Screening method for various environmental pollutants in raw milk

Abstract

The invention discloses a method for screening various environmental pollutants in raw milk. The method comprises the steps of pollutant database construction, sample pretreatment, data acquisition, screening confirmation and the like. The method disclosed by the invention is simple to operate and good in accuracy and reproducibility, is already used for large-batch actual sample detection, and provides accurate and reliable technical support for quality safety of milk and dairy products.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing technology, specifically, it relates to a method for screening multiple environmental pollutants in raw milk. Background Technology

[0002] Food safety is crucial to the health and safety of the people and the future of the Chinese nation. Milk and dairy products are a vital industry for people's livelihood in my country. As consumers increasingly focus on the quality and nutritional value of milk and dairy products, they crave authentic and nutritious dairy products, thus preferring products made directly from fresh milk. High-quality raw milk helps improve the taste, nutritional value, and safety of these products. Furthermore, the quality of raw milk directly impacts the quality of dairy products such as milk, milk powder, cheese, and yogurt. High-quality raw milk contributes to improved taste, nutritional value, and safety, placing higher demands on the quality of raw milk.

[0003] Currently, the main direct threats to the safety of raw milk include microbial hazards, chemically toxic and harmful substances, and risks arising from new technologies. Among these, chemically toxic and harmful substances (such as veterinary drugs, pesticides, biotoxins, environmental pollutants, food additives, and industrial chemicals) have become the primary cause of raw milk contamination. There are currently over 270 million registered chemicals globally. my country is the world's largest producer and consumer of chemical products. According to the "Global Chemicals Outlook II," my country's chemical sales account for approximately 40% of the global total, and this figure is projected to reach 50% by 2030. Some of these high-volume toxic and harmful chemicals may be released into the environment during production, processing, use, consumption, and disposal, posing a significant threat to the health, survival, and reproduction of organisms, and harming human health through bioaccumulation in plants and animals and dietary intake. Typically, these chemicals exist at trace levels in the environment and within organisms. The complexity of food matrices and the diversity of environments further increase the difficulty of responding to food safety emergencies, controlling food risk factors, regulating food production, and effectively ensuring food safety. Therefore, high-throughput, multi-residue detection of environmental pollutants in raw milk is of great significance for ensuring the quality and safety of milk and dairy products. However, due to the wide variety of environmental pollutants, and the fact that these pollutants usually exist in trace amounts in raw milk, current methods for detecting trace pollutants and multi-residues typically employ gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). However, traditional detection methods have significant limitations in detection coverage and screening throughput, making it difficult to meet the needs of comprehensive monitoring of multiple organic pollutants. Furthermore, they often suffer from slow analysis speed and low screening throughput. Therefore, high-throughput rapid screening technology is the main technical means for pollutant monitoring in the future, and how to optimize detection technology is an urgent problem to be solved. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a high-throughput screening method for typical environmental pollutants in raw milk based on gas chromatography-high-resolution mass spectrometry (GC-MS). Furthermore, this invention provides a chromatographic-mass spectrometry sample pretreatment technique more suitable for raw milk, thereby simplifying the steps of extracting and purifying pollutants from complex matrices and laying the foundation for broad-spectrum screening of environmental pollutants in raw milk.

[0005] In one aspect, the present invention provides a method for screening multiple environmental pollutants in raw milk, the method comprising the following steps:

[0006] S-0: Constructing a pollutant database: Measured using gas chromatography and high-resolution mass spectrometry, and collected total ion chromatograms of typical environmental pollutant standard solutions to obtain relevant data;

[0007] S-1: Sample pretreatment: Mix the raw milk sample with the extraction reagent, and perform extraction, purification, centrifugation, nitrogen blowing dry the supernatant, resolution and membrane filtration in sequence to obtain the sample to be tested;

[0008] S-2: Data acquisition: The sample to be tested is introduced into a gas chromatograph-high resolution mass spectrometer to obtain a total ion chromatogram based on high resolution mass spectrometry.

[0009] S-3: Screening and Confirmation: Screening parameters are set for the test results to identify peaks, extract them, and compare information based on a self-built database to screen for contaminants present in the samples.

[0010] In another aspect, the present invention provides a pretreatment method for raw milk, the method comprising: mixing a raw milk sample with an extraction reagent, and sequentially performing extraction, purification, centrifugation, drying the supernatant with nitrogen, reconstitution, and membrane filtration to obtain a sample to be tested.

