Polycyclic aromatic hydrocarbon detection method based on deep eutectic solvent extraction

Abstract

The invention particularly relates to a polycyclic aromatic hydrocarbon detection method based on deep eutectic solvent extraction. The invention firstly provides a deep eutectic solvent composed of DL-menthol and thymol, and provides application of the deep eutectic solvent as an extraction agent in the field of detection of four polycyclic aromatic hydrocarbons. The main innovation point of the detection method provided by the invention lies in establishment of a sample pretreatment method, in the final detection method provided by the invention, related reagents mainly comprise acetonitrile, salt, menthol and thymol, the components are non-toxic and harmless, the cost is low, and high-sensitivity, high-accuracy and environment-friendly determination of trace and ultra-trace PAH4 in special medical food is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of deep eutectic solvent extraction, and particularly to the preparation of a phenolic nonionic hydrophobic natural deep eutectic solvent and the application of the combined liquid-liquid microextraction technology in the detection of polycyclic aromatic hydrocarbons. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily to be regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Polycyclic aromatic hydrocarbons (PAHs) are one of the most widely distributed environmental and food processing pollutants. A large number of studies have shown that frequent exposure will increase the risk of cardiovascular diseases such as thrombosis and myocardial infarction, and they have strong carcinogenicity, mutagenicity and teratogenicity. The International Agency for Research on Cancer (IARC) classifies benzo[a]pyrene as a Group 1 carcinogen, and benzo[a]anthracene, chrysene, and benzo[b]fluoranthene as Group 2 carcinogens. The European Food Safety Authority believes that analyzing only benzo[a]pyrene cannot fully reflect all the genotoxic and carcinogenic PAHs present in food, and recommends evaluating the sum of the above four PAHs, namely benzo[a]pyrene, benzo[a]anthracene, chrysene, and benzo[b]fluoranthene as a polycyclic aromatic hydrocarbon marker.

[0004] Special medical purpose formula foods are formula foods specifically processed and prepared to meet the special needs of nutrients or diets for people in specific disease states, and are foods that are the focus of domestic and foreign supervision. However, vegetable oils in their raw materials and ingredients are susceptible to PAHs contamination, and polycyclic aromatic hydrocarbons may also be produced during the heat treatment process of production and processing. The new EU regulation on the maximum levels of contaminants in food (EU) 2023 / 915 stipulates that the content of benzo[a]pyrene in special medical purpose formula foods shall not exceed 1.0 μg / kg, and the total content of 4 polycyclic aromatic hydrocarbon markers (PAH4) shall not exceed 1.0 μg / kg. This maximum limit only applies to products in the ready-to-eat state. China's GB 2762—2022 National Food Safety Standard - Maximum Levels of Contaminants in Foods does not stipulate the limits of PAHs in special medical purpose formula foods. In recent years, the detection of PAHs in infant formula milk powder has been a hot topic in food safety around the world, but there is little research on PAHs in special medical purpose formula foods, which are also special foods. Special medical purpose formula foods have a complex matrix and low PAHs content. Therefore, it is necessary to improve the enrichment efficiency and minimize matrix interference to improve the detection sensitivity. The pretreatment techniques for this type of food matrix usually include steps such as fat extraction, saponification, and purification with solid-phase extraction columns. This method has problems such as cumbersome operation, low detection efficiency, and large solvent consumption, which limit its application in high-throughput analysis. Therefore, the development of more efficient and environmentally friendly pretreatment techniques has become an important research direction in the field of polycyclic aromatic hydrocarbon analysis in special medical purpose formula foods.

[0005] Considering the analytical concept of green chemistry, in recent years, the dispersive liquid-liquid microextraction (DLLME) technique based on deep eutectic solvents has made great progress in the analysis of polycyclic aromatic hydrocarbons. Deep eutectic solvents are mainly formed by two substances: one is a hydrogen bond donor and the other is a hydrogen bond acceptor. They form a new solvent under a given temperature and molar ratio. The deep eutectic solvent is simple to prepare, has low toxicity, and good biodegradability. This method is simple to operate, the extraction solvent volume is in microliters, has a high preconcentration factor, is fast, has low cost, and is more environmentally friendly. Deep eutectic solvents can be divided into five categories according to their components. Types I-IV of deep eutectic solvents generally belong to ionic deep eutectic solvents, which are essentially hydrophilic. Type V deep eutectic solvents are non-ionic substances composed of phenolic hydrogen bonds, which is a new type of deep eutectic solvent proposed by Abranches et al. It has hydrophobicity and low viscosity and density, and is more suitable for gas chromatography-mass spectrometry analysis.

