Pretreatment method for detecting short-chain chlorinated paraffin and detection method matched with pretreatment method

Abstract

The invention relates to the technical field of short-chain chlorinated paraffin detection. The invention provides a pretreatment method for detecting short-chain chlorinated paraffin and a detection method matched with the pretreatment method, the pretreatment method comprises the following steps: (1) mixing a sample to be detected, florisil and an extraction reagent, and extracting to obtain extract liquor; (2) purifying and eluting the extracting solution by using a silica gel-florisil composite column to obtain an eluent; and (3) removing the extraction reagent in the eluent to obtain a treated sample. By adjusting and optimizing pretreatment steps and instrument analysis conditions, the SCCPs extraction efficiency of different reagents, extraction method selection, solid-phase extraction column filling amount and reagent usage amount are researched, and the SCCPs analysis method suitable for the traditional Chinese medicinal material matrix is established.

Description

Technical Field

[0001] The present invention relates to the technical field of short-chain chlorinated paraffin detection, and in particular to a pretreatment method for short-chain chlorinated paraffin detection and a detection method coordinated with the pretreatment method. Background Art

[0002] Chlorinated paraffins are a mixture of polychlorinated normal alkanes, among which short-chain chlorinated paraffins with a carbon chain length of 10 to 13 carbon atoms have become a global environmental threat. The characteristics of persistent organic pollutants such as toxicity, persistence, long-range migration and bioaccumulation have attracted great attention. At present, the inconsistent standards for pollutant limits in Chinese medicinal materials and the imperfect grading management standards have hindered the development of Chinese medicinal materials internationally. Previous studies have found that Chinese medicinal materials are generally contaminated by heavy metals and pesticide residues. The hepatotoxicity, developmental toxicity, endocrine and metabolic disruption of short-chain chlorinated paraffins have caused great harm to human health. Therefore, it is necessary to investigate the pollution characteristics and health risks of short-chain chlorinated paraffins in Chinese medicinal materials.

[0003] Among the current analytical methods for short-chain chlorinated paraffins, gas chromatography electron capture low-resolution mass spectrometry is one of the most widely used analytical techniques, but it is difficult to identify chlorinated paraffin monomers; high-performance liquid chromatography time-of-flight mass spectrometry requires the addition of dichloromethane after the column, which is cumbersome to operate; and the current detection methods still have the problem of interference from chlorinated paraffin homologues and organic halogen compounds. Summary of the invention

[0004] The purpose of the present invention is to provide a pretreatment method for the detection of short-chain chlorinated paraffins and a detection method coordinated with the pretreatment method, to establish a simple, efficient and reliable analysis method for short-chain chlorinated paraffins, to detect short-chain chlorinated paraffins in traditional Chinese medicines, to analyze the pollution characteristics of short-chain chlorinated paraffins in different traditional Chinese medicine matrices, and to scientifically evaluate the dietary exposure level of short-chain chlorinated paraffins and the health risks to the population, in order to provide a reference for the establishment of a standard method for instrumental analysis of short-chain chlorinated paraffins and to provide data support for the setting of chlorinated paraffin limit standards.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a pretreatment method for detecting short-chain chlorinated paraffins, comprising the following steps:

[0007] (1) mixing a sample to be tested, floral silica and an extraction reagent, extracting and obtaining an extract;

[0008] (2) purifying the extract with a silica gel-florisil composite column and eluting to obtain an eluate;

[0009] (3) Removing the extraction reagent from the eluent to obtain a processed sample.

[0010] Preferably, the ratio of the sample to be tested, Florisil and extraction reagent in step (1) is 3-8 g: 3-8 g: 45-55 mL;

[0011] The extraction reagent includes n-hexane and dichloromethane, and the volume ratio of n-hexane to dichloromethane is 1:0.8-1.2;

[0012] The extraction conditions are: extraction temperature 95-105° C., heating time 4-6 min, static extraction time 8-12 min, flushing volume 45-55%, nitrogen purge time 50-70 s, and the number of extractions is 2-4 times.

[0013] Preferably, the silica gel-florisil composite column in step (2) is filled with floral silica, silica gel, acidic silica gel and anhydrous sodium sulfate in order from bottom to top, and the mass ratio of floral silica, silica gel, acidic silica gel and anhydrous sodium sulfate is 2.3-2.7:2.3-2.7:3.3-3.7:1.3-1.7;

[0014] The purification method comprises: eluting the silica gel-florisil composite column with n-hexane and adding an extracting liquid for purification;

[0015] The elution method is: adding n-hexane to the silica gel-florisil composite column to elute impurities, and then adding an extract to obtain an eluent.

[0016] Preferably, nitrogen gas is used to dry the eluent in step (3), and the treated sample is re-dissolved in methanol before use.

[0017] The present invention also provides application of the pretreatment method in reducing background interference in a detection process of short-chain chlorinated paraffins, wherein the object of detection is traditional Chinese medicine.

[0018] The present invention also provides a detection method used in conjunction with the pretreatment method, comprising the following steps: re-dissolving the treated sample with methanol, and then detecting with ultra-high performance liquid chromatography-tandem mass spectrometry.

