Method for simultaneously determining various nitrophenol compounds in different food matrices

Abstract

The method for simultaneously determining multiple nitrophenol compounds in different food matrices according to the present invention involves preparing a series of mixed solutions with different concentrations of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol using acetonitrile-acetic acid solution as standard samples; the food matrices to be tested are then subjected to extraction and purification treatment. The sample was obtained through a process; then, high-performance liquid chromatography-tandem mass spectrometry was used to detect the sample and standard to obtain the concentrations of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in the sample. The content of each nitrophenol compound in the food matrix was then calculated based on the concentrations of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in the sample.

Description

Technical Field

[0001] This invention belongs to the field of nitrophenol compound detection technology, and relates to a method for the simultaneous determination of multiple compounds such as 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in different food matrices. Background Technology

[0002] Nitrophenol compounds mainly include 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol. Among them, 2,4-dinitrophenol (2,4-DNP) is a typical mitochondrial oxidative phosphorylation uncoupling agent, which can enhance metabolism. In the 1930s, it was widely used as a "dieting aid" in weight loss drugs. However, long-term use can produce a series of side effects. Acute poisoning can cause nausea, vomiting, sweating, dizziness, and fever. Chronic poisoning can lead to cataracts, skin damage, and varying degrees of damage to the bone marrow, central nervous system, and cardiovascular system.

[0003] Current research on the detection of nitrophenol compounds largely focuses on pollution control in soil and water resources. Detection methods are limited to total nitrophenol content, lacking qualitative and quantitative methods for specific nitrophenol compounds (e.g., 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, 3,5-dinitrophenol). In the field of weight-loss foods, such as tea, chocolate, coffee, compressed candies, and solid beverages, my country currently lacks detection methods for nitrophenol compounds, and even more so, corresponding limit standards. Therefore, establishing detection methods for nitrophenol compounds in the matrix of weight-loss foods is particularly necessary. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for simultaneously determining multiple nitrophenol compounds in different food matrices, so as to achieve quantitative detection of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol contained in food, especially weight loss food, with good accuracy and precision, while reducing analysis costs.

[0005] The method for simultaneously determining multiple nitrophenol compounds in different food matrices according to the present invention involves preparing a series of mixed solutions with different concentrations of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol using an acetonitrile-acetic acid solution as a series of standard samples. The volume ratio of acetonitrile to acetic acid in the acetonitrile-acetic acid solution is 99:1. The food matrices to be tested are then subjected to extraction and purification treatment to obtain the sample solution. Then, liquid chromatography-mass spectrometry was used to detect a series of standard samples and sample solutions to obtain the concentrations of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in the sample. The content of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in the tested food matrix was calculated based on the concentrations of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in the sample.

[0006] In the above method, the extraction and processing procedures for the food matrix being tested are as follows:

[0007] (1) The food matrix tested was tea and its products.

[0008] When tea and its products are solid, the sample is pulverized and then added to deionized water and allowed to stand until fully swollen. When tea products are liquid, the sample is directly added to deionized water, followed by acetonitrile-acetic acid solution and ceramic homogenizer, and mixed thoroughly by shaking. Then, anhydrous magnesium sulfate and anhydrous sodium acetate are added and mixed thoroughly by shaking. Subsequently, the sample is completely extracted by ultrasound. The supernatant obtained after centrifugation after ultrasound is the extract. The mass ratio of pulverized tea and its products or liquid tea products to deionized water is 1:5-6. In the acetonitrile-acetic acid solution, the volume ratio of acetonitrile to acetic acid is 99:1. The amount of acetonitrile-acetic acid solution added should simultaneously meet the requirements of completely extracting the five nitrophenol compounds in the sample and reaching the detection limit. The mass ratio of pulverized tea and its products or liquid tea products to anhydrous magnesium sulfate is 1:3-4, and the mass ratio of pulverized tea and its products or liquid tea products to anhydrous sodium acetate is 1:0.7-1.

[0009] (2) The food matrix being tested is other foods besides tea and its products.

[0010] When other foods are solids that are easily melted when heated and crushed at room temperature, the sample is pulverized and then added to an acetonitrile-acetic acid solution and ceramic homogenizers. When other foods are solids that are easily melted when heated and crushed at room temperature, the sample is cooled and then pulverized before adding the acetonitrile-acetic acid solution and ceramic homogenizers. When other foods are liquids, the sample is directly added to an acetonitrile-acetic acid solution and ceramic homogenizers, then mixed evenly by shaking, and then extracted completely by sonication. The supernatant obtained after centrifugation after sonication is the extract. In the acetonitrile-acetic acid solution, the volume ratio of acetonitrile to acetic acid is 99:1. The amount of acetonitrile-acetic acid solution added should simultaneously meet the requirements of completely extracting the five nitrophenol compounds in the sample and reaching the detection limit.

