Method for detecting content of chlorophenol substances in bamboo materials and products for food contact

Abstract

The invention discloses a method for detecting the content of chlorophenol substances in a bamboo material and a product for food contact, and belongs to the technical field of detection. The method specifically comprises the steps of standard liquid preparation, sample treatment and sample testing. According to the method, a content and migration quantity pretreatment mode without derivatization treatment is adopted, and an ultra-high performance liquid chromatography-tandem mass spectrometry detection technology is matched, so that the method for testing the content of the chlorophenol substances in the bamboo and wood materials and products for food contact, which is simple in pretreatment process, accurate in test, efficient, rapid, high in sensitivity and high in practicability, is realized.

Description

Technical Field

[0001] The invention belongs to the field of detection technology, and particularly relates to a method for detecting the content of chlorophenol-containing substances in bamboo and wood materials for food contact and products. Background Art

[0002] Chlorophenols have been used for bamboo timber protection for over 50 years and are very effective in suppressing the blueing, mildewing, and decay of timber. However, as preservatives and bactericides, they also have significant biotoxicity to the human body. Inhalation or percutaneous absorption can cause damage to the central nervous system, liver, and kidneys, which can be fatal. Chlorophenols also have a cumulative and strong adsorptive property, posing a certain threat to the environment. Currently, the harm of chlorophenols has attracted global attention.

[0003] At present, the methods for measuring chlorophenol-containing substances reported in domestic and foreign literature mainly focus on gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), but these methods still have defects. For example, national standard GB 4806.12-2022 adopts GC-MS to measure the migration amount of pentachlorophenol. This method soaks wood products with food simulants. The food simulants after soaking are added with potassium carbonate and converted into potassium carbonate solution. The pentachlorophenol soaked out is extracted with n-hexane after acetylation with acetic anhydride, concentrated and fixed to volume with a nitrogen blowdown instrument, and finally measured with gas chromatography-mass spectrometry, and quantified with aldrin as internal standard. The method involves multiple steps in the pre-treatment process, which is very cumbersome, especially the acetylation step. If the acetylation is incomplete, it will lead to inaccurate test results. At the same time, the existing method adopts gas chromatography-mass spectrometry to test, and the whole test process is time-consuming, requiring about half an hour or more, and the test efficiency is low. Although the method for determining pentachlorophenol residues in food uses LC-MS, due to the influence of its matrix, it needs to be extracted with alkaline acetonitrile aqueous solution, purified, concentrated, and fixed to volume with a MAX solid-phase extraction column, and then detected and confirmed by LC-MS. The process is still cumbersome.

[0004] Therefore, it is necessary to invent a testing method with simple pretreatment and high detection efficiency, which can accurately determine the content of high-polarity, high-boiling-point chlorophenol-containing substances in bamboo and wood materials and products for food contact, thereby overcoming the shortcomings of the existing methods. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for a method for detecting the content of chlorophenols in bamboo and wood materials and products for food contact. The method achieves the goals of simple pretreatment process, accurate testing, high efficiency, rapidity, high sensitivity and strong practicality.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for detecting the content of chlorophenol-containing substances in bamboo and wood materials for food contact and products, which comprises the following steps:

[0008] 1) Preparation of standard solution

[0009] Weigh the chlorinated phenol-containing substance standard to be tested respectively, and dilute to volume with solvent to obtain a standard stock solution; pipette the standard stock solution, and dilute to volume with solvent to obtain a mixed standard intermediate solution of each chlorinated phenol-containing substance; pipette the mixed standard intermediate solution of different gradient masses, dilute to volume with solvent, and mix well to obtain a mixed aqueous food simulant standard working solution;

[0010] 2) Sample processing

[0011] Weigh the crushed sample, add methanol solvent for ultrasonic treatment, filter and collect the clear liquid after ultrasonication, add methanol to the solid residue and repeat the treatment once, filter and combine the extracts, and filter the combined extracts through a 0.22 μm filter membrane into a sampling bottle to obtain the test solution;

[0012] 3) Sample testing

[0013] Ultra-high performance liquid chromatography-tandem mass spectrometry was used for determination, and the specific parameters were as follows:

[0014] Chromatographic conditions:

