Purification column and detection method for p-phenylenediamine and product in aquatic product

Abstract

The invention relates to a purification column and a detection method for p-phenylenediamine and a product in an aquatic product, a filler layer is composed of a C18 filler and a Florisil filler, and the filler and the Florisil filler are mixed in proportion and filled in a column body. The invention also provides a method for detecting p-phenylenediamine and a product in an aquatic product. The method comprises the following steps: preparing a sample; adding an interior label and an antioxidant; an extraction process; a purification process; sample concentration and detection analysis; by optimizing extraction, purification and detection conditions, the detection sensitivity and accuracy of the p-phenylenediamine antioxidant and the degradation product thereof in aquatic products are remarkably improved, and the problems that in an existing method, the extraction efficiency is low, the detection sensitivity is not high, and a target compound is easy to degrade are solved. The high efficiency and repeatability of the method are verified, and the method is suitable for large-scale detection of aquatic product samples.

Description

Technical Field

[0001] The invention relates to the technical field of food safety detection, in particular to a purification column and a detection method for p-phenylenediamine and products in aquatic products. Background Art

[0002] Paraphenylenediamine antioxidants (PPDs) are an important class of antioxidants, which are widely used in tires, rubber shoes, sports equipment, wires and cables. Common types include N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), N-isopropyl-N'-phenyl-p-phenylenediamine (IPPD), N-cyclohexyl-N'-phenyl-p-phenylenediamine (CPPD), N-(1,4-dimethylpentyl)-N'-phenyl-p-phenylenediamine (7PPD), etc. These compounds enter the environment directly or indirectly, and are oxidized or degraded into a series of other compounds, causing secondary environmental pollution.

[0003] In 2021, literature confirmed that the oxidation product N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediaminequinone (6PPDQ) generated by the reaction of 6PPD with ozone is acutely toxic to coho salmon. This research result has attracted the attention of many scientists to the environmental residues, biological toxicity and metabolic mechanisms of p-phenylenediamine antioxidants (PPDs) and their transformation products. In addition to its high toxicity to coho salmon, 6PPDQ has also been shown to be acutely toxic to brook trout, rainbow trout and white-striped trout. In addition, 6PPD and 6PPDQ can cause neurotoxicity and developmental toxicity to zebrafish, and also have adverse effects on mice, nematodes, etc. Therefore, p-phenylenediamine antioxidants and their products, p-phenylenediamine and their products, may also be potentially toxic to humans, which requires timely understanding of the residual amounts of PPDs and their products in aquatic products, timely control of risks, and reduction of hazards.

[0004] At present, although PPDs and their transformation products have been widely detected in environmental media such as sediments, air and water, there are few studies on them in organisms and food. Since aquatic products are in direct contact with environmental water and sediments, PPDs and their products may be enriched in aquatic products, so it is necessary to monitor the residues of PPDs and their products in aquatic products. Although the existing literature briefly describes the detection methods of PPDs and their products in water and sediments, these methods have problems such as few types of detection products, low sensitivity, and simple purification process. However, the matrix of aquatic products is complex and the matrix effect is strong, so simple extraction and purification methods are difficult to apply. In addition, this type of compound is easily oxidized and decomposed, and its decomposition needs to be controlled to improve detection accuracy.

[0005] Therefore, a sensitive, stable and antioxidant detection method for detecting p-phenylenediamine antioxidants and their products in aquatic products is proposed.

[0006] Therefore, it is necessary to establish a sensitive, stable and anti-oxidation detection method in aquatic products. This patent selects 6PPD, IPPD and its products 6PPDQ, IPPDQ, 4-hydroxydiphenylamine (4-HDPA), 4-aminodiphenylamine (4-ADPA), 4-nitrosodiphenylamine (4-NDPA), 4- as the detection targets. The detection technology focuses on slowing down the degradation of the target substance, reducing the matrix effect, and improving the detection sensitivity of the compound to obtain more accurate detection results. Summary of the invention

[0007] The object of the present invention is to provide a purification column and a detection method for p-phenylenediamine and its products in aquatic products, which have the advantages of high efficiency, stability, sensitivity and antioxidant properties, and solve the problems in the background technology.

[0008] To achieve the above-mentioned purpose, the present invention provides the following technical solution: a purification column for p-phenylenediamine and products in aquatic products, comprising a packing layer and a column body, and also comprising a column bolt disposed in the column body, wherein the packing layer is located in the middle and lower space of the column body, a sieve plate is provided on the inner bottom wall of the column body, the bottom of the column body is penetrated and fixedly connected with a liquid outlet, and the packing layer is composed of C18 packing and Florisil packing, which are mixed in proportion and filled in the column body.