[0006] The inventors believe that at present, deep eutectic solvents are mainly applied to the detection of polycyclic aromatic hydrocarbons (PAHs) in single-component samples with simple sample matrices. However, the composition of foods for special medical purposes is complex, and it is more difficult to achieve accurate detection. Therefore, providing a detection method with good selectivity, high sensitivity, and strong anti-interference ability is of great significance for controlling the quality and safety of foods for special medical purposes. In addition, the detection of PAHs mainly relies on gas chromatography-mass spectrometry (GC-MS) technology. However, type I-IV deep eutectic solvents reported in the prior art essentially belong to hydrophilic reagents and are not suitable for GC-MS analysis. Summary of the Invention

[0007] Aiming at the problems existing in the above prior art, the present invention aims to establish a liquid-liquid microextraction technology based on type V deep eutectic solvents, combined with gas chromatography-triple quadrupole tandem mass spectrometry with good selectivity, high sensitivity, and strong anti-interference ability, to achieve highly sensitive, highly accurate, and green determination of trace and ultra-trace PAH4 in foods for special medical purposes. Combining type V deep eutectic solvents with liquid-liquid microextraction technology can achieve the detection of trace PAHs in complex matrices, and this technology has advantages such as high enrichment efficiency and environmental friendliness.

[0008] Based on the above technical effects achieved, the present invention provides the following solutions: The present invention provides a method for detecting polycyclic aromatic hydrocarbons based on deep eutectic solvent extraction. The main feature of the detection method is that deep eutectic solvents are used to extract polycyclic aromatic hydrocarbons in the sample to be detected. The deep eutectic solvents are composed of DL -menthol and thymol.

[0009] The deep eutectic solvents are mainly applied to the liquid-liquid extraction process in the pretreatment of polycyclic aromatic hydrocarbons. Among them, DL- menthol is a hydrogen bond acceptor, and thymol is a hydrogen bond donor. DL- The molar ratio of menthol to thymol directly affects the properties of the deep eutectic solvents, affects the hydrogen bonds and π-π interactions between them and PAHs, and affects the solubility of PAHs in the solvents. DL- The higher the proportion of menthol, the stronger the hydrogen bond interaction with thymol. The higher the proportion of thymol, the stronger the π-π interaction between its aromatic ring and PAHs, and its phenolic properties can improve the solubility of PAHs as aromatic compounds.

[0010] In a preferred embodiment verified in the present invention, the DL- molar ratio of menthol to thymol is 3:1, 2:1, 1:1, 1:2, or 1:3, and the best ratio is 1:1. The preparation method of the deep eutectic solvents is as follows: Mix appropriate molar ratios of DL- menthol and thymol, and stir and heat at 55-65 °C until a transparent and homogeneous solution is obtained.

[0011] Furthermore, the above detection method comprises the following steps: (1) Add water, acetonitrile and salt to the sample to be tested. After sufficient extraction, retain the acetonitrile part; (2) Add brine and the deep eutectic solvent to the acetonitrile part in step (1), mix well, retain the organic phase part, dry it, and then re-dissolve it in acetonitrile for volume fixation; (3) Detect by gas chromatography-triple quadrupole mass spectrometry method.

[0012] The above detection method also has the following preferred technical solutions: In step (1): For the sample to be tested, the detection samples applicable to the above detection method include solid samples and liquid samples. According to the common polycyclic aromatic hydrocarbon detection objects in the art, the samples to be tested further applicable to the above detection method are foods or beverages, especially samples with complex components can be detected. In an implementation mode verified by the present invention, the object to be tested is food, specifically, a food for special medical purposes, hereinafter referred to as a special medical food. The production process of special medical foods is complex, rich in components such as fat and protein, and polycyclic aromatic hydrocarbons often coexist with fat compounds. Therefore, how to efficiently extract PAHs from special medical foods has become a key problem to be solved urgently. The Soxhlet extraction method is a classic method for extracting PAHs from foods. Although it has good effects, it takes a long time and has low efficiency. In recent years, a variety of efficient extraction methods have emerged and been applied, such as ultrasonic extraction, pressurized liquid extraction, QuEChERS method, etc. Among them, the QuEChERS method has the advantages of green energy consumption reduction, environmental friendliness, high efficiency, etc., and is widely used in the pretreatment field of PAHs in foods.

