A method for accurately and rapidly detecting a plurality of perfluoroalkyl and polyfluoroalkyl substances in honey

Abstract

This invention belongs to the field of food testing and analytical chemistry, specifically relating to a method for the accurate and rapid detection of multiple perfluorinated and polyfluoroalkyl substances (PFAS) in honey based on an improved QuEChERS pretreatment technique coupled with liquid chromatography-tandem mass spectrometry (LC-MS / MS). The method involves adding an internal standard to the honey sample, followed by dissolution and extraction with water, acetonitrile, and formic acid. After shaking, salting out, and centrifugation, the supernatant is purified using a composite purification material. The supernatant is then collected again, dried, and the resulting residue is reconstituted. The content of multiple PFAS in the honey sample is determined using the internal standard method with high-performance liquid chromatography-triple quadrupole mass spectrometry. This invention overcomes the problems of significant interference from the honey matrix in PFAS detection, low recovery rate, and poor result stability in existing technologies. It can accommodate the extraction and detection needs of PFAS with different chain lengths and structures, achieving simultaneous and stable analysis of multiple PFAS, and is rapid and accurate.

Description

Technical Field

[0001] This invention relates to the field of testing and analytical chemistry, specifically to a method for the accurate and rapid detection of multiple perfluorinated and polyfluoroalkyl substances in honey based on an improved QuEChERS pretreatment technique coupled with liquid chromatography-tandem mass spectrometry (LC-MS / MS). Background Technology

[0002] Honey is a typical natural food matrix, primarily composed of high levels of monosaccharides and oligosaccharides, along with small amounts of protein, organic acids, and trace active substances. Overall, it exhibits high sugar content, high viscosity, and a complex matrix composition. These characteristics make honey prone to significant matrix effects during sample pretreatment and instrumental analysis, severely impacting the extraction efficiency and detection accuracy of target contaminants.

[0003] Per- and perfluorinated alkyl substances (PFAS) are a class of novel organic pollutants characterized by stable structures, high polarity, and a wide range of carbon chain lengths, typically ranging from C4 to C18. Because PFAS molecules readily interact non-specifically with sugars and proteins in honey, conventional sample pretreatment conditions often result in low extraction recoveries, poor reproducibility, and unstable quantitative results. Existing general-purpose QuEChERS methods are mostly designed for matrices such as fruits, vegetables, and grains, making them difficult to apply directly to the complex, high-sugar, and high-viscosity matrix of honey, and thus failing to meet the requirements for trace and simultaneous detection of multiple PFAS in honey. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an accurate and rapid method for detecting multiple perfluorinated and polyfluoroalkyl substances in honey, which addresses the shortcomings of the existing technology. By effectively purifying the complex honey matrix and reducing the matrix effect caused by co-extractants on the detection system, the method combines high performance liquid chromatography-triple quadrupole mass spectrometry to effectively improve the peak shape, resolution and response stability of multiple PFAS, enabling the simultaneous, rapid and accurate detection of multiple PFAS in honey.

[0005] The technical solution adopted by the present invention to solve the above-mentioned problems is as follows: A method for accurately and rapidly detecting multiple PFAS in honey includes the following steps: (1) Add internal standard to the honey sample to be tested, let it stand, add an appropriate amount of water to dissolve the honey, then add acetonitrile and formic acid for extraction, and then take the supernatant (first supernatant) after shaking, salting out and centrifugation. (2) The supernatant obtained in step (1) is purified by composite purification material, then centrifuged to obtain the supernatant (second supernatant), dried with nitrogen and then reconstituted with methanol solution to obtain the sample loading solution of the honey sample to be tested. (3) Prepare a mixed standard solution of various PFAS, add an internal standard, and use it as a standard sample solution; (4) The standard sample solution in step (3) was analyzed using a high performance liquid chromatography-triple quadrupole mass spectrometer. After separation by high performance liquid chromatography, data was collected in the multiple reaction monitoring mode of mass spectrometry to obtain the MRM chromatogram. Then, the peak area ratio of each PFAS standard to the corresponding internal standard in the standard sample solution was used as the ordinate, and the concentration ratio of each PFAS standard to the internal standard was used as the abscissa to obtain the standard curve of each PFAS. (5) Under the same detection conditions as in step (4), the sample obtained in step (2) is analyzed by high performance liquid chromatography-triple quadrupole mass spectrometry to obtain the MRM spectrum. Then, combined with the standard curve of the corresponding PFAS in step (4), the concentration of each PFAS in the sample is obtained, and the content of each PFAS in the honey sample to be tested can be calculated.

