Method for detecting meduronium, trimetazidine and clomiphen in dairy product

Abstract

The invention provides a method for detecting medulamide, trimetazidine and clomiphen in a dairy product. According to the detection method, by optimizing the components and the proportion of a pretreatment reagent and setting liquid chromatography conditions including a chromatographic column, a mobile phase, elution conditions and the like, the medulamide, the trimetazidine and the clomiphen in the dairy product can be detected at the same time. According to the method, acidified methanol acetonitrile is used for extraction, ethylenediamine-N-propyl silanized silica gel and octadecyl silane bonded silica gel are used for purification and filtration respectively, a microporous filter membrane is used for purification and filtration, and optimized liquid chromatography-tandem mass spectrometry conditions are adopted for simultaneous detection of medulamide, trimetazidine and clomiphen in the dairy product. The three metabolism regulators can be effectively separated and detected.

Description

Technical Field

[0001] The invention relates to a detection method, in particular to a detection method for medochloramine, trimetazidine and clomiphene in dairy products, belonging to the field of food detection. Background Art

[0002] The World Anti-Doping Agency has included medomonium, trimetazidine and clomiphene in the "Prohibited List", and the Anti-Doping Center of the General Administration of Sport of China has issued the "Guidelines for the Prevention and Control of Foodborne Stimulants in Major Events", which classifies medomonium, trimetazidine and clomiphene as similar metabolic regulators, and also clearly stipulates that the control limit of medomonium, trimetazidine and clomiphene in competition food is not allowed to be detected.

[0003] Medochloramine, trimetazidine and clomiphene are all clinical drugs for treating diseases, but they can also promote animal growth and are used in animal breeding, which may result in the presence of the above three metabolic regulators in animal-derived foods. If athletes consume foods containing the above three metabolic regulators, they may be misdiagnosed as having taken banned stimulants, which may affect the athletes' eligibility to participate in the competition, personal reputation, etc. Therefore, it is very necessary to be able to simultaneously detect whether these three metabolic regulators are contained in food.

[0004] However, the molecular structures of medochlorpionate, trimetazidine and clomiphene are very different, and the polarity is greatly different, which brings challenges to the simultaneous detection of medochlorpionate, trimetazidine and clomiphene. There is no method for the simultaneous detection of medochlorpionate, trimetazidine and clomiphene in the existing public information. Summary of the invention

[0005] The invention provides a method for detecting medodium, trimetazidine and clomiphene in dairy products. The molecular structures of medodium, trimetazidine and clomiphene are greatly different, and the polarity difference is large, which brings challenges to the simultaneous detection of medodium, trimetazidine and clomiphene, and there is no method for simultaneously detecting medodium, trimetazidine and clomiphene in dairy products, and the problem of being unable to meet regulatory requirements is solved. For the first time, a high performance liquid chromatography tandem mass spectrometry detection method for medodium, trimetazidine and clomiphene in dairy products is systematically established, and medodium, trimetazidine and clomiphene in dairy products can be accurately detected simultaneously. The detection method is simple to operate, low in cost, and accurate in results, and various technical indicators such as recovery rate, quantitative limit and precision meet the requirements, thereby filling the gap in relevant detection methods and having important significance in the field of doping detection in sports events.

[0006] The present invention provides a method for detecting medochlor, trimetazidine and clomiphene in dairy products, comprising the following steps:

[0007] 1) After adding a treatment agent to the dairy product to be tested, the system is subjected to an oscillation treatment and a solid-liquid separation treatment in sequence to obtain a first treatment liquid; the treatment agent comprises formic acid, methanol and acetonitrile, and the volume ratio of the formic acid, methanol and acetonitrile is (0.05-2): (0-50): (50-100);

[0008] 2) subjecting the first treatment liquid to a silica gel purification filler adsorption treatment to obtain a second treatment liquid;

[0009] 3) performing membrane filtration on the second treatment liquid to obtain an analyte, and performing liquid chromatography-mass spectrometry analysis on the analyte to obtain an ion peak area of ​​the target substance in the dairy product to be tested;

[0010] 4) comparing the ion peak area with a standard curve of ion peak area-mass concentration to obtain the mass concentration of the target substance in the dairy product to be tested;

[0011] In the liquid chromatography-mass spectrometry analysis, the conditions of liquid chromatography are:

[0012] HILIC columns;

[0013] Mobile phase A is a mixture of 0.025%-0.075% formic acid and 5-20mM ammonium acetate aqueous solution, mobile phase B is acetonitrile, the eluent flow rate is 0.25-0.45mL / min, and the elution gradient is: 0-1min, 5% mobile phase A; 1-7min, 5%-40% mobile phase A; 7-9min, 40% mobile phase A; 9-9.1min, 40%-5% mobile phase A; 9.1-10min, 5% mobile phase A.

[0014] In the detection method as described above, the volume ratio of formic acid, methanol and acetonitrile is (0.1-1):(0-20):(80-100).

[0015] In the detection method as described above, the volume ratio of formic acid, methanol and acetonitrile is 0.1:10:90.

[0016] The detection method as described above, wherein the HILIC chromatographic column is a Waters ACQUITY BEHHILIC 1.7μm 100×2.1mm chromatographic column.

[0017] In the above detection method, in the liquid chromatography-mass spectrometry analysis, the mass spectrometry conditions are:

[0018] Ionization mode: electrospray positive ion (ESI+); in positive ion mode, interface voltage: 4000 V; interface temperature: 300°C; drying gas flow rate: 10 L / min; nebulizing gas flow rate: 3 L / min.

[0019] The detection method as described above, wherein step 1) comprises: after the dairy product to be tested is mixed with water, adding the treatment agent to the mixed system;

[0020] Wherein, the mass ratio of the dairy product to be tested to water is (1-2):(3-5).

[0021] In the detection method as described above, the volume ratio of the mixed system to the treatment agent is (4-7): (20-25).

[0022] In the detection method as described above, in step 2), the silica gel purification filler adsorption treatment includes oscillating the first treatment liquid using a mixed system of PSA and C18, and then centrifuging the oscillation system to obtain the second treatment liquid.

[0023] In the detection method as described above, the volume mass ratio of the first treatment liquid, PSA and C18 is 5 mL:50 mg:50 mg.

[0024] In the detection method as described above, in step 3), the filter membrane is a 0.22 μm organic filter membrane.

[0025] The present invention systematically establishes a detection method for medochloramine, trimetazidine and clomiphene in dairy products for the first time. By optimizing the formula of the treating agent, medochloramine, trimetazidine and clomiphene in dairy products can be extracted simultaneously, and the separation effect from interfering impurities in dairy products is good; by adjusting the conditions of high performance liquid chromatography, including optimizing the chromatographic column, mobile phase and elution conditions, the above three components in dairy products can be qualitatively and quantitatively detected simultaneously, and the recovery rate of medochloramine, trimetazidine and clomiphene in dairy products is significantly improved. The detection method is simple to operate and the results are accurate. Various technical indicators such as recovery rate, quantitative limit and precision meet the requirements, meet regulatory needs, and fill the gap in related detection methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 To use ACQUITY Spectrum obtained with BEH C18 1.7μm 2.1*100mm column;

[0027] Figure 2 For use Spectrum obtained with HSS T3 2.5μm 2.1*100mm column;

[0028] Figure 3 To use ACQUITY Spectrum obtained with BEH HILIC 1.7μm 2.1*100mm column;

[0029] Figure 4 This is the spectrum obtained when mobile phase A is 20 mM ammonium acetate aqueous solution;