[0013] In the detection method provided by the present invention, the extraction solvent of the QuEChERS method will be used as the dispersant of the next dispersive liquid-liquid microextraction (DLLME) system. Its selection is crucial and it needs to have strong extraction ability and dispersing ability at the same time. In the DLLME system, the volume ratio of the dispersant is a key factor affecting the extraction efficiency. Its action mechanism is complex and diverse, and has a significant impact on the recovery rate of the target analyte. It has been verified that the present invention finds that acetonitrile as an extraction solvent has good recovery effects on 4 PAHs (benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[a]pyrene). Usually, acetonitrile and water can be miscible in any proportion. In order to achieve the separation of acetonitrile, the present invention also adds salt to the aqueous solution of the sample to be tested, so that acetonitrile and brine are stratified.

[0014] Furthermore, the salt is preferably a salt with high solubility in water, such as sodium chloride. In an implementation mode with better effects, the dosage ratio of the sample to be tested, water, acetonitrile, and salt is 1 g: 4-6 mL: 4-6 mL: 0.8-1.2 g.

[0015] In the DLLME procedure, the volume of the extraction solvent is one of the key parameters affecting the extraction efficiency. The volume of the extraction solvent directly determines the amount of micro-droplets generated, and the number of micro-droplets is closely related to the mass transfer process of the target from the aqueous phase to the organic phase, thereby significantly affecting the extraction efficiency. In addition, when the extraction volume is too large, a dilution effect will occur. The ionic strength is another important parameter affecting the extraction efficiency in the DLLME procedure. In the DLLME distribution system, salts can significantly change the distribution behavior of the analyte between the extraction phase and the aqueous phase and affect its recovery rate. As the ionic strength increases, the density and viscosity of the aqueous solution show an increasing trend, which will reduce the mass transfer efficiency process of the target analyte. Verified by the present invention, as the salt concentration increases, the volume of the upper organic phase gradually increases, and more acetonitrile will dissolve into the HNADES phase. When the salt concentration is 15%-30%, the solution after nitrogen blowing and re-dissolution is turbid, and a small amount of salt precipitates. Therefore, considering the extraction efficiency and experimental phenomena comprehensively, the more preferred salt concentration is 10%.

[0016] In step (2): Acetonitrile is used as the extraction solvent in the extraction process and also serves as a dispersant in the DLLME system. Therefore, the more preferred dosage ratio of the acetonitrile part, brine, and deep eutectic solvent is 5 mL: 7-9 mL: 150-250 μL, and the concentration of the brine is 8-12 wt.%.

[0017] During the process of volume fixation by adding acetonitrile again after drying, the amount of acetonitrile added should be able to fully dissolve the target analyte to be measured and at the same time reach the detection limit of the subsequent gas chromatography-mass spectrometry detection method, which belongs to the technical content that can be routinely determined by those skilled in the art.

[0018] In step (3): The gas chromatography detection methods applicable to the present invention include but are not limited to headspace gas chromatography (HS-GC), gas chromatography-mass spectrometry (GC-MS), gas chromatography-flame ionization detection (GC-FID), gas chromatography-electron capture detection (GC-ECD), fast gas chromatography (Fast GC). In one implementation mode verified by the present invention, the detection method is a gas chromatography-triple quadrupole tandem mass spectrometry (GC-MS / MS) analysis method, and the specific parameters are as follows: Chromatographic conditions: Chromatographic column: DB-EUPAH capillary column, inlet temperature 280 - 320 °C; carrier gas is high-purity inert gas; injection volume 1 μL, splitless injection; solvent delay 16.5 min. Temperature programming: initial temperature 80 °C, hold for 2 min, increase to 250 °C at 8 - 12 °C / min, hold for 2 min, increase to 315 °C at 7 - 9 °C / min, hold for 5 min, increase to 320 °C at 18 - 22 °C / min, hold for 5 min.

[0019] Mass spectrometry conditions: Ionization method is electron impact ionization source (EI), ionization energy 70 eV; determination method: multiple reaction monitoring mode (MRM); ion source temperature 320 °C; transfer line temperature 280 °C; quadrupole temperature 150 °C.

