Rapid screening method and screening database for food-borne stimulants in racing food based on high-throughput technology

Abstract

The invention provides a method for rapidly screening food-borne stimulants in racing food based on a high-throughput technology. According to the method, 64 food-borne stimulants are detected by adopting a phase chromatography-high resolution mass spectrometry. According to the method, the detection efficiency is remarkably improved, synchronous detection of 64 food-borne stimulant drugs can be completed within 6 hours, and the speed is increased by 4-12 times compared with that of a traditional method; the detection cost is greatly reduced, all detection items can be completed by only one device and one detection method, accurate qualitative screening can be carried out without a standard substance, the use of expensive isotope interior labels is completely abandoned, the operation process is simplified, and the device investment and the operation cost are remarkably reduced; environmental pollution is reduced, and the usage amount of chemical reagents is reduced by optimizing a reagent formula and a detection process; according to the food-borne stimulant detection method, the detection sensitivity is equal to or even superior to that of a traditional national standard method, the lowest detection limit meets the strict requirements of an international anti-stimulant institution (WADA), the detection indexes comprehensively cover all food-borne stimulant items required by current sports events, and the strict requirements of domestic major event guarantee are completely met.

Description

Technical Field

[0001] The present invention relates to a rapid screening method and a screening database for foodborne stimulants in food for competition based on high-throughput technology, and belongs to the technical field of food detection. Background Art

[0002] Foodborne stimulant drugs refer to the stimulant components in food, and these foods may be general foods, edible agricultural products or health foods. Their sources may be naturally present during the production and processing of food, or may be residues deliberately added by humans. When athletes ingest foods containing stimulant drugs, it will not only undermine the fairness of the competition, stain personal reputations, but also may damage the national image and have an adverse impact on the reputation of food safety supervision. Currently, foodborne stimulant drug problems caused by the contamination of typical foods such as meat or the accidental ingestion of drugs are common. Among them, most of the accidental ingestion incidents occur during large-scale sports competitions, and have occurred in events such as football, swimming, track and field, basketball, and tennis. In view of the increasing number of doping positive incidents caused by food contamination in recent years and the increasingly prominent food safety problems caused by stimulants, food safety supervision and management departments, especially those responsible for ensuring food safety in sports events, have attached increasing importance to ensuring food safety in events and strictly controlling foodborne stimulant drugs.

[0003] Currently, the detection methods for stimulant drugs mainly include immunoassay, chromatography and mass spectrometry, etc. Immunoassay is an analytical method based on the specific binding of antigen and antibody. Generally used as a screening method, it provides too little structural information of the analyte, is prone to false positives, and other methods such as chromatography or mass spectrometry techniques are needed to quantify and confirm the analyte. Chromatography mainly includes gas chromatography and liquid chromatography. Liquid chromatography has wide applicability and is usually used in combination with ultraviolet, diode array and fluorescence detectors, etc. However, these detectors usually cannot provide the structural information of compounds and have insufficient discrimination ability for compounds with similar retention characteristics, thus limiting its application scope. Gas chromatography is applicable to thermally stable volatile compounds, and derivatization steps are required for most stimulants. Liquid chromatography-tandem mass spectrometry has been widely used in the detection of various drug residues in food due to its wide practicability, high sensitivity and selectivity. Most of the currently effective national foodborne stimulant drug detection standard methods are this method. Traditional tandem mass spectrometry methods can obtain the mass-to-charge ratio information of compounds, but due to their low mass resolution and mass accuracy, they have deficiencies in the non-targeted analysis and high-throughput screening of compounds.

[0004] On November 23, 2021, the Anti-Doping Center of the General Administration of Sport issued the "Guidelines for the Prevention and Control of Foodborne Doping Drugs in Large-scale Events" (Letter No. 584 of the General Administration of Sport Anti-Doping

[2021] , hereinafter referred to as Document No. 584), updating the prevention and control guidelines for foodborne doping drugs. The number of indicators has been expanded to 55, including 6 categories such as β2-agonists, β-blockers, anabolic agents, glucocorticoids, diuretics, and metabolic regulators. Currently, the detection standards and methods at home and abroad only cover the detection of 43 target substances, and there are no corresponding detection standards or methods for 5 target substances such as atenolol, metoprolol, clenproperol, bendroflumethiazide, and tretoquinol. In addition, the existing detection standards or methods also have the defect of incomplete scope of application. High-resolution mass spectrometry has higher mass accuracy, higher mass resolution, and the ability to analyze unknown compounds, providing a better solution for the extensive and high-throughput screening of foodborne doping drugs.

[0005] In recent years, how to quickly and effectively avoid the food safety problem of foodborne doping in sports events has become one of the key points of the work of the organizers. The traditional detection methods for foodborne doping drugs mainly target single indicators, unable to represent all the information in the sample, with low detection efficiency and unable to meet the requirements of timeliness and accuracy of detection for various events. Document No. 584 clearly requires that to ensure the timeliness of food supply for events, the organizers of large-scale events should require the testing agency to issue a test report for the sampled products within 48 hours, and the test report for fresh products should be issued within 24 hours. According to the current detection methods, more than a dozen standard methods need to be applied for detection. If all items are detected simultaneously, the cycle is long, unable to meet the detection time limit for emergency samples, and the costs of personnel and instruments are high.

[0006] CN202311011333.X, Invention Title: A Method for Simultaneously Detecting 24 Foodborne Stimulants in Foods. A sample pretreatment method for simultaneously detecting 24 foodborne stimulants in foods is provided. The present invention also provides a method for simultaneously detecting 24 foodborne stimulants in foods. The present invention combines 11 β2-agonists (salbutamol, ractopamine, terbutaline, fenoterol, tulobuterol, penbutolol, cimaterol, clenproperol, clenbuterol, salmeterol, zilpaterol), 4 β-blockers (propranolol, metoprolol, carteolol, atenolol), 2 anabolic agents (dehydroepiandrosterone, metenolone), 1 glucocorticoid (cortisone), and 6 mycotoxins (zeranol, β-zearalenol, α-zearalenol, β-zearalenol, zearalanone, zearalenone), a total of 24 foodborne stimulants. According to their physical and chemical properties, by investigating the extractant, extraction time, etc., a pretreatment method for 24 foodborne stimulants in foods with strong generality, good reproducibility, and good adaptability to complex matrices is established; a liquid chromatography-triple quadrupole mass spectrometer with strong specificity is selected for determination.

[0007] CN202311018839.3, Invention Title: A Method for Simultaneously Detecting 34 Foodborne Stimulants in Foods. A method for simultaneously detecting 34 foodborne stimulants in foods is provided. It uses a high performance liquid chromatography-mass spectrometry combined method to detect the sample to be tested. According to the physical and chemical properties of 34 foodborne stimulants, by investigating the extractant, extraction time, etc., a pretreatment method for 34 foodborne stimulants in foods with strong generality and good reproducibility is established; a liquid chromatography-triple quadrupole mass spectrometer with strong specificity is selected for determination.

