Method for rapidly detecting content of levamisole in children health food

Abstract

The invention discloses a method for rapidly detecting the content of levamisole in children health food. The method comprises the following steps: firstly, extracting powdery health food to prepare a to-be-detected solution; detecting the levamisole standard working solutions with different concentrations by adopting a liquid chromatography-mass spectrometry method, and drawing a standard curve taking the levamisole standard working solutions as abscissas and the response values as ordinates; and finally, detecting the liquid to be detected by adopting a liquid chromatography-mass spectrometry method to obtain a response value, substituting the response value into the standard curve, calculating to obtain the concentration of the levamisole, and converting to obtain the content of the levamisole in the health food. According to the method, the content of the levamisole in the child health food is rapidly detected through the liquid chromatography-mass spectrometry, the detection limit is low, and the accuracy is high.

Description

Technical Field

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

[0002] Levamisole is mainly used to treat gastrointestinal nematodes, gastric nematodes, lung nematodes and swine kidney nematodes in livestock and poultry. The anthelmintic mechanism of levamisole is mainly to inhibit the activity of succinate dehydrogenase and cholinesterase in the muscles of the worm body, so that the energy metabolism of the worm body is blocked and the muscles are paralyzed, thus achieving the anthelmintic effect.

[0003] However, levamisole is easily absorbed and metabolized by the body when used. Irrational use of levamisole will cause animal residues. The main potential harm of residual levamisole to the human body is teratogenicity and mutagenicity. In order to ensure the safety of animal-derived products, many countries or international organizations have established strict limit standards, generally 10 to 100 μg / kg. my country stipulates that the maximum residue limit (MRL) of levamisole in cattle, sheep, pigs and poultry products is 10 μg / kg.

[0004] Market research has found that levamisole may be present in children's health foods. In addition to illegal additions by unscrupulous businesses, the main source of levamisole in children's health foods is the residues of anthelmintics and immunostimulants used in animal production and veterinary clinics. In order to ensure the health of children, it is very necessary to test levamisole in children's health foods.

[0005] The traditional detection method of levamisole is titration, which has large errors due to human factors, large matrix interference, low accuracy, certain limitations on the detection limit, and cannot meet the needs of trace detection. High performance liquid chromatography, liquid chromatography tandem mass spectrometry, gas chromatography combined with mass spectrometry and high performance liquid chromatography tandem mass spectrometry have the advantages of high sensitivity and accuracy and a wide range of applications. In recent years, people have used liquid chromatography-ultraviolet detection and gas chromatography-nitrogen-phosphorus detection to detect levamisole. These methods have low detection limits, but the detection time is long and the operation is cumbersome. In addition, as the regulations on the residue limit of levamisole in various countries around the world become more and more stringent, ordinary chromatographic detection methods have gradually failed to fully meet the detection requirements of levamisole residues. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for rapidly detecting the content of levamisole in children's health food.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for rapidly detecting the content of levamisole in children's health food comprises the following steps: firstly extracting powdered health food to prepare a test solution; then using liquid chromatography-mass spectrometry to detect levamisole standard working solutions of different concentrations, and drawing a standard curve with the levamisole standard working solution as the abscissa and the response value as the ordinate; finally using liquid chromatography-mass spectrometry to detect the test solution to obtain the response value, substituting the response value into the standard curve, calculating the levamisole concentration, and converting it to obtain the levamisole content in the health food.

[0009] Preferably, the health food includes but is not limited to: protein powder and vitamin powder.

[0010] Preferably, the test solution is prepared by the following method: first, uniformly mix the powdered health food with sodium hydroxide solution, acetonitrile and ethyl acetate, perform ultrasonic oscillation treatment for the first time, centrifuge to obtain the supernatant, then mix the supernatant with the formic acid solution, perform ultrasonic oscillation treatment for the second time, centrifuge to obtain the lower aqueous phase, pass through an MCX solid phase extraction column, elute, blow dry with nitrogen, constant volume, and pass through a 0.22 μm filter membrane to obtain the test solution; wherein the dosage ratio of the powdered health food, sodium hydroxide solution, acetonitrile, ethyl acetate and formic acid solution is 1 g: 0.1-0.2 mL: 2-3 mL: 20-22 mL: 1-2 mL, and the concentrations of the sodium hydroxide solution and the formic acid solution are 8-10 mol / L and 0.1-0.2 mol / L respectively.