[0011] In one or more embodiments, in step S-0, the relevant data includes the target peak (expressed as molecular weight), fragment ion peak (expressed as molecular weight), retention time, screening limit, relative standard deviation (RSD), and spiked recovery.

[0012] In one or more embodiments, in step S-0, the environmental pollutant includes one or more combinations of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), bromodiphenyl ethers (PBDEs), polychlorinated dibenzofurans (PCDFs), polychlorinated dibenzo-p-dioxins (PCDDs), organochlorine pesticides (OCPs), phthalates (PAEs), or chlorobenzenes (CBs).

[0013] Preferably, the environmental pollutant is selected from one or more combinations of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), bromodiphenyl ethers (PBDEs), polychlorinated dibenzofurans (PCDFs), polychlorinated dibenzo-p-dioxins (PCDDs), organochlorine pesticides (OCPs), phthalates (PAEs), or chlorobenzenes (CBs).

[0014] More preferably, the environmental pollutant is selected from Benz(a)anthracene, Benzo(a)pyrene, Benzo(b)fluoranthene, Benzo(k)fluoranthene, Benzo[k]fluoranthene, and Chrysene. Dibenz[a,h]anthracene, Indeno[1,2,3-cd]pyrene, Benzo[c]fluorene, Cyclopenta[c,d]pyrene, 5-Methylchrysene ), Benzo[j]fluoranthene, Dibenzo[a,l]pyrene, Dibenzo[a,e]pyrene, Dibenzo[a,i]pyrene, Dibenzo[a,h]pyrene, PCB 18, PCB 28, PCB 33, PCB 44, PCB 52, PCB 70, PCB 101, PCB 105, PCB118, PCB 128, PCB 138, PCB 153, PCB 170, PCB 180, PCB 187, PCB 194, PCB 195, PCB 199, PCB 206, PCB 209, PBDE 28, PBDE 47, PBDE 99, PBDE 100, PBDE 153, PBDE 154, PBDE 183, 1,2,3,4,6,7,8-HpCDF (1,2,3,4,6,7,8-heptachlorodibenzofuran), 1,2,3,4,7,8-HxCDF (1,2,3,4,7,8-hexachlorodibenzofuran), 1,2,3,7,8-PeCDF (1,2,3,7,8-pentachlorodibenzofuran), 2,3,7,8-TCDF (2,3,7, 8-Tetrachlorodibenzofuran), OCDF (octachlorodibenzofuran), 2,3,4,7,8-PeCDF (2,3,4,7,8-pentachlorodibenzofuran), 1,2,3,6,7,8-HxCDF (1,2,3,6,7,8-hexachlorodibenzofuran), 1,2,3,7,8,9-HxCDF (1,2,3,7,8,9-hexachlorodibenzofuran), 2,3 4,6,7,8-HxCDF(2,3,4,6,7,8-hexachlorodibenzofuran), 1,2,3,4,7,8,9-HpCDF(1,2,3,4,7,8,9-heptachlorodibenzofuran), 1,2,3,4,6,7,8-HpCDD(1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin), 1,2,3,4,7,8-HxC DD (1,2,3,4,7,8-hexachlorodibenzo-p-dioxin), 1,2,3,7,8-PeCDD (1,2,3,7,8-pentachlorodibenzo-p-dioxin), 2,3,7,8-TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), OCDD (octachlorodibenzo-p-dioxin), 1,2,3,7,8,9-HxCDD (1,2,3,7,8,9-Hexachlorocyclohexane (α-HCH), α-Hexachlorocyclohexane (α-HCH), Hexachlorobenzene, β-Hexachlorocyclohexane (β-HCH), γ-Hexachlorocyclohexane (γ-HCH), δ-Hexachlorocyclohexane (δ-HCH), Heptachlor, Aldrin, Dichloropropionic acid, Heptachlorepoxide (Isomer B), α-Chlordane, Endosulfan I (A-endodendron), g-Chlordane (trans-Chlordron), Dieldrin, p,p'-DDE (p,p'-DDD), Endrin, Endosulfan II (B-endodendron), p,p'-DDD (p,p'-DDD), o,p'-DDT (o,p'-DDT), Endrinaldehyde, Endosulfan sulfate, p,p'-DDT (p,DDT, Endrin ketone, Methoxychlor, Mirex, Dimethylphthalate (DMP), Diethyl Phthalate (DEP), Diallyl phthalate (DAP), Bis(2-methylpropyl)ester (DIBP), Dibutyl phthalate (DBP), Bis(2-methoxyethyl)phthalate (DMEP), Bis(4-methylpentyl)ester (BMPP), Bis(2-ethoxyethyl)ester (DEEP), Diamyl Diphenyl phthalate (DPP), Dihexyl ester (DHXP), Benzyl butyl phthalate (BBP), Bis(2-butoxyethyl)phthalate (DBEP), Dicyclohexyl phthalate (DCHP), Bis(2-ethylhexyl)phthalate (DEHP), Diphenyl ester (DPhP), Di-n-octyl phthalate (DNOP), Diisononyl phthalate (DINP), Dinonyl phthalate ester (DNP) (dinonyl phthalate), 2-Chlorotoluene (2-chlorotoluene), 4-Chlorotoluene (4-chlorotoluene), 1,3-Dichlorobenzene (1,3-dichlorobenzene), 1,2-Dichlorobenzene (1,2-dichlorobenzene), 1,4-Dichlorobenzene (1,4-dichlorobenzene), 2,6-Dichlorotoluene (2,6-dichlorotoluene), 2,3-Dichlorotoluene (2,3-dichlorotoluene), 1,3,5-Trichlorobenzene (1,3,5-trichlorobenzene), 2,5-Dichlorotoluene (2,The following are combinations of one or more of the following: 5-dichlorotoluene, 1,2,4-trichlorobenzene, 1,2,3-trichlorobenzene, 2,3,6-trichlorotoluene, 2,4,5-trichlorotoluene, 1,2,4,5-Tetrachlorobenzene, 1,2,3,5-Tetrachlorobenzene, 1,2,3,4-Tetrachlorobenzene, Pentachlorobenzene, or 2,3,4,5,6-Pentachlorotoluene.

