A purification column and its use

Abstract

The application provides a purification column and application thereof, and the purification column comprises a hollow column body and a first sieve plate, an alkaline alumina layer, an activated silica gel microsphere layer, an acidified silica gel layer and a second sieve plate which are sequentially filled in the hollow column body from bottom to top. The purification column is suitable for application in mass spectrometric analysis of indicative polychlorinated biphenyls and dioxin polychlorinated biphenyls. The high-efficiency purification column integrates a composite silica gel column and an alkaline alumina column into one high-efficiency purification column, realizes high-efficiency purification of polychlorinated biphenyls in food, and achieves that the solvent usage amount of each sample is only 1 / 6 of that of a traditional method, the purification cycle time length can be reduced to 1 / 9 of that of the traditional method, and the usage amount of the filler is only 1 / 7 of that of the traditional method.

Description

Technical Field

[0001] This invention belongs to the field of analysis and detection technology of organic pollutants in food, and specifically relates to a purification column and its application. Background Technology

[0002] Polychlorinated biphenyls (PCBs) are a class of synthetic organochlorine compounds. Based on the number and position of chlorine atoms on the biphenyl ring, there are 209 homologues. They were among the first persistent organic pollutants (POPs) listed in the Stockholm Convention and have received widespread international and domestic attention. These compounds are characterized by persistence, high toxicity, semi-volatility, and accumulation. They easily accumulate in the fat of organisms and can be amplified along the food chain. The WHO considers 90% of common human exposure to PCBs to be through dietary exposure. Based on their toxicological properties, PCBs can be divided into dioxin-type PCBs (composed of 12 homologues similar in structure to dioxins) and non-dioxin-type PCBs (the other 197 PCBs). Seven PCBs (PCB28, 52, 101, 118, 138, 153, and 180) are used to monitor PCB contamination levels in food samples and are known as indicator PCBs. my country's National Food Safety Standard GB 2762-2022 specifies the limits for seven indicator polychlorinated biphenyls (PCBs) in aquatic animals and their products, as well as aquatic animal fats and oils. Internationally, the European Union has set limits for indicator PCBs and dioxin-type PCBs in different types of food, including meat, fish, dairy products, eggs, aquatic animals, fats and oils, and infant formula (COMMISSION REGULATION (EU) No 1259 / 2011).

[0003] The pretreatment method for polychlorinated biphenyls (PCBs) in food includes four steps: extraction, degreasing, purification, and instrumental detection. However, due to the complexity of the food matrix, the extracted solution may contain fats, pigments, fat-soluble proteins, and other small molecule impurities, interfering with the analytes and affecting the accuracy and reliability of subsequent instrumental detection. Therefore, the purification step in the sample pretreatment process is crucial, directly affecting the qualitative and quantitative determination of the target compounds. Currently, the existing national food safety standards GB 5009.190-2014 "Determination of Indicative Polychlorinated Biphenyls in Food" and GB 5009.205-2013 "Determination of Toxicity Equivalents of Dioxins and Their Analogs in Food" specify that the purification method for PCBs employs 2-3 chromatography purification columns, including purification columns prepared with different packing materials, such as acidic silica columns, composite silica columns, and basic alumina columns, to achieve the desired sample purification effect.

[0004] There are two standardized purification methods in the existing technology. One method is to pass the sample extract through (1) a composite silica column (20mm inner diameter * 30cm length, filled with glass wool, 1.5g silver nitrate silica, 1g activated silica, 2g alkaline silica, 1g activated silica, 4g acidified silica, 2g activated silica, 2g anhydrous sodium sulfate) and (2) an alkaline alumina column (8mm inner diameter * 20cm length, filled with glass wool, 2.5g alkaline alumina, 2g anhydrous sodium sulfate) from bottom to top. The purification column (1) was pre-eluted with 30 mL of a hexane / dichloromethane mixed solvent with a volume ratio of 97:3, then 1 mL of polychlorinated biphenyl extract was loaded onto the column, followed by elution with 50 mL of a hexane / dichloromethane mixed solvent with a volume ratio of 97:3. The purification column (2) was pre-eluted with 15 mL of hexane, then the eluent from column (1) was concentrated to 1 mL and loaded onto the column, followed by elution with 30 mL of hexane and 25 mL of a hexane / dichloromethane mixed solution with a volume ratio of 95:5. This purification method is suitable for gas chromatography-mass spectrometry, which provides accurate qualitative and quantitative analysis with minimal matrix interference and high sensitivity. However, it suffers from problems such as cumbersome preparation, large material consumption, long processing cycle, complex operation, and high requirements for analysts.