[0011] More preferably, diphenhydramine hydrochloride is added to the powdered health food as an internal standard.

[0012] More preferably, the process conditions for the first ultrasonic oscillation treatment are: 500-600W ultrasonic oscillation treatment for 5-8 minutes; the process conditions for the second ultrasonic oscillation treatment are: 300-400W ultrasonic oscillation treatment for 8-10 minutes.

[0013] More preferably, the MCX solid phase extraction column is activated sequentially using 3 mL of acetonitrile, 2 mL of ultrapure water, and 1 mL of 0.2 mol / L hydrochloric acid solution.

[0014] Further preferably, elution is performed using a mixed solution obtained by uniformly mixing concentrated ammonia water with a mass concentration of 28%, acetonitrile and ultrapure water in a volume ratio of 1:2-3:8-10; finally, the eluate is fixed to volume using a formic acid-acetonitrile aqueous solution; the volume concentrations of formic acid and acetonitrile in the formic acid-acetonitrile aqueous solution are 1-2% and 8-10%, respectively.

[0015] Preferably, the liquid chromatography conditions are as follows:

[0016] Chromatographic column: XBridge BEH C18 2.5μm, 50*2.1mm;

[0017] Column temperature: 40°C;

[0018] Flow rate: 0.2 mL / min;

[0019] Mobile phase: (A) 0.2% formic acid aqueous solution; (B) ammonium acetate-formic acid-acetonitrile solution, wherein the ratio of ammonium acetate, formic acid and acetonitrile is 0.1 g: 0.4 mL: 100 mL;

[0020] Injection volume: 10 μL.

[0021] Further preferably, the liquid chromatography adopts gradient elution, and the specific procedure is shown in Table 1;

[0022] Table 1. Gradient elution program

[0023] Time (min) A(%,volume) B (%, volume) 0 95 5 1 85 15 2 30 70 4 10 90 6 95 5

[0024] Preferably, the mass spectrometry conditions are as follows:

[0025] Ionization mode: electrospray ionization, positive ion;

[0026] Capillary voltage: 2.5kV;

[0027] Cone voltage: 39V;

[0028] Drying gas temperature: 150℃;

[0029] Drying gas flow rate: 15L / min;

[0030] Sheath gas temperature: 350℃;

[0031] Sheath gas flow rate: 13 L / min;

[0032] Electron multiplier voltage: 650V;

[0033] Scanning mode: multiple ion reaction monitoring;

[0034] Ion selection parameters were: parent ion 205.1, product ions 91.1 and 178.1, collision energies were 36 eV and 21 eV, and dwell time was 0.05 min.

[0035] 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, 4.0 ng / mL, 6.0 ng / mL, and 10 ng / mL.

[0036] Preferably, the levamisole content in the health food is calculated by the following formula:

[0037] X = C × V / M × 1000 / 1000,

[0038] 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.

[0039] Beneficial effects of the present invention:

[0040] The present invention is aimed at powdered children's health foods such as protein powder and vitamin powder, and proposes a method for quickly detecting the content of levamisole in children's health foods. First, the powdered health foods are extracted to prepare a test solution; then, liquid chromatography-mass spectrometry is used to detect standard working solutions of levamisole with different concentrations, and a standard curve with the levamisole standard working solution as the horizontal coordinate and the response value as the vertical coordinate is drawn; finally, liquid chromatography-mass spectrometry is used to detect the test solution, and the response value is substituted into the standard curve to calculate the levamisole concentration, and the levamisole content in the health food is converted. The present invention realizes the rapid detection of the levamisole content in children's health foods by liquid chromatography-mass spectrometry, with low detection limit and high accuracy.

