Pretreatment method for detecting levamisole in children health food

Abstract

The invention discloses a pretreatment method for detecting levamisole in health-care food for children, so as to obtain a to-be-detected solution which can be used for liquid chromatography-mass spectrometry detection. The method comprises the following specific steps: firstly, performing vacuum freeze drying on the children health food, and crushing to obtain a powder sample; then, ultrasonically dispersing the powder sample into ammonium bicarbonate-ammoniated methanol-acetonitrile mixed liquid, treating by a high-voltage pulse electric field, and centrifuging to take supernatant; and drying with nitrogen, fixing the volume by using a trichloroacetic acid acetonitrile aqueous solution, carrying out vortex mixing uniformly, and passing through a 0.22 [mu] m filter membrane to obtain a to-be-detected solution capable of being subjected to liquid chromatography-mass spectrometry. The pretreatment method is simple, can be directly used for liquid chromatography-mass spectrometry detection, and is high in detection accuracy.

Description

Technical Field

[0001] The invention relates to a levamisole detection method, in particular to a pretreatment method for detecting levamisole in children's health food, belonging to the technical field of analysis and detection. Background Art

[0002] Levamisole, also known as levamisole and anthelmintic, mainly exists in the form of hydrochloride. Levamisole hydrochloride is the levorotatory form of tetramisole, and its activity is about 1 to 2 times that of tetramisole (racemic form), but its toxic side effects are lower. Levamisole hydrochloride is used in animal breeding for antibacterial and anti-inflammatory, antiviral, and growth promotion, and plays an immunomodulatory role. Levamisole hydrochloride mainly enhances the body's immunity by increasing the activity of phagocytes and neutrophils, stimulating the production or secretion of lymphocytes, increasing the metabolic capacity of white blood cells and the activity of serum lysozyme, inducing the production of antibodies and the generation of complement. Levamisole hydrochloride is a broad-spectrum, highly effective imidazole anthelmintic commonly used in agricultural and animal husbandry production and veterinary clinics, and has a good effect on roundworms, hookworms, pinworms, and strongyloides.

[0003] When levamisole forms residues in animals due to unreasonable use, the concentration of levamisole in the human body will continue to increase with the accumulation of the food chain, thus causing harm to the human body. Studies have shown that levamisole has certain toxic side effects on the human digestive system, nervous system, cardiovascular system, etc., and can cause teratogenesis and mutagenesis.

[0004] Market research has found that some unscrupulous merchants illegally add levamisole to children's health foods. In addition, since many raw materials for enhanced children's health foods come from animal organs or dairy products, animal residues of levamisole enter the final children's health foods. In order to ensure the health of children, it is very necessary to test levamisole in children's health foods.

[0005] At present, the detection method for levamisole is mainly based on chromatography, such as liquid chromatography-ultraviolet detection and gas chromatography-nitrogen-phosphorus detection. These methods have low detection limits and are complicated and cumbersome to operate. Complex sample pretreatment has become the key to restricting the detection effect of liquid chromatography.

[0006] Nutritious gummies are a popular type of children's health food. Since nutritious gummies often contain sticky ingredients such as fat, protein, and polysaccharides, it is even more difficult to pretreat this type of health food. Therefore, it is very necessary to study the pretreatment method for the detection of levamisole in children's health food. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a pretreatment method for detecting levamisole in children's health food.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The pretreatment method for the detection of levamisole in children's health food is as follows:

[0010] (1) First, the children's health food is vacuum freeze-dried and crushed into 80-100 meshes to obtain a powder sample;

[0011] (2) then ultrasonically dispersing the powder sample in a mixture of ammonium bicarbonate-ammonia methanol-acetonitrile, treating it with a high-voltage pulse electric field, and centrifuging to obtain the supernatant;

[0012] (3) Dry with nitrogen gas, make up to volume with trichloroacetic acid acetonitrile aqueous solution, vortex mix, and filter through a 0.22 μm filter membrane to obtain a test solution that can be subjected to liquid chromatography-mass spectrometry.

[0013] Preferably, the children's health food is nutritious soft candy.

[0014] Preferably, in step (1), the process conditions for vacuum freeze drying are: first freezing at -30 to -40°C for 12 to 14 hours, evacuating to 10 to 20 Pa, heating to 30 to 35°C, and drying for 10 to 12 hours.