[0005] Another method involves adding concentrated sulfuric acid to the sample extract (1), shaking for 1 minute, then centrifuging to separate the sulfuric acid layer and the organic layer. The upper colorless solution is then extracted and passed through an alkaline alumina column (6 mm inner diameter * 20 cm length, filled sequentially from bottom to top with glass wool, 2.5 g alkaline alumina, and 2 g anhydrous sodium sulfate). The purified column (2) is pre-washed with 15 mL of n-hexane, then 5 mL of polychlorinated biphenyl extract is loaded onto the column, followed by elution with 30 mL of n-hexane and 25 mL of a 95:5 mixture of n-hexane and dichloromethane. This purification method is suitable for gas chromatography, but suffers from poor qualitative and quantitative analysis, poor separation and purification effects, significant matrix interference, low sensitivity (unable to analyze dioxin-type polychlorinated biphenyls), high requirements for analysts, and some comparability issues with mass spectrometry.

[0006] Therefore, there is an urgent need to develop a purification method for polychlorinated biphenyls (PCBs) in food samples that is suitable for mass spectrometry, and that is simple to operate, cost-effective, has a short processing cycle, high accuracy, and good recovery rate. Summary of the Invention

[0007] This invention provides a highly efficient purification column suitable for mass spectrometry, capable of simultaneously purifying indicator polychlorinated biphenyls (PCBs) and dioxin-type PCBs, and its application, to address the aforementioned technical problems. This invention is simple to operate, time-saving, labor-saving, economical, and efficient; it also minimizes sample interference from impurities, achieves high detection accuracy, and has good recovery rates. It helps improve the analytical efficiency of PCB samples in the food industry, especially those with complex matrices.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A purification column, the purification column comprising a hollow column and, from bottom to top, a first sieve plate, an alkaline alumina layer, an activated silica microsphere layer, an acidified silica layer, and a second sieve plate sequentially filled in the hollow column.

[0010] Preferably, the preparation of the acidified silica gel in the acidified silica gel layer is as follows: the silica microspheres are baked at 500-800℃ for 8-12 hours, concentrated sulfuric acid is added and mixed and stirred evenly, and the mixture is placed in a sealed container and placed in a shaking box and shaken until it reaches a uniform flow state, thus obtaining the acidified silica gel, wherein the mass ratio of concentrated sulfuric acid to silica microspheres is 44:56.

[0011] Preferably, the activated silica microspheres in the activated silica microsphere layer are prepared by baking the silica microspheres at 80-120℃ for 2-4 hours.

[0012] Preferably, the silica microspheres are refined chromatographic silica microspheres with a particle size of 0.063-0.100 mm and a pH of 6.5-7.5.

[0013] Preferably, the alkaline alumina in the alkaline alumina layer is prepared by baking refined chromatographic alkaline activated alumina with an activity grade of I and a particle size of 0.063-0.200 mm at 500-800℃ for 8-12 hours, wherein the pH of the refined chromatographic alkaline activated alumina is 9-10.

[0014] Preferably, the mass ratio of the alkaline alumina layer, the activated silica microsphere layer, and the acidified silica layer is 2:1:2.

[0015] Preferably, the volume of the hollow cylinder is 8-12 ml.

[0016] The purification column is applicable to the mass spectrometry analysis of indicative PCBs and dioxin-type PCBs.

[0017] Preferably, the food sample to be tested is pretreated, and the pretreatment includes extraction, degreasing, and purification in sequence; the purified sample is detected by GC-MS / MS; the purification step involves activating, loading, and eluting the purification column in sequence.