[0015] In one or more embodiments, the gas chromatography detection conditions are as follows: the chromatographic column is a Thermo TG-5Sil MS 30m*0.25mm*0.25μm, or other equivalent; the temperature program is: 60℃ for 2 min, ramp to 180℃ at 20℃ / min, ramp to 220℃ at 3℃ / min, ramp to 310℃ at 8℃ / min for 5 min, and ramp to 320℃ at 20℃ / min for 5 min; the carrier gas is helium with a purity ≥99.999% and a flow rate of 1.20 mL / min; the injection port temperature is 300℃; the injection volume is 1 μL; and the injection method is splitless injection.

[0016] In one or more embodiments, the detection conditions of the mass spectrometer are: electron impact source: 70 Ev; transfer line temperature: 260 °C; ion source temperature: 280 °C; scanning mode: SCAN full scan, scanning range m / z 75~1050; resolution: 70000.

[0017] Preferably, the number of the target peak and fragment ions is 2 to 5.

[0018] Preferably, the number of target peaks is 1 and the number of fragment ions is 3.

[0019] In one or more embodiments, the extraction reagent is selected from acetonitrile or acetonitrile-ethyl acetate (a mixture of acetic acid and ethyl acetate); wherein, in the acetonitrile-ethyl acetate mixture, the volume ratio of acetonitrile to ethyl acetate is 1:1.5 to 1.5:1; the volume ratio of the liquid milk to the extraction reagent is 1:5 to 1:1, and the extraction is performed once.

[0020] Preferably, in the acetonitrile-ethyl acetate mixture, the volume ratio of acetonitrile to ethyl acetate is 1:1.

[0021] Preferably, the volume ratio of the liquid milk to the solvent is 1:4.

[0022] In one or more embodiments, the purification method is low-temperature freezing degreasing.

[0023] In one or more embodiments, in step S-2, the temperature program of the gas chromatograph is as follows: hold at 60°C for 2 min, increase to 180°C at 20°C / min, increase to 220°C at 3°C / min, increase to 310°C at 8°C / min and hold for 5 min, and increase to 320°C at 20°C / min and hold for 5 min.

[0024] Preferably, in step S-2, the gas chromatography detection conditions are as follows: the chromatographic column is a Thermo TG-5Sil MS 30m*0.25mm*0.25μm, or other equivalent; the temperature program is: 60℃ for 2 min, 20℃ / min to 180℃, 3℃ / min to 220℃, 8℃ / min to 310℃ for 5 min, and 20℃ / min to 320℃ for 5 min; the carrier gas is helium with a purity ≥99.999% and a flow rate of 1.20 mL / min; the injection port temperature is 300℃; the injection volume is 1 μL; and the injection method is splitless injection.

