Method for detecting migration of chlorophenol substances in bamboo wood material for food contact and articles

Abstract

The application discloses a kind of bamboo wood material and the detection method of the migration amount of chlorophenol substance in food contact product, belongs to detection technical field.The application specifically includes:1) standard solution preparation;2) sample processing;3) sample test.The application carries out migration test to sample using aqueous simulant, and immersion liquid does not need to be handled and can be directly analyzed by machine, does not need to carry out derivatization treatment, and pre-treatment process is simple.The application analyzes to-be-measured substance using ultra-high performance liquid chromatography tandem mass spectrometry analysis technology, significantly improves detection sensitivity and speed.The combination of migration processing technology and ultra-high performance liquid chromatography tandem mass spectrometry analysis technology reduces detection steps, greatly reduces the loss of target substance, and improves the accuracy of detection result.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to a method for detecting the migration of chlorophenol substances in bamboo and wood materials and products for food contact. Background Technology

[0002] Chlorinated phenols (such as pentachlorophenol) have been used in the protection of bamboo and wood for over fifty years, effectively inhibiting blue stain, mold, and decay in wood. However, these substances have significant biotoxicity to humans (damaging the central nervous system and liver and kidney function, and in severe cases, causing death), and pose a threat to the environment due to their accumulative and strong adsorption properties, drawing global attention to their harmful effects. Therefore, the Chinese National Standard GB 4806.12-2022, "National Food Safety Standard for Bamboo and Wood Materials and Products for Food Contact," clearly stipulates the migration limits of pentachlorophenol and its salts in bamboo and wood products for food contact.

[0003] Currently, methods for determining this type of migration (such as GC, GC-MS, HPLC, LC-MS) still have significant limitations:

[0004] Applicability limitations of simulants: Existing methods (such as SN / T 2204-2015) mostly use water-based or solvent-based simulants to replace food immersion tests, failing to directly use oil-based food simulants. This may result in test results that do not accurately reflect the migration risk of chlorophenol when it comes into actual contact with oil-based foods.

[0005] The pretreatment process is cumbersome and prone to errors: the sample pretreatment steps are complex, especially the critical acetylation derivatization step. Incomplete execution of this step directly affects subsequent extraction efficiency and the accuracy of the final detection results. Furthermore, it typically involves multiple steps such as extraction and nitrogen blowing concentration, which are time-consuming and prone to loss or contamination.

[0006] Low detection efficiency: Existing mainstream methods (such as GC-MS) take a long time for a single test (usually more than 30 minutes), which is difficult to meet the needs of high-throughput detection.

[0007] Therefore, there is an urgent need to develop a new detection method that should be able to: more realistically assess risks by directly using simulants of oily foods for migration testing; simplify operations and improve accuracy by significantly simplifying the pretreatment process, avoiding complex derivatization steps, and reducing error risks; improve detection efficiency by shortening the detection time for a single sample and increasing analytical throughput; and accurately determine target substances by achieving accurate and efficient determination of the migration of highly polar, high-boiling-point chlorophenolic substances in bamboo and wood materials and products used in food contact, thereby overcoming the shortcomings of existing technologies. Summary of the Invention

[0008] To address the problems existing in the prior art, the purpose of this invention is to design a technical solution for detecting the migration amount of chlorophenol substances in bamboo and wood materials and products for food contact.

[0009] The present invention is specifically implemented using the following technical solutions:

[0010] This invention provides a method for detecting the migration of chlorophenol substances in bamboo and wood materials and products for food contact, comprising the following steps:

[0011] 1) Preparation of standard solution

[0012] Preparation of standard stock solution: Weigh out the standard of the chlorophenol-containing substance to be tested and dilute to volume with methanol to obtain the standard stock solution;

[0013] Preparation of mixed standard intermediate solution: Take the standard stock solution separately and dilute to volume with methanol to obtain mixed standard intermediate solution;

[0014] Preparation of aqueous food simulant standard working solution: Transfer mixed standard intermediate solutions of different gradient masses, dilute to volume with aqueous food simulant, mix well to obtain mixed aqueous food simulant standard working solution, to be tested;

[0015] Preparation of standard working solution for simulants of oily foods: Weigh the simulant of oily foods, add mixed intermediate standard solutions of different gradient masses to obtain the standard working solution of simulants of oily foods, extract multiple times with solvent, take the supernatant and it is ready for analysis;

[0016] 2) Sample processing

[0017] According to the sample contact area specified in GB 5009.156-2016 standard, the area in contact with the food simulant in the sample was accurately determined; according to the food simulants and migration conditions specified in GB 31604.1-2023 standard, the test samples were soaked in aqueous food simulants and oily food simulants respectively.