[0006] According to the above scheme, the various PFAS mentioned in this invention include perfluorobutyric acid (PFBA), perfluorovalerate (PFPA), perfluorohexanoic acid (PFHxA), perfluorobutanesulfonic acid (PFBS), perfluoroheptanoic acid (PFHpA), and perfluoropentanesulfonic acid (PFPeS). 3H-Perfluoro-4,8-dioxonanoic acid (ADONA), perfluorooctanoic acid (PFOA), perfluorohexanesulfonic acid (PFHxS), perfluorononanoic acid (PFNA), perfluoroheptanesulfonic acid (PFHpS), perfluorodecanoic acid (PFDA), perfluorooctanesulfonic acid (PFOS), perfluoroundecanoic acid (PFUnDA), perfluorononanesulfonic acid (PFNS), 9-chlorohexadecano-3-oxanonane-1-sulfonic acid (9Cl-PF3ONS), perfluorododecanic acid (PFDoA), perfluorodecanesulfonic acid (PFDS), perfluorotridecanoic acid (PFTriDA), 11-chloroeicosicofluoro-3-oxaundecane-1-sulfonic acid (11Cl-PF3OUdS), perfluorotetradecanoic acid (PFTeDA), perfluorohexadecanoic acid (PFHxDA), perfluorooctadecanic acid (PFODA).

[0007] According to the above scheme, the internal standards include ¹³C4(MPFBA), ¹³C5(M5PFPeA), ¹³C5(M5PFHxA), ¹³C4(M4PFHpA), ¹³C8(MPFOA), ¹³C9(M9PFNA), ¹³C6(M6PFDA), ¹³C7(M7PFUdA), ¹³C2(MPFDoA), ¹³C2(M2PFTeDA), ¹³C3(M3PFBS), ¹³C3(M3PFHxS), and ¹³C8(M8PFOS).

[0008] According to the above scheme, the amount of each internal standard added to the honey in step (1) is calculated as 1~1.5 ng of internal standard per gram of honey.

[0009] According to the above scheme, in step (1), the ratio of honey to each extraction reagent is honey:water:acetonitrile:formic acid = 1 g:(2~4) mL:(4~6) mL:(40~60) μL; magnesium sulfate and sodium chloride are used for salting out, and the mass ratio of honey:magnesium sulfate:sodium chloride is 1:(1.5~2.5):(0.4~0.6).

[0010] According to the above scheme, the composite purification material in step (2) is composed of magnesium sulfate (MgSO4), octadecyl bonded silica gel (C18), graphitized carbon black (GCB), and N-propylethylenediamine (PSA). Further, the ratio of the first supernatant to the composite purification material is 1 mL: (200-250) mg; the composite purification material, by mass fraction, consists of 80-100 parts MgSO4, 20-40 parts C18, 10-20 parts PSA, and 1-2 parts GCB. This invention employs a composite purification material synergistically composed of PSA, C18, GCB, and MgSO4 in the purification step to achieve the graded removal of interfering components in the complex matrix of honey, thereby effectively reducing the matrix effect. Specifically, PSA effectively adsorbs sugars, organic acids, and some polar impurities in the sample, reducing interference from high-sugar matrices on the detection of target analytes; C18 is mainly used to remove hydrophobic lipids and non-polar impurities from the sample, reducing the impact of non-polar co-extractants on instrument response; GCB has a strong adsorption capacity for pigments and some planar interferences, further improving sample purification and chromatographic baseline stability. These composite purification materials, through targeted removal of different types of matrix interferences, achieve effective purification of complex honey matrices, reducing the ion inhibition or enhancement effects caused by co-extractants entering the detection system, thereby improving the accuracy, stability, and repeatability of PFAS detection.

[0011] According to the above scheme, in step (2), the methanol content of the methanol solution is 70%~100%, and the remainder is water; the temperature of nitrogen blowing is 30~50 ℃.

[0012] According to the above scheme, in step (2), the standard sample solution is prepared with methanol as the solvent, the concentration of each PFAS standard is in the range of 0~20 ng / mL, and the concentration of each internal standard is in the range of 5~15 ng / mL.