[0009] Preferably, the particle size of the C18 filler is 40-60 μm, and the particle size of the Florisil filler is 60-100 μm.

[0010] Preferably, the mass ratio of the C18 filler to the Florisil filler is 2:1.

[0011] Preferably, the purification column is suitable for a liquid chromatography-tandem triple quadrupole mass spectrometry detection system.

[0012] A method for detecting p-phenylenediamine and its products in aquatic products comprises the following steps: S1. Sample preparation: Take more than 200g of the edible part of the aquatic product and homogenize it; Accurately weigh 5.0 g of aquatic product sample into a 50 mL plastic centrifuge tube; S2. Add antioxidants: Add 50 μL of 6-PPDQ internal standard at a concentration of 1 mg / L; Add 1 mL of antioxidant solution; S3, extraction process: Shake well, let stand for 10 minutes, then add 10.0mL acetonitrile (containing 0.1% NaOH (1mol / L)); After vortex extraction for 5 minutes, 3 g of sodium chloride was added and shaken for 1 minute; Centrifuge at 4400 r / min for 5 minutes; S4, purification process: Take 6 mL of the supernatant and add it to the penetration purification column. Use positive pressure to make the extract pass through the purification column. After the extract has completely penetrated the purification column, use a graduated test tube to collect the above-mentioned outflow liquid and add 1 mL of antioxidant solution at the same time; In a 35°C water bath, blow with nitrogen until the volume is below 1 mL; S5. Sample concentration: Make up to 1 mL with acetonitrile; Pass through a 0.22 μm organic filter membrane into a sample injection vial; S6. Detection and analysis: Liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS / MS) was used for detection; The internal standard method was used for quantitative analysis.

[0013] Preferably, the antioxidant solution is composed of a mixed solution of 10 g / L ascorbic acid and 10 g / L 3,5-di-tert-butylphenol dissolved in 100 mL acetonitrile / water (1 / 1).

[0014] Preferably, the temperature of the nitrogen drying is 35°C.

[0015] Preferably, the mobile phase of the liquid chromatography is acetonitrile and 5 mmol ammonium acetate aqueous solution.

[0016] Preferably, the concentrations of L-ascorbic acid and 3,5-di-tert-butylphenol in the antioxidant solution are 10 g / L respectively.

[0017] Preferably, the mobile phase A in the liquid chromatography-tandem triple quadrupole mass spectrometry detection is acetonitrile, the mobile phase B is 5 mmol ammonium acetate aqueous solution, the flow rate is 0.3 mL / min, the gradient program is: 0.0∼1.5 minutes 5% A, 1.5∼6.0 minutes 5∼95% A, 6.0∼7.0 minutes 95% A, 7.0∼7.4 minutes 95∼5% A, 7.4∼8.0 minutes 5% A, the injection volume is 5 μL, and the column temperature is 30°C.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention significantly improves the recovery rate and purification efficiency of the target compound by setting an optimized C18 and Florisil mixed filler purification column. The optimized purification column can effectively remove interfering substances in aquatic products, especially phospholipids, carnitine and glycerides, ensure the purity of the sample, and thus improve the accuracy and sensitivity of the detection results.

[0019] The present invention extracts the target compound to the maximum extent by selecting an appropriate extraction solvent and extraction method. Acetonitrile is used as the extraction solvent, combined with a salting-out assisted extraction method, which significantly improves the extraction efficiency, reduces the degradation of the target compound in the sample, and ensures the efficiency and stability of the extraction process.

[0020] The present invention adopts a combined antioxidant to prevent the oxidative degradation of the target compound, thereby improving the recovery rate. The combined antioxidant of L-ascorbic acid and 3,5-di-tert-butylphenol is used to effectively prevent the oxidative degradation of the target compound during the extraction and purification process, thereby ensuring a high recovery rate and the stability of the detection results.