[0018] Preferably, the purification column is activated with 8-12 mL of dichloromethane and 8-12 mL of n-hexane, and after sample loading, it is eluted with 6-10 mL of a 1:1 n-hexane / dichloromethane mixed solution.

[0019] The working principle of the high-efficiency purification column for indicator polychlorinated biphenyls (PCBs) and dioxin-type PCBs in this invention is as follows:

[0020] (1) Existing methods for PCB purification use composite silica gel columns and alkaline alumina columns, requiring two or even three columns for each purification process. Each column requires collecting the filtrate, concentrating, and reloading the sample, which is cumbersome, time-consuming, and requires a large amount of solvent for each sample. The high-efficiency purification column provided by this invention can achieve high-efficiency purification of PCBs in food using only one column. It is simple and efficient to operate, eliminating the need for multiple collection, concentration, and reloading processes of the filtrate. The amount of solvent used per sample is only 1 / 6 of that of traditional methods, and the purification cycle time can be reduced from 3 hours to only 20 minutes.

[0021] (2) Existing composite silica gel columns and alkaline alumina columns use glass columns with an inner diameter of 20mm and a length of 30cm and 8mm and a length of 20cm, respectively. These not only require customization but also require the use of six different filling materials (including silver nitrate silica gel, alkaline silica gel, activated silica gel, acidified silica gel, anhydrous sodium sulfate, and alkaline alumina columns). Among these, the production of silver nitrate silica gel requires highly skilled personnel and has poor reproducibility. The high-efficiency purification column provided by this invention uses a more readily available empty column tube with an inner diameter of 15mm and a length of 8cm. It only requires filling with three filling materials (alkaline alumina, activated spherical silica gel, and 44% acidified silica gel) to achieve the purification of polychlorinated biphenyls (PCBs) in food. The amount of filling material used is only 1 / 7 of that of traditional methods, making it economical, efficient, and greatly reducing the difficulty of operation for personnel.

[0022] (3) In existing purification methods, indicator PCBs are mainly used for detection by gas chromatography and gas chromatography-mass spectrometry, while dioxin-type PCBs are mainly used for detection by high-resolution gas chromatography-high-resolution magnetic mass spectrometry. Furthermore, for the internationally common gas chromatography-tandem triple quadrupole mass spectrometer, there is a lack of corresponding pre-purification methods and high-efficiency purification columns. The PCB purification column provided by this invention can simultaneously analyze indicator PCBs and dioxin-type PCBs, and is applicable to gas chromatography-tandem triple quadrupole mass spectrometry.

[0023] (4) The polychlorinated biphenyl (PCB) purification column of the present invention is simple to prepare, the packing material is easy to obtain and the cost is extremely low. It can be industrialized and promoted on a large scale within the industry to improve the stability and reproducibility of the purification process, which has strong practical significance. Moreover, the column can be further used in conjunction with commercially available automated solid-phase extraction instruments to achieve automation.

[0024] (5) The detection method using this purification column has a simple and rapid pretreatment process, little matrix interference, good accuracy, high sensitivity, low detection limit, good recovery rate and repeatability, and can accurately and quickly obtain the content of indicator polychlorinated biphenyls and dioxin-type polychlorinated biphenyls in food.

[0025] Compared with the prior art, the advantages and positive effects of this invention are:

[0026] The present invention provides a high-efficiency purification column applicable to indicator polychlorinated biphenyls (PCBs) and dioxin-type PCBs. (1) By integrating a composite silica gel column and an alkaline alumina column into a single high-efficiency purification column, high-efficiency purification of PCBs in food is achieved, reducing the solvent usage per sample to only 1 / 6 of that of traditional methods and reducing the purification cycle time to 1 / 9 of the original. (2) By adjusting the ratio of the packing material in the composite silica gel column and the alkaline alumina column, the amount of packing material used is reduced to only 1 / 7 of that of traditional methods. (3) The PCB purification column provided by the present invention can simultaneously separate and purify indicator PCBs and dioxin-type PCBs, and can be applied to sample pretreatment in gas chromatography-tandem triple quadrupole mass spectrometry. (4) The PCB purification column provided by the present invention can be commercially mass-produced and used in conjunction with commercially available automated solid-phase extraction instruments to achieve automation. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the purification column structure provided in Embodiment 1 of the present invention;