[0008] CN202210880285.7, Invention Title: A Detection Method for Metabolic Modulators in Foods. A detection method for metabolic modulators in foods is disclosed. a. Preparation of the standard working solution; b. Preparation of the test sample solution; c. Respectively draw the standard working solution and the test sample solution and inject them into a liquid chromatography-triple quadrupole mass spectrometer. The detection method for metabolic modulators in foods of the present invention can simultaneously detect three metabolic modulators in foods, clomiphene, trimetazidine, and mepipramide, through specific sample treatment, chromatographic conditions, and mass spectrometry conditions.

[0009] By deeply analyzing the existing patented technologies (CN202311011333, CN202311018839, CN202210880285.7), it can be found that due to the significant differences in the physical and chemical properties of different categories of foodborne stimulants, the existing technologies have to adopt multiple detection methods for separate detections. This technical route has the following prominent problems: 1) The detection efficiency is low. Completing all detection items requires multiple injections and repeated operations on different instruments, taking as long as 24 - 48 hours; 2) The detection cost is high. It is necessary to configure multiple detection devices, significantly increasing the equipment investment and maintenance costs; 3) The operation process is complex. Within the limited detection time, each type of method requires multiple different professional and technical personnel to cooperate in the operation, resulting in high labor costs; 4) The environmental pollution is serious. Multiple detection methods lead to a substantial increase in the usage of organic solvents and chemical reagents.

[0010] CN202111178328.9, Invention Title: A Method for Simultaneously Determining the Contents of Multiple Foodborne Stimulants in Livestock and Poultry Meat. A method for simultaneously determining the contents of multiple foodborne stimulants in livestock and poultry meat is provided. The method includes: after pre - treating the sample based on the QuEChERS method, detecting it by liquid chromatography - tandem mass spectrometry; the pre - treatment includes the extraction and purification of the sample. Among them, the extraction solvent is acetonitrile containing 0.4 - 0.6% acetic acid, and the purification agent is PSA, C18, neutral alumina, and anhydrous magnesium sulfate with a mass ratio of (0.9 - 1.1):(0.9 - 1.1):(0.9 - 1.1):(5 - 6). This invention adopts the QuEChERS sample pre - treatment technology combined with the liquid chromatography - mass spectrometry combined detection method, with stable recovery rates and precision, having the advantages of high throughput and high sensitivity, being able to meet the detection requirements, and can quickly and accurately screen 47 kinds of foodborne stimulant residues in livestock and poultry meat simultaneously. This patent uses low - resolution mass spectrometry and has the following inherent defects: 1) Standard substances must be used for each detection, and the use validity periods of different standard substances need to be ensured, increasing the detection cost and operation complexity; 2) It must be paired with 11 expensive isotope internal standards, with a high single - detection cost; 3) The detection indicators are few and the coverage is insufficient, unable to meet the full - item requirements for foodborne stimulant detection in current sports events. Summary of the Invention

[0011] In response to the above - mentioned technical problems, the present invention provides an innovative solution.

[0012] The present invention provides a rapid screening method for foodborne stimulants in competition - supplied foods based on high - throughput technology. It uses gas chromatography - high - resolution mass spectrometry to detect 64 kinds of foodborne stimulants, and its chromatographic conditions are:

[0013] Chromatographic column: Waters XBridge C 18Chromatographic column (2.1×150 mm, 2.5 μm);

[0014] Column temperature: 40 °C;

[0015] Flow rate: 0.2 mL / min;

[0016] Sample injection volume: 10 μL;

[0017] Gradient elution was carried out separately in the same chromatographic column using acidic mobile phase and basic mobile phase.

[0018] Among them, the acidic mobile phase is: Mobile phase I (acidic): 1 mmol / L ammonium formate aqueous solution (containing 0.1% formic acid) - acetonitrile;

[0019] The basic mobile phase is: Mobile phase II (basic): 0.01% ammonia aqueous solution - acetonitrile;

[0020] The acidic and basic gradient elution programs are shown in Table 1 and Table 2.

[0021] Among them, the elution process is a tandem process, and the elution order of the two elution mobile phases can be interchanged.

[0022] Table 1 Mobile phase gradient elution program (acidic)

[0023]

[0024] Table 2 Mobile phase gradient elution program (basic)

[0025]

[0026] The mass spectrometry conditions are as follows:

[0027] Scan mode: Full Scan / dd-MS 2 ; Resolution: 120,000 for the first stage and 15,000 for the second stage.

[0028] Among them, the 64 foodborne stimulants are:

[0029] Clenbuterol, Salbutamol, Ractopamine, Terbutaline, Fenoterol, Tulobuterol, Penbutolol, cimaterol, Salmeterol, Epinephrine, Clorprenaline, Tretoquinol, Demethylcoclaurine, Propranolol, Atenolol, Metoprolol, carteolol, Methandienone, Stanozolol, Methyltestosterone, Testosterone Propionate, Nandrolone, Nandrolone Propionate, Nandrolone Phenylpropionate, Boldenone, Trenbolone, Testosterone, Metenolone, Dehydroepiandrosterone, Zilpaterol, Prednisone, Prednisolone, Dexamethasone, Betamethasone, Fludrocortisone, Methylprednisolone, Beclomethasone, Hydrocortisone, Cortisone, Acetazolamide, Canrenone, Chlortalidone, Furosemide, Spironolactone, Bendroflumethiazide, Chlorothiazide, Hydrochlorothiazide, Triamterene, 4-Amino-6-chloro-1,3-benzenedisulfonamide, Bumetanide, Torasemide, Trimetazidine, Clomiphene, Mecamylamine, Dihydrotestosterone, Oxymetholone, Clorprenaline, Methoxytyramine, Zeranol (Zeranol), β-Zeranol, α-Zearalenol, β-Zearalenol, Zearalanone, Zearalenone.

[0030] Among them, the mass spectrometry conditions are as follows:

[0031] Ion source: HESI; Spray voltage: 3.0 kV in positive ion mode, -2.5 kV in negative ion mode; Ion transfer tube temperature: 325 °C; Sheath gas, auxiliary gas and purge gas are all N2, with flow rates of 50, 10 and 0 arb respectively; Auxiliary gas temperature: 350 °C; Scanning mode: Positive and negative ion simultaneous scanning mode; Acquisition method: Full Scan / dd-MS2; Scanning range: 100–450 m / z; Resolution: 120,000 for the first stage, 15,000 for the second stage; RF lens voltage: 35%; Acquisition data type: Profile map; AGC target: Standard; MIT: Auto; Number of cycles: 3 times for the second stage; Number of MSX: 1; Isolation width: 2 m / z; Dynamic exclusion: 3.0 s; Vertex triggering: Not enabled; Turn on the EASY-IC mass axis real-time correction function.

[0032] Specifically, it includes the following steps:

[0033] a. Sample pretreatment:

[0034] Weigh the sample to be tested, add water and mix evenly, add 1% formic acid acetonitrile, vortex and oscillate, extract by ultrasonic, and centrifuge at high speed; Take the supernatant and pass it through a Waters PRiME HLB solid phase extraction column, collect the effluent, and concentrate it to dryness under a gentle nitrogen stream at 40 °C, then dissolve it in a 5% methanol aqueous solution containing 0.1% formic acid, and filter it through a 0.22 μm microporous filter membrane made of polytetrafluoroethylene to obtain the sample to be tested;

[0035] b. Detect according to the chromatographic conditions and mass spectrometry conditions described in claim 1.