[0030] Figure 5 The spectrum obtained when mobile phase A is a mixture of 0.05% formic acid and 20 mM ammonium acetate aqueous solution;

[0031] Figure 6 This is a spectrum obtained when mobile phase A is a mixture of 0.1% formic acid and 20 mM ammonium acetate aqueous solution. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] The present invention provides a method for detecting medochlor, trimetazidine and clomiphene in dairy products, comprising the following steps:

[0034] 1) After adding a treatment agent to the dairy product to be tested, the system is subjected to an oscillation treatment and a solid-liquid separation treatment in sequence to obtain a first treatment liquid; the treatment agent comprises formic acid, methanol and acetonitrile, and the volume ratio of formic acid, methanol and acetonitrile is (0.05-2): (0-50): (50-100);

[0035] 2) subjecting the first treatment liquid to silica gel purification filler adsorption treatment to obtain a second treatment liquid;

[0036] 3) performing membrane filtration on the second treatment liquid to obtain an analyte, and performing liquid chromatography-mass spectrometry analysis on the analyte to obtain an ion peak area of ​​the target substance in the dairy product to be tested;

[0037] 4) comparing the above ion peak area with the ion peak area-mass concentration standard curve to obtain the mass concentration of the target substance in the dairy product to be tested;

[0038] In the above-mentioned liquid chromatography-mass spectrometry analysis, the conditions of liquid chromatography are:

[0039] HILIC columns;

[0040] Mobile phase A is a mixture of 0.025%-0.075% formic acid and 5-20mM ammonium acetate aqueous solution, mobile phase B is acetonitrile, the eluent flow rate is 0.25-0.45mL / min, and the elution gradient is: 0-1min, 5% mobile phase A; 1-7min, 5%-40% mobile phase A; 7-9min, 40% mobile phase A; 9-9.1min, 40%-5% mobile phase A; 9.1-10min, 5% mobile phase A.

[0041] The present invention does not specifically limit the types of the above-mentioned dairy products, such as cow's milk, cow's milk, goat's milk, sheep's milk, fermented milk, milk powder, frozen drinks, cheese, etc.

[0042] The role of the treatment agent is to better separate the three target substances of medochloramine, trimetazidine and clomiphene in dairy products from the interfering impurities in dairy products. In order to better separate the three target substances from the interfering impurities in dairy products, so as to efficiently recover the three target substances from dairy products, the present invention defines that the treatment agent includes formic acid, methanol and acetonitrile, and the volume ratio of formic acid, methanol and acetonitrile is (0.05-2): (0-50): (50-100); it should be noted that because the content of formic acid in the mixed system of formic acid, methanol and acetonitrile is very small, the volume ratio of formic acid, methanol and acetonitrile is (0.05-2): (0-50): (50-100) It can be understood that the volume sum of methanol and acetonitrile is 100 and the volume ratio of the two is (0-50): (50-100), and the volume percentage of formic acid in the mixed system of formic acid, methanol and acetonitrile is 0.05%-2%. Under the action of the above treatment agents, the average recovery rates of medochloramine, trimetazidine and clomiphene were not less than 60.0%.

[0043] The present invention does not specifically limit the method and conditions of the above-mentioned oscillation treatment, as long as the dairy product to be tested and the treatment agent are fully mixed and uniform, for example, a vortex oscillator is used to vortex the mixed system of the dairy product to be tested and the treatment agent for 1-5 minutes.

[0044] The solid-liquid separation can be carried out by the centrifugal separation method commonly used in the art. The centrifugal separation conditions are set as follows: temperature 4-25°C, rotation speed 5000-10000rpm / min, time 5-10min, preferably temperature 4°C, rotation speed 7800rpm / min, time 5min, the purpose is to separate the solid and liquid in the mixed system of the dairy product to be tested and the treatment agent, and obtain the supernatant, which is the first treatment liquid. The three target substances to be tested, such as medochloramine, trimetazidine and clomiphene, are dissolved in the first treatment liquid under the action of the treatment agent.

[0045] It is understandable that in order to quantitatively determine the three target substances in the dairy product to be tested, after the solid-liquid separation to obtain the liquid part in step 1), the liquid part can be transferred to a volumetric flask for constant volume to obtain the above-mentioned first treatment liquid.

[0046] The first treatment liquid is further purified using purification filler silica gel to obtain a second treatment liquid. The purification treatment can further remove interfering impurities, improve the purity of medochloramine, trimetazidine and clomiphene in the second treatment liquid, avoid the damage of interfering impurities to the liquid chromatography-tandem mass spectrometry instrument, and more importantly, help to improve the accuracy of the test results.

[0047] The above process is a process of pre-treating the dairy product to be tested, which is simple to operate and low in cost.

[0048] The second treatment liquid obtained in the above pretreatment process is transferred to an injection bottle after filtering through a filter membrane, and subjected to liquid chromatography-mass spectrometry analysis to obtain the ion peak areas of the three target substances in the dairy products to be tested; the above ion peak areas are compared with the ion peak area-mass concentration standard curve to obtain the mass concentrations of the three target substances in the dairy products to be tested.

[0049] Among them, the present invention limits the chromatographic column used in liquid chromatography to a HILIC chromatographic column. This is because through analysis of the chemical structural formulas of medochloramine, trimetazidine and clomiphene, it is found that the structure of medochloramine is very different from the latter two, and there is a great difference in polarity, resulting in most chromatographic columns being unable to retain and separate the above three targets at the same time. For example, conventional C4, C18, and T3 chromatographic columns have poor retention and separation capabilities for the above three targets. However, after a large number of exploratory experiments, the inventors compared the separation and retention effects of different chromatographic columns on the three targets and found that the use of a HILIC chromatographic column can better separate and retain the above three metabolic regulators at the same time. This is because the HILIC chromatographic column is a hydrophilic chromatographic column and has the ability to separate and retain the three metabolic regulators at the same time.

[0050] The mobile phase A of the liquid chromatography is a mixture of 0.025%-0.075% formic acid and 5-20mM ammonium acetate aqueous solution, and the mobile phase B is acetonitrile, which solves the problem of clomiphene chromatographic peak bifurcation and the problem that medofen is unable to be separated from impurities in the dairy matrix, resulting in large chromatographic peak errors.

[0051] The elution conditions of the above-mentioned liquid chromatography are as follows: the flow rate of the eluent is 0.25-0.45 mL / min, and the elution gradient is: 0-1 min, 5% mobile phase A; 1-7 min, 5%-40% mobile phase A; 7-9 min, 40% mobile phase A; 9-9.1 min, 40%-5% mobile phase A; 9.1-10 min, 5% mobile phase A. In combination with the chemical structures and polarity characteristics of medochloramine, trimetazidine and clomiphene, the elution conditions are scientifically set, so that no matter the components that elute first or later, their peak shapes can basically be guaranteed to be sharp and symmetrical; and strongly retained interferences can be eluted, so that impurity peaks can be well separated to prevent them from affecting data collection, thereby improving the accuracy of the detection results.

[0052] Furthermore, the mobile phase A can also be a mixture of 0.05% formic acid and 20 mM ammonium acetate aqueous solution; the flow rate of the eluent can also be 300 μL, and the accuracy of the detection result is higher.

[0053] The above-mentioned limitations on liquid chromatography conditions, the coordinated use of HILIC chromatographic columns, mobile phase composition, and elution conditions enable the three target substances in dairy products to be detected simultaneously with good detection effects.