[0019] Preferably, the ultra-high performance liquid chromatography process uses a Thermo Scientific Accucore C18 (2.1×100 mm, 2.6 μm) chromatographic column, with a column temperature of 38 to 42° C., an injection volume of 1 to 3 μL, a nitrogen flow rate of 0.25 to 0.35 mL / min, and an ionization source set to ESI negative ion mode.

[0020] Preferably, in the ultra-high performance liquid chromatography process, water is used as phase A, and 0.03-0.05 mmol / L tetramethylammonium chloride methanol solution is used as phase B, and the elution procedure is: at 0 min, phase A: phase B is 80:20;

[0021] At 4 min, the ratio of phase A to phase B was 80:20;

[0022] At 6 min, the ratio of phase A to phase B was 0:100;

[0023] At 11 min, the ratio of phase A to phase B was 0:100;

[0024] At 13 min, the ratio of phase A to phase B was 80:20;

[0025] At 15 minutes, the ratio of phase A to phase B was 80:20.

[0026] Preferably, the eluent in the ultra-high performance liquid chromatography process is injected into the mass spectrometer at 6 to 10 minutes of elution.

[0027] Preferably, the Full MS mode is used in the mass spectrometry process: scanning range 100-1200 m / z, resolution 35000-140000, negative ion mode, sheath gas flow rate 48-52, auxiliary flow rate 9-11, spray voltage 2.2-2.4 kV.

[0028] The present invention provides a pretreatment method for detecting short-chain chlorinated paraffins and a detection method used in conjunction with the pretreatment method, wherein the pretreatment method comprises the following steps: (1) mixing a sample to be tested, Florisil and an extraction reagent, extracting to obtain an extract; (2) purifying the extract with a silica gel-Florisil composite column, eluting to obtain an eluent; (3) removing the extraction reagent from the eluent to obtain a treated sample. The present invention adjusts and optimizes the pretreatment steps and instrumental analysis conditions, studies the SCCPs extraction efficiency of different reagents, the selection of extraction methods, the amount of solid phase extraction column filler and the amount of reagent used, and establishes a SCCPs analysis method suitable for a Chinese medicinal material matrix. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the recovery rate of different extraction reagents;

[0030] Figure 2 is the total ion current of SCCPs in the blank sample;

[0031] Figure 3 is the total ion current of SCCPs in the spiked sample;

[0032] Figure 4 The chromatograms of C10 monomers are C 10 H 17Cl5, C 10 H 16 Cl6, C 10 H 15 Cl7, C 10 H 14 Cl8, C 10 H 13 Cl9, C 10 H 12 Cl 10 ;

[0033] Figure 5 The chromatograms of the C11 monomers are C 11 H 19 Cl5, C 11 H 18 Cl6, C 11 H 17 Cl7, C 11 H 16 Cl8, C 11 H 15 Cl9, C 11 H 14 Cl 10 ;

[0034] Figure 6 The chromatograms of C12 monomers are C 12 H 21 Cl5, C 12 H 20 Cl6, C 12 H 19 Cl7, C 12 H 18 Cl8, C 12 H 17 Cl9, C 12 H 16 Cl 10 ;

[0035] Figure 7 The chromatograms of C13 monomers are C 13 H 23 Cl5, C 13 H 22 Cl6, C 13 H 21 Cl7, C 13 H 20 Cl8, C 13 H 19 Cl9, C 13 H 18 Cl 10 . DETAILED DESCRIPTION

[0036] The present invention provides a pretreatment method for detecting short-chain chlorinated paraffins, comprising the following steps:

[0037] (1) mixing a sample to be tested, floral silica and an extraction reagent, extracting and obtaining an extract;

[0038] (2) purifying the extract with a silica gel-florisil composite column and eluting to obtain an eluate;

[0039] (3) Removing the extraction reagent from the eluent to obtain a processed sample.

[0040] In the present invention, the dosage ratio of the sample to be tested, Florisil and the extraction reagent in step (1) is preferably 3-8 g: 3-8 g: 45-55 mL, and more preferably 5 g: 5 g: 50 mL.

[0041] The extraction reagent preferably includes n-hexane and dichloromethane, and the volume ratio of n-hexane to dichloromethane is preferably 1:0.8-1.2, and more preferably 1:1;

[0042] The extraction conditions are preferably: extraction temperature 95-105°C, heating time 4-6 min, static extraction time 8-12 min, flushing volume 45-55%, nitrogen purge time 50-70 s, and more preferably: extraction temperature 100°C, heating time 5 min, static extraction time 10 min, flushing volume 50%, nitrogen purge time 60 s. The number of extractions is preferably 2-4 times, and more preferably 3 times.