[0020] In another embodiment of the present invention, isotope internal standard method is used for detection. Therefore, the characteristic ion pairs and collision energies of 4 PAHs and internal standard are shown in Table 1: Table 1 Characteristic ion pairs and collision energies of 4 PAHs and internal standard under multiple reaction detection (MRM) mode Compared with the prior art, the beneficial effects of the present invention are: 1. Special medical foods are usually special nutritional supplements for disease populations. Ensuring the safety of such special foods is of great significance. Moreover, the components of special medical foods are complex, the detection matrix effect is large, and the detection difficulty is high. The method of the present invention has been verified to be applicable to the detection of four common harmful polycyclic aromatic hydrocarbons in special medical foods, filling the technical gap in the quality control of special medical foods.

[0021] 2. The present invention proposes a phenolic nonionic hydrophobic natural deep eutectic solvent based on DL- menthol and thymol for liquid-liquid microextraction of 4 polycyclic aromatic hydrocarbons in foods for special medical purposes, providing an optimized pretreatment method. The synthesis cost of this deep eutectic solvent is low and the reagent is environmentally friendly. As an extractant, the volume consumption is only 5 - 6 mL. Traditional methods such as GB 5009.265 - 2021 and EU EN 16619:2015 etc. standards require 50 - 60 mL and 200 - 300 mL of extractant respectively when determining polycyclic aromatic hydrocarbons in infant formula milk powder. The dispersive liquid-liquid microextraction technology adopted in the present invention has a high enrichment factor, reducing the extractant dosage by more than 90%, avoiding the large-volume solvent consumption of traditional pretreatment methods, overcoming problems such as easy loss of target substances and generation of blank interference caused by large-volume solvent concentration, avoiding the multiple nitrogen blowing concentration process, and being more green and friendly to the environment.

[0022] On the other hand, the optimized pretreatment method of the present invention is simple and fast. It only takes 20 - 30 minutes to analyze one sample, while traditional methods such as GB 5009.265 - 2021 and EU EN 16619:2015 etc. require 1.5 - 2 hours and 8 - 10 hours respectively for the determination of polycyclic aromatic hydrocarbons in infant formula milk powder. The optimization of the pretreatment process of the present invention has achieved a reduction in analysis time by more than 1 hour, overcome the pain points of the traditional method such as cumbersome operation and long time consumption in the pretreatment process, and improved work efficiency.

[0023] 3. Based on the above pretreatment method, the present invention uses gas chromatography - triple quadrupole tandem mass spectrometry in the multiple reaction monitoring mode for detection, and has established a method for detecting polycyclic aromatic hydrocarbons based on deep eutectic solvent extraction - gas chromatography tandem mass spectrometry. This detection method has a good linear relationship in the concentration range of 0.2 - 20 ng / mL, and the correlation coefficient r 2 > 0.99, the detection limit is 0.1 μg / kg, the quantification limit is 0.25 μg / kg, the spiked recoveries at three different concentration addition levels are 96.6% - 113.2%, and the RSD is 2.4% - 8.3%. The quantification limits of the traditional methods GB 5009.265 - 2021 and EU EN 16619:2015 are 0.5 μg / kg and 0.9 μg / kg. The method of the present invention significantly improves the detection sensitivity and selectivity compared with the above standards, and has the advantages of green environmental protection, precision and reliability, etc.

[0024] In addition, the present invention uses gas chromatography - triple quadrupole tandem mass spectrometry technology in the multiple reaction monitoring mode for determination, which has stronger selectivity and better specificity, and improves the detection sensitivity by reducing background noise.

[0025] To sum up, the present invention has the advantages of simplicity, rapidity, environmental friendliness, low quantification limit, good specificity, etc., is suitable for the determination of trace and ultra - trace components in the matrix of special medical foods, and has broad application prospects in the determination of actual samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The attached drawings of the specification, which form a part of the present invention, are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0027] Figure 1 is the chemical structure of the compound used to prepare the deep eutectic solvent in Example 1; Figure 2 is the histogram of the extraction solvent effect in Example 1; Figure 3 is the histogram of the extraction effect of 4 PAHs under different HNADES extraction conditions in Example 1; Figure 4is a histogram of the recovery rates of four polycyclic aromatic hydrocarbons under the HNADES extraction conditions of different molar ratios described in Example 1; Figure 5 is a histogram of the recoveries of four PAHs under different HNADES volume extraction conditions described in Example 1; Figure 6 is a histogram showing the effect of different dispersant volumes on the recovery rates of four polycyclic aromatic hydrocarbons described in Example 1; Figure 7 This is the total ion current diagram of the standard working solution described in Example 1. DETAILED DESCRIPTION

[0028] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0029] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0030] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0031] Example 1 1 Methods and Materials 1.1 Instruments and reagents Gas chromatography-triple quadrupole tandem mass spectrometry (Agilent 8890-7010B, Agilent, USA), vortex mixer (IKA, Germany), centrifuge (Sigma, Germany), analytical balance (precision 0.0001 g, Beijing Sartorius Balance Co., Ltd.), ultrasonic cleaning machine-KQ-800DE, (Kunshan Ultrasonic Instrument Co., Ltd.).