[0025] In one or more embodiments, in step S-2, the detection conditions of the high-resolution mass spectrometry include: electron impact source: 70 Ev; transfer line temperature: 260 °C; ion source temperature: 280 °C; scanning mode: SCAN full scan, scanning range m / z 75~1050; resolution: 70000.

[0026] In one or more embodiments, in step S-3, the pollutant identification index in the test sample is: the presence of at least two precisely matched fragment ions, with a mass accuracy deviation of less than 5 ppm; the chromatographic retention time deviates from the corresponding retention time in the database within ±0.1 min; and the signal-to-noise ratio is greater than or equal to 3.

[0027] This invention provides a method for the detection and screening of multiple pollutants based on gas chromatography-high resolution mass spectrometry (GC-HRMS). First, using full-scan HRMS data, a GC-HRMS database of various typical environmental pollutants was constructed. Second, an effective pretreatment method for pollutants in raw milk was established based on cryogenic freezing-liquid extraction. Compared with other pretreatment techniques, this method does not require any additives such as salts, sugars, or polymers, thus avoiding the introduction of new impurities. Finally, the established database of typical environmental pollutants was used for spectral library retrieval, matching information of compounds in the database, including precise molecular weight and chromatographic peak retention time, to screen and confirm various environmental pollutants in raw milk samples.

[0028] Since most typical environmental pollutants are weakly polar or nonpolar compounds, this invention employs GC-HRMS for screening typical environmental pollutants. HRMS can acquire mass-to-charge ratios accurate to five decimal places. This invention obtains retention times and fragment ion information for various pollutants by performing a full scan of a raw milk matrix containing various pollutant standards, thus constructing a pollutant database. The data from the collected test samples are then compared with the database as a basic condition for high-throughput screening. The data obtained from the test samples are then searched within the database to match compounds with similar data, thereby confirming the screening results. The high-resolution full-scan mode of the electrostatic field orbital trap high-resolution mass spectrometry (Orbitrap HRMS) used in this invention can simultaneously acquire information on all target compounds in the pollutants, thus enabling broad-spectrum high-throughput screening and qualitative identification of a large number of pollutants in complex matrices.

[0029] The detection and screening method of this invention has instrument screening limits of 2–10 μg / L for most environmental pollutants in raw milk. The recoveries of 104 out of 119 typical environmental pollutants range from 50% to 130%, and the recoveries of 78 pollutants range from 80% to 120%. The linear regression coefficients of the matrix-matched standard curves range from 0.97 to 0.999, confirming the feasibility of using the detection method of this invention to screen and quantify multiple types of pollutants in raw milk corresponding to the database. Overall, the method of this invention is simple to operate, has good accuracy and reproducibility, and has been used for large-scale actual sample testing, providing accurate and reliable technical support for the quality and safety of milk and dairy products. Attached Figure Description

[0030] Figure 1Figure 1 shows the extraction ion chromatograms of various mixed standards. Figure A is the extraction ion chromatogram of a mixed standard of 16 polycyclic aromatic hydrocarbons (PAHs); Figure B is the extraction ion chromatogram of a mixed standard of 20 polychlorinated biphenyls (PCBs); Figure C is the extraction ion chromatogram of a mixed standard of 7 polybrominated diphenyl ethers (PBDEs); Figure D is the extraction ion chromatogram of a mixed standard of 10 polychlorinated dibenzofurans (PCDFs); Figure E is the extraction ion chromatogram of a mixed standard of 6 polychlorinated dibenzo-p-dioxins (PCDDs); Figure F is the extraction ion chromatogram of a mixed standard of 24 organochlorine pesticides (OCPs); Figure G is the extraction ion chromatogram of a mixed standard of 18 phthalate esters (PAEs); and Figure H is the extraction ion chromatogram of a mixed standard of 18 chlorobenzenes (CBs).

[0031] Figure 2 This is a total ion current chromatogram of raw milk processed by different extraction and purification methods in full scan mode.

[0032] Figure 3 Figure A shows the recovery results under different solvent conditions, different volume ratios of liquid emulsion to extraction solvent, and different extraction times. Figure B shows the recovery results under different solvent conditions; Figure C shows the recovery results under different volume ratios of liquid emulsion to extraction solvent; and Figure C shows the recovery results under different extraction times.

[0033] Figure 4 This is a cumulative probability distribution of the method screening limits for 119 compound standards.