[0011] In the above method, the purification process of the food matrix being tested is as follows:

[0012] (1) The food matrix tested was tea and its products.

[0013] The extract of tea leaves and their products is added to a container containing a dehydrating agent and a purifying agent, mixed thoroughly, and then centrifuged to obtain the supernatant. The supernatant is then filtered through an organic filter membrane, and the resulting filtrate is the sample. The dehydrating agent is anhydrous magnesium sulfate, and the purifying agent is C18 (octadecylsilane bond and silica gel) and PSA (ethylenediamine-N-propylsilyl silica gel). The mass ratio of anhydrous magnesium sulfate, C18, and PSA is 3:1:1. The mass ratio of the extract of tea leaves and their products to the total mass of the dehydrating agent and purifying agent is 4:0.8 to 1.2.

[0014] (2) The food matrix being tested is other foods besides tea and its products.

[0015] The enhanced lipid removal purifying agent (EMR-Lipid) was activated with deionized water at a mass ratio of 1:5 to 8. Extracts from other foods and the activated purifying solution were weighed at a volume ratio of 1.3 to 2:1, mixed thoroughly, and then centrifuged to obtain a first supernatant. The first supernatant was then added to a container containing sodium chloride and anhydrous sodium sulfate, mixed thoroughly, and centrifuged again to obtain a second supernatant. The second supernatant was filtered through an organic filter membrane, and the resulting filtrate was the sample. The mass ratio of sodium chloride to other food samples was 1 to 2:1, the mass ratio of anhydrous sodium sulfate to other food samples was 1 to 2:1, and the mass ratio of the first supernatant to the total mass of sodium chloride and anhydrous sodium sulfate was 1:0.2 to 0.3.

[0016] The above method involves the following steps for detecting samples and standards using liquid chromatography-mass spectrometry:

[0017] (1) Qualitative detection

[0018] The samples and a series of standards were determined according to the high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) conditions. The chromatographic retention times of each nitrophenol compound in the samples and standards were recorded. The relative ionic abundance of the qualitative ion pair was taken as the percentage relative to the abundance of the strongest ion. The relative ionic abundance of the corresponding nitrophenol compounds in the samples and standards with equivalent concentrations was also recorded. When a chromatographic peak was detected in the sample whose chromatographic retention time varied within ±2.5% of that of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in a certain standard, and the allowable deviation of the relative ionic abundance did not exceed the specified range, it was determined that the sample contained the corresponding nitrophenol compound. Otherwise, the sample did not contain the corresponding nitrophenol compound.

[0019] (2) Quantitative detection

[0020] ① Construction of Quasi-curves

[0021] According to the high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) conditions, a series of standard samples were determined to obtain the quantitative ion chromatographic peak areas of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in each standard sample. The mass concentration of nitrophenol compounds in the standard samples was plotted on the x-axis, and the peak area of ​​the quantitative ion chromatographic peak was plotted on the y-axis.

[0022] ② Detection of the sample

[0023] The sample was analyzed under high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) conditions to obtain the quantitative chromatographic peak areas of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in the sample. Then, based on the standard curves of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol, the concentrations of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in the sample were obtained.

[0024] The response values ​​of the analyte in the sample should all be within the linear range measured by the instrument. If the concentration of the analyte in the sample exceeds the range of the standard curve, the sample should be diluted accordingly before measurement.

[0025] In the above method, the content of each of the above-mentioned nitrophenol compounds in the tested food matrix is ​​calculated by the following formula based on the concentrations of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in the sample:

[0026]

[0027] In the formula,

[0028] X represents the content of nitrophenolic compounds in the tested food matrix.

[0029] C, The concentration of nitrophenol compounds in the obtained sample was detected.

[0030] V, the volume of acetonitrile-acetic acid solution added during the extraction and processing of the food matrix being tested.

[0031] m is the sample size of the food matrix sample being tested.

[0032] The present invention has the following beneficial effects:

[0033] 1. The method described in this invention fills the gap in the food field, especially in the field of weight-loss foods, where there are currently no detection methods and standards for specific nitrophenol compounds such as 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol, both domestically and internationally. It also optimizes the extraction, purification, and separation conditions of the food matrix and the qualitative and quantitative methods, providing technical support for combating the illegal addition of nitrophenol compounds to weight-loss foods.

[0034] 2. The method described in this invention has good accuracy and precision. The examples show that the recoveries and precision of the blank samples of tea and chocolate at different levels were 83.3% to 94.8% and 0.75% to 6.21% respectively [(n=6), see Tables 5 and 10]. Therefore, the method described in this invention meets the detection requirements.