[0043] In the present invention, the silica gel-florisil composite column in step (2) is filled with floral silica, silica gel, acidic silica gel and anhydrous sodium sulfate in order from bottom to top, and the mass ratio of floral silica, silica gel, acidic silica gel and anhydrous sodium sulfate is preferably 2.3-2.7:2.3-2.7:3.3-3.7:1.3-1.7, and more preferably 2.5:2.5:3.5:1.5;

[0044] In the present invention, the florisil, silica gel and anhydrous sodium sulfate are baked in a muffle furnace at 550° C. for 5 hours before the experiment to remove the interference of chlorinated paraffin in the filler; 44% acidic silica gel is prepared by adding 44g of concentrated sulfuric acid to 100g of silica gel.

[0045] The purification method is preferably: eluting the silica gel-florisil composite column with n-hexane and adding the extract to purify;

[0046] The elution method is preferably: adding n-hexane to a silica gel-florisil composite column to elute impurities, and then adding an extract to obtain an eluent.

[0047] In the present invention, nitrogen is preferably used to blow dry the eluent in step (3), and the treated sample is preferably re-dissolved in methanol before use.

[0048] The present invention also provides application of the pretreatment method in reducing background interference in a detection process of short-chain chlorinated paraffins, wherein the object of detection is traditional Chinese medicine.

[0049] The present invention also provides a detection method used in conjunction with the pretreatment method, comprising the following steps: re-dissolving the treated sample with methanol, and then detecting with ultra-high performance liquid chromatography-tandem mass spectrometry.

[0050] In the present invention, the ultra-high performance liquid chromatography process preferably uses a Thermo Scientific AccucoreC18 (2.1×100mm, 2.6μm) chromatographic column, with a column temperature of 38-42°C, an injection volume of 1-3μL, and a nitrogen flow rate of 0.25-0.35mL / min. It is further preferred that the column temperature is 40°C, the injection volume is 2μL, the nitrogen flow rate is 0.3mL / min, and the ionization source is preferably set in ESI negative ion mode.

[0051] Preferably, in the ultra-high performance liquid chromatography process, water is used as phase A and 0.03-0.05 mmol / L tetramethylammonium chloride methanol solution is used as phase B, and more preferably water is used as phase A and 0.04 mmol / L tetramethylammonium chloride methanol solution is used as phase B. The elution procedure is preferably: at 0 min, phase A: phase B is 80:20;

[0052] At 4 min, the ratio of phase A to phase B was 80:20;

[0053] At 6 min, the ratio of phase A to phase B was 0:100;

[0054] At 11 min, the ratio of phase A to phase B was 0:100;

[0055] At 13 min, the ratio of phase A to phase B was 80:20;

[0056] At 15 minutes, the ratio of phase A to phase B was 80:20.

[0057] In the present invention, the eluent in the ultra-high performance liquid chromatography process is preferably injected into the mass spectrometer at 6 to 10 minutes of elution.

[0058] In the present invention, the Full MS mode is preferably used in the mass spectrometry process: scanning range 100-1200m / z, resolution 35000-140000, negative ion mode, sheath gas flow rate 48-52, auxiliary flow rate 9-11, spray voltage 2.2-2.4kV, further preferably: scanning range 200-1000m / z, resolution 70000, negative ion mode, sheath gas flow rate 50, auxiliary flow rate 10, spray voltage 2.3kV.

[0059] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0060] Materials used in the following examples:

[0061] 1. Experimental Materials

[0062] The Chinese medicinal materials in this study were collected from the authentic medicinal materials planting base in Shandong Province from September to November 2022. The samples were collected from the planting base and processing plant. Among them, 104 samples of American ginseng were produced in Weihai, 100 samples of Ganoderma lucidum were produced in Liaocheng, and 103 samples of Dendrobium candidum were collected from Dezhou. In the experiment, commercial products such as medicinal wine and ointment were not included in this study. The samples were crushed into a homogenate by a multi-functional grinder and sealed and frozen for later use.

[0063] 2. Experimental reagents are shown in Table 1.

[0064] Table 1 Main reagents used in the experiment

[0065]

[0066] Florisil, silica gel, and anhydrous sodium sulfate were baked in a muffle furnace at 550°C for 5 hours before the experiment to remove the interference of chlorinated paraffin in the filler; 44% acidic silica gel was prepared by adding 44g of concentrated sulfuric acid to 100g of silica gel.

[0067] 3. Experimental instruments are shown in Table 2

[0068] Table 2 Main instruments used in the experiment

[0069]

[0070]

[0071] Example 1 Selection of extraction method

[0072] 1. Accelerated solvent extraction

[0073] Weigh 5g of Dendrobium candidum sample, add 10μL of 63% chlorine SCCPs standard stock solution to the sample matrix, set up a blank group at the same time, mix the sample and 5g of Florisil and transfer to a 34mL extraction cell, and use n-hexane-dichloromethane (1:1, v / v) as the extraction solvent. The ASE instrument conditions are set as follows: extraction temperature 100℃, heating time 5 minutes, static extraction time 10 minutes, flushing volume 50%, nitrogen purge time 60 seconds, and the whole process is cycled 3 times; collect the extract; and blow the liquid nitrogen in the extraction collection tube to concentrate it to 2mL for standby use.