[0013] According to the above scheme, the analytical conditions for high performance liquid chromatography in steps (4) and (5) include: a. Mobile phase A: 5~10 mM ammonium acetate, Mobile phase B: Methanol; b. Analytical procedure: A gradient elution program was used, with a sample loading volume of 1~3 μL, a flow rate of 0.2~0.5 mL / min, and an analysis time of 8~15 min; c. Chromatographic column: ACQUITY UPLC BEH C18 reversed-phase column, column temperature: 35~40 ℃; d. A trapping column is installed before the high-performance liquid chromatography column to eliminate interference from the environmental matrix.

[0014] Furthermore, the gradient elution program for high performance liquid chromatography is as follows (by volume percentage): 0–0.3 min, 95% mobile phase A (balance is mobile phase B); 0.9–9 min, 5% mobile phase A; 9–12 min, 5% mobile phase A; 12.0–12.1 min, 95% mobile phase A; 12.1–14.0 min, 95% mobile phase A.

[0015] According to the above scheme, in steps (4) and (5), triple quadrupole mass spectrometry uses electron bombardment ionization, employs multiple reaction monitoring mode for detection, sets the ion source temperature to 120~150℃, selects PFAS bulk fragments as specific precursor ions for collision-induced dissociation, and uses negative ion mode for both the target analyte and the internal standard.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention addresses the matrix characteristics of honey, such as high sugar content, high viscosity, and complex composition, by specifically improving the traditional QuEChERS pretreatment method. Firstly, water is introduced during sample pretreatment to dilute the honey, fully dissolving it, reducing system viscosity, and improving mass transfer efficiency between the target analyte and the extraction solvent. Secondly, considering the structural characteristics of PFAS molecules, which possess both hydrophobic fluorocarbon chains and hydrophilic functional groups, an acetonitrile-water system is used as the extraction solvent. Combined with the QuEChERS salting-out effect, this promotes the distribution of PFAS from the aqueous phase to the organic phase, achieving effective separation of PFAS from the complex honey matrix and enabling efficient extraction of various perfluorinated and polyfluoroalkyl substances. Thirdly, based on the high content of polar interfering substances such as sugars in honey, a composite purification material is formed by rationally combining PSA, C18, and GCB during the purification process. This effectively removes different types of interfering substances, such as sugars, organic acids, lipids, and pigments, reducing matrix effects and ion inhibition effects, and improving detection stability and repeatability. This effectively overcomes the problems of low recovery rate and poor stability in the complex honey matrix of traditional pretreatment methods.

[0017] 2. While PFAS molecules generally possess hydrophobic fluorocarbon chain structures, long-chain PFAS exhibit stronger hydrophobicity, making them more readily absorbed into the organic phase. Conversely, some short-chain PFAS, due to their relatively weaker hydrophobicity, are more prone to loss or incomplete partitioning during extraction. This invention further incorporates an internal standard correction strategy, using isotopic internal standards to correct for potential losses during sample pretreatment and detection, thereby effectively improving the quantitative accuracy and method stability of short-chain PFAS. Based on this design, this invention can accommodate the extraction and detection needs of PFAS with different chain lengths and structures, achieving simultaneous applicability and stable analysis of various PFAS.

[0018] 3. Based on the above, this invention improves the matching of chromatographic and mass spectrometric conditions, giving full play to the high efficiency of liquid chromatography and the high sensitivity and high selectivity of tandem mass spectrometry, effectively improving the peak shape, resolution and response stability of multiple PFAS, and finally constructing an analytical method suitable for the simultaneous rapid and accurate detection of multiple PFAS in honey samples. Attached Figure Description

[0019] Figure 1 The MRM spectra of each PFAS standard in Example 1 at a concentration of 5 ppb are shown.

[0020] Figure 2 The images show the MRM spectra of the actual samples after spiking in Example 2.

[0021] Figure 3 Figure showing the spiked recovery results for different PSA usage amounts.

[0022] Figure 4Distribution map of measured ΣPFAS values ​​for honey samples from various provinces.

[0023] Figure 5 The PFAS composition spectrum of honey samples from various provinces is shown. Detailed Implementation

[0024] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the present invention is not limited to the following embodiments.

