A method for detecting a metabolic modulator in a foodstuff

Abstract

The application discloses a kind of detection methods of metabolic regulator in food, a, preparation of standard working solution;B, preparation of test solution;C, respectively, standard working solution and test solution are injected into liquid chromatography-triple quadrupole mass spectrometer.The application discloses a kind of detection methods of metabolic regulator in food, through specific sample processing and chromatographic conditions and mass spectrometry conditions, three kinds of metabolic regulator in food chloromifene, trimetazidine and medoxomil can be simultaneously detected, the result is accurate, degree of automation is high, fast, sensitivity is high, detection limit is low, and it has very important significance to food safety control.

Description

Technical Field

[0001] This invention relates to the field of doping detection, and more specifically to a method for detecting metabolic regulators in food. Background Technology

[0002] The World Anti-Doping Agency (WADA) classifies metabolic modifiers as S4 category hormones and metabolic modifiers, prohibiting their use both in and out of competition. Clomiphene is an anti-estrogen substance; these drugs can significantly enhance muscle explosiveness and anaerobic performance in a short period, but their use can severely impact performance in events such as swimming and track and field. Trimetazidine and melidomide are two metabolic modifiers that can improve myocardial energy metabolism, thus enhancing athletes' athletic performance. Furthermore, metabolic modifiers are sometimes added to animal feed as veterinary drugs to promote animal growth, posing a risk of accidental ingestion by athletes. Therefore, the China Anti-Doping Agency recommends testing for clomiphene, trimetazidine, and melidomide in food-borne doping events at Level 1 competitions. Testing should be conducted on pork, beef, lamb, poultry, eggs, dairy products, and aquatic products. In addition to animal-derived foods, trimetazidine testing should also include all processed cooking seasonings (such as oil, salt, soy sauce, vinegar, cooking wine, MSG, chicken essence, oyster sauce, etc.).

[0003] Currently, most detection methods for clomiphene, trimetazidine, and melidomide are based on drug, urine, and blood matrices, with few reported methods for detection in food. Due to the complexity of food samples and significant matrix interference, accurate detection is difficult, and there is currently no method using liquid chromatography-mass spectrometry (LC-MS) for accurate detection of these three metabolic regulators. Summary of the Invention

[0004] To address the above problems, this invention provides a method for detecting metabolic regulators in food, comprising the following steps:

[0005] a. Preparation of standard working solution: Take the metabolic regulator standard, dissolve and dilute it with methanol to form a solution, then add the internal standard solution and acetonitrile aqueous solution containing formic acid and mix well to obtain the solution;

[0006] b. Preparation of test solution: Take the solid or semi-solid sample to be tested, add internal standard solution, water and acetonitrile solution containing formic acid to extract, centrifuge, take the supernatant and pass it through a solid phase extraction column, collect the filtrate and dry it, then add acetonitrile aqueous solution containing formic acid to dissolve it, and the test solution is obtained.

[0007] Alternatively: Take the liquid sample to be tested, add internal standard solution and acetonitrile solution containing formic acid to extract, centrifuge, take the supernatant and pass it through a solid phase extraction column, collect the filtrate, dry it, and then dissolve it in an aqueous solution of acetonitrile containing formic acid to obtain the final product;

[0008] c. Inject the standard working solution and the test solution into the liquid chromatography-triple quadrupole mass spectrometer, respectively.

[0009] The chromatographic conditions are as follows:

[0010] Column: Ethylene-bridged hybrid particles as packing material; 20 mmol / L ammonium formate solution as mobile phase A, 0.1% formic acid acetonitrile solution as mobile phase B, gradient elution program as follows:

[0011]

[0012] Mass spectrometry conditions: Ion source: electrospray ionization source; Mass spectrometry detection mode: multi-ion reaction monitoring; Scan mode: positive ion mode scan;

[0013] The internal standard solution is a methanol solution of a deuterated metabolism regulator standard;

[0014] The metabolic regulator standards are clomiphene citrate, trimetazidine, and / or metronidazole;

[0015] The deuterium-labeled metabolic regulators are clomiphene-d5, trimetazidine-d8, and / or melidomine-d3.

[0016] Further, the volume ratio of the solution, internal standard solution, and acetonitrile aqueous solution containing formic acid in step a is 0.05–2:0.5:7.5–9.45; the concentration of the solution is 0.2 μg / mL to 1 mg / mL.

[0017] Further, in step b, the mass-to-volume ratio of the solid or semi-solid sample to be tested, the internal standard solution, water, and the acetonitrile solution containing formic acid is 1–5 g: 0.1 ml: 1–5 ml: 8–9 ml, preferably 2 g: 0.1 ml: 1 ml: 9 ml; the mass-to-volume ratio of the liquid sample to be tested, the internal standard solution, and the acetonitrile solution containing formic acid is 1–5 g: 0.1 ml: 8–10 ml, preferably 2 g: 0.1 ml: 8 or 10 ml; the concentration of formic acid in the acetonitrile solution containing formic acid is 0.5%–5%, v / v, preferably 1%, v / v.

[0018] Furthermore, the deuterated metabolism regulator standard is clomiphene hydrochloride-d5, trimetazidine dihydrochloride-d8, and / or melidomine-d3; the concentration of the deuterated metabolism regulator standard in the internal standard solution is 0.2-1 μg / mL, preferably clomiphene-d5 0.2 μg / mL, trimetazidine-d8 0.2 μg / mL, and melidomine-d3 1.0 μg / mL.

[0019] Further, the extraction in step b involves vortexing for 5 minutes followed by sonication for 30 minutes; centrifugation for 3–8 minutes at a speed of 5000–10000 r / min, preferably 5 minutes at a speed of 8000 r / min; the solid-phase extraction column is a Prime HLB column; the drying is carried out by nitrogen blowing at 40°C; and the amount of acetonitrile aqueous solution containing formic acid added is 1 / 4 to 1 / 5 of the filtrate volume.

[0020] Furthermore, the concentration of formic acid in the formic acid-containing acetonitrile aqueous solution is 0.05% to 5% v / v, preferably 0.1% v / v, and the volume ratio of acetonitrile to water is 9:1.

[0021] Further, the chromatographic column in step c is an ACQUITY UPLC BEH Amide, 3.0×100mm, 1.7μm; the chromatographic conditions are: flow rate 0.4mL / min, column temperature 40℃, injection volume 2μL, and mobile phase A is a 20mmol / L ammonium formate solution containing 0.1% formic acid.

[0022] Further, in step c, the mass spectrometry conditions are as follows: drying gas temperature 300℃, drying gas flow rate 11L / min, nebulizing gas pressure 35psi, sheath gas temperature 250℃, sheath gas flow rate 11L / min, and capillary voltage 3500V.

[0023] Further, in the mass spectrometry conditions described in step c, the qualitative detection ion pair of clomiphene is m / z 406.2→m / z 71.9, fragmentation voltage 120V, collision energy 40V; the quantitative detection ion pair is m / z 406.2→m / z 99.8, fragmentation voltage 120V, collision energy 24V; and the quantitative detection ion pair of clomiphene-d5 is m / z 411.1→m / z 58.1, fragmentation voltage 120V, collision energy 40V.

[0024] Trimetazidine qualitative detection of ion pairs: m / z 267.2→m / z 165.8, fragmentation voltage 120V, collision energy 28V; quantitative detection of ion pairs: m / z 267.2→m / z 180.8, fragmentation voltage 120V, collision energy 12V; Trimetazidine-d8 quantitative detection of ion pairs: m / z 275.3→m / z 181, fragmentation voltage 120V, collision energy 12V.

[0025] For qualitative detection of ion pairs using mediocrene, the m / z range is 147.1 to 58.9, with a fragmentation voltage of 120V and a collision energy of 20V. For quantitative detection of ion pairs, the m / z range is 147.1 to 57.9, with a fragmentation voltage of 120V and a collision energy of 32V. For quantitative detection of ion pairs using mediocrene-d3, the m / z range is 149.8 to 61.1, with a fragmentation voltage of 120V and a collision energy of 32V.

[0026] Furthermore, if the chromatogram of the test sample solution shows chromatographic peaks with retention times consistent with those of the qualitative and quantitative ions in the chromatogram of the standard working solution, and the deviation of the relative abundance of the qualitative and quantitative ions of the chromatographic peaks from that of the chromatograms of the equivalent concentration standard working solution does not exceed the maximum permissible deviation range, then the sample to be tested is confirmed to contain a metabolic regulator; the maximum permissible deviation range is as follows:

[0027]

[0028] Furthermore, the content of metabolic regulators in the solid, semi-solid, or liquid samples to be tested is calculated by peak area using the external standard method.

[0029] The "equivalent concentration standard solution" mentioned in this invention refers to a metabolic regulator whose concentration is equal to the metabolic regulator concentration in the test sample solution. + 30% standard working solution.

[0030] The term "semi-solid" as used in this invention is a very common and widely used professional term in the food industry. Semi-solid foods have a higher viscosity than liquid foods, but unlike solids, they are fluid substances. Examples include mayonnaise, salad dressing, nut butter, seed puree, chili sauce, hot pot base, spicy hot pot base, chili oil, ketchup, and shrimp paste.

[0031] This invention discloses a method for detecting metabolic regulators in food. Through specific sample processing, chromatographic conditions, and mass spectrometry conditions, it can simultaneously detect three metabolic regulators—clomiphene, trimetazidine, and meldane—in animal-derived foods. The method is accurate, highly automated, rapid, highly sensitive, and has a low detection limit. It provides an effective guarantee for the accurate detection of metabolic regulator content in animal-derived foods, ensuring food safety and showing promising application prospects.

[0032] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0033] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0034] Figure 1 Technical route for the determination of clomiphene, trimetazidine, and meldamine in food products

[0035] Figure 2Multiple reaction monitoring (MRM) chromatograms of clomiphene and clomiphene-d5

[0036] Figure 3 Multiple reaction monitoring (MRM) chromatograms of trimetazidine and trimetazidine-d8

[0037] Figure 4 Multiple reaction monitoring (MRM) chromatograms of meldonium and meldonium-d3

[0038] Figure 5 Investigation of clomiphene extraction solvent

[0039] Figure 6 Investigation of extracting solvents for trimetazidine

[0040] Figure 7 Study on the extraction solvent of methamphetamine

[0041] Figure 8 chlormiphene extraction solvent volume study

[0042] Figure 9 Investigation of the extraction solvent volume of trimetazidine

[0043] Figure 10 Investigation of the volume of solvent for the extraction of methamphetamine

[0044] Figure 11 Clomiphen extraction time study

[0045] Figure 12 Investigation of trimetazidine extraction time

[0046] Figure 13 Study on extraction time of methaqualone

[0047] Figure 14 Investigation of clomiphene purification methods

[0048] Figure 15 Investigation of Trimetazidine Purification Methods

[0049] Figure 16 Investigation of Ammonium Purification Methods

[0050] Figure 17 Matrix effect investigation Detailed Implementation

[0051] Example 1: Detection of metabolic regulators in livestock and poultry meat and their products, poultry egg products, dairy products (excluding liquid milk), aquatic products, and cooking seasonings (solid and semi-solid).