[0041] The technical key of the present invention lies in the screening of liquid chromatography conditions and mass spectrometry conditions. The mobile phase of the liquid chromatography (A) is a formic acid aqueous solution with a volume concentration of 0.2%; and (B) is an ammonium acetate-formic acid-acetonitrile solution. Combined with a specific gradient elution program, the detection of the levamisole content in the test liquid is achieved.

[0042] The mass spectrometry ion selection parameters of the present invention are: parent ion 205.1, daughter ions 91.1 and 178.1, collision energies 36eV and 21eV, and a residence time of 0.05min. Combined with the above-mentioned liquid chromatography conditions, rapid detection of levamisole content in children's health food is achieved. DETAILED DESCRIPTION

[0043] 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.

[0044] Reference Example 1:

[0045] A certain brand of children's health protein powder was extracted to prepare a test solution. The specific method is as follows: first, the protein powder is evenly mixed with sodium hydroxide solution, acetonitrile and ethyl acetate, subjected to ultrasonic oscillation for the first time, and the supernatant is centrifuged and mixed with formic acid solution, subjected to ultrasonic oscillation for the second time, and the lower aqueous phase is centrifuged and passed through an MCX solid phase extraction column, eluted, dried with nitrogen, fixed to volume, and passed through a 0.22 μm filter membrane to obtain the test solution; wherein, the amount ratio of protein powder, sodium hydroxide solution, acetonitrile, ethyl acetate and formic acid solution is 1 g: 0.1 mL: 2 mL: 20 mL: 1 mL, and the concentrations of sodium hydroxide solution and formic acid solution are 8 mol / L and 0.1 mol / L respectively.

[0046] The process conditions of the first ultrasonic oscillation treatment are: 500W ultrasonic oscillation treatment for 5 minutes; the process conditions of the second ultrasonic oscillation treatment are: 300W ultrasonic oscillation treatment for 8 minutes.

[0047] The MCX solid phase extraction column was activated with 3 mL of acetonitrile, 2 mL of ultrapure water, and 1 mL of 0.2 mol / L hydrochloric acid solution in sequence.

[0048] The method comprises eluting the eluent by uniformly mixing concentrated ammonia water with a mass concentration of 28%, acetonitrile and ultrapure water in a volume ratio of 1:2:8; finally, the eluent is fixed to volume with a formic acid-acetonitrile aqueous solution; the volume concentrations of formic acid and acetonitrile in the formic acid-acetonitrile aqueous solution are 1% and 8%, respectively.

[0049] Reference Example 2:

[0050] A certain brand of children's health protein powder was extracted to prepare a test solution. The specific method is as follows: first, the protein powder is evenly mixed with sodium hydroxide solution, acetonitrile and ethyl acetate, subjected to ultrasonic oscillation for the first time, and the supernatant is centrifuged and mixed with formic acid solution, subjected to ultrasonic oscillation for the second time, and the lower aqueous phase is centrifuged and passed through an MCX solid phase extraction column, eluted, dried with nitrogen, fixed to volume, and passed through a 0.22 μm filter membrane to obtain the test solution; wherein, the amount ratio of protein powder, sodium hydroxide solution, acetonitrile, ethyl acetate and formic acid solution is 1 g: 0.2 mL: 3 mL: 22 mL: 2 mL, and the concentrations of sodium hydroxide solution and formic acid solution are 10 mol / L and 0.2 mol / L respectively.

[0051] The process conditions of the first ultrasonic oscillation treatment are: 600W ultrasonic oscillation treatment for 8 minutes; the process conditions of the second ultrasonic oscillation treatment are: 400W ultrasonic oscillation treatment for 10 minutes.

[0052] The MCX solid phase extraction column was activated with 3 mL of acetonitrile, 2 mL of ultrapure water, and 1 mL of 0.2 mol / L hydrochloric acid solution in sequence.

[0053] The method comprises eluting the eluent by uniformly mixing concentrated ammonia water with a mass concentration of 28%, acetonitrile and ultrapure water in a volume ratio of 1:3:10; finally, the eluent is fixed to volume with a formic acid-acetonitrile aqueous solution; the volume concentrations of formic acid and acetonitrile in the formic acid-acetonitrile aqueous solution are 2% and 10%, respectively.