[0028] Figure 2 Multiple reaction monitoring (MRM) chromatograms of the purification effect of Example 1 on fish oil samples;

[0029] Figure 3 Multiple reaction monitoring (MRM) chromatograms of the purification effect of Comparative Example 1 were used for fish oil samples;

[0030] Figure 4 Multiple reaction monitoring (MRM) chromatograms of the purification effect of Comparative Example 2 were used for fish oil samples;

[0031] Figure 5 Total ion (TIC) chromatogram of a large yellow croaker sample purified using conventional methods;

[0032] Figure 6 Total ion (TIC) chromatogram of a large yellow croaker sample using the purification column of Example 1.

[0033] Figure 7 The MRM spectra of PCB-28 on three different chromatographic columns in Example 4 are shown.

[0034] Figure 8 The MRM chromatograms of PCB-180 on three different chromatographic columns in Example 4 are shown.

[0035] Figure 9 This shows the relationship between different temperatures and the average recovery rate of PCBs in Example 4. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0037] Example 1

[0038] like Figure 1 As shown, this is a purification column provided in this embodiment. The purification column includes a hollow column 1 and a first sieve plate 2, an alkaline alumina layer 3, an activated silica microsphere layer 4, an acidified silica layer 5, and a second sieve plate 6, which are sequentially filled from bottom to top in the hollow column.

[0039] The preparation of the acidified silica gel in the acidified silica gel layer 5 is as follows: the silica gel microspheres are baked at 600°C for 8 hours, concentrated sulfuric acid (mass ratio of silica gel to silica gel 44:56) is added and mixed and stirred evenly, and then placed in a sealed container and placed in a shaking box and shaken until it reaches a uniform flow state.

[0040] The activated silica microspheres in the activated silica microsphere layer 4 are prepared by baking silica microspheres (0.063-0.100 mm) at 180°C for 2 hours.

[0041] The alkaline alumina in the alkaline alumina layer 3 is prepared by baking refined chromatographic alkaline activated alumina with an activity level of I and a particle size of 0.063-0.200 mm at 600°C for 8 hours. The pH of the refined chromatographic alkaline activated alumina is 9-10.

[0042] The aforementioned silica microspheres are refined chromatographic silica microspheres with a particle size of 0.063-0.100 mm, a pore size of 6 nm, and a pH of 6.5-7.5.

[0043] The mass ratio of the alkaline alumina layer 3, the activated silica microsphere layer 4, and the acidified silica layer 5 is 2:1:2.

[0044] The purification column was prepared as follows: using a 12mL hollow solid-phase extraction column 1 as a carrier, the following components were filled sequentially from bottom to top: a first sieve plate, 1g of basic alumina, 0.5g of activated silica microspheres, 1g of 44% acidified silica, and a second sieve plate. After filling, the packing material was fully compacted using a packing rod.

[0045] The hollow cylinder 1 has a volume of 12ml, an inner diameter of 15mm, and a length of 8cm.

[0046] To verify the recovery rate of the purification column of the present invention, the standard solutions of 18 indicative polychlorinated biphenyls (PCBs) and dioxin-type PCBs were purified and detected using the purification column, including the following steps:

[0047] Step 1: Activation of the high-efficiency purification column. The aforementioned purification column is first activated with 10 mL of dichloromethane and 10 mL of n-hexane, and the effluent is discarded.

[0048] Step 2: Sample loading. Load approximately 5 mL of a hexane solution containing 18 indicative PCBs and dioxin-type PCB isotope standards, and discard the eluent.

[0049] Step 3: Elution. Elute with 8 mL of a 1:1 mixture of n-hexane and dichloromethane and collect the liquid.

[0050] Step 4: Detection. Blow the eluent down to 1 mL with nitrogen, then transfer it to a gas chromatograph vial and continue blowing with nitrogen until nearly dry. Add 100 μl of nonane and 10 μl of isotope-labeled internal standard for recovery, and detect using a gas chromatography-tandem triple quadrupole mass spectrometer.