[0036] 5. A high-resolution mass spectrometry screening database for foodborne stimulants in competition foods, characterized in that it contains high-resolution screening information of 64 target compounds obtained by the rapid screening method for foodborne stimulants in competition foods based on high-throughput technology described in any one of claims 1-4.

[0037] Among them, the high-resolution screening information of the 64 target compounds is shown in the following table:

[0038] Table 3 Chromatographic and mass spectrometric information table of 64 target compounds (primary level)

[0039]

[0040]

[0041]

[0042] Table 4 Chromatographic and mass spectrometric information table of 64 target compounds (secondary level)

[0043]

[0044]

[0045]

[0046]

[0047] * There are no fragments or the fragment responses are not representative. Qualification is carried out by combining the primary parent ion with retention time and isotope distribution.

[0048] The present invention provides a screening method for foodborne stimulants in competition foods. Based on the high-resolution mass spectrometry screening database for foodborne stimulants in competition foods, TraceFinder software is used for screening and confirmation. The key parameter settings are as follows:

[0049] Primary parent ion: The minimum detection intensity of the peak is not enabled; the signal-to-noise ratio threshold is 5; the error of the primary accurate mass number does not exceed ±5 ppm;

[0050] Retention time: The retention time error ≤ ±15 s;

[0051] Isotope distribution: The similarity score ≥ 80%; the error of the accurate mass number ≤ 10 ppm; the allowable intensity error ≤ 20%;

[0052] Secondary fragment ions: The number of matching fragments ≥ 2, except those not qualified by secondary level; the minimum detection intensity is 5000; the error of the secondary accurate mass number does not exceed ±10 ppm;

[0053] If all four conditions are met in the screening results, the presence can be confirmed; if none of them are met, it can be determined that it is not detected; if only two or three conditions are met, it is judged as suspected and further confirmation with reference standards is required. The present invention provides specific detection techniques and optimized detection processes, achieving the following breakthroughs:

[0054] 1) Significantly improve the detection efficiency, enabling the simultaneous detection of 64 food-derived doping drugs within 6 hours, which is 4 - 12 times faster than traditional methods;

[0055] 2) Greatly reduce the detection cost. Only one device and one detection method are required to complete all detection items, and accurate qualitative screening can be carried out without reference standards, completely eliminating the use of expensive isotope internal standards, simplifying the operation process, and significantly reducing equipment investment and operating costs;

[0056] 3) Reduce environmental pollution. By optimizing the reagent formula and detection process, the usage amount of chemical reagents is reduced;

[0057] 4) The detection sensitivity is equal to or even better than traditional national standard methods, and the lowest detection limits all meet the strict requirements of the World Anti-Doping Agency (WADA). The detection indicators comprehensively cover all food-derived doping items required in current sports events and fully meet the strict requirements for major domestic sports event guarantee. Brief Description of the Drawings

[0058] Figure 1 Workflow of high-throughput screening of food-derived doping drugs

[0059] Figure 2 Extracted ion chromatograms of cimaterol separated under two different chromatographic column conditions (a is Vanquish C 18 , b is XBridge C 18 )

[0060] Figure 3 Extracted ion chromatograms of bumetanide under 4 different acid-base and scanning mode conditions

[0061] Figure 4 Extracted ion chromatograms of tulobuterol under 4 different acid-base and scanning mode conditions

[0062] Figure 5 Extracted ion chromatograms of betamethasone and dexamethasone

[0063] Figure 6 Secondary fragment mass spectrum of salbutamol at the optimal collision energy (NCE = 30%)

[0064] Figure 7 Secondary fragment mass spectrum of triamterene at the optimal collision energy (NCE = 80%)

[0065] Figure 8 Secondary fragment mass spectrum of mepiquat chloride at the optimal collision energy (NCE = 60%)

[0066] Figure 9 Secondary fragment mass spectrum of boldenone at the optimal collision energy (NCE = 25%)

[0067] Figure 10 Secondary fragment mass spectrum of propranolol at the optimal collision energy (NCE = 50%)

[0068] Figure 11 Chromatograms of mixed standard solution and single standard solution of prednisolone and cortisone

[0069] Figure 12 Chromatograms of mixed standard solution and single standard solution of atenolol and trimetazidine

[0070] Figure 13 Optimization of extraction solvent

[0071] Figure 14 Optimization of SPE column Specific implementation manner

[0072] In accordance with the latest Document No. 584, the present invention has established a rapid detection method for 64 kinds of foodborne doping drugs in food based on high-resolution mass spectrometry high-throughput screening technology, which can achieve one-time high-throughput high-sensitivity rapid screening without reference standards and high-sensitivity rapid quantification with reference standards, providing strong support for the risk detection of foodborne doping drugs. The high-throughput screening workflow of foodborne doping drugs is as Figure 1 shown.

[0073] Example 1 Rapid screening method for foodborne doping drugs in competition food based on high-throughput technology of the present invention

[0074] 1. Experimental part

[0075] 1.1 Reagents and materials

[0076] Acetonitrile (mass spectrometry grade), methanol (mass spectrometry grade), formic acid (mass spectrometry grade) and ammonia water (chromatography grade) were provided by Thermo Fisher Scientific (USA); pure water (resistivity ≥ 18.2 MΩ·cm at 25°C) came from Milli-Q Integral water purification system (USA); mass spectrometry tuning calibration solution (Pierce Flex Mix Calibration, positive and negative ion mixed tuning solution) was purchased from Thermo Fisher Scientific (USA).

[0077] 64 foodborne doping drug reference materials were purchased from Alta Scientific (Tianjin, China), and the specifications were all single standard solutions.

[0078] 1.2 Analytical instruments and processing software

[0079] Ultra-high performance liquid chromatography-quadrupole / electrostatic field orbitrap mass spectrometry bench-top combined high-resolution mass spectrometry system (UHPLC-Q / Orbitrap HRMS, Thermo Fisher Scientific, USA), including: Vanquish Horizen ultra-high performance liquid chromatograph (binary high-pressure gradient, maximum pressure resistance 22000 psi) and Orbitrap Exploris 480 high-resolution mass spectrometer (equipped with HESI heated electrospray ionization source, maximum resolution 480,000); Avanti J-E floor-standing high-speed centrifuge (Beckman Coulter, USA); N1-28 automatic nitrogen concentrator (Yiyao, China); Milli-Q Direct ultrapure water system (resistivity 18.2 MΩ·cm, Merck Millipore, USA); ME204 electronic balance (accurate to 0.1 mg, one in ten thousand balance, Mettler Toledo, Switzerland); DTC-8 ultrasonic cleaner (Dingtai Hengsheng, China).

[0080] The instrument tuning software uses Orbitrap Exploris Tune 3.1, the data acquisition software uses Xcalibur 4.4, the qualitative software uses Freestyle 1.8, the screening confirmation and quantitative processing software uses TraceFinder 5.1 SP3, and the spectral library construction software uses mzVault 2.3 SP1. All the above software is provided by Thermo Fisher Scientific (USA).

