A method for detecting polycyclic aromatic hydrocarbons based on deep eutectic solvent extraction

Abstract

The application specifically relates to a polycyclic aromatic hydrocarbon detection method based on deep eutectic solvent extraction. DL The application provides a deep eutectic solvent composed of menthol and thymol, and provides application of the deep eutectic solvent as an extractant in the field of detection of four polycyclic aromatic hydrocarbons. The main innovation point of the detection method provided by the application lies in establishment of a sample pretreatment method. In the detection method finally provided by the application, the reagents mainly include acetonitrile, salt, menthol and thymol, the components are non-toxic and economical in cost, and high-sensitivity, high-accuracy, green and environment-friendly determination of trace and ultratrace PAH4 in special medical food is achieved.

Description

Technical Field

[0001] This invention relates to the field of deep eutectic solvent extraction technology, specifically to the preparation of a phenolic nonionic hydrophobic natural deep eutectic solvent and its application in the detection of polycyclic aromatic hydrocarbons (PAHs) using liquid-liquid microextraction technology. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Polycyclic aromatic hydrocarbons (PAHs) are among the most widely distributed environmental and food processing contaminants. Numerous studies have shown that frequent exposure increases the risk of cardiovascular diseases such as thrombosis and myocardial infarction, and they are highly carcinogenic, mutagenic, and teratogenic. The International Agency for Research on Cancer (IARC) classifies benzo[a]pyrene as a Group 1 carcinogen, and benzo[a]anthracene, benzo[b]fluoranthracene, and benzo[b]fluoranthracene as Group 2 carcinogens. The European Food Safety Authority (EFSA) believes that analyzing only benzo[a]pyrene is insufficient to reflect all genotoxic and carcinogenic PAHs present in food, and recommends assessing the sum of the four PAHs mentioned above—benzo[a]pyrene, benzo[a]anthracene, benzo[b]fluoranthracene, and benzo[a]fluoranthracene—as a marker for PAHs.

[0004] Foods for Special Medical Purposes (FSMP) are specially formulated foods designed to meet the specific nutritional or dietary needs of individuals with specific disease states. They are a key focus of regulatory attention both domestically and internationally. However, vegetable oils in their raw materials and ingredients are susceptible to PAH contamination, and heat treatment during production and processing can also generate polycyclic aromatic hydrocarbons (PAHs). The EU's new Regulation (EU) 2023 / 915 on the Limits of Contaminants in Foods for Special Medical Purposes stipulates that benzo[a]pyrene in FSMP cannot exceed 1.0 μg / kg, and the total amount of the four PAH markers (PAH4) cannot exceed 1.0 μg / kg. This maximum limit applies only to ready-to-eat products. my country's GB 2762—2022, "National Food Safety Standard: Limits of Contaminants in Foods," has not yet set limits for PAHs in FSMP. In recent years, the detection of PAHs in infant formula has been a global focus of food safety concerns, but research on PAHs in FSMP, which is also a special food category, is scarce. Special medical purpose foods have complex matrices and low PAH content, thus requiring improved enrichment efficiency and minimized matrix interference to enhance detection sensitivity. Pretreatment techniques for these matrices typically involve fat extraction, saponification, and solid-phase extraction column purification. However, these methods are cumbersome, have low detection efficiency, and consume large amounts of solvent, limiting their application in high-efficiency analysis. Therefore, developing more efficient and environmentally friendly pretreatment techniques has become an important research direction in the analysis of polycyclic aromatic hydrocarbons (PAHs) in special medical purpose foods.

[0005] Considering the analytical principles of green chemistry, dispersive liquid-liquid microextraction (DLLME) technology based on deep eutectic solvents has made significant progress in the analysis of polycyclic aromatic hydrocarbons (PAHs) in recent years. Deep eutectic solvents are mainly formed by two substances: a hydrogen bond donor and a hydrogen bond acceptor. Under given temperature and molar ratio, they co-configure to form a novel solvent. This deep eutectic solvent is simple to prepare, has low toxicity, and good biodegradability. The method is simple to operate, extracts solvent in microliters, has a high pre-enrichment coefficient, is rapid, low-cost, and more environmentally friendly. Deep eutectic solvents can be classified into five types according to their composition. Types I-IV deep eutectic solvents are generally ionic deep eutectic solvents, which are essentially hydrophilic. Type V deep eutectic solvents are nonionic substances composed of phenolic hydrogen bonds. Proposed by Abranches et al., this novel deep eutectic solvent is hydrophobic and has low viscosity and density, making it more suitable for gas chromatography-mass spectrometry (GC-MS) analysis.