[0035] 3. Using the method described in this invention, the content of five nitrophenol compounds can be obtained simultaneously in one operation using a set of instruments and a reagent system, which can reduce the workload of testing and save analytical reagents, thereby reducing the analysis cost. Attached Figure Description

[0036] Figure 1The total ion chromatogram of one of the series of standards measured in Examples 1 and 2 (the concentrations of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol are 0.25 ng / mL, 5 ng / mL, 2 ng / mL, 0.5 ng / mL, and 0.05 ng / mL, respectively).

[0037] Figure 2 The total ion chromatogram of the sample prepared by spiked blank tea with five nitrophenol compounds in Example 1 is shown below (the concentrations of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol are 0.25 ng / mL, 5 ng / mL, 2 ng / mL, 0.5 ng / mL, and 0.05 ng / mL, respectively).

[0038] Figure 3 The total ion chromatogram of the sample prepared by spiked blank chocolate with five nitrophenol compounds as standards in Example 2 is shown below (the concentrations of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol are 0.25 ng / mL, 5 ng / mL, 2 ng / mL, 0.5 ng / mL, and 0.05 ng / mL, respectively).

[0039] Figure 4 This is a standard curve of 2,4-dinitrophenol plotted by measuring a series of standard samples in Examples 1 and 2.

[0040] Figure 5 This is a standard curve of 2,5-dinitrophenol plotted by measuring a series of standard samples in Examples 1 and 2.

[0041] Figure 6 This is a standard curve of 2,6-dinitrophenol plotted by measuring a series of standard samples in Examples 1 and 2.

[0042] Figure 7 This is a standard curve of 3,4-dinitrophenol plotted by measuring a series of standard samples in Examples 1 and 2.

[0043] Figure 8 This is a standard curve of 3,5-dinitrophenol plotted by measuring a series of standard samples in Examples 1 and 2.

[0044] Figure 9 This is a distribution diagram of 2,4-dinitrophenol content obtained from the detection of green tea in Example 1.

[0045] Figure 10This is a distribution diagram of 2,4-dinitrophenol content obtained from jasmine tea in Example 1.

[0046] Figure 11 This is a distribution diagram of the 2,4-dinitrophenol content obtained from the black tea in Example 1.

[0047] Figure 12 This is a distribution diagram of the 2,4-dinitrophenol content obtained from the Pu-erh tea tested in Example 1.

[0048] Figure 13 This is a distribution diagram of 2,4-dinitrophenol content obtained from the white tea tested in Example 1.

[0049] Figure 14 This is a distribution map of 2,4-dinitrophenol content obtained from the detection of green tea from provinces A and B in Example 1. Detailed Implementation

[0050] The method for simultaneously determining multiple nitrophenolic compounds in different food matrices according to the present invention will be further described below through examples and in conjunction with the accompanying drawings. Obviously, the described examples are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of the present invention.

[0051] In the following examples, the standards for 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol were all purchased commercially; other chemical reagents were of chromatographic purity and were all purchased commercially.

[0052] Example 1

[0053] In this embodiment, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was used to simultaneously determine 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in tea.

[0054] I. Determination of blank tea leaves

[0055] The blank tea leaves are tea leaves that do not contain 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, or 3,5-dinitrophenol, and are used for verification of the method described in this invention. In this embodiment, the blank tea leaves are green tea purchased from the market.

[0056] The steps are as follows:

[0057] 1. Preparation of standard samples

[0058] (1) Preparation of intermediate solution

[0059] Measure 100 μL of each of the following standards (2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol) with a concentration of 100 μg / mL and place them in five 10 mL volumetric flasks. Dilute to the mark with acetonitrile and mix well to obtain five intermediate solutions of nitrophenols with a concentration of 1.0 μg / mL. Store these solutions in brown storage bottles at -20°C for later use.

[0060] (2) Preparation of mixed standard solutions

[0061] Take 0.25 mL of 2,4-dinitrophenol intermediate solution, 5 mL of 2,5-dinitrophenol intermediate solution, 2 mL of 2,6-dinitrophenol intermediate solution, 0.5 mL of 3,4-dinitrophenol intermediate solution, and 0.05 mL of 3,5-dinitrophenol intermediate solution, and place them in the same 10 mL volumetric flask. Dilute to the mark with acetonitrile and mix well to obtain a mixed standard solution. Store this solution in a brown stock bottle at -20℃ for later use. The concentrations of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in the mixed standard solution are 0.025 μg / mL, 0.5 μg / mL, 0.2 μg / mL, 0.05 μg / mL, and 0.005 μg / mL, respectively.

[0062] (3) Preparation of a series of standard samples

[0063] Measure 1 μL, 2 μL, 5 μL, 10 μL, 20 μL, and 50 μL of the mixed standard solution into six volumetric flasks, respectively, and dilute to 1.00 mL with acetonitrile-acetic acid solution (acetonitrile:acetic acid = 99:1, volume ratio), shake well, and you will get a series of standard solutions composed of five nitrophenol compounds.