[0054] The mass concentration of the target substance in the dairy product to be tested can be obtained by comparing the above ion peak area with the ion peak area-mass concentration standard curve. Specifically, the present invention uses an external standard method to perform qualitative and quantitative determination of the above three target substances, and uses the pure product of the target substance to draw a standard curve. The ion peak area of ​​the sample to be tested is compared with the standard curve and then converted to obtain the mass concentration of the target substance in the sample to be tested. The external standard method has the advantages of low cost and high practical application value. The process of drawing the standard curve and obtaining the mass concentration of the target substance in the dairy product to be tested by the external standard method includes the following steps:

[0055] 1) Accurately weigh a certain amount of medochloramine, trimetazidine and clomiphene into different brown volumetric flasks, dissolve them in methanol, and make up to volume to prepare a standard stock solution of a specific concentration;

[0056] 2) Pipette 1 mL of each of the above standard stock solutions into a 100 mL brown volumetric flask, and dilute to volume with methanol to prepare a mixed intermediate solution I;

[0057] 3) Pipette 1 mL of mixed intermediate solution I into a 100 mL brown volumetric flask and dilute to volume with methanol to prepare mixed intermediate solution II;

[0058] 4) treating the negative sample according to steps 1) and 2) of the above-mentioned detection method for medochloramine, trimetazidine and clomiphene in dairy products, and filtering the sample with a membrane to obtain a negative second treatment solution; then taking a certain volume of mixed intermediate solution II, diluting the volume to 1 mL with the above-mentioned negative second treatment solution, and preparing a series of standard working solutions with concentrations of 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, and 5 ng / mL, respectively;

[0059] 5) analyzing the above series of standard working solutions by liquid chromatography-mass spectrometry to obtain the ion peak areas of the three target substances in the series of standard working solutions; plotting the quantitative ion peak area-concentration to obtain a standard curve, regression equation and correlation coefficient;

[0060] 6) Compare the ion peak areas of the three metabolic regulators in the dairy products to be tested with the ion peak area-mass concentration standard curve to obtain the on-machine mass concentrations of the three metabolic regulators in the dairy products to be tested, and then convert the on-machine mass concentrations to obtain the mass concentrations of the three metabolic regulators in the dairy products to be tested.

[0061] Among them, the concentration of the standard stock solution is generally 1 mg / mL. Correspondingly, the concentrations of the three targets in the mixed intermediate solution I are all 10 μg / mL, and the concentrations of the three targets in the mixed intermediate solution II are all 100 ng / mL; the standard stock solution can be prepared in advance and stored at -18°C away from light, with a shelf life of 6 months; the mixed intermediate solution I can be prepared in advance and stored at -18°C away from light, with a shelf life of 3 months; the mixed intermediate solution II is prepared on demand.

[0062] When the correlation coefficient of the above standard curve regression equation is greater than 0.9900, the quantitative ion peak area-concentration presents a good linear relationship, indicating that the standard curve meets the operating requirements.

[0063] It is understandable that the detection method of medochlor, trimetazidine and clomiphene in the above-mentioned dairy products must be carried out strictly in the order specified in the present invention to ensure that better detection results are obtained.

[0064] The present invention systematically establishes for the first time a detection method for medoamine, trimetazidine and clomiphene in dairy products. The detection method can simultaneously quickly and accurately qualitatively and quantitatively detect the above three target substances in dairy products, fully separate them from interfering substances in dairy products, and significantly improve the recovery rates of medoamine, trimetazidine and clomiphene in dairy products, filling the gap in related detection methods and having important significance in the field of doping detection in sports events. The detection method is simple and easy to operate, low in cost, and accurate in results, and various technical indicators such as recovery rate, quantitative limit and precision all meet the requirements.

[0065] The above detection method also includes quantitative limit verification, which specifically includes the following steps: preparing a series of standard solutions of three target substances with different concentrations; adding the above standard solutions to a series of negative samples (or blank samples) respectively to obtain a series of sample solutions containing different concentrations of target substances; performing liquid chromatography-mass spectrometry analysis on the above series of sample solutions containing different concentrations of target substances according to the above detection method for medochloramine, trimetazidine and clomiphene in dairy products, and determining the concentration corresponding to a signal-to-noise ratio greater than or equal to 10, which is the quantitative limit.

[0066] In some embodiments, when the dairy product to be tested is liquid milk, fermented milk or frozen drink, the quantification limits of the three targets are all 1 μg / kg; when the dairy product to be tested is milk powder or cheese, the quantification limits of the three targets are all 2 μg / kg, indicating that the detection method of the present invention has high sensitivity.

[0067] In some embodiments, the detection method of the present invention was used to detect three target substances in some dairy products, and the results were all undetected, indicating that these dairy products do not contain the above target substances, are in compliance with national food safety standards, and can be provided to athletes as safe foods.

[0068] In other embodiments, the present invention determines the recovery rate and relative standard deviation of three target substances through a spike test to verify the stability, reliability and precision of the above-mentioned detection method. The specific process is that by adding a certain concentration of the three target substances to the negative pure milk sample, for example, the spike concentration is 1 μg / kg, 2 μg / kg and 10 μg / kg, and the recovery rate of the three target substances detected by the above-mentioned detection method is in the range of 75.0% to 119%, and the relative standard deviation (RSD) is 1.31% to 8.79%, which proves that the recovery rate and relative standard deviation of the above-mentioned detection method meet the requirements of GB / T 27404-2008, indicating that the above-mentioned detection method of the present invention is stable, reliable and has high precision.

[0069] Furthermore, when the volume ratio of formic acid, methanol and acetonitrile in the above-mentioned treatment agent is (0.1-1): (0-20): (80-100), similarly, the above-mentioned volume ratio can be understood as that the volume sum of methanol and acetonitrile is 100 and the volume ratio is (0-20): (80-100), the volume percentage of formic acid in the mixed system of formic acid, methanol and acetonitrile is 0.1%-1%. Under the action of the above-mentioned treatment agent, the recovery rates of medochloramine, trimetazidine and clomiphene can be further improved, and the average recovery rates of the three metabolic regulators are not less than 66.5%.

[0070] Furthermore, the volume ratio of formic acid, methanol and acetonitrile in the above-mentioned treatment agent is 0.1:10:90, which further improves the recovery rates of medochloramine, trimetazidine and clomiphene, and the average recovery rates of medochloramine, trimetazidine and clomiphene are not less than 85.0%, respectively.

[0071] In some embodiments, the HILIC column is a Waters ACQUITY The BEH HILIC 1.7μm 100×2.1mm chromatographic column has good separation and retention effects on the three metabolic regulators.

[0072] In some embodiments, the mass spectrometry conditions are: ionization mode: electrospray positive ion (ESI+); in positive ion mode, interface voltage: 4000V; interface temperature: 300°C; drying gas flow rate: 10L / min; nebulizing gas flow rate: 3L / min. By setting the mass spectrometry conditions as above, the characteristic ion pairs of the three metabolic regulators are determined, and the three metabolic regulators are accurately qualitatively and quantitatively detected.

[0073] In order to further improve the treatment effect of the treatment agent, the dairy product to be tested can be mixed with water first, and then the treatment agent can be added to the mixed system; wherein the mass volume ratio of the dairy product to be tested to water is (1-2): (3-5). The above mass volume ratio is the ratio of the mass of the dairy product to be tested to the volume of water, that is, (1-2) g: (3-5) mL.