[0006] The inventors believe that current applications of deep eutectic solvents are mainly used for 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, making accurate detection much more difficult. 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. Furthermore, the detection of PAHs mainly relies on gas chromatography-mass spectrometry (GC-MS), while the type I-IV deep eutectic solvents reported in existing technologies are essentially hydrophilic reagents and are unsuitable for GC-MS analysis. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention aims to establish a liquid-liquid microextraction technique based on V-type deep eutectic solvents, combined with gas chromatography-triple quadrupole tandem mass spectrometry (GC-MS / MS), which offers high selectivity, high sensitivity, and strong anti-interference capabilities. This enables highly sensitive, accurate, and environmentally friendly determination of trace and ultra-trace PAH4 in foods formulated for special medical purposes. Combining V-type deep eutectic solvents with liquid-liquid microextraction technology allows for the detection of trace PAHs in complex matrices, offering advantages such as high enrichment efficiency and environmental friendliness.

[0008] Based on the above-mentioned technical effects, the present invention provides the following solution:

[0009] This invention provides a method for detecting polycyclic aromatic hydrocarbons (PAHs) based on deep eutectic solvent extraction. The main feature of this method is the use of a deep eutectic solvent to extract PAHs from the sample. The deep eutectic solvent is composed of… DL - Composed of menthol and thymol.

[0010] The deep eutectic solvent is mainly used in the liquid-liquid extraction process of polycyclic aromatic hydrocarbon pretreatment, wherein... 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 solvent, influencing the hydrogen bonds and π-π interactions between menthol and PAHs, and thus the solubility of PAHs in the solvent. DL- The higher the proportion of menthol, the stronger the hydrogen bonding between it and thymol. The higher the proportion of thymol, the stronger the π-π interaction between its aromatic ring and PAHs. Its phenolic properties can improve the solubility of PAHs as aromatic compounds.

[0011] In a preferred embodiment of the verification effect of the present invention, the following... DL- The molar ratio of menthol to thymol is 3:1, 2:1, 1:1, 1:2, or 1:3, with 1:1 being the most effective. The deep eutectic solvent is prepared as follows: [The appropriate molar ratio of...] DL-Menthol and thymol were mixed and heated at 55-65°C with stirring until a clear and homogeneous solution was obtained.

[0012] Furthermore, the steps of the above detection method are as follows:

[0013] (1) Add water, acetonitrile and salt to the sample to be tested, extract fully and retain the acetonitrile portion;

[0014] (2) Add salt water and the deep eutectic solvent to the acetonitrile portion in step (1) and mix thoroughly. After drying the organic phase portion, add acetonitrile again to make up the volume.

[0015] (3) Detection was performed by gas chromatography-triple quadrupole mass spectrometry.

[0016] The above detection method also has the following preferred technical solutions:

[0017] In step (1):

[0018] The test sample, applicable to the above detection method, includes solid and liquid samples. Based on common polycyclic aromatic hydrocarbon (PAH) detection targets in the field, the above detection method is further applicable to food or beverage samples, especially those with complex components. In one embodiment verified by this invention, the test target is food, specifically, a food for special medical purposes (hereinafter referred to as medical food). Medical foods have complex production processes and are rich in fats, proteins, and other components; PAHs often coexist with fatty compounds. Therefore, how to efficiently extract PAHs from medical foods has become a critical problem to be solved. Soxhlet extraction is a classic method for extracting PAHs from food, which, while effective, is time-consuming and inefficient. In recent years, various efficient extraction methods have emerged and been applied, such as ultrasonic extraction, pressurized liquid extraction, and the QuEChERS method. Among them, the QuEChERS method has advantages such as green energy saving, environmental friendliness, and high efficiency, and is widely used in the pretreatment of PAHs in food.