[0018] The aqueous food simulant that had been soaked in the sample to be tested was filtered through a filter membrane and then tested.

[0019] The oily food simulant soaked in the test sample was first mixed with hexane solvent, then extracted with methanol solvent containing 0.5% formic acid. After vortexing and standing, the upper solvent layer was collected. Then, the extraction was repeated with methanol solvent containing 0.5% formic acid. The upper solvent layers were combined and filtered through a filter membrane for testing.

[0020] 3) Sample testing

[0021] The determination was performed using ultra-high performance liquid chromatography-tandem mass spectrometry;

[0022] The standard working solution in step 1) is injected sequentially from low concentration to high concentration for determination. The retention time of the target compound and the response value of the quantitative ion are recorded. A standard curve is established with the quantitative ion response value of the target compound on the ordinate and the concentration of the target compound on the abscissa.

[0023] The test solution obtained in step 2) is measured to obtain the quantitative ion response value of the target compound. The concentration of chlorophenol is calculated by substituting the value into the standard curve. The migration amount of chlorophenol is obtained by converting the sample-to-immersion solution contact area to the immersion solution volume, expressed in μg / kg or μg / dm³. 2 express.

[0024] Furthermore, the chlorophenol standard in step 1) is 2,3,4,5-tetrachlorophenol, 2,3,4,6-tetrachlorophenol, pentachlorophenol, and 2,3,5,6-tetrachlorophenol.

[0025] Furthermore, in step 1), the concentration of the standard stock solution is 20 μg / mL; and the concentration of the mixed standard intermediate solution is 1.0 μg / mL.

[0026] Furthermore, in step 1), the aqueous food simulant is 4% acetic acid or 10% ethanol, and the oily food simulant is olive oil.

[0027] Furthermore, the multiple solvent extractions of the standard working solution for the simulant of oily food in step 1) are specifically as follows: first, add hexane and mix evenly, then add methanol solvent containing 0.5% formic acid, vortex and shake, let stand, and transfer the supernatant; then add methanol solvent containing 0.5% formic acid to repeat the extraction once, and combine the supernatant.

[0028] Furthermore, in step 3), the chromatographic conditions for ultra-high performance liquid chromatography-tandem mass spectrometry are as follows: column: 1.6 μm octadecyl bonded silica column, 2.0 mm × 75 mm; column temperature: 40 °C; injection volume: 5 μL; elution conditions: gradient elution of 5 mM ammonium acetate: methanol = (40%-10%): (60%-90%) for 15 min, flow rate: 0.3 mL / min.

[0029] Furthermore, in step 3), the ultra-high performance liquid chromatography-tandem mass spectrometry conditions are as follows: ion source: electrospray ion source; scanning mode: positive and negative ion scanning; detection mode: multiple reaction monitoring; ionization voltage: 5500V, -4500V; ion source temperature: 550℃; CUR: 25.0; CAD: Medium; GS1: 55; GS2: 50.

[0030] The present invention has the following beneficial effects:

[0031] (1) In this invention, the oil simulants are used to conduct migration tests on the samples. The soaking solution can be directly analyzed after solvent extraction without derivatization. The pretreatment process is simple.

[0032] (2) The present invention uses aqueous simulants to conduct migration tests on samples. The soaking solution can be directly analyzed without treatment and without derivatization. The pretreatment process is simple.

[0033] (3) This invention employs ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) to analyze the analytes, significantly improving detection sensitivity and speed. The combination of migration processing technology and UHPLC-MS / MS reduces detection steps, greatly minimizes target analyte loss, and improves the accuracy of detection results. Attached Figure Description

[0034] Figure 1 The extracted ion chromatograms are of four chlorophenol-containing substances in bamboo and wood materials and products for food contact; namely, 1,2,3,4,5-tetrachlorophenol (3.92 min), 2,2,3,4,6-tetrachlorophenol (4.18 min), 3,5-pentachlorophenol (4.43 min), and 4,2,3,5,6-tetrachlorophenol (5.65 min).