[0074] After the silica gel-florisil composite purification column was eluted with 20 mL of n-hexane, the concentrate was added; 20 mL of n-hexane was used to elute the impurities; finally, 30 mL of n-hexane-dichloromethane (1:1, v / v) mixed reagent was added to elute and this part of the eluate was collected. The eluted liquid nitrogen was blown to dryness, re-dissolved with 1 mL of methanol and transferred to the injection vial for testing. A total of 5 parallel experiments and 1 blank experiment were set up.

[0075] 2. Ultrasonic extraction

[0076] Weigh 5g of Dendrobium candidum sample in a conical flask, add 10μL of 63% SCCPs standard stock solution to the sample matrix, use n-hexane-dichloromethane (1:1, v / v) as the extraction solvent, and set up a blank group at the same time. Set the ultrasonic temperature to 50℃ and the ultrasonic power to 80%. After ultrasonic extraction for 30 minutes, centrifuge at 8000r / min for 10 minutes, take the supernatant, and concentrate the collected supernatant to 2mL by nitrogen blowing for later use.

[0077] After the silica gel-florisil composite purification column was eluted with 20 mL of n-hexane, the concentrate was added; 20 mL of n-hexane was used to elute the impurities; finally, 30 mL of n-hexane-dichloromethane (1:1, v / v) mixed reagent was added to elute and this part of the eluate was collected. After the eluted liquid nitrogen was blown to dryness, it was re-dissolved with 1 mL of methanol and transferred to the injection vial. A total of 5 parallel experiments and 1 blank experiment were set up.

[0078] Example 2 Selection of extraction reagents

[0079] Weigh 5g of Dendrobium officinale sample, add 10μL of 63% chlorine SCCPs standard stock solution to the sample matrix, and set a blank group as a blank control. Mix the sample with an appropriate amount of Florisil and fill it into a 34mL extraction cell. Replace the A phase, B phase, C phase extraction reagents and ASE extraction procedures of the accelerated solvent extractor in sequence, and use methanol, n-hexane, dichloromethane, acetone, n-hexane-dichloromethane (1:1, v / v), and n-hexane-acetone (1:1, v / v) as extraction solvents respectively. Extraction was performed by the accelerated solvent extractor, with an extraction temperature of 100℃, a heating time of 5 minutes, a static extraction time of 10 minutes, a flushing volume of 50%, a nitrogen purge time of 60 seconds, and the whole process was cycled 3 times; the extract was collected; and the liquid nitrogen in the extraction collection tube was blown and concentrated to 2mL for standby use.

[0080] After the silica gel-florisil composite purification column was eluted with 20 mL of n-hexane, the concentrate was added; impurities were eluted with 20 mL of n-hexane; finally, 30 mL of n-hexane-dichloromethane (1:1, v / v) mixed reagent was added to elute and this part of the eluate was collected. The eluted liquid nitrogen was blown to dryness, re-dissolved with 1 mL of methanol and transferred to the injection vial for testing. Five parallel experiments and one blank experiment were performed for each reagent.

[0081] Example 3 Silica gel-florisil composite column filler amount

[0082] In order to effectively remove the interference of lipids, proteins, pigments, chlorine-containing pollutants and other macromolecular substances, this study selected a silica-florisil composite column filled with floral silica, silica gel, acidic silica gel and anhydrous sodium sulfate. Florisil, silica gel, acidic silica gel and anhydrous sodium sulfate were used as the objects of investigation, and the spiked recovery rate of 63% SCCPs standard with chlorine content was used as the evaluation index. A 4-factor 5-level orthogonal experimental design was used to design and optimize the filling amount of the four components of the composite column.

[0083] Florisil content ranged from 0g to 3g, silica gel content ranged from 0g to 3g, acid silica gel content ranged from 2g to 4g, and anhydrous sodium sulfate content ranged from 1.5g to 3.5g. The factor level design is shown in Table 3.

[0084] After the silica gel-florisil composite purification column was eluted with 20 mL of n-hexane, 10 μL of 63% chlorine-containing SCCPs standard stock solution was directly added; 20 mL of n-hexane was used to elute impurities; finally, 30 mL of n-hexane-dichloromethane (1:1, v / v) mixed reagent was added to elute and this part of the eluate was collected. The eluted liquid nitrogen was blown to dryness, re-dissolved with 1 mL of methanol and transferred to the injection vial for testing.

[0085] Table 3 Orthogonal experimental design scheme of composite silica gel column

[0086]

[0087]

[0088] Example 4 Silica gel-florisil composite column elution, elution volume

[0089] Weigh 5g of Dendrobium officinale sample, add 10μL of 63% chlorine content SCCPs standard stock solution to the sample matrix, and set up a blank group at the same time. Use n-hexane-dichloromethane as the extraction solvent, with a volume ratio of 1:1. Extraction is performed using an accelerated solvent extractor, with an extraction temperature of 100℃, a heating time of 5 minutes, a static extraction time of 10 minutes, a flushing volume of 50%, a nitrogen purge time of 60 seconds, and the whole process is cycled 3 times; collect the extract; and blow the liquid nitrogen in the extraction collection tube to concentrate it to 2mL for standby use.