[0025] In the following examples, the high-performance liquid chromatography-triple quadrupole mass spectrometry system used was a XevoTQ-S micro, manufactured by Waters Corporation, USA. Specifically, the high-performance liquid chromatography used an ACQUITY UPLC BEH C18 reversed-phase column with a particle size of 1.7 µm, a pore size of 130 Å, a column size of 2.1 mm × 50 mm, a column temperature of 40 °C, a column flow rate of 0.3 mL / min, and a BEH C18 reversed-phase trapping column size of 2.1 × 50 mm. The mobile phase A was 5 mM ammonium acetate, and B was pure methanol. The gradient elution program was: 0–0.3 min, 95% A; 0.9–9 min, 5% A; 9–12 min, 5% A; 12.0–12.1 min, 95% A; 12.1–14.0 min, 95% A; and the injection volume was 2 μL.

[0026] Triple quadrupole mass spectrometry uses an electron impact ionization (EI) source and multiple reaction monitoring (MRM) mode. The ion source temperature is set to 150℃, and selected specific precursor ions are subjected to collision-induced dissociation. Mass spectrometry signals are acquired only for selected specific daughter ions. The MRM parameters are shown in Table 1.

[0027] Table 1

[0028] Example 1 A method for accurately and rapidly detecting multiple PFAS in honey includes the following steps: (1) A mixed standard solution of 13 PFAS internal standards was prepared using pure methanol, and the concentration of each internal standard was 100 ng / mL.

[0029] (2) Standard sample solutions were prepared using pure methanol. The concentration gradient of each PFAS standard was in the range of 0.05-20 ng / mL, and the concentration of each internal standard was 10 ng / mL.

[0030] (3) The honey samples to be tested (actual sample 1 and actual sample 2, respectively) were thoroughly mixed by gentle heating (temperature below 40℃, around 30℃) and stirring. 2 g was placed in a 50 mL PP centrifuge tube, and then 250 μL of the internal standard solution from step (1) was accurately added. The mixture was allowed to stand for 10 min, then 5 mL of ultrapure water was added and shaken for 5 min. Then 10 mL of acetonitrile and 100 μL of analytical grade formic acid were added and shaken for 2 min. 4 g of MgSO4 and 1 g of NaCl were added and shaken for 8 min. The mixture was then centrifuged at 4500 rpm for 5 min. 6 mL of the supernatant was accurately pipetted into a purification tube, and 900 mg of MgSO4, 300 mg of C18, 150 mg of PSA and 15 mg of GCB were added sequentially. The mixture was shaken for 5 min again and then centrifuged at 4500 rpm for 5 min. 4 mL of the supernatant was then transferred to a new 15 mL centrifuge tube. The sample was purged with nitrogen at 40°C in a PP centrifuge tube, then reconstituted with 1 mL of pure methanol, ultrasonically vibrated for 1 min, and filtered into a sample vial using a 0.22 μm nylon needle filter to obtain the sample loading solution for the honey to be tested.

[0031] (4) The standard sample solution from step (3) was analyzed by high performance liquid chromatography-triple quadrupole mass spectrometry to obtain the corresponding MRM spectrum. Figure 1 (The MRM spectra of each PFAS standard at a concentration of 5 ppb are shown in Table 2.) Then, standard curves for various PFAS are plotted with the concentration ratio x of each PFAS standard to the corresponding internal standard as the abscissa and the peak area ratio y of the corresponding quantitative ion pair as the ordinate.

[0032] Table 2

[0033] (5) The sample solution of the honey sample obtained in step (3) was analyzed by high performance liquid chromatography-triple quadrupole mass spectrometry to obtain the corresponding MRM spectrum. The peak area ratio of the quantitative ion pair of the analyte and the corresponding internal standard was substituted into the standard curve and multiplied by the concentration of the corresponding internal standard to obtain the concentration of each target analyte in the sample solution. Then, the content of each target analyte in the honey sample was calculated. In this embodiment, two actual samples were tested, and the results are detailed in Table 3. All four types of PFAS were detected.

[0034] Table 3

[0035] Example 2 To examine the accuracy of the method for detecting the content of multiple PFAS provided by this invention, this embodiment uses actual honey samples for spiked recovery experiments. Specifically, the sample loading solution is prepared according to step (3) in Example 1, with the only difference being that after adding the internal standard solution, 250 μL of a mixed standard solution of PFAS (each PFAS standard concentration is 50 ng / mL) is added. Subsequent pretreatment procedures are the same as step (3) in Example 1. Then, high-performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-MS / MS) is used for determination to obtain the corresponding MRM spectrum, as shown below. Figure 2 The assay was repeated six times, and the average recovery rate ranged from 74.3% to 121.8%, with an RSD of <10%. Detailed recovery results are shown in Table 4. The recovery results indicate that the trace PFAS spiked in this invention exhibits high and stable recovery.