[0015] Chromatographic column: Waters ACQUITY Premier BEH C18 column, 1.7 μm, 2.1 mm × 100 mm; column temperature: 30°C; injection volume: 2 μL; elution conditions: mobile phase A: 5 mM ammonium acetate, mobile phase B: methanol, gradient elution (5 mM ammonium acetate: methanol = (4-1): (6-9) for 10 min, flow rate: 0.15 mL / min;

[0016] Mass spectrometry conditions:

[0017] Ion source: electrospray ionization source; Scan mode: negative ion scanning; Detection method: multiple reaction monitoring; Ionization voltage: -4500 V; Ion source temperature: 550°C;

[0018] The standard working solution in step 2) is sequentially injected from low concentration to high concentration, and 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 as the vertical axis and the concentration of the target compound as the horizontal axis;

[0019] The test solution obtained in step 3) is measured to obtain the quantitative ion response value of the target compound, which is inserted into the standard curve to calculate the concentration of the chlorophenol-containing substance. The concentration of the chlorophenol-containing substance in the sample is converted according to the weighed sample mass and expressed in mg / kg.

[0020] Furthermore, the chlorophenol-containing substances in step 1) include 2,3,4,5-tetrachlorophenol, 2,3,4,6-tetrachlorophenol, pentachlorophenol and 2,3,5,6-tetrachlorophenol.

[0021] Furthermore, 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.

[0022] Furthermore, the ultrasonic treatment conditions in step 2) are as follows: ultrasonic temperature of 50° C., frequency of 100 Hz, and ultrasonic duration of 40 min.

[0023] Furthermore, the gradient elution in step 3) is specifically as follows:

[0024] Time / min A / ％ B / ％ 0 40 60 5 10 90 8 10 90 10 40 60

[0025] Furthermore, the mass spectrometry parameters of the chlorophenol-containing substance in step 3) are specifically:

[0026]

[0027]

[0028] The present invention has the following beneficial effects:

[0029] 1) The present invention uses a pretreatment technology combining methanol and ultrasonic extraction to extract chlorophenol-containing substances. The extract can be sampled and analyzed after filtration without the need for derivatization treatment, and the pretreatment process is simple.

[0030] 2) The present invention uses ultra-high performance liquid chromatography-tandem mass spectrometry to analyze the substance to be tested, which significantly improves the detection sensitivity and speed.

[0031] 3) The combination of the ultrasonic extraction pretreatment technology of the present invention and the ultra-performance liquid chromatography tandem mass spectrometry analysis technology reduces the detection steps, greatly reduces the loss of the target object, and improves the accuracy of the detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the extracted ion chromatogram of four chlorophenol-containing substances in bamboo and wood materials and products for food contact, including 1.2,3,4,5-tetrachlorophenol (3.92min), 2.2,3,4,6-tetrachlorophenol (4.18min), 3. pentachlorophenol (4.42min), and 4.2,3,5,6-tetrachlorophenol (5.63min).

[0033] Figure 2 The recoveries of four chlorophenol-containing substances at different ultrasonic treatment temperatures are shown in Figure 2.

[0034] Figure 3 The recovery rates of four chlorophenol-containing substances at different ultrasonic treatment times. DETAILED DESCRIPTION

[0035] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0036] Example 1:

[0037] 1. Reagents and Materials

[0038] Unless otherwise specified, the reagents used below are all analytical grade reagents; the water is grade 2 water that complies with GB / T6682.

[0039] Standards: 4 chlorophenol standard substances (2,3,4,5-tetrachlorophenol, 2,3,4,6-tetrachlorophenol, pentachlorophenol, 2,3,5,6-tetrachlorophenol).

[0040] Methanol (chromatographic grade), formic acid (analytical grade), ammonium acetate (analytical grade);

[0041] II. Instruments and Equipment

[0042] Ultra-high performance liquid chromatography-mass spectrometry, electronic balance (sensitivity 0.0001 g), digitally controlled ultrasonic device;

[0043] 3. Measurement steps

[0044] 1. Preparation of standard solution

[0045] Preparation of standard stock solution: Accurately measure a certain amount of 2,3,4,5-tetrachlorophenol, 2,3,4,6-tetrachlorophenol, pentachlorophenol, and 2,3,5,6-tetrachlorophenol in a 10 mL volumetric flask, 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°C to 8°C.