[0081] 1.3 Sample collection

[0082] According to Document No. 584, literature, standards, market research and expert consultation, the main objects of foodborne doping drugs in food include meat products, eggs and egg products, and aquatic products. Therefore, 161 groups of representative samples (30 groups of meat products, 80 groups of eggs and egg products, 51 groups of aquatic products) were collected in this study. The specific sample types are shown in Tables 5 to 7.

[0083] Table 5 Sample information of 30 groups of meat products

[0084]

[0085] Table 6 Sample information of 80 groups of eggs and egg products

[0086]

[0087] Table 7 Sample information of 51 groups of aquatic products

[0088]

[0089] 1.4 Sample pretreatment

[0090] Accurately weigh 5 g of the sample (accurate to 0.01 g), add 5 mL of water and mix well to disperse the matrix. Then add 20 mL of 1% formic acid acetonitrile, vortex for 5 min, ultrasonically extract for 10 min, and centrifuge at 9500 r / min for 5 min. Take the supernatant and pass it through a 6 cc 200 mg Waters PRiME HLB solid phase extraction column or equivalent. Accurately collect 5 mL of the eluate in a 10 mL graduated tube. Concentrate it to near dryness under a gentle nitrogen stream at 40 °C. Dilute it to 1 mL with the initial mobile phase (5% methanol aqueous solution containing 0.1% formic acid), and pass it through a 0.22 μm microporous filter membrane made of polytetrafluoroethylene for liquid chromatography-high resolution mass spectrometry analysis.

[0091] 1.5 Chromatographic conditions

[0092] Gradient elution is carried out using acidic mobile phase and basic mobile phase respectively. Chromatographic column: Waters XBridge C 18 Chromatographic column (2.1×150 mm, 2.5 μm); Column temperature: 40 °C; Flow rate: 0.2 mL / min; Injection volume: 10 μL; Mobile phase I (acidic): 1 mmol / L ammonium formate aqueous solution (containing 0.1% formic acid)-acetonitrile, Mobile phase II (basic): 0.01% ammonia aqueous solution-acetonitrile; The acidic and basic gradient elution program information is shown in Table 8 and Table 9.

[0093] Table 8 Mobile phase gradient elution program (acidic)

[0094]

[0095] Table 9 Mobile phase gradient elution program (basic)

[0096]

[0097] 1.6 Mass spectrometry conditions

[0098] Ion source: HESI; Spray voltage: 3.0 kV in positive ion mode, -2.5 kV in negative ion mode; Ion transfer tube temperature: 325 °C; Sheath gas, auxiliary gas and purge gas are all N2, with flow rates of 50, 10 and 0 arb respectively; Auxiliary gas temperature: 350 °C; Scanning mode: Positive and negative ion simultaneous scanning mode; Acquisition method: Full Scan / dd-MS 2; Scan range: 100–450 m / z; Resolution: 120,000 for MS1, 15,000 for MS2; RF lens voltage: 35%; Data acquisition type: Profile; AGC target: Standard; MIT: Auto; Number of cycles: 3 times for MS2; Number of MSX: 1; Isolation width: 2 m / z; Dynamic exclusion: 3.0 s; Vertex triggering: Not enabled; Enable the EASY-IC real-time mass axis correction function.

[0099] 1.7 High-resolution mass spectrometry screening database

[0100] The present invention establishes a high-resolution mass spectrometry database and an actual spectral library (mzVault Library) for 64 food-derived doping drugs based on UHPLC-Q / Orbitrap HRMS. Analyze the standard solutions (about 100 ng / mL) of 64 food-derived doping drugs under the optimal chromatographic conditions and mass spectrometry conditions in the previous section, including Chinese and English names, CAS numbers, molecular formulas, adduct states, mobile phase selection (acidic or basic), retention times, theoretical accurate mass numbers of MS1 precursor ions, theoretical accurate mass numbers and molecular formulas of MS2, optimal collision energies, isotope distributions, actual standard spectra (mzVault spectra), and other data information. The high-resolution screening information tables for 64 target compounds are shown in Tables 10 and 11. The specific construction method is as follows.

[0101] 1.7.1 Basic information

[0102] Collect and organize the basic chemical information of 64 target compounds, such as Chinese and English names, molecular formulas, CAS numbers, etc., import the information into the Compound Database of TraceFinder, calculate the accurate mass numbers accurate to five decimal places, and automatically generate theoretical isotope distribution information.

[0103] 1.7.2 MS1 high-resolution mass spectrometry database

[0104] Prepare a mixed standard solution of 64 target compounds with a concentration of 100 ng / mL using 50% methanol aqueous solution. Based on the optimal chromatographic and mass spectrometry conditions, collect full-scan data to obtain the actual accurate mass numbers and retention times of MS1. Determine the optimal adduct state to ensure the optimal sensitivity of MS1 precursor ions. There are 4 selected adduct states, including [M+H] + , [M-H] - , [M-H2O+H] + , [M-CH3COS] - . Enter the above information into the Compound Database and the acquisition method of Xcalibur.

[0105] 1.7.3 MS2 high-resolution mass spectrometry database

[0106] In the acquisition method, set the NCE gradient value with a range of 5–100% and a step size of 5%. The prepared 100 ng / mL mixed standard solution was collected for Full Scan / dd-MS using the chromatographic conditions and mass spectrometry conditions optimized in the previous section. 2 Data, perform qualitative analysis using FreeStyle software. Based on the optimal adduct state selected in Table 3-6 as the parent ion, select the collision energy under the fragment ion conditions with high response, rich fragments, and relative mass deviation <10 ppm as the optimal collision energy. Finally, write the secondary fragment information formed under the optimal adduct state and optimal collision energy conditions into the Compound Database and the acquisition method.

[0107] 1.7.4 mzVault spectral library

[0108] Record the secondary fragment mass spectrometry spectra formed under the optimal adduct state and optimal collision energy conditions, write them into the mzVault software, and form a characteristic secondary spectral library (mzVault Library).

[0109] Table 10 Chromatographic and mass spectrometry information table of 64 target compounds (primary)

[0110]

[0111]

[0112]

[0113]

[0114] Table 11 Chromatographic and mass spectrometry information table of 64 target compounds (secondary)

[0115]

[0116]

[0117]

[0118]

[0119] *No fragments or fragment responses are not representative. Qualitative analysis is performed using the primary parent ion combined with retention time and isotope distribution.

[0120] 1.8 High-resolution mass spectrometry screening method

[0121] This qualitative screening method is based on a high-resolution mass spectrometry screening database of 64 foodborne doping drugs constructed, and the TraceFinder software is used for screening and confirmation. The key parameter settings are as follows:

[0122] For the primary parent ion: the minimum peak detection intensity is not enabled; the signal-to-noise ratio threshold is 5; the error of the primary accurate mass number does not exceed ±5 ppm.

[0123] Retention time: the retention time error ≤ ±15 s.

[0124] Isotope distribution: similarity score ≥ 80%; accurate mass number error ≤ 10 ppm; allowable intensity error ≤ 20%.