[0035] Figure 2 Schematic diagram of recovery rates for different extraction solvents.

[0036] Figure 3 This diagram illustrates the recovery rates of extracts spiked with different formic acid concentrations. Detailed Implementation

[0037] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0038] Example 1

[0039] I. Reagents and Materials

[0040] Unless otherwise specified, all reagents used below are analytical grade; the water is grade II water as specified in GB / T6682.

[0041] Standards: Four chlorophenol-containing standard substances (2,3,4,5-tetrachlorophenol, 2,3,4,6-tetrachlorophenol, pentachlorophenol, and 2,3,5,6-tetrachlorophenol).

[0042] Methanol (chromatographic grade), acetonitrile (chromatographic grade), formic acid (analytical grade), ethanol (analytical grade), acetic acid (analytical grade), ammonium acetate (analytical grade), refined olive oil.

[0043] II. Instruments and Equipment

[0044] Ultra-high performance liquid chromatography-mass spectrometry, electronic balance (sensitivity 0.0001g), digitally controlled ultrasonic instrument, migration cell, ultrapure water system.

[0045] III. Measurement Procedure

[0046] 1. Preparation of standard solutions

[0047] Preparation of standard stock solutions: Accurately measure 2,3,4,5-tetrachlorophenol, 2,3,4,6-tetrachlorophenol, pentachlorophenol, and 2,3,5,6-tetrachlorophenol into 10 mL volumetric flasks, and dilute to the mark with methanol to obtain a standard stock solution with a concentration of 20 μg / mL. Store in a refrigerator at 4℃~8℃.

[0048] Preparation of mixed standard intermediate solution: Accurately pipette 0.5 mL of the above standard stock solution into a 10 mL volumetric flask, and dilute to the mark with methanol to obtain a standard intermediate solution with a concentration of 1.0 μg / mL;

[0049] Preparation of aqueous food simulant standard working solutions: 0 μL, 10 μL, 20 μL, 50 μL, 100 μL, and 200 μL of standard intermediate solution were placed in six 10 mL volumetric flasks. The solutions were then diluted to the mark with aqueous food simulants (4% acetic acid, 10% ethanol) and mixed thoroughly to obtain mixed standard working solutions with concentrations of 0 ng / mL, 1.0 ng / mL, 2.0 ng / mL, 5.0 ng / mL, 10.0 ng / mL, and 20.0 ng / mL, respectively, for testing.

[0050] Preparation of standard working solutions for oil and fat food simulants: Accurately weigh 10g (accurate to 0.01g) of oil and fat food simulant (olive oil) into six 25mL stoppered test tubes. Add 0μL, 10μL, 20μL, 50μL, 100μL, and 200μL of mixed standard intermediate stock solution respectively to obtain mixed standard working solutions with contents of 0ng / g, 1.0ng / g, 2.0ng / g, 5.0ng / g, 10.0ng / g, and 20.0ng / g. Add 3mL of n-hexane to each test tube, mix well, then add 5mL of methanol + formic acid (0.5%) mixed solvent, vortex for 5min, let stand for 5min, transfer the supernatant, and repeat the extraction once with 5mL of methanol + formic acid (0.5%). Combine the supernatants, draw up the test solution with a syringe, filter it through a 0.22μm PTFE filter membrane, and use it for determination.

[0051] 2. Sample processing

[0052] Based on the intended use and conditions of the samples to be tested, and referring to the migration test methods and conditions of GB 5009.156-2016 and GB 31604.1-2023, samples were soaked in 4% acetic acid (food simulant A), 10% ethanol (food simulant B), and olive oil (food simulant C), respectively.

[0053] The aqueous food simulant was prepared by injecting 1 mL of dilution solution into a syringe, filtering it through a 0.45 μm PTFE membrane, and then adding it to the sample for testing.