[0046] Preparation of mixed standard intermediate solution: Accurately pipette 0.5 mL of each of the above standard stock solutions into a 10 mL volumetric flask and dilute to the mark with methanol to obtain a mixed standard intermediate solution with a concentration of 1.0 μg / mL.

[0047] Preparation of standard working solution: Pipette 0 μL, 10 μL, 20 μL, 50 μL, 100 μL, and 200 μL of the standard intermediate solution into six 10 mL volumetric flasks, dilute to the mark with methanol, and mix well 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 for testing.

[0048] 2. Sample processing

[0049] Crush food contact bamboo and wood products, weigh 1g of the crushed sample, add 5mL of methanol, and sonicate at 50°C, 100Hz, and 40min. After sonication, filter the supernatant and repeat the process with 5mL of methanol to the solid residue. Filter the combined extracts and filter through a 0.22μm filter membrane into a vial for testing.

[0050] 3. Instrument conditions

[0051] (1) Liquid chromatography conditions

[0052] Chromatographic column: Waters ACQUITY Premier BEH C18 column (1.7 μm) 2.1 mm × 100 mm;

[0053] Column temperature: 30°C;

[0054] Injection volume: 2 μL;

[0055] The gradient elution conditions are shown in Table 1: mobile phase A is 5 mM ammonium acetate, and mobile phase B is methanol.

[0056] Table 1 Liquid chromatography gradient elution reference conditions

[0057] Time / min A / ％ B / ％ 0 40 60 5 10 90 8 10 90 10 40 60

[0058] (2) Mass spectrometry conditions:

[0059] Ion source: electrospray ion source; Scan mode: negative ion scan; Detection method: multiple reaction monitoring; Ionization voltage: -4500 V; Ion source temperature: 550°C.

[0060] The mass spectrometry parameters of specific substances are shown in Table 2 below.

[0061] Table 2 Reference mass spectrometry parameters of four chlorophenol-containing substances

[0062]

[0063] Figure 1This is the extracted ion chromatogram of four chlorophenol-containing substances in bamboo and wood materials and products for food contact, including 1.2,3,4,5-tetrachlorophenol (3.92min), 2.2,3,4,6-tetrachlorophenol (4.18min), 3. pentachlorophenol (4.42min), and 4.2,3,5,6-tetrachlorophenol (5.63min).

[0064] 4. Measurement results

[0065] (1) Linear experiment

[0066] Table 3 Standard curve, detection limit and quantification limit of the method

[0067]

[0068] (2) Accuracy and precision experiments of detection methods

[0069] Table 4 Accuracy and precision of the method

[0070]

[0071] Example 2: Sample extraction conditions and mobile phase optimization

[0072] Crush the food contact bamboo and wood products, weigh 1g of the crushed sample, and optimize the following conditions:

[0073] (1) Ultrasonic temperature

[0074] Methanol was used as the extraction solvent, the extraction time was 30 min, and the ultrasonic temperature was set at 30°C, 40°C, 50°C, and 60°C respectively. The spike recovery test was carried out. The results are shown in the figure. Figure 2 .

[0075] Figure 2 The results show that the recoveries of the four chlorophenol-containing substances all showed an upward trend with increasing ultrasonic temperature. However, the upward trend slowed down when the temperature exceeded 50°C, and at 60°C, the recovery of pentachlorophenol even decreased. Therefore, 50°C was selected as the optimal ultrasonic temperature.

[0076] (2) Ultrasound duration

[0077] Methanol was used as the extraction solvent, the ultrasonic temperature was 30°C, and the ultrasonic time was set to 20 min, 30 min, 40 min, 50 min, and 60 min, respectively. Spiked recovery tests were carried out, and the results are shown in the table. Figure 3 .

[0078] The results showed that the recovery rates of the four chlorophenol-containing substances gradually increased with increasing ultrasonication time, indicating that increasing ultrasonication time can maximize the dissolution of the target compounds. When the ultrasonication time reaches 40-50 minutes, the recovery rate tends to stabilize. Therefore, 40 minutes is the preferred ultrasonication time.