[0052] (I) Reagent Preparation

[0053] 1% Formic Acid Acetonitrile Solution: Transfer 10.0 mL of formic acid and dilute to 1000 mL with acetonitrile, then mix well.

[0054] 0.1% Formic Acid Acetonitrile Solution: Transfer 1.0 mL of formic acid and dilute to 1000 mL with acetonitrile, then mix well.

[0055] 0.1% formic acid aqueous solution: Transfer 1.0 mL of formic acid, dilute with water to 1000 mL, and mix well.

[0056] 0.1% Formic Acid Acetonitrile Aqueous Solution (9+1): Measure 900 mL of 0.1% Formic Acid Acetonitrile and 100 mL of 0.1% Formic Acid Aqueous Solution, and mix well.

[0057] Internal standard solutions: Accurately weigh clomiphene hydrochloride-d5, trimetazidine dihydrochloride-d8 and melidomine-d3 standards (accurate to 0.01 mg) into different 10 mL volumetric flasks, dissolve them in methanol and dilute to volume to prepare internal standard stock solutions with a concentration of 100 μg / mL for each of clomiphene-d5, trimetazidine-d8 and melidomine-d3.

[0058] Accurately pipette 0.2 mL, 0.2 mL, and 1.0 mL of clomiphene-d5, trimetazidine-d8, and medocalonium-d3 internal standard stock solutions into the same 10 mL volumetric flask, respectively, and dilute to the mark with methanol. Shake well to prepare mixed internal standard intermediate solution I with concentrations of 2.0 μg / mL, 2.0 μg / mL, and 10.0 μg / mL for clomiphene-d5, trimetazidine-d8, and medocalonium-d3, respectively.

[0059] Accurately pipette 1.0 mL of the mixed internal standard intermediate solution I into a 10 mL volumetric flask, dilute to the mark with methanol, shake well, and prepare a mixed internal standard solution with concentrations of 0.2 μg / mL for clomiphene-d5, 0.2 μg / mL for trimetazidine-d8, and 1.0 μg / mL for methaqualone-d3.

[0060] (II) Preparation of standard working solutions

[0061] Accurately weigh clomiphene citrate, trimetazidine, and melidomide standards (accurate to 0.01 mg) into different 10 mL volumetric flasks, dissolve them in methanol, and dilute to volume to prepare standard stock solutions with a concentration of 1 mg / mL for each of clomiphene, trimetazidine, and melidomide.

[0062] Accurately pipette each standard stock solution into different 10 mL volumetric flasks, dilute to the mark with methanol, shake well, and prepare standard intermediate solutions with a concentration of 10 μg / mL for clomiphene, trimetazidine, and methaqualone.

[0063] Accurately pipette 0.2 mL, 0.2 mL, and 1.0 mL of the intermediate standard solutions of clomiphene, trimetazidine, and melidomide into the same 10 mL volumetric flask, respectively, and dilute to the mark with methanol. Shake well to prepare a mixed intermediate standard solution with concentrations of 0.2 μg / mL, 0.2 μg / mL, and 1.0 μg / mL of clomiphene, trimetazidine, and melidomide, respectively.

[0064] Pipette 0.05 mL, 0.1 mL, 0.2 mL, 0.5 mL, 1.0 mL, and 2.0 mL of the mixed standard intermediate solution into 10 mL volumetric flasks, respectively. Add 0.5 mL of the mixed internal standard solution, and dilute to the mark with 0.1% formic acid acetonitrile aqueous solution (9+1). Shake well to obtain standard working solutions with concentrations of clomiphene and trimetazidine of 1 ng / mL, 2 ng / mL, 4 ng / mL, 10 ng / mL, 20 ng / mL, and 40 ng / mL, and melidomide concentrations of 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL. The concentrations of clomiphene-d5 and trimetazidine-d8 are both 10 ng / mL, and the concentration of melidomide-d3 is 50 ng / mL.

[0065] (III) Preparation of the test solution

[0066] Accurately weigh 2g of the pulverized and homogenized sample (accurate to 0.01g) into a centrifuge tube, add 0.10mL of mixed internal standard solution, add two ceramic homogenates, add 1mL of water, shake well, then add 9mL of 1% formic acid acetonitrile solution, vortex for 5min, sonicate for 30min, shaking twice during extraction, centrifuge at 8000r / min for 5min, take the supernatant and pass it through a Prime HLB column, collect the filtrate in a nitrogen blow-off tube, mix well, take 5mL of the supernatant and blow it with nitrogen at 40℃ until nearly dry, make up to 1mL with 0.1% formic acid acetonitrile aqueous solution, mix well, pass through a 0.22μm organic filter membrane, and then test on the instrument.

[0067] (iv) Testing

[0068] Inject the standard working solution and the test solution into the liquid chromatography-triple quadrupole mass spectrometer, respectively.

[0069] The chromatographic conditions are as follows:

[0070] Chromatographic column: ACQUITY UPLC BEH Amide, 3.0 × 100 mm, 1.7 μm; mobile phase A: 20 mmol / L ammonium formate solution (containing 0.1% formic acid); mobile phase B: 0.1% formic acid acetonitrile solution; flow rate: 0.4 mL / min; column temperature: 40℃; injection volume: 2 μL; gradient elution program:

[0071]

[0072]

[0073] Mass spectrometry conditions: Ion source: electrospray ionization source; Mass spectrometry detection mode: multiple ion reaction monitoring; Scanning method:

[0074] Positive ion mode scanning; Drying gas temperature: 300℃; Drying gas flow rate: 11L / min;

[0075] Nebulizer gas pressure: 35 psi; sheath gas temperature: 250℃; sheath gas flow rate: 11 L / min; capillary voltage: 3500 V; monitored ion pairs and corresponding mass spectrometry parameters are as follows:

[0076]

[0077] Note: 1. "*" indicates quantitative ions.

[0078] (V) Qualitative Determination

[0079] If the chromatogram of the test sample solution shows chromatographic peaks with retention times consistent with those of the qualitative and quantitative ions in the chromatogram of the standard working solution, and the deviation of the relative abundance ratio of the qualitative and quantitative ions in the chromatogram of the peaks from that in the chromatogram of the standard working solution of equivalent concentration does not exceed the specified maximum permissible deviation range, then the test sample contains the metabolic regulators clomiphene, trimetazidine, and / or methaqualone. The specified maximum permissible deviation ranges are as follows:

[0080]

[0081] (vi) Quantitative determination

[0082] When the test sample is found to contain the metabolic regulators clomiphene, trimetazidine, and / or melidomine, a standard curve is plotted with the ratio of the concentration of the analyte to the concentration of the corresponding deuterated internal standard in the standard working solution as the x-axis and the ratio of the peak area of ​​the quantitative ion of the analyte to the peak area of ​​the corresponding deuterated internal standard as the y-axis, thus obtaining a linear regression equation. The concentration of the metabolic regulator in the test sample solution is calculated based on the linear regression equation, and then the content of the metabolic regulator in the test sample is calculated using the following formula.

[0083]

[0084] In the formula:

[0085] X—The content of the metabolic regulator to be tested in the sample, in micrograms per kilogram (μg / kg);

[0086] ρ—The concentration of the metabolic regulator to be tested in the test solution obtained from the standard curve, in nanograms per milliliter (ng / mL);

[0087] V1—Extractant volume (10), in milliliters (mL);

[0088] V2—Volume of extractant transferred (5), in milliliters (mL);

[0089] V3—The final volume of the purified and concentrated sample solution (1), in milliliters (mL);

[0090] m—mass of the sample (2), in grams (g);

[0091] f—Dilution factor (1).

[0092] Example 2: Detection of metabolic regulators in poultry eggs, liquid milk, and cooking seasonings (liquid)

[0093] (I) Reagent Preparation

[0094] 1% Formic Acid Acetonitrile Solution: Transfer 10.0 mL of formic acid and dilute to 1000 mL with acetonitrile, then mix well.

[0095] 0.1% Formic Acid Acetonitrile Solution: Transfer 1.0 mL of formic acid and dilute to 1000 mL with acetonitrile, then mix well.

[0096] 0.1% formic acid aqueous solution: Transfer 1.0 mL of formic acid, dilute with water to 1000 mL, and mix well.

[0097] 0.1% Formic Acid Acetonitrile Aqueous Solution (9+1): Measure 900 mL of 0.1% Formic Acid Acetonitrile and 100 mL of 0.1% Formic Acid Aqueous Solution, and mix well.

[0098] Internal standard solutions: Accurately weigh clomiphene hydrochloride-d5, trimetazidine dihydrochloride-d8 and melidomine-d3 standards (accurate to 0.01 mg) into different 10 mL volumetric flasks, dissolve them in methanol and dilute to volume to prepare internal standard stock solutions with a concentration of 100 μg / mL for each of clomiphene-d5, trimetazidine-d8 and melidomine-d3.

[0099] Accurately pipette 0.2 mL, 0.2 mL, and 1.0 mL of clomiphene-d5, trimetazidine-d8, and medocalonium-d3 internal standard stock solutions into the same 10 mL volumetric flask, respectively, and dilute to the mark with methanol. Shake well to prepare mixed internal standard intermediate solution I with concentrations of 2.0 μg / mL, 2.0 μg / mL, and 10.0 μg / mL for clomiphene-d5, trimetazidine-d8, and medocalonium-d3, respectively.

[0100] Accurately pipette 1.0 mL of the mixed internal standard intermediate solution I into a 10 mL volumetric flask, dilute to the mark with methanol, shake well, and prepare a mixed internal standard solution with concentrations of 0.2 μg / mL for clomiphene-d5, 0.2 μg / mL for trimetazidine-d8, and 1.0 μg / mL for methaqualone-d3.

[0101] (II) Preparation of standard working solutions

[0102] Accurately weigh clomiphene citrate, trimetazidine, and melidomide standards (accurate to 0.01 mg) into different 10 mL volumetric flasks, dissolve them in methanol, and dilute to volume to prepare standard stock solutions with a concentration of 1 mg / mL for each of clomiphene, trimetazidine, and melidomide.

[0103] Accurately pipette each standard stock solution into different 10 mL volumetric flasks, dilute to the mark with methanol, shake well, and prepare standard intermediate solutions with a concentration of 10 μg / mL for clomiphene, trimetazidine, and methaqualone.

