Analysis method for determining perfluorinated and polyfluoroalkyl substances in traditional Chinese medicine pseudo-ginseng based on ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry

Abstract

The invention belongs to the technical field of detection of perfluoroalkyl and polyfluoroalkyl substances in traditional Chinese medicines, and particularly relates to an analysis method for determining perfluoroalkyl and polyfluoroalkyl substances (PFAS) in a traditional Chinese medicine pseudo-ginseng on the basis of ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry). According to the method, QuEChERS extraction is adopted, then through-type purification is performed through an enhanced matrix removal (EMR) small column, and finally LC / MS / MS detection is utilized. According to the analysis method provided by the invention, the sample pretreatment process is simple and efficient, the LC / MS / MS detection sensitivity is high, and reliable quantification is realized by using a matrix labeling curve. The analysis method provided by the invention is suitable for quantitative screening of 20 common PFAS in the plant-derived traditional Chinese medicine pseudo-ginseng.

Description

Technical Field

[0001] This invention belongs to the field of detection technology of perfluorinated and polyfluoroalkyl substances in traditional Chinese medicine, specifically involving an analytical method for determining perfluorinated and polyfluoroalkyl substances in Panax notoginseng based on ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry. Background Technology

[0002] Perfluoroalkyl and polyfluoroalkyl substances (PFAS) and their derivatives are highly fluorinated aliphatic compounds widely used in various industrial and chemical fields, such as paper, textiles, packaging materials, leather, fabric manufacturing, and interior decoration. Some PFAS, due to their high surface activity, high thermal stability, and chemical stability, are used in the production of fire-extinguishing foams and surfactants. In recent years, scientists have discovered high concentrations of PFAS residues in various food categories (including grains, vegetables, starches, roots and tubers, fruits, meats, fish, and milk) and biological samples (such as animal liver, blood, breast milk, and serum). Epidemiological studies have shown that PFAS compounds are a class of toxic substances that affect the health of organisms, exhibiting immunotoxicity, hepatotoxicity, neurobehavioral toxicity, and reproductive toxicity. Animal toxicology studies have also verified the genotoxicity and carcinogenicity of these substances. Furthermore, PFAS are considered persistent organic pollutants that can accumulate in the human body through the food chain. Therefore, the development of sensitive, accurate, and rapid PFAS analysis techniques is urgently needed.

[0003] Panax notoginseng is the dried root and rhizome of Panax notoginseng, a plant in the Araliaceae family. It is mainly produced in Wenshan, Yunnan Province. Also known as Tianqi or Jinbuhuan, it is a representative Chinese medicinal material for stopping bleeding, dispersing blood stasis, reducing swelling and relieving pain. It is widely used in clinical practice and daily conditioning in traditional Chinese medicine.

[0004] Currently, there are no reports on the detection methods for PFAS in Panax notoginseng. Summary of the Invention

[0005] The purpose of this invention is to provide an analytical method for determining perfluorinated and polyfluoroalkyl substances in Panax notoginseng based on ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry. The analytical method provided by this invention can analyze 20 perfluorinated and polyfluoroalkyl substances in Panax notoginseng and has good accuracy and precision.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides an analytical method for determining perfluorinated and polyfluoroalkyl substances in the traditional Chinese medicine Panax notoginseng based on ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry, comprising the following steps: The Chinese herb Panax notoginseng was pretreated to obtain the sample to be tested. The pretreatment included sequential QuEChERS extraction and enhanced matrix removal (EMR) column pass-through purification. The sample to be tested was subjected to ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry for detection. Based on the detection results, the perfluorinated and polyfluoroalkyl substances in Panax notoginseng were qualitatively and quantitatively analyzed. The ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (UHPLC-MS / MS) conditions for detection include: mobile phases A and B, where mobile phase A is an aqueous solution of ammonium acetate and mobile phase B is acetonitrile; the elution method is gradient elution, and the gradient elution program is as follows: 0–3 min, the volume percentage of mobile phase B changes uniformly from 15% to 30%; 3–8 min, the volume percentage of mobile phase B changes uniformly from 30% to 55%; 8–9 min, the volume percentage of mobile phase B changes uniformly from 55% to 75%; 9–13 min, the volume percentage of mobile phase B changes uniformly from 75% to 100%; 13–13.8 min, the volume percentage of mobile phase B remains at 100%; 13.8–14 min, the volume percentage of mobile phase B changes uniformly from 100% to 15%; 14–18 min, the volume percentage of mobile phase B remains at 15%. The mass spectrometry conditions for the ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (UHPLC-MS / MS) detection include: negative ion ES mode, dynamic multiple reaction monitoring, drying gas temperature of 200-250 °C, drying gas flow rate of 5-10 L / min, nebulizer pressure of 30-35 psi, sheath gas temperature of 300-350 °C, sheath gas flow rate of 10-11 L / min, capillary voltage of 2000-2500 V, and nozzle voltage of 0 V. The perfluorinated and polyfluoroalkyl substances include perfluorobutyric acid (PFBA), perfluoropentanoic acid (PFPeA), perfluorohexanoic acid (PFHxA), perfluorobutane sulfonic acid (PFBS), perfluoroheptanoic acid (PFHpA), perfluoropentane sulfonic acid (PFPeS), perfluorooctanoic acid (PFOA), perfluorohexane sulfonic acid (PFHxS), perfluorononanoic acid (PFNA), perfluoroheptane sulfonic acid (PFHpS), perfluorodecanoic acid (PFDA), perfluorooctane sulfonic acid (PFOS), perfluoroundecanoic acid (PFUnDA), perfluorononane sulfonic acid (PFNS), perfluorododecanic acid (PFDoDA), perfluorodecane sulfonic acid (PFDS), perfluorotridecanoic acid (PFTrDA), perfluorotetradecanoic acid (PFTeDA), perfluorohexadecanoic acid (PFHxDA), and perfluorooctadecanic acid (PFODA).