[0125] For the secondary fragment ions: the number of matching fragments ≥ 2, except those not using secondary qualitative analysis; the minimum detection intensity is 5000; the error of the secondary accurate mass number does not exceed ±10 ppm.

[0126] If all four conditions are met, the presence can be confirmed. If none of them are met, it can be determined that it is not detected. If some are met, manual comprehensive comparison is required to avoid the presence of impurity peaks in the isotope distribution of the primary or secondary ions, which may interfere with the result determination.

[0127] 2. Results and Discussion

[0128] 2.1 Optimization of Chromatographic Conditions

[0129] Most of the foodborne doping drugs in foods represented by meat products are veterinary drug residues, with low contents, and there are also isomers, which have high requirements for both sensitivity and separation. Therefore, the correct selection of chromatographic conditions is of great significance for the simultaneous analysis of foodborne doping drugs with different properties. The composition and proportion of the mobile phase will affect the retention time of the analyte, the separation of peaks, and the formation of the mass spectrometry parent ion adduct. The present invention optimizes key chromatographic parameters, such as the selection of the chromatographic column and the composition of the mobile phase, to ensure the best separation and sensitivity of the target analytes, and to maximize the savings in analysis time and reduce the use of the mobile phase.

[0130] 2.1.1 Selection of Chromatographic Column

[0131] Two ultra-high performance liquid chromatography columns, Thermo Accucore Vanquish C 18 (2.1×150 mm, 1.5 μm) and Waters XBridge C 18 (2.1×150 mm, 2.5 μm), were compared. It was found that the peak shape of some analytes tailed severely on the Vanquish C 18 chromatographic column, while the peak shape of XBridge C 18 was better. For example, cimaterol, see Figure 2, a is Vanquish C 18 , b is XBridge C 18 . Therefore, Waters XBridge C 18 (2.1×150 mm, 2.5 μm) chromatographic column was selected as the analytical column. In addition, considering the continuous expansion of foodborne stimulants, in order to reduce the optimization of chromatographic conditions after adding new foodborne stimulants in the later stage, a longer 150 mm chromatographic column was selected for separation in this study.

[0132] 2.2.2 Selection of mobile phase

[0133] To ensure that the primary parent ions of the target compounds reach the highest possible sensitivity, two mobile phases, acidic and basic, were used respectively in the selection of the mobile phase, and the strategy of simultaneous positive and negative scanning was combined to detect 64 target compounds with high sensitivity and high resolution. For example Figure 3 and Figure 4 , a is acidic (positive ion mode), b is acidic (negative ion mode), c is basic (positive ion mode), d is basic (negative ion mode). For bumetanide, acidic (negative ion mode) was selected to obtain the highest sensitivity of the primary adduct state. Toltrazuril can only peak under positive ion conditions and does not peak in the negative ion mode. Therefore, acidic (positive ion mode) was selected to achieve the optimal sensitivity.

[0134] The effects of methanol and acetonitrile as organic phases on the chromatographic separation of 64 target compounds were investigated. The 64 target compounds have different chemical properties, and many have isomers, such as dexamethasone / betamethasone, zearalenone / α-zearalenol / β-zearalenol. When methanol was used as the organic phase, the typical isomers (optical isomers) betamethasone and dexamethasone were difficult to achieve baseline separation. Therefore, acetonitrile was selected as the organic phase. The optimized chromatograms of betamethasone and dexamethasone are shown in Figure 5 , a is a mixed standard of betamethasone and dexamethasone, b is a single standard of dexamethasone, c is a single standard of betamethasone.

[0135] The selection of acidic aqueous mobile phase was investigated. The addition of an appropriate amount of formic acid not only helps the ionization of the target substances, improves the positive ion sensitivity, but also can improve the peak shape and stability. When 0.1% formic acid water + acetonitrile was used as the mobile phase, due to the large number of compound types, some compounds showed co-elution phenomena and did not peak. When an appropriate amount of ammonium formate was added to the aqueous solution, the co-elution phenomena could be improved.

[0136] Further investigate the content of ammonium formate in the aqueous solution. Select the mobile phases of 5 mmol / L ammonium formate (containing 0.1% formic acid) solution + acetonitrile, 2 mmol / L ammonium formate (containing 0.1% formic acid) solution + acetonitrile, and 1 mmol / L ammonium formate (containing 0.1% formic acid) solution + acetonitrile. The experiment found that the content of ammonium formate in the aqueous solution has an impact on the baseline and response of some compounds. The greater the content of ammonium formate, the higher the noise and the lower the signal-to-noise ratio.

[0137] Considering the elution conditions of 64 compounds comprehensively, select 1 mmol / L ammonium formate (containing 0.1% formic acid) solution + acetonitrile as the acidic mobile phase (Mobile Phase I), and 0.01% ammonia aqueous solution + acetonitrile as the basic mobile phase (Mobile Phase II).

[0138] 2.2 Optimization of Mass Spectrometry Conditions

[0139] 2.2.1 Selection of Scanning Acquisition Mode

[0140] In this experiment, three scanning modes were compared: full scan (Full Scan), full scan / data-dependent MS / MS (Full Scan / dd-MS 2 ), and selected ion monitoring / data-dependent MS / MS (Target-SIM / dd-MS 2 ). Full Scan only identifies the parent ion by its exact mass number and retention time, that is, it only has a screening function; Target-SIM / dd-MS 2 can obtain the daughter ion fragment information, but it is non-targeted information and cannot be retrospectively analyzed later. The Full Scan / dd-MS 2 scanning mode can simultaneously screen and confirm the target substances and accurately quantify them. When new analysis objects appear later, they can also be traced in the original data file. Therefore, it was finally selected as the scanning acquisition mode for this study.

[0141] 2.2.2 Selection of Resolution

[0142] As a core parameter of high-resolution mass spectrometry technology, the resolution directly affects the mass accuracy of target compounds, which in turn affects the accurate inference of molecular formulas to avoid false positive results. The higher the resolution, the lower the scanning speed, which ultimately leads to too few chromatographic peak points and inaccurate quantification. Therefore, when determining the scanning resolution, sensitivity and repeatability should be considered. The study found that when the resolution is adjusted to the highest (R = 480,000), the scanning speed will be significantly reduced, and the number of points collected will also be small, resulting in too few points to form a peak shape and difficult to quantify, and some compounds will also be missed. When repeating the injection, the results are inconsistent and key information in the mass spectrum is lost, resulting in a significant reduction in repeatability and increasing the uncertainty of the detection results. When the resolution of the first-stage full scan is R = 120,000, all analytes can be baseline separated from the interfering substances in the matrix, and the response value is also significantly improved. The number of collected points for each chromatographic peak is greater than 12. Therefore, considering the accuracy of both qualitative and quantitative analysis, the resolution of the first-stage mass spectrometry parent ion full scan is selected as 120,000 (mass error ppm ≤ 5), and the resolution of the second-stage mass spectrometry fragment ion scan is selected as 15,000 (mass error ppm ≤ 10).