[0104] Accurately pipette 0.2 mL, 0.2 mL, and 1.0 mL of the intermediate standard solutions of clomiphene, trimetazidine, and melidomide into the same 10 mL volumetric flask, respectively, and dilute to the mark with methanol. Shake well to prepare a mixed intermediate standard solution with concentrations of 0.2 μg / mL, 0.2 μg / mL, and 1.0 μg / mL of clomiphene, trimetazidine, and melidomide, respectively.

[0105] Pipette 0.05 mL, 0.1 mL, 0.2 mL, 0.5 mL, 1.0 mL, and 2.0 mL of the mixed standard intermediate solution into 10 mL volumetric flasks, respectively. Add 0.5 mL of the mixed internal standard solution, and dilute to the mark with 0.1% formic acid acetonitrile aqueous solution (9+1). Shake well to obtain standard working solutions with concentrations of clomiphene and trimetazidine of 1 ng / mL, 2 ng / mL, 4 ng / mL, 10 ng / mL, 20 ng / mL, and 40 ng / mL, and melidomide concentrations of 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL. The concentrations of clomiphene-d5 and trimetazidine-d8 are both 10 ng / mL, and the concentration of melidomide-d3 is 50 ng / mL.

[0106] (III) Preparation of the test solution

[0107] Accurately weigh 2g of the pulverized and homogenized sample (accurate to 0.01g) into a centrifuge tube, add 0.10mL of mixed internal standard solution, add two ceramic homogenates, add 8mL of 1% formic acid acetonitrile solution, vortex for 5min, sonicate for 30min, shaking twice during extraction, centrifuge at 8000r / min for 5min, take the supernatant and pass it through a Prime HLB column, collect the filtrate in a nitrogen blow-off tube, mix well, take 5mL of the supernatant and blow it with nitrogen at 40℃ until nearly dry, make up to 1mL with 0.1% formic acid acetonitrile aqueous solution, mix well, pass through a 0.22μm organic filter membrane, and then test on the instrument.

[0108] (iv) Testing

[0109] Inject the standard working solution and the test solution into the liquid chromatography-triple quadrupole mass spectrometer, respectively.

[0110] The chromatographic conditions are as follows:

[0111] Chromatographic column: ACQUITY UPLC BEH Amide, 3.0 × 100 mm, 1.7 μm; mobile phase A: 20 mmol / L ammonium formate solution (containing 0.1% formic acid); mobile phase B: 0.1% formic acid acetonitrile solution; flow rate: 0.4 mL / min; column temperature: 40℃; injection volume: 2 μL; gradient elution program:

[0112]

[0113] Mass spectrometry conditions: Ion source: electrospray ionization source; Mass spectrometry detection mode: multiple ion reaction monitoring; Scanning method:

[0114] Positive ion mode scanning; Drying gas temperature: 300℃; Drying gas flow rate: 11L / min;

[0115] Nebulizer gas pressure: 35 psi; sheath gas temperature: 250℃; sheath gas flow rate: 11 L / min; capillary voltage: 3500 V; monitored ion pairs and corresponding mass spectrometry parameters are as follows:

[0116]

[0117] Note: 1. "*" indicates quantitative ions.

[0118] (V) Qualitative Determination

[0119] If the chromatogram of the test sample solution shows chromatographic peaks with retention times of qualitative and quantitative ions consistent with those of the standard working solution, and the relative abundance ratio of qualitative and quantitative ions in the chromatogram peaks deviates from that of the equivalent concentration standard working solution within the specified maximum permissible deviation range, then the test sample contains the metabolic regulators clomiphene, trimetazidine, and / or methaqualone. The specified maximum permissible deviation ranges are as follows:

[0120]

[0121] (vi) Quantitative determination

[0122] When the test sample is found to contain the metabolic regulators clomiphene, trimetazidine, and / or melidomine, a standard curve is plotted with the ratio of the concentration of the analyte to the concentration of the corresponding deuterated internal standard in the standard working solution as the x-axis and the ratio of the peak area of ​​the quantitative ion of the analyte to the peak area of ​​the corresponding deuterated internal standard as the y-axis, thus obtaining a linear regression equation. The concentration of the metabolic regulator in the test sample solution is calculated based on the linear regression equation, and then the content of the metabolic regulator in the test sample is calculated using the following formula.

[0123]

[0124] In the formula:

[0125] X—The content of the metabolic regulator to be tested in the sample, in micrograms per kilogram (μg / kg);

[0126] ρ—The concentration of the metabolic regulator to be tested in the test solution obtained from the standard curve, in nanograms per milliliter (ng / mL);

[0127] V1—Extractant volume (8), in milliliters (mL);

[0128] V2—Volume of extractant transferred (5), in milliliters (mL);

[0129] V3—The final volume of the purified and concentrated sample solution (1), in milliliters (mL);

[0130] m—mass of the sample (2), in grams (g);

[0131] f—Dilution factor (1).

[0132] Example 3: Detection of metabolic regulators in edible vegetable oils

[0133] (I) Reagent Preparation

[0134] 1% Formic Acid Acetonitrile Solution: Transfer 10.0 mL of formic acid and dilute to 1000 mL with acetonitrile, then mix well.

[0135] 0.1% Formic Acid Acetonitrile Solution: Transfer 1.0 mL of formic acid and dilute to 1000 mL with acetonitrile, then mix well.

[0136] 0.1% formic acid aqueous solution: Transfer 1.0 mL of formic acid, dilute with water to 1000 mL, and mix well.

[0137] 0.1% Formic Acid Acetonitrile Aqueous Solution (9+1): Measure 900 mL of 0.1% Formic Acid Acetonitrile and 100 mL of 0.1% Formic Acid Aqueous Solution, and mix well.

[0138] Internal standard solutions: Accurately weigh the standards (accurate to 0.01 mg) into different 10 mL volumetric flasks, dissolve them in methanol and dilute to volume to prepare internal standard stock solutions with a concentration of 100 μg / mL for clomiphene-d5, trimetazidine-d8 and melidomine-d3.

[0139] Accurately pipette 0.2 mL, 0.2 mL, and 1.0 mL of clomiphene-d5, trimetazidine-d8, and medocalonium-d3 internal standard stock solutions into the same 10 mL volumetric flask, respectively, and dilute to the mark with methanol. Shake well to prepare mixed internal standard intermediate solution I with concentrations of 2.0 μg / mL, 2.0 μg / mL, and 10.0 μg / mL for clomiphene-d5, trimetazidine-d8, and medocalonium-d3, respectively.

[0140] Accurately pipette 1.0 mL of the mixed internal standard intermediate solution I into a 10 mL volumetric flask, dilute to the mark with methanol, shake well, and prepare a mixed internal standard solution with concentrations of 0.2 μg / mL for clomiphene-d5, 0.2 μg / mL for trimetazidine-d8, and 1.0 μg / mL for methaqualone-d3.

[0141] (II) Preparation of standard working solutions

[0142] Accurately weigh the standards (accurate to 0.01 mg) into different 10 mL volumetric flasks, dissolve them in methanol and dilute to volume to prepare standard stock solutions of clomiphene, trimetazidine and methaqualone, each with a concentration of 1 mg / mL;

[0143] Accurately pipette each standard stock solution into different 10 mL volumetric flasks, dilute to the mark with methanol, shake well, and prepare standard intermediate solutions with a concentration of 10 μg / mL for clomiphene, trimetazidine, and methaqualone.

[0144] Accurately pipette 0.2 mL, 0.2 mL, and 1.0 mL of the intermediate standard solutions of clomiphene, trimetazidine, and melidomide into the same 10 mL volumetric flask, respectively, and dilute to the mark with methanol. Shake well to prepare a mixed intermediate standard solution with concentrations of 0.2 μg / mL, 0.2 μg / mL, and 1.0 μg / mL of clomiphene, trimetazidine, and melidomide, respectively.

[0145] Pipette 0.05 mL, 0.1 mL, 0.2 mL, 0.5 mL, 1.0 mL, and 2.0 mL of the mixed standard intermediate solution into 10 mL volumetric flasks, respectively. Add 0.5 mL of the mixed internal standard solution, and dilute to the mark with 0.1% formic acid acetonitrile aqueous solution (9+1). Shake well to obtain standard working solutions with concentrations of clomiphene and trimetazidine of 1 ng / mL, 2 ng / mL, 4 ng / mL, 10 ng / mL, 20 ng / mL, and 40 ng / mL, and melidomide concentrations of 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL. The concentrations of clomiphene-d5 and trimetazidine-d8 are both 10 ng / mL, and the concentration of melidomide-d3 is 50 ng / mL.

[0146] (III) Preparation of the test solution

[0147] Accurately weigh 2g of mixed edible vegetable oil (accurate to 0.01g) into a centrifuge tube, add 0.10mL of mixed internal standard intermediate solution II, add two ceramic homogenants, add 10mL of 1% formic acid acetonitrile solution, vortex for 5min, sonicate for 30min, shaking twice during extraction, centrifuge at 8000r / min for 5min, take the supernatant and pass it through a Prime HLB column, collect the filtrate in a nitrogen blow-off tube, mix well, take 5mL of the supernatant and blow it with nitrogen at 40℃ until nearly dry, make up to 1mL with 0.1% formic acid acetonitrile aqueous solution, mix well, pass through a 0.22μm organic filter membrane, and then test.

[0148] (iv) Testing

[0149] Inject the standard working solution and the test solution into the liquid chromatography-triple quadrupole mass spectrometer, respectively.

[0150] The chromatographic conditions are as follows:

[0151] Chromatographic column: ACQUITY UPLC BEH Amide, 3.0 × 100 mm, 1.7 μm; mobile phase A: 20 mmol / L ammonium formate solution (containing 0.1% formic acid); mobile phase B: 0.1% formic acid acetonitrile solution; flow rate: 0.4 mL / min; column temperature: 40℃; injection volume: 2 μL; gradient elution program:

[0152]

[0153] Mass spectrometry conditions: Ion source: electrospray ionization source; Mass spectrometry detection mode: multiple ion reaction monitoring; Scanning method:

[0154] Positive ion mode scanning; Drying gas temperature: 300℃; Drying gas flow rate: 11L / min;

[0155] Nebulizer gas pressure: 35 psi; sheath gas temperature: 250℃; sheath gas flow rate: 11 L / min; capillary voltage: 3500 V; monitored ion pairs and corresponding mass spectrometry parameters are as follows:

[0156]

[0157]

[0158] Note: 1. "*" indicates quantitative ions.