[0019] In the detection method provided by this invention, the extraction solvent of the QuEChERS method serves as the dispersant in the subsequent dispersible liquid-liquid microextraction (DLLME) system. Its selection is crucial, requiring both strong extraction and dispersing capabilities. In the DLLME system, the volume percentage of the dispersant is a key factor affecting extraction efficiency, with a complex and multifaceted mechanism of action that significantly impacts the recovery rate of the target analyte. This invention has verified that acetonitrile, as the extraction solvent, exhibits excellent recovery performance for four PAHs (benzo[a]anthracene, benzo[b]fluoranthracene, and benzo[a]pyrene). While acetonitrile and water are typically miscible in any proportion, this invention also adds salt to the aqueous solution of the sample to achieve acetonitrile separation, causing the acetonitrile and salt water to separate into layers.

[0020] Furthermore, the salt is preferably a salt with high solubility in water, such as sodium chloride. In a more effective embodiment, the dosage ratio of the test sample, water, acetonitrile, and salt is 1g:4~6mL:4~6mL:0.8~1.2g.

[0021] In the DLLME process, 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 fine droplets generated, and the number of fine droplets is closely related to the mass transfer process of the target analyte from the aqueous phase to the organic phase, thus significantly affecting the extraction efficiency. Furthermore, a dilution effect occurs when the extraction volume is too large. Ionic strength is another important parameter affecting the extraction efficiency in the DLLME process. In the DLLME partitioning system, salt significantly alters the partitioning behavior of the analyte between the extraction and aqueous phases, affecting its recovery rate. As the ionic strength increases, the density and viscosity of the aqueous solution tend to increase, which reduces the mass transfer efficiency of the target analyte. This invention has verified that as the salt concentration increases, the volume of the upper organic phase gradually increases, resulting in more acetonitrile dissolving into the HNADES phase. When the salt concentration is 15%-30%, the solution after nitrogen blowing and resolution exhibits turbidity, and a small amount of salt precipitates. Therefore, considering both extraction efficiency and experimental phenomena, a salt concentration of 10% is preferred.

[0022] In step (2):

[0023] Acetonitrile is used as the extraction solvent in the extraction process and also acts as a dispersant in the DLLME system. Therefore, the preferred dosage ratio of the acetonitrile fraction, brine and deep eutectic solvent is 5 mL: 7~9 mL: 150~250 μL, and the concentration of the brine is 8~12 wt.%.

[0024] During the process of adding acetonitrile to make up the volume after drying, the amount of acetonitrile added should be sufficient to fully dissolve the target analyte and reach the detection limit of the subsequent gas chromatography-mass spectrometry detection method. This is a technical content that can be routinely determined by those skilled in the art.

[0025] In step (3):

[0026] The gas chromatography detection methods applicable to this 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), and fast gas chromatography (Fast GC). In one embodiment verified by this invention, the detection method is gas chromatography-triple quadrupole tandem mass spectrometry (GC-MS / MS), with specific parameters as follows:

[0027] Chromatographic conditions:

[0028] Chromatographic column: DB-EUPAH capillary column, injection port temperature 280~320℃; carrier gas: high-purity inert gas; injection volume: 1μL, splitless injection; solvent delay: 16.5 min. Temperature program: 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.

[0029] Mass spectrometry conditions:

[0030] The ionization method was electron impact source (EI), with an ionization energy of 70 eV; the measurement method was multiple reaction monitoring (MRM); the ion source temperature was 320℃; the transfer line temperature was 280℃; and the quadrupole temperature was 150℃.

[0031] In another embodiment of the present invention, the isotope internal standard method is used for detection. Therefore, the characteristic ion pairs and collision energies of the four PAHs and the internal standard are shown in Table 1:

[0032] Table 1. Characteristic ion pairs and collision energies of four PAHs and internal standards under multiple reaction detection (MRM) mode.