[0090] The silica gel-florisil composite column was first eluted with 20 mL of n-hexane, and the eluent was collected in a glass centrifuge tube; after adding the reserved concentrated solution, the impurities were eluted with 20 mL of n-hexane, and the n-hexane eluent was collected in a glass centrifuge tube; finally, 30 mL of n-hexane-dichloromethane (1:1, v / v) mixed reagent was added for elution, and the columns were collected in 15 mL glass centrifuge tubes. The eluent and elution liquid were blown dry with liquid nitrogen, then re-dissolved with 1 mL of methanol and transferred to the injection vial for testing. A total of 5 parallel experiments and 1 blank experiment were set up. The amount of the eluent and eluent was evaluated by the recovery effect of the spiked eluent and eluent.

[0091] Example 5 Quality Control

[0092] All glassware in this study was soaked and ultrasonically cleaned with Decon 90 detergent, anhydrous ethanol, and n-hexane, rinsed with ultrapure water, and dried in an oven at 105°C for later use. A full-process blank experiment was set up in each group as a blank control to check whether there was contamination in the instrument, sample, and operation process. The glassware was rinsed with n-hexane and dichloromethane three times before use. The pretreatment method and instrumental analysis method established in this study were verified by parameters such as specificity, detection limit, precision, accuracy, recovery rate, and stability.

[0093] 1. Specificity

[0094] The samples of Dendrobium officinale, Ganoderma lucidum, and American ginseng, and the spiked samples of Dendrobium officinale, Ganoderma lucidum, and American ginseng were pretreated and detected using the method established in this study, and the total ion current (TIC) and SCCPs monomer mass spectrometry response intensity diagram were obtained. The specificity was evaluated by comparing the spectra.

[0095] 2. Detection limit

[0096] Based on the ratio between the detection signal and the noise, the instrument detection limit was determined with a three-fold signal-to-noise ratio (S / N=3), and the method detection limit was determined according to the sample weight and quantitative volume.

[0097] 3. Precision and accuracy

[0098] 100 ng / mL, 500 ng / mL, and 1000 ng / mL SCCPs standards were added to the blank matrix solutions of Dendrobium officinale, Ganoderma lucidum, and American ginseng, respectively, as low-concentration, medium-concentration, and high-concentration groups, and the instrumental analysis method established in this study was used for determination, with 6 parallel experiments set up for each concentration. Precision was evaluated by calculating the relative standard deviation (RSD) of parallel experiments, and accuracy was evaluated by comparing the relative deviation (RE) between the calculated concentration and the configured standard concentration.

[0099] 4. Recovery rate

[0100] Samples of Dendrobium officinale, Ganoderma lucidum and American ginseng were added with 63% chlorine content SCCPs standard stock solution, and blank samples and spiked samples were pretreated and measured according to the method established in this study. Five parallel experiments and one blank experiment were set up for each matrix. After deducting the blank concentration, the recovery rate was obtained by calculating the ratio of the measured value to the spiked amount.

[0101] Example 6 Instrumental Analysis Method

[0102] 1. Instrument conditions

[0103] The instrumental analysis used an Ultimate 3000 ultra-high performance liquid chromatograph and a Q-Exactive Orbitrap MS mass spectrometer. The chromatographic column was Thermo Scientific Accucore C18 (2.1×100mm, 2.6μm), and the column oven was set at 40°C. The injection volume was 2μL. The nitrogen flow rate was 0.3mL / min. The ionization source was set to ESI negative ion mode. Gradient elution was performed using water (A) and a methanol solution of tetramethylammonium chloride (B). As a surfactant, tetramethylammonium chloride may cause instrument contamination when injected into the mass spectrometer. Therefore, in this study, the six-way valve was adjusted only at the 6th to 10th minute of the elution of the analyte to inject the liquid chromatography eluent into the mass spectrometer to minimize the contamination of the sample matrix on the ion source. The gradient elution program is shown in Table 4.

[0104] Table 4 Liquid chromatography gradient elution program

[0105] time Phase A Phase B 0 80 20 4 80 20 6 0 100 11 0 100 13 80 20 15 80 20

[0106] The Q-Exactive Orbitrap MS used the Full MS mode: scan range 100-1200 m / z, resolution 70000, negative ion mode, sheath gas flow rate 50, auxiliary flow rate 10, and spray voltage 2.3 kV.

[0107] 2. Qualitative and quantitative analysis of monomers

[0108] The group with the highest abundance of halogenated adducts [M+Cl]-signals was selected as the scanning monitoring ions for qualitative and quantitative analysis of SCCPs. The detection range of SCCPs monomers includes 24 monomers with carbon chain lengths of 10 to 13 carbon atoms and chlorine substitution numbers of 5 to 10 chlorine atoms. The precise molecular weights of specific ions are shown in Table 5.

[0109] Table 5 SCCPs qualitative and quantitative ion information

[0110]

[0111]

[0112] The quantitative analysis adopts the deconvolution quantitative method, and the quantitative analysis of SCCPs is realized by constructing the Isqnonneg function. The SCCPs standards used for quantitative analysis are SCCPs standards with chlorine content of 51.5%, 55.5%, and 63.0%, and the standard concentration is 1 ppm.