[0159] (V) Qualitative Determination

[0160] If the chromatogram of the test sample solution shows chromatographic peaks with retention times of qualitative and quantitative ions consistent with those of the standard working solution, and the relative abundance ratio of qualitative and quantitative ions in the chromatogram peaks deviates from that of the equivalent concentration standard working solution within the specified maximum permissible deviation range, then the test sample contains the metabolic regulators clomiphene, trimetazidine, and / or methaqualone. The specified maximum permissible deviation ranges are as follows:

[0161]

[0162] (vi) Quantitative determination

[0163] When the test sample is found to contain the metabolic regulators clomiphene, trimetazidine, and / or melidomine, a standard curve is plotted with the ratio of the concentration of the analyte to the concentration of the corresponding deuterated internal standard in the standard working solution as the x-axis and the ratio of the peak area of ​​the quantitative ion of the analyte to the peak area of ​​the corresponding deuterated internal standard as the y-axis, thus obtaining a linear regression equation. The concentration of the metabolic regulator in the test sample solution is calculated based on the linear regression equation, and then the content of the metabolic regulator in the test sample is calculated using the following formula.

[0164]

[0165] In the formula:

[0166] X — The content of the metabolic regulator to be tested in edible vegetable oil, in micrograms per kilogram (μg / kg);

[0167] ρ—The concentration of the metabolic regulator to be tested in the test solution obtained from the standard curve, in nanograms per milliliter (ng / mL);

[0168] V1—Extractant volume (10), in milliliters (mL);

[0169] V2—Volume of extractant transferred (5), in milliliters (mL);

[0170] V3—The final volume of the purified and concentrated sample solution (1), in milliliters (mL);

[0171] m—mass of edible vegetable oil (2), in grams (g);

[0172] f—Dilution factor (1).

[0173] The following experimental examples further illustrate the beneficial effects of the present invention.

[0174] Experimental Example 1: Study on the detection method of metabolic regulators in food

[0175] (I) Main Research Content of Methodology

[0176] 1. Main information of the test object

[0177] The Chinese names, English names, CAS numbers, molecular formulas, relative molecular masses, and structural formulas of clomiphene, trimetazidine, and methaqualone are shown in Table 1.

[0178] Table 1. Chinese Name, English Name, CAS Number, Molecular Formula, Relative Molecular Mass, Structural Formula

[0179]

[0180] 2. Research Technical Route

[0181] In this study, a versatile extraction reagent was selected for sample pretreatment for extraction and concentration. Qualitative and quantitative determination were then performed using liquid chromatography-triple quadrupole mass spectrometry (LC-MS / MS). For detailed technical procedures, please refer to [link to technical details]. Figure 1 .

[0182] II) Research Process

[0183] 1. Determination of conditions for liquid chromatography-triple quadrupole mass spectrometry

[0184] In this study, an Agilent 1290+6470 liquid chromatography-triple quadrupole mass spectrometry (LC-MS / MS) system (Agilent Technologies, USA) was selected to optimize and establish the LC-MS / MS conditions. Two validation personnel then used Waters TQ-S and AB5500 instruments respectively for method validation, and the method suitability met the requirements in both cases.

[0185] 1.1 Preparation of standard solutions

[0186] 1.1.1 Standards: Clomiphene citrate, purity ≥99%; Trimetazidine, purity ≥99%; Melidomine, purity ≥99%; Clomiphene-D5 hydrochloride, purity ≥98%; Trimetazidine dihydrochloride-D8, purity ≥98%; Melidomine-D3, purity ≥98%.

[0187] 1.1.2 Standard Stock Solutions (1 mg / mL): Accurately weigh appropriate amounts of the standards (accurate to 0.1 mg) into 10 mL volumetric flasks, dissolve them in methanol, and dilute to volume to prepare standard stock solutions of clomiphene (after citrate conversion), trimetazidine, and melidomine, each with a concentration of 1 mg / mL. Transfer the solutions to brown glass containers and store below -18°C. Shelf life is 3 months.

[0188] 1.1.3 Isotope Internal Standard Stock Solutions (100 μg / mL): Accurately weigh appropriate amounts of the standards (accurate to 0.1 mg) into 10 mL volumetric flasks, dissolve them in methanol, and dilute to volume to prepare isotope internal standard stock solutions with concentrations of 100 μg / mL for clomiphene-d5 (after conversion to hydrochloric acid), trimetazidine-d8, and meldane-d3. Transfer the solutions to amber glass containers and store below -18°C. The shelf life is 3 months.

[0189] 1.1.4 Standard Intermediate Working Solutions (10 μg / mL): Accurately pipette appropriate amounts of each standard stock solution (1.1.2) into 10 mL volumetric flasks, dilute to the mark with methanol, and mix well to prepare standard intermediate working solutions with a concentration of 10 μg / mL for clomiphene, trimetazidine, and melidomide. Transfer the solutions to amber glass containers and store below -18°C. Shelf life is one month.

[0190] 1.1.5 Mixed Standard Intermediate Working Solution: Accurately pipette 0.2 mL, 0.2 mL, and 1.0 mL of clomiphene, trimetazidine, and melidomide standard intermediate working solutions (1.1.5) into the same 10 mL volumetric flask, dilute to the mark with methanol, and shake well to prepare mixed standard intermediate working solutions with concentrations of 0.2 μg / mL, 0.2 μg / mL, and 1.0 μg / mL for clomiphene, trimetazidine, and melidomide, respectively. Prepare fresh before use.

[0191] 1.1.6 Mixed Isotope Internal Standard Intermediate Working Solution I: Accurately pipette 0.2 mL, 0.2 mL, and 1.0 mL of the clomiphene-d5, trimetazidine-d8, and medocyanine-d3 isotope internal standard stock solutions (1.1.3) into the same 10 mL volumetric flask. Dilute to the mark with methanol, shake well, and prepare mixed isotope internal standard intermediate working solution I with concentrations of 2.0 μg / mL for clomiphene-d5, 2.0 μg / mL for trimetazidine-d8, and 10.0 μg / mL for medocyanine-d3. Transfer the solution to a brown glass container and store below -18°C. Shelf life is 1 month.

[0192] 1.1.7 Mixed Isotope Internal Standard Intermediate Working Solution II: Accurately pipette 1 mL of Mixed Isotope Internal Standard Intermediate Working Solution I (1.1.6) into a 10 mL volumetric flask, dilute to the mark with methanol, shake well, and prepare Mixed Isotope Internal Standard Intermediate Working Solution II with concentrations of 0.2 μg / mL for clomiphene-d5, 0.2 μg / mL for trimetazidine-d8, and 1.0 μg / mL for methaqualone-d3. Prepare fresh before use.

[0193] 1.1.8 Standard Working Solutions: Pipette 0.5 mL, 0.1 mL, 0.2 mL, 0.5 mL, 1.0 mL, and 2.0 mL of the mixed standard intermediate working solution (1.1.5) into 10 mL volumetric flasks, respectively. Add 0.5 mL of the mixed isotope internal standard intermediate working solution II (1.1.7) to each volumetric flask. Dilute to the mark with 0.1% formic acid acetonitrile aqueous solution (9+1), and mix well. This yields trimetazidine and clomiphene concentrations of 1 ng / mL and 2 ng / mL, respectively. Standard curves with concentrations of L, 4 ng / mL, 10 ng / mL, 20 ng / mL, and 40 ng / mL, and melidomine concentrations of 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL, respectively. The concentrations of clomiphene-d5 and trimetazidine-d8 are 10 ng / mL, and the concentration of melidomine-d3 is 50 ng / mL. Standards should be prepared fresh before use or according to the instrument response.

[0194] 1.2 Selection of Chromatographic Column

[0195] Conventional liquid chromatography (LC) columns typically use packing materials with a particle size of 5 μm, characterized by long equilibration times, large mobile phase volumes, and broad peak widths. Ultra-high performance liquid (UHPLC) columns, on the other hand, use packing materials with a particle size of less than 2 μm, offering advantages such as lower reagent consumption, faster detection speeds, and higher column efficiency. Therefore, this study also selected UHPLC columns as the research object to maximize the savings in analysis time and mobile phase. This study investigated the separation performance of three columns: ACQUITY UPLC BEH C18 (3.0 mm × 100 mm, 1.7 μm), ACQUITY UPLC BEH HILIC (3.0 mm × 100 mm, 1.7 μm), and ACQUITY UPLC BEH Amide (3.0 mm × 100 mm, 1.7 μm). After multiple experiments, under the same mobile phase composition, using a C18 column, ammonium methyl ether showed no retention, eluted at 0.9 min, and remained indistinguishable from endogenous impurities (such as lysine); using a HILIC column, clomiphene showed peak bifurcation; and using an Amide column, the peak shape and resolution of all three target analytes met the requirements. Therefore, the ACQUITY UPLCBEH Amide column (3.0 mm × 100 mm, 1.7 μm) was ultimately selected as the chromatographic column for target analyte separation.

[0196] 1.3 Selection of mobile phase and establishment of elution program

[0197] Acetonitrile and methanol were both investigated as organic phases for the mobile phase. Both solvents achieved the required peak elution and separation of the target analyte. However, considering that methanol can dissolve various proteins in animal-derived foods in trace amounts, long-term use may lead to crystallization and pipeline blockage. Therefore, acetonitrile was selected as the organic phase for the mobile phase in this method, and 0.1% formic acid was added to optimize the peak shape. Four different concentrations of ammonium formate solutions (5 mM, 10 mM, 20 mM, and 50 mM, containing 0.1% formic acid) were investigated as aqueous phases for the mobile phase. All four solutions achieved the required peak elution and separation of the target analyte. Considering peak shape, symmetry, and salt content of the mobile phase, 20 mM ammonium formate (containing 0.1% formic acid) was selected as the aqueous phase for the mobile phase in this invention. The chromatogram is shown below. Figures 2-4 .

[0198] The final chromatographic conditions are:

[0199] Column: ACQUITY UPLC BEH Amide (3.0 mm × 100 mm, 1.7 μm), or a column with equivalent performance;

[0200] Mobile phase: 20 mmol / L ammonium formate solution (containing 0.1% formic acid) (A): acetonitrile (B) (containing 0.1% formic acid);

[0201] Flow rate: 0.4 mL / min;

[0202] Column temperature: 40℃;

[0203] Injection volume: 2 μL;

[0204] Elution procedure: see Table 2.

[0205] Table 2 Gradient elution program

[0206]

[0207] Note: When co-eluting components affect the detection of the target compound, the mobile phase ratio can be adjusted appropriately to separate them from interfering components as much as possible and reduce interference.

[0208] 1.4 Establishment of mass spectrometry conditions

[0209] By optimizing parameters such as capillary voltage, drying gas temperature, drying gas flow rate, nebulizing gas pressure, sheath gas temperature, and sheath gas flow rate, the final mass spectrometry conditions were obtained:

[0210] Ion source: Electrospray ionization source (ESI+);

[0211] Detection method: Multiple response monitoring (MRM);

[0212] Drying gas temperature: 300℃;

[0213] Drying gas flow rate: 11 L / min;

[0214] Nebulizer gas pressure: 35 psi;

[0215] Sheath gas temperature: 250℃;

[0216] Sheath gas flow rate: 11 L / min;

[0217] Capillary voltage: 3500V;

[0218] The reference values ​​for the monitored ion pairs and mass spectrometry parameters are shown in Table 3.