[0033]

[0034] Compared with the prior art, the beneficial effects of the present invention are:

[0035] 1. Medical foods are typically special nutritional supplements for people with specific diseases, making it crucial to ensure their safety. Furthermore, medical foods have complex compositions, leading to significant matrix effects and challenging detection. The method described in this invention has been validated for detecting four common harmful polycyclic aromatic hydrocarbons (PAHs) in medical foods, filling a technological gap in the quality control of these foods.

[0036] 2. This invention proposes a method based on DL-A phenolic nonionic hydrophobic natural deep eutectic solvent, composed of menthol and thymol, is used for liquid-liquid microextraction of four polycyclic aromatic hydrocarbons (PAHs) in food formulations for special medical purposes, providing an optimized pretreatment method. This deep eutectic solvent has low synthesis cost and is environmentally friendly, requiring only 5-6 mL of extractant volume. Traditional methods, such as GB 5009.265-2021 and EU EN 16619:2015, require 50-60 mL and 200-300 mL of extractant volume, respectively, for the determination of PAHs in infant formula. The dispersion-liquid microextraction technology used in this invention achieves high enrichment, reducing extractant consumption by more than 90%, avoiding the large solvent consumption of traditional pretreatment methods, overcoming problems such as target analyte loss and blank interference caused by large-volume solvent concentration, and avoiding multiple nitrogen blowing concentration processes, making it more environmentally friendly.

[0037] On the other hand, the optimized pretreatment method of this invention is simple and rapid, requiring only 20-30 minutes to analyze a single sample, while traditional methods such as GB 5009.265-2021 and EU EN 16619:2015 require 1.5-2 hours and 8-10 hours respectively to determine polycyclic aromatic hydrocarbons in infant formula milk powder. The optimized pretreatment process of this invention reduces the analysis time by more than one hour, overcoming the pain points of cumbersome and time-consuming pretreatment procedures in traditional methods, and improving work efficiency.

[0038] 3. Based on the above pretreatment method, this invention employs gas chromatography-triple quadrupole tandem mass spectrometry (GC-MS) in multiple reaction monitoring mode to establish a method for detecting polycyclic aromatic hydrocarbons (PAHs) based on deep eutectic solvent extraction-GC-MS. This method exhibits good linearity in the concentration range of 0.2–20 ng / mL, with a correlation coefficient r0. 2 The detection limit (LOD) is >0.99 μg / kg, the LOD is 0.1 μg / kg, and the LOD is 0.25 μg / kg. The recoveries at three different spiking levels ranged from 96.6% to 113.2%, with RSDs ranging from 2.4% to 8.3%. The LODs of the traditional methods GB 5009.265-2021 and EN 16619:2015 are 0.5 μg / kg and 0.9 μg / kg, respectively. Compared to the aforementioned standards, the method of this invention significantly improves the sensitivity and selectivity of detection and has advantages such as being environmentally friendly, accurate, and reliable.

[0039] In addition, this invention uses gas chromatography-triple quadrupole tandem mass spectrometry in multiple reaction monitoring mode, which has stronger selectivity and better specificity, and improves detection sensitivity by reducing background noise.

[0040] In summary, this invention has the advantages of being simple, rapid, environmentally friendly, having a low limit of quantitation and good specificity, and is suitable for the determination of trace and ultra-trace components in the matrix of special medical foods. It has broad application prospects in the determination of actual samples. Attached Figure Description

[0041] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0042] Figure 1 The chemical structure of the compound used to prepare the deep eutectic solvent as described in Example 1;

[0043] Figure 2 The histogram of the extraction solvent effect described in Example 1;

[0044] Figure 3 The histogram shows the extraction effects of four PAHs under different HNADES extraction conditions described in Example 1.

[0045] Figure 4 The histograms show the recovery rates of four polycyclic aromatic hydrocarbons under different molar ratios of HNADES extraction conditions as described in Example 1.

[0046] Figure 5 The histogram shows the recovery rates of four polycyclic aromatic hydrocarbons under different HNADES volume extraction conditions described in Example 1.

[0047] Figure 6 The histogram shows the effect of different dispersant volumes on the recovery rates of the four polycyclic aromatic hydrocarbons described in Example 1.