[0064] 2. Sample preparation

[0065] (1) Extraction and processing of the green tea being tested

[0066] Weigh 2g of green tea sample, pulverize it, and place it in a 50mL centrifuge tube. Add 10mL of deionized water, mix well, and let it stand for 30min to allow it to swell completely. Then add 15mL of acetonitrile-acetic acid solution (acetonitrile:acetic acid = 99:1, volume ratio) and 1 ceramic homogenant, shake vigorously for 1min to mix well, then add 6g of anhydrous magnesium sulfate and 1.5g of anhydrous sodium acetate, shake vigorously for 1min to mix well, then sonicate for 10min, and centrifuge at 8000r / min for 5min. The resulting supernatant is the green tea extract.

[0067] (2) Purification treatment of green tea extract

[0068] Pipette 2.0 mL of green tea extract into a centrifuge tube containing anhydrous magnesium sulfate (dehydrating agent), C18, and PSA (purifying agent). Vortex for 2 min, then centrifuge at 8000 r / min for 5 min. Pass the supernatant through a 0.22 μm organic filter membrane. The filtrate obtained after filtration through the organic filter membrane is the sample. The centrifuge tube contains 300 mg of anhydrous magnesium sulfate, 100 mg of C18, and 100 mg of PSA.

[0069] 3. Instrument conditions

[0070] (1) Liquid phase conditions

[0071] Chromatographic column: Waters ACQUITY UPLC BEH Shield RP18 (100 mm x 3.0 mm, 1.7 μm); mobile phase: A was 10 mmol / L ammonium formate aqueous solution (containing 0.1% formic acid by volume), B was acetonitrile (containing 0.1% formic acid by volume), gradient elution program: 0–10.0 min, 25% B–70% B; 10.0–10.01 min, 70% B–25% B; 10.01–13.0 min, 25% B. Column temperature: 30℃; flow rate: 0.25 mL / min; injection volume: 5 μL.

[0072] (2) Mass spectrometry conditions

[0073] Ionization mode: electrospray ionization, negative ion mode (ESI-); Mass spectrometry scanning mode: multiple reaction pair monitoring (MRM); Curtain gas pressure (CUR): 55 kPa, Collision gas pressure (CAD): 55 kPa, Spray voltage (IS): -4500 V, Ion source temperature (TEM): 500 °C, Nebulizer gas pressure (GS1): 207 kPa, Auxiliary heating gas pressure 2 (GS2): 207 kPa. The optimized mass spectrometry parameters are shown in Table 1.

[0074] Table 1. Main mass spectrometry parameters of the compounds

[0075]

[0076] Note: * indicates a quantitative ion. Parameters may vary depending on the mass spectrometer; mass spectrometry parameters should be optimized before measurement.

[0077] 4. Qualitative determination

[0078] The samples and a series of standards were determined according to high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) conditions. The chromatographic retention times of nitrophenolic compounds in the samples and standards were recorded. The relative abundance of the qualitative ion pair was taken as the percentage relative to the abundance of the strongest ion. The relative ion abundance of the corresponding components in the samples and standards with equivalent concentrations was also recorded. When a chromatographic peak was detected in the sample whose retention time varied within ±2.5% of that of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in a certain standard, and the allowable deviation of the relative ion abundance did not exceed the range specified in Table 2, then the sample was determined to contain the corresponding nitrophenolic compound. Otherwise, the sample did not contain the corresponding nitrophenolic compound.

[0079] Table 2. Permissible deviation range of relative ion abundance in the sample during qualitative analysis.

[0080] Relative ion abundance (%) Maximum permissible deviation (%) ＞50 ±20 20～50 ±25 10～20 ±30 ≤10 ±50

[0081] Since the green tea tested was a blank tea leaf, the sample did not contain the corresponding nitrophenol compounds.

[0082] 5. Quantitative determination

[0083] (1) Construction of standard curve

[0084] Under high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) conditions, a series of standards consisting of six nitrophenol compounds were analyzed. The quantitative ion chromatography (QIC) peak areas of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in each standard were obtained. Standard curves for 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol were plotted with the mass concentration of the nitrophenol compounds in the standards on the x-axis and the peak area on the y-axis. The plotted standard curves are shown below. Figures 4 to 8 .according to Figures 4 to 8 The linear equations and correlation coefficients obtained from the standard curves shown are presented in Table 3.

[0085] Table 3. Linear equations and linear ranges of the five compounds in acetonitrile-acetic acid solution.