[0051] Table 1 shows the recovery rates of 18 indicator polychlorinated biphenyls (PCBs) and dioxin-type PCBs as internal standards. The recovery rates were evaluated according to the requirements of GB 5009.190 "Determination of Indicator PCBs in Food" and GB 5009.205 "Determination of Toxicity Equivalents of Dioxins and Their Analogs in Food". The average recovery rate of the 18 PCB internal standards was 97.9%, and the recovery rates of individual samples ranged from 76% to 123%, meeting and exceeding the requirements of the national standards.

[0052] Table 1 shows the recoveries of 18 indicator polychlorinated biphenyls (PCBs) and dioxin-type PCBs as quantitative internal standards.

[0053]

[0054]

[0055] To verify the recovery rate of the purification column of the present invention, the standard solutions of 18 indicative polychlorinated biphenyls (PCBs) and dioxin-type PCBs in fish oil samples were also purified and detected using the purification column, including the following steps:

[0056] Step 1: Activation of the high-efficiency purification column. The aforementioned purification column is first activated with 10 mL of dichloromethane and 10 mL of n-hexane, and the effluent is discarded.

[0057] Step 2: Sample loading. Dissolve 0.1g of the oil sample in 5mL of n-hexane, then add 18 indicative PCB and dioxin-type PCB isotope standard solutions, load the sample, and discard the eluent.

[0058] Step 3: Elution. Elute with 8 mL of a 1:1 mixture of n-hexane and dichloromethane and collect the liquid.

[0059] Step 4: Detection. Blow the eluent down to 1 mL with nitrogen, then transfer it to a gas chromatograph vial and continue blowing with nitrogen until nearly dry. Add 100 μl of nonane and 10 μl of isotope-labeled internal standard for recovery, and detect using a gas chromatography-tandem triple quadrupole mass spectrometer.

[0060] Table 2 shows the recovery rates of 18 indicator polychlorinated biphenyls (PCBs) and dioxin-type PCBs as internal standards in fish oil samples after purification column treatment. The average recovery rate was 86.3%, and the recovery rates of the internal standards for individual samples ranged from 52% to 114%, which met and exceeded the requirements of the national standard.

[0061] Table 2 Recovery rates of 18 indicator polychlorinated biphenyls (PCBs) and dioxin-type PCBs as internal standards in fish oil

[0062]

[0063]

[0064] Comparative Example 1

[0065] This comparative example provides a purification column. The purification column includes a hollow column and, from bottom to top, a first sieve plate, a 1g alkaline alumina layer, a 0.5g activated silica microsphere layer, and a second sieve plate sequentially filled into the hollow column.

[0066] Comparative Example 2

[0067] This comparative example provides a purification column. The purification column includes a hollow column and, from bottom to top, a first sieve plate, a 1g alkaline alumina layer, a 1g alkaline silica gel layer, a 0.5g activated silica gel microsphere layer, a 1g acidified silica gel layer, and a second sieve plate sequentially filled into the hollow column.

[0068] Using the application steps in Example 1, the purified columns of Example 1, Comparative Example 1, and Comparative Example 2 were analyzed using gas chromatography-tandem triple quadrupole mass spectrometry. The purification results of Example 1, Comparative Example 1, and Comparative Example 2 are as follows: Figure 2 , Figure 3 and Figure 4 As shown. The purification columns of Example 1 and Comparative Example 2 can accurately separate and quantify the target compound, while Comparative Example 1 cannot purify and separate the target compound; its target peaks PCB 28 and PCB 52 are too severely interfered with by impurities, making accurate qualitative and quantitative analysis impossible. However, Comparative Example 2 uses more types of packing materials than Example 1, increasing costs.

[0069] Comparative Example 3

[0070] For the large yellow croaker sample, both the traditional large column and the purification column of Example 1 were used. The TIC chromatograms of their purification methods are shown below. Figure 5 and Figure 6As shown. Although the purification effect and qualitative and quantitative effects of these two methods are the same, among them... Figure 5 The traditional method uses large columns, which requires more packing material, solvents, and time.

