Liquid chromatography-mass spectrometry detection method for thaumatin in food

Abstract

The invention provides a liquid chromatography-mass spectrometry detection method for thaumatin in food, and relates to the technical field of analysis and detection. According to the method, the trifluoroacetic acid aqueous solution is used as an extraction solvent to extract thaumatin in the to-be-detected food sample, and the thaumatin extraction rate is high. In the solid-phase extraction column purification and enrichment process, a PEP extraction column or a cation retin CX column is adopted, the thaumatin retention effect is good, 60-75 v / v% methanol-ammonia water serves as an eluent, the thaumatin recovery rate is high, the enrichment effect is good, and the impurity removal effect is good. Trypsin is used for carrying out enzymolysis on the purified liquid obtained through nitrogen blowing ammonia removal and methanol constant volume, obtained enzymatic hydrolysate is subjected to solid-phase extraction column desalination, and a characteristic peptide fragment of thaumatin can be obtained; by adopting liquid chromatography-mass spectrometry detection, accurate qualitative and quantitative detection of I and thaumatin II in food can be realized.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, specifically to a liquid chromatography-mass spectrometry (LC-MS) method for detecting semathys in food. Background Technology

[0002] Saumatran, also known as African sucralose, sucralose protein, or tamarind, is classified as sweetener 957 in the International Food Additives Code. It boasts numerous advantages, including being natural, low in calories, safe, non-toxic, and biodegradable into essential amino acids. Its sweetness is approximately 1600 times that of the same weight of sucrose, and its sweetness threshold is extremely low, even when diluted to 10%. -8 Its sweetness can still be perceived even at mol / L. Its main uses are enhancing aroma, modifying unpleasant flavors, providing a good mouthfeel, and synergistic effects with other ingredients. Currently, the total permitted levels of sematrandine in food are 0.5–300 mg / kg.

[0003] The main components of sematriline are sematriline I and sematriline II, and their amino acid composition is shown in Table 1.

[0004] Table 1 Amino acid composition of sematriol

[0005] Both sematrandine I and sematrandine II contain a single chain of 207 amino acids, with an N-terminus of alanine, lacking histidine, and both linked by eight disulfide bonds; however, sematrandine II differs from sematrandine I in six positions. The molecular weight of sematrandine is approximately 2.1 × 10⁻⁶. 4 Based on known amino acids, the molecular weight of sematrandine I is calculated to be 22209 g / mol, and the molecular weight of sematrandine II is 22293 g / mol. Existing detection methods for the two main components of sematrandine are prone to false positives, resulting in poor accuracy. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a liquid chromatography-mass spectrometry (LC-MS) method for detecting semaphore in food. The method provided by this invention can accurately detect the content of both semaphore I and semaphore II.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a high-performance liquid chromatography-tandem mass spectrometry method for the detection of semathys in food, comprising the following steps: Semamate was extracted from the food sample using an extraction solvent to obtain an extract; the extraction solvent was an aqueous solution of trifluoroacetic acid, and the volume fraction of trifluoroacetic acid in the aqueous solution was 0.1-1%. The extract was purified and enriched using a solid-phase extraction column. The eluent was collected, and after nitrogen stripping to remove ammonia, it was diluted to volume with methanol to obtain a purified solution. The conditions for the solid-phase extraction column purification and enrichment included: the extraction column being a PEP extraction column or a cation retainer CX column; the eluent being ammonia-methanol, wherein the volume fraction of ammonia in the ammonia-methanol mixture was 25-40%. The purified solution was enzymatically hydrolyzed using liquid trypsin, and the resulting hydrolysate was desalted using a solid-phase extraction column to obtain the sample solution to be tested. The test sample solution was subjected to liquid chromatography-mass spectrometry (LC-MS) to obtain the detection result of sematran. The sematriline is a mixture of sematriline I and sematriline II; The liquid chromatography-mass spectrometry (LC-MS) detection includes high performance liquid chromatography-mass spectrometry / mass spectrometry detection or high performance liquid chromatography-high resolution mass spectrometry / mass spectrometry detection. The high-performance liquid chromatography (HPLC) separation conditions for the liquid chromatography-mass spectrometry (LC-MS) detection include: a reversed-phase C18 column; mobile phase A is 0.1 v / v% formic acid-acetonitrile; mobile phase B is 0.1 v / v% formic acid-water; and the elution method is gradient elution. The gradient elution program includes: 0-1 min, the volume fraction of mobile phase A increases from 5% to 10%; 1-25 min, the volume fraction of mobile phase A increases from 10% to 50%; 25-27 min, the volume fraction of mobile phase A is 50%; 27-27.1 min, the volume fraction of mobile phase A decreases from 50% to 5%; and 27.1-30 min, the volume fraction of mobile phase A is 5%. The high-resolution mass spectrometry (HPLC-MS / MS) detection conditions include: ionization mode is electrospray ionization, positive ion; scanning mode is full scan data-dependent secondary mass spectrometry identification mode; scan range is 300~2000 m / z; optimized voltage is 30~50 eV; full scan resolution is 35000; sematrandine is a mixture of sematrandine I and sematrandine II; the unique characteristic peptide of sematrandine I is QLNSGESWTINVEPGTNGGK; the unique characteristic peptide of sematrandine II is QLNSGESWTINVEPGTK; the common characteristic peptides of sematrandine I and sematrandine II include FGRPPTTLAEFSLNQYGK, DYIDISNIK, and GFNVPMDFSPTTR; The mass spectrometry detection conditions for the high performance liquid chromatography-mass spectrometry / mass spectrometry detection include: ionization mode is electrospray ionization, positive ion; scanning mode is multiple reaction monitoring; electrospray voltage is 3800V; ion source temperature is 350℃; capillary temperature is 350℃; sheath gas pressure is 35psi; auxiliary gas pressure is 10psi; the common characteristic peptides of semaphore I and semaphore II include FGRPPTTLAEFSLNQYGK and DYIDISNIK.

[0008] Preferably, the column temperature of the high-performance liquid phase separator is 40°C and the mobile phase flow rate is 0.3 mL / min.

[0009] Preferably, the reversed-phase C18 column has a packing particle size of 5 μm, a packing pore size of 300 Å, and a column size of 100 mm × 2.1 mm.

[0010] Preferably, the enzymatic hydrolysis includes enzymatic hydrolysis using a SMART Digest trypsin kit; The enzymatic hydrolysis is performed at a temperature of 30-90℃ for 30-50 minutes.