[0032] Acetonitrile (chromatographic grade, Merck KGaA, Germany); Sodium chloride (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); DL -Menthol, thymol, undecanol, decanoic acid (analytical grade). Mixed standard solution of 4 PAHs (benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[a]pyrene) (purity ≥ 99%, purchased from Beijing Manhag Biotechnology Co., Ltd.), 4 internal standard products: D 12-Benzo[a]anthracene, D 12 - Qu, D 12 -Benzo[b]fluoranthene, D 12 -Benzo[a]pyrene (purity ≥99%, purchased from Beijing Manhag Biotechnology Co., Ltd.).

[0033] 1.2 Preparation of deep eutectic solvent by DL -Menthol is a hydrogen bond acceptor, thymol, undecanol or decanoic acid is a hydrogen bond donor, appropriate amounts of hydrogen bond donors and hydrogen bond acceptors are weighed into a glass bottle according to a molar ratio of 1:1, and a non-ionic hydrophobic natural deep eutectic solvent (HNADES) is prepared by a simple heating and stirring method, that is, magnetic stirring is performed at 60 °C until a uniform, clear and transparent solution is formed, and the mixture is set aside.

[0034] Similarly, five different molar ratios were synthesized according to the molar ratios of 3:1, 2:1, 1:1, 1:2, and 1:3. DL -Menthol-thymol deep eutectic solvent.

[0035] 1.3 Pre-treatment methods 1.3.1 Sample extraction Accurately weigh 1g (accurate to 0.0001g) of FSMP sample into a 15 mL centrifuge tube, add 5 mL of ultrapure water, and vortex for 2 minutes to dissolve the sample. Then, add 5 mL of acetonitrile and 1 g of sodium chloride, vortex for 2 minutes for extraction, centrifuge at 8000 rpm for 5 minutes, and transfer the acetonitrile layer to a 15 mL centrifuge tube for later use.

[0036] 1.3.2 DLLME Program Add 8 mL of 10 wt.% saline and 200 μL of DL- HNADES with a molar ratio of menthol to thymol of 1:1 was vortexed for 2 min. Centrifuged at 8000 rpm for 2 min, the upper organic phase was nitrogen purged and then diluted to 0.5 mL with acetonitrile for GC-MS / MS analysis.

[0037] 1.4 Analysis conditions Chromatographic conditions: chromatographic column DB-EUPAH capillary column (20 m×0.18 mm×0.14μm); injection port temperature 300℃; carrier gas is high-purity helium, purity ≥99.999%; injection volume 1μL, splitless injection; solvent delay 16.5 min. Program temperature rise: initial temperature 80℃, hold for 2 min, increase to 250℃ at 10℃ / min, hold for 2 min, increase to 315℃ at 8℃ / min, hold for 5 min, increase to 320℃ at 20℃ / min, hold for 5 min.

[0038] Mass spectrometry conditions: The ionization method is electron impact ionization source (EI), and the ionization energy is 70 eV ; Determination method: multiple reaction monitoring mode (MRM); Ion source temperature is 320 °C; Transfer line temperature is 280 °C; Quadrupole temperature is 150 °C; The characteristic ion pairs and collision energies of 4 PAHs and internal standards are shown in Table 2.

[0039] Table 2 Characteristic ion pairs and collision energies of 4 PAHs and internal standards under multiple reaction detection (MRM) mode 2 Results and discussion 2.1 Selection of extraction solvent In this example, a special medical purpose formula food added with 5 μg / kg PAHs was used as the test sample, and the extraction effects of 3 extraction solvents, namely acetonitrile, isopropanol, and acetone, on 4 PAHs in the special medical purpose formula food were investigated. The results showed that when acetonitrile was used as the extraction solvent, the extraction efficiency of 4 PAHs was the highest, and the recovery rate was between 80.0% and 109.2% (see Figure 2 ). This may be because acetonitrile belongs to a high-polarity-index organic solvent with a high dipole moment. The π bond and linear structure of acetonitrile have a strong hydrophobic interaction with non-polar PAHs, making acetonitrile more selective, with better extraction efficiency and being more suitable for the extraction of PAHs. Based on the above results, acetonitrile was finally selected as the extraction solvent in this example.

