enhanced resonance Raman spectroscopy according to claim 1, characterized in that The preparation of the aromatic diazonium salt in step II: the p-aminophenol hydrochloride solution with a concentration of 0.7 mg / mL and the sodium nitrite solution with a concentration of 0.03 g / mL are mixed at a volume ratio of 1:1, then reacted in an ice-salt bath at 0-5℃ until the yellow aromatic diazonium salt is generated, and then the pH is adjusted to 8-10 with a 50% NaOH solution, stirred for 5 min, and ready for use.

3. The method for detecting clenbuterol based on thin layer chromatography and surface enhanced resonance Raman spectroscopy according to claim 1, characterized in that The amount of the aromatic diazonium salt solution used in step II is 4 μL; the amount of the surface enhancer silver sol used is 8 μL.

4. The method for detecting clenbuterol based on thin layer chromatography and surface enhanced resonance Raman spectroscopy according to claim 1, characterized in that The preparation of the surface enhancer silver sol in step II: 100 mL of silver nitrate solution with a concentration of 0.37 mg / mL is heated to micro-boiling in a microwave oven, then 2.6 mL of 1% sodium citrate solution is added, mixed and heated for another 2 min, and then cooled to obtain a gray-green sol, which is the surface enhancer silver sol.

5. The method for detecting clenbuterol based on thin layer chromatography and surface enhanced resonance Raman spectroscopy according to claim 1, characterized in that The detection conditions of surface-enhanced resonance Raman spectroscopy in step three: microscope magnification was 10 times, image pixel was 540 μm, scanning range was 100-3300 cm -1 , 5.0 μm confocal pinhole diaphragm, scanning mode was area surface scanning, laser power was 10.0 mW, exposure time was 0.05000 s, scanning times were 30, data collection and analysis software was OMNICxi, automatic baseline correction, and drawing software was Origin 6.

1.

6. The method for detecting clenbuterol based on thin layer chromatography and surface enhanced resonance Raman spectroscopy according to claim 1, characterized in that In step III, if the surface enhanced resonance Raman spectrum at the same Rf value of the pig liver sample solution is the same as that of the reference substance solution, that is, the peak shape and peak position of the azo compound component spot are consistent with those of the azo compound component spot generated by the clenbuterol component reference substance, it indicates that the pig liver sample contains salbutamol and terbutaline components.

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