[0054] Weigh 10g (accurate to 0.01g) of olive oil food simulant into a 25mL stoppered glass centrifuge tube, add 3mL of n-hexane and mix well, add 5mL of methanol + formic acid (0.5%) solvent for extraction, vortex for 5min and let stand for 5min, transfer the supernatant; the sample is then extracted again with 5mL of methanol + formic acid (0.5%), the supernatants are combined, mixed well, and filtered through a 0.22μm PTFE filter into a sample vial for analysis.

[0055] 3. Instrument conditions

[0056] (1) Liquid chromatography conditions

[0057] Chromatographic column: Octadecyl bonded silica column (1.6μm), 2.0mm × 75mm or equivalent;

[0058] Column temperature: 40℃;

[0059] Injection volume: 5 μL;

[0060] The gradient elution conditions are shown in Table 1: mobile phase A is 5 mM ammonium acetate, and mobile phase B is methanol.

[0061] Table 1 Reference conditions for gradient elution in liquid chromatography

[0062] 0 40 60 5 10 90 8 10 90 10 40 60

[0063] (2) Mass spectrometry conditions:

[0064] Ion source: electrospray ion source; scanning method: negative ion scanning; detection method: multiple reaction monitoring; ionization voltage: -4500V; ion source temperature: 550℃; CUR: 25.0; CAD: Medium; GS1: 55; GS2: 50.

[0065] The mass spectrometry parameters of the specific substances are shown in Table 2 below.

[0066] Table 2 Reference mass spectrometry parameters for four chlorophenol-containing substances

[0067]

[0068] Figure 1 The extracted ion chromatograms are of four chlorophenol-containing substances in bamboo and wood materials and products for food contact; namely, 1,2,3,4,5-tetrachlorophenol (3.92 min), 2,2,3,4,6-tetrachlorophenol (4.18 min), 3,5-pentachlorophenol (4.43 min), and 4,2,3,5,6-tetrachlorophenol (5.65 min).

[0069] 4. Measurement Results

[0070] (1) Linear Experiment

[0071] The standard working solution in step 1) is injected sequentially from low concentration to high concentration for determination. The retention time of the target compound and the response value of the quantitative ion are recorded. A standard curve is established with the quantitative ion response value of the target compound on the ordinate and the concentration of the target compound on the abscissa.

[0072] The test solution obtained in step 2) is measured to obtain the quantitative ion response value of the target compound. The concentration of chlorophenol is calculated by substituting the value into the standard curve. The migration amount of chlorophenol is obtained by converting the sample-to-immersion solution contact area to the immersion solution volume, expressed in μg / kg or μg / dm³. 2 express.

[0073] Table 3 Standard curve, limit of detection, and limit of quantitation for the method.

[0074]

[0075]

[0076] 5. Accuracy and precision experiments of the detection method

[0077] Table 4. Accuracy and precision of the method

[0078]

[0079] Example 2: Optimization of Extraction Conditions for Oil Simulators

[0080] Target analytes in immersion solutions of oil-based simulants were extracted using organic solvent extraction. Methanol, acetonitrile, 50% methanol-water solution, 50% acetonitrile-water solution, and 0.1% formic acid-methanol solution were selected for extraction. Spike recoveries were calculated, and the results are shown in [Figure number missing]. Figure 2 .

[0081] Figure 2The results showed that 50% methanol aqueous solution and 50% acetonitrile aqueous solution could hardly extract chlorophenol-containing substances; acetonitrile could partially extract chlorophenol-containing substances, but the extraction efficiency was low; methanol and 0.1% formic acid methanol solution had relatively better extraction effects, and the extraction efficiency was even higher when 0.1% formic acid was added to methanol, especially for pentachlorophenol, which significantly enhanced the extraction efficiency, reflecting that acidic conditions are more conducive to the extraction of chlorophenol-containing substances.

[0082] To investigate the effect of acidity on extraction efficiency, a spiked recovery experiment was conducted to test the extraction efficiency of methanol solutions with five different formic acid concentrations (0.05%, 0.1%, 0.5%, 1%, and 2%) for four chlorophenol-containing substances. The results are shown in [Figure number missing]. Figure 3 .

[0083] The experimental results showed that the extraction efficiency of the four target compounds was optimal when the acidity increased from 0.05% to 0.5%. As the acidity continued to increase, the extraction efficiency decreased. Therefore, a 0.5% formic acid-methanol solution is preferred for organic solvent extraction of oily simulants.