[0074] Specifically, when the dairy product to be tested is liquid milk, fermented milk or frozen drink, the mass volume ratio is 2:3, that is, 2g of liquid milk, fermented milk or frozen drink is mixed with 3mL of water; when the dairy product to be tested is milk powder or cheese, the mass volume ratio is 1:5, that is, 1g of milk powder or cheese is mixed with 5mL of water. In this way, the dairy product to be tested can be fully mixed with water to obtain a mixed system with an appropriate concentration, which is conducive to the treatment agent to play a role, thereby improving the recovery rate of medochloramine, trimetazidine and clomiphene, and improving the accuracy of the test results.

[0075] When the volume ratio of the mixed system of the dairy product to be tested and water to the treating agent is (4-7): (20-25), the treating agent can fully exert its effect on the dairy product to be tested, so that medochloramine, trimetazidine and clomiphene are fully separated from the interfering impurities in the dairy product to be tested, which helps to improve the recovery rate of medochloramine, trimetazidine and clomiphene and improve the accuracy of the test results.

[0076] Furthermore, depending on the type of dairy product to be tested, the above volume ratio can also be 4:20 or 6:20. Adjusting the appropriate volume ratio based on the characteristics of the type of dairy product to be tested is beneficial to improving the recovery rate of the three metabolic regulators in the dairy product to be tested and improving the accuracy of the detection method of the present invention.

[0077] In some embodiments, the silica gel purification filler adsorption treatment in step 2) of the above detection method includes oscillating the first treatment liquid using a mixed system of PSA and C18, and then centrifuging the oscillation system to obtain a second treatment liquid.

[0078] Wherein, PSA is ethylenediamine-N-propyl silane silica gel, 40-60 μm; C18 is octadecylsilane bonded silica gel, 40-60 μm; the present invention does not specifically limit the brands of the above-mentioned PSA and C18.

[0079] The present invention does not specifically limit the method and conditions of the above-mentioned oscillation treatment, as long as the first treatment liquid and the mixed system of PSA and C18 are fully mixed and uniformly mixed, for example, a vortex oscillator is used for vortex oscillation for 1-5 minutes. The above-mentioned centrifugal separation conditions can be: temperature 4-25°C, speed 5000-10000rpm / min, time 10min, the purpose is to separate the first treatment liquid from the solid and liquid in the mixed system of PSA and C18, and obtain the supernatant, which is the second treatment liquid.

[0080] PSA and C18 are both dispersed solid phase extraction agents. In the detection method of the present invention, they are used as adsorbents to purify the first treatment liquid, which has the advantages of being economical, simple to operate, saving operation time, having good adsorption effect of interfering substances, and high purification efficiency.

[0081] In some embodiments, the volume mass ratio of the first treatment liquid, PSA and C18 is 5 mL: 50 mg: 50 mg. Specifically, the volume of the first treatment liquid is 5 mL, and the masses of PSA and C18 are both 50 mg. Under this condition, the recovery rates of medocaine, trimetazidine and clomiphene are all high, and the usage of PSA and C18 is relatively small, thereby achieving savings in the cost of purification consumables.

[0082] In some embodiments, the filter membrane in step 3) is a 0.22 μm organic filter membrane, which can remove interfering impurities in the second treatment liquid. The present invention does not specifically limit the brand of the above 0.22 μm filter membrane.

[0083] The technical solution of the present invention is described in more detail below through specific embodiments and comparative examples.

[0084] The reagents used in the following examples and comparative examples are as follows:

[0085] Water: Grade 1 water in accordance with GB / T 6682;

[0086] Methanol, formic acid, acetonitrile: LC-MS grade;

[0087] Example 1

[0088] This embodiment provides a method for detecting medochlor, trimetazidine and clomiphene in a certain brand of pure milk spiked on the market, comprising:

[0089] 1. The quantitative limit verification of the above detection method includes the following steps:

[0090] 1) Take four equal amounts of pure milk, add three target substances, medochloramine, trimetazidine and clomiphene, to each of the pure milk to prepare four test samples, and the mass ratio of each target substance to the pure milk to which it belongs in the four test samples is 0.5 μg / kg, 1 μg / kg, 2 μg / kg, and 3 μg / kg, respectively;

[0091] 2) Take 2 g of the sample to be tested in a 50 mL centrifuge tube, add 3 mL of water to mix, then add 20 mL of formic acid, methanol, acetonitrile (v:v:v, 0.1:10:90), vortex oscillate for 5 min, and then centrifuge at 4 ° C, 7800 rpm / min for 10 min to obtain a supernatant, transfer the supernatant to a 25 mL volumetric flask and dilute to volume to obtain a first treatment solution;

[0092] 3) Take 5 mL of the first treatment solution, add 50 mg PSA and 50 mg C18, vortex and oscillate for 5 min, then centrifuge at 4°C, 7800 rpm / min for 5 min to obtain a supernatant, and then filter the supernatant using a 0.22 μm filter membrane to obtain a second treatment solution;

[0093] 4) The second treated liquid was analyzed by liquid chromatography-mass spectrometry to determine the signal-to-noise ratio. The concentration of the sample to be tested corresponding to a signal-to-noise ratio greater than or equal to 10 was the quantitative limit. When the sample was measured to be pure milk, the quantitative limit of the detection method was 1 μg / kg. At the same time, the characteristic ion pair parameters of the three target substances were determined, as shown in Table 1.

[0094] 2. Drawing of the standard curve includes the following steps:

[0095] 1) Accurately weigh 0.01 g of medocain, trimetazidine and clomiphene into different 10 mL brown volumetric flasks, dissolve and dilute with methanol to prepare 1 mg / mL standard stock solutions I of the above three target substances;

[0096] 2) Pipette 1 mL of the standard stock solutions of the three target substances mentioned above into a 100 mL brown volumetric flask, and dilute to volume with methanol to prepare a mixed intermediate solution II with a concentration of 10 μg / mL for each of the three target substances;

[0097] 3) Pipette 1 mL of mixed intermediate solution I into a 100 mL brown volumetric flask and dilute to volume with methanol to prepare mixed intermediate solution II with a concentration of 100 ng / mL for the above three target substances;

[0098] 4) 2 g of pure milk was placed in a 50 mL centrifuge tube, 3 mL of water was added and mixed, and then 20 mL of formic acid, methanol, and acetonitrile (v:v:v, 0.1:10:90) was added, vortexed for 5 min, and then centrifuged at 4° C. and 7800 rpm / min for 10 min to obtain a supernatant, and the supernatant was transferred to a 25 mL volumetric flask and fixed to volume to obtain a first treatment solution;

[0099] 5) Take 5 mL of the first treatment solution, add 50 mg PSA and 50 mg C18, vortex and oscillate for 5 min, then centrifuge at 4°C, 7800 rpm / min for 5 min to obtain a supernatant, and then filter the supernatant using a 0.22 μm filter membrane to obtain a second treatment solution;

[0100] 6) Take a certain volume of mixed intermediate solution II, dilute it to 1 mL with the above second treatment solution, and prepare a series of standard working solutions with concentrations of 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, and 5 ng / mL, respectively, for liquid chromatography-tandem mass spectrometry determination, and plot the quantitative ion peak area-concentration to obtain the regression equation and correlation coefficient of the standard curve. The specific results are shown in Table 2.