[0007] Preferably, the molar concentration of ammonium acetate in the aqueous solution of ammonium acetate is 4-5 mM.

[0008] Preferably, the ultra-high performance liquid chromatography conditions further include: the analytical column is a C18 column with dimensions of 2.1×100 mm and 2.7 µm, and the pressure of the analytical column is 1000 bar; the retardation column is a C18 retardation column with dimensions of 4.6×30 mm.

[0009] Preferably, the ultra-high performance liquid chromatography conditions further include a mobile phase flow rate of 0.1~0.3 mL / min.

[0010] Preferably, the ultra-high performance liquid chromatography conditions also include a column temperature of 25~35℃.

[0011] Preferably, the ultra-high performance liquid chromatography conditions further include an injection volume of 3~5 μL.

[0012] Preferably, the mass spectrometry conditions further include a detector gain factor of 5.

[0013] Preferably, the mass spectrometry acquisition conditions for the ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (UHPLC-MS / MS) detection are as follows:

[0015] Preferably, the qualitative method is as follows: qualitative analysis is performed based on the retention time of the chromatographic peaks and the characteristic ion pairs of the mass spectrometry in the obtained chromatogram; The quantitative method is as follows: the content of perfluorinated and polyfluoroalkyl substances in Panax notoginseng is calculated based on the peak area of ​​the chromatographic peaks in the obtained chromatogram and the linear regression equation; the vertical axis of the linear regression equation is the chromatographic peak area of ​​the quantitative ions of perfluorinated and polyfluoroalkyl substances, and the horizontal axis is the concentration of the standard sample in the matrix blank solution of perfluorinated and polyfluoroalkyl substances.

[0016] Preferably, the standard curves for the perfluorinated and polyfluoroalkyl substances include:

[0018] This invention provides an analytical method for determining perfluorinated and polyfluoroalkyl substances in the traditional Chinese medicine Panax notoginseng using ultra-high performance liquid chromatography (UHPLC) coupled with quadrupole mass spectrometry (MS / MS). The method involves pretreatment of the Panax notoginseng using QuEChERS extraction followed by pass-through purification via an EMR column. The resulting sample is then analyzed using LC / MS / MS. The pretreatment process provided by this invention is simple and efficient, and the LC / MS / MS, through optimized detection conditions, achieves high detection sensitivity. Therefore, the analytical method provided by this invention can analyze 20 perfluorinated and polyfluoroalkyl substances in Panax notoginseng with good accuracy and precision.

[0019] Furthermore, this invention utilizes matrix spiking curves to achieve reliable quantification of perfluorinated and polyfluoroalkyl substances (PFAS) in the traditional Chinese medicine Panax notoginseng. The method provided by this invention is applicable to the quantitative screening of 20 common PFAS in Panax notoginseng. Attached Figure Description