[0054] Reference Example 3:

[0055] A certain brand of children's health protein powder was extracted to prepare a test solution. The specific method is as follows: first, the protein powder is evenly mixed with sodium hydroxide solution, acetonitrile and ethyl acetate, subjected to ultrasonic oscillation for the first time, and the supernatant is centrifuged and mixed with formic acid solution, subjected to ultrasonic oscillation for the second time, and the lower aqueous phase is centrifuged and passed through an MCX solid phase extraction column, eluted, dried with nitrogen, fixed to volume, and passed through a 0.22 μm filter membrane to obtain the test solution; wherein, the dosage ratio of protein powder, sodium hydroxide solution, acetonitrile, ethyl acetate and formic acid solution is 1 g: 0.15 mL: 2.5 mL: 21 mL: 1.5 mL, and the concentrations of sodium hydroxide solution and formic acid solution are 9 mol / L and 0.15 mol / L respectively.

[0056] The process conditions of the first ultrasonic oscillation treatment are: 600W ultrasonic oscillation treatment for 6 minutes; the process conditions of the second ultrasonic oscillation treatment are: 400W ultrasonic oscillation treatment for 9 minutes.

[0057] The MCX solid phase extraction column was activated with 3 mL of acetonitrile, 2 mL of ultrapure water, and 1 mL of 0.2 mol / L hydrochloric acid solution in sequence.

[0058] The method comprises eluting the eluent by uniformly mixing concentrated ammonia water with a mass concentration of 28%, acetonitrile and ultrapure water in a volume ratio of 1:2.5:9; finally, the eluent is fixed to volume with a formic acid-acetonitrile aqueous solution; the volume concentrations of formic acid and acetonitrile in the formic acid-acetonitrile aqueous solution are 1.5% and 9%, respectively.

[0059] Example:

[0060] A method for quickly detecting the content of levamisole in children's health food, using liquid chromatography-mass spectrometry to detect different concentrations of levamisole standard working solutions, drawing a standard curve with the levamisole standard working solution as the horizontal coordinate and the response value as the vertical coordinate; using liquid chromatography-mass spectrometry to detect the test solution obtained in reference example 1, obtaining the response value, substituting the response value into the standard curve, calculating the levamisole concentration, and converting the levamisole content in the health food. Diphenhydramine hydrochloride is also added to the powdered health food as an internal standard, and the internal standard concentration in the test solution is 2.0 ng / mL.

[0061] The liquid chromatography conditions were as follows:

[0062] Chromatographic column: XBridge BEH C18 2.5μm, 50*2.1mm;

[0063] Column temperature: 40°C;

[0064] Flow rate: 0.2 mL / min;

[0065] Mobile phase: (A) 0.2% formic acid aqueous solution; (B) ammonium acetate-formic acid-acetonitrile solution, wherein the ratio of ammonium acetate, formic acid and acetonitrile is 0.1 g: 0.4 mL: 100 mL;

[0066] Injection volume: 10 μL.

[0067] Further preferably, the liquid chromatography adopts gradient elution, and the specific procedure is shown in Table 1;

[0068] Table 1. Gradient elution program

[0069]

[0070]

[0071] Preferably, the mass spectrometry conditions are as follows:

[0072] Ionization mode: electrospray ionization, positive ion;

[0073] Capillary voltage: 2.5kV;

[0074] Cone voltage: 39V;

[0075] Drying gas temperature: 150℃;

[0076] Drying gas flow rate: 15L / min;

[0077] Sheath gas temperature: 350℃;

[0078] Sheath gas flow rate: 13 L / min;

[0079] Electron multiplier voltage: 650V;

[0080] Scanning mode: multiple ion reaction monitoring;

[0081] Ion selection parameters were: parent ion 205.1, product ions 91.1 and 178.1, collision energies were 36 eV and 21 eV, and dwell time was 0.05 min.

[0082] Levamisole standard working solution was prepared using levamisole standard with concentrations of 0.0 ng / mL, 1.0 ng / mL, 2.0 ng / mL, 4.0 ng / mL, 6.0 ng / mL, and 10 ng / mL.