[0011] Preferably, the food sample to be tested comes from general food samples, high-protein food samples, or high-fat food samples. The general food samples include one or more of the following: beverages, hard candy, powdered sugar, table sweeteners, betel nuts, and biscuits; The high-fat food samples include one or more of the following: chocolate, dried fruit, hot pot ingredients, sandwich cookies, and cakes; The high-protein food samples include one or more of the following: milkshakes, reconstituted milk, ice cream, and milk powder.

[0012] Preferably, the high-fat food sample is defatted before extraction to obtain the food sample to be tested; The ratio of the mass of the high-fat food sample to the volume of the extraction solvent is 1g:5~10mL.

[0013] Preferably, when the food sample to be tested is a high-protein food sample, the extraction includes: mixing the high-protein food sample with an extraction solvent, performing a first extraction, separating the solid and liquid components to obtain a first liquid component and a solid component respectively; mixing the solid component with the extraction solvent, performing a second extraction to obtain a second liquid component; and combining the first liquid component and the second liquid component as an extract. The ratio of the mass of the high-protein food sample to the volume of the extraction solvent used for the first extraction is 1g: 5~20mL; The ratio of the mass of the high-protein food sample to the volume of the extraction solvent used for the second extraction is 1g:1~4mL.

[0014] Preferably, the solid-liquid separation includes centrifugal separation or glass fiber filter paper filtration; When using glass fiber filter paper for filtration, after the second extraction is completed, the glass fiber filter paper is washed with the extraction solvent to obtain a washing solution. The extraction solution includes the washing solution, a first liquid component, and a second liquid component. The ratio of the mass of the high-protein food sample to the volume of the extraction solvent used for washing is 1g:0.5~2mL.

[0015] Preferably, the ratio of the mass of the general food sample to the volume of the extraction solvent is 1g:2~10mL.

[0016] Preferably, the extraction includes ultrasonic extraction, and the extraction time for a single extraction of the general food sample is 10-15 min; the extraction time for a single extraction of the high-protein food sample is 20-25 min; and the extraction time for a single extraction of the high-fat food sample is 20-25 min.

[0017] This invention utilizes trifluoroacetic acid aqueous solution as the extraction solvent to extract semamate from food samples, achieving a high extraction rate. During the solid-phase extraction (SPE) column purification and enrichment process, a PEP extraction column or a cationic retainer CX column is used, resulting in good semamate retention. Using 25-40 v / v% ammonia-methanol as the eluent, the semamate recovery rate is high, the enrichment effect is good, and the removal of impurities is effective. The purified solution, obtained after nitrogen stripping of ammonia and methanol dilution, is enzymatically hydrolyzed with trypsin. The resulting hydrolysate is then desalted using a SPE column to obtain characteristic semamate peptides. Liquid chromatography-mass spectrometry (LC-MS) detection enables accurate qualitative and quantitative detection of semamate I and semamate II in food. Attached Figure Description

[0018] Figure 1 The full scan chromatogram of the semathy hydrolysate obtained by enzymatic hydrolysis at 60℃ for 50 min is shown. Figure 2 Total ion chromatogram and mass spectrum of semathys characteristic peptides detected by high performance liquid chromatography-mass spectrometry / mass spectrometry; Figure 3 Ion chromatogram and mass spectrum of the common characteristic peptide FGRPPTTLAEFSLNQYGK of semamate I and semamate II detected by high performance liquid chromatography-mass spectrometry / mass spectrometry; Figure 4 Ion chromatogram and mass spectrum of DYIDISNIK, a common characteristic peptide of semaphore I and semaphore II, detected by high performance liquid chromatography-mass spectrometry / mass spectrometry. Detailed Implementation

[0019] This invention provides a liquid chromatography-mass spectrometry (LC-MS) method for detecting semathys in food, comprising the following steps: Semamate was extracted from the food sample using an extraction solvent to obtain an extract; the extraction solvent was an aqueous solution of trifluoroacetic acid, and the volume fraction of trifluoroacetic acid in the aqueous solution was 0.1-1%. The extract was purified and enriched using a solid-phase extraction column. The eluent was collected, and after nitrogen stripping to remove ammonia, it was diluted to volume with methanol to obtain a purified solution. The conditions for the solid-phase extraction column purification and enrichment included: the extraction column being a PEP extraction column or a cation retainer CX column; the eluent being ammonia-methanol, wherein the volume fraction of ammonia in the ammonia-methanol mixture was 25-40%. The purified solution was enzymatically hydrolyzed using liquid trypsin, and the resulting hydrolysate was desalted using a solid-phase extraction column to obtain the sample solution to be tested. The test sample solution was subjected to liquid chromatography-mass spectrometry (LC-MS) to obtain the detection result of sematran. The sematriline is a mixture of sematriline I and sematriline II; The liquid chromatography-mass spectrometry (LC-MS) detection includes high performance liquid chromatography-mass spectrometry / mass spectrometry detection or high performance liquid chromatography-high resolution mass spectrometry / mass spectrometry detection. The high-performance liquid chromatography (HPLC) separation conditions for the liquid chromatography-mass spectrometry (LC-MS) detection include: a reversed-phase C18 column; mobile phase A is 0.1 v / v% formic acid-acetonitrile; mobile phase B is 0.1 v / v% formic acid-water; and the elution method is gradient elution. The gradient elution program includes: 0-1 min, the volume fraction of mobile phase A increases from 5% to 10%; 1-25 min, the volume fraction of mobile phase A increases from 10% to 50%; 25-27 min, the volume fraction of mobile phase A is 50%; 27-27.1 min, the volume fraction of mobile phase A decreases from 50% to 5%; and 27.1-30 min, the volume fraction of mobile phase A is 5%. The high-resolution mass spectrometry (HPLC-MS / MS) detection conditions include: ionization mode is electrospray ionization, positive ion; scanning mode is full scan data-dependent secondary mass spectrometry identification mode; scan range is 300~2000 m / z; optimized voltage is 30~50 eV; full scan resolution is 35000; sematrandine is a mixture of sematrandine I and sematrandine II; the unique characteristic peptide of sematrandine I is QLNSGESWTINVEPGTNGGK; the unique characteristic peptide of sematrandine II is QLNSGESWTINVEPGTK; the common characteristic peptides of sematrandine I and sematrandine II include FGRPPTTLAEFSLNQYGK, DYIDISNIK, and GFNVPMDFSPTTR; The mass spectrometry detection conditions for the high performance liquid chromatography-mass spectrometry / mass spectrometry detection include: ionization mode is electrospray ionization, positive ion; scanning mode is multiple reaction monitoring; electrospray voltage is 3800V; ion source temperature is 350℃; capillary temperature is 350℃; sheath gas pressure is 35psi; auxiliary gas pressure is 10psi; the common characteristic peptides of semaphore I and semaphore II include FGRPPTTLAEFSLNQYGK and DYIDISNIK.