[0020] Figure 1 The solutions were acetonitrile-10 mM ammonium acetate and methanol-10 mM ammonium acetate, with a PFAS standard concentration of 10 μg / L. Figure 2 A comparison chart of PFAS standard concentrations of 0.5 μg / L in standard solutions of acetonitrile-10 mM ammonium acetate (20250311-002.d) and acetonitrile-5 mM ammonium acetate (20250311-010.d); Figure 3 The image shows a superimposed graph of acetonitrile-10 mM ammonium acetate (20250311-002.d) and acetonitrile-5 mM ammonium acetate (20250311-010.d) with a PFAS standard concentration of 0.5 μg / L in the standard solutions. Figure 4 The extraction MRM chromatogram of a matrix blank spiked sample containing 20 PFAS target analytes at a concentration of 0.2 ng / mL is shown. Figure 5 A bar chart comparing solvent blank / EMR-MB (0.9 ng / mL eluent diluted 1-fold, original eluent and nitrogen blown concentrated 1-fold) / WAX-MB / 0.05 ng / mL solvent standard MRM; Figure 6 Peak areas for solvent blank, matrix blank, lowest concentration (0.05 μg / L) of matrix-matched calibration curve, and final concentration (0.09 μg / L) of LOQ determination for the four core PFAS target analytes (PFOS, PFOA, PFNA, and PFHxS); Figure 7 Flowchart of sample pretreatment using Bond Elut PFAS WAX; Figure 8 This is a flowchart illustrating the sample pretreatment process using the Agilent Captiva EMR PFAS Food II column for this invention. Detailed Implementation

[0021] This invention provides an analytical method for determining perfluorinated and polyfluoroalkyl substances in the traditional Chinese medicine Panax notoginseng based on ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry, comprising the following steps: The Chinese herb Panax notoginseng is pretreated to obtain the sample to be tested. The pretreatment includes sequential QuEChERS extraction and EMR column pass-through purification. The sample to be tested was subjected to ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry for detection. Based on the detection results, the perfluorinated and polyfluoroalkyl substances in Panax notoginseng were qualitatively and quantitatively analyzed. The ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (UHPLC-MS / MS) conditions for detection include: mobile phases A and B, where mobile phase A is an aqueous solution of ammonium acetate and mobile phase B is acetonitrile; the elution method is gradient elution, and the gradient elution program is as follows: 0–3 min, the volume percentage of mobile phase B changes uniformly from 15% to 30%; 3–8 min, the volume percentage of mobile phase B changes uniformly from 30% to 55%; 8–9 min, the volume percentage of mobile phase B changes uniformly from 55% to 75%; 9–13 min, the volume percentage of mobile phase B changes uniformly from 75% to 100%; 13–13.8 min, the volume percentage of mobile phase B remains at 100%; 13.8–14 min, the volume percentage of mobile phase B changes uniformly from 100% to 15%; 14–18 min, the volume percentage of mobile phase B remains at 15%. The mass spectrometry conditions for the ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (UHPLC-MS / MS) detection include: negative ion ES mode, dynamic multiple reaction monitoring, drying gas temperature of 250 °C, drying gas flow rate of 10 L / min, nebulizer pressure of 35 psi, sheath gas temperature of 350 °C, sheath gas flow rate of 11 L / min, capillary voltage of 2500 V, and nozzle voltage of 0 V. The perfluorinated and polyfluoroalkyl substances include perfluorobutyric acid (PFBA), perfluoropentanoic acid (PFPeA), perfluorohexanoic acid (PFHxA), perfluorobutane sulfonic acid (PFBS), perfluoroheptanoic acid (PFHpA), perfluoropentane sulfonic acid (PFPeS), perfluorooctanoic acid (PFOA), perfluorohexane sulfonic acid (PFHxS), perfluorononanoic acid (PFNA), perfluoroheptane sulfonic acid (PFHpS), perfluorodecanoic acid (PFDA), perfluorooctane sulfonic acid (PFOS), perfluoroundecanoic acid (PFUnDA), perfluorononane sulfonic acid (PFNS), perfluorododecanic acid (PFDoDA), perfluorodecane sulfonic acid (PFDS), perfluorotridecanoic acid (PFTrDA), perfluorotetradecanoic acid (PFTeDA), perfluorohexadecanoic acid (PFHxDA), and perfluorooctadecanic acid (PFODA).

[0022] In this invention, unless otherwise specified, all raw materials / components used in the preparation are commercially available products well known to those skilled in the art.

[0023] This invention pre-processes the traditional Chinese medicine Panax notoginseng to obtain a sample to be tested. The pre-processing includes sequential QuEChERS extraction and EM column pass-through purification.

[0024] In this invention, the Panax notoginseng is a plant-derived Panax notoginseng. The raw material for the pretreatment is preferably Panax notoginseng powder. The Panax notoginseng powder is preferably obtained by sequentially pulverizing and sieving Panax notoginseng, with pulverization preferably performed using a cell wall breaker. The sieving is preferably performed using a 60-mesh sieve. The sieving process preferably removes the undersize material to obtain the Panax notoginseng powder.

[0025] In this invention, the QuEChERS extraction preferably comprises: mixing the Panax notoginseng powder, water, acetic acid-acetonitrile solution, and QuEChERS extraction salt for extraction, followed by centrifugation to obtain a supernatant. The volume fraction of acetic acid in the acetic acid-acetonitrile solution is preferably 1%. The volume ratio of water to the acetic acid-acetonitrile solution is preferably 1:1. In this invention, the extraction is carried out under stirring conditions, preferably using a ceramic homogenizer. The preferred ratio of Panax notoginseng powder to water is 5 g: 10 mL.