[0036] Table 4

[0037] Example 3 To examine the precision of the accurate and rapid method for detecting multiple PFAS in honey provided by this invention, a repeatability analysis experiment was conducted. The difference from Example 2 was that low and high concentrations of PFAS standard solutions were added, with spike concentrations of 0.625 ng / g and 25 ng / g, respectively. After sample pretreatment according to step (3) of Example 1, the samples were measured using high-performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-MS / MS). Each concentration was measured three times. The precision RSD results are shown in Table 5 below. Table 5 shows that the repeatability RSD at different spike concentrations was <10%, indicating good method precision.

[0038] Table 5

[0039] Comparative Example 1 The extraction system used in the pretreatment process of the actual honey samples in this invention plays a crucial role in the accuracy of the detection results. The sample pretreatment process of Comparative Example 1 differs from that of Example 2, as shown in Table 6. The remaining parameters and pretreatment process are the same as in Example 2. It should be noted that, considering the certain differences between different batches of reagent honey samples, in order to ensure the reliability of the comparison results, the pretreatment conditions of Example 2 were used simultaneously during the experiment of Comparative Example 1 for comparison with Comparative Example 1.

[0040] Table 6

[0041] After pretreatment under the above conditions, the two honey samples in Comparative Example 1 were analyzed by high performance liquid chromatography-triple quadrupole mass spectrometry (under the conditions of Example 1). The recovery rates were significantly lower than those under the conditions of Example 2, as shown in Tables 7 and 8.

[0042] Table 7

[0043] Table 8

[0044] Comparative Example 2 The composition of the purification tube packing material used in the pretreatment process of the actual samples in this invention plays a crucial role in the accuracy of the detection results. Comparative Example 2 was conducted in accordance with Example 2, with the difference in experimental conditions being that the amount of PSA used in the purification tube packing material in step (3) was 300 mg, as shown in Table 9. It should be noted that, considering the certain differences between different batches of actual honey samples, in order to ensure the reliability of the comparison results, the actual honey samples used in Comparative Example 2 were simultaneously subjected to the pretreatment conditions of Example 2 for comparison with Comparative Example 2.

[0045] Example 4 Example 4 was conducted in accordance with Example 2, except that the amount of PSA used in the purification tube packing in step (3) was 100 mg and 200 mg, respectively, as shown in Table 9. The actual honey sample used in Example 4 was the same as that in Comparative Example 2.

[0046] Table 9

[0047] The recovery rate of the honey sample in Comparative Example 2 was significantly lower than that in Example 2 after analysis by a high-performance liquid chromatography-triple quadrupole mass spectrometry system (e.g., Figure 3 As shown in the figure): Compared with the 100 mg PSA group, the recovery rate of the 150 mg PSA group was 76.1%-100.2%, and the overall recovery rate was higher than that of the 100 mg PSA group; as the PSA dosage was further increased to 200 mg, the recovery rate of PFAS decreased, but still met the basic requirements. However, in Comparative Example 2, when the PSA dosage was 300 mg, the recovery rates of 15 PFAS were all below 80%, which could not meet the requirements for the detection of multiple PFAS.

[0048] Example 5 The method established in this invention was used to determine the content of multiple PFAS in honey from ten different provinces. The specific process is as follows: Take 2 g of honey sample into a 50 mL PP centrifuge tube, then accurately add 250 μL of internal standard solution (each internal standard concentration is 10 ng / mL), let stand for 10 min, add 5 mL of ultrapure water, shake for 5 min, add 10 mL of acetonitrile and 100 μL of formic acid, shake for 2 min, then add 4 g of MgSO4 and 1 g of NaCl, shake for 8 min, and centrifuge at 4500 rpm for 5 min; then accurately pipette 6 mL of supernatant into a purification tube, the purification tube packing is 900 mg MgSO4, 300 mg C18, 150 mg PSA, 15 mg GCB, shake again for 5 min, and centrifuge at 4500 rpm for 5 min; then take 4 mL of supernatant into a new 15 mL PP centrifuge tube, perform nitrogen blowing at 40℃, finally redissolve with 1 mL of pure methanol, sonicate for 1 min, and filter through a 0.22 μm nylon needle filter into a sample vial to obtain the honey sample loading solution. After analysis using high-performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-MS / MS), and based on the standard curve and conversion, the content of each target analyte in the honey sample was obtained. Specific detection results in honey samples from different provinces are as follows: Figure 3 , Figure 4 As shown.