[0015] Preferably, in step (2), the ratio of ammonium bicarbonate, ammoniated methanol and acetonitrile in the ammonium bicarbonate-ammoniomethanol-acetonitrile mixture is 1 mg: 3-5 mL: 85-90 mL.

[0016] Preferably, in step (2), the process conditions of the high-voltage pulse electric field treatment are: electric field intensity 20-30 kV / cm, electric field frequency 2000-3000 Hz, pulse number 10-15, pulse width 4-5 μs, and treatment time 30-40 μs.

[0017] Preferably, in step (3), the mass concentration of trichloroacetic acid in the trichloroacetic acid-acetonitrile aqueous solution is 1-2%, and the volume concentration of acetonitrile is 10-12%.

[0018] Preferably, the test liquid is subjected to liquid chromatography-mass spectrometry to detect levamisole in children's health food. The specific method is: first, liquid chromatography-mass spectrometry is used to detect levamisole standard working solutions of different concentrations, and a standard curve is drawn with the levamisole standard working solution as the horizontal axis and the response value as the vertical axis; then liquid chromatography-mass spectrometry is used to detect the test liquid to obtain the response value, and the response value is substituted into the standard curve to calculate the levamisole concentration, and the levamisole content in the health food is obtained by conversion.

[0019] Further preferably, the liquid chromatography conditions are as follows:

[0020] Chromatographic column: XBridge BEH C18 2.5μm, 50*2.1mm;

[0021] Column temperature: 40°C;

[0022] Flow rate: 0.3 mL / min;

[0023] Mobile phase: (A) 0.1% trichloroacetic acid aqueous solution; (B) formic acid-acetonitrile solution, wherein the volume ratio of formic acid to acetonitrile is 0.7-0.8:100;

[0024] Injection volume: 10 μL.

[0025] More preferably, the liquid chromatography adopts gradient elution, and the specific procedure is shown in Table 1;

[0026] Table 1. Gradient elution program

[0027] Time (min) A(%,volume) B (%, volume) 0 80 20 5 80 20

[0028] Further preferably, the mass spectrometry conditions are as follows:

[0029] Ionization mode: electrospray ionization, positive ion;

[0030] Capillary voltage: 3kV;

[0031] Cone voltage: 40V;

[0032] Drying gas temperature: 170℃;

[0033] Drying gas flow rate: 18L / min;

[0034] Sheath gas temperature: 360℃;

[0035] Sheath gas flow rate: 14 L / min;

[0036] Electron multiplier voltage: 650V;

[0037] Scanning mode: multiple ion reaction monitoring;

[0038] Ion selection parameters were: parent ion 205.1, product ions 91.1 and 178.1, collision energies were 38 eV and 20 eV, and dwell time was 0.05 min.

[0039] Further preferably, the levamisole standard working solution is prepared using the levamisole standard substance, and the concentrations are: 0.0 ng / mL, 1.0 ng / mL, 2.0 ng / mL, 5.0 ng / mL, 10.0 ng / mL, and 20 ng / mL.

[0040] Further preferably, the levamisole content in the health food is calculated by the following formula:

[0041] X = C × V / M × 1000 / 1000,

[0042] Wherein, X is the content of levamisole in the health food, μg / kg; C is the concentration of levamisole in the test solution (obtained by substituting into the standard curve), ng / mL; M is the mass of the health food, g; V is the fixed volume, mL.

[0043] Beneficial effects of the present invention:

[0044] The present invention provides a pretreatment method for detecting levamisole in children's health foods such as nutritious soft candies, thereby obtaining a test liquid that can be used for liquid chromatography-mass spectrometry detection. The specific steps of the present invention are as follows: first, the children's health food is vacuum freeze-dried and crushed to obtain a powder sample; then the powder sample is ultrasonically dispersed in a mixed solution of ammonium bicarbonate-ammoniation methanol-acetonitrile, treated with a high-voltage pulse electric field, and centrifuged to obtain a supernatant; nitrogen is blown dry, and the volume is constant using a trichloroacetic acid acetonitrile aqueous solution, and after vortex mixing, it is filtered through a 0.22μm filter membrane to obtain a test liquid that can be subjected to liquid chromatography-mass spectrometry. The pretreatment method of the present invention is simple, can be directly used for liquid chromatography-mass spectrometry detection, and has high detection accuracy.