[0021] The present invention optimizes the detection conditions of liquid chromatography-tandem triple quadrupole mass spectrometry and improves the detection sensitivity and accuracy. By selecting a suitable mobile phase and gradient program and optimizing the detection conditions, the detection limit of the target compound in a complex matrix is ​​significantly reduced, and the detection results are more accurate and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is an analysis diagram of the effects of different extraction conditions on the extraction efficiency of the target compound of the present invention; Figure 2 This is an analysis diagram of the effects of different antioxidants on the recovery rate and degradation stability of the target compound of the present invention; Figure 3 This is an analysis diagram of the effects of different purification fillers on the recovery rate of target compounds and matrix effects of the present invention; Figure 4 This is a diagram showing the effect of the self-made purification column of the present invention on the removal of phospholipid interfering substances; Figure 5 This is a diagram showing the effect of the homemade purification column of the present invention on the removal of carnitine and glyceride interferences; Figure 6 This is a diagram showing the purification effect of the homemade purification column of the present invention on the target substances 4-HDPA and 4-NDPA; Figure 7 This is a diagram showing the purification effect of the self-made purification column of the present invention on the crab extract; Figure 8 The chromatogram of 6PPD and IPPD detected in the white shrimp sample of the present invention; Fig. 9 It is a schematic diagram of the composition and structure analysis of the purification column of the present invention; Fig.10 This is a flow chart of the method for detecting p-phenylenediamine and products in aquatic products according to the present invention; Fig.11 Schematic diagram of specific parameters and conditions of each key step in the detection method of the present invention; Fig.12 It is a schematic diagram of the structure of the purification column of the present invention.

[0023] In the figure: 1, column bolt; 2, column body; 3, sieve plate; 4, packing layer; 5, sieve plate; 6, liquid outlet. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] See also Figures 1 to 12 The present invention provides a technical solution: a purification column for p-phenylenediamine and products in aquatic products, comprising a packing layer 4 and a column 2, characterized in that: it also includes a column plug 1 placed in the column 2, the packing layer 4 is in the middle and lower space of the column 2, a sieve plate 5 is provided on the inner bottom wall of the column 2, the bottom of the column 2 is penetrated and fixedly connected with a liquid outlet 6, the packing layer is composed of C18 packing and Florisil packing, which are mixed in proportion and filled in the column; The present invention provides a purification column, which is used to detect the residues of p-phenylenediamine and products in aquatic products. The core component of the purification column is a filler layer, which is composed of C18 filler and Florisil filler. The C18 filler has good hydrophobicity and can effectively adsorb and separate target compounds. The Florisil filler has good polar separation performance and can further remove impurities in the sample. The two fillers are mixed in proportion and filled in the column to form an efficient purification system, which can significantly reduce the matrix effect during the detection process and improve the detection sensitivity and accuracy; The particle size of C18 filler is 40-60 μm, and the particle size of Florisil filler is 60-100 μm; To further optimize the purification effect, the present invention specifies the particle size range of C18 filler and Florisil filler. The particle size of C18 filler is 40-60 μm, ensuring that it has sufficient specific surface area to effectively adsorb the target compound while maintaining a low pressure drop. The particle size range of Florisil filler is 60-100 μm. The selection of this range takes into account the separation performance and fluidity of the filler, ensuring that impurities can be efficiently removed during the sample purification process and improving the recovery rate of the target compound; The mass ratio of C18 filler to Florisil filler is 2:1; The present invention optimizes the mass ratio of C18 filler and Florisil filler and determines that the optimal ratio is 2:1. The selection of this ratio is based on experimental data, which shows that at this ratio, the purification column can most effectively remove interfering substances in the sample while maintaining a high recovery rate of the target compound. The filler layer configured in this ratio can show a consistent purification effect in different types of aquatic product matrices; The cleanup column is suitable for liquid chromatography-tandem triple quadrupole mass spectrometry detection system. The cleanup column is designed for use with liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS / MS) detection system. This detection system has high sensitivity and high selectivity and can accurately quantify the target compounds in the sample. During the sample pretreatment stage, the cleanup column can effectively remove matrix interferences to ensure that the target compounds are accurately detected after entering the LC-MS / MS system. This configuration can significantly improve the detection accuracy and stability of p-phenylenediamine and its products in aquatic products; A method for detecting p-phenylenediamine and its products in aquatic products comprises the following steps: S1. Sample preparation: Take more than 200g of the edible part of the aquatic product and homogenize it; Accurately weigh 5.0 g of aquatic product sample into a 50 mL plastic centrifuge tube; Sample preparation is the first critical step in the testing process to ensure that the sample is representative. To obtain accurate test results, more than 200g of the edible part of the aquatic product needs to be taken and homogenized. Homogenization ensures the uniformity of the sample, making the test results more representative. Then, accurately weigh 5.0g of the homogenized sample and place it in a 50mL plastic centrifuge tube in preparation for subsequent processing.