[0084] Example 3: Determination of actual samples

[0085] The method of Example 1 was applied to test five bamboo and wood samples collected from the market. The migration amount of chlorophenol was calculated based on the ratio of the sample's contact area with the soaking solution to the volume of the soaking solution, and expressed as (μg / kg).

[0086]

Claims

1. A method for detecting the migration of chlorophenol substances in bamboo and wood materials and products for food contact, characterized in that, Includes the following steps: 1) Preparation of standard solution Preparation of standard stock solutions: Weigh the chlorinated phenol standard to be tested and dilute to volume with methanol to obtain the standard stock solution. The chlorinated phenol standard is 2,3,4,5-tetrachlorophenol, 2,3,4,6-tetrachlorophenol, pentachlorophenol and 2,3,5,6-tetrachlorophenol. Preparation of mixed standard intermediate solution: Take the standard stock solution separately and dilute to volume with methanol to obtain mixed standard intermediate solution; Preparation of aqueous food simulant standard working solution: Transfer mixed standard intermediate solutions of different gradient masses, dilute to volume with aqueous food simulant, mix well to obtain mixed aqueous food simulant standard working solution, to be tested; Preparation of standard working solution for simulants of oily foods: Weigh the simulant of oily foods, add mixed intermediate standard solutions of different gradient masses to obtain the standard working solution of simulants of oily foods, extract multiple times with solvent, take the supernatant and it is ready for analysis; The aqueous food simulant is 4% acetic acid or 10% ethanol, and the oil-based food simulant is olive oil; The specific steps for multiple solvent extractions of the standard working solution for the simulants of oily foods are as follows: first, add n-hexane and mix thoroughly, then add methanol solvent containing 0.5% formic acid, vortex, let stand, and transfer the supernatant; then, add methanol solvent containing 0.5% formic acid and repeat the extraction once more, and combine the supernatants. 2) Sample processing According to the sample contact area specified in GB 5009.156-2016 standard, the area in contact with the food simulant in the sample was accurately determined; according to the food simulants and migration conditions specified in GB 31604.1-2023 standard, the test samples were soaked in aqueous food simulants and oily food simulants respectively. The aqueous food simulant that had been soaked in the sample to be tested was filtered through a filter membrane and then tested. The oily food simulant soaked in the test sample was first mixed with hexane solvent, then extracted with methanol solvent containing 0.5% formic acid. After vortexing and standing, the upper solvent layer was collected. Then, the extraction was repeated with methanol solvent containing 0.5% formic acid. The upper solvent layers were combined and filtered through a filter membrane for testing. 3) Sample testing The determination was performed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS). The chromatographic conditions were as follows: column: 1.6 μm octadecyl-bonded silica column, 2.0 mm × 75 mm; column temperature: 40 °C; injection volume: 5 μL; elution conditions: gradient elution of 5 mM ammonium acetate: methanol = (40%-10%):(60%-90%) for 15 min, flow rate: 0.3 mL / min. The mass spectrometric conditions were as follows: ion source: electrospray ionization source; scanning mode: positive and negative ion scanning; detection mode: multiple reaction monitoring; ionization voltage: 5500 V, -4500 V; ion source temperature: 550 °C; CUR: 25.0; CAD: Medium; GS1: 55; GS2:

50. The standard working solution in step 1) is injected sequentially from low concentration to high concentration for determination. The retention time of the target compound and the response value of the quantitative ion are recorded. A standard curve is established with the response value of the quantitative ion of the target compound on the ordinate and the concentration of the target compound on the abscissa. The test solution obtained in step 2) is measured to obtain the quantitative ion response value of the target compound. The concentration of chlorophenol is calculated by substituting the value into the standard curve. The migration amount of chlorophenol is obtained by converting the sample-to-immersion solution contact area to the immersion solution volume, expressed in μg / kg or μg / dm³. 2 express.

2. The method for detecting the migration of chlorophenol in bamboo and wood materials and products for food contact as described in claim 1, characterized in that, In step 1), the concentration of the standard stock solution is 20 μg / mL; the concentration of the mixed standard intermediate solution is 1.0 μg / mL.

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