[0101] 3. The detection method of medochlor, trimetazidine and clomiphene in spiked pure milk comprises the following steps:

[0102] 1) Take three equal portions of pure milk, add three target substances, medochloramine, trimetazidine and clomiphene, to each portion of pure milk to prepare three portions of spiked pure milk, and the addition amount of each target substance in the three portions of spiked pure milk (i.e., the spiked concentration Y i ) are 1 μg / kg, 2 μg / kg, and 10 μg / kg, respectively;

[0103] 2) The three portions of spiked pure milk were subjected to the following pretreatments respectively: 2 g of spiked pure milk was placed in a 50 mL centrifuge tube, 3 mL of water was added and mixed, and then 20 mL of formic acid, methanol, and acetonitrile (v:v:v, 0.1:10:90) was added, vortexed for 5 min, and then centrifuged at 4°C and 7800 rpm / min for 10 min to obtain a supernatant, and the supernatant was transferred to a 25 mL volumetric flask and fixed to volume to obtain a first treatment solution;

[0104] 3) Take 5 mL of the first treatment solution, add 50 mg PSA and 50 mg C18, vortex and oscillate for 5 min, then centrifuge at 4°C, 7800 rpm / min for 5 min to obtain a supernatant, and then filter the supernatant using a 0.22 μm filter membrane to obtain a second treatment solution;

[0105] 4) performing liquid chromatography-mass spectrometry analysis on the second treated liquid to obtain the ion peak areas of medochloramine, trimetazidine and clomiphene in the spiked pure milk;

[0106] The conditions of liquid chromatography are:

[0107] Waters ACQUITY BEH HILIC 1.7 μm 100 × 2.1 mm column;

[0108] Mobile phase A is a mixture of 0.05% formic acid and 20 mM ammonium acetate aqueous solution, mobile phase B is acetonitrile, the eluent flow rate is 300 μL / min, and the elution gradient is: 0-1 min, 5% mobile phase A, 95% mobile phase B; 1-7 min, 5%-40% mobile phase A, 95% mobile phase B; 7-9 min, 40% mobile phase A, 60% mobile phase B; 9-9.1 min, 40%-5% mobile phase A, 60% mobile phase B; 9.1-10 min, 5% mobile phase A;

[0109] The conditions for mass spectrometry were:

[0110] Ionization mode: electrospray positive ion (ESI+); in positive ion mode, interface voltage: 4000 V; interface temperature: 300 °C; drying gas flow rate: 10 L / min; nebulizing gas flow rate: 3 L / min;

[0111] The parallel determination was performed 6 times;

[0112] 5) Compare the above ion peak areas with the standard curve obtained above, and convert according to the following formula to obtain the mass concentrations of medochloramine, trimetazidine and clomiphene in the spiked pure milk,

[0113]

[0114] Wherein, i is one of medochloramine, trimetazidine and clomiphene; X i is the mass concentration of i in spiked pure milk, μg / kg; c i is the concentration of i in the second treatment solution obtained by comparison with the standard curve, ng / mL; V is the fixed volume, mL; 1000 is the conversion factor; m is the mass of spiked pure milk, g.

[0115] 6) Calculate the above X i With spike concentration Y i The ratio is the recovery rate, and the relative standard deviation is calculated at the same time. The specific results are shown in Table 3.

[0116] Example 2

[0117] This embodiment is basically the same as Embodiment 1, except that:

[0118] 1) The sample to be tested is a certain brand of fermented milk on the market;

[0119] 2) In the detection method of medochlor, trimetazidine and clomiphene in fermented milk, only the spiked concentration Y is measured. i The recoveries and relative standard deviations of medochloramine, trimetazidine and clomiphene in fermented milk spiked with 2 μg / kg;

[0120] See Table 4-5 for specific results.

[0121] Example 3

[0122] This embodiment is basically the same as Embodiment 1, except that:

[0123] 1) The sample to be tested is a certain brand of milk powder on the market;

[0124] 2) In the quantitative limit verification test, the quantitative limit of the detection method for medochloramine, trimetazidine and clomiphene in milk powder was 2 μg / kg;

[0125] 3) In the detection method of medochlor, trimetazidine and clomiphene in spiked milk powder, only the spiked concentration Y is measured. i The recoveries and relative standard deviations of medochloramine, trimetazidine and clomiphene in spiked milk powder at 4 μg / kg;

[0126] See Tables 6-7 for specific results.

[0127] Example 4

[0128] This embodiment is basically the same as Embodiment 1, except that:

[0129] 1) The sample to be tested is a certain brand of frozen drink on the market;

[0130] 2) In the detection method of medochlor, trimetazidine and clomiphene in spiked frozen drinks, only the spiked concentration Y is measured i The recoveries and relative standard deviations of medochloramine, trimetazidine and clomiphene in fermented milk spiked with 2 μg / kg;

[0131] See Tables 8-9 for specific results.

[0132] Example 5

[0133] This embodiment is basically the same as Embodiment 1, except that:

[0134] 1) The sample to be tested is a certain brand of processed cheese on the market;

[0135] 2) In the quantitative limit verification test, the quantitative limit of the detection method for medochloramine, trimetazidine and clomiphene in milk powder was 2 μg / kg;

[0136] 3) In the detection method of medochlor, trimetazidine and clomiphene in spiked cheese, only the spiked concentration Y is measured. i The recoveries and relative standard deviations of medochloramine, trimetazidine and clomiphene in spiked milk powder at 4 μg / kg;

[0137] See Tables 10-11 for specific results.

[0138] Example 6

[0139] This embodiment is a method for detecting medochlor, trimetazidine and clomiphene in dairy products, wherein the dairy products include the five samples in Examples 1-5, namely, a certain brand of pure milk, a certain brand of fermented milk, a certain brand of milk powder, a certain brand of frozen beverage and a certain brand of processed cheese, a total of 25 batches of samples; the above detection method specifically includes the following steps:

[0140] 1) Take 2 g of the sample to be tested in a 50 mL centrifuge tube, add 3 mL of water to mix, then add 20 mL of formic acid, methanol, acetonitrile (v:v:v, 0.1:10:90), vortex and oscillate for 5 min, then centrifuge at 4 ° C, 7800 rpm / min for 10 min to obtain a supernatant, transfer the supernatant to a 25 mL volumetric flask and dilute to volume to obtain a first treatment solution;

[0141] 2) Take 5 mL of the first treatment solution, add 50 mg PSA and 50 mg C18, vortex and oscillate for 5 min, then centrifuge at 4°C, 7800 rpm / min for 5 min to obtain a supernatant, and then filter the supernatant using a 0.22 μm filter membrane to obtain a second treatment solution;

[0142] 3) performing liquid chromatography-mass spectrometry analysis on the second treatment liquid to obtain the ion peak areas of medochloramine, trimetazidine and clomiphene in the sample to be tested;

[0143] The conditions of liquid chromatography are:

[0144] Waters ACQUITY BEH HILIC 1.7 μm 100 × 2.1 mm column;

[0145] Mobile phase A was a mixture of 0.05% formic acid and 20 mM ammonium acetate aqueous solution, mobile phase B was acetonitrile, the eluent flow rate was 300 μL / min, and the elution gradient was: 0-1 min, 5% mobile phase A; 1-7 min, 5%-40% mobile phase A; 7-9 min, 40% mobile phase A; 9-9.1 min, 40%-5% mobile phase A; 9.1-10 min, 5% mobile phase A;

[0146] The conditions for mass spectrometry were:

[0147] Ionization mode: electrospray positive ion (ESI+); in positive ion mode, interface voltage: 4000 V; interface temperature: 300 °C; drying gas flow rate: 10 L / min; nebulizing gas flow rate: 3 L / min;

[0148] The parallel determination was performed 6 times;

[0149] 5) Compare the above ion peak areas with the standard curves of the corresponding samples in Examples 1-5, and convert according to the following formula to obtain the mass concentrations of medochloramine, trimetazidine and clomiphene in the sample to be tested,

[0150]

[0151] Wherein, i is one of medochloramine, trimetazidine and clomiphene; X i is the mass concentration of i in spiked pure milk, μg / kg; c i is the concentration of i in the second treatment solution obtained by comparison with the standard curve, ng / mL; V is the fixed volume, mL; 1000 is the conversion factor; m is the mass of spiked pure milk, g; the specific results are shown in Table 12.