[0026] S2. Add antioxidants: Add 1 mL of antioxidant solution; In sample processing, adding antioxidants is an important step to ensure the recovery rate of target compounds. Antioxidant solution (a mixed solution of 10 g / L ascorbic acid and 10 g / L 3,5-di-tert-butylphenol dissolved in 100 mL acetonitrile / water (1 / 1)) can effectively prevent the target compounds from oxidative decomposition during extraction and purification, thereby improving detection accuracy.

[0027] S3, extraction process: Shake well, let stand for 10 minutes, then add 10.0mL acetonitrile (containing 0.1% NaOH (1mol / L)); After vortex extraction for 5 minutes, 3 g of sodium chloride was added and shaken for 1 minute; Centrifuge at 4400 r / min for 5 minutes; The extraction process is a key step to separate the target compound from the sample. First, shake the sample in the centrifuge tube slightly, let it stand for 10 minutes, and then add 10.0mL of acetonitrile containing 0.1% NaOH. NaOH helps to improve the extraction efficiency, and acetonitrile as an organic solvent can effectively extract the target compound. Then, vortex the extraction for 5 minutes to ensure that the compound is fully dissolved, then add 3g of sodium chloride and shake for 1 minute to further separate impurities through the salting-out effect. Finally, centrifuge at 4400 r / min for 5 minutes to separate the supernatant.

[0028] S4, purification process: Take 6 mL of the supernatant and add it to a penetration purification column (composed of 1 g of C18 filler and 0.5 g of Florisil filler), and use positive pressure to make the extract pass through the purification column; After the extract has completely penetrated the purification column, use a graduated test tube to collect the above-mentioned outflow liquid and add 1 mL of antioxidant solution at the same time; In a 35°C water bath, blow with nitrogen until the volume is below 1 mL; The purification process is a key step to remove impurities in the sample and obtain a pure extract. Take 6 mL of the supernatant and add it to the penetration purification column. The purification column consists of 1 gram of C18 filler and 0.5 grams of Florisil filler. This combination of fillers can effectively remove interfering substances in the sample. The extract passes through the purification column through positive pressure to ensure that impurities in the sample are effectively removed. After the extract completely penetrates the purification column, use a graduated test tube to receive the outflowing liquid and add 1 mL of antioxidant solution to prevent oxidation of the target compound. Finally, blow nitrogen to less than 1 mL in a 35°C water bath to concentrate the sample.

[0029] S5. Sample concentration: Make up to 1 mL with acetonitrile; Pass through a 0.22 μm organic filter membrane into a sample injection vial; The concentrated sample was diluted to 1 mL with acetonitrile to make the sample volume suitable for LC-MS / MS analysis. Then, it was filtered through a 0.22 μm organic filter to ensure the purity of the sample and avoid clogging the chromatographic column, providing good sample conditions for subsequent analysis.

[0030] S6. Detection and analysis: Liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS / MS) was used for detection; The internal standard method was used for quantitative analysis.

[0031] Detection analysis is the final step of quantitative analysis of target compounds. Liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS / MS) is used for detection. This highly sensitive and selective detection method can accurately detect target compounds in samples. The internal standard method is used for quantitative analysis. By comparing the response values ​​of the target compound in the sample and the internal standard, the concentration of the target compound in the sample can be accurately calculated.

[0032] The present invention selects 10 g / L ascorbic acid and 10 g / L 3,5-di-tert-butylphenol as antioxidants, which are dissolved in a 100 mL acetonitrile / water (1 / 1) mixed solution. Ascorbic acid is an effective reducing agent that can prevent the target compound from being oxidized during the extraction and purification process; 3,5-di-tert-butylphenol is an antioxidant that can provide additional protection to further prevent oxidation. The combination of the two ensures the stability of the target compound and improves the accuracy and sensitivity of detection. Experimental data show that this antioxidant solution can significantly improve the recovery rate and stability of the target compound; During the sample concentration process, the nitrogen drying temperature was set to 35°C. This temperature can effectively evaporate the solvent while preventing the target compound from decomposing due to high temperature. The experimental results show that under the condition of 35°C, the nitrogen drying process can quickly and efficiently concentrate the sample while maintaining the integrity and stability of the target compound; In liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS / MS) detection, the choice of mobile phase is crucial. Acetonitrile, as an organic phase, has good solubility and low viscosity, which helps to improve separation efficiency; 5 mmol ammonium acetate aqueous solution is used as the aqueous phase to provide appropriate ionic strength and pH value to ensure good separation and detection of target compounds. Experimental data show that this mobile phase combination can significantly improve the detection sensitivity and separation of target compounds; The specific antioxidant solution of the present invention is configured to contain 10 g / L of L-ascorbic acid and 10 g / L of 3,5-di-tert-butylphenol per 100 mL of solution. Such concentration selection is based on the optimization results of experimental data and can provide the best antioxidant protection during the extraction and purification process. When the concentrations of L-ascorbic acid and 3,5-di-tert-butylphenol are 10 g / L respectively, the oxidative degradation of the target compound can be significantly prevented, ensuring the accuracy and repeatability of the detection; In the liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS / MS) detection process, the selection of mobile phase and the setting of gradient program are crucial. The present invention uses acetonitrile as mobile phase A, 5 mmol ammonium acetate aqueous solution as mobile phase B, and the flow rate is set to 0.3 mL / min. The gradient program is specifically: 0.0 to 1.5 minutes 5% A, 1.5 to 6.0 minutes 5 to 95% A, 6.0 to 7.0 minutes 95% A, 7.0 to 7.4 minutes 95 to 5% A, 7.4 to 8.0 minutes 5% A, injection volume is 5 μL, column temperature is 30 ° C. This setting can ensure good separation and high-sensitivity detection of target compounds. The experimental data support the selection of this gradient program and conditions, showing good repeatability and accuracy.