[0113] Use formula 1 to integrate the 24 groups of homologues and calculate the proportion y of each homologue i,j

[0114]

[0115] Where i and j represent the number of homologues and the number of standard samples used, i = 1 to 24, j = 1 to 3; A i,j represents the peak area of ​​each homologue; It represents the sum of the peak areas of 24 homologues.

[0116] Matlab software was used to establish variable table A of SCCPs standard in the workspace, which represented standard samples, and the ratios of each homologue in the three SCCPs standard samples were brought into the variable table; variable table B of samples was established in the workspace, which represented samples, and the ratios of each homologue in the samples were brought into the variable table; x=lsqnonneg function was constructed, and three values ​​were obtained by operation, which respectively represented the proportions of three standard samples with different chlorine contents in the samples. The three values ​​were brought into Excel for further normalization to obtain the normalization coefficient.

[0117] Total content of sample = (total peak area of ​​sample × normalization coefficient of standard sample 1 / total peak area of ​​standard sample 1 + total peak area of ​​sample × normalization coefficient of standard sample 2 / total peak area of ​​standard sample 2 + total peak area of ​​sample × normalization coefficient of standard sample 3 / total peak area of ​​standard sample 3) × standard sample concentration;

[0118] Calculation of SCCPs monomer content = ratio of monomer peak area × total sample content.

[0119] 3. Data processing

[0120] Xcalibur software was used to perform qualitative and quantitative analysis on the instrument analysis results. Matlab software (MathWorks, USA) and Excel (Microsoft, USA) were used to calculate the normalization coefficient and concentration of SCCPs. SPSS21 (International Business Machines Corporation, USA) was used for statistical analysis, and Origin (OriginLab, USA) and Prism (GraphPad Software, USA) were used to visualize the analysis results.

[0121] result

[0122] 1. Extraction effect

[0123] (1) Extraction method

[0124] The extraction effect of the analyte mainly depends on the compatibility between the properties of the analyte and the extraction method. The optimal extraction method of SCCPs in Chinese herbal medicine matrix was explored by comparing the traditional ultrasonic extraction method and the currently widely used accelerated solvent extraction method. The recovery rate comparison between accelerated solvent extraction and ultrasonic extraction is shown in Table 6.

[0125] The spike recovery rate of the accelerated solvent extraction method was 79.93%, and the spike recovery efficiency of the ultrasonic extraction method was 63.84%. The ASE method had a higher extraction efficiency. The relative standard deviation of the ASE method was 1.53%, and the relative standard deviation of the ultrasonic extraction method was 6.71%. It can be seen that the ASE method is more stable and has better precision during extraction.

[0126] Table 6 Comparison of accelerated solvent extraction and ultrasonic extraction efficiency

[0127] Extraction method Recovery rate Relative standard deviation Accelerated Solvent Extraction 79.93％ 1.53％ Ultrasonic extraction 63.84％ 6.71％

[0128] (2) Extraction reagent

[0129] Different reagents have different extraction efficiencies during the extraction process. In order to select the reagent with the best extraction efficiency, a full-process spike recovery experiment was conducted on 4 single reagents and 2 mixed reagents. After deducting the concentration of the blank control group from the concentration of the spiked group, the spiked recovery rate of each group was calculated. The recovery rate results of different extraction reagents are shown in Figure 2. Figure 1 As shown. Among them, when n-hexane-dichloromethane and n-hexane-acetone were used as extraction reagents, the spike recovery rates were the highest, which were 73.84% and 66.94% respectively. It can be seen that the mixed reagents have a higher extraction efficiency; when using a single reagent for extraction, n-hexane has a higher spike recovery rate of 43.85%; the recovery rate of acetone is second, which is 37.81%; while the extraction efficiency of dichloromethane and methanol is low when used alone, which are 16.86% and 12.97% respectively.

[0130] 2. Purification effect

[0131] (1) Filling amount of silica gel-florisil composite column

[0132] A 20mL solid phase extraction empty column was selected and filled with Florisil, silica gel, acidic silica gel, and anhydrous sodium sulfate. The four fillers play different roles in the extraction process. In order to explore the optimal filler combination, an orthogonal design scheme was adopted to conduct a spike recovery experiment.

[0133] The results of variance analysis of orthogonal experimental design are shown in Table 7. It can be seen that the effects of floral silica, silica gel, acidic silica gel, and anhydrous sodium sulfate on the purification effect of the composite silica gel column are statistically significant (P<0.05), indicating that the composite silica gel column composed of floral silica, silica gel, acidic silica gel, and anhydrous sodium sulfate is suitable for the purification of SCCPs.