[0040] 2.2 Optimization of DLLME extraction conditions 2.2.1 Selection of extractant In this example, a special medical food added with 5 μg / kg PAHs was used as the test sample, and the recovery rate of PAHs and chromatographic behavior were used as indicators to investigate DL- menthol-thymol, DL- menthol-undecanol, DL- menthol-decanoic acid, 3 different types of HNADES for the extraction effects of 4 PAHs in special medical foods. The results showed that all 3 HNADES had certain extraction effects on the target substances, DL- the extraction efficiency of menthol-decanoic acid was the lowest, about 60%; DL- menthol-thymol and DL- menthol-undecanol had higher extraction efficiencies, 84.8% - 89.3% and 84.6% - 96.4% respectively (see Figure 3 ). However, DL- when menthol-undecanol was used as the extractant, the chromatographic peaks of benzo[a]anthracene and chrysene were not sharp, with poor symmetry, and there were phenomena such as fronting peaks and split peaks, DL-When menthol - thymol is used as an extractant, it not only has good extraction ability, but also the chromatographic peaks of the target substances are sharp, with good symmetry and good chromatographic behavior, and each peak can achieve baseline separation. Therefore, in this example, DL- menthol - thymol is finally selected as the extraction solvent for the subsequent steps.

[0041] 2.2.2 Molar ratio composition of HNADES In this example, DL- The molar ratio of menthol - thymol is optimized. Five kinds of HNADES with different molar ratios (i.e., 3:1, 2:1, 1:1, 1:2, 1:3) are synthesized respectively to extract polycyclic aromatic hydrocarbons in special medical foods, and the extraction efficiency of 4 kinds of PAHs is investigated. The results show that the extraction recovery rates of HNADES with different molar ratios are all good (>60 %), and the extraction efficiency is the highest when the molar ratio is 1:1 (see Figure 4 ). In the process of the molar ratio from 3:1 to 1:3, the viscosity of HNADES gradually increases with the increase of the molar ratio of thymol, which is not conducive to the mass transfer of the target substances. Among them, when the molar ratio is 1:3, it is unstable and easy to solidify at room temperature. Therefore, in this example, DL- the molar ratio of menthol to thymol is finally selected as 1:1.

[0042] 2.2.3 Volume of phenol - based HNADES To determine the optimal dosage of the extractant, in this example, the extraction efficiencies at extraction volumes of 50 μL, 100 μL, 150 μL, 200 μL, and 250 μL are investigated. The results show that with the increase of the extractant dosage, the extraction efficiency shows a gradually increasing trend. When the extraction volume reaches 200 μL, the extraction efficiencies of 4 kinds of PAHs reach the highest, between 86.3% and 93.7%. Therefore, in this example, DL- the extraction volume of menthol and thymol is finally selected as 200 μL.

[0043] 2.2.4 Influence of ionic strength on extraction efficiency In this example, the extraction effects under seven different salt concentrations (0 wt.%, 5 wt.%, 10 wt.%, 15 wt.%, 20 wt.%, 25 wt.%, 30 wt.%) are compared. The research results show that the addition of salt significantly improves the extraction efficiency. With the increase of the sodium chloride concentration in the system, that is, the increase of the ionic strength, the response of the target analyte shows an upward trend and reaches the highest when the salt concentration is 10 wt.%. Therefore, considering the extraction efficiency and experimental phenomena comprehensively, the sodium chloride concentration is finally selected as 10 wt.%.

[0044] 2.2.5 Influence of the proportion of dispersant on extraction efficiency In this example, by adding different volumes of brine (4, 5, 6, 8, 10 mL), the proportion of the dispersant acetonitrile in the DLLME system was changed to explore the influence law of this factor on the extraction effect. The experimental results showed that as the volume of the added brine increased, the recoveries of the 4 PAHs gradually increased. When the volume was 8 mL, the recoveries of the 4 PAHs reached the peak value, which was 81.6% - 88.7%. At this time, the proportion of the dispersant acetonitrile to water in the DLLME system reached an optimized balance point, making the extraction efficiency of the system for the 4 PAHs reach the best state. Therefore, the volume of the added brine was finally selected as 8 mL.