[0034] Figure 5 This is a cumulative probability distribution of the peak area RSD for multiple injections of 119 compound standards. Detailed Implementation

[0035] The following embodiments are merely illustrative of implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims.

[0036] In this invention, the preferred detection conditions for gas chromatography are as follows:

[0037] Chromatographic column: Thermo TG-5Sil MS 30m*0.25mm*0.25μm, or equivalent; Temperature program: 60℃ for 2 min, ramp to 180℃ at 20℃ / min, ramp to 220℃ at 3℃ / min, ramp to 310℃ at 8℃ / min and hold for 5 min, ramp to 320℃ at 20℃ / min and hold for 5 min. Carrier gas: Helium, purity ≥99.999%, flow rate: 1.20 mL / min; Injector temperature: 300℃; Injection volume: 1 μL; Injection method: Splitless injection.

[0038] In this invention, the preferred detection conditions for mass spectrometry are as follows:

[0039] Electron impact source: 70 Ev; Transmission line temperature: 260℃; Ion source temperature: 280℃; Scan mode: SCAN full scan, scan range m / z 75~1050; Resolution: 70000.

[0040] Example 1: Construction of a high-resolution mass spectrometry database for multiple types of environmental pollutants

[0041] Accurate characterization of environmental pollutants in milk and dairy products is crucial for ensuring the reliability of test results. This embodiment uses raw milk as a matrix with mixed standard solutions of various pollutants. After gas chromatography-high resolution mass spectrometry (GC-HMS), extractable ion chromatography (XIC) chromatograms with good isomer separation were obtained, and the retention times of each pollutant standard were recorded. In the XIC chromatograms, the ion with the strongest intensity was selected as the target ion, and 2-5 fragment ions with the highest abundance were selected as characteristic fragment ions. Based on these data, a high-resolution mass spectrometry (HRMS) database of pollutants was established, involving a total of 119 standards from various types of pollutants.

[0042] The various pollutant standards include: mixed standards for polycyclic aromatic hydrocarbons (PAHs) (containing a mixed solution of 16 PAH standards), mixed standards for polychlorinated biphenyls (PCBs) (containing a mixed solution of 20 PCB standards), mixed standards for polybrominated diphenyl ethers (PBDEs) (containing a mixed solution of 7 PBDE standards), and mixed standards for polychlorinated dibenzofurans (PCDFs) (containing a mixed solution of 10 PCDF standards). ), polychlorinated dibenzo-p-dioxins (PCDDs) mixed standards (containing a mixed solution of 6 PCDDs), organochlorine pesticides (OCPs) mixed standards (containing a mixed solution of 24 organochlorine pesticides), phthalates (PAEs) mixed standards (containing a mixed solution of 18 PAEs), and chlorobenzenes (CBs) mixed standards (containing a mixed solution of 18 chlorobenzenes).

[0043] Figure 1 The extracted ion chromatograms of different types of pollutants are shown, demonstrating that this chromatographic method has good separation effect on isomers of various organic pollutants. The names, CAS numbers, target peaks (expressed as molecular weight), three fragment ion peaks (expressed as molecular weight), and retention times (RT) of each standard are shown in Table 1 below.

[0044] Table 1: Gas Chromatography-High Resolution Mass Spectrometry Database of 119 Typical Environmental Pollutants

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051] Example 2: Optimization of pretreatment method for raw milk

[0052] The general pretreatment process before injection chromatography-mass spectrometry mainly includes two steps: extraction and purification. This example explores efficient methods for simultaneous extraction and purification of different types of pollutants, thereby optimizing the pretreatment process.

[0053] Purification method optimization:

[0054] Environmental pollutants in raw liquid milk were extracted using acetonitrile to obtain raw milk extracts. These extracts were then processed using Florisil material, resin, and C... 18 The raw milk extract was further purified using adsorption materials and low-temperature cryogenic defatting to obtain test samples 1#-3#. Additionally, environmental pollutants in the liquid raw milk were extracted using a mixed solvent of acetonitrile and ethyl acetate (1:1) to obtain raw milk extracts, which were then further purified using low-temperature treatment to obtain test sample 4#. Thus, raw milk underwent different extraction and purification methods to obtain test samples 1#-4#. The sample numbers and conditions for each raw milk sample during the purification method optimization process are shown in Table 2 below.