[0134] Table 7 Analysis of variance of orthogonal design experiment

[0135] factor Mean Square Degrees of Freedom F Significance Florisil 67.67 4 3.94 0.047 Silicone 150.578 4 8.768 0.005 Acidic silica gel 66.475 4 3.871 0.049 Anhydrous sodium sulfate 108.283 4 6.305 0.014 error 137.396 8

[0136] The results of the orthogonal test design range analysis are shown in Table 8. It can be seen that the range of the composite silica gel column filler is Ra 6.947, Rb is 14.244, Rc is 9.620, and Rd is 11.514. The order of the range is Rb>Rd>Rc>Ra, indicating that the order of influence of each factor on the purification effect is silica gel>anhydrous sodium sulfate>acidic silica gel>florisil. The k value shows that the filler amount groups with the highest spiked recoveries are floral silica, silica gel, acidic silica gel, and anhydrous sodium sulfate. The average recovery rate of k4 in the floral silica group is the highest, which is 49.602%. The average recovery rate of k4 in the silica gel group is the highest, which is 52.179%. The average recovery rate of k4 in the acidic silica gel group is the highest, which is 51.968%. The average recovery rate of k1 in the anhydrous sodium sulfate group is the highest, which is 54.149%. This indicates that the optimal combination for extraction effect is A4B4C4D1, that is, the composite silica gel column is filled with 2.5g floral silica, 2.5g silica gel, 3.5g acidic silica gel, and 1.5g anhydrous sodium sulfate from bottom to top.

[0137] Table 8 Orthogonal experimental design range analysis results of composite silica gel column

[0138]

[0139]

[0140]

[0141] (2) Silica gel-florisil composite column elution and elution volume

[0142] A 20 mL self-filled solid phase extraction column was used to explore the optimal elution and elution volumes. The eluents of each step were collected separately and the spike recovery was calculated. The results are shown in Table 9. The total recovery of the spiked group was 76.27%, and the extraction efficiency was similar to the results of the previous part, indicating that the experimental results were reliable. The blank experimental concentration was similar to the experimental results of the SCCPs survey in the diet of residents in Jinan City

[66] , indicating that the errors caused by the experimental operation and reagents were within an acceptable range. Among them, the SCCPs concentrations of n-hexane eluent and eluent were 1.69 ng / mL and 2.41 ng / mL, respectively, which were similar to the blank group, indicating that n-hexane had a better effect in activating and eluting impurities; the SCCPs concentration of 1-15 mL n-hexane-dichloromethane eluent was 738.47 ng / mL, indicating that 15 mL n-hexane-dichloromethane could elute most of the SCCPs in the solid phase extraction column; the SCCPs concentration of 15-30 mL n-hexane-dichloromethane eluent was 20.08 ng / mL, indicating that a small amount of SCCPs were still eluted at this time.

[0143] Table 9: Concentration of washing and eluent

[0144]

[0145] The composition of the recovered liquid was analyzed, and the total recovery concentration was 762.65 ng / mL, of which the concentrations of elution, n-hexane elution, 1-15 mL n-hexane-dichloromethane eluent, and 15-30 mL n-hexane-dichloromethane eluent accounted for 0.22%, 0.32%, 96.83%, and 2.63% of the total recovery concentration, respectively, indicating that n-hexane elution can fully activate the column and the reagent will not cause interference; after spiked, n-hexane will not cause interference when eluting impurities such as lipids and pigments The elution volume of 30 mL of n-hexane-dichloromethane accounts for 99.46% of the total recovery concentration, indicating that the elution volume of 30 mL can meet the elution requirements of the solid phase extraction column in this experiment. Therefore, the elution and elution procedures of this study are: elution with 20 mL of n-hexane, addition of concentrated solution, elution with 20 mL of n-hexane, and finally elution and collection with 30 mL of n-hexane-dichloromethane (1:1, v / v) mixed reagent.

[0146] 3. Methodological evaluation

[0147] (1) Specificity

[0148] The total ion current and monomer response signal mass spectra of SCCPs in blank samples and spiked samples are shown in Figures 2-3 As shown. It can be seen that the liquid chromatography has a certain separation effect in the 7th to 10th minute; the mass spectrum of the standard solution monomer response signal has a good response intensity at the corresponding mass-to-charge ratio, and the mass spectrum of the blank sample monomer response signal has obvious baseline noise and low monomer response signal intensity. Therefore, the method established in this study has good specificity.

[0149] (2) Detection limit

[0150] Taking the concentration corresponding to three times the signal-to-noise ratio (S / N=3) as the instrument detection limit, when the sample weight is 10g and the quantitative volume is 1mL, the detection limit of the method in this study is 0.1ng / g.

[0151] (3) Precision and accuracy

[0152] The accuracy of the matrix solutions spiked with 100ng / mL, 500ng / mL, and 1000ng / mL of Dendrobium officinale was 94.25%, 95.81%, and 98.11%; the accuracy of the matrix solutions spiked with 100ng / mL, 500ng / mL, and 1000ng / mL of Ganoderma lucidum was 93.46%, 96.11%, and 97.64%; the accuracy of the matrix solutions spiked with 100ng / mL, 500ng / mL, and 1000ng / mL of American ginseng was 95.12%, 97.21%, and 99.18%, showing good accuracy. The RSDs of Dendrobium officinale matrix were 5.15%, 4.45% and 3.52% respectively; the RSDs of Ganoderma lucidum matrix were 6.46%, 3.21% and 3.48% respectively; the RSDs of American ginseng matrix were 5.92%, 2.77% and 2.98% respectively, showing good precision.