[0020] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0021] This invention utilizes an extraction solvent to extract semathys from the food sample to be tested, thereby obtaining an extract.

[0022] In this invention, the source of the food sample to be tested preferably includes general food samples, high-protein food samples, or high-fat food samples. In this invention, the general food sample refers to food samples other than high-protein and high-fat food samples. The general food sample preferably includes one or more of beverages, hard candy, powdered sugar, table sweeteners, betel nuts, and biscuits; the beverage includes fruit juice; the fruit juice includes orange juice; the table sweetener preferably includes ketchup and jam. In this invention, the high-protein food sample preferably includes one or more of milkshakes, reconstituted milk, ice cream, and milk powder. In this invention, the high-fat food sample preferably includes one or more of chocolate, dried fruit, hot pot ingredients, sandwich biscuits, and cakes; the dried fruit preferably includes walnuts and / or almonds.

[0023] In this invention, when the food sample to be tested is a high-protein food sample, the extraction preferably includes: mixing the high-protein food sample with an extraction solvent, performing a first extraction, separating the solid and liquid components to obtain a first liquid component and a solid component; mixing the solid component with the extraction solvent, performing a second extraction to obtain a second liquid component; and combining the first liquid component and the second liquid component as an extract. In this invention, the mixing preferably includes vortex mixing. This invention does not have a specific limitation on the vortex mixing, as long as it can mix the raw materials uniformly. During the mixing process, if viscous, undispersible clumps are formed in the mixing system instead of a uniform suspension, the amount of extractant can be increased. In this invention, the mass ratio of the high-protein food sample to the volume of the extraction solvent used for the first extraction is preferably 1g:5~20mL, specifically 1g:5mL, 1g:10mL, 1g:15mL, or 1g:20mL. In this invention, the preferred ratio of the mass of the high-protein food sample to the volume of the extraction solvent for the second extraction is 1g:2-4mL, specifically 1g:2mL, 1g:2.5mL, 1g:3mL, or 1g:4mL. In this invention, the solid-liquid separation includes centrifugation or glass fiber filter paper filtration. In this invention, the preferred centrifugation speed is 12000-16000 r / min, specifically 12000 r / min, 13000 r / min, 14000 r / min, 15000 r / min, or 16000 r / min; the preferred centrifugation time is 5-15 min, specifically 5 min, 8 min, 10 min, 12 min, or 15 min. In this invention, when using glass fiber filter paper filtration, after the second extraction is completed, the glass fiber filter paper is washed with the extraction solvent to obtain a washing liquid, which includes the washing liquid, a first liquid component, and a second liquid component. In this invention, the ratio of the mass of the high-protein food sample to the volume of the extraction solvent used for washing is 1g:0.5~2mL, specifically 1g:0.5mL, 1g:1mL, 1g:1.5mL or 1g:2mL.

[0024] In this invention, the high-fat food sample is preferably defatted before extraction to obtain the food sample to be tested. In this invention, defatting preferably includes defatting with a defatting solvent, and the defatting solvent preferably includes n-hexane. In this invention, the mass ratio of the high-fat food sample to the volume of the defatting solvent is preferably 1g:5~10mL, specifically 1g:5mL, 1g:6mL, 1g:7mL, 1g:8mL, 1g:9mL, or 1g:10mL. In this invention, the mass ratio of the high-fat food sample to the volume of the extraction solvent used for a single extraction is preferably 1g:5~10mL, specifically 1g:5mL, 1g:6mL, 1g:7mL, 1g:8mL, 1g:9mL, or 1g:10mL.

[0025] In this invention, the ratio of the mass of the general food sample to the volume of the extraction solvent is preferably 1g:2~10mL, specifically 1g:2mL, 1g:5mL, 1g:8mL or 1g:10mL.

[0026] In this invention, the extraction solvent is an aqueous solution of trifluoroacetic acid, and the volume fraction of trifluoroacetic acid in the aqueous solution is 0.1-1%, specifically 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%.

[0027] In this invention, the extraction preferably includes ultrasonic extraction, and the temperature of the ultrasonic extraction is preferably 20-30℃, more preferably 25℃. This invention does not specifically limit the power and frequency of the ultrasonic extraction; ultrasonic extraction power and frequency conditions well known to those skilled in the art can be used. The extraction time for a single extraction of the general food sample is preferably 10-15 min, specifically 10 min, 11 min, 12 min, 13 min, 14 min, or 15 min. The number of extractions of the general food sample is preferably 1-4 times, specifically 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 10 times, 11 times, 12 times, 13 times, 14 times, or 15 times. The extraction time for the high-protein food sample is preferably 20-25 min, specifically 20 min, 21 min, 22 min, 23 min, 24 min, or 25 min; the extraction number of the high-protein food sample is preferably 2 times; the extraction time for the high-fat food sample is preferably 20-25 min, specifically 20 min, 21 min, 22 min, 23 min, 24 min, or 25 min; the extraction number of the high-fat food sample is preferably 1-4 times, specifically 1 time, 2 times, 3 times, or 4 times.

[0028] After obtaining the extract, the present invention purifies and enriches the extract by solid-phase extraction column, collects the eluent, removes ammonia by nitrogen stripping the eluent and then makes up to volume with methanol to obtain the purified solution.

[0029] In this invention, the solid-phase extraction column used for purification and enrichment includes a PEP extraction column or a cationic retain-CX column. Preferably, the solid-phase extraction column is activated before use, which preferably includes sequential activation with methanol and water, followed by negative pressure removal of the eluent. The eluent used for purification and enrichment in this invention is ammonia-methanol, wherein the volume fraction of ammonia in the ammonia-methanol mixture is 25-40%, specifically 25%, 30%, 35%, or 40%. The mass concentration of the ammonia is preferably 25-30%, specifically 25%, 26%, 27%, 28%, 29%, or 30%.

[0030] In this invention, the ratio of the mass of the food sample to be tested to the volume of the purification liquid can be 1~2g:2mL, specifically 1g:2mL, 1.5g:2mL or 2g:2mL.