[0143] 2.2.3 Selection of the range of accurate mass error

[0144] Among the 64 foodborne doping drugs, 48 compounds have higher responses in the positive ion mode, and the parent ions of 3 target compounds are [M - H2O + H] - , and the parent ions of 45 compounds are [M + H] + ; the remaining 16 compounds all have responses in the negative ion mode. The parent ion of 1 compound (spironolactone) is [M - CH3COS] - , and the parent ions of 15 compounds are [M - H] - . Tables 10 and 11 list the theoretical accurate masses of the first and second stages of the 64 target compounds. Due to the use of the Easy IC real-time internal standard to correct the mass axis function during the mass spectrometry acquisition process, the first-stage mass deviation of the 64 target compounds is less than 0.5 ppm, and the second-stage relative mass deviation is less than 5 ppm, far better than the requirements for mass deviation of ordinary high-resolution mass spectrometry (first stage ≤ 5 ppm, second stage ≤ 10 ppm), proving that the mass accuracy of the target compounds is very good and can meet the needs of screening, confirmation and accurate quantification.

[0145] 2.2.4 Second-stage mass spectrometry conditions

[0146] The accurate mass of the target compound parent ion is obtained through full scan. According to the obtained accurate mass, a target list is set. Only the parent ions in the list are found during the first-stage mass spectrometry scan, and when their intensity reaches the set threshold (1×10 5When (condition not specified in the original) occurs, it can automatically collect the second-level mass spectrometry scan. Through the optimization of parameters such as TopN, apex triggering, and dynamic exclusion, high-quality second-level mass spectrometry spectra can be obtained. First, by adjusting and optimizing the dynamic exclusion, it is found that the optimal dynamic exclusion time is the full width at half maximum. However, due to the different separation degrees of 64 target compounds, the dynamic exclusion time is finally determined to be 3 s according to most peaks. Secondly, there are many compounds in this study, and the peak widths are not completely consistent. Therefore, setting a certain period of time cannot guarantee that all compounds are excited to the second level at the peak apex, so the apex triggering function is not enabled. Thus, the analysis results contain both the first-level accurate mass number and retention time, as well as high-quality second-level mass spectrometry information, which can effectively improve the accuracy and reliability of target compound judgment.

[0147] 2.2.5 Target Ionization and Ion Adduct Modes

[0148] The correct selection of the parent ion adduct state is a prerequisite for ensuring the first-level quantitative sensitivity and high-quality second-level mass spectrometry spectra. In this study, analysis was carried out by simultaneously collecting positive and negative ions in acidic and basic mobile phases respectively to find the optimal parent ions corresponding to foodborne doping drugs to ensure the best sensitivity. Four adduct states were selected for comparison, including [M+H] + , [M-H] - , [M-H2O+H] + , [M-CH3COS] - . The optimal selection results are shown in Table 10.

[0149] 2.2.6 Optimization of Collision Energy

[0150] The exact mass number of the secondary fragment ions is the key information required for confirmation. Different from the one-to-one relationship between the parent ion and the daughter ion in the multiple reaction monitoring (MRM) mode of traditional triple quadrupole mass spectrometry, the secondary mass spectrometry map of high-resolution mass spectrometry shows a relationship where one parent ion corresponds to multiple daughter ions. A high-quality secondary mass spectrometry map requires abundant, characteristic, and highly sensitive fragments, so the selection of collision energy is extremely important. In this study, the smallest molecular mass is less than 150 Da, while the highest molecular mass is about 450 Da. However, with the increase in food-derived doping drugs, new compounds will appear in the later stage. Using the same collision energy cannot make all compounds exhibit the best fragmentation state. The multi-gradient normalized collision energy combination is a simple and effective method widely used in most high-resolution mass spectrometry analyses at present. It allows the input of multiple different energy values and finally obtains an adduct map of fragment ions with multiple energies. In theory, this method can ensure the uniform distribution of the mass spectrometry map at the low mass number end and the high mass number end. However, through experiments, it is found that this method cannot dissociate all parent ions of food-derived doping drugs with the optimal collision efficiency. For some compounds, the optimal collision energy is much greater than 50%, such as triamterene (80%); for some compounds, fragmentation will occur at a lower collision energy, such as dexamethasone (15%). Therefore, in this study, different NCE energy gradients such as 0, 5%, 10%... 95% and 100% were used, and a 100 μg / L mixed standard solution of 64 food-derived doping drugs was injected into the UHPLC-Q / OrbitrapMS under acidic and alkaline conditions. The target compounds were fragmented at different NCE collision voltages, and mass spectrometry information was collected in real time. In this way, it can be ensured that each compound in different adduct states has a spectrum with different fragmentation energies. Later, the spectra with different fragmentation energies were analyzed by Free Style qualitative software, and the NCE collision voltage was optimized one by one. The NCE collision voltage with better fragmentation and rich characteristic fragments was selected as the best collision energy for the compound. Then, the best collision energy was input into the inclusion list information of the instrument acquisition method to obtain the best-quality secondary mass spectrometry information. For a high-quality secondary mass spectrometry map, usually, the intensity of the best parent ion should account for about one-third of the fragment ions with the highest response. If the parent ion is completely fragmented, the situation of multiple fragmentations of fragment ions caused by excessive energy cannot be excluded. This situation may cause the characteristic daughter ions to tend to the low mass number end and lack specificity. The collision energies set for 64 target compounds are shown in Table 11. The spectra of typical compounds at the optimal collision energy are shown in Figures 6 to 10 . In addition, based on the optimal secondary fragments, the mass error of the fragments was calculated. The mass errors are all less than 5 ppm, and most of the fragments are less than 1 ppm, far lower than the requirement of ≤10 ppm for the secondary error of conventional high-resolution mass spectrometry, indicating that the qualitative results are reliable.

[0151] 2.2.7 Identification of Isomers

[0152] To distinguish the isomers among 64 target compounds, in this study, a method of separately injecting the mixed standard and single standard of isomers was adopted, and they were identified by the different retention times in the chromatogram. There are multiple groups of isomeric substances in this study, as shown in Table 12 for details, and typical chromatograms are shown in Figure 5 , Figure 11 and Figure 12 . Figure 3-11 Among them, the RT of prednisolone is 16.29 min, and the RT of cortisone is 16.77 min. Figure 12 Among them, the RT of atenolol is 6.15 min, and the RT of trimetazidine is 6.52 min. Among them, atenolol and trimetazidine, testosterone and dehydroepiandrosterone, prednisolone and cortisone can be further identified by selecting different primary adduct states or the same primary adduct state but different secondary characteristic fragments, so as to ensure the reliability of screening confirmation and quantification.

[0153] Table 12 Foodborne doping agents with isomers

[0154]

[0155]

[0156] 2.2.8 Discrimination of Isotope Distribution Characteristics

[0157] The isotope distribution characteristics of high-resolution mass spectrometry can improve the reliability of the screening method. In the TraceFinder software of the present invention, the abundance ratio can be directly calculated by the software, and the measured isotope distribution of the target compound is compared with the theoretical value. If the comprehensive score of the isotope distribution is consistent with the pre-calculated data, it can be judged that the compound may be contained. However, if only part of the isotope distribution of the analyte is consistent, more information, such as fragment ion data, etc., is needed for further verification and confirmation. In addition, the isotope distribution belongs to the primary analysis and is easily interfered by other miscellaneous peaks. If the isotope discrimination fails, it is necessary to further confirm whether it is detected from the primary mass spectrum and secondary fragment identification and other methods.