[0152] Example 7

[0153] The detection methods of medochlor, trimetazidine and clomiphene in spiked pure milk in this embodiment are basically the same as those in Example 1, except that:

[0154] 1) Only pure milk spiked with a concentration of 10 μg / kg was tested;

[0155] 2) replacing formic acid, methanol, acetonitrile (v:v:v, 0.1:10:90) with formic acid, methanol, acetonitrile (v:v:v, 0.5:10:90) in step 2;

[0156] See Table 13 for specific results.

[0157] Example 8

[0158] The detection methods of medochlor, trimetazidine and clomiphene in spiked pure milk in this embodiment are basically the same as those in Example 1, except that:

[0159] 1) Only pure milk spiked with a concentration of 10 μg / kg was tested;

[0160] 2) replacing formic acid, methanol, acetonitrile (v:v:v, 0.1:10:90) with formic acid, methanol, acetonitrile (v:v:v, 1:10:90) in step 2;

[0161] See Table 13 for specific results.

[0162] Example 9

[0163] The detection methods of medochlor, trimetazidine and clomiphene in spiked pure milk in this embodiment are basically the same as those in Example 1, except that:

[0164] 1) Only pure milk spiked with a concentration of 10 μg / kg was tested;

[0165] 2) replacing formic acid, methanol, acetonitrile (v:v:v, 0.1:10:90) with formic acid, methanol, acetonitrile (v:v:v, 1:20:80) in step 2;

[0166] See Table 13 for specific results.

[0167] Example 10

[0168] The detection methods of medochlor, trimetazidine and clomiphene in spiked pure milk in this embodiment are basically the same as those in Example 1, except that:

[0169] 1) Only pure milk spiked with a concentration of 10 μg / kg was tested;

[0170] 2) replacing formic acid, methanol, acetonitrile (v:v:v, 0.1:10:90) with formic acid, methanol, acetonitrile (v:v:v, 0.1:0:100) in step 2;

[0171] See Table 13 for specific results.

[0172] Embodiment 11

[0173] This embodiment is basically the same as Embodiment 10, except that:

[0174] In step 2, formic acid, methanol, acetonitrile (v:v:v, 0.1:0:100) was replaced with formic acid, methanol, acetonitrile (v:v:v, 2:50:50);

[0175] See Table 13 for specific results.

[0176] Example 12

[0177] This embodiment is basically the same as Embodiment 10, except that:

[0178] In step 2, formic acid, methanol, acetonitrile (v:v:v, 0.1:0:100) was replaced with formic acid, methanol, acetonitrile (v:v:v, 0.05:0:100);

[0179] See Table 13 for specific results.

[0180] Embodiment 13

[0181] The detection methods of medochlor, trimetazidine and clomiphene in spiked pure milk in this embodiment are basically the same as those in Example 1, except that:

[0182] 1) Only pure milk spiked with a concentration of 10 μg / kg was tested;

[0183] 2) Replace 50 mg C18 with 100 mg C18 in step 3;

[0184] See Table 14 for specific results.

[0185] Embodiment 14

[0186] The detection methods of medochlor, trimetazidine and clomiphene in spiked pure milk in this embodiment are basically the same as those in Example 1, except that:

[0187] 1) Only pure milk spiked with a concentration of 10 μg / kg was tested;

[0188] 2) Replace 50 mg C18 with 150 mg C18 in step 3;

[0189] See Table 14 for specific results.

[0190] Embodiment 15

[0191] The detection methods of medochlor, trimetazidine and clomiphene in spiked pure milk in this embodiment are basically the same as those in Example 1, except that:

[0192] 1) Only pure milk spiked with a concentration of 10 μg / kg was tested;

[0193] 2) Replace 50 mg C18 with 200 mg C18 in step 3;

[0194] See Table 14 for specific results.

[0195] Example 16

[0196] The detection method steps 1) to 4) of medochloramine, trimetazidine and clomiphene in spiked pure milk in this embodiment are basically the same as those in embodiment 1, except that:

[0197] Only spiked pure milk with a spike concentration of 10 μg / kg was tested;

[0198] For specific results, see Figure 3 .

[0199] Embodiment 17

[0200] The detection method steps 1) to 4) of medochloramine, trimetazidine and clomiphene in spiked pure milk in this embodiment are basically the same as those in embodiment 1, except that:

[0201] Only spiked pure milk with a spike concentration of 10 μg / kg was tested;

[0202] For specific results, see Figure 5.

[0203] Comparative Example 1

[0204] The detection method steps 1) to 4) of medochloramine, trimetazidine and clomiphene in spiked pure milk in this comparative example are basically the same as those in Example 1, except that:

[0205] 1) Only pure milk spiked with a concentration of 10 μg / kg was tested;

[0206] 2) Liquid chromatography columns are used in sequence HSS T3 2.5μm 2.1*100mm;

[0207] For specific results, see Figure 2 .

[0208] Comparative Example 2

[0209] This comparative example is basically the same as comparative example 1, except that:

[0210] The liquid chromatography column is ACQUITY BEH C18 1.7μm 2.1*100mm;

[0211] For specific results, see Figure 1 .

[0212] Comparative Example 3

[0213] The detection method steps 1) to 4) of medochloramine, trimetazidine and clomiphene in spiked pure milk in this comparative example are basically the same as those in Example 1, except that:

[0214] 1) Only pure milk spiked with a concentration of 10 μg / kg was tested;

[0215] 2) Mobile phase A is 20 mM ammonium acetate aqueous solution;

[0216] For specific results, see Figure 4 .

[0217] Comparative Example 4

[0218] This comparative example is basically the same as comparative example 3, except that:

[0219] Mobile phase A was a mixture of 0.1% formic acid and 20 mM ammonium acetate aqueous solution;

[0220] For specific results, see Figure 6 .

[0221] Comparative Example 5

[0222] The detection methods of medochlor, trimetazidine and clomiphene in spiked pure milk in this comparative example are basically the same as those in Example 1, except that:

[0223] 1) Only pure milk spiked with a concentration of 10 μg / kg was tested;

[0224] 2) In step 2, formic acid, methanol, and acetonitrile (v:v:v, 0.1:10:90) were replaced with methanol;

[0225] See Table 13 for specific results.

[0226] Comparative Example 6

[0227] This comparative example is basically the same as comparative example 5, except that:

[0228] In step 2, methanol is replaced by acetonitrile;

[0229] See Table 13 for specific results.

[0230] Comparative Example 7

[0231] This comparative example is basically the same as comparative example 5, except that:

[0232] In step 2, methanol was replaced with methanol-acetonitrile (v:v, 90:10);

[0233] See Table 13 for specific results.

[0234] Comparative Example 8

[0235] This comparative example is basically the same as comparative example 5, except that:

[0236] In step 2, methanol was replaced with methanol-acetonitrile (v:v, 50:50);

[0237] See Table 13 for specific results.

[0238] Comparative Example 9

[0239] This comparative example is basically the same as comparative example 5, except that:

[0240] In step 2, methanol was replaced with methanol-acetonitrile (v:v, 10:90);

[0241] See Table 13 for specific results.