[0045] 2.3 Methodological parameters Isotope dilution mass spectrometry has the characteristics of high precision and good accuracy of detection results, and can effectively eliminate matrix interference brought by mass spectrometry signals and sample pretreatment processes. It has unique technical advantages in trace analysis. In this example, the isotope dilution internal standard method was used to determine polycyclic aromatic hydrocarbons in special medical foods, and methodological verification was carried out by evaluating the linear range, accuracy, precision, limit of quantification, and limit of detection of the method. A series of standard working solutions of 4 PAHs isotopes with mass concentrations of 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0 ng / mL and an internal standard with a mass concentration of 5.0 ng / mL were determined according to the instrument analysis conditions in 1.4. Taking the mass concentration as the abscissa (x, ng / mL) and the ratio of the peak areas of the 4 PAHs to the internal standard as the ordinate (y), a standard curve was plotted to obtain a linear equation. The total ion chromatogram of the standard working solution is shown in Figure 7 At the same time, different levels of PAHs were added to blank samples of special medical foods. The added level corresponding to a signal-to-noise ratio (S / N) ≥ 3 was taken as the limit of detection (LODs), and the added level corresponding to S / N ≥ 10 was taken as the limit of quantification (LOQs).

[0046] The results of the method verification showed that the 4 PAHs had a good linear relationship in the range of 0.2 - 20.0 ng / mL, and the correlation coefficients were all greater than 0.99. The LODs of the 4 PAHs were 0.1 μg / kg, and the LOQs were 0.25 μg / kg. Blank matrices of special medical foods were taken, and low, medium, and high levels of the target analytes were added respectively. Each spiked level was repeated 6 times for experiments. According to the sample pretreatment method in 1.3, the recoveries and relative standard deviations (RSDs) were calculated. The average recoveries of the 4 PAHs were 96.6% - 113.2%, and the RSDs were 2.4% - 8.3%, indicating that the method had good accuracy and precision. The relevant data are shown in Table 3.

[0047] Table 3 Results of precision and recovery experiments (n = 6) 3 Analysis of actual samples Using the established method, 29 batches of commercially available special medical samples were detected. The results showed that 5 batches of samples were detected with benzo[a]anthracene, and the detection rate was 17.2%, and the detected values were respectively in the range of 0.29 - 0.38 μg / kg; 10 batches were detected with chrysene, and the detection rate was 34.5%, and the detected values were respectively in the range of 0.26 - 0.73 μg / kg; neither benzo[a]pyrene nor benzo[b]fluoranthene was detected, and the detected values of the sum of the 4 polycyclic aromatic hydrocarbons were in the range of 0.26 - 1.11 μg / kg. It can be seen from the detection results that the polycyclic aromatic hydrocarbons with higher detection frequencies in special medical samples are benzo[a]anthracene and chrysene, and their detected values are all lower than the EU limit value of 1.0 μg / kg for special medical foods.

[0048] Example 2 In this example, another method for detecting polycyclic aromatic hydrocarbons based on deep eutectic solvent extraction is provided. The preparation method of the deep eutectic solvent is as follows: Mix DL-menthol and thymol with a molar ratio of 1:1, and stir and heat at 55 °C until a transparent and homogeneous solution is obtained for standby; The steps of the detection method are as follows: (1) Accurately weigh 1 g (accurate to 0.0001 g) of the special medical sample into a 15 mL centrifuge tube, add 4 mL of ultrapure water thereto, and vortex for 2 min to dissolve the sample. Subsequently, add 4 mL of acetonitrile and 0.8 g of sodium chloride, vortex for 2 min for extraction, centrifuge at 8000 rpm for 5 min, and transfer the acetonitrile layer to a 15 mL centrifuge tube for standby.

[0049] (2) Add 7 mL of 12 wt.% brine and 250 μL of HNADES with a molar ratio of 1:1 of DL-menthol to thymol to the above acetonitrile extract, and vortex for 2 min. Centrifuge at a speed of 8000 rpm for 2 min, take the upper organic phase, blow it to dry with nitrogen, and then dilute it to 0.5 mL with acetonitrile for GC-MS / MS analysis; (3) Detect by gas chromatography-triple quadrupole mass spectrometry method, and the specific parameters are the same as those in "1.4 Analysis conditions" in Example 1.