[0158] 2.2.9 Optimization of Screening Method

[0159] In this study, the blank matrix standard solution and the solvent standard solution were first used for pre-screening, and the screening parameters were continuously adjusted until all the compounds in the mixed standard solution were successfully screened out. The optimization of the screening method is based on the requirements of the European Union (SANCO / 12495 / 2011) for the confirmation of the analysis results of drug residues such as foodborne doping agents, with at least 2 confirmation ions and a mass error of ≤5 ppm.

[0160] Ion peak intensity threshold: not set for the first stage, set to 5000 for the second stage. When the content of foodborne doping drugs is low or the matrix effect is strong, their response may be less than 5000. To avoid missed detections, this threshold is not set for the first stage. The second-stage fragment is set to 5000 to exclude impurity interference.

[0161] The retention time window is set to 30 s, that is, ±15 s.

[0162] Accurate mass error: ≤5 ppm for the first stage, ≤10 ppm for the second stage. The electrostatic field orbitrap high-resolution mass spectrometer used in this study adopts the RunStart EASY-IC mass axis internal standard calibration method (fluoranthene, C 16 H 10 ). The experimental results show that the difference between the theoretical accurate mass and the measured value does not exceed 2 ppm for the first stage and 5 ppm for the second stage. However, to avoid the deviation of the mass axis caused by environmental and matrix factors, it is set to ≤5 ppm for the first stage and ≤10 ppm for the second stage, which can ensure the accurate qualitative determination of target compounds.

[0163] 2.3 Pretreatment optimization

[0164] There are relatively high requirements for the timeliness of the detection of foodborne doping drugs. To improve the detection efficiency, this study intends to develop a simple, rapid, efficient, and unified pretreatment method to ensure the rapid and accurate detection of 64 foodborne doping drugs in 3 types of typical foods.

[0165] 2.3.1 Optimization of extraction solvents

[0166] Select 0.2% formic acid acetonitrile, 0.5% formic acid acetonitrile, 1% formic acid acetonitrile, 2% formic acid acetonitrile, and 5% formic acid acetonitrile as extraction solvents. Under the condition of ensuring that other conditions such as the purification method remain unchanged, the recoveries of 64 foodborne doping drugs in 3 major types of samples are investigated. The results are shown in Figure 13 . As can be seen from the figure, when the compound recovery rate is above 50%, the proportion of 1% formic acid acetonitrile extraction solvent is generally greater than that of other extraction solvents. Therefore, 1% formic acid acetonitrile is selected as the extraction solvent for the pretreatment method.

[0167] 2.3.2 Optimization of purification cartridges

[0168] In this invention, SPE cartridges suitable for high-throughput analysis are selected, including Biotage EXPRESSABN (150 mg 6 mL), Comma Biotech HLB SPE Cartridge (200 mg 6 mL), Phenomenex Strata-X PRO (200 mg 6 mL), Agilent QuEChERS dSPE EMR-Lipid, and Waters PRiME HLB (6 cc 200 mg), and the recoveries of 64 target compounds in 3 types of samples are investigated. The results are shown in Figure 14 , as can be seen from the figure, among the ideal recovery range of 80–120%, Waters PRiME HLB has the largest proportion. Considering comprehensively, Waters PRiME HLB is selected as the purification cartridge for sample pretreatment.

[0169] 2.4 Methodology validation

[0170] Although high-resolution mass spectrometry technology has excellent qualitative and quantitative capabilities, at the ESI ion source, there are still competitive matrix effects, which affect the sensitivity and accuracy of the method. Therefore, in this study, a standard curve is established using a blank matrix-matched standard solution to avoid matrix effects. The standard working solution of 64 target compounds is prepared with the blank pork matrix extract and determined under the optimized analysis conditions. The standard curve is plotted with the target chromatographic peak area (y) and its corresponding component concentration (x), and the linear regression equation and its correlation coefficient (r) of the analyte are calculated. Since most of the EIC spectra of high-resolution mass spectrometry have no noise and the detection limit cannot be calculated by S / N, the concentration with a signal of 10,000 is used as the instrument detection limit, and the method detection limit and method quantification limit are calculated. The results are shown in Table 13.

[0171] As can be seen from the table, within the range of the response mass concentration, the matrix-matched standard curves of 64 foodborne stimulant drugs can show good linear relationships, and the corresponding correlation coefficients r are all greater than 0.9950. The detection limits and quantification limits of the method all meet the experimental requirements. Among them, the performance of 36 target compounds is better than that of traditional national standards and other methods. These include GB 31658.22-2022 Determination of β-agonist residues in animal-derived foods - Liquid chromatography-tandem mass spectrometry method, SN / T 1924-2011 Determination of clenbuterol, ractopamine, salbutamol and terbutaline residues in imported and exported animal-derived foods - Liquid chromatography-mass spectrometry / mass spectrometry method, GB / T 23218-2008 Determination of zeranol residues in foods - Liquid chromatography-tandem mass spectrometry method, GB / T 21982-2008 Determination of zeranol, β-zeranol, α-zearalenol, β-zearalenol, zearalanone and zearalenone residues in animal-derived foods - Liquid chromatography-mass spectrometry / mass spectrometry method, SN / T 5171-2019 Determination of demethylcoclaurine in exported plant-derived foods - Liquid chromatography-mass spectrometry / mass spectrometry method, Announcement No. 1031-2-2008 of the Ministry of Agriculture and Rural Affairs Determination of multi-residues of glucocorticoid drugs in foods - Liquid chromatography-tandem mass spectrometry method, GB / T 21981-2008 Determination of multi-residues of hormones in animal-derived foods - Liquid chromatography-mass spectrometry / mass spectrometry method, etc. The mixed standard solution of 64 foodborne stimulant drugs was added to the blank matrices of pork, eggs and fish respectively for the spiked recovery experiment. A total of 3 concentration levels were set, and each concentration level was tested 6 times. The results showed that the average recoveries of pork, eggs and fish were 72.5–103.1%, 73.3–107.3% and 75.8–105.8% respectively, and the relative standard deviations were 0.8–7.9%, 0.5–7.6% and 1.1–8.3% respectively.

[0172] Table 13 Method detection limit and method quantification limit

[0173]

[0174]

[0175]

[0176] 2.5 Analysis of actual samples

[0177] Based on the method established in this study, 161 groups of typical foods (30 groups of meat products, 80 groups of eggs and egg products, 51 groups of aquatic products) were pretreated and measured on the instrument. TraceFinder was used to screen, confirm and accurately quantify the data. The screening results showed that endogenous component boldenone (0.6-5 μg / kg) was detected in 11 groups of eggs and egg products, and no other substances were detected.