[0031] After obtaining the purified solution, the present invention uses liquid trypsin to enzymatically hydrolyze the purified solution, and the resulting enzymatic hydrolysate is desalted by solid-phase extraction column to obtain the sample solution to be tested.

[0032] In this invention, the enzymatic hydrolysis preferably includes enzymatic hydrolysis using a SMART Digest trypsin kit. In this invention, the enzymatic hydrolysis preferably includes: mixing the purification solution, liquid trypsin, and buffer solution, and then performing enzymatic hydrolysis. In this invention, the volume ratio of the purification solution to the liquid trypsin is preferably 1:0.1 to 0.4, specifically 1:0.1, 1:0.2, 1:0.3, or 1:0.4. In this invention, the volume ratio of the purification solution to the buffer solution is preferably 1:2 to 4, specifically 1:2, 1:2.5, 1:2.8, 1:3, 1:3.5, or 1:4.

[0033] In this invention, the enzymatic hydrolysis temperature is preferably 30~90℃, specifically 30℃, 40℃, 50℃, 60℃, 70℃, 80℃ or 90℃; the enzymatic hydrolysis time is preferably 30~50min, specifically 30min, 35min, 40min, 45min or 50min; the enzymatic hydrolysis pH value is preferably 7.5~8.5, specifically 7.5, 7.8, 8, 8.2 or 8.5.

[0034] In this invention, the solid phase extraction column is further activated before use. The activation preferably includes sequential activation with acetonitrile and 0.1 v / v% formic acid water, followed by negative pressure to discard the effluent.

[0035] In this invention, the desalting process of the solid-phase extraction column preferably includes sequential washing with formic acid aqueous solution and elution with formic acid-acetonitrile-aqueous solution, with the collected formic acid-acetonitrile-aqueous solution serving as the sample solution to be tested. In this invention, the volume fraction of formic acid in the formic acid aqueous solution is preferably 0.1-1%, specifically 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%. In this invention, the volume fraction of formic acid in the formic acid-acetonitrile-aqueous solution is preferably 0.1-1%, specifically 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%; the volume fraction of acetonitrile in the formic acid-acetonitrile-aqueous solution is preferably 60-80%, specifically 60%, 65%, 70%, 75%, or 80%.

[0036] After obtaining the sample solution to be tested, the present invention performs liquid chromatography-mass spectrometry (LC-MS) on the sample solution to obtain the detection result of sematran; the sematran is a mixture of sematran I and sematran II.

[0037] In this invention, the sample solution to be tested can be filtered through a 0.45 μm organic phase membrane before injection.

[0038] In this invention, the liquid chromatography-mass spectrometry (LC-MS) detection includes high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) detection or high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-MS / MS) detection; the HPLC separation conditions for the LC-MS detection include: the chromatographic column includes a reversed-phase C18 column, specifically a BioBasic column. TM The 18 LC column, preferably with a packing particle size of 5 μm, a pore size of 300 Å, and a column size of 100 mm × 2.1 mm (inner diameter); the column temperature is preferably 40 °C; mobile phase A is 0.1 v / v% trifluoroacetic acid aqueous solution, and mobile phase B is acetonitrile; the mobile phase flow rate is preferably 0.3 mL / min; the elution method is gradient elution, and the gradient elution program is shown in Table 2.

[0039] Table 2 Gradient elution program for mobile phase

[0040] In this invention, the conditions for high-resolution mass spectrometry detection using high-performance liquid chromatography-high-resolution mass spectrometry / mass spectrometry detection include: ionization mode is electrospray ionization, positive ions; scanning mode is full scan data-dependent secondary mass spectrometry identification mode (Full ms-DDMS). 2 Discovery); the scanning range is 300~2000 m / z; the optimized voltage is 30~50 eV; the full scan resolution R is 35000; the characteristic peptides of the semathymide and its main reference mass spectrometry parameters are shown in Table 3.

[0041] Table 3. Characteristic peptides of semaphore and their main reference mass spectrometry parameters

[0042] In this invention, the mass spectrometry detection conditions for the high performance liquid chromatography-mass spectrometry / mass spectrometry detection include: ionization mode is electrospray ionization, positive ion; scanning mode is multiple reaction monitoring; electrospray voltage is 3800V; ion source temperature is 350℃; capillary temperature is 350℃; sheath gas pressure is 35psi; auxiliary gas pressure is 10psi; the common characteristic peptides of semaphore I and semaphore II and their main reference mass spectrometry parameters are shown in Table 4.

[0043] Table 4. Characteristic peptides of semaphore and their main reference mass spectrometry parameters

[0044] To further illustrate the present invention, the method for detecting semathys in food using liquid chromatography-mass spectrometry (LC-MS) provided by the present invention is described in detail below with reference to embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0045] In the following examples, the reagents and materials are as follows: (1) Microporous membrane: Aqueous low-protein adsorption membrane, 0.22µm. (2) Centrifuge tubes: 1.5mL low-protein adsorption centrifuge tubes. (3) 100µL and 1000µL low-protein adsorption pipette tips. (4) SMART Digest liquid trypsin with buffer. (5) SOLAμ HRP solid-phase extraction column: 2mg / 1mL. (6) 0.1% formic acid-water solution: Measure 1mL of formic acid and dilute to 1L with water. (7) 0.1% formic acid-70% acetonitrile-water solution: Measure 1mL of formic acid, add 700mL of acetonitrile, and dilute to 1L with water. (8) Liquid trypsin and buffer: SMART Digest trypsin kit.

[0046] The instruments and equipment are as follows: (1) TSQ Ultra liquid chromatography-mass spectrometry: equipped with an electrospray ionization (ESI) source. (2) QE liquid chromatography-high resolution mass spectrometry: equipped with an electrospray ionization (ESI) source and proteomics software: Proteome Discoverer 3.0. (3) Constant temperature water bath.

[0047] Liquid chromatography conditions: Column: BioBasic TM18 LC column (100 mm × 2.1 mm (inner diameter), packing particle size of 5 μm, pore size of 300 Å); column temperature of 40 °C; mobile phase A of 0.1 v / v% formic acid-acetonitrile, mobile phase B of 0.1 v / v% formic acid-water, gradient elution as shown in Table 2; mobile phase flow rate of 0.3 mL / min; injection volume of 5 μL.

[0048] High-resolution mass spectrometry conditions: ionization mode was electrospray ionization, positive ion; scanning mode was full scan (full ms-ddms2 discovery); scan range was 300~2000 m / z; optimized voltages were 30 eV, 40 eV, and 50 eV; full scan resolution R=35000. The characteristic peptides of semathymide and their main reference mass spectrometry parameters are shown in Table 3.