[0045] Children's health foods such as soft candies contain protein, fat, polysaccharides, etc., which wrap levamisole and often have high viscosity, making it difficult to detect the levamisole content. The present invention first performs vacuum freeze drying to reduce the difficulty of crushing, and after crushing into a powder sample, the levamisole component is fully released by selecting a specific solvent composition and combining high-voltage pulse electric field treatment, so as to be used for subsequent detection.

[0046] The present invention adopts a mixed solution of ammonium bicarbonate-ammoniation methanol-acetonitrile, and a specific ratio of ammonium bicarbonate, ammonia methanol and acetonitrile is used to make the powder sample fully dispersed, which is beneficial to the high-voltage pulse electric field treatment effect. Finally, the trichloroacetic acid acetonitrile aqueous solution is used to make the volume constant, which is also beneficial to further improve the subsequent liquid chromatography-mass spectrometry detection effect. DETAILED DESCRIPTION

[0047] The present invention is further described below in conjunction with embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its contents.

[0048] Embodiment 1:

[0049] The pretreatment method for the detection of levamisole in children's health food is as follows:

[0050] (1) First, the children's health food is vacuum freeze-dried and crushed into 80 meshes to obtain a powder sample;

[0051] (2) then ultrasonically dispersing the powder sample in a mixture of ammonium bicarbonate-ammonia methanol-acetonitrile, treating it with a high-voltage pulse electric field, and centrifuging to obtain the supernatant;

[0052] (3) Dry with nitrogen gas, make up to volume with trichloroacetic acid acetonitrile aqueous solution, vortex mix, and filter through a 0.22 μm filter membrane to obtain a test solution that can be subjected to liquid chromatography-mass spectrometry.

[0053] The children's health food is a certain brand of children's nutritional gummies.

[0054] In step (1), the process conditions for vacuum freeze drying are: first freezing at -30°C for 12 hours, evacuating to 10 Pa, heating to 30°C, and drying for 10 hours.

[0055] In step (2), the ratio of ammonium bicarbonate, ammoniated methanol and acetonitrile in the ammonium bicarbonate-ammoniomethanol-acetonitrile mixture is 1 mg: 3 mL: 85 mL.

[0056] In step (2), the process conditions of the high-voltage pulse electric field treatment are: electric field intensity 20 kV / cm, electric field frequency 2000 Hz, pulse number 10, pulse width 4 μs, and treatment time 30 μs.

[0057] In step (3), the mass concentration of trichloroacetic acid in the trichloroacetic acid-acetonitrile aqueous solution is 1%, and the volume concentration of acetonitrile is 10%.

[0058] Embodiment 2:

[0059] The pretreatment method for the detection of levamisole in children's health food is as follows:

[0060] (1) First, the children's health food is vacuum freeze-dried and crushed into 100 meshes to obtain a powder sample;

[0061] (2) then ultrasonically dispersing the powder sample in a mixture of ammonium bicarbonate-ammonia methanol-acetonitrile, treating it with a high-voltage pulse electric field, and centrifuging to obtain the supernatant;

[0062] (3) Dry with nitrogen gas, make up to volume with trichloroacetic acid acetonitrile aqueous solution, vortex mix, and filter through a 0.22 μm filter membrane to obtain a test solution that can be subjected to liquid chromatography-mass spectrometry.

[0063] The children's health food is a certain brand of children's nutritional gummies.

[0064] In step (1), the process conditions for vacuum freeze drying are: first freezing at -40°C for 14 hours, evacuating to 20 Pa, heating to 35°C, and drying for 12 hours.

[0065] In step (2), the ratio of ammonium bicarbonate, ammoniated methanol and acetonitrile in the ammonium bicarbonate-ammoniomethanol-acetonitrile mixture is 1 mg: 5 mL: 90 mL.

[0066] In step (2), the process conditions of the high-voltage pulse electric field treatment are: electric field intensity 30 kV / cm, electric field frequency 3000 Hz, pulse number 15, pulse width 5 μs, and treatment time 40 μs.

[0067] In step (3), the mass concentration of trichloroacetic acid in the trichloroacetic acid-acetonitrile aqueous solution is 2%, and the volume concentration of acetonitrile is 12%.