[0033] Example 1: Materials and methods and their optimization Standards and samples 6PPD, IPPD, 6PPDQ, IPPDQ, 4-ADPA, 4-NDPA, 4-HDPA and 6PPDQ-d5 were stored at -18 °C.

[0034] Reagents and tools Reagents: Acetonitrile and methanol (HPLC, 99.9%).

[0035] Ammonium acetate, formic acid (HPLC, 99.0%).

[0036] Sodium chloride (AR, 99.8%).

[0037] Reduced glutathione (HPLC, 98.0%), ethylenediaminetetraacetic acid, butylated hydroxyanisole, L-ascorbyl palmitate, 3,5-di-tert-butylbenzene, L-ascorbic acid, anhydrous sodium sulfite, 1,4-dithiothreonine.

[0038] Oasis C18 solid phase extraction column (500 mg, 6 mL), PSA solid phase extraction column (500 mg, 6 mL) and Oasis PRiMEHLB (200 mg, 6 mL) solid phase extraction column EMR-Lipid solid phase extraction column (300 mg, 3 mL) Graphitized carbon black filler (GCB, 40-60μm), PSA filler (40-60μm) and C18 filler (40-60μm). Florisil (60-100μm). Multi-walled carbon nanotubes (CMWANTs, length: ∼50μm).

[0039] equipment: MX-S Vortexer.

[0040] D-37520 Refrigerated Centrifuge.

[0041] SK8300GT ultrasonic cleaner.

[0042] Milli-Q series ultrapure water analyzer.

[0043] N-EVAP-12 Nitrogen Evaporation Apparatus.

[0044] PB936 Oaks food processor.

[0045] UPLC-MS / MS analysis ACQUITY UPLC ultra-high performance liquid chromatograph equipped with a BEH C18 column (100 mm × 2.1 mm, 1.7 μm).

[0046] The mobile phase A was acetonitrile, the mobile phase B was 5 mmol ammonium acetate aqueous solution, and the flow rate was 0.3 mL / min. The gradient program was as follows: 0.0-1.5 min 5% A, 1.5-6.0 min 5%-95% A, 6.0-7.0 min 95% A, 7.0-7.4 min 95%-5% A, 7.4-8.0 min 5% A. Injection volume: 5 μL, column temperature 30°C.

[0047] AB5500 triple quadrupole mass spectrometer was purchased from AB SCIEX, equipped with an electrospray ionization source (ESI). All compounds were determined in ESI+ mode, with an ionization voltage of 5 kV, an ion source temperature of 500°C, a spray gas of 55 psi, and a curtain gas of 35 psi.

[0048] Table 1 shows the parameters of the target analytes in mass spectrometry

[0049] Figure 1 (A) Extraction efficiency of the three solvents for the target compounds.

[0050] Figure 1 (B) Extraction efficiency of target compounds by three extraction methods.

[0051] Figure 1 (C) Extraction efficiency of target compounds at three extraction volumes.

[0052] Figure 1 (D) Effect of acidity and alkalinity on the extraction efficiency of target compounds.