[0026] In a specific embodiment of the present invention, the QuEChERS extraction specifically includes the following steps: premixing the Panax notoginseng powder and water, then adding an acetic acid-acetonitrile solution for a first mixing, wherein the volume content of acetic acid in the acetic acid-acetonitrile solution is preferably 1%. Then, adding a QuEChERS extraction salt packet and using a ceramic homogenizer for a second mixing, and finally centrifuging to obtain the supernatant, yielding the QuEChERS extraction solution. The preferred ratio of the Panax notoginseng powder to water is 5g:5~10mL. The preferred ratio of the Panax notoginseng powder to the acetic acid-acetonitrile solution is 5g:5~10mL. The amount of the QuEChERS extraction salt packet used in this invention can be any amount known to those skilled in the art. The premixing is carried out under vortex conditions for a time preferably 5~10min. The first mixing is carried out under vortex conditions for a time preferably 10~20s. The second mixing is carried out under shaking conditions for a time preferably 3~5min. The centrifugation speed is preferably 5000~8000r / min for a time preferably 3~5min.

[0027] The preferred method for purification of the EMR column includes: mixing the supernatant (QuEChERS extraction solution) with water to obtain a supernatant mixture; sequentially washing, equilibrating and drying the EMR column, loading the sample, and performing gravity elution, collecting the eluent; and centrifuging the eluent to obtain the supernatant sample. In this invention, the volume ratio of the supernatant to the water is preferably 5.4:0.6. The washing uses a mixture of 1 vol% acetic acid in an acetic acid-acetonitrile solution and methanol, with the volume ratio of the 1 vol% acetic acid in the acetic acid-acetonitrile solution to methanol preferably being 1:1. Equilibration is performed using the supernatant mixture. After loading the sample, gravity elution is performed, followed by vacuum drying of the column, preferably at a pressure of 0.02 MPa.

[0028] In this invention, the mass concentration of the supernatant obtained by the EMR column through-process purification is preferably 0.09 ng / mL.

[0029] After obtaining the sample to be tested, the present invention performs ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry on the sample to be tested, and performs qualitative and quantitative analysis on the perfluorinated and polyfluoroalkyl substances in Panax notoginseng based on the detection results.

[0030] In this invention, the molar concentration of ammonium acetate in the aqueous solution of ammonium acetate is preferably 5 mM.

[0031] In this invention, the ultra-high performance liquid chromatography conditions further include: the analytical column is preferably a C18 column with a size of 2.1 × 100 mm and a diameter of 2.7 µm, and the pressure of the analytical column is preferably 1000 bar; the retardation column is preferably a C18 retardation column with a size of 4.6 × 30 mm.

[0032] In this invention, the ultra-high performance liquid chromatography conditions preferably further include: a mobile phase flow rate of 0.3 mL / min.

[0033] In this invention, the ultra-high performance liquid chromatography conditions preferably include a column temperature of 25~35℃.

[0034] In this invention, the ultra-high performance liquid chromatography conditions preferably further include: an injection volume of 3~5 μL.

[0035] In this invention, the mass spectrometry conditions preferably further include: under negative mode conditions, the detector gain factor is 5.

[0036] In this invention, the mass spectrometry acquisition conditions for the ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (UHPLC-MS / MS) detection are shown in Table 4.

[0037] In this invention, the qualitative method is preferably: qualitative analysis is performed based on the retention time and mass spectrometry characteristics of the chromatographic peaks in the obtained chromatogram.

[0038] In this invention, the preferred quantitative method is to calculate the content of perfluorinated and polyfluoroalkyl substances in Panax notoginseng based on the peak area of ​​the chromatographic peaks in the obtained chromatogram and the linear regression equation; the vertical axis of the linear regression equation is the peak area of ​​perfluorinated and polyfluoroalkyl substances, and the horizontal axis is the content of perfluorinated and polyfluoroalkyl substances.

[0039] Preferably, the standard curves of the perfluorinated and polyfluoroalkyl substances are shown in Table 5.

[0040] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1 This embodiment provides an analytical method for determining perfluorinated and polyfluoroalkyl substances in Panax notoginseng using ultra-high performance liquid chromatography (UHPLC)-tandem quadrupole mass spectrometry (MS / MS). The analytical method provided in this embodiment can determine 20 PFAS in Panax notoginseng.