[0049] Figure 4 This study presents the detection results of ΣPFAS in actual honey samples from 10 provinces in the Yangtze River Basin (Anhui, Hubei, Hunan, Jiangsu, Jiangxi, Qinghai, Sichuan, Yunnan, Zhejiang, and Chongqing). Figure 4 As shown, the total PFAS concentration varies among honey samples from different origins. Geographically, the PFAS content in East China is generally higher than that in Central and Western China. This result indicates that the method of this invention has good detection capability for actual samples from different origins, and the detection sensitivity is sufficient to reflect the differences in pollution levels between regions.

[0050] Figure 5 The relative percentage composition of the top four PFAS components in honey samples from the aforementioned provinces is further provided. Figure 5 It can be seen that the composition of samples from different origins exhibits certain regional characteristics: for example, the proportion of PFHxA components in samples from Anhui Province differs distinguishably from that in neighboring provinces such as Hubei and Hunan. Experimental results demonstrate that the method of this invention can reliably obtain the compositional information of each monomer, providing strong data support for traceability analysis and origin determination.

[0051] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A method for accurately and rapidly detecting multiple perfluorinated and polyfluoroalkyl substances in honey, characterized in that, An internal standard was added to the honey sample, followed by the addition of water, acetonitrile, and formic acid for dissolution and extraction. After salting out, the supernatant was purified using a composite purification material. The supernatant was collected again after purification and dried. The resulting residue was reconstituted, and the contents of various perfluorinated and polyfluoroalkyl substances in the honey sample were determined by internal standard method using high performance liquid chromatography-triple quadrupole mass spectrometry. The composite purification material consisted of magnesium sulfate, octadecyl bonded silica gel, graphitized carbon black, and N-propylethylenediamine.

2. The method for accurately and rapidly detecting multiple perfluorinated and polyfluoroalkyl substances in honey according to claim 1, characterized in that, The composite purification material is composed of 80-100 parts magnesium sulfate, 20-40 parts octadecyl bonded silica gel, 10-20 parts N-propylethylenediamine, and 1-2 parts graphitized carbon black by mass.

3. The method for accurately and rapidly detecting multiple perfluorinated and polyfluoroalkyl substances in honey according to claim 1, characterized in that, The ratio of honey to water, acetonitrile, and formic acid is 1 g:(2~4) mL:(4~6) mL:(40~60) μL; magnesium sulfate and sodium chloride are used for salting out, and the mass ratio of honey, magnesium sulfate, and sodium chloride is 1:(1.5~2.5):(0.4~0.6).

4. The method for accurately and rapidly detecting multiple perfluorinated and polyfluoroalkyl substances in honey according to claim 1, characterized in that, The high-performance liquid chromatography (HPLC) used a gradient elution program, which, by volume percentage, was as follows: 0–0.3 min, 90%–95% mobile phase A, with the remainder being mobile phase B; 0.9–9 min, 5%–10% mobile phase A; 9–12 min, 5%–10% mobile phase A. 12.0~12.1 min, 90%~95% mobile phase A; 12.1~14.0 min, 90%~95% mobile phase A; wherein, mobile phase A is 5~10 mM ammonium acetate aqueous solution, and mobile phase B is methanol.

5. The method for accurately and rapidly detecting multiple perfluorinated and polyfluoroalkyl substances in honey according to claim 1, characterized in that, Triple quadrupole mass spectrometry uses electron impact ionization and employs multiple reaction monitoring (MRM) mode. The ion source temperature is set to 120–150 °C. PFAS bulk fragments are selected as specific precursor ions for collision-induced dissociation. Both the target analyte and the internal standard are in negative ion mode.