[0053] This study studies the effects of different extraction solvents, different extraction methods, different extraction volumes and different acidity and alkalinity on the extraction efficiency. The extraction study used crucian carp samples as the pretreatment experimental matrix. First, the extraction efficiency of three extraction solvents, methanol, acetone and acetonitrile, on PPDs and their products was compared. The results are shown above. Figure 1A, In the case of direct extraction with three solvents, acetonitrile has the highest extraction efficiency overall.

[0054] Then three extraction methods were tested: one acetonitrile extraction, two acetonitrile extractions, and NaCl salting-out extraction (salting-out assisted extraction). The results showed that the response value of the salting-out assisted extraction method was the highest ( Figure 1 B).

[0055] On this basis, the effects of different extraction volumes on the extraction effect were compared. The results showed that there was no significant difference in the extraction efficiency of the target substance when 5 mL, 10 mL and 20 mL of acetonitrile were used for extraction ( Figure 1 C).

[0056] However, the extract needs to be purified and concentrated later, so 10 mL of extraction volume was selected. Finally, the effects of acidity and alkalinity on the extraction efficiency were compared. Acetonitrile, 1% formic acid acetonitrile, 0.1% NaOH (1N) acetonitrile and 0.2% NaOH (1N) acetonitrile solutions were used for extraction, and the results are shown in Figure 1 D.

[0057] After adding 1% formic acid solution to acetonitrile, the recovery rates of 6PPD and 4-ADPA decreased significantly. After adding NaOH solution, the recovery rates of the two increased significantly. The effects of 0.1% NaOH solution and 0.2% NaOH solution on the target substances were not significantly different, so 0.1% NaOH solution was selected to adjust the acidity and alkalinity. Example 2: Selection of Antioxidants Figure 2 (A) Effect of different antioxidants on the recovery of seven target compounds; (B) Recovery of four easily degradable compounds in different matrices over time and the effect of adding antioxidants.

[0058] Since p-phenylenediamine antioxidants are themselves a type of antioxidant, they are prone to undergo redox reactions with the matrix or oxidative free radicals in the air, which leads to low recovery rates and inaccurate detection during the detection and analysis process.

[0059] After optimization of the above conditions, the recovery rates of 6PPD, IPPD, 4-ADPA and 4-HDPA were still lower than 60%, which may be due to their own oxidative degradation.

[0060] In order to investigate the protective effects of different antioxidants on the target compounds, antioxidants glutathione, ethylenediaminetetraacetic acid, L-ascorbic acid palmitic acid, 3,5-di-tert-butylphenol, L-ascorbic acid, anhydrous sodium sulfite, and 1,4-dithiothreose were added in the addition recovery test.

[0061] The addition of L-ascorbyl palmitic acid, 3,5-di-tert-butylphenol, L-ascorbic acid and 1,4-dithiothreose can increase the recovery rates of 6PPD, IPPD, 4-ADPA and 4-HDPA to more than 60%, among which L-ascorbic acid has the most outstanding effect.

[0062] L-ascorbic acid is a highly polar substance. After salting out and stratification, L-ascorbic acid is distributed in the water phase but not in the acetonitrile layer, so the target substance cannot be protected any more. It is necessary to compound another antioxidant that is easily soluble in acetonitrile. 3,5-di-tert-butylphenol and L-ascorbic acid are selected to be used together to protect the whole process. From the results, it can be seen that the combined use of the two is more effective than the single use, and the recovery rates of the eight target substances are between 65.80-89.37%.

[0063] The degradation of four easily oxidizable compounds in shrimp and crucian carp matrix within 4 hours was compared. Figure 2 A. It was found that the four compounds in the shrimp matrix were basically not degraded within 4 hours, while the four compounds in the crucian carp matrix had been degraded by more than 50% within 4 hours, indicating that the degradation of these four compounds in different aquatic products is different.

[0064] However, after adding the above antioxidants to the crucian carp matrix, it was found that the four easily degradable compounds did not further degrade, further indicating that the antioxidant combination effectively protected the four compounds.

[0065] Example 3: Preparation of purification column, selection of filler and method verification Figure 3 (A) Recovery rate of target compounds under different purification fillers; (B) Matrix effect of target compounds under different purification materials.

[0066] During the pretreatment process, interfering substances such as proteins and lipids in aquatic products will be extracted together with the target compounds. These substances will interfere with the detection of the target compounds and will also cause contamination and damage to the chromatographic columns and instruments. Purification is required, and traditional purification columns require activation, loading, elution, and other steps, which are cumbersome and reduce the recovery rate. In order to reduce the purification steps, PSA, PRiME-HLB, EMR-Lipid, C18, CARB, CWCNTs, and Florisil purification columns are selected or made, and the added matrix standard solution is directly applied to the column for purification. The external standard method is used to compare the recovery rate and matrix effect after purification of each purification column.