[0219] Table 3 Reference values ​​for qualitative, quantitative ion, and mass spectrometry analysis parameters of analytes

[0220]

[0221] Note: 1. "*" indicates quantitative ions.

[0222] 1.5 Determination of Qualitative Ion Abundance Ratio

[0223] The retention time of the target compound chromatographic peak in the sample should vary within ±2.5% compared to the retention time of the corresponding standard chromatographic peak.

[0224] Based on the characteristics of liquid chromatography-triple quadrupole mass spectrometry (LC-MS / MS) and the standards for qualitative judgment of LC-MS / MS in drug residue determination in EU document 2002 / 657 / EC, the maximum permissible deviation range of the relative abundance ratio of qualitative and quantitative ions was finally determined, as shown in Table 4.

[0225] Table 4. Maximum permissible deviation of relative ion abundance for qualitative determination.

[0226] Maximum allowable deviation ±20％ ±25％ ±30％ ±50％

[0227] 2. Research on sample pretreatment methods

[0228] 2.1 Examination of sample preparation

[0229] Animal meat and its products, poultry egg products, dairy products (excluding liquid milk), aquatic products, cooking seasonings (solid and semi-solid): Accurately weigh 2g of sample (accurate to 0.01g) into a centrifuge tube, add 0.10mL of mixed isotope internal standard intermediate working solution II (1.1.7), add two ceramic homogenants, add 1mL of water, shake well to disperse the sample matrix, and set aside.

[0230] For poultry eggs, liquid milk, cooking seasonings (liquid), etc.: accurately weigh 2g of sample (accurate to 0.01g) into a centrifuge tube, add 0.10mL of mixed isotope internal standard intermediate working solution II (1.1.7), add two ceramic homogenants, shake well, and set aside.

[0231] Edible vegetable oil: Accurately weigh 2g of sample (accurate to 0.01g) into a centrifuge tube, add 0.10mL of mixed isotope internal standard intermediate working solution II (1.1.7), add two ceramic homogenants, shake well, and set aside.

[0232] 2.2 Investigation of Extraction Solvents

[0233] The extraction efficiencies of five extraction solvents (acetonitrile matrix, 0.1% formic acid acetonitrile, 0.5% formic acid acetonitrile, 1% formic acid acetonitrile, and 5% formic acid acetonitrile) on clomiphene, trimetazidine, and meldane in the samples were investigated. The results are shown in [Figure number missing]. Figures 5-7 The results showed that adding formic acid to acetonitrile improved the extraction rate of the three target compounds, and the recovery rate of mediocream increased significantly with increasing formic acid content. However, the recovery rate did not change significantly when the formic acid content exceeded 1%. The method of this invention selected 1% formic acid acetonitrile as the extraction solvent.

[0234] 2.3 Investigation of Extraction Solvent Volume

[0235] The extraction efficiency was investigated when different sample matrices were treated with 1% formic acid acetonitrile as the extraction solvent, with total solution volumes of 5 mL, 10 mL, 15 mL, and 20 mL. The results are shown in [Figure number missing]. Figures 8-10 The results showed that when the total solution volume reached 10 mL, the dissolution rates of the three target compounds, clomiphene, trimetazidine, and methaqualone, all reached equilibrium, and further increasing the extraction solvent volume did not change the extraction rate. Therefore, considering cost and environmental factors, the method of this invention selects an extraction condition of 10 mL with the addition of 1% formic acid acetonitrile as the extraction solvent.

[0236] 2.4 Examination of extraction time

[0237] The effect of different extraction times on the extraction efficiency of the target analyte was investigated. Under the condition that other factors remained constant, the effects of vortexing for 5 min + ultrasound for 5 min, vortexing for 5 min + ultrasound for 10 min, vortexing for 5 min + ultrasound for 15 min, vortexing for 5 min + ultrasound for 20 min, vortexing for 5 min + ultrasound for 25 min, vortexing for 5 min + ultrasound for 30 min, vortexing for 5 min + ultrasound for 35 min, and vortexing for 5 min + ultrasound for 40 min on the extraction efficiency of the target analyte were examined. The results are shown below. Figures 11-13The results showed that, within a certain time frame, the recovery rate of the target analytes increased with increasing sonication time. Clomiphene's recovery rate stabilized after 20 minutes of sonication, showing no significant change. Trimetazidine and methaqualone's recovery rates stabilized after 30 minutes of sonication, and trimetazidine exhibited a significant matrix enhancement effect after 40 minutes of sonication. The method of this invention uses a 5-minute vortex followed by 30 minutes of sonication. Due to the longer sonication time, to prevent prolonged sample deposition at the bottom of the centrifuge tube, two shaking operations were performed during sonication.

[0238] 2.5 Examination of Purification Methods

[0239] The effects of three purification methods—C18+PSA+graphitized carbon, Agilent Elut purification pack, and Prime HLB column (200 mg / 6 mL)—and the lack of purification of the extract solution on the extraction efficiency of the target analyte were investigated. Results are shown below. Figures 14-16 The results showed that the three purification methods had little difference in their impact on the recovery rate of clomiphene. The recovery rate of clomiphene without purification was lower compared to the three purification methods, but all still met the recovery rate requirements of GB / T 27404-2008 Laboratory Quality Control Standard. Using two purification methods—C18+PSA+graphitized carbon and Agilent Elut purification package—the recovery rates of trimetazidine in various sample matrices were greater than 120%. The recovery rates of meldamine in all sample matrices were high, with some even exceeding 200%, but the peak shape was poor, indicating interference from endogenous impurities, and thus did not meet the recovery rate requirements of GB / T 27404-2008 Laboratory Quality Control Standard. When using a PrimeHLB column (200 mg / 6 mL) for purification, the recovery rates of all three target analytes met the recovery rate requirements of GB / T 27404-2008 Laboratory Quality Control Standard. Therefore, the method of this invention uses a PrimeHLB column (200 mg / 6 mL) to purify the extract.

[0240] 2.6 Investigation of matrix effect

[0241] Weigh a blank matrix sample and conduct the experiment according to the selected pretreatment conditions. Combine the resulting test solutions to form the blank sample extract. Prepare a matrix standard curve using the blank sample extract and a solvent standard curve using a 0.1% formic acid-acetonitrile aqueous solution. The matrix effect of the three target analytes is examined by the ratio of the slope of the matrix standard curve to the slope of the solvent standard curve. If the ratio of the slope of the matrix standard curve to the slope of the solvent standard curve is between 0.85 and 1.15, the matrix effect is considered to be absent. Results are shown in […]. Figure 17 The results showed that the slope ratio of the curves for the three target compounds in all sample matrices was less than 0.85, indicating the presence of matrix effects. To reduce the interference of matrix effects on the determination of actual samples, the method of this invention uses the isotope internal standard method to correct for matrix effects.

[0242] 2.7 Pre-treatment methods after investigation

[0243] For livestock and poultry meat and their products, poultry egg products, dairy products (excluding liquid milk), aquatic products, and cooking seasonings (solid and semi-solid): accurately weigh 2g of sample (accurate to 0.01g) into a centrifuge tube, add 0.10mL of mixed isotope internal standard intermediate working solution II (1.1.7), add two ceramic homogenants, add 1mL of water, shake well to disperse the sample matrix, add 9mL of 1% formic acid acetonitrile solution, vortex for 5min, sonicate for 30min, shake twice during extraction, centrifuge at 8000r / min for 5min, take the supernatant and pass it through a Prime HLB column (200mg / 6mL), collect the filtrate in a nitrogen blow-off tube, mix well, take 5mL of supernatant and blow it with nitrogen at 40℃ until nearly dry, make up to 1mL with 0.1% formic acid acetonitrile aqueous solution, mix well, pass through a 0.22μm organic filter membrane, and then test on the instrument.

[0244] For poultry eggs, liquid milk, cooking seasonings (liquid), etc.: Accurately weigh 2g of sample (accurate to 0.01g) into a centrifuge tube, add 0.10mL of mixed isotope internal standard intermediate working solution II (1.1.7), add two ceramic homogenants, shake well, add 9mL of 1% formic acid acetonitrile solution, vortex for 5min, sonicate for 30min, shake twice during extraction, centrifuge at 8000r / min for 5min, take the supernatant and pass it through a Prime HLB column (200mg / 6mL), collect the filtrate in a nitrogen blow-off tube, mix well, take 5mL of supernatant and blow it with nitrogen at 40℃ until nearly dry, make up to 1mL with 0.1% formic acid acetonitrile aqueous solution, mix well, pass through a 0.22μm organic filter membrane, and then test.

[0245] Edible vegetable oil: Accurately weigh 2g of sample (accurate to 0.01g) into a centrifuge tube, add 0.10mL of mixed isotope internal standard intermediate working solution II (1.1.7), add two ceramic homogenants, shake well, add 10mL of 1% formic acid acetonitrile solution, vortex for 5min, sonicate for 30min, shake twice during extraction, centrifuge at 8000r / min for 5min, take the supernatant and pass it through a Prime HLB column (200mg / 6mL), collect the filtrate in a nitrogen blow-off tube, mix well, take 5mL of supernatant and blow it with nitrogen at 40℃ until nearly dry, make up to 1mL with 0.1% formic acid acetonitrile aqueous solution, mix well, pass through a 0.22μm organic filter membrane, and then test on the instrument.

[0246] 3. Methodological Examination

[0247] 3.1 Sample Source

[0248] All samples were purchased from local supermarkets and underwent baseline screening.

[0249] 3.2 Stability Test

[0250] 3.2.1 Investigation on stability of standard stock solutions

[0251] The prepared clomiphene standard stock solution, trimetazidine standard stock solution, meldonium standard stock solution, clomiphene-d5 internal standard stock solution, trimetazidine-d8 internal standard stock solution and meldonium-d3 internal standard stock solution were separately dispensed into storage bottles and stored at -18°C. One batch of the stock solutions was taken out at regular intervals, in which clomiphene, trimetazidine and meldonium were respectively diluted to a concentration at 10 times the limit of quantitation (the concentrations of clomiphene and trimetazidine were 20 ng / mL, and the concentration of meldonium was 100 ng / mL), clomiphene-d5 and trimetazidine-d8 were diluted to 10 ng / mL, and meldonium-d3 was diluted to 50 ng / mL. Before each measurement, each standard substance and internal standard substance were re-weighed to prepare standard solutions and internal standard solutions for measuring the above diluted stock solutions. A liquid chromatography-tandem mass spectrometer was used for 6 parallel determinations, and quantification was performed by the external standard method, as shown in Table 5. The results showed that there was no significant change in the concentration of all analyte stock solutions within 3 months, and 3 months was selected as the validity period of the stock solutions. The t-test method was adopted, and the calculated t (clomiphene) = 1.24, t (trimetazidine) = 0.92, t (meldonium) = 1.38, t (clomiphene-d5) = 1.44, t (trimetazidine-d8) = 0.70, t (meldonium-d3) = 1.43. By checking the t-test critical value table, t(0.05,11) = 2.201 was obtained, and t < t(0.05,11), indicating that there was no significant difference in the characteristic quantity values of each standard stock solution and internal standard stock solution, which were consistent with the standard values.