[0086]

[0087] (2) Determination of sample solution

[0088] The sample was analyzed under high performance liquid chromatography-tandem mass spectrometry conditions. The chromatographic peak areas of the five nitrophenol compounds in the sample were 0, and the concentrations of the five nitrophenol compounds in this tea were all 0.

[0089] 6. Calculation of the content of nitrophenolic compounds

[0090] Calculate the contents of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol using the following formula:

[0091]

[0092] X represents the content of nitrophenolic compounds in green tea, expressed in μg / kg.

[0093] C, the concentration of nitrophenol compounds in the sample obtained by detection, in ng / mL;

[0094] V represents the volume of acetonitrile-acetic acid solution added during the extraction and processing of green tea samples, in mL.

[0095] m represents the sample size of green tea, expressed in grams.

[0096] In the blank tea test, the volume of acetonitrile-acetic acid solution added during the green tea sample extraction process was 15 mL, and the sample weight of green tea was 2 g. However, since the concentration of the five nitrophenol compounds in the sample was zero, the calculated contents of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in the tested green tea were all 0.

[0097] 7. Method Validation

[0098] (1) Matrix effect

[0099] Because mass spectrometry responses are easily affected by the sample matrix, the relative response value method was used to investigate the matrix effect (ME) in green tea during the experiment. The matrix effect was evaluated by calculating the slope ratio of the matrix-matched standard curve and the pure solvent standard curve. Here, the pure solvent standard curve represents the standard curve obtained by detecting the matrix-matched standard curve under high performance liquid chromatography-tandem mass spectrometry conditions using a series of standard samples prepared with acetonitrile-acetic acid solution (acetonitrile:acetic acid = 99:1, volume ratio) consisting of a mixture of five nitrophenol compounds with different concentrations (the same number and concentration as the series of standard samples prepared in the "Determination of Blank Tea" section of this example). Figures 4 to 8(Same as above) The matrix-matched standard curve represents the standard curve obtained by preparing a series of standard samples (the number of standard samples and the concentration of the five nitrophenol compounds in the standard samples are the same as those in the pure solvent standard curve) using the purified filtrate obtained from blank tea (green tea) according to the above-described green tea extraction and purification methods, and detecting them under high performance liquid chromatography-tandem mass spectrometry conditions. Generally, when the ME value is between 80% and 120%, it indicates that the matrix effect is within an acceptable range and can be ignored in actual detection; otherwise, the influence of the matrix effect on actual detection should be considered. The ME calculations for the five nitrophenol compounds in green tea are shown in Table 4. The results show that these compounds do not have a strong matrix effect and can be ignored. In actual detection, acetonitrile-acetic acid solution (acetonitrile:acetic acid = 99:1, volume ratio) can be used to prepare standard samples for quantitative detection.

[0100] Table 4. Matrix effects of five nitrophenol compounds in blank tea leaves.

[0101]

[0102] (2) Linear range and correlation coefficient

[0103] Based on the responses of five nitrophenol compounds in high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), a series of standards for 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol were prepared using acetonitrile-acetic acid solution (acetonitrile:acetic acid = 99:1, volume ratio). Under optimized chromatographic conditions, standard curves were plotted by comparing peak area (Y) against the corresponding mass concentration (X), and the linear regression equations and correlation coefficients for each analyte nitrophenol compound were calculated. (Appendix) Figure 4 ~Attached Figure 8 Table 3 shows that the linear relationships are good for 2,4-dinitrophenol concentrations in the ranges of 0.025–1.25 ng / mL, 2,5-dinitrophenol concentrations in the ranges of 0.5–25 ng / mL, 2,6-dinitrophenol concentrations in the ranges of 0.2–10 ng / mL, 3,4-dinitrophenol concentrations in the ranges of 0.05–2.5 ng / mL, and 3,5-dinitrophenol concentrations in the ranges of 0.005–0.25 ng / mL, with correlation coefficients r > 0.999 for all of them.

[0104] (3) Recovery rate and precision

[0105] Following steps 1-6 of the blank tea sample determination procedure, a spiked recovery test was conducted on the blank tea sample containing the tested nitrophenol compounds using five nitrophenol compound standards. Six samples were prepared for each different spike level to examine the accuracy and precision of the method described in this invention. The results are shown in Table 5. The results show that the average recoveries of the five nitrophenol compounds at each spike level were between 65.0% and 115.0%, and the precision was <10%. The accuracy and precision of the method basically meet the requirements for quantitative analysis. Additionally, samples with the limit of quantitation (LOQ) level added to the blank tea sample were measured at 0, 2, 4, and 7 days, and the recoveries were expressed as recoveries. As shown in Table 6, the standard solutions of 2,4-dinitrophenol and 2,6-dinitrophenol began to degrade significantly after one day of storage; therefore, fresh solutions should be prepared immediately before use during the experiment.

[0106] Table 5. Recovery and precision results of five nitrophenol compounds.