[0067] From the results ( Figure 3B) shows that the matrix inhibition rate of 6PPD, IPPD and 4-ADPA is more than 70%, and Florisil has the strongest ability to remove matrix interference. From the perspective of removing oil and pigment, C18, GCB and CWCNTs have the best removal effect, but GCB and CWCNTs have strong adsorption capacity for the target, resulting in the recovery rate of all compounds being less than 40% ( Figure 3 A). The recovery rate of C18 is higher than that of GCB and CWCNTs. Therefore, C18 and Florisil purification filler are used in combination, and the purification effect is compared with the previous 7 purification columns ( Figure 3 A and 3B), the matrix effect of the compound was less than 30% and the recovery rate was higher than 60% after the combination of the two, which was better than the use of either alone. Therefore, the C18+Florisil composite column was finally selected as the rapid filtration purification column.

[0068] At the same time, the homemade purification column was investigated for its effect on removing typical lipid interfering substances such as phospholipids, carnitine, and glyceride interfering substances, as well as its purification effect on the target substances. The results showed that the homemade purification column can effectively remove 4 phospholipid interfering substances in the matrix ( Figure 4 A and 4B) can also remove some carnitine and glyceride impurities ( Figure 5 A and 5B). In addition, the homemade purification column has the effect of removing the interference peaks of the target substances 4-HDPA and 4-NDPA ( Figure 6 A and 6B). The obvious purification effect of the homemade purification column on impurities can also be seen with the naked eye ( Figure 7 A and 7B).

[0069] Figure 4 The effect of the homemade purification column in removing phospholipid interferences; (A) Chromatographic response values ​​of the four phospholipids before purification. (B) Chromatographic response values ​​of the four phospholipids after purification.

[0070] Figure 5 Effect of homemade cleanup column in removing carnitine and glyceride interferences; (A) Chromatographic response values ​​of the three interfering substances before purification. (B) Chromatographic response values ​​of the three interfering substances after purification.

[0071] Figure 6 (A) The purification effect of the homemade purification column on the target substance 4-HDPA; (B) The purification effect of the homemade purification column on the target substance 4-NDPA.

[0072] Figure 7 (A) Loading liquid of crab extract before purification; (B) Loading liquid of crab extract after purification by homemade purification column.

[0073] Under the optimized chromatographic and mass spectrometric conditions, the prepared solvent standard solution and matrix standard solution were measured, and the mass concentration of the target compound was used as the horizontal axis, and the ratio of the target compound to the internal standard compound was used as the vertical axis to draw a standard curve. As shown in Table 2, the 7 compounds had a good linear relationship in the range of 0.1-10 ng / mL, and the correlation coefficient (r 2 ) are all greater than 0.995.

[0074] The matrix effects of fish, crab and shrimp were 70%-210%, 80%-130% and 140%-590%, respectively, which indicates that even after purification and isotope correction, some compounds still have strong matrix effects, so matrix standards are needed for quantification. At three spike levels (0.2, 0.5 and 1 μg / kg), the recoveries of fish, crab and shrimp were 70.1%-111.5%, 62.1%-111.3% and 62.7%-114.5%, respectively. The relative standard deviations were all less than 13.9%, indicating that the method has good accuracy and precision. By stepwise dilution of each matrix standard solution, the method detection limit and method quantification limit were determined with S / N≥3 and S / N≥10, and the detection limits were 0.003-0.4μg / kg and 0.01-0.8μg / kg, respectively. The specific results are shown in Table 2. Compared with the existing published methods, this method has a lower detection limit and can detect more types of compounds. It shows that this method is suitable for the analysis and detection of p-phenylenediamine and its products in aquatic products.

[0075] Table 2 shows the accuracy, precision, sensitivity, linearity and matrix effect in fish, shrimp and crab matrices

[0076] The above-established detection method was used to analyze 95 aquatic product samples in Shanghai. The results showed that 6PPD was detected in some samples with a concentration range of 0.06-1.59 μg / kg, and IPPD was detected in 2 samples with a detection concentration of 0.20 and 0.38 μg / kg. The mass spectrometry peaks of the samples are shown in Figure 8 ; This scheme establishes a UPLC / MS-MS detection method for seven compounds in aquatic products, including p-phenylenediamine antioxidants 6PPD, IPPD and their products 6PPDQ, IPPDQ, 4-ADPA, 4-HDPA and 4-NDPA. The experiment optimizes the chromatographic and mass spectrometric conditions to achieve high response values ​​for the compounds; selects suitable antioxidants to prevent the degradation of some target compounds and improve the recovery rate; compares different extraction solvents and extraction methods to obtain a higher recovery rate; and uses a rapid filtration cleanup method (multi-plugfiltration cleanup (m-PFC)) to greatly improve the purification efficiency. The final method was validated, and the precision, accuracy and sensitivity of the seven targets met the detection requirements of routine tests and evaluations. This method was used to screen 95 aquatic products, and 6PPD and IPPD were detected.