[0252] Table 5 Investigation Table of Stock Solution Stability

[0253]

[0254] 3.2.2 Investigation on stability of standard intermediate solutions

[0255] The prepared standard intermediate working solutions with clomifene, trimetazidine, and meldonium each at a concentration of 10 μg / mL, and the mixed isotope internal standard intermediate working solution with clomifene-d5, trimetazidine-d8, and meldonium-d3 at concentrations of 10.0 μg / mL, 2.0 μg / mL, and 2.0 μg / mL respectively are aliquoted into storage bottles and stored at below -18°C. At regular intervals, one batch of the intermediate solutions is taken out, in which clomifene, trimetazidine, and meldonium are respectively diluted to the concentration at 10 times the limit of quantitation (the concentrations of clomifene and trimetazidine are 20 ng / mL, and the concentration of meldonium is 100 ng / mL), clomifene-d5 and trimetazidine-d8 are diluted to 10 ng / mL, and meldonium-d3 is diluted to 50 ng / mL. Before each measurement, each standard substance and internal standard substance are re-weighed to prepare a standard solution and an internal standard solution, which are used to measure the above diluted intermediate solutions. A liquid chromatography-tandem mass spectrometer is used for 6 parallel determinations, and quantitation is performed by the external standard method, as shown in Table 6. The results show that within 5 weeks, there is no significant change in the concentrations of all the standard intermediate working solutions with clomifene, trimetazidine, and meldonium each at 10 μg / mL, and the mixed isotope internal standard intermediate working solution with clomifene-d5, trimetazidine-d8, and meldonium-d3 at concentrations of 10.0 μg / mL, 2.0 μg / mL, and 2.0 μg / mL respectively. Finally, 1 month is selected as the validity period for the standard intermediate working solutions with clomifene, trimetazidine, and meldonium each at a concentration of 10 μg / mL, and the mixed isotope internal standard intermediate working solution with clomifene-d5, trimetazidine-d8, and meldonium-d3 at concentrations of 10.0 μg / mL, 2.0 μg / mL, and 2.0 μg / mL respectively. Using the t-test method, the calculated t values are t(clomifene) = 1.63, t(trimetazidine) = 0.37, t(meldonium) = 0.44, t(clomifene-d5) = 0.11, t(trimetazidine-d8) = 1.46, t(meldonium-d3) = 0.97. By looking up the t-test critical value table, t(0.05,4) = 2.776, and t < t(0.05,4), which indicates that there is no significant difference in the characteristic quantity values of the standard intermediate working solutions of clomifene, trimetazidine, and meldonium, and the mixed isotope internal standard intermediate working solution of clomifene-d5, trimetazidine-d8, and meldonium-d3.

[0256] Table 6 Intermediate solution stability investigation table

[0257]

[0258] 3.2.3 Investigation on stability of standard working solution

[0259] The standard curve working solution is divided into 4 portions, placed in sample vials and then set on the sample tray at room temperature. Samples are injected at 0 h, 8 h, 24 h, and 48 h respectively, and the response values of the analytes are counted, as shown in Table 7. The results show that there is no significant change in the standard curve working solution within 48 h.

[0260] Table 7. Stability Study of Working Fluid

[0261]

[0262]

[0263] 3.3 Precision Examination

[0264] 3.3.1 Precision assessment within one day

[0265] Standard working solutions of clomiphene and trimetazidine at concentrations of 2 ng / mL and melidomide at concentration of 10 ng / mL were injected six times within the same day under the established chromatographic and mass spectrometric conditions. The repeatability of chromatographic peak areas was used as the indicator for intra-day method precision testing. The intra-day precision RSD values ​​for clomiphene, trimetazidine, and melidomide were 5.2, 3.7, and 4.8, respectively, and the absolute difference between any two independent determinations did not exceed 20% of the arithmetic mean. The experimental results meet the method requirements.

[0266] 3.3.2 Daytime Precision Assessment

[0267] The prepared standard working solutions of clomiphene and trimetazidine at concentrations of 2 ng / mL and melidomine at concentration of 10 ng / mL were stored in a sealed, light-protected environment at 2–6 °C. Under the established chromatographic and mass spectrometric conditions, the samples were injected six times on different days. The reproducibility of the chromatographic peak area was used as the indicator for inter-day precision experiments. The inter-day precision RSD values ​​for the three substances were 9.4, 6.5, and 8.0, respectively, and the absolute difference between any two independent determinations did not exceed 20% of the arithmetic mean. The experimental results meet the method requirements.

[0268] 3.4 Linear Range

[0269] The standard working curve for clomiphene has an accuracy of r = 0.9992 in the concentration range of 0–40 ng / mL; the standard working curve for trimetazidine has an accuracy of r = 0.9992 in the concentration range of 0–40 ng / mL; and the standard working curve for methaqualone has an accuracy of r = 0.9996 in the concentration range of 0–200 ng / mL, which meets the requirement of GB / T 27404-2008 that r should not be less than 0.99 in the calibration curve confirmation.

[0270] 3.5 Limit of detection and limit of quantitation

[0271] The limits of detection (LOD) and quantitation (LOQ) were determined by progressively decreasing the spiked concentration in blank samples. The LOD for clomiphene and trimetazidine in this method was found to be 1.00 μg / kg, and the LOQ for trimetazidine was 2.00 μg / kg. The LOD for meldranium was 5.00 μg / kg, and the LOQ for meldranium was 10.0 μg / kg. 10 μL of a mixed standard intermediate working solution (clomiphene and trimetazidine concentrations of 0.2 μg / mL, and meldranium concentration of 1.0 μg / mL) was added to samples of pork, beef, braised eggs, bacon, sausage, cheese, silver carp, scallops, freshwater shrimp, chicken, broad bean paste, oyster sauce, chicken essence, yogurt, milk, eggs, and rapeseed oil. After pretreatment according to the method, the qualitative and quantitative ion-pair signal-to-noise ratios were both greater than 3. 20 μL of a mixed standard intermediate working solution (clomiphene and trimetazidine concentrations of 0.2 μg / mL, and melidomine concentration of 1.0 μg / mL) was added to pork, beef, braised eggs, bacon, sausage, cheese, silver carp, scallops, freshwater shrimp, chicken, broad bean paste, oyster sauce, chicken essence, yogurt, milk, eggs, and rapeseed oil samples according to the method pretreatment and instrument detection. The signal-to-noise ratios of qualitative and quantitative ion pairs were both greater than 10.

[0272] 3.6 Recovery rate and precision

[0273] According to GB / T 27404-2008 Laboratory Quality Control Standard (Food Physicochemical Testing), blank matrix samples were weighed according to the method text. Three-level spiking tests were performed at the limit of quantitation (LOQ), twice the LOQ, and ten times the LOQ, with six replicates for each level. The methods were followed to determine the LOQ of pork, beef, sausage, yogurt, bacon, cheese, eggs, freshwater shrimp, broad bean paste, oyster sauce, and chicken essence. The recovery rate was examined, and the recovery rate was within the range of 80%–110%, meeting the recovery rate and precision requirements of GB / T 27404-2008. Further laboratory repeatability tests were performed using Grubbs' test. The calculated G values ​​were all less than the upper 5% critical value of 1.887, indicating statistically significant validity, meeting the statistical requirements for detection data in GB / T 6379.2-2004.

[0274] 4. Verification Results

[0275] In accordance with the method validation requirements, the method development unit organized three institutions—Chengdu Customs Technology Center, Guangzhou Quality Supervision and Inspection Institute, and Qingdao Customs Technology Center—to participate in the method validation. Instrument parameters, pretreatment methods, and all performance indicators met the method requirements. Each participating institution selected pork, chicken, fish, eggs, yogurt, and bacon as sample matrices for spiking, recovery, and precision experiments. Based on GB / T 27404-2008 Laboratory Quality Control Standard (Food Physicochemical Testing), six copies of each sample were prepared at spiking levels of 1, 2, and 10 times the limit of quantitation (LOQ). Repeatability experiments were conducted using this method, and the average recovery rate and relative standard deviation (RSD) were calculated. The average recovery rate and precision at each spiking level by the three validation institutions met the requirements of GB / T 27404-2008 Laboratory Quality Control Standard. The average recovery rate at each spiking level by the three validation institutions ranged from 78.5% to 112.1%, and the RSD ranged from 2.34% to 6.36%. Further analysis using the Cochrane test to statistically analyze the spiked recovery rates of the three validation units showed that the calculated C values ​​were all less than the 5% critical value (0.707), indicating that the statistical results were significantly effective. The repeatability and reproducibility of the method met the accuracy requirements of GB / T6379.2 and complied with the technical requirements for the development of national food safety standards. The interlaboratory validation results were satisfactory.

[0276] 5. Determination methods for three metabolic regulators in food.

[0277] (1) Scope

[0278] The present invention specifies a method for determining clomiphene, trimetazidine, and meldamine in food.

[0279] The method of this invention is applicable to the determination of clomiphene, trimetazidine and meldamine in livestock and poultry meat and products, poultry eggs and their products, dairy products, aquatic products, edible vegetable oils and condiments (salt, soy sauce, vinegar, cooking wine, monosodium glutamate, chicken essence, oyster sauce).

[0280] (2) Principle

[0281] The sample was added to the extraction solvent, extracted by vortexing and ultrasonication, centrifuged and purified, and then analyzed by liquid chromatography-mass spectrometry / mass spectrometry.

[0282] (3) Reagents and materials

[0283] All reagents used in the method of this invention are of analytical grade, and the water is Grade I water as specified in GB / T 6682.

[0284] 3.1 Reagents

[0285] 3.1.1 Acetonitrile (C2H3N): chromatographic grade.

[0286] 3.1.2 Methanol (CH3OH): chromatographic grade.

[0287] 3.1.3 Formic acid (HCOOH): chromatographic grade.

[0288] 3.2 Reagent Preparation

[0289] 3.2.1 1% Formic Acid Acetonitrile Solution: Transfer 10.0 mL of formic acid and dilute to 1000 mL with acetonitrile, then mix well.

[0290] 3.2.2 0.1% Formic Acid Acetonitrile Solution: Transfer 1.0 mL of formic acid and dilute to 1000 mL with acetonitrile, then mix well.

[0291] 3.2.3 0.1% formic acid aqueous solution: Transfer 1.0 mL of formic acid, dilute with water to 1000 mL, and mix well.