[0083] The levamisole content in health foods is calculated using the following formula:

[0084] X = C × V / M × 1000 / 1000,

[0085] 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.

[0086] Through the analysis of the results, the regression equation is as follows:

[0087] Y = 0.8135x - 0.0051,

[0088] Correlation coefficient R: 0.9999;

[0089] Detection limit: 0.01 μg / kg;

[0090] Limit of quantification: 0.03μg / kg.

[0091] 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 method for rapidly detecting the content of levamisole in children's health food, characterized in that: First, the powdered health food is extracted to prepare a test solution; then, the levamisole standard working solutions of different concentrations are detected by liquid chromatography-mass spectrometry, and a standard curve is drawn with the levamisole standard working solution as the horizontal axis and the response value as the vertical axis; finally, the test solution is detected by liquid chromatography-mass spectrometry 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 converted.

2. The method according to claim 1, characterized in that The test solution is prepared by the following method: firstly, powdered health food is uniformly mixed with sodium hydroxide solution, acetonitrile and ethyl acetate, subjected to ultrasonic oscillation treatment for the first time, centrifuged to obtain supernatant, then the supernatant is mixed with formic acid solution, subjected to ultrasonic oscillation treatment for the second time, centrifuged to obtain lower aqueous phase, passed through MCX solid phase extraction column, eluted, dried with nitrogen, constant volume, and passed through 0.22 μm filter membrane to obtain the test solution; wherein, the dosage ratio of powdered health food, sodium hydroxide solution, acetonitrile, ethyl acetate and formic acid solution is 1 g: 0.1-0.2 mL: 2-3 mL: 20-22 mL: 1-2 mL, and the concentrations of sodium hydroxide solution and formic acid solution are 8-10 mol / L and 0.1-0.2 mol / L respectively.

3. The method according to claim 2, characterized in that The process conditions of the first ultrasonic oscillation treatment are: 500-600W ultrasonic oscillation treatment for 5-8 minutes; the process conditions of the second ultrasonic oscillation treatment are: 300-400W ultrasonic oscillation treatment for 8-10 minutes.

4. The method according to claim 2, characterized in that: The MCX solid phase extraction column was activated with 3 mL of acetonitrile, 2 mL of ultrapure water, and 1 mL of 0.2 mol / L hydrochloric acid solution in sequence.

5. The method according to claim 2, characterized in that: The method comprises the following steps: using a mixed solution obtained by uniformly mixing concentrated ammonia water with a mass concentration of 28%, acetonitrile and ultrapure water in a volume ratio of 1:2-3:8-10 for elution; and finally fixing the eluate to a fixed volume with a formic acid-acetonitrile aqueous solution; wherein the volume concentrations of formic acid and acetonitrile in the formic acid-acetonitrile aqueous solution are 1-2% and 8-10%, respectively.

6. The method according to claim 1, 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: 0.2 mL / min; Mobile phase: (A) 0.2% formic acid aqueous solution; (B) ammonium acetate-formic acid-acetonitrile solution, wherein the ratio of ammonium acetate, formic acid and acetonitrile is 0.1 g: 0.4 mL: 100 mL; Injection volume: 10 μL.

7. The method according to claim 6, characterized in that Liquid chromatography used gradient elution, and the specific procedure is shown in Table 1; Table 1. Gradient elution program 。 8. The method according to claim 1, characterized in that The mass spectrometry conditions were as follows: Ionization mode: electrospray ionization, positive ion; Capillary voltage: 2.5kV; Cone voltage: 39V; Drying gas temperature: 150℃; Drying gas flow rate: 15L / min; Sheath gas temperature: 350℃; Sheath gas flow rate: 13 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 36 eV and 21 eV, and dwell time was 0.05 min.

9. The method according to claim 1, characterized in that: Levamisole standard working solution was prepared using levamisole standard, the concentrations of which were: 0.0ng / mL, 1.0ng / mL, 2.0ng / mL, 4.0ng / mL, 6.0ng / mL, 10ng / mL.

10. The method according to claim 1, 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.

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