[0042] Experimental Section Reagents and Samples All reagents and solvents used in this embodiment were of chromatographic or analytical grade. Chromatographic grade acetonitrile and methanol were purchased from Bailingwei; 20 PFAS standards were purchased from Tianjin Alta Technology Co., Ltd.; the experimental water was high-purity deionized water prepared fresh from the Millipore Milli-Q ultrapure water system. The test sample was Panax notoginseng, a traditional Chinese medicine.

[0043] Instruments and Materials The ultra-high performance liquid chromatography (UHPLC)-tandem quadrupole mass spectrometry (MS / MS) used in this embodiment had the following chromatographic columns: C18, 2.1 × 100 mm, 2.7 µm, 1000 bar.

[0044] QuEChERS extraction kit (4 g MgSO4, 1 g NaCl, 1 g sodium citrate, 0.5 g disodium citrate).

[0045] EMR column, 6 mL, 750 mg.

[0046] All consumables used in this study were tested and validated to ensure that the cleanliness of the PFAS met the requirements.

[0047] Preparation of standard solutions Three PFAS spiked solutions (I, II, and III) were prepared by diluting the PFAS mixed standard solution with methanol, covering 20 PFAS targets at concentrations of 100, 20, and 10 ng / mL, respectively.

[0048] Mixed standard working solutions were prepared using PFAS spiking solutions with concentrations of 0.05, 0.1, 0.2, 0.5, 1.0, and 5.0 ng / mL, respectively, in acetonitrile:water (v:v) = 9:1. All standards were stored at 4 °C and used within two weeks.

[0049] Sample pretreatment The Chinese herb Panax notoginseng was purchased from the market. After being pulverized using a high-speed blender, it was passed through a 60-mesh sieve and then pre-processed.

[0050] like Figure 8 As shown, the pretreatment process was as follows: 5g of sieved Panax notoginseng sample was placed in a 50mL tube, 10mL of water was added, and the mixture was vortexed for 10min. 10mL of an acetic acid-acetonitrile solution containing 1 vol% acetic acid was added, and the mixture was vortexed for 20s. Two ceramic homogenants and a QuEChERS extraction salt packet were added, mixed well, and shaken for 5min. The mixture was centrifuged at 8000r / min for 5min. 5.4mL of the supernatant was transferred to a 15mL centrifuge tube and mixed with 0.6mL of water to obtain the supernatant mixture.

[0051] The EMR column (Agilent Captiva EMR PFAS Food II column) was washed with 5 mL of an acetic acid-acetonitrile solution containing 1 vol% acetic acid:methanol (v:v = 1:1). The column was then equilibrated with 0.8 mL of the supernatant mixture, followed by gravity elution. The eluent was discarded, and the column was dried completely under vacuum. 5 mL of the supernatant mixture was transferred to the column, and gravity elution was performed. At the end of the elution, pressure (vacuum pressure, 0.02 MPa) was applied for 2 min to ensure the adsorbent bed was completely dry. The eluent (target concentration 0.09 ng / mL) was collected, vortexed for 20 s, and centrifuged at 8000 rpm for 5 min. 0.5 mL of the supernatant (target concentration 0.09 ng / mL) was analyzed using an LC-MS-MS system.

[0052] Take 9 mL of commercially available ammonia water (37 wt%) and dilute to 100 mL with methanol.

[0053] This embodiment verifies the accuracy of the analytical method by adding a standard solution.

[0054] LC / MS / MS instrument conditions in this embodiment: The conditions for the binary pump in liquid chromatography are listed in Table 1, the Multisampler program is listed in Table 2, the parameters of the mass spectrometry ion source are listed in Table 3, and the dMRM acquisition parameters are listed in Table 4.

[0055] Table 1. Liquid chromatography pump conditions for LC / MS / MS

[0056] Table 2 LC Multisampler Program for LC / MS / MS

[0057] Table 3 Mass spectrometry conditions for LC / MS / MS

[0058] Table 4 MS acquisition conditions for PFAS target monitoring (simultaneous acquisition is possible at one time)

[0059] Performance evaluation of the analysis method provided in this embodiment: This embodiment uses a through-feed purification method with EMR columns to evaluate matrix removal, target analyte recovery, and repeatability during sample purification. The entire method was then validated, including calibration curves, determination of the limit of quantitation (LOQ) for the method pair, and accuracy and precision of recovery. Samples at three pre-spiked quality control (QC) concentrations were evaluated, with six replicates at each concentration. Additionally, three matrix blanks were prepared in this embodiment for the quantitative analysis of the target analyte in matrix control samples. This is crucial for accuracy assessment because the matrix's influence on certain PFAS is unavoidable.