6. A method for accurately and rapidly detecting multiple perfluorinated and polyfluoroalkyl substances in honey according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) Add internal standard to the honey sample to be tested, let it stand, add an appropriate amount of water to dissolve the honey, then add acetonitrile and formic acid for extraction, and then obtain the first supernatant after shaking, salting out and centrifugation. (2) The first supernatant obtained in step (1) is purified by composite purification material, then centrifuged to obtain the second supernatant, dried with nitrogen and reconstituted with methanol solution to obtain the sample loading solution of the honey sample to be tested. (3) Prepare a mixed standard solution of various PFAS, add an internal standard, and use it as a standard sample solution; (4) The standard sample solution in step (3) was analyzed using a high performance liquid chromatography-triple quadrupole mass spectrometer. After separation by high performance liquid chromatography, data was collected in the multiple reaction monitoring mode of mass spectrometry to obtain the MRM chromatogram. Then, the peak area ratio of each PFAS standard to the corresponding internal standard in the standard sample solution was used as the ordinate, and the concentration ratio of each PFAS standard to the internal standard was used as the abscissa to obtain the standard curve of each PFAS. (5) Under the same detection conditions as in step (4), the sample obtained in step (2) is analyzed by high performance liquid chromatography-triple quadrupole mass spectrometry to obtain the MRM spectrum. Then, combined with the standard curve of the corresponding PFAS in step (4), the concentration of each PFAS in the sample is calculated, and the content of each PFAS in the honey sample to be tested is obtained.

7. The method for accurately and rapidly detecting multiple perfluorinated and polyfluoroalkyl substances in honey according to claim 6, characterized in that, The various PFAS include perfluorobutyric acid, perfluoropentanoic acid, perfluorohexanoic acid, perfluorobutane sulfonic acid, perfluoroheptanoic acid, perfluoropentane sulfonic acid, 3H-perfluoro-4,8-dioxonanoic acid, perfluorooctanoic acid, perfluorohexane sulfonic acid, perfluorononanoic acid, perfluoroheptane sulfonic acid, perfluorodecanoic acid, perfluorooctane sulfonic acid, perfluoroundecanoic acid, perfluorononane sulfonic acid, 9-chlorohexadecano-3-oxanonane-1-sulfonic acid, perfluorododecanic acid, perfluorodecane sulfonic acid, perfluorotridecanoic acid, 11-chloroeicosico-3-oxaundecane-1-sulfonic acid, perfluorotetradecanoic acid, perfluorohexadecanoic acid, and perfluorooctadecanic acid. Internal standards include ¹³C4(MPFBA), ¹³C5(M5PFPeA), ¹³C5(M5PFHxA), ¹³C4(M4PFHpA), ¹³C8(MPFOA), ¹³C9(M9PFNA), ¹³C6(M 6PFDA), ¹³C7(M7PFUdA), ¹³C2(MPFDoA), ¹³C2(M2PFTeDA), ¹³C3(M3PFBS), ¹³C3(M3PFHxS), ¹³C8(M8PFOS).

8. The method for accurately and rapidly detecting multiple perfluorinated and polyfluoroalkyl substances in honey according to claim 6, characterized in that, In step (1), the amount of each internal standard added to the honey is calculated as 1~1.5 ng of internal standard per gram of honey; in step (2), the ratio of the first supernatant to the composite purification material is 1 mL: (200-250) mg; the methanol content of the methanol solution is 70%~100%, and the remainder is water; the temperature of nitrogen blowing is 30~50℃.

9. The method for accurately and rapidly detecting multiple perfluorinated and polyfluoroalkyl substances in honey according to claim 6, characterized in that, In step (2), the standard sample solution is prepared using methanol as the solvent, the concentration of each PFAS standard is in the range of 0~20 ng / mL, and the concentration of each internal standard is in the range of 5~15 ng / mL.

10. The method for accurately and rapidly detecting multiple perfluorinated and polyfluoroalkyl substances in honey according to claim 6, characterized in that, In steps (4) and (5), the analytical conditions for high-performance liquid chromatography include: a. Mobile phase A: 5~10 mM ammonium acetate aqueous solution, mobile phase B: methanol; b. Analytical procedure: A gradient elution program was used, with a sample loading volume of 1~3 μL, a flow rate of 0.2~0.5 mL / min, and an analysis time of 8~15 min; c. Chromatographic column: ACQUITY UPLC BEH C18 reversed-phase column, column temperature: 35~40 ℃; d. A trapping column is installed before the high-performance liquid chromatography column.

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