[0050] Example 3 In this example, another method for detecting polycyclic aromatic hydrocarbons based on deep eutectic solvent extraction is provided. The preparation method of the deep eutectic solvent is as follows: Mix DL-menthol and thymol with a molar ratio of 1:1, and stir and heat at 65 °C until a transparent and homogeneous solution is obtained for standby; The steps of the detection method are as follows: (1) Accurately weigh 1 g (accurate to 0.0001 g) of the special medical sample into a 15 mL centrifuge tube, add 6 mL of ultrapure water thereto, and vortex for 2 min to dissolve the sample. Subsequently, add 6 mL of acetonitrile and 1.2 g of sodium chloride, vortex for 2 min for extraction, centrifuge at 8000 rpm for 5 min, and transfer the acetonitrile layer to a 15 mL centrifuge tube for standby.

[0051] (2) Add 9 mL of 8 wt.% brine and 150 μL of HNADES with a molar ratio of DL-menthol to thymol of 1:1 to the above acetonitrile extract, and vortex for 2 min. Centrifuge at a speed of 8000 rpm for 2 min, take the upper organic phase, blow it with nitrogen, and then dilute it to 0.5 mL with acetonitrile for GC-MS / MS analysis; (3) Detect by gas chromatography-triple quadrupole mass spectrometry method, and the specific parameters are the same as those in "1.4 Analysis Conditions" in Example 1.

[0052] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for detecting polycyclic aromatic hydrocarbons based on deep eutectic solvent extraction, characterized in that: The polycyclic aromatic hydrocarbons are benzo[a]anthracene, chrysene, benzo[b]fluoranthene and benzo[a]pyrene; the detection method uses a deep eutectic solvent to extract the polycyclic aromatic hydrocarbons in the sample to be tested, and the deep eutectic solvent is prepared as follows: a molar ratio of 3:1, 2:1, 1:1, 1:2 or 1:3 is added to DL -Menthol and thymol are mixed and heated at 55-65°C with stirring until a transparent uniform solution is obtained; The detection method steps are as follows: (1) Add water, acetonitrile and salt to the sample to be tested, and retain the acetonitrile part after sufficient extraction. The dosage ratio of the sample to be tested, water, acetonitrile and salt is 1g:4~6mL:4~6mL:0.8~1.2g; (2) adding saline and the deep eutectic solvent to the acetonitrile portion in step (1) and mixing thoroughly, retaining the organic phase and blowing it dry, then adding acetonitrile to the fixed volume; (3) Detection was performed by gas chromatography-triple quadrupole mass spectrometry.

2. The method for detecting polycyclic aromatic hydrocarbons based on deep eutectic solvent extraction according to claim 1, characterized in that: Said DL- The molar ratio of menthol to thymol is 1:

1.

3. The polycyclic aromatic hydrocarbons detection method based on deep eutectic solvent extraction according to claim 1, characterized in that: In step (1): the sample to be tested is a food for special medical purposes.

4. The polycyclic aromatic hydrocarbons detection method based on deep eutectic solvent extraction as claimed in claim 1, characterized in that: The salt is sodium chloride.

5. The polycyclic aromatic hydrocarbons detection method based on deep eutectic solvent extraction according to claim 1, characterized in that: In step (2), the dosage ratio of the acetonitrile part, the brine and the deep eutectic solvent is 5 mL:7-9 mL:150-250 μL, and the concentration of the brine is 8-12 wt.%.

6. The polycyclic aromatic hydrocarbons detection method based on deep eutectic solvent extraction according to claim 1, characterized in that: In step (3): the detection method is a gas chromatography-triple quadrupole tandem mass spectrometry analysis method, and the specific parameters are as follows: Chromatographic conditions: Chromatographic column: DB-EUPAH capillary column, injection port temperature 280~320℃; carrier gas is high-purity inert gas; injection volume 1μL, splitless injection; solvent delay 16.5 min; program temperature rise: initial temperature 80℃, hold for 2 min, increase to 250℃ at 8~12℃ / min, hold for 2 min, increase to 315℃ at 7~9℃ / min, hold for 5 min, increase to 320℃ at 18~22℃ / min, hold for 5 min; Mass spectrometry conditions: The ionization method was electron bombardment source with ionization energy of 70 eV; the measurement method was multiple reaction monitoring mode; the ion source temperature was 320°C; the transfer line temperature was 280°C; and the quadrupole temperature was 150°C.

Images