[0055] Table 2: Optimization conditions for purification methods

[0056] Sample number to be tested Extraction methods Purification methods 1# Acetonitrile Floris silica materials 2# Acetonitrile <![CDATA[Resin and C 18 Adsorbent material]]> 3# Acetonitrile Low-temperature freezing degreasing 4# Acetonitrile-ethyl acetate Low-temperature freezing degreasing

[0057] Each sample to be tested was injected into a GC-HRMS to obtain the following results: Figure 2The total ion current chromatography (TIC) chromatogram is shown. The sample #1, purified with Floris silica after acetonitrile extraction, exhibits the highest total peak area in its TIC chromatogram. Based on deconvolution and database matching, it is inferred that this sample contains the highest levels of 2-palmitoylglycerol, cholesterol, and glycerides. The sample purified with resin and C20 after acetonitrile extraction... 18 The total peak area of ​​the TIC spectrum for sample #2, purified by the adsorption material, was the second largest. However, after deconvolution and database matching, it was found that the content of oleic acid amide in this sample was higher than that in other samples. The peak area of ​​the TIC spectrum for sample #4, extracted with a mixed solution of acetonitrile and ethyl acetate (1:1, v / v), was slightly larger than that for sample #3, extracted with acetonitrile. In the TIC spectrum of sample #4, the peak detected at elution time 36.2 min was mainly 283.19037 (C 16 H 27 O4) and 383.31559 (C 23 H 43 Fragments of O4 were found, presumably belonging to the glycerol ester family; no fragment ions of the target pollutant were detected. Overall, combined with... Figure 2 As can be seen from the above analysis, the smaller the total peak area of ​​the TIC diagram, the better the degreasing and purification effect. Therefore, the degreasing and purification effect of each pretreatment method is ranked as 3# > 4# > 2# > 1#.

[0058] Extraction method optimization:

[0059] To further optimize the recovery rate of the pretreatment method, this embodiment investigated the effects of different extraction solvents, different volume ratios of liquid milk to raw milk and extraction reagent, and different extraction times on the recovery rate. 13 C-mark 13 C 12 -PCB101 as 13 C 12 -PCB18, 13 C 12 -PCB28, 13 C 12 -PCB52 and 13 C 12 - PCB153 recovery rate internal standard 13 C 12 -PCB194 as 13 C 12 -PCB194, 13 C 12 -PCB180, 13 C 12 -PCB202 and 13 C 12 -PCB206, 13 C 12-The recovery rate internal standard of PCB209 was used to examine the recovery rate of each target analyte, and the results are as follows: Figure 3 As shown; the recovery rate R is calculated according to the following formula: In the formula, R is the recovery rate of the target analyte; A s The peak area of ​​the quantitative internal standard; m r The amount of the internal standard for recovery added to the sample, in ng; A r is the peak area of ​​the internal standard for recovery; RRF is the relative response factor of the target analyte to the internal standard for recovery; m s The amount of the target substance added to the sample is expressed in ng; the relative response factor (RRF) is calculated according to the following formula: In the formula A s C represents the peak area of ​​the target object. r For the internal standard concentration of recovery, A r C represents the peak area of ​​the internal standard for recovery. s The concentration of the target substance.

[0060] Depend on Figure 3 As shown in A, extraction of each PCB using acetonitrile-ethyl acetate (1:1, v / v) yielded better recoveries than extraction using acetonitrile (ACN), with recoveries ranging from 76% to 102% using acetonitrile-ethyl acetate (1:1, v / v). Figure 3 As shown in B, except for PCB 28, for the volume ratio of liquid milk to extraction reagent, a 1:4 ratio results in better recovery than a 1:2 ratio; Figure 3 As can be seen from C, for every 1 mL of liquid raw milk, using 4 mL of extraction reagent, the recovery rate of one extraction and three extractions is roughly the same.

[0061] In summary, the subsequent extraction method employed was to extract once with 4 mL of acetonitrile-ethyl acetate (1:1, v / v) per 1 mL of liquid raw milk. Considering the recovery rate, the effectiveness and simplicity of the degreasing method, the final pretreatment method employed was to use an extraction method of 4 mL of acetonitrile-ethyl acetate (1:1, v / v) per 1 mL of liquid raw milk, along with a purification method of low-temperature cryogenic degreasing.