[0048] Figure 7 The total ion chromatogram is shown for the standard working solution described in Example 1. Detailed Implementation

[0049] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0050] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0051] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0052] Example 1

[0053] 1. Methods and Materials

[0054] 1.1 Instruments and Reagents

[0055] Gas chromatography-triple quadrupole tandem mass spectrometry (Agilent 8890-7010B, Agilent Technologies, USA), vortex mixer (IKA GmbH, Germany), centrifuge (Sigma GmbH, Germany), analytical balance (accuracy 0.0001 g, Sartorius Balance Co., Ltd., Beijing), ultrasonic cleaner - KQ-800DE (Kunshan Ultrasonic Instrument Co., Ltd.).

[0056] Acetonitrile (chromatographic grade, Merck AG, Germany); Sodium chloride (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); DL - Menthol, thymol, undecyl alcohol, decanoic acid (analytical grade). A mixed standard solution of four polycyclic aromatic hydrocarbons (benzo[a]anthracene, β-carboxylic acid, benzo[b]fluoranthene, benzo[a]pyrene) (purity ≥99%, purchased from Beijing Manhag Biotechnology Co., Ltd.), and four internal standard standards: 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.).

[0057] 1.2 Preparation of Deep Eutectic Solvents

[0058] by DL - Menthol is the hydrogen bond acceptor, and thymol, undecanoic acid or decanoic acid is the hydrogen bond donor. Weigh appropriate amounts of hydrogen bond donor and hydrogen bond acceptor into glass bottles at a molar ratio of 1:1. Prepare nonionic hydrophobic natural deep eutectic solvent (HNADES) using a simple heating and stirring method, i.e., magnetically stir at 60 °C until a homogeneous, clear and transparent solution is formed for later use.

[0059] Similarly, five different molar ratios were synthesized according to molar ratios of 3:1, 2:1, 1:1, 1:2, and 1:3. DL -Menthol-thymol deep eutectic solvent.

[0060] 1.3 Pretreatment Methods

[0061] 1.3.1 Sample Extraction

[0062] Accurately weigh 1 g (accurate to 0.0001 g) of the special medical sample into a 15 mL centrifuge tube, add 5 mL of ultrapure water, and vortex for 2 min to dissolve the sample. Then, add 5 mL of acetonitrile and 1 g of sodium chloride, vortex for 2 min to extract, centrifuge at 8000 rpm for 5 min, and transfer the acetonitrile layer to a 15 mL centrifuge tube for later use.

[0063] 1.3.2 DLLME program

[0064] Add 8 mL of 10 wt.% saline and 200 μL of [unclear] to the above acetonitrile extract. DL- HNADES was prepared with a 1:1 molar ratio of menthol to thymol and vortexed for 2 min. After centrifugation at 8000 rpm for 2 min, the upper organic phase was collected, purged with nitrogen, and diluted to 0.5 mL with acetonitrile for GC-MS / MS analysis.

[0065] 1.4 Analysis Conditions

[0066] Chromatographic conditions: DB-EUPAH capillary column (20 m × 0.18 mm × 0.14 μm); injection port temperature 300℃; carrier gas: high-purity helium, purity ≥99.999%; injection volume 1 μL, splitless injection; solvent delay 16.5 min. Temperature program: 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.

[0067] Mass spectrometry conditions: ionization method: electron impact source (EI), ionization energy: 70 eV Measurement method: Multiple reaction monitoring (MRM); Ion source temperature 320℃; Transfer line temperature 280℃; Quadrupole temperature 150℃; Characteristic ion pairs and collision energies of the four PAHs and internal standard are shown in Table 2.

[0068] Table 2 Characteristic ion pairs and collision energies of four PAHs and internal standards under multiple reaction detection (MRM) mode.