[0049] Mass spectrometry conditions: ionization mode was electrospray ionization, positive ion; scanning mode was multiple reaction monitoring; electrospray voltage was 3800 V; ion source temperature was 350 °C; capillary temperature was 350 °C; sheath gas pressure was 35 psi; auxiliary gas pressure was 10 psi; the characteristic peptides of semathys and their main reference mass spectrometry parameters are shown in Table 4.

[0050] Example 1 Sample pretreatment 1. Sample selection The study tested semathymide in commercially available foods and obtained 8 positive samples and 22 negative samples. The samples included beverages, juices, processed nuts, baked goods, candies, table sweeteners, frozen drinks, dairy products, chocolates and other foods. The results can be used to optimize the extraction method of standard methods, determine the limit of detection, limit of quantitation, recovery rate, precision, accuracy and laboratory validation.

[0051] 2. Determining the extraction method Semamate is highly soluble in water and soluble in 60 v / v% ethanol aqueous solution, but insoluble in organic solvents such as acetone. Based on the properties of sesame and the SPE purification conditions, experiments were conducted on the extraction process of different matrices.

[0052] 2.1 Pretreatment of general food samples 2.1.1 Optimization of extraction solvent General food samples: beverages, orange juice, hard candy, powdered sugar, ketchup, jam, betel nuts, and biscuits. Hard candy, betel nuts, and biscuits should be crushed before use.

[0053] Extraction solvents: 0.1 v / v% trifluoroacetic acid aqueous solution, 0.2 v / v% trifluoroacetic acid aqueous solution, 1 v / v% trifluoroacetic acid aqueous solution or 60 v / v% ethanol aqueous solution.

[0054] Extraction solvents of trifluoroacetic acid aqueous solution and 60 v / v% ethanol aqueous solution at different concentrations were used. The mixtures were vortexed and a recovery experiment was conducted to compare the recovery rates of sematrandrolite extracted by room temperature ultrasonication in different extracts. The specific experimental steps are as follows: Weigh 2 g (accurate to 0.001 g) of food sample into a 15 mL stoppered centrifuge tube, add 5 mL of extraction solvent, vortex and mix, ultrasonically extract for 10 min, centrifuge at 14000 r / min for 10 min, and collect the supernatant. Repeat the extraction once with 5 mL of extraction solvent, centrifuge at 14000 r / min for 10 min, and collect the supernatant. Combine the two supernatants as the extract. The recovery rates of sematrandrolite under different extraction solvents are shown in Table 5.

[0055] Table 5. Recovery rates (%) of semastem using different extraction solvents.

[0056] Results: Trifluoroacetic acid aqueous solution was superior to 60 v / v% ethanol aqueous solution in terms of extraction efficiency and applicability. The content of trifluoroacetic acid in aqueous solution had little effect on the extraction rate of the target compound semathymide. Finally, 0.1 v / v% trifluoroacetic acid aqueous solution was selected as the extraction solvent for subsequent experiments.

[0057] 2.1.2 Optimization of extraction time The positive samples (sugar powder, areca nut 1#, areca nut 2#, areca nut 3#, areca nut 4#, and areca nut 5#) were tested using a 0.1 v / v% trifluoroacetic acid aqueous solution. The extract was prepared according to 2.1.1, with ultrasonic extraction times of 5 min, 10 min, and 15 min, respectively. The effect of ultrasonic extraction time (5~15 min) on the extraction rate of the target compound semathymide was compared, and the results are shown in Table 6.

[0058] Table 6. Test values ​​(mg / kg) of sematrandole at different extraction times in 0.1 v / v% trifluoroacetic acid aqueous solution.

[0059] Results: After ultrasonic extraction for more than 10 minutes, the test value of semathymide in the sample no longer changed significantly. Therefore, an ultrasonic extraction time of 10 minutes was finally selected for subsequent experiments.

[0060] 2.2 Pretreatment of High-Protein Food Samples The target compound, sematrandine, is a large protein molecule. During the removal of matrix proteins using methods such as potassium ferrocyanide and zinc acetate precipitation, sematrandine aggregates and cannot be recovered. For high-protein food samples that can be removed by centrifugation, centrifugation is used to remove insoluble matter. For samples with poor centrifugation results, the sample weight should be reduced or filtration with glass fiber filter paper should be performed. If the sample is viscous, the amount of 0.1 v / v% trifluoroacetic acid aqueous solution can be increased. If filtration with glass fiber filter paper is performed, the filter paper should be rinsed with 2 mL of 0.1% trifluoroacetic acid solution after two extractions.

[0061] The specific steps are as follows: Weigh 2g (accurate to 0.001g) of food sample into a 15mL stoppered centrifuge tube, add 10mL of extraction solvent, vortex to mix, extract ultrasonically for 20min, centrifuge at 14000r / min for 10min, and collect the supernatant. Add 5mL of extraction solvent and repeat the extraction once, centrifuge at 14000r / min for 10min, collect the supernatant, and combine the two supernatants as the extract.

[0062] 2.2.1 Optimization of extraction solvent High-protein food samples: milkshakes, reconstituted milk, ice cream, milk powder.

[0063] Extraction solvent: 0.1 v / v% trifluoroacetic acid aqueous solution or 60 v / v% ethanol aqueous solution.

[0064] The content of trifluoroacetic acid in aqueous solution has little effect on the extraction efficiency of the target compound. Extraction was performed using 0.1% trifluoroacetic acid aqueous solution and 60 v / v% ethanol aqueous solution, respectively, under isothermal ultrasonic extraction. Addition and recovery experiments were conducted to compare the recovery rate of sematrandil in high-protein samples. The specific steps are as follows: 2g (accurate to 0.001g) of food sample was defatted with 10mL of n-hexane, 20mL of extraction solvent was added, and the mixture was ultrasonically extracted at room temperature for 20min. The mixture was filtered through glass fiber filter paper, and the glass fiber filter paper was washed with 3mL of extraction solvent to obtain the first liquid component and the solid component. The solid component was mixed with 15mL of extraction solvent, and ultrasonically extracted at room temperature for 20min. The mixture was filtered through glass fiber filter paper, and the glass fiber filter paper was washed with 2mL of extraction solvent to obtain the second liquid component. The first and second liquid components were combined as the extraction solution. The extraction solvents were 0.1 v / v% trifluoroacetic acid aqueous solution and 60 v / v% ethanol aqueous solution, respectively.