[0062] Example 3: Performance parameter verification of a screening method for multiple types of environmental pollutants

[0063] The GC-HRMS-based targeted screening technology is applicable to existing pollutant control lists in various regions. Rapid screening is conducted using a self-built database based on these lists, and accurate quantitative methods are then established for monitoring after confirmation. The screening parameters are set as follows: at least two precisely matched fragment ions are present, with a mass accuracy deviation of less than 5 ppm; the chromatographic retention time deviates from the corresponding retention time in the database within ±0.1 min; and the compound is detected positively in five consecutive repeated tests of the sample, indicating the presence of the corresponding pollutant (specific parameters are shown in Table 3).

[0064] Table 3: Qualitative Judgment Indicators for the Detection of Typical Environmental Pollutants

[0065] Serial Number Judgment Indicators Result range 1 Retention time deviation ±0.1min 2 Signal-to-noise ratio ≥3 3 Precise mass deviation <±5ppm 4 Positive detection rate 100％

[0066] The performance parameters of the screening method were verified by selecting 119 standard samples of different categories of pollutants mentioned above.

[0067] Screening limits:

[0068] The screening limit of this analytical method was evaluated using a stepwise dilution method. Each standard was serially diluted with raw bovine milk white matrix extract to obtain standard solutions with concentrations of 2 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 25 ng / mL, 50 ng / mL, and 100 ng / mL. For each standard, solutions were injected for screening in descending order of concentration until the instrument could detect the lowest concentration of the standard solution. This lowest concentration was determined as the screening limit for that standard. The screening limits for each standard are shown in Table 4. Figure 4 As can be seen, at six different concentrations of 2 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 25 ng / mL, 50 ng / mL, and 100 ng / mL, the percentages of pollutants that this screening method could detect were 42.9%, 79.8%, 91.6%, 93.3%, 97.5%, 99.2%, and 100%, respectively. This indicates that the instrumental screening limit for most environmental pollutants in raw milk using this screening method is at the 2–10 μg / L level.

[0069] For each standard, the linear range is defined as a range with the screening limit as the lower limit and 100 ng / mL as the upper limit; within the linear range, the linear regression coefficient R of the matrix-matched standard curve for each standard is... 2 Between 0.97 and 0.999.

[0070] stability:

[0071] The stability of the screening method was assessed by repeatedly injecting the same sample and using the relative standard deviation (RSD) of the peak area. Each standard was diluted with raw bovine milk white matrix extract to obtain a standard solution with a concentration of 100 ng / mL. Each standard solution was injected 5 times (n=5), the peak area was measured, and the RSD of the peak area was calculated. The RSDs of the peak areas for each standard are shown in Table 4. Figure 5 It can be seen that at a concentration of 100 ng / mL (i.e. 100 μg / L), 95% of the peak areas of the substances have a deviation of less than 20%, indicating that the screening method has good stability.

[0072] Spike recovery rate:

[0073] Using raw milk as the blank spiking matrix, various standards were added to the raw milk at a spiking level of 50 ng / mL. The raw milk containing the added contaminant standards was treated according to the pretreatment method finally selected in Example 2 to extract and recover the standards. The spiked recovery rate of the recovered standards was then detected, and the spiked recovery rate was calculated according to the following formula: The spiked recoveries of each standard are shown in Table 4. As shown in Table 4, the recoveries of the standards for 78 pollutants ranged from 80% to 120%, and the recoveries of the standards for 104 typical environmental pollutants ranged from 50% to 130%. Overall, this pretreatment method can achieve a high recovery rate.

[0074] Table 4: High-resolution mass spectrometry database of environmental pollutants

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082] Application example:

[0083] Using the final determination pretreatment method described in Example 2, and employing GC-Exactive Orbitrap HRMS, a multi-target simultaneous screening of 119 typical environmental pollutants was conducted on 119 raw milk samples from different production areas in China (Guangxi, Qinghai, Xinjiang, Hebei, Henan, Jiangsu, and Sichuan). The analytical results were statistically analyzed. Since impurities may be introduced during sample collection, transportation, storage, preparation, extraction, and analysis, a blank control experiment was added throughout the sample screening process to improve qualitative accuracy. If the abundance of a compound in a sample was more than three times that in a blank sample, the compound was retained; otherwise, it was discarded. A total of six pollutants were screened and confirmed from the 119 raw milk liquid samples, including five phthalates and one chlorobenzene compound. The detection rate of 1,2-Dichlorobenzene was 88%; the detection rate of DMP was 100%; the detection rate of DEP was 52%; the detection rate of DIBP was 46%; the detection rate of DBP was 62%; and the detection rate of DEHP was 24%. Other typical environmental pollutants were not detected.