[0060] Results and discussion of this embodiment: Optimization of chromatographic conditions In this embodiment, PFAS compounds were analyzed using a C18 column (2.1 × 100 mm, 2.7 µm). Adjustment of the mobile phase gradient resulted in excellent compound separation and a short analysis time, achieving efficient separation of 20 target compounds within 18 minutes. The mobile phase composition affects the retention time, peak shape, and ionization efficiency of the target analytes, thus influencing detection sensitivity and result accuracy. This embodiment compared mobile phase systems of methanol-10 mM ammonium acetate aqueous solution, acetonitrile-10 mM ammonium acetate aqueous solution, and acetonitrile-5 mM ammonium acetate aqueous solution. Figure 1 Acetonitrile-10 mM ammonium acetate ( Figure 1 (See the image below), methanol-10mM ammonium acetate ( Figure 1 (See the image above). The concentration of the PFAS standard in the standard solution is 10 μg / L. Figure 2A comparison chart of PFAS standard concentrations of 0.5 μg / L in standard solutions of acetonitrile-10 mM ammonium acetate (20250311-002.d) and acetonitrile-5 mM ammonium acetate (20250311-010.d); Figure 3 The image shows a superimposed graph of acetonitrile-10 mM ammonium acetate (20250311-002.d) and acetonitrile-5 mM ammonium acetate (20250311-010.d), with a PFAS standard concentration of 0.5 μg / L in the standard solutions.

[0061] Figure 1 , Figure 2 and Figure 3 The results showed that in the methanol-10 mM ammonium acetate aqueous solution system, the compounds were mainly concentrated in the high-proportion organic phase for co-elution, while methanol had poor elution ability. In the acetonitrile-10 mM ammonium acetate aqueous solution system, the target compounds could be effectively separated. There was a certain difference in ionization efficiency between the acetonitrile-10 mM ammonium acetate aqueous solution and the acetonitrile-5 mM ammonium acetate aqueous solution mobile phase. Considering all factors, the acetonitrile-5 mM ammonium acetate aqueous solution system was chosen as the mobile phase.

[0062] In this embodiment, Figures 1-3 In the graph: the horizontal axis represents time, and the vertical axis represents the response intensity.

[0063] Figure 4 The extraction MRM chromatogram is shown for a matrix blank spiked sample containing 20 PFAS target analytes at a concentration of 0.2 ng / mL.

[0064] Preprocessing screening and optimization in this embodiment: Effective removal of matrix co-extractants during sample pretreatment and applicability to different types of sample matrices are key objectives in establishing PFAS analysis methods for food. Weak anion exchange (WAX) solid-phase extraction is a commonly used method. It relies on the retention of carboxylic or sulfonic acid groups in the PFAS structure, followed by elution to remove impurities and concentration via nitrogen blowing. This process involves numerous steps and carries the potential to introduce more background interference. In addition, dispersive solid-phase extraction (dSPE) is also used for PFAS detection in food. While simple, it cannot effectively remove complex matrix interferences, posing a challenge to achieving high-sensitivity analysis.

[0065] This embodiment uses two types of purification columns, WAX and EMR, to process a sample of Panax notoginseng. The purification process using WAX columns is as follows: Figure 7 As shown. When using EMR, the effects of 1-fold dilution of the eluent and 1-fold concentration by nitrogen blowing on the spike recovery results were investigated simultaneously. Figure 4The extraction MRM chromatogram of a matrix blank spiked sample containing 20 PFAS target analytes at a concentration of 0.2 ng / mL is shown. Figure 5 Solvent blank / EMR-MB (matrix blank sample purified by EMR column) (diluted 1-fold, original eluent and concentrated 1-fold by nitrogen blowing) / WAX-MB (according to Figure 7 The matrix blank sample purified by the WAX ​​column obtained by the method / 0.05 ng / mL solvent standard MRM comparison column chart, Figure 5 The 0.2 ng / g in the figure represents the sample concentration, while 1.0 ng / mL, 0.045 ng / mL, 0.09 ng / mL, and 0.18 ng / mL represent the theoretically calculated concentrations of the analyte obtained after a series of treatments. Spectra show that the two purification columns exhibit different removal effects on the matrix that interferes with the early-eluting target compounds. WAX purification resulted in less interference with PFBA and PFHxA, superior to EMR, but the opposite was true for PFPeA and PFBS. Considering the recovery results of the 0.2 ng / g matrix standard sample, WAX effectively removes the matrix, but also suffers significant loss of the target compounds.

[0066] Comparing the results of receiving the eluent with and without further processing revealed that dry plant-derived matrices like Panax notoginseng are typically very complex, containing a large number of interfering substances ranging from polar to nonpolar. Even with enhanced cleanliness through EMR washing, many interfering substances still remained in the final eluent. Dilution can reduce matrix effects to some extent, but the sensitivity of the instrument must be considered; while post-concentration steps significantly induce matrix effects and lead to inhibition, especially for polar compounds.