[0065] The recovery rates of semastem under different extraction solvents are shown in Table 7.

[0066] Table 7. Recovery rates (%) of sematrandrine with different extraction solvents

[0067] Results: High-protein samples showed better extraction efficiency when 0.1% trifluoroacetic acid solution was used as the extraction solvent.

[0068] 2.2.2 Optimization of extraction time Following the experimental procedure in 2.2.1, the positive sample milkshake was tested using a 0.1 v / v% trifluoroacetic acid aqueous solution to compare the effect of ultrasonic extraction time (5~25 min) on the extraction efficiency of the target analyte. The results are shown in Table 8.

[0069] Table 8. Test values ​​(mg / kg) of sematrandole at different extraction times in 0.1 v / v% trifluoroacetic acid aqueous solution.

[0070] Results: After ultrasonic extraction for more than 20 minutes, the test value of semathy in high-protein samples no longer changed significantly. Therefore, the ultrasonic extraction time of 20 minutes was finally selected.

[0071] 2.3 High-fat food samples 2.3.1 Optimization of Extraction Solvent High-fat samples: chocolate, walnuts, hot pot ingredients, sandwich cookies, cakes.

[0072] Extraction solvent: 0.1 v / v% trifluoroacetic acid aqueous solution or 60 v / v% ethanol aqueous solution.

[0073] High-oil samples were dissolved in n-hexane. Experiment 2.1 showed that the content of trifluoroacetic acid in the aqueous solution had little effect on the extraction efficiency of the target compound. Extraction was performed by ultrasonic extraction in a water bath using either 0.1% trifluoroacetic acid aqueous solution or 60 v / v% ethanol aqueous solution. After centrifugation, the organic phase was discarded. The recovery rate of thomaline was compared. The specific steps are as follows: 2 g (accurate to 0.001 g) of food sample was mixed with 20 mL of extraction solvent and ultrasonically extracted at room temperature for 20 min. Centrifugation was performed at 14000 r / min for 10 min, and the supernatant was collected to obtain the first liquid component and the solid component. The solid component was mixed with 15 mL of extraction solvent and ultrasonically extracted at room temperature for 20 min. Centrifugation was performed at 14000 r / min for 10 min, and the supernatant was collected to obtain the second liquid component. The first and second liquid components were combined as the extract. The extraction solvents were 0.1 v / v% trifluoroacetic acid aqueous solution and 60 v / v% ethanol aqueous solution, respectively.

[0074] The results are shown in Table 9.

[0075] Table 9. Recovery rates (%) of semastem using different extraction solvents

[0076] Results: Semamate extraction was better when 0.1% trifluoroacetic acid solution was used as the extraction solvent.

[0077] 2.3.2 Optimization of extraction time Following the experimental procedure in 2.3.1, a 0.1 v / v% trifluoroacetic acid aqueous solution was used to conduct experiments on the positive sample dried fruit (commercially available flavored almonds) to compare the effect of ultrasonic extraction time (5~25 min) on the extraction efficiency of the target substance. The results are shown in Table 10.

[0078] Table 10. Test values ​​(mg / kg) of sematrandole at different extraction times in 0.1 v / v% trifluoroacetic acid aqueous solution.

[0079] Results: After ultrasonic extraction for more than 20 minutes, the test value of sematrandine in high-oil samples no longer changed significantly. Therefore, the ultrasonic extraction time of 20 minutes was finally selected.

[0080] 3. Selection of purification conditions Sample solution: 5 mL of 0.1 v / v% trifluoroacetic acid aqueous solution containing 0.5 mg sematrandezine.

[0081] 3.1 Optimization of Solid Phase Extraction Column Literature reports on the purification of sematrandrolone indicate that solid-phase extraction (SPE) or ultrafiltration can be used, although ultrafiltration is time-consuming. For additive purification, SPE is frequently used in relevant standards, with C18, PEP, and MCX columns being the most common. C18 columns are known for their ability to remove impurities. This study investigates the limits for sematrandrolone in food products both domestically and internationally, and their detection capabilities using liquid chromatography. This invention purifies and concentrates the sample solution simultaneously. Using a 25% ammonia-methanol solution as the eluent, the extract is purified and enriched using Hypersep Retain PEP and cationic retain CX columns, respectively. The eluent is collected, and after nitrogen purging to remove ammonia, it is diluted to 2 mL with methanol to obtain the purified solution. Before use, the Hypersep Retain PEP and cationic retain CX columns are activated sequentially with 5 mL of methanol and 5 mL of water, respectively. The SPE results for different columns are shown in Table 11.

[0082] Table 11 Recovery rates (%) of sematran under different SPE solid-phase extraction columns

[0083] Results: Both the PEP column and the cation retain CX column showed no flow-through and good retention of the target analyte, with the PEP column showing better recovery.

[0084] 3.2 Optimization of eluent for solid phase extraction columns Using Hypersep Retain PEP solid-phase extraction columns, the elution effects of different eluents (2~60 v / v% ammonia-methanol) were investigated to assess the solubility of sematrandrolone. The relevant recovery rates are shown in Table 12.

[0085] Table 12 Recovery rates of sematran with different elution solvents

[0086] Results: The elution effect of 25-35% ammonia-methanol solution was better. Considering the amount of ammonia used, the present invention selected 25% ammonia-methanol solution as the eluent.

[0087] 4. Enzymatic hydrolysis of semaprotein The purified solution obtained in "3. Selection of Purification Conditions" was enzymatically digested using the SMART Digest trypsin kit. The specific steps are as follows: The purified solution was placed in a 1.5 mL low-protein adsorption centrifuge tube, and 10 μL of liquid trypsin and 140 μL of buffer were added. After mixing, the solution was placed at enzymatic digestion temperatures of 30℃, 60℃, 70℃, and 90℃ for 30 min, 40 min, and 50 min, respectively. The digested solution was then transferred to a solid-phase extraction column (SOLA μHRP solid-phase extraction column, 2 mg / 1 mL). After the sample solution had completely eluted, it was washed with 500 μL of 0.1 v / v% formic acid-water. After the liquid in the column had completely drained, it was eluted with 50 μL of 0.1 v / v% formic acid-70 v / v% acetonitrile-water solution. The eluent was collected and filtered through a 0.45 μm organic phase filter membrane to obtain the sample solution for testing. Before use, the solid-phase extraction column was activated sequentially with 500 μL of acetonitrile and 500 μL of 0.1 v / v% formic acid solution, and the effluent was discarded under negative pressure. The semathymide recovery results are shown in Table 13.