[0153] (4) Recovery rate

[0154] The recoveries of spiked samples in the experimental process of Dendrobium officinale, Ganoderma lucidum, and American ginseng were 75.68%, 78.20%, and 82.61%, respectively. The recoveries were slightly low, which may be related to the decomposition of some SCCPs monomer compounds under high temperature and high pressure conditions during the accelerated solvent extraction process. Secondly, losses may also be caused during the elution and washing process of the solid phase extraction column. However, the recovery rate in this experiment was relatively stable.

[0155] As can be seen from the above embodiments, the present invention provides a pretreatment method for the detection of short-chain chlorinated paraffins and a detection method used in conjunction with the pretreatment method, wherein the pretreatment method comprises the following steps: (1) mixing a sample to be tested, Florisil and an extraction reagent, extracting to obtain an extract; (2) purifying the extract with a silica gel-Florisil composite column, eluting to obtain an eluent; (3) removing the extraction reagent from the eluent to obtain a treated sample. The present invention adjusts and optimizes the pretreatment steps and instrumental analysis conditions, studies the SCCPs extraction efficiency of different reagents, the selection of extraction methods, the amount of solid phase extraction column filler and the amount of reagent used, and establishes a SCCPs analysis method suitable for Chinese medicinal materials matrix.

[0156] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A pretreatment method for detecting short-chain chlorinated paraffins, characterized in that: The steps include: (1) mixing a sample to be tested, floral silica and an extraction reagent, extracting and obtaining an extract; (2) purifying the extract with a silica gel-florisil composite column and eluting to obtain an eluate; (3) Removing the extraction reagent from the eluent to obtain a processed sample.

2. The pre-treatment method according to claim 1, characterized in that: The ratio of the sample to be tested, Florisil and extraction reagent in step (1) is 3-8 g: 3-8 g: 45-55 mL; The extraction reagent includes n-hexane and dichloromethane, and the volume ratio of n-hexane to dichloromethane is 1:0.8-1.2; The extraction conditions are: extraction temperature 95-105° C., heating time 4-6 min, static extraction time 8-12 min, flushing volume 45-55%, nitrogen purge time 50-70 s, and the number of extractions is 2-4 times.

3. The pre-treatment method according to claim 1, characterized in that: The silica gel-florisil composite column in step (2) is filled with floral silica, silica gel, acidic silica gel and anhydrous sodium sulfate in order from bottom to top, and the mass ratio of floral silica, silica gel, acidic silica gel and anhydrous sodium sulfate is 2.3-2.7:2.3-2.7:3.3-3.7:1.3-1.7; The purification method is: washing the silica gel-florisil composite column with n-hexane, adding an extracting liquid for purification; The elution method is: adding n-hexane to the silica gel-florisil composite column to elute impurities, and then adding an extract to obtain an eluent.

4. The pre-treatment method according to claim 1, characterized in that: In step (3), nitrogen gas is used to blow dry the eluent, and the treated sample is re-dissolved in methanol before use.

5. Use of the pretreatment method according to any one of claims 1 to 4 in reducing background interference in the detection process of short-chain chlorinated paraffins, characterized in that: The object of detection is traditional Chinese medicine.

6. A detection method used in conjunction with the pretreatment method according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: re-dissolving the treated sample with methanol, and then detecting with ultra-high performance liquid chromatography-tandem mass spectrometry.

7. The detection method according to claim 6, characterized in that: The ultra-high performance liquid chromatography process uses a Thermo Scientific Accucore C18 (2.1×100 mm, 2.6 μm) chromatographic column, with a column temperature of 38-42° C., an injection volume of 1-3 μL, a nitrogen flow rate of 0.25-0.35 mL / min, and an ionization source set to ESI negative ion mode.

8. The detection method according to claim 6, characterized in that: In the ultra-high performance liquid chromatography process, water is used as phase A, and 0.03-0.05 mmol / L tetramethylammonium chloride methanol solution is used as phase B. The elution procedure is: at 0 min, phase A: phase B is 80:20; At 4 min, the ratio of phase A to phase B was 80:20; At 6 min, the ratio of phase A to phase B was 0:100; At 11 min, the ratio of phase A to phase B was 0:100; At 13 min, the ratio of phase A to phase B was 80:20; At 15 minutes, the ratio of phase A to phase B was 80:

20.

9. The detection method according to claim 6, characterized in that: The eluent in the ultra-high performance liquid chromatography process is injected into the mass spectrometer at the 6th to 10th minute of elution.

10. The detection method according to claim 6, characterized in that: The FullMS mode was used in the mass spectrometry process: scanning range 100-1200 m / z, resolution 35000-140000, negative ion mode, sheath gas flow rate 48-52, auxiliary flow rate 9-11, spray voltage 2.2-2.4 kV.

Images