[0069]

[0070] 2 Results and Discussion

[0071] 2.1 Selection of extraction solvent

[0072] In this embodiment, a special medical purpose food product containing 5 μg / kg PAHs was used as the test sample. The extraction efficiency of acetonitrile, isopropanol, and acetone as three extraction solvents for four PAHs in the special medical purpose food product was investigated. The results showed that acetonitrile had the highest extraction efficiency for the four PAHs when used as the extraction solvent, with recoveries ranging from 80.0% to 109.2% (see [link to sample]). Figure 2 This is likely because acetonitrile is a high polarity index organic solvent with a high dipole moment. Its π-bonds and linear structure have a strong hydrophobic interaction with nonpolar PAHs, resulting in higher selectivity, better extraction efficiency, and making it more suitable for PAH extraction. Based on the above results, acetonitrile was ultimately selected as the extraction solvent in this embodiment.

[0073] 2.2 Optimization of DLLME Extraction Conditions

[0074] 2.2.1 Selection of Extractant

[0075] In this embodiment, a special medical food containing 5 μg / kg PAHs was used as the test sample. The recovery rate and chromatographic behavior of PAHs were used as indicators to investigate the effects of PAHs on the chromatographic behavior of PAHs. DL- Menthol-thymol DL- Menthol-Undecyl alcohol DL- Extraction effects of three different types of HNADES (menthol-decanoic acid) on four PAHs in medical foods. The results showed that all three HNADES had a certain extraction effect on the target analytes. DL- The extraction efficiency of menthol-decanoic acid is the lowest, around 60%. DL- Menthol-thymol and DL- Menthol-undecanol had higher extraction efficiencies, ranging from 84.8% to 89.3% and from 84.6% to 96.4%, respectively (see...). Figure 3 ).but DL- When menthol-undecyl alcohol is used as the extractant, the chromatographic peaks of benzo[a]anthracene and β are not sharp, have poor symmetry, and exhibit phenomena such as leading peaks and bifurcated peaks. DL- Menthol-thymol, when used as an extractant, not only exhibits excellent extraction ability but also produces sharp, symmetrical peaks of the target analyte, demonstrating good chromatographic behavior and achieving baseline separation. Therefore, this embodiment ultimately selected... DL- Menthol-thymol was used as the subsequent extraction solvent.

[0076] 2.2.2 Molar composition of HNADES

[0077] This embodiment is... DL-The molar ratio of menthol to thymol was optimized, and HNADES with five different molar ratios (3:1, 2:1, 1:1, 1:2, and 1:3) were synthesized for the extraction of polycyclic aromatic hydrocarbons (PAHs) from foods for special medical use. The extraction efficiency of four PAHs was investigated. The results showed that the extraction recoveries of HNADES with different molar ratios were all good (>60%), with the highest extraction efficiency achieved at a molar ratio of 1:1 (see [link to relevant documentation]). Figure 4 As the molar ratio decreased from 3:1 to 1:3, the viscosity of HNADES gradually increased with the increase of the thymol molar ratio, which was not conducive to the mass transfer of the target analyte. The 1:3 molar ratio was unstable and easily solidified at room temperature. Therefore, this embodiment ultimately selected... DL- The molar ratio of menthol to thymol is 1:1.

[0078] 2.2.3 Volume of phenolic HNADES

[0079] To determine the optimal extractant dosage, this example investigated the extraction efficiency at extraction volumes of 50 μL, 100 μL, 150 μL, 200 μL, and 250 μL. The results showed that the extraction efficiency gradually increased with increasing extractant dosage. The highest extraction efficiency for the four PAHs was achieved at an extraction volume of 200 μL, ranging from 86.3% to 93.7%. Therefore, this example ultimately selected... DL- The extraction volume of menthol and thymol was 200 μL.

[0080] 2.2.4 Effect of ionic strength on extraction efficiency

[0081] This embodiment compared the extraction effects at seven different salt concentrations (0 wt.%, 5 wt.%, 10 wt.%, 15 wt.%, 20 wt.%, 25 wt.%, and 30 wt.%). The results showed that the addition of salt significantly improved the extraction efficiency. As the sodium chloride concentration in the system increased, i.e., the ionic strength increased, the response of the target analyte showed an upward trend, reaching its peak at a salt concentration of 10 wt.%. Therefore, considering both the extraction efficiency and experimental phenomena, a sodium chloride concentration of 10 wt.% was ultimately selected.