[0242] Comparative Example 10

[0243] This comparative example is basically the same as comparative example 5, except that:

[0244] In step 2, methanol was replaced with formic acid, methanol, and acetonitrile (v:v:v, 5:20:80);

[0245] See Table 13 for specific results.

[0246] Test results

[0247] 1. Test results of Example 1

[0248] Table 1 Mass spectrometry characteristic ion pair parameters of medochloramine, trimetazidine and clomiphene

[0249]

[0250] Note: * indicates quantitative ion pair.

[0251] Table 1 Mass spectrometry characteristic ion pair parameters of medochloramine, trimetazidine and clomiphene show that the present invention determines the mass spectrometry characteristic ion pair parameters of three metabolic regulators, medochloramine, trimetazidine and clomiphene.

[0252] Table 2 Regression equations and correlation coefficients of standard curves of medochlor, trimetazidine and clomiphene in spiked pure milk

[0253] Compound Standard curve regression equation Correlation coefficient Medocaproin Y=795464X+1792.32 0.9998 Trimetazidine Y=1553780X-1209.05 0.9999 Clomiphene Y=207761X-1411.65 0.9994

[0254] The results of Table 2 standard curve regression equation and correlation coefficient show that the concentrations X of medochloramine, trimetazidine and clomiphene in the spiked pure milk of Example 1 and the quantitative ion peak area response value Y have a good linear relationship, and the correlation coefficients are all greater than 0.9900, which meets the requirements.

[0255] Table 3 Recoveries and relative standard deviations (RSD) of medochloramine, trimetazidine and clomiphene in spiked pure milk (n=6)

[0256]

[0257] The results of recovery and relative standard deviation (RSD) in Table 3 show that the recovery of medochloramine, trimetazidine and clomiphene in pure milk with spiked concentrations of 1 μg / kg, 2 μg / kg and 10 μg / kg ranges from 75.0% to 119%; the RSD is from 1.31% to 8.79%; the recovery and RSD of the detection method of the present invention are in compliance with GB / T 27404-2008, and have high accuracy and precision.

[0258] 2. Test results of Example 2

[0259] Table 4 Regression equations and correlation coefficients of standard curves of medochloramine, trimetazidine and clomiphene in spiked fermented milk

[0260]

[0261]

[0262] The results of the standard curve regression equation and correlation coefficient in Table 4 show that the concentrations X of medochlorpamide, trimetazidine and clomiphene in the spiked fermented milk of Example 2 and the quantitative ion peak area response value Y have a good linear relationship, and the correlation coefficients are all greater than 0.9900, which meets the requirements.

[0263] Table 5 Recovery and relative standard deviation (RSD) of medochloramine, trimetazidine and clomiphene in spiked fermented milk (n=6)

[0264]

[0265] The results of recovery rate and relative standard deviation (RSD) in Table 5 show that the recovery rate of medochloramine, trimetazidine and clomiphene in fermented milk with a spiked concentration of 2 μg / kg ranges from 85.0% to 98.1%; the RSD is between 2.91% and 3.38%; the recovery rate and RSD of the detection method of the present invention are in line with GB / T27404-2008, and have high accuracy and precision.

[0266] 3. Test results of Example 3

[0267] Table 6 Regression equations and correlation coefficients of standard curves of medochlor, trimetazidine and clomiphene in spiked milk powder

[0268] Compound Standard curve regression equation Correlation coefficient Medocaproin Y=555958X-1148.38 0.9996 Trimetazidine Y=526362X-3956.54 0.9998 Clomiphene Y=86989.3X-89.4433 0.9992

[0269] The results of standard curve regression equation and correlation coefficient in Table 6 show that the concentrations X of medochlorpamide, trimetazidine and clomiphene in the spiked milk powder of Example 3 and the quantitative ion peak area response value Y have a good linear relationship, and the correlation coefficients are all greater than 0.9900, which meets the requirements.

[0270] Table 7 Recoveries and relative standard deviations (RSD) of medochloramine, trimetazidine and clomiphene in spiked milk powder (n=6)

[0271]

[0272] The recovery rate and relative standard deviation (RSD) results in Table 7 show that the recovery rate of medochlor, trimetazidine and clomiphene in the spiked milk powder with a spiked concentration of 4 μg / kg ranges from 78.8% to 115%; the RSD is between 2.42% and 6.56%; the recovery rate and RSD of the detection method of the present invention are in line with GB / T27404-2008, and have high accuracy and precision.

[0273] 4. Test results of Example 4

[0274] Table 8 Regression equations and correlation coefficients of standard curves of medochloramine, trimetazidine and clomiphene in spiked frozen drinks

[0275] Compound Standard curve regression equation Correlation coefficient Medocaproin Y=702939X+230.598 0.9998 Trimetazidine Y=996185X-5365.95 0.9996 Clomiphene Y=238427X+70.1969 0.9996

[0276] The results of standard curve regression equation and correlation coefficient in Table 8 show that the concentrations X of medochloramine, trimetazidine and clomiphene in the spiked frozen drinks of Example 4 and the quantitative ion peak area response value Y have a good linear relationship, and the correlation coefficients are all greater than 0.9900, which meets the requirements.

[0277] Table 9 Recoveries and relative standard deviations (RSD) of medochloramine, trimetazidine and clomiphene in spiked frozen drinks (n=6)

[0278]

[0279]

[0280] The recovery rate and relative standard deviation (RSD) results in Table 9 show that the recovery rate of medochloramine, trimetazidine and clomiphene in the spiked frozen drinks with a spiked concentration of 2 μg / kg ranges from 75.0% to 101%; the RSD is between 3.10% and 4.69%; the recovery rate and RSD of the detection method of the present invention are in compliance with GB / T 27404-2008, and have high accuracy and precision.

[0281] 5. Test results of Example 5

[0282] Table 10 Regression equations and correlation coefficients of standard curves of medochloramine, trimetazidine and clomiphene in spiked cheese

[0283] Compound Standard curve regression equation Correlation coefficient Medocaproin Y=606151X+1743.07 0.9997 Trimetazidine Y=408057X-352.236 0.9999 Clomiphene Y = 145889X - 2016.58 0.9976

[0284] The results of standard curve regression equation and correlation coefficient in Table 10 show that the concentration X of medochloramine, trimetazidine and clomiphene in the spiked cheese in Example 5 has a good linear relationship with the quantitative ion peak area response value Y, and the correlation coefficients are all greater than 0.9900, which meets the requirements.

[0285] Table 11 Recoveries and relative standard deviations (RSD) of medochloramine, trimetazidine and clomiphene in spiked cheese (n=6)

[0286]

[0287] The recovery rate and relative standard deviation (RSD) results in Table 11 show that the recovery rate of medochlor, trimetazidine and clomiphene in spiked cheese with a spiked concentration of 4 μg / kg ranges from 74.4% to 94.4%; the RSD is between 3.40% and 6.28%; the recovery rate and RSD of the detection method of the present invention are in compliance with GB / T27404-2008, and have high accuracy and precision.

[0288] 6. Test results of Example 6

[0289] Table 12 Mass concentration of medochlor, trimetazidine and clomiphene in dairy products

[0290]

[0291] The results of mass concentration in Table 12 show that through the testing of 25 batches of samples of 5 kinds of dairy products, medochloramine, trimetazidine and clomiphene were not detected.