[0107]

[0108] Table 6. Daytime precision results for five nitrophenol compounds.

[0109]

[0110] (4) Limit of detection and limit of quantitation

[0111] The limit of detection (LOD) was determined by adding spikes to blank tea leaves and diluting them stepwise. Spiked samples were obtained by extracting and purifying blank tea leaves and detected by high performance liquid chromatography-tandem mass spectrometry. The limits of detection (LOD) and limits of quantitation (LOQ) were determined based on the spiked levels corresponding to the signal-to-noise ratios (S / N) of the characteristic chromatographic peaks of each nitrophenol compound at S / N = 3 and S / N = 10. The LOD and LOQ were ultimately determined as follows: 0.075 μg / kg for 2,4-dinitrophenol and 0.1875 μg / kg for LOQ; 1.50 μg / kg for 2,5-dinitrophenol and 3.75 μg / kg for LOQ; 0.375 μg / kg for 2,6-dinitrophenol and 1.50 μg / kg for LOQ; 0.15 μg / kg for 3,4-dinitrophenol and 0.375 μg / kg for LOQ; and 0.015 μg / kg for LOQ and 0.0375 μg / kg for LOQ.

[0112] II. Actual Tea Measurement

[0113] The actual tea samples were tested using the same method as the blank tea samples. 423 batches of tea purchased from the market and online were tested, including 290 batches of green tea, 85 batches of jasmine tea, 33 batches of black tea, 9 batches of Pu-erh tea, and 6 batches of white tea. At least one sample (2g) was taken from each batch. Only 2,4-dinitrophenol was detected in all samples; the other four nitrophenol compounds were not detected. The specific detection results of 2,4-dinitrophenol in the actual tea samples are shown in Table 7. Figures 9 to 13 The test results showed that the content of 2,4-dinitrophenol varied considerably among different types of tea. Green tea had a relatively high content of 2,4-dinitrophenol, while all six batches of white tea tested positive for 2,4-dinitrophenol. The content ranges of 2,4-dinitrophenol in jasmine tea, black tea, and Pu-erh tea were relatively similar. This may be related to the different growth habits of the teas, the degree of enrichment of 2,4-dinitrophenol, and the soil and water environment.

[0114] Table 7. Content of 2,4-dinitrophenol in tea leaves

[0115]

[0116] III. Comparison of 2,4-dinitrophenol in green tea from different provinces

[0117] Using the same method as for the blank tea sample test, 230 batches of green tea purchased from markets and online in Province A and 60 batches of green tea purchased from markets and online in Province B were tested. At least one sample of 2g was taken from each batch. The test results are shown below. Figure 14 ,from Figure 14 It can be seen that only 2,4-dinitrophenol was detected in green tea from both provinces A and B; the other four nitrophenol compounds were not detected. The content of 2,4-dinitrophenol in green tea from province A ranged from 0 to 18.81 μg / kg, while that from province B ranged from 0 to 2.49 μg / kg. The test results indicate a significant difference in the 2,4-dinitrophenol content between the two provinces, with the content in green tea from province B being much lower than that from province A. This difference may be related to the soil and water conditions used for tea cultivation in those regions.

[0118] Example 2

[0119] In this embodiment, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was used to simultaneously determine 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in chocolate.

[0120] I. Determination of blank chocolate

[0121] The blank chocolate is a chocolate that does not contain 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, or 3,5-dinitrophenol, and is used for verification of the method described in this invention. In this embodiment, the blank chocolate is purchased from the market.

[0122] The operation is as follows:

[0123] 1. Preparation of standard samples

[0124] The preparation method is the same as that of the standard sample in Example 1.

[0125] 2. Sample preparation

[0126] (1) Extraction and processing of the tested chocolate

[0127] After freezing the chocolate sample at -20℃ for 12 hours, it was crushed and mixed using a tissue homogenizer. 1 g of the crushed and mixed chocolate sample was weighed and placed in a 50 mL centrifuge tube. 10 mL of acetonitrile-acetic acid solution (acetonitrile:acetic acid = 99:1, volume ratio) and 1 ceramic homogenate were added. The mixture was shaken vigorously for 1 min to mix evenly. Then, it was sonicated for 10 min and centrifuged at 8000 r / min for 5 min. The supernatant obtained was the chocolate extract.

[0128] (2) Purification treatment of chocolate extract

[0129] Activate 600 mg of enhanced lipid removal and purification agent (EMR-Lipid) with 3 mL of deionized water. Add 5 mL of chocolate extract to the activated purification solution and mix for 2 min. Then centrifuge at 8000 r / min for 5 min at 4 °C to obtain the first supernatant. Add the first supernatant to a centrifuge tube containing 1 g of sodium chloride and 1 g of anhydrous sodium sulfate, vortex and mix for 2 min. Then centrifuge at 8000 r / min for 5 min at 4 °C to obtain the second supernatant. Filter the second supernatant through a 0.22 μm organic filter membrane. The filtrate obtained after filtration through the organic filter membrane is the sample.