[0082] 2.2.5 Effect of dispersant ratio on extraction efficiency

[0083] In this embodiment, the ratio of acetonitrile dispersant to water in the DLLME system was varied by adding different volumes of saline (4, 5, 6, 8, and 10 mL) to investigate the effect of this factor on the extraction efficiency. The experimental results showed that the recovery rates of the four PAHs gradually increased with increasing saline volume. The recovery rates of the four PAHs reached their peak at 8 mL, ranging from 81.6% to 88.7%. At this point, the ratio of acetonitrile to water in the DLLME system reached an optimized equilibrium, resulting in the best extraction efficiency for the four PAHs. Therefore, a saline volume of 8 mL was ultimately selected.

[0084] 2.3 Methodological Parameters

[0085] Isotope dilution mass spectrometry (IDMS) offers advantages such as high precision and accuracy, effectively eliminating matrix interference from mass spectrometry signals and pretreatment processes, making it uniquely advantageous in trace analysis. This embodiment employs the Isotope dilution internal standard method to determine polycyclic aromatic hydrocarbons (PAHs) in special medical foods. Methodological validation was conducted by evaluating the linear range, accuracy, precision, limit of quantitation, and limit of detection. Four PAH isotopes with mass concentrations of 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, and 20.0 ng / mL, along with a series of standard working solutions with an internal standard concentration of 5.0 ng / mL, were analyzed according to the instrumental analysis conditions described in section 1.4. A standard curve was plotted with mass concentration on the x-axis (x, ng / mL) and the ratio of the peak areas of the four PAHs to the internal standard (y) on the y-axis, yielding the linear equation. The total ion chromatogram of the standard working solutions is shown in [reference needed]. Figure 7 Meanwhile, different levels of PAHs were added to blank samples of special medical foods. The addition level corresponding to a signal-to-noise ratio (S / N) ≥ 3 was used as the limit of detection (LOD), and the addition level corresponding to an S / N ≥ 10 was used as the limit of quantification (LOQ).

[0086] Method validation results showed that the four PAHs exhibited good linearity in the range of 0.2–20.0 ng / mL, with correlation coefficients all greater than 0.99. The limits of detection (LODs) for the four PAHs were 0.1 μg / kg, and the limits of quantitation (LOQs) were 0.25 μg / kg. Blank medical food matrix was used, and three different levels of the analytes (low, medium, and high) were added. Each spiked level was repeated six times. The recovery rate and relative standard deviation (RSDs) were calculated according to the sample pretreatment method in section 1.3. The average recovery rate of the four PAHs was 96.6%–113.2%, and the RSD was 2.4%–8.3%, indicating good accuracy and precision of the method. Relevant data are shown in Table 3.

[0087] Table 3. Precision and recovery experiment results (n=6)

[0088]

[0089] 3. Analysis of actual samples

[0090] Using the established method, 29 batches of commercially available medical food samples were tested. The results showed that benzo[a]anthracene was detected in 5 batches of samples, with a detection rate of 17.2% and detected values ​​ranging from 0.29 to 0.38 μg / kg; benzo[a]pyrene was detected in 10 batches, with a detection rate of 34.5% and detected values ​​ranging from 0.26 to 0.73 μg / kg; benzo[a]pyrene and benzo[b]fluoranthene were not detected. The total detected values ​​of the four polycyclic aromatic hydrocarbons ranged from 0.26 to 1.11 μg / kg. The test results indicate that benzo[a]anthracene and benzo[b]fluoranthene were the most frequently detected polycyclic aromatic hydrocarbons in the medical food samples, and their detected values ​​were all below the EU limit of 1.0 μg / kg for medical foods.

[0091] Example 2

[0092] In this embodiment, another method for detecting polycyclic aromatic hydrocarbons based on deep eutectic solvent extraction is provided. The deep eutectic solvent is prepared as follows: DL-menthol and thymol are mixed in a molar ratio of 1:1 and then stirred and heated at 55°C until a transparent and homogeneous solution is obtained for later use. The detection method steps are as follows:

[0093] (1) Accurately weigh 1g (accurate to 0.0001g) of special medical sample into a 15 mL centrifuge tube, add 4 mL of ultrapure water, and vortex for 2 min to dissolve the sample. Then, add 4 mL of acetonitrile and 0.8g of sodium chloride, vortex for 2 min to extract, centrifuge at 8000 rpm for 5 min, and transfer the acetonitrile layer to a 15 mL centrifuge tube for later use.