[0079] (3) Mobile phase selection

[0080] A mixed standard solution of four chlorophenol-containing compounds was tested using four mobile phase systems: methanol + water (90%), methanol + ammonium acetate (5 mmol / L), acetonitrile + water (90%), and acetonitrile + ammonium acetate (5 mmol / L). The results showed that the methanol + ammonium acetate (5 mmol / L) mobile phase system effectively separated the four target compounds, with the retention times shown below.

[0081] Compound 2,3,4,5-Tetrachlorophenol 2,3,4,6-Tetrachlorophenol 2,3,5,6-Tetrachlorophenol Pentachlorophenol Retention time (min) 3.92 4.18 5.63 4.42

[0082] The other three mobile phase systems could not completely separate the four target compounds; therefore, methanol + ammonium acetate (5 mmol / L) system was selected as the preferred mobile phase.

[0083] Example 3: Measurement of actual samples

[0084] This method was applied to five commercially available bamboo and wood samples. The concentration of chlorophenols in the commercially available samples was calculated by dividing the concentration obtained by the weighed sample mass (mg / kg).

[0085] Table 5 Blind sample test results

[0086]

Claims

1. A method for detecting the content of chlorophenol in bamboo and wood materials for food contact and their products, characterized in that: The following steps are involved: 1) Preparation of standard solution Weigh the chlorinated phenol-containing substance standard to be tested respectively, and dilute to volume with solvent to obtain a standard stock solution; pipette the standard stock solution, and dilute to volume with solvent to obtain a mixed standard intermediate solution of each chlorinated phenol-containing substance; pipette the mixed standard intermediate solution of different gradient masses, dilute to volume with solvent, and mix well to obtain a mixed aqueous food simulant standard working solution; 2) Sample processing Weigh the crushed sample, add methanol solvent for ultrasonic treatment, filter and collect the clear liquid after ultrasonication, add methanol to the solid residue and repeat the treatment once, filter and combine the extracts, and filter the combined extracts through a 0.22 μm filter membrane into a sampling bottle to obtain the test solution; 3) Sample testing Ultra-high performance liquid chromatography-tandem mass spectrometry was used for determination, and the specific parameters were as follows: Chromatographic conditions: Chromatographic column: Waters ACQUITY Premier BEH C18 column, 1.7 μm, 2.1 mm × 100 mm; column temperature: 30°C; injection volume: 2 μL; elution conditions: mobile phase A: 5 mM ammonium acetate, mobile phase B: methanol, gradient elution (5 mM ammonium acetate: methanol = (4-1): (6-9) for 10 min, flow rate: 0.15 mL / min; Mass spectrometry conditions: Ion source: electrospray ionization source; Scan mode: negative ion scanning; Detection method: multiple reaction monitoring; Ionization voltage: -4500 V; Ion source temperature: 550°C; The standard working solution in step 2) is sequentially injected from low concentration to high concentration, and 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 as the vertical axis and the concentration of the target compound as the horizontal axis; The test solution obtained in step 3) is measured to obtain the quantitative ion response value of the target compound, which is inserted into the standard curve to calculate the concentration of the chlorophenol-containing substance. The concentration of the chlorophenol-containing substance in the sample is converted according to the weighed sample mass and expressed in mg / kg.

2. The method for detecting the content of chlorophenol in bamboo and wood materials for food contact and products according to claim 1, wherein: The chlorophenol-containing substances in step 1) include 2,3,4,5-tetrachlorophenol, 2,3,4,6-tetrachlorophenol, pentachlorophenol and 2,3,5,6-tetrachlorophenol.

3. The method for detecting the content of chlorophenol in bamboo and wood materials for food contact and products according to claim 1, wherein: 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.

4. The method for detecting the content of chlorophenol in a food contact bamboo or wood material and its products according to claim 1, wherein: The ultrasonic treatment conditions in step 2) are as follows: ultrasonic temperature of 50° C., frequency of 100 Hz, and ultrasonic duration of 40 min.

5. The method for detecting the content of chlorophenol in bamboo and wood materials for food contact and products according to claim 1, wherein: The gradient elution in step 3) is specifically as follows:

6. The method for detecting the content of chlorophenol in bamboo and wood materials for food contact and products according to claim 1, wherein: The mass spectrometry parameters of the chlorophenol-containing substance in step 3) are specifically: 。

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