[0130] 3. Instrument conditions

[0131] Same as Example 1.

[0132] 4. Qualitative determination

[0133] The samples and a series of standards were determined according to the high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) conditions. The chromatographic retention times of nitrophenolic compounds in the samples and standards were recorded. The relative abundance of the qualitative ion pair was taken as the percentage relative to the abundance of the strongest ion. The relative ion abundance of the corresponding components in the samples and standards with equivalent concentrations was also recorded. When a chromatographic peak was detected in the sample whose retention time varied within ±2.5% of that of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in a certain standard, and the allowable deviation of the relative ion abundance did not exceed the range specified in Table 8, then the sample was determined to contain the corresponding nitrophenolic compound. Otherwise, the sample did not contain the corresponding nitrophenolic compound.

[0134] Table 8. Permissible deviation range of relative ion abundance in the sample during qualitative analysis.

[0135] Relative ion abundance (%) Maximum permissible deviation (%) ＞50 ±20 20～50 ±25 10～20 ±30 ≤10 ±50

[0136] Since the tested chocolate was blank chocolate, the sample did not contain the corresponding nitrophenol compounds.

[0137] 5. Quantitative determination

[0138] (1) Construction of standard curve

[0139] Same as Example 1.

[0140] (2) Determination of sample solution

[0141] The sample was analyzed under high performance liquid chromatography-tandem mass spectrometry conditions. The chromatographic peak areas of the five nitrophenol compounds in the sample were 0, and the concentrations of the five nitrophenol compounds in this chocolate were all 0.

[0142] 6. Calculation of the content of nitrophenolic compounds

[0143] Calculate the contents of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol using the following formula:

[0144]

[0145] X represents the content of nitrophenolic compounds in chocolate, expressed in μg / kg.

[0146] C, the concentration of nitrophenol compounds in the sample obtained by detection, in ng / mL;

[0147] V represents the volume of acetonitrile-acetic acid solution added during the extraction and processing of the chocolate sample, in mL.

[0148] m represents the sample weight of the chocolate, expressed in grams.

[0149] In the blank chocolate test, the volume of acetonitrile-acetic acid solution added during chocolate sample extraction was 10 mL, and the sample weight was 1 g. However, since the concentration of the five nitrophenol compounds in the sample was zero, the calculated content of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in the tested chocolate was all 0.

[0150] 7. Method Validation

[0151] (1) Matrix effect

[0152] Because mass spectrometry responses are easily affected by the sample matrix, the relative response value method was used to investigate the matrix effect (ME) in chocolate during the experiment. The matrix effect was evaluated by calculating the slope ratio of the matrix-matched standard curve and the pure solvent standard curve. Here, the pure solvent standard curve represents the standard curve obtained by detecting a series of standard samples prepared with acetonitrile-acetic acid solution (acetonitrile:acetic acid = 99:1, volume ratio) containing a mixture of five nitrophenol compounds at different concentrations (the same number and concentration of standard samples prepared in the "Determination of Blank Chocolate" section of this example) under high performance liquid chromatography-tandem mass spectrometry conditions (as shown in the figure). Figures 4 to 8 (Same as above) The matrix-matched standard curve represents the standard curve obtained by preparing a series of standard samples (the number of standard samples and the concentration of the five nitrophenol compounds in the standard samples are the same as those in the standard curve for determining pure solvent) using the purified filtrate obtained from blank chocolate according to the above-described chocolate extraction and purification methods, and detecting them under high performance liquid chromatography-tandem mass spectrometry conditions. Generally, when the ME value is between 80% and 120%, it indicates that the matrix effect is within an acceptable range and can be ignored in actual detection; otherwise, the influence of the matrix effect on actual detection should be considered. The ME calculations for the five compounds in chocolate are shown in Table 9. The results show that these compounds do not have a strong matrix effect and can be ignored. In actual detection, acetonitrile-acetic acid solution (acetonitrile:acetic acid = 99:1, volume ratio) can be used to prepare standard samples for quantitative detection.

[0153] Table 9. Matrix effects of five compounds in blank chocolate.

[0154]

[0155] (2) Linear range and correlation coefficient

[0156] Same as Example 1.

[0157] (3) Recovery rate and precision

[0158] Following steps 1-6 of the blank chocolate assay, a spiked recovery test was conducted on a blank chocolate sample containing no nitrophenol compounds to be tested using five nitrophenol compound standards. Six samples were prepared for each different spike level to examine the accuracy and precision of the method described in this invention. The results are shown in Table 10. The results indicate that the average recoveries of the five nitrophenol compounds at each spike level ranged from 65.0% to 115.0%, and the precision was <10% for all samples. The accuracy and precision of the method basically meet the requirements for quantitative analysis.