[0084] In summary, this invention optimizes the pretreatment method for raw milk. By performing a full GC-HRMS scan on each mixed standard, data such as target peaks, fragment ions, retention times, screening limits, spiked recoveries, and RSDs for each contaminant compound are obtained, thereby constructing a database containing a total of 119 contaminants. This facilitates high-throughput screening of multiple contaminants in raw milk. The detection method utilizing this database is simple to operate, accurate, and reproducible, and has been used for large-scale testing of actual samples, providing accurate and reliable technical support for the quality and safety of milk and dairy products.

Claims

1. A method for screening of multiple classes of environmental pollutants in raw cow milk, characterized in that, The method comprises the following steps: S-0: Constructing a pollutant database: determining by gas chromatography and high-resolution mass spectrometry, and collecting total ion flow chromatograms of standard solutions of typical environmental pollutants to obtain relevant data; S-1: Sample pretreatment: mixing raw milk samples with extraction reagents, and sequentially performing extraction, purification, centrifugation, nitrogen blowing of supernatant, redissolution and membrane filtration to obtain samples to be tested; S-2: Data acquisition: introducing the samples to be tested into a gas chromatography-high resolution mass spectrometer to obtain total ion flow chromatograms based on high-resolution mass spectrometry; S-3: Screening and verification: performing peak identification on the total ion flow chromatograms of the test samples, and identifying the pollutants present in the samples based on the self-built database.

2. The method of claim 1, wherein, In step S-0, the relevant data includes target peaks (represented by molecular weight), fragment ion peaks (represented by molecular weight), retention time, screening limit, relative standard deviation (RSD) and standard addition recovery.

3. The method of claim 1, wherein, In step S-0, the environmental pollutants include one or more combinations of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), brominated diphenyl ethers (PBDEs), polychlorinated dibenzofurans (PCDFs), polychlorinated dibenzo-p-dioxins (PCDDs), organochlorine pesticides (OCPs), phthalate esters (PAEs) or chlorobenzenes (CBs).

4. The method of claim 1, wherein, The detection conditions of the gas chromatography are as follows: the chromatographic column is Thermo TG-5Sil MS 30m*0.25mm*0.25μm, or other equivalent columns; the temperature rising program is 60℃ for 2min, 20℃ / min to 180℃, 3℃ / min to 220℃, 8℃ / min to 310℃ for 5min, 20℃ / min to 320℃ for 5min; The carrier gas is helium with a purity of ≥99.999% and a flow rate of 1.20mL / min; the injection port temperature is 300℃; the injection amount is 1μL; and the injection mode is splitless injection; The mass spectrometry detection conditions are as follows: electron impact source: 70Ev; transmission line temperature: 260℃; ion source temperature: 280℃; scanning mode: SCAN full scan, scanning range m / z 75~1050; The resolution is 70000.

5. The method of claim 1, wherein, The extraction reagent is selected from acetonitrile or acetonitrile-ethyl acetate; in the acetonitrile-ethyl acetate, the volume ratio of acetonitrile to ethyl acetate is 1:1.5~1.5:1; the volume ratio of the liquid raw milk to the extraction reagent is 1:5~1:1, and the extraction is performed once.

6. The method of claim 1, wherein, The purification mode is low-temperature refrigeration lipid removal.

7. The method of claim 1, wherein, In step S-2, the temperature rising program of the gas chromatography is as follows: 60℃ for 2min, 20℃ / min to 180℃, 3℃ / min to 220℃, 8℃ / min to 310℃ for 5min, 20℃ / min to 320℃ for 5min.

8. The method of claim 1, wherein, In step S-2, the detection condition of the high-resolution mass spectrometer includes: electron impact source: 70 Ev; transmission line temperature: 260 DEG C; ion source temperature: 280 DEG C; scanning mode: SCAN full scan, scanning range m / z 75-1050; resolution: 60000.

9. The method of claim 1, wherein, In step S-3, the identification index of the contaminant in the detection sample is: at least two matched accurate mass number ions exist, the mass accuracy deviation is less than 5ppm; the chromatographic retention time and the corresponding retention time in the database deviate within ±0.1 min; and the signal-to-noise ratio is greater than or equal to 3.

10. A method for pre-treating raw cow's milk, characterized in that, The method comprises: mixing the raw milk sample with an extraction reagent, sequentially performing extraction, purification, centrifugation, nitrogen blowing of the supernatant, redissolution and membrane filtration to obtain a sample to be detected.

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