[0067] In summary, for Panax notoginseng samples, EMR sample purification method achieves superior purification effect and higher sensitivity compared to WAX. In this experiment, EMR columns were used for sample pretreatment, and the eluent was directly detected after high-speed centrifugation.

[0068] Method Validation The analytical method provided by this invention was verified according to AOAC SMPR 2023.003 guidelines. For Panax notoginseng, a Chinese herbal medicine matrix with complex components, there is currently no relevant classification. Referring to the "coffee" matrix category in the AOAC SMPR guidelines, the LOQ of the four core PFAS target analytes (PFOS, PFOA, PFNA, and PFHxS) is required to be ≤ 0.3 µg / kg, and the LOQ of the remaining PFAS target analytes is required to be ≤ 3.0 µg / kg.

[0069] The LOQ of the analysis method provided in this embodiment.

[0070] In this embodiment, the Panax notoginseng matrices evaluated all showed positive results for the detection of a few PFAS target analytes in the matrix blank. The method was validated for target compound recoveries using matrix background correction. According to AOAC SMPR 2023.003 guidelines, a matrix blank is considered appropriate if the concentration of the target analyte in the matrix blank sample does not exceed 30% of the target limit of quantitation (LOQ). Three matrix blank parallel samples and six matrix-spiked parallel samples were prepared. By comparing the results of the matrix-spiked samples with those of the matrix blank samples, the concentration of the matrix blank samples was found to be less than 30% of the concentration of the matrix-spiked samples, thus determining the method LOQ. Figure 6 This section compares the peak areas of four core PFAS target analytes (PFOS, PFOA, PFNA, and PFHxS) at solvent blank, matrix blank, lowest concentration (0.05 μg / L) in the matrix-matched calibration curve, and final concentration (0.09 μg / L) at the LOQ determination. PFOA was detectable in the matrix blank, but the matrix-spiked sample response in the LOQ met the matrix blank response by more than three times, corresponding to a spiked amount of 0.2 µg / kg, which satisfies the relevant matrix LOQ requirements in the AOAC SMPR 2023.003 guideline.

[0071] Standard curve and correlation coefficient The calibration curve range was determined based on the required LOQ in the food matrix, the concentration factor introduced by sample pretreatment, and the sensitivity of the instrument method. In this embodiment, some target compounds are affected by the matrix; therefore, matrix-matched calibration curves were used. Matrix-matched standard curves were plotted with the concentration of the standard sample in the matrix blank solution as the abscissa and the chromatographic peak area of ​​the quantitative ion as the ordinate. The obtained linear regression equations and correlation coefficients are shown in Table 5. Table 5 shows that the 20 PFAS exhibit good linearity in the concentration range of 0.05–5.0 ng / mL, with correlation coefficients all greater than 0.999.

[0072] Table 5. Standard curve concentration ranges, linear regression equations, and correlation coefficients for 20 PFAS.

[0073] Spike recovery and precision The recovery and precision of the analytical method provided by this invention were validated. Referring to AOAC SMPR 2023.003, the acceptable recoveries vary for different target compounds. The acceptable recoveries for the four core PFAS target analytes (PFOS, PFOA, PFNA, and PFHxS) are 65%–135% with RSD ≤ 25%, while the acceptable recoveries for other indicators are 40%–1140% with RSD ≤ 30%.

[0074] According to the analytical method provided in this embodiment, spiked samples of Panax notoginseng matrix at three concentration levels (0.2 µg / kg, 0.4 µg / kg, and 2.0 µg / kg) were prepared, and six parallel samples were prepared at each spike concentration. The samples were analyzed using the pretreatment method described above, and quantification was performed using an external standard. The results are shown in Table 6. The average recoveries and RSDs of the 20 PFAS in Panax notoginseng were all within 10%, indicating that the analytical method provided in this embodiment has good accuracy and precision.

[0075] Table 6. Average recoveries and concentrations of 20 PFAS in Panax notoginseng (a traditional Chinese medicine).

[0076] In this embodiment, PFAS in two commercially available Panax notoginseng samples were analyzed and tested. The results are shown in Tables 7 and 8.

[0077] Table 7. Detection results of PFAS in Panax notoginseng samples.

[0078] Table 8. Detection results of PFAS in Panax notoginseng samples.

[0079] As shown in the above embodiments, the analytical method for determining perfluorinated and polyfluoroalkyl substances in Panax notoginseng based on ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry provided by this invention employs QuEChERS extraction, followed by pass-through purification using an EMR column, and finally detection using LC / MS / MS. The sample pretreatment process of the analytical method provided by this invention is simple and efficient, the LC / MS / MS detection sensitivity is high, and reliable quantification is achieved using matrix spiking curves. This invention has validated the applicability, sensitivity, accuracy, and precision of the method according to AOAC Standard Method Performance Requirements (SMPR) 2023.003. The analytical method provided by this invention is suitable for quantitative screening of 20 common PFAS in Panax notoginseng, a plant-derived traditional Chinese medicine.