[0071] The purification column provided by this invention has the following advantages over traditional national standard analysis methods and other SPE methods mentioned in the background art, as shown in Table 3.

[0072] Table 3

[0073]

[0074]

[0075] Example 2

[0076] This embodiment provides a method for determining indicator polychlorinated biphenyls (PCBs) in food, including the following steps:

[0077] The food sample to be tested is pretreated, and the pretreatment includes extraction, degreasing and purification in sequence.

[0078] The purified samples were analyzed using GC-MS / MS.

[0079] The specific process of sample pretreatment is as follows:

[0080] 1) Extraction

[0081] Solid sample: Place 5g of sample in a ceramic mortar, add an appropriate amount of anhydrous sodium sulfate, dry and mix until flowable, then mix evenly with an appropriate amount of diatomaceous earth and transfer to an extraction tank. Add 10μL of PCBs isotope-labeled quantitative internal standard solution, seal and place on an accelerated solvent extractor for extraction. The extraction reference conditions are: extraction solvent: n-hexane:dichloromethane:acetone (2:1:1, volume ratio); pressure: 10.3MPa; temperature: 130℃; static extraction time: 10min; 1 cycle. Then, evaporate the solvent to near dryness using a rotary evaporator, and dissolve in 50ml of n-hexane.

[0082] Oil sample: Weigh 1.0g of sample (accurate to 0.01g) directly into a round-bottom flask, add 50mL of n-hexane to dissolve it, then add 10μL of PCBs isotope-labeled quantitative internal standard solution and mix well.

[0083] PCBs isotope-labeled quantitative internal standard solutions (7 types) 13 C 12 -PCBs solution) containing; PCBs isotope labeling recovery internal standard solution (containing 13 C 12 -PCB 70, 13 C12 -PCB 111 and 13 C 12 -PCB 170 solution); and a series of calibration curve standard solutions for PCBs containing natural and isotopic labels were purchased from Wellingtong, Canada. Details of each component are shown in Tables 1-3.

[0084] Table 1. Quantitative Internal Standard Solutions for Isotope Labeling of PCBs

[0085]

[0086] Table 2 Recovery rates of isotope labeling in PCBs (internal standard solutions)

[0087]

[0088] Table 3 Standard solutions for PCB calibration

[0089]

[0090] 2) Degreasing

[0091] Dissolve the sample (5.2 or 5.1.2) in 50 mL of n-hexane, add 10 g of acidified silica gel (44%, mass fraction), and shake in a water bath at 50°C for 30 min. After standing for 5-10 min, pour the supernatant into a clean round-bottom flask. Rinse the acidified silica gel in the flask twice with 10 mL of n-hexane, and combine the supernatants into the round-bottom flask. If the silica gel is dark in color, repeat the above process. Concentrate the degreased supernatant to about 5 mL using a rotary evaporator for further purification.

[0092] 3) Purification

[0093] The purification column was activated sequentially with 10 mL of dichloromethane and 10 mL of n-hexane. The extract was then transferred entirely to the purification column and loaded onto the column. Finally, elution was performed with 8 mL of a 1:1 (v / v) hexane / dichloromethane mixture, and the extract was collected. The volume was reduced to approximately 50 μL by a nitrogen stream at 50 °C. 10 μL of the PCBs isotope-labeled recovery internal standard solution was added, and the mixture was vortexed before GC-MS / MS analysis. If the sample was not to be analyzed on the same day, it was stored at <-10 °C in the dark.

[0094] The specific process of GC-MS / MS is as follows:

[0095] Gas chromatography conditions: DB-XLB capillary column (30m × 0.25mm × 0.25μm); injection port temperature 290℃; injection mode splitless injection, constant flow mode; injection volume 1μL. Transfer line temperature 290℃; temperature program: initial temperature 80℃; increase to 200℃ at 15℃ / min (hold for 1 min); then increase to 270℃ at 2.5℃ / min; increase to 310℃ at 20℃ / min (hold for 4 min). Carrier gas flow rate: high-purity helium (>99.999%), 1.2mL / min.