[0292] 7. Example 1 (spiked concentration of 10 μg / kg), Examples 7-12 and Comparative Examples 5-10 are to optimize the treatment agent in the detection method, and investigate the effects of different treatment agents on the recovery rates of medochloramine, trimetazidine and clomiphene in spiked pure milk (spiked concentration of 10 μg / kg). The test results are as follows:

[0293] Table 13 Recovery and relative standard deviation (RSD) of medochloramine, trimetazidine and clomiphene in spiked pure milk (n=6)

[0294]

[0295]

[0296] The recovery rate and relative standard deviation results in Table 13 show that the recovery rate and relative standard deviation results of the above examples are in line with GB / T 27404-2008, while the recovery rate of the comparative examples is low and does not meet GB / T27404-2008; among them, by comparing comparative examples 5-9 horizontally, it is found that when acetonitrile is used for extraction, the recovery rates of the three compounds are better than those of methanol. In the experiment, it was observed that when the volume ratio of methanol was 50%-100%, the precipitation supernatant was turbid and the precipitation effect was poor. In contrast, the extraction with methanol-acetonitrile (v:v, 10:90) was mixed, the extract was clear, and the extraction recovery effect was better. Good; by comparing Example 1 (spiked concentration of 10 μg / kg), Examples 7-12 and each comparative example, it was found that the addition of formic acid significantly improved the recovery rate of clomiphene, but when the addition amount of formic acid reached 5%, the recovery rate decreased instead, and when the addition amount of formic acid was 0.1%, the recovery rates of the three metabolic regulators were better, the recovery rate of medoamine was 92.4-94.6%; the recovery rate of trimetazidine was 90.4-95.6%; the recovery rate of clomiphene was 99.3-104%.

[0297] 8. Example 1 (spiked concentration of 10 μg / kg) and Examples 13-15 are to optimize the silica gel purification filler in the detection method, and investigate the effects of different silica gel purification filler adsorption treatments on the recovery rates of medochloramine, trimetazidine and clomiphene in spiked pure milk (spiked concentration of 10 μg / kg). The test results are as follows:

[0298] Table 14 Recovery and relative standard deviation (RSD) of medochloramine, trimetazidine and clomiphene in spiked pure milk (n=6)

[0299]

[0300] The results of recovery rate and relative standard deviation in Table 14 show that the silica gel fillers used in the adsorption treatment in the above embodiments are all PSA+C18, and the mass ratios of the two are 1:1, 1:2, 1:3, and 1:4, respectively. The recovery rates of the three metabolic regulators in the above embodiments are very close, and the difference is not significant; and when 50 mg PSA+50 mg C18 is used for purification in Example 1, the recovery rate of medocaine is 92.4-94.6%; the recovery rate of trimetazidine is 90.4-95.6%; and the recovery rate of clomiphene is 99.3-104%; therefore, based on the principle of saving the cost of purification consumables, 50 mg PSA+50 mg C18 is selected to purify the first treatment liquid extracted.

[0301] 9. Example 16. Comparative Examples 1 and 2 are to optimize the chromatographic column in the detection method, and investigate the effects of different chromatographic columns on the separation and retention of medochloramine, trimetazidine and clomiphene in spiked pure milk (spiked concentration of 10 μg / kg). The test results are as follows: Figure 1 , Figure 2 , Figure 3 As shown;

[0302] Figure 1 , Figure 2 , Figure 3 It shows that the retention effect of medochloramine on the chromatographic column of Comparative Examples 1 and 2 is poor, and the retention effect on the chromatographic column of Example 16 is good.

[0303] 10. Example 17, Comparative Examples 3 and 4 are to optimize the mobile phase in the detection method, and investigate the influence of different mobile phases on the separation and retention of medochloramine, trimetazidine and clomiphene in spiked pure milk (spiked concentration is 10 μg / kg). The detection results are as follows Figure 4 , Figure 5 , Figure 6 As shown;

[0304] Figure 4 , Figure 5 , Figure 6 The results show that when formic acid is not added, clomiphene will have a bifurcation phenomenon in the chromatographic peak; when 0.05% formic acid is added, the bifurcation problem of clomiphene chromatographic peak is solved; and when the formic acid concentration is further increased to 0.1%, medochloramine cannot be separated from impurities. Therefore, selecting 20mM ammonium acetate + 0.05% formic acid as the mobile phase A composition can solve the bifurcation problem of clomiphene chromatographic peak and the matrix interference problem of medochloramine at the same time.

[0305] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting medochlor, trimetazidine and clomiphene in dairy products, characterized in that: The following steps are involved: 1) After adding a treatment agent to the dairy product to be tested, the system is subjected to an oscillation treatment and a solid-liquid separation treatment in sequence to obtain a first treatment liquid; the treatment agent comprises formic acid, methanol and acetonitrile, and the volume ratio of the formic acid, methanol and acetonitrile is (0.05-2): (0-50): (50-100); 2) subjecting the first treatment liquid to a silica gel purification filler adsorption treatment to obtain a second treatment liquid; 3) performing membrane filtration on the second treatment liquid to obtain an analyte, and performing liquid chromatography-mass spectrometry analysis on the analyte to obtain an ion peak area of ​​the target substance in the dairy product to be tested; 4) comparing the ion peak area with a standard curve of ion peak area-mass concentration to obtain the mass concentration of the target substance in the dairy product to be tested; In the liquid chromatography-mass spectrometry analysis, the conditions of liquid chromatography are: HILIC columns; Mobile phase A is a mixture of 0.025%-0.075% formic acid and 5-20mM ammonium acetate aqueous solution, mobile phase B is acetonitrile, the eluent flow rate is 0.25-0.45mL / min, and the elution gradient is: 0-1min, 5% mobile phase A; 1-7min, 5%-40% mobile phase A; 7-9min, 40% mobile phase A; 9-9.1min, 40%-5% mobile phase A; 9.1-10min, 5% mobile phase A.

2. The detection method according to claim 1, characterized in that: The volume ratio of formic acid, methanol and acetonitrile is (0.1-1):(0-20):(80-100).

3. The detection method according to claim 2, characterized in that: The volume ratio of formic acid, methanol and acetonitrile is 0.1:10:

90.

4. The detection method according to any one of claims 1 to 3, characterized in that: The HILIC column is Waters ACQUITY BEH HILIC 1.7μm 100×2.1mm column.

5. The detection method according to any one of claims 1 to 4, characterized in that: In the liquid chromatography-mass spectrometry analysis, the mass spectrometry conditions are: Ionization mode: electrospray positive ion (ESI+); in positive ion mode, interface voltage: 4000 V; interface temperature: 300°C; drying gas flow rate: 10 L / min; nebulizing gas flow rate: 3 L / min.

6. The detection method according to any one of claims 1 to 5, characterized in that: Step 1) comprises: after the dairy product to be tested is mixed with water, the treatment agent is added to the mixed system; Wherein, the mass volume ratio of the dairy product to be tested and water is (1-2):(3-5).

7. The detection method according to claim 6, characterized in that: The volume ratio of the mixed system to the treatment agent is (4-7):(20-25).

8. The detection method according to any one of claims 1 to 7, characterized in that: In step 2), the silica gel purification filler adsorption treatment includes oscillating the first treatment liquid using a mixed system of PSA and C18, and then centrifuging the oscillation system to obtain the second treatment liquid.

9. The detection method according to claim 8, characterized in that: The volume mass ratio of the first treatment liquid, PSA and C18 is 5 mL:50 mg:50 mg.

10. The detection method according to any one of claims 1 to 9, characterized in that: In step 3), the filter membrane is a 0.22 μm organic filter membrane.

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