[0068] Embodiment 3:

[0069] The pretreatment method for the detection of levamisole in children's health food is as follows:

[0070] (1) First, the children's health food is vacuum freeze-dried and crushed into 90 meshes to obtain a powder sample;

[0071] (2) then ultrasonically dispersing the powder sample in a mixture of ammonium bicarbonate-ammonia methanol-acetonitrile, treating it with a high-voltage pulse electric field, and centrifuging to obtain the supernatant;

[0072] (3) Dry with nitrogen gas, make up to volume with trichloroacetic acid acetonitrile aqueous solution, vortex mix, and filter through a 0.22 μm filter membrane to obtain a test solution that can be subjected to liquid chromatography-mass spectrometry.

[0073] The children's health food is a certain brand of children's nutritional gummies.

[0074] In step (1), the process conditions for vacuum freeze drying are: first freezing at -35°C for 13 hours, evacuating to 15 Pa, heating to 33°C, and drying for 11 hours.

[0075] In step (2), the ratio of ammonium bicarbonate, ammoniated methanol and acetonitrile in the ammonium bicarbonate-ammoniomethanol-acetonitrile mixture is 1 mg:4 mL:88 mL.

[0076] In step (2), the process conditions of the high-voltage pulse electric field treatment are: electric field intensity 25 kV / cm, electric field frequency 2500 Hz, pulse number 12, pulse width 4 μs, and treatment time 35 μs.

[0077] In step (3), the mass concentration of trichloroacetic acid in the trichloroacetic acid-acetonitrile aqueous solution is 1.5%, and the volume concentration of acetonitrile is 11%.

[0078] Application examples:

[0079] The test solution obtained in Example 1 was used for the detection of levamisole in children's health food by liquid chromatography-mass spectrometry. The specific method is as follows: firstly, different concentrations of levamisole standard working solutions were detected by liquid chromatography-mass spectrometry, and a standard curve was drawn with the levamisole standard working solution as the horizontal axis and the response value as the vertical axis; then, the test solution was detected by liquid chromatography-mass spectrometry to obtain the response value, and the response value was substituted into the standard curve to calculate the levamisole concentration, and the levamisole content in the health food was obtained by conversion.

[0080] The liquid chromatography conditions were as follows:

[0081] Chromatographic column: XBridge BEH C18 2.5μm, 50*2.1mm;

[0082] Column temperature: 40°C;

[0083] Flow rate: 0.3 mL / min;

[0084] Mobile phase: (A) 0.1% trichloroacetic acid aqueous solution; (B) formic acid-acetonitrile solution, wherein the volume ratio of formic acid to acetonitrile is 0.7-0.8:100;

[0085] Injection volume: 10 μL.

[0086] Liquid chromatography used gradient elution, and the specific procedure is shown in Table 1;

[0087] Table 1. Gradient elution program

[0088] Time (min) A(%,volume) B (%, volume) 0 80 20 5 80 20

[0089] The mass spectrometry conditions were as follows:

[0090] Ionization mode: electrospray ionization, positive ion;

[0091] Capillary voltage: 3kV;

[0092] Cone voltage: 40V;

[0093] Drying gas temperature: 170℃;

[0094] Drying gas flow rate: 18L / min;

[0095] Sheath gas temperature: 360℃;

[0096] Sheath gas flow rate: 14 L / min;

[0097] Electron multiplier voltage: 650V;

[0098] Scanning mode: multiple ion reaction monitoring;

[0099] Ion selection parameters were: parent ion 205.1, product ions 91.1 and 178.1, collision energies were 38 eV and 20 eV, and dwell time was 0.05 min.

[0100] Levamisole standard working solution was prepared using levamisole standard with concentrations of 0.0 ng / mL, 1.0 ng / mL, 2.0 ng / mL, 5.0 ng / mL, 10.0 ng / mL, and 20 ng / mL.

[0101] The levamisole content in health foods is calculated using the following formula:

[0102] X = C × V / M × 1000 / 1000,

[0103] Wherein, X is the content of levamisole in the health food, μg / kg; C is the concentration of levamisole in the test solution (obtained by substituting into the standard curve), ng / mL; M is the mass of the health food, g; V is the fixed volume, mL.