[0096] Mass spectrometry conditions: The quadrupole resolution should be better than or equal to the resolution per unit mass; the ionization mode is electron impact ionization (EI) at an energy of 70 eV. An EI ionization source is used at a temperature of 280 °C; the data acquisition mode is multiple reaction ion monitoring (MRM), monitoring two specific precursor ions and one daughter ion produced by each precursor ion for all compounds.

[0097] Specific information on the ion pairs of each compound can be found in Appendix Table 4.

[0098] Table 4. Monitoring ion pairs of PCBs using GC-MS / MS methods

[0099]

[0100] The applicant compared three chromatographic columns: DB-XLB (30m × 0.25mm × 0.25μm), DB-5MS (30m × 0.25mm × 0.25μm), and DB-5MS (60m × 0.25mm × 0.25μm). It was found that when using standard substances for quantitative analysis, the DB-5MS (30m × 0.25mm × 0.25μm) and DB-5MS (60m × 0.25mm × 0.25μm) columns set excessively high values ​​for PCB-28 and PCB-180, especially PCB-28, which showed values ​​approximately 1.8 times higher than the reference values. This indicates that there are interfering peaks from isomers of PCB-28 and PCB-180, which cannot be separated on either the DB-5MS (30m × 0.25mm × 0.25μm) or DB-5MS (60m × 0.25mm × 0.25μm) columns. Separation requires a DB-XLB (30m × 0.25mm × 0.25μm) column, therefore the DB-XLB column is preferred. See below for details. Figure 7 and Figure 8 It can be seen that in Figure 7 In the MRM chromatogram of PCB-28, DB-XLB could separate the chromatographic peaks of three isomers, while the two columns of DB-5MS only separated the peaks of two isomers; similarly, in Figure 8As can be seen from the MRM chromatogram of PCB-180, DB-XLB can separate two isomer peaks, while the two columns of DB-5MS can only separate one chromatographic peak.

[0101] The applicant compared accelerated solvent extraction (CSE) and Soxhlet extraction. CSE is simple, fast, and saves solvent, taking about 30 minutes per sample and consuming less than 100 ml of solvent; while Soxhlet extraction requires 18-24 hours per sample and consumes more than 200 ml of solvent.

[0102] The selection of accelerated solvent extraction instrument temperature was investigated, and the effects of different extraction temperatures (100℃, 110℃, 120℃, 130℃, 140℃, 150℃) on the average recovery rate of PCBs were compared. For example... Figure 9 As shown, the average recovery rate was around 80% at 6 different extraction temperatures, but the average recovery rate was highest at an extraction temperature of 130℃. Therefore, we finally chose an extraction temperature of 130℃ for the accelerated solvent extractor.

[0103] The linear range of the standard curve obtained by this method is:

[0104] The linear range of the polychlorinated biphenyl (PCB) correction standard solutions CS1-CS6 was 0.5-1000 ng / mL, with PCB 118 also ranging from 0.5-1000 ng / mL. The R² values ​​were between 0.9998 and 0.9999, the RRF values ​​ranged from 1.05 to 1.19, and the RSD (n=6) ranged from 1.97% to 4.36%. The standard curve showed good linearity and met the experimental requirements. Specific parameters are shown in Table 5.

[0105] Table 5 Calibration curve parameters for polychlorinated biphenyls

[0106] Linear regression equation <![CDATA[R 2 ]]> RRF RSD% PCB-28 y = 1.0599x - 0.1142 0.9999 1.05 2.31 PCB-52 y = 1.2248x - 0.1794 0.9999 1.19 1.97 PCB-101 y = 1.2115x - 0.2360 0.9999 1.17 3.02 PCB-118 y = 1.1180x - 0.1500 0.9999 1.07 4.16 PCB-138 y = 1.1080x - 0.2647 0.9999 1.06 4.36 PCB-153 y = 1.1272x - 0.0783 0.9999 1.11 4.15 PCB-180 y = 1.2100x - 0.3052 0.9998 1.16 2.52