[0088] Table 13 Recovery rate of semaster under different enzymatic hydrolysis conditions

[0089] As shown in Table 13, the recovery rate of sematrandrine was highest when the enzymatic hydrolysis temperature was 70℃. This invention performs enzymatic hydrolysis at 70℃, simultaneously denaturing the protein and cleaving it, thus significantly improving the recovery rate of sematrandrine.

[0090] 5. Liquid chromatography-high resolution mass spectrometry / mass spectrometry detection The sample solution was analyzed by liquid chromatography-high resolution mass spectrometry / mass spectrometry to obtain the detection results of semathys.

[0091] Figure 1 The full scan chromatogram of the semaphore hydrolysate obtained by enzymatic hydrolysis at 60℃ for 50 min shows that, by analyzing the high-resolution mass spectrometry data using the proteomics software Proteome Discoverer 3.0 and the semaphore protein structure, characteristic peptide sequence information was obtained (Table 3). One peptide is unique to semaphore I, one peptide is unique to semaphore II, and the remaining three peptides are shared by semaphore I and semaphore II.

[0092] 6. Liquid Chromatography-Mass Spectrometry / Mass Spectrometry Detection The sample solution was analyzed by liquid chromatography-mass spectrometry (LC-MS / MS) to obtain the detection results of semamate. The acquisition parameters for the two characteristic peptides with high response values ​​were optimized using TSQULtra LC-MS / MS tune software; the optimization results are shown in Table 4. Measurements were performed under optimized conditions, and the total ion chromatogram and mass spectrum of the semamate characteristic peptides were obtained. Figure 2 The ion chromatogram and mass spectrum of the common characteristic peptide FGRPPTTLAEFSLNQYGK of semathys I and semathys II are shown in [reference needed]. Figure 3 The ion chromatogram and mass spectrum of DYIDISNIK, a common characteristic peptide of semaphore I and semaphore II, are shown in [reference needed]. Figure 4 From Table 4 and Figures 2-4 As can be seen, the characteristic peptide sequence information was obtained (Table 3), one of which is a unique peptide of samamethoxam I. The common peptides FGRPPTTLAEFSLNQYGK and DYIDISNIK of samamethoxam I and samamethoxam II were detected by liquid chromatography-mass spectrometry / mass spectrometry.

[0093] Example 2 Methodological Validation 1. Specificity test The 0.1 v / v% formic acid-acetonitrile, 0.1 v / v% formic acid-water and 0.1 v / v% trifluoroacetic acid aqueous solution were detected under the detection conditions of high performance liquid chromatography-tandem mass spectrometry. The results showed that there was no interference from the above solutions, indicating that the method has high specificity.

[0094] 2. Linear range Preparation of 100% semaphore standard enzymatic hydrolysate: Semaphore was dissolved in 0.1 v / v% trifluoroacetic acid aqueous solution to obtain a semaphore stock solution with a concentration of 1 mg / mL. The semaphore stock solution was serially diluted with 0.1 v / v% trifluoroacetic acid aqueous solution to obtain semaphore solutions with concentrations of 0.001 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, and 0.8 mg / mL. Using 25% ammonia-methanol solution as the elution solvent, the semaphore solutions with concentrations from 0.0001 to 1 mg / mL were purified using a Hypersep Retain PEP solid-phase extraction column to obtain purified solutions. The purified solutions were then enzymatically hydrolyzed according to the semaphore protein enzymatic hydrolysis method described in Example 2, Section 4, to obtain the semaphore standard enzymatic hydrolysate. The detection was performed according to the detection conditions of high performance liquid chromatography-tandem mass spectrometry. The enzyme digests of each samaram standard were measured separately. A standard curve was established with the concentration of the samaram standard enzyme digest as the abscissa and the corresponding peak area as the ordinate. The results showed that the linearity was good in the concentration range of 1~1000 mg / L.

[0095] 3. Precision Preparation of semamate standard enzymatic hydrolysate: Soak semamate was dissolved in 0.1 v / v% trifluoroacetic acid aqueous solution to obtain a semamate solution with a concentration of 0.1 mg / mL. The semamate solution was purified using a Hypersep Retain PEP solid-phase extraction column with 25% ammonia-methanol solution as the elution solvent to obtain a purified solution. The purified solution was then enzymatically hydrolyzed according to the semamate protein enzymatic hydrolysis method described in Example 2, Section 4, to obtain the semamate standard enzymatic hydrolysate.

[0096] The semaphore standard enzymatic hydrolysate was repeatedly detected 6 times under the detection conditions of high performance liquid chromatography-tandem mass spectrometry. The RSD was 1.4%, indicating that the detection method provided by the present invention has high precision.

[0097] 4. Spike recovery rate Recovery and precision experiments were conducted using negative sample spiking recovery experiments (n=6), with orange juice, powdered sugar, tomato sauce, areca nut, biscuits, milk powder, and walnut kernels used as negative samples. Spiking recovery experiments were performed at three concentration levels (25 mg / kg, 50 mg / kg, and 100 mg / kg), with pretreatment performed under the optimal pretreatment conditions described in Examples 2-3. Each concentration level was replicated six times. The average recovery rate and relative standard deviation are shown in Table 14. The average recovery rate of sematrandine in the seven matrices of general food samples, high-protein food samples, and high-fat food samples was 92.1-99.2%, with a relative standard deviation (RSD) below 4.1%, indicating high accuracy and precision. This demonstrates that the detection method provided by this invention is feasible for the analysis and detection of sematrandine in food samples.

[0098] Table 14 Results of spiked recovery of sematriol in food samples (n=6)

[0099] 5. Limit of detection and limit of quantitation The semaphore standard enzymatic hydrolysate prepared with precision in step 2 was detected under the detection conditions of high performance liquid chromatography-tandem mass spectrometry. The limit of quantitation was determined for the concentrations where the ratio of the analyte signal (S) to baseline noise (N) was ≥10 (S / N≥10), and the limit of detection was determined for the concentrations where S / N≥3. The results are shown in Table 15.

[0100] Table 15 Results of Limit of Detection and Limit of Quantification (mg / kg)

[0101] As shown in Table 15, the detection method provided by the present invention has a detection limit of 0.3~0.5 mg / kg, a quantitation limit of 1~1.7 mg / kg, and high detection sensitivity.