[0080] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An analytical method for determining perfluorinated and polyfluoroalkyl substances in Panax notoginseng based on ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry, characterized in that, Includes the following steps: The Chinese herb Panax notoginseng is pretreated to obtain the sample to be tested. The pretreatment includes sequential QuEChERS extraction and EMR column pass-through purification. The sample to be tested was subjected to ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry for detection. Based on the detection results, the perfluorinated and polyfluoroalkyl substances in Panax notoginseng were qualitatively and quantitatively analyzed. The ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (UHPLC-MS / MS) conditions for detection include: mobile phases A and B, where mobile phase A is an aqueous solution of ammonium acetate and mobile phase B is acetonitrile; the elution method is gradient elution, and the gradient elution program is as follows: 0–3 min, the volume percentage of mobile phase B changes uniformly from 15% to 30%; 3–8 min, the volume percentage of mobile phase B changes uniformly from 30% to 55%; 8–9 min, the volume percentage of mobile phase B changes uniformly from 55% to 75%; 9–13 min, the volume percentage of mobile phase B changes uniformly from 75% to 100%; 13–13.8 min, the volume percentage of mobile phase B remains at 100%; 13.8–14 min, the volume percentage of mobile phase B changes uniformly from 100% to 15%; 14–18 min, the volume percentage of mobile phase B remains at 15%. The mass spectrometry conditions for the ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (UHPLC-MS / MS) detection include: negative ion ES mode, dynamic multiple reaction monitoring, drying gas temperature of 200-250 °C, drying gas flow rate of 5-10 L / min, nebulizer pressure of 30-35 psi, sheath gas temperature of 300-350 °C, sheath gas flow rate of 10-11 L / min, capillary voltage of 2000-2500 V, and nozzle voltage of 0 V. The perfluorinated and polyfluoroalkyl substances include perfluorobutyric acid, perfluorovalerate, perfluorohexanoic acid, perfluorobutane sulfonic acid, perfluoroheptanoic acid, perfluoropentane sulfonic acid, perfluorooctanoic acid, perfluorohexane sulfonic acid, perfluorononanoic acid, perfluoroheptane sulfonic acid, perfluorodecanoic acid, perfluorooctane sulfonic acid, perfluoroundecanoic acid, perfluorononane sulfonic acid, perfluorododecanic acid, perfluorodecane sulfonic acid, perfluorotridecanoic acid, perfluorotetradecanoic acid, perfluorohexadecanoic acid, and perfluorooctadecanic acid.

2. The analytical method according to claim 1, characterized in that, The molar concentration of ammonium acetate in the aqueous solution is 4-5 mM.

3. The analytical method according to claim 1, characterized in that, The ultra-high performance liquid chromatography conditions also include: the analytical column is a C18 column with dimensions of 2.1 × 100 mm and 2.7 µm, and the pressure of the analytical column is 1000 bar; the retardation column is a C18 retardation column with dimensions of 4.6 × 30 mm.

4. The analytical method according to claim 1, characterized in that, The ultra-high performance liquid chromatography conditions also include: a mobile phase flow rate of 0.1~0.3 mL / min.

5. The analytical method according to claim 1, characterized in that, The ultra-high performance liquid chromatography conditions also include a column temperature of 25~35℃.

6. The analytical method according to claim 1, characterized in that, The ultra-high performance liquid chromatography conditions also include an injection volume of 3~5 μL.

7. The analytical method according to claim 1, characterized in that, The mass spectrometry conditions also include: a detector gain factor of 5.

8. The analytical method according to claim 1, characterized in that, The mass spectrometry acquisition conditions for the ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (UHPLC-MS / MS) detection are as follows: 。 9. The analytical method according to claim 1, characterized in that, The qualitative method is as follows: qualitative analysis is performed based on the retention time of the chromatographic peaks and the characteristic ions of the mass spectrometry in the chromatogram obtained from the detection. The quantitative method is as follows: the content of perfluorinated and polyfluoroalkyl substances in Panax notoginseng is calculated based on the peak area of ​​the chromatographic peaks in the obtained chromatogram and the linear regression equation; the vertical axis of the linear regression equation is the chromatographic peak area of ​​the quantitative ions of perfluorinated and polyfluoroalkyl substances, and the horizontal axis is the concentration of the standard sample in the matrix blank solution of perfluorinated and polyfluoroalkyl substances.

10. The analytical method according to claim 9, characterized in that, The standard curves for the perfluorinated and polyfluoroalkyl substances include: 。

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