[0159] Table 10 Recovery and precision results of five nitrophenol compounds

[0160]

[0161] (4) Limit of detection and limit of quantitation

[0162] The limit of detection (LOD) was determined by adding spikes to blank chocolate and diluting it stepwise. Spiked samples were obtained by extracting and purifying blank chocolate and detected by high performance liquid chromatography-tandem mass spectrometry. The limits of detection (LOD) and limits of quantitation (LOQ) were determined based on the spiked levels corresponding to the signal-to-noise ratios (S / N) of the characteristic chromatographic peaks of each nitrophenol compound at S / N = 3 and S / N = 10. The LOD and LOQ were ultimately determined as follows: 0.10 μg / kg for 2,4-dinitrophenol and 0.25 μg / kg for 2,5-dinitrophenol; 1.5 μg / kg and 5.00 μg / kg for 2,6-dinitrophenol; 0.50 μg / kg and 2.00 μg / kg for 3,4-dinitrophenol; 0.15 μg / kg and 0.50 μg / kg for 3,5-dinitrophenol; and 0.015 μg / kg and 0.05 μg / kg for 3,5-dinitrophenol.

[0163] II. Actual Chocolate Measurement

[0164] The actual chocolate samples were tested using the same method as the blank chocolate samples. Twenty batches of chocolate purchased from the market and online were tested. At least one sample (1g) was taken from each batch. The results showed that 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol were not detected in any of the 20 batches of chocolate.

Claims

1. A method for simultaneously determining multiple nitrophenolic compounds in different food matrices, characterized in that, The various nitrophenol compounds are 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol. A series of mixed solutions with different concentrations of the five nitrophenol compounds were prepared using an acetonitrile-acetic acid solution as standards, wherein the volume ratio of acetonitrile to acetic acid in the acetonitrile-acetic acid solution was 99:

1. The food matrix to be tested was extracted and purified to obtain the sample. Then, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was used to detect the sample and the series of standards to obtain the concentration of the five nitrophenol compounds in the sample. The content of the five nitrophenol compounds in the food matrix to be tested was then calculated based on the concentration of the five nitrophenol compounds in the sample. The HPLC-MS / MS conditions included: column: Waters ACQUITY UPLC BEH Shield RP18; mobile phase: A with a concentration of 10... The solution is a mmol / L ammonium formate aqueous solution containing 0.1% formic acid by volume; B is acetonitrile containing 0.1% formic acid by volume. The gradient elution program is as follows: 0–10.0 min, 25% B–70% B; 10.0–10.01 min, 70% B–25% B; 10.01–13.0 min, 25% B.

2. The method for simultaneously determining multiple nitrophenolic compounds in different food matrices according to claim 1, characterized in that... The extraction and processing procedures for the food matrix being tested are as follows: (1) The food matrix being tested is tea and its products. When tea and its products are solid, the sample is pulverized and then added to deionized water and allowed to stand until fully swollen. When tea products are liquid, the sample is directly added to deionized water, followed by acetonitrile-acetic acid solution and ceramic homogenizer, and mixed thoroughly by shaking. Then anhydrous magnesium sulfate and sodium acetate are added and mixed thoroughly by shaking. Subsequently, the sample is completely extracted by ultrasound. The supernatant obtained after centrifugation after ultrasound is the extract. The mass ratio of pulverized tea and its products or liquid tea products to deionized water is 1:5~6. In the acetonitrile-acetic acid solution, the volume ratio of acetonitrile to acetic acid is 99:

1. The amount of acetonitrile-acetic acid solution added should simultaneously meet the requirements of completely extracting the five nitrophenol compounds in the sample and reaching the detection limit. The mass ratio of pulverized tea and its products or liquid tea products to anhydrous magnesium sulfate is 1:3~4, and the mass ratio of pulverized tea and its products or liquid tea products to anhydrous sodium acetate is 1:0.7~1. (2) The food matrix being tested is other foods besides tea and its products. When other foods are solids that are easily melted when heated and crushed at room temperature, the sample is pulverized and then added to an acetonitrile-acetic acid solution and ceramic homogenizers. When other foods are solids that are easily melted when heated and crushed at room temperature, the sample is cooled and then pulverized before adding the acetonitrile-acetic acid solution and ceramic homogenizers. When other foods are liquids, the sample is directly added to an acetonitrile-acetic acid solution and ceramic homogenizers, then mixed evenly by shaking, and then extracted completely by sonication. The supernatant obtained after centrifugation after sonication is the extract. In the acetonitrile-acetic acid solution, the volume ratio of