[0102] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A high-performance liquid chromatography-tandem mass spectrometry method for the detection of semathys in food, comprising the following steps: Semamate was extracted from the food sample using an extraction solvent to obtain an extract; the extraction solvent was an aqueous solution of trifluoroacetic acid, and the volume fraction of trifluoroacetic acid in the aqueous solution was 0.1-1%. The extract was purified and enriched by solid-phase extraction column, the eluent was collected, and the eluent was subjected to nitrogen stripping to remove ammonia and then diluted with methanol to obtain a purified solution. The conditions for purification and enrichment by the solid-phase extraction column include: the extraction column includes a PEP extraction column or a cationic retain CX column, and the eluent is ammonia-methanol, wherein the volume fraction of ammonia in the ammonia-methanol is 25-40%; The purified solution was enzymatically hydrolyzed using liquid trypsin, and the resulting hydrolysate was desalted using a solid-phase extraction column to obtain the sample solution to be tested. The test sample solution was subjected to liquid chromatography-mass spectrometry (LC-MS) to obtain the detection result of sematran. The sematriline is a mixture of sematriline I and sematriline II; The liquid chromatography-mass spectrometry (LC-MS) detection includes high performance liquid chromatography-mass spectrometry / mass spectrometry detection or high performance liquid chromatography-high resolution mass spectrometry / mass spectrometry detection. The high-performance liquid chromatography (HPLC) separation conditions for the liquid chromatography-mass spectrometry (LC-MS) detection include: a reversed-phase C18 column; mobile phase A is 0.1 v / v% formic acid-acetonitrile; mobile phase B is 0.1 v / v% formic acid-water; and the elution method is gradient elution. The gradient elution program includes: 0-1 min, the volume fraction of mobile phase A increases from 5% to 10%; 1-25 min, the volume fraction of mobile phase A increases from 10% to 50%; 25-27 min, the volume fraction of mobile phase A is 50%; 27-27.1 min, the volume fraction of mobile phase A decreases from 50% to 5%; and 27.1-30 min, the volume fraction of mobile phase A is 5%. The high-resolution mass spectrometry (HPLC-MS / MS) detection conditions include: ionization mode is electrospray ionization, positive ion; scanning mode is full scan data-dependent secondary mass spectrometry identification mode; scan range is 300~2000 m / z; optimized voltage is 30~50 eV; full scan resolution is 35000; sematrandine is a mixture of sematrandine I and sematrandine II; the unique characteristic peptide of sematrandine I is QLNSGESWTINVEPGTNGGK; the unique characteristic peptide of sematrandine II is QLNSGESWTINVEPGTK; the common characteristic peptides of sematrandine I and sematrandine II include FGRPPTTLAEFSLNQYGK, DYIDISNIK, and GFNVPMDFSPTTR; The mass spectrometry detection conditions for the high performance liquid chromatography-mass spectrometry / mass spectrometry detection include: ionization mode is electrospray ionization, positive ion; scanning mode is multiple reaction monitoring; electrospray voltage is 3800V; ion source temperature is 350℃; capillary temperature is 350℃; sheath gas pressure is 35psi; auxiliary gas pressure is 10psi; the common characteristic peptides of semaphore I and semaphore II include FGRPPTTLAEFSLNQYGK and DYIDISNIK.

2. The liquid chromatography-mass spectrometry (LC-MS) detection method according to claim 1, characterized in that, The column temperature for the high-performance liquid chromatography (HPLC) separation is 40°C, and the mobile phase flow rate is 0.3 mL / min.

3. The liquid chromatography-mass spectrometry (LC-MS) detection method according to claim 1, characterized in that, The reversed-phase C18 column has a packing particle size of 5 μm, a packing pore size of 300 Å, and a column size of 100 mm × 2.1 mm.

4. The liquid chromatography-mass spectrometry (LC-MS) detection method according to claim 1, characterized in that, The enzymatic hydrolysis includes enzymatic hydrolysis using the SMARTDigest trypsin kit. The enzymatic hydrolysis is performed at a temperature of 30-90℃ for 30-50 minutes.

5. The method according to claim 1, characterized in that, The sources of the food samples to be tested include general food samples, high-protein food samples, or high-fat food samples; The general food samples include one or more of the following: beverages, hard candy, powdered sugar, table sweeteners, betel nuts, and biscuits; The high-fat food samples include one or more of the following: chocolate, dried fruit, hot pot ingredients, sandwich cookies, and cakes; The high-protein food samples include one or more of the following: milkshakes, reconstituted milk, ice cream, and milk powder.

6. The liquid chromatography-mass spectrometry (LC-MS) detection method according to claim 5, characterized in that, The high-fat food sample was defatted before extraction to obtain the food sample to be tested. The ratio of the mass of the high-fat food sample to the volume of the extraction solvent is 1g:5~10mL.

7. The liquid chromatography-mass spectrometry (LC-MS) detection method according to claim 5, characterized in that, When the food sample to be tested is a high-protein food sample, the extraction includes: mixing the high-protein food sample with an extraction solvent, performing a first extraction, separating the solid and liquid components to obtain a first liquid component and a solid component respectively; mixing the solid component with the extraction solvent, performing a second extraction to obtain a second liquid component; and combining the first liquid component and the second liquid component as an extract. The ratio of the mass of the high-protein food sample to the volume of the extraction solvent used for the first extraction is 1g: 5~20mL; The ratio of the mass of the high-protein food sample to the volume of the extraction solvent used for the second extraction is 1g:1~4mL.

8. The detection method according to claim 7, characterized in that, The solid-liquid separation includes centrifugal separation or glass fiber filter paper filtration; When using glass fiber filter paper for filtration, after the second extraction is completed, the glass fiber filter paper is washed with the extraction solvent to obtain a washing solution. The extraction solution includes the washing solution, a first liquid component, and a second liquid component. The ratio of the mass of the high-protein food sample to the volume of the extraction solvent used for washing is 1g:0.5~2mL.

9. The liquid chromatography-mass spectrometry (LC-MS) detection method according to claim 5, characterized in that, The ratio of the mass of the general food sample to the volume of the extraction solvent is 1g:2~10mL.

10. The liquid chromatography-mass spectrometry (LC-MS) detection method according to any one of claims 5 to 9, characterized in that, The extraction includes ultrasonic extraction. The extraction time for a single extraction of the general food sample is 10-15 min; the extraction time for a single extraction of the high-protein food sample is 20-25 min; and the extraction time for a single extraction of the high-fat food sample is 20-25 min.

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