[0292] 3.2.4 0.1% Formic Acid Acetonitrile Aqueous Solution (9+1): Measure 900 mL of 0.1% Formic Acid Acetonitrile and 100 mL of 0.1% Formic Acid Aqueous Solution, and mix well.

[0293] 3.3 Standard Products

[0294] Clomiphene citrate, purity ≥99%; Trimetazidine, purity ≥99%; Melidomine, purity ≥99%; Clomiphene hydrochloride-d5, purity ≥98%; Trimetazidine dihydrochloride-d8, purity ≥98%; Melidomine-d3, purity ≥98%.

[0295] 3.4 Preparation of Standard Solutions

[0296] 3.4.1 Standard stock solution (1 mg / mL)

[0297] Accurately weigh appropriate amounts of the standards (accurate to 0.01 mg) into separate 10 mL volumetric flasks, dissolve them in methanol, and dilute to volume to prepare standard stock solutions of clomiphene, trimetazidine, and melidomine, each with a concentration of 1 mg / mL. Transfer the solutions to brown glass containers and store below -18°C. The shelf life is 3 months.

[0298] 3.4.2 Internal standard stock solution (100 μg / mL)

[0299] Accurately weigh appropriate amounts of the standards (accurate to 0.01 mg) into separate 10 mL volumetric flasks, dissolve them in methanol, and dilute to volume to prepare internal standard stock solutions of clomiphene-d5, trimetazidine-d8, and melidomine-d3, each with a concentration of 100 μg / mL. Transfer the solutions to brown glass containers and store below -18°C. The shelf life is 3 months.

[0300] 3.4.3 Standard intermediate solution (10 μg / mL)

[0301] Accurately pipette appropriate amounts of each standard stock solution (3.4.1) into separate 10 mL volumetric flasks, dilute to the mark with methanol, and mix well to prepare standard intermediate solutions of clomiphene, trimetazidine, and methaqualone, each with a concentration of 10 μg / mL. Transfer the solutions to brown glass containers and store below -18°C for one month.

[0302] 3.4.4 Mixed Standard Intermediate Solution

[0303] Accurately pipette 0.2 mL, 0.2 mL, and 1.0 mL of the clomiphene, trimetazidine, and medocalmium standard intermediate solutions (3.4.3) into the same 10 mL volumetric flask, dilute to the mark with methanol, shake well, and prepare mixed standard intermediate solutions with concentrations of 0.2 μg / mL, 0.2 μg / mL, and 1.0 μg / mL for clomiphene, trimetazidine, and medocalmium, respectively. Prepare fresh before use.

[0304] 3.4.5 Mixed Internal Standard Intermediate Solution I

[0305] Accurately pipette 0.2 mL, 0.2 mL, and 1.0 mL of clomiphene-d5, trimetazidine-d8, and medocyanine-d3 internal standard stock solutions (3.4.2) into the same 10 mL volumetric flask, respectively. Dilute to the mark with methanol, shake well, and prepare a mixed internal standard intermediate solution I with concentrations of 2.0 μg / mL for clomiphene-d5, 2.0 μg / mL for trimetazidine-d8, and 10.0 μg / mL for medocyanine-d3. Transfer the solution to a brown glass container and store below -18°C. Shelf life is one month.

[0306] 3.4.6 Mixed Internal Standard Intermediate Solution II

[0307] Accurately pipette 1.0 mL of mixed internal standard intermediate solution I (3.4.5) into a 10 mL volumetric flask, dilute to the mark with methanol, shake well, and prepare mixed internal standard intermediate solution II with concentrations of 0.2 μg / mL for clomiphene-d5, 0.2 μg / mL for trimetazidine-d8, and 1.0 μg / mL for methaqualone-d3. Prepare fresh before use.

[0308] 3.4.7 Standard working solution

[0309] Pipette 0.05 mL, 0.1 mL, 0.2 mL, 0.5 mL, 1.0 mL, and 2.0 mL of the mixed standard intermediate solution (3.4.4) into 10 mL volumetric flasks, respectively. Add 0.5 mL of the mixed internal standard intermediate solution II (3.4.6), and dilute to the mark with 0.1% formic acid acetonitrile aqueous solution (9+1) (3.2.4). Shake well to obtain standard curves with clomiphene and trimetazidine concentrations of 1 ng / mL, 2 ng / mL, 4 ng / mL, 10 ng / mL, 20 ng / mL, and 40 ng / mL, and melidomide concentrations of 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL. The concentrations of clomiphene-d5 and trimetazidine-d8 are both 10 ng / mL, and the concentration of melidomide-d3 is 50 ng / mL. Prepare fresh before use, or prepare standard working solutions of appropriate concentrations according to the instrument response.

[0310] 3.5 Materials

[0311] 3.5.1 Purification column: Prime HLB, 200mg, 6cc.

[0312] 3.5.2 Centrifuge tubes: 50 mL.

[0313] 3.5.3 Organic filter membrane: 0.22μm, Nylon or PTFE material.

[0314] 3.5.4 Ceramic homogeneous protons.

[0315] 3.5.5 Nitrogen blow-off tube, 10 mL.

[0316] (4) Instruments and equipment

[0317] 4.1 Liquid chromatography-mass spectrometry / mass spectrometer, equipped with an electrospray ionization source (ESI).

[0318] 4.2 Balance: Sensitivity is 0.01 mg and 0.01 g respectively.

[0319] 4.3 Ultrasonic generator.

[0320] 4.4 Multi-tube vortex oscillator.

[0321] 4.5 Nitrogen blowing device.

[0322] 4.6 Tissue homogenizer.

[0323] 4.7 High-speed centrifuge.

[0324] (5) Sample preparation and preservation

[0325] 5.1 Sample Preparation Requirements

[0326] During sample preparation, it is important to prevent the sample from becoming contaminated or experiencing changes in residue content.

[0327] 5.2 Livestock and poultry meat and their products, aquatic products

[0328] Take about 500g of muscle tissue, homogenize it using a tissue homogenizer, mix it thoroughly, and then put it into a clean container. Seal and label the container. Store livestock and poultry meat and aquatic products at -18℃ in the dark, and store other samples at 4℃ in the dark.

[0329] 5.3 Poultry eggs and their products

[0330] Take about 500g of sample, wash and remove the shell, mix thoroughly, put into a clean container, seal and label, and store at -18℃ in the dark; weigh about 500g of other samples, homogenize them with a tissue homogenizer, mix thoroughly, put into a clean container, seal and label, and store at 4℃ in the dark.

[0331] 5.4 Liquid milk such as cow's milk and fermented milk, edible vegetable oils, and cooking seasonings (liquid)

[0332] Take about 500g of sample, stir thoroughly, put it into a clean container, seal and label it, and store at 4℃ away from light.

[0333] 5.5 Cheese and other dairy products

[0334] Take about 500g of sample, heat to 50℃–60℃ to melt, stir thoroughly while hot, then put into a clean container, seal and label, and store at 4℃ away from light.

[0335] 5.6 Cooking seasonings (solid, semi-solid)

[0336] Take about 500g of sample, homogenize it using a tissue homogenizer, mix thoroughly, put it into a clean container, seal and label it, and store it at 4℃ away from light.

[0337] (6) Measurement steps

[0338] 6.1 Sample preparation

[0339] 6.1.1 Livestock and poultry meat and their products, poultry egg products, dairy products (excluding liquid milk), aquatic products, and cooking seasonings (solid and semi-solid).

[0340] Accurately weigh 2g of the mixed sample (accurate to 0.01g) into a centrifuge tube, add 0.10mL of mixed internal standard intermediate solution II (3.4.6), add two ceramic homogenates (3.5.4), add 1mL of water, shake well, then add 9mL of 1% formic acid acetonitrile solution (3.2.1), vortex for 5min, sonicate for 30min, shaking twice during extraction, centrifuge at 8000r / min for 5min, take the supernatant and pass it through a Prime HLB column (3.5.1), collect the filtrate in a nitrogen blow-off tube (3.5.5), mix well, take 5mL of the supernatant and blow it with nitrogen at 40℃ until nearly dry, make up to 1mL with 0.1% formic acid acetonitrile aqueous solution (3.2.4), mix well, pass through a 0.22μm organic filter membrane (3.5.3), and then test on the instrument.

[0341] 6.1.2 Poultry eggs, liquid milk, cooking seasonings (liquid), etc.

[0342] Accurately weigh 2g of the mixed sample (accurate to 0.01g) into a centrifuge tube, add 0.10mL of mixed internal standard intermediate solution II (3.4.6), add two ceramic homogenates (3.5.4), add 8mL of 1% formic acid acetonitrile solution (3.2.1), vortex for 5min, sonicate for 30min, shake twice during extraction, centrifuge at 8000r / min for 5min, take the supernatant and pass it through a Prime HLB column (3.5.1), collect the filtrate in a nitrogen blow-off tube (3.5.5), mix well, take 5mL of the supernatant and blow it with nitrogen at 40℃ until nearly dry, make up to 1mL with 0.1% formic acid acetonitrile aqueous solution (3.2.4), mix well, pass through a 0.22μm organic filter membrane (3.5.3), and test on the instrument.

[0343] 6.1.3 Edible vegetable oils

[0344] Accurately weigh 2g of the mixed sample (accurate to 0.01g) into a centrifuge tube, add 0.10mL of mixed internal standard intermediate solution II (3.4.6), add two ceramic homogenates (3.5.4), add 10mL of 1% formic acid acetonitrile solution (3.2.1), vortex for 5min, sonicate for 30min, shake twice during extraction, centrifuge at 8000r / min for 5min, take the supernatant and pass it through a Prime HLB column (3.5.1), collect the filtrate in a nitrogen blow-off tube (3.5.5), mix well, take 5mL of the supernatant and blow it with nitrogen at 40℃ until nearly dry, make up to 1mL with 0.1% formic acid acetonitrile aqueous solution (3.2.4), mix well, pass through a 0.22μm organic filter membrane (3.5.3), and test on the instrument.

[0345] 6.2 Measurement

[0346] 6.2.1 Chromatographic Reference Conditions

[0347] 6.2.1.1 Column: ACQUITY UPLC BEH Amide, 3.0×100mm, 1.7μm, or a column with equivalent performance;

[0348] 6.2.1.2 Mobile phase: A is 20 mmol / L ammonium formate solution (containing 0.1% formic acid), B is 0.1% formic acid acetonitrile solution;

[0349] 6.2.1.3 Flow rate: 0.4 mL / min;

[0350] 6.2.1.4 Column temperature: 40℃;

[0351] 6.2.1.5 Injection volume: 2 μL;

[0352] 6.2.1.6 Elution procedure: See Table 8.

[0353] Table 8 Gradient Elution Procedure

[0354] 0.0 5 95 1.0 5 95 3.0 15 85 5.0 50 50 5.5 50 50 5.6 5 95 7.5 5 95

[0355] Note: When co-eluting components affect the detection of the target compound, the mobile phase ratio can be adjusted appropriately to separate the target compound from the interfering components as much as possible and reduce interference.