[0107] The accuracy and precision of this method

[0108] This method uses the national primary standard reference material salmon meal (GBW10040), the European Commission certified reference material Mussel Organization (BCR-682), and the Norwegian Public Health Organization's comparison sample fish oil to evaluate the precision and accuracy of the method. Each sample was measured six times. After six parallel determinations of salmon meal (GBW10040), the mean values ​​of PCBs were all within the reference range, with relative standard deviations (RSDs) of 1.7%–4.3%. For mussel tissue (BCR-682), the mean values ​​after six parallel determinations were also within the reference range, with RSDs of 0.9%–3.8%. For fish oil samples, the Z-values ​​after six parallel determinations were all less than 2, with RSDs of 0.8%–2.7%. The precision and accuracy of this method meet the requirements for PCB analysis. See Tables 6-8 for details.

[0109] Table 6 Precision and accuracy tests of salmon meal (GBW10040)

[0110]

[0111] Table 7 Precision and accuracy tests of BCR-682 mussel tissue samples.

[0112]

[0113] Table 8 Precision and accuracy tests of fish oil samples

[0114]

[0115] Limit of quantitation for this method:

[0116] The EU Regulation (EU) 2017 / 644, Sampling and Analysis Methods for Dioxins, Dioxin-like PCBs and Non-Dioxin-like PCBs in Food, specifies the following principles for determining the limit of quantitation (LOQ) of PCBs by GC-MS / MS: The deviation between the RRF at the LOQ concentration point and the average RRF calculated from the calibration standard curve should be less than 30%, and then the LOQ of PCBs should be calculated according to formula (3).

[0117]

[0118] In the formula:

[0119] m — the amount of sample taken;

[0120] V—Volume at constant volume;

[0121] R – Recovery rate of the sample.

[0122] This method selects 0.5 ng / ml of PCBs as the limit of quantitation concentration and substitutes it into the standard curve (it can be seen that the deviation will be less than 30% within the linear range of the standard curve). Then, the lowest recovery rate (50%) of the sample is calculated from this lowest concentration point. The average sample amount (5g) and the fixed volume (50μL) are substituted into formula (3) to calculate that the limit of quantitation of PCB28, PCB52, PCB101, PCB118, PCB138, PCB153 and PCB180 is 10 ng / kg.

[0123] The above embodiments are merely illustrative examples and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations; therefore, any obvious variations or modifications derived therefrom remain within the scope of protection of this invention.

Claims

1. A purification column, characterized in that, The purification column comprises a hollow column and, from bottom to top, a first sieve plate, an alkaline alumina layer, an activated silica microsphere layer, an acidified silica layer, and a second sieve plate. The volume of the hollow column is 8-12 ml. The acidified silica in the acidified silica layer is prepared by baking silica microspheres at 500-800 ℃ for 8-12 hours, adding concentrated sulfuric acid, mixing and stirring evenly, placing in a sealed container and shaking in a shaker until a uniform flow state is achieved, thus obtaining the acidified silica. The mass ratio of concentrated sulfuric acid to silica microspheres is 44:

56. The activated silica microspheres in the activated silica microsphere layer are prepared by baking silica microspheres at 80-120 ℃ for 2-4 hours. The silica microspheres are refined chromatography silica microspheres with a particle size of 0.063-0.

100. The alkaline alumina in the alkaline alumina layer is prepared by baking refined chromatographic alkaline activated alumina with an activity grade of I and a particle size of 0.063-0.200 mm at 500-800 ℃ for 8-12 hours. The pH of the refined chromatographic alkaline activated alumina is 9-10. The mass ratio of the alkaline alumina layer, the activated silica microsphere layer, and the acidified silica layer is 2:1:

2.

2. The application of the purification column as described in claim 1 in mass spectrometry analysis of indicative polychlorinated biphenyls (PCBs) and dioxin-type PCBs.

3. The application as described in claim 2, characterized in that, The food sample to be tested is pretreated, which includes extraction, degreasing, and purification in sequence; the purified sample is detected by GC-MS / MS; the purification step involves activating, loading, and eluting the purification column in sequence.

4. The application as described in claim 3, characterized in that, The GC column used in the GC-MS / MS is DB-XLB.

Images