[0094] (2) Add 7 mL of 12 wt.% saline and 250 μL of HNADES (DL-menthol to thymol in a molar ratio of 1:1) to the above acetonitrile extract, and vortex for 2 min. Centrifuge at 8000 rpm for 2 min, take the upper organic phase, blow it with nitrogen, and make up the volume to 0.5 mL with acetonitrile for GC-MS / MS analysis;

[0095] (3) The detection was performed by gas chromatography-triple quadrupole mass spectrometry, with the same parameters as in “1.4 Analytical conditions” in Example 1.

[0096] Example 3

[0097] In this embodiment, another method for detecting polycyclic aromatic hydrocarbons based on deep eutectic solvent extraction is provided. The deep eutectic solvent is prepared as follows: DL-menthol and thymol are mixed in a molar ratio of 1:1 and then stirred and heated at 65°C until a transparent and homogeneous solution is obtained for later use. The detection method steps are as follows:

[0098] (1) Accurately weigh 1g (accurate to 0.0001g) of special medical sample into a 15 mL centrifuge tube, add 6 mL of ultrapure water, and vortex for 2 min to dissolve the sample. Then, add 6 mL of acetonitrile and 1.2g of sodium chloride, vortex for 2 min to extract, centrifuge at 8000 rpm for 5 min, and transfer the acetonitrile layer to a 15 mL centrifuge tube for later use.

[0099] (2) Add 9 mL of 8wt.% saline and 150 μL of HNADES (DL-menthol to thymol molar ratio of 1:1) to the above acetonitrile extract, and vortex for 2 min. Centrifuge at 8000 rpm for 2 min, take the upper organic phase, blow it with nitrogen, and make up the volume to 0.5 mL with acetonitrile for GC-MS / MS analysis;

[0100] (3) The detection was performed by gas chromatography-triple quadrupole mass spectrometry, with the same parameters as in “1.4 Analytical conditions” in Example 1.

[0101] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting polycyclic aromatic hydrocarbons based on deep eutectic solvent extraction, characterized by, The polycyclic aromatic hydrocarbons are benzo[a]anthracene, chrysene, benzo[b]fluoranthene and benzo[a]pyrene; the detection method extracts the polycyclic aromatic hydrocarbons in the sample to be measured by using a deep eutectic solvent, and the deep eutectic solvent is prepared in the following manner: 1:1 molar ratio of DL - After mixing menthol and thymol, heating and stirring at 55~65℃ until a transparent and uniform solution is obtained, it is obtained; The sample to be tested is a special medical use formula food; The detection method is as follows: (1) The sample to be tested is added with water, acetonitrile and sodium chloride, and after being fully extracted, the acetonitrile part is reserved, and the dosage ratio of the sample to be tested, water, acetonitrile and salt is 1g:4-6mL:4-6mL:0.8-1.2g; (2) The acetonitrile part in step (1) is added with salt water and the deep eutectic solvent and fully mixed, and the organic phase part is dried, then acetonitrile is added for constant volume; the dosage ratio of the acetonitrile part, salt water and deep eutectic solvent is 5mL:7-9mL:150-250μL, and the concentration of the salt water is 8-12wt.%; (3) The detection is 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, wherein, In step (3), the detection method is gas chromatography-triple quadrupole 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, no split injection; solvent delay 16.5min; programmed temperature: initial temperature 80℃, holding for 2min, increasing to 250℃ at 8-12℃ / min, holding for 2min, increasing to 315℃ at 7-9℃ / min, holding for 5min, increasing to 320℃ at 18-22℃ / min, holding for 5min; Mass spectrometry conditions: Ionization mode is electron impact source, ionization energy 70eV; determination mode: multiple reaction monitoring mode; ion source temperature 320℃; transmission line temperature 280℃; quadrupole temperature 150℃.

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