[0178] 3. Summary

[0179] The present invention has established a rapid screening method for 64 kinds of foodborne stimulant drugs in 6 categories, such as β2-agonists, β-blockers, anabolic agents, glucocorticoids, diuretics and metabolic regulators in foods, by ultra-high performance liquid chromatography-quadrupole / electrostatic field orbitrap high-resolution mass spectrometry. The pretreatment of typical samples such as meat products, eggs and egg products, and aquatic products is rapidly processed by using a through-type solid-phase extraction column. The target compounds are separated by gradient elution with acidic and basic mobile phases respectively. The mass spectrometry collects data in the Full Scan / dd-MS 2 mode, establishes a high-resolution mass spectrometry screening database and a spectral library for the target compounds, and finally uses the TraceFinder software combined with the self-built spectral library to achieve rapid qualitative screening and high-sensitivity quantification of samples. The results show that the results of methodological investigations such as the linear relationship, method detection limit, method quantification limit, and accuracy of the 64 kinds of foodborne stimulant drugs are ideal. Among them, the comprehensive performance of 36 target compounds is better than that of the traditional national standard method. This method has strong specificity, high sensitivity, fast and efficient, can significantly shorten the detection time (completing the detection of 64 kinds of foodborne stimulant drugs within 6 hours), reduce the detection cost (1 device, 1 method), and reduce environmental pollution (less use of chemical reagents), and is applicable to the rapid screening and confirmation of multiple types of foodborne stimulants in foods, and is of great significance for maintaining the fairness of sports events and ensuring the successful holding of events.

Claims

1. A rapid screening method for foodborne stimulants in competition food based on high-throughput technology, characterized in that: It uses phase chromatography-high resolution mass spectrometry to detect 64 food-borne stimulants, and its chromatographic conditions are: Column: Waters XBridge C 18 Chromatographic column (2.1×150 mm, 2.5 μm); Column temperature: 40°C; Flow rate: 0.2 mL / min; Injection volume: 10 μL; Acidic mobile phase and alkaline mobile phase were used in the same chromatographic column for gradient elution. Wherein, the acid mobile phase is: mobile phase I (acidic): 1mmol / L ammonium formate aqueous solution (containing 0.1% formic acid)-acetonitrile; The basic mobile phases were: Mobile phase II (basic): 0.01% aqueous ammonia solution-acetonitrile; The acidic and alkaline gradient elution programs are shown in Tables 1 and 2 ; Table 1 Mobile phase gradient elution program (acidic) Table 2 Mobile phase gradient elution program (alkaline) The mass spectrometry conditions were: Scan mode: Full Scan / dd-MS 2 ; Resolution: 120000 for level 1, 15000 for level 2.

2. The method for rapid screening of foodborne stimulants in competition food based on high-throughput technology according to claim 1, characterized in that: The 64 food-borne stimulants are: Clenbuterol, salbutamol, ractopamine, terbutaline, fenoterol, tolbuterol, penbuterol, cimaterol, salmeterol, epinephrine, clenproperol, trotoquinol, higenamine, propranolol, atenolol, metoprolol, carteolol, methandrostenolone, stanozolol, methyltestosterone, testosterone propionate, nandrolone, nandrolone propionate, nandrolone phenylpropionate, boldenone, trenbolone, testosterone, methenolone, dehydroepiandrosterone, zilpaterol, prednisone, prednisolone, dexamethasone, betamethasone, fludrocortisone, methyl Prednisolone, beclomethasone, hydrocortisone, cortisone, acetazolamide, canrenone, chlorthalidone, furosemide, spironolactone, bendroflumethiazide, chlorothiazide, hydrochlorothiazide, triamterene, 4-amino-6-chloro-1,3-benzenedisulfonamide, bumetanide, torasemide, trimetazidine, clomiphene, medochloramine, dihydrotestosterone, oxymetholone, chlorprenaline, methoxytyramine, zearalanol (zearalanol), β-zearalanol, α-zearalanenol, β-zearalanenol, zearalanone, zearalanenone.

3. The method for rapid screening of foodborne stimulants in competition food based on high-throughput technology according to claim 1, characterized in that: The mass spectrometry conditions are: Ion source: HESI; spray voltage: 3.0 kV in positive ion mode, -2.5 kV in negative ion mode; ion transfer tube temperature: 325 °C; sheath gas, auxiliary gas and sweep gas are all N2, with flow rates of 50, 10 and 0 arb respectively; auxiliary gas temperature: 350 °C; scan mode: positive and negative ion simultaneous scan mode; acquisition method: Full Scan / dd-MS2; scan range: 100–450 m / z; Resolution: 120000 for primary, 15000 for secondary; RF lens voltage: 35%; Acquisition data type: Contour; AGC target: Standard; MIT: Auto; Number of cycles: 3 times for secondary; Number of MSX: 1; Isolation width: 2m / z; Dynamic exclusion: 3.0s; Vertex trigger: Not enabled; Enable the EASY-IC mass axis real-time correction function.

4. The method for rapid screening of foodborne stimulants in competition food based on high-throughput technology according to any one of claims 1 to 3, characterized in that: It includes the following steps: a. Sample pretreatment: Weigh the sample to be tested, add water to mix, add 1% formic acid acetonitrile, vortex oscillation, ultrasonic extraction, and high-speed centrifugation; take the supernatant and pass it through the Waters PRiME HLB solid phase extraction column, collect the effluent, concentrate to dryness under a gentle nitrogen flow at 40°C, and then make up to volume with 5% methanol aqueous solution containing 0.1% formic acid, and pass it through a 0.22μm polytetrafluoroethylene microporous filter membrane to obtain the sample to be tested; b. Detection according to the chromatographic conditions and mass spectrometry conditions described in claim 1.

5. A high-resolution mass spectrometry screening database for food-borne stimulants in competition foods, characterized in that it contains high-resolution screening information of 64 target compounds obtained by the rapid screening method for food-borne stimulants in competition foods based on high-throughput technology as described in any one of claims 1-4.

6. The high-resolution mass spectrometry screening database for food-borne stimulants in competition foods according to claim 5, characterized in that: the high-resolution screening information of the 64 target compounds is shown in the following table: Table 64 chromatographic and mass spectrometry information table of target compounds (primary) Table 6 Chromatographic and mass spectrometric information of 4 target compounds (secondary) *If there is no fragment or the fragment response is not representative, the primary precursor ion combined with retention time and isotope distribution is used for qualitative analysis.

7. A method for screening food-borne stimulants in competition food, characterized in that: It is based on the high-resolution mass spectrometry screening database of food-borne stimulants in competition foods as described in claim 6, and uses TraceFinder software for screening and confirmation, and the key parameters are set as follows: Primary parent ion: minimum peak detection intensity is not enabled; signal-to-noise ratio threshold is 5; primary accurate mass error does not exceed ±5ppm; Retention time: Retention time error ≤ ±15s; Isotope distribution: similarity score ≥ 80%; accurate mass error ≤ 10ppm; allowable intensity error ≤ 20%; Secondary fragment ions: matching fragments ≥ 2, except for those without secondary qualitative analysis; minimum detection intensity is 5000; secondary accurate mass error does not exceed ±10ppm; If the screening results meet all four conditions, it can be confirmed that the disease exists; if none of them are met, it can be determined that the disease has not been detected; If only two or three items are met, it is considered suspected.

Images