[0104] Through the analysis of the results, the regression equation is as follows:

[0105] Y = 0.8133x - 0.0049,

[0106] Correlation coefficient R: 0.9999;

[0107] Detection limit: 0.01 μg / kg;

[0108] Limit of quantification: 0.02μg / kg.

[0109] Although the above describes the specific implementation methods of the present invention, it does not limit the scope of protection of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. A pretreatment method for detecting levamisole in children's health food, characterized in that: The specific steps are as follows: (1) First, the children's health food is vacuum freeze-dried and crushed into 80-100 meshes to obtain a powder sample; (2) then ultrasonically dispersing the powder sample in a mixture of ammonium bicarbonate-ammoniamethanol-acetonitrile, treating it with a high-voltage pulse electric field, and centrifuging to obtain the supernatant; (3) Dry with nitrogen gas, make up to volume with trichloroacetic acid acetonitrile aqueous solution, vortex mix, and filter through a 0.22 μm filter membrane to obtain a test solution that can be subjected to liquid chromatography-mass spectrometry.

2. The pretreatment method according to claim 1, characterized in that In step (1), the process conditions of vacuum freeze drying are: first freezing at -30 to -40°C for 12 to 14 hours, evacuating to 10 to 20 Pa, heating to 30 to 35°C, and drying for 10 to 12 hours.

3. The pretreatment method according to claim 1, characterized in that: In step (2), the ratio of ammonium bicarbonate, ammoniated methanol and acetonitrile in the ammonium bicarbonate-ammoniomethanol-acetonitrile mixture is 1 mg: 3-5 mL: 85-90 mL.

4. The pretreatment method according to claim 1, characterized in that: In step (2), the process conditions of the high-voltage pulse electric field treatment are: electric field intensity 20-30 kV / cm, electric field frequency 2000-3000 Hz, pulse number 10-15, pulse width 4-5 μs, and treatment time 30-40 μs.

5. The pretreatment method according to claim 1, characterized in that: In step (3), the mass concentration of trichloroacetic acid contained in the trichloroacetic acid-acetonitrile aqueous solution is 1-2%, and the volume concentration of acetonitrile is 10-12%.

6. The pretreatment method according to claim 1, characterized in that: The liquid to be tested is subjected to liquid chromatography-mass spectrometry to detect levamisole in children's health food. The specific method is: firstly, the liquid chromatography-mass spectrometry is used to detect the standard working solutions of levamisole with different concentrations, and a standard curve is drawn with the standard working solution of levamisole as the horizontal axis and the response value as the vertical axis; then, the liquid chromatography-mass spectrometry is used to detect the liquid to be tested, and the response value is obtained, and the response value is substituted into the standard curve to calculate the levamisole concentration, and the levamisole content in the health food is obtained by conversion.

7. The pretreatment method according to claim 6, characterized in that: The liquid chromatography conditions were as follows: Chromatographic column: XBridge BEH C18 2.5μm, 50*2.1mm; Column temperature: 40°C; Flow rate: 0.3 mL / min; Mobile phase: (A) 0.1% trichloroacetic acid aqueous solution; (B) formic acid-acetonitrile solution, wherein the volume ratio of formic acid to acetonitrile is 0.7-0.8:100; Injection volume: 10 μL.

8. The pretreatment method according to claim 7, characterized in that: Liquid chromatography used gradient elution, and the specific procedure is shown in Table 1; Table 1. Gradient elution program 。 9. The pretreatment method according to claim 6, characterized in that: The mass spectrometry conditions were as follows: Ionization mode: electrospray ionization, positive ion; Capillary voltage: 3kV; Cone voltage: 40V; Drying gas temperature: 170℃; Drying gas flow rate: 18L / min; Sheath gas temperature: 360℃; Sheath gas flow rate: 14 L / min; Electron multiplier voltage: 650V; Scanning mode: multiple ion reaction monitoring; Ion selection parameters were: parent ion 205.1, product ions 91.1 and 178.1, collision energies were 38 eV and 20 eV, and dwell time was 0.05 min.

10. The pretreatment method according to claim 6, characterized in that: The levamisole content in health foods is calculated using the following formula: X = C × V / M × 1000 / 1000, Wherein, X is the content of levamisole in the health food, μg / kg; C is the concentration of levamisole in the test solution (obtained by substituting into the standard curve), ng / mL; M is the mass of the health food, g; V is the fixed volume, mL.