[0077] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A purification column for p-phenylenediamine and products in aquatic products, comprising a packing layer (4) and a column body (2), characterized in that: It also includes a column bolt (1) placed in the column (2), the packing layer (4) is located in the middle and lower space of the column (2), a sieve plate (5) is provided on the inner bottom wall of the column (2), and a liquid outlet (6) is penetrated and fixedly connected to the bottom of the column (2); The packing layer is composed of C18 packing and Florisil packing, which are mixed in proportion and filled in the column.

2. A purification column for p-phenylenediamine and products in aquatic products according to claim 1, characterized in that: The particle size of the C18 filler is 40-60 μm, and the particle size of the Florisil filler is 60-100 μm.

3. A purification column for p-phenylenediamine and products in aquatic products according to claim 1, characterized in that: The mass ratio of the C18 filler to the Florisil filler is 2:

1.

4. A purification column for p-phenylenediamine and products in aquatic products according to claim 1, characterized in that: The purification column is suitable for a liquid chromatography-tandem triple quadrupole mass spectrometry detection system.

5. A method for detecting p-phenylenediamine and its products in aquatic products, applied to the purification column for p-phenylenediamine and its products in aquatic products according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. Sample preparation: Take more than 200g of the edible part of the aquatic product and homogenize it; Accurately weigh 5.0 g of aquatic product sample into a 50 mL plastic centrifuge tube; S2. Add antioxidants: Add 50 μL of 6-PPDQ internal standard at a concentration of 1 mg / L; Add 1 mL of antioxidant solution; S3, extraction process: Shake well, let stand for 10 minutes, then add 10.0 mL of acetonitrile containing 0.1% NaOH; After vortex extraction for 5 minutes, 3 g of sodium chloride was added and shaken for 1 minute; Centrifuge at 4400 r / min for 5 minutes; S4, purification process: Take 6 mL of the supernatant and add it to the penetration purification column. Use positive pressure to make the extract pass through the purification column. Use a graduated test tube to collect the above-mentioned outflowing liquid. After the extract completely penetrates the purification column, add 1 mL of antioxidant solution; In a 35°C water bath, blow with nitrogen until the volume is below 1 mL; S5. Sample concentration: Make up to 1 mL with acetonitrile; Pass through a 0.22 μm organic filter membrane into a sample injection vial; S6. Detection and analysis: Liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS / MS) was used for detection; The internal standard method was used for quantitative analysis.

6. A method for detecting p-phenylenediamine and products in aquatic products according to claim 5, characterized in that: The antioxidant solution is composed of a mixed solution of 10 g / L ascorbic acid and 10 g / L 3,5-di-tert-butylphenol dissolved in 100 mL of acetonitrile / water (1 / 1).

7. A method for detecting p-phenylenediamine and products in aquatic products according to claim 6, characterized in that: The temperature of the nitrogen drying is 35°C.

8. A method for detecting p-phenylenediamine and products in aquatic products according to claim 7, characterized in that: The mobile phase of the liquid chromatography is acetonitrile and 5 mmol ammonium acetate aqueous solution.

9. A method for detecting p-phenylenediamine and products in aquatic products according to claim 8, characterized in that: The concentrations of L-ascorbic acid and 3,5-di-tert-butylphenol in the antioxidant solution are 10 g / L respectively.

10. A method for detecting p-phenylenediamine and products in aquatic products according to claim 9, characterized in that: The mobile phase A in the liquid chromatography-tandem triple quadrupole mass spectrometry detection was acetonitrile, the mobile phase B was 5 mmol ammonium acetate aqueous solution, the flow rate was 0.3 mL / min, the gradient program was: 0.0∼1.5 min 5% A, 1.5∼6.0 min 5∼95% A, 6.0∼7.0 min 95% A, 7.0∼7.4 min 95∼5% A, 7.4∼8.0 min 5% A, the injection volume was 5 μL, and the column temperature was 30°C.

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