[0356] 6.2.2 Mass Spectrometry Reference Conditions

[0357] 6.2.2.1 Ion source: Electrospray ionization (ESI) source;

[0358] 6.2.2.2 Detection method: Multiple reaction monitoring (MRM);

[0359] 6.2.2.3 Scanning mode: Positive ion mode scan (ESI) + )

[0360] 6.2.2.4 Drying gas temperature: 300℃;

[0361] 6.2.2.5 Drying gas flow rate: 11 L / min;

[0362] 6.2.2.6 Atomizing gas pressure: 35 psi;

[0363] 6.2.2.7 Sheath gas temperature: 250℃;

[0364] 6.2.2.8 Sheath gas flow rate: 11 L / min;

[0365] 6.2.2.9 Capillary voltage: 3500V;

[0366] 6.2.2.10 The reference values ​​for monitoring ion pairs and mass spectrometry parameters are shown in Table 9.

[0367] Table 9. Reference values ​​for qualitative ion pairs, quantitative ion pairs, and mass spectrometry parameters of the analytes.

[0368]

[0369] Note: 1. "*" indicates quantitative ions; 2. Instrument parameters may vary for different mass spectrometers. Mass spectrometry parameters should be optimized to the best before measurement.

[0370] 6.3 Qualitative determination

[0371] The sample and standard working solutions were determined according to the high performance liquid chromatography-mass spectrometry / mass spectrometry conditions. The chromatographic retention times of each compound in the sample solution and standard solution were recorded. When a chromatographic peak with the same retention time as the qualitative and quantitative ion chromatographic peaks of the target compound was detected in the sample solution (within ±2.5%), and the deviation of the relative abundance ratio of the qualitative and quantitative ions of the chromatographic peaks in the sample solution from the relative abundance ratio of the qualitative and quantitative ions of the standard solution of equivalent concentration did not exceed the range specified in Table 10, the target compound could be determined to be detected in the sample.

[0372] Table 10 Maximum permissible deviation of relative ion abundance during qualitative confirmation.

[0373] Maximum allowable deviation ±20％ ±25％ ±30％ ±50％

[0374] 6.4 Quantitative determination

[0375] 6.4.1 Construction of Standard Curve

[0376] The standard working solution (3.4.7) was tested according to the instrument reference conditions (6.2). The ratio of the concentration of the analyte to the concentration of the corresponding isotope internal standard was used as the abscissa, and the ratio of the peak area of ​​the analyte to the peak area of ​​the corresponding isotope internal standard was used as the ordinate to plot the standard curve and obtain the linear regression equation.

[0377] 6.4.2 Determination of Sample Solution

[0378] The sample solution (6.1) was measured according to the instrument reference conditions (6.2) to obtain the chromatographic peak area of ​​the sample solution. The response value of the target analyte in the sample solution should be within the linear range of the instrument detection. If it exceeds the linear range, it can be appropriately diluted to the linear range of the standard curve according to the actual concentration.

[0379] 6.5 Parallel Tests

[0380] Perform parallel tests on the same sample following the steps above.

[0381] 6.6 Blank Test

[0382] Except for cases where no sample is weighed, blank tests should be performed according to 6.1. The sample should not contain any substances that may interfere with the analyte.

[0383] 6.7 Calculation of Results

[0384] The content of the analyte in the sample is calculated according to formula (1).

[0385]

[0386] In the formula:

[0387] X—The content of the analyte in the sample, expressed in micrograms per kilogram (μg / kg);

[0388] ρ—The concentration of the analyte in the sample solution obtained from the standard curve, in nanograms per milliliter (ng / mL);

[0389] V1—Volume of extractant, in milliliters (mL);

[0390] V2—Volume of extractant transferred, in milliliters (mL);

[0391] V3—The final volume of the purified and concentrated sample solution, in milliliters (mL);

[0392] m—mass of the sample, in grams (g);

[0393] f—Dilution factor.

[0394] Blank values ​​should be deducted from the calculation results. The measurement results are expressed as the arithmetic mean of parallel measurements and are retained to three significant figures.

[0395] In summary, this invention can simultaneously detect three metabolic regulators—clomiphene, trimetazidine, and melidomide—in animal-derived foods with accurate results. It is highly automated, rapid, sensitive, and has a low detection limit, providing effective assurance for the accurate detection of metabolic regulator content in animal-derived foods, ensuring food safety, and has promising application prospects.

Claims

1. A method for detecting metabolic regulators in food, characterized in that: It includes the following steps: a. Preparation of standard working solution: Take the metabolic regulator standard, dissolve and dilute it with methanol to form a solution, then add the internal standard solution and acetonitrile aqueous solution containing formic acid and mix well to obtain the solution; b. Preparation of test solution: Take the solid or semi-solid sample to be tested, add internal standard solution, water and 1% formic acid acetonitrile solution to extract, centrifuge, take the supernatant and pass it through a Prime HLB column, collect the filtrate and dry it, then add formic acid acetonitrile aqueous solution to dissolve it, and the test solution is obtained. Alternatively: Take the liquid sample to be tested, add internal standard solution and acetonitrile solution containing formic acid to extract, centrifuge, take the supernatant and pass it through a PrimeHLB column, collect the filtrate and dry it, then add acetonitrile aqueous solution containing formic acid to dissolve it, and the result is obtained; c. Inject the standard working solution and the test solution into the liquid chromatography-triple quadrupole mass spectrometer, respectively. The chromatographic conditions are as follows: Chromatographic column: ACQUITY UPLC BEH Amide, 3.0 × 100 mm, 1.7 μm; mobile phase A: 20 mmol / L ammonium formate solution containing 0.1% formic acid; mobile phase B: 0.1% formic acid acetonitrile solution; flow rate: 0.4 mL / min; column temperature: 40℃; injection volume: 2 μL; gradient elution program: Mass spectrometry conditions: Ion source: electrospray ionization source; Mass spectrometry detection mode: multi-ion reaction monitoring; Scanning mode: positive ion mode; Drying gas temperature: 300℃, Drying gas flow rate: 11 L / min, Nebulizing gas pressure: 35 psi, Sheath gas temperature: 250℃, Sheath gas flow rate: 11 L / min, Capillary voltage: 3500V. The internal standard solution is a methanol solution of a deuterated metabolism regulator standard; The metabolic regulator standards are clomiphene citrate, trimetazidine, and metronidazole.

2. The detection method according to claim 1, characterized in that: The volume ratio of the solution, internal standard solution, and acetonitrile aqueous solution containing formic acid in step a is 0.05~2:0.5:7.5~9.45; the concentration of the metabolic regulator in the solution is 0.2μg / mL~1mg / mL.

3. The detection method according to claim 1, characterized in that: In step b, the mass-to-volume ratio of the solid or semi-solid sample to be tested, the internal standard solution, water, and the acetonitrile solution containing formic acid is 1~5g:0.1ml:1~5ml:8~9ml; the mass-to-volume ratio of the liquid sample to be tested, the internal standard solution, and the acetonitrile solution containing formic acid is 1~5g:0.1ml:8~10ml.

4. The detection method according to claim 3, characterized in that: The mass-to-volume ratio of the solid or semi-solid sample to be tested, the internal standard solution, water, and the acetonitrile solution containing formic acid is 2g:0.1ml:1ml:9ml; the mass-to-volume ratio of the liquid sample to be tested, the internal standard solution, and the acetonitrile solution containing formic acid is 2g:0.1ml:8 or 10ml.

5. The detection method according to claim 1, characterized in that: The deuterated metabolism regulator standard is clomiphene hydrochloride-d5, trimetazidine dihydrochloride-d8 and / or melidomine-d3; the concentration of the deuterated metabolism regulator standard in the internal standard solution is 0.2 ~ 1 μg / mL.

6. The detection method according to claim 5, characterized in that: The internal standard solution contained clomiphene hydrochloride-d5 0.2 μg / mL, trimetazidine dihydrochloride-d8 0.2 μg / mL, and melidomine-d3 1.0 μg / mL.

7. The detection method according to claim 1, characterized in that: The extraction in step b involves vortexing for 5 minutes followed by sonication for 30 minutes; the centrifugation time is 3-8 minutes at a speed of 5000-10000 r / min; the drying is carried out by nitrogen blowing at 40℃; and the amount of acetonitrile aqueous solution containing formic acid added is 1 / 4 to 1 / 5 of the filtrate volume.

8. The detection method according to claim 7, characterized in that: The centrifugation time was 5 minutes and the speed was 8000 r / min.

9. The detection method according to claim 1, characterized in that: The concentration of formic acid in the acetonitrile aqueous solution containing formic acid is 0.05%~5%, v / v.

10. The detection method according to claim 9, characterized in that: The formic acid concentration in the acetonitrile aqueous solution is 0.1%, v / v, and the volume ratio of acetonitrile to water is 9:

1.

11. The detection method according to claim 1, characterized in that: In step c, the mass spectrometry conditions for clomiphene qualitative detection are m / z 406.2→m / z 71.9, fragmentation voltage 120V, collision energy 40V, and for quantitative detection of ion pairs m / z 406.2→m / z 99.8, fragmentation voltage 120V, collision energy 24V. Clomiphene-D5 quantitative detection of ion pairs m / z 411.1→m / z 58.1, fragmentation voltage 120V, collision energy 40V; Trimetazidine qualitative detection of ion pairs: m / z 267.2→m / z 165.8, fragmentation voltage 120V, collision energy 28V; quantitative detection of ion pairs: m / z 267.2→m / z 180.8, fragmentation voltage 120V, collision energy 12V; Trimetazidine-d8 quantitative detection of ion pairs: m / z 275.3→m / z 181, fragmentation voltage 120V, collision energy 12V. For qualitative detection of ion pairs using mediocrene, the m / z range is 147.1 to 58.9, with a fragmentation voltage of 120V and a collision energy of 20V. For quantitative detection of ion pairs, the m / z range is 147.1 to 57.9, with a fragmentation voltage of 120V and a collision energy of 32V. For quantitative detection of ion pairs using mediocrene-d3, the m / z range is 149.8 to 61.1, with a fragmentation voltage of 120V and a collision energy of 32V.

12. The detection method according to claim 1, characterized in that: The chromatogram of the test sample solution shows chromatographic peaks with retention times of qualitative and quantitative ions consistent with those of the standard working solution, and the deviation of the relative abundance ratio of qualitative and quantitative ions of the chromatographic peaks from that of the standard working solution of equivalent concentration does not exceed the specified maximum permissible deviation range, thus confirming that the test sample contains a metabolic regulator; the specified maximum permissible deviation range is as follows:

13. The detection method according to claim 12, characterized in that: The content of metabolic regulators in the solid, semi-solid or liquid samples to be tested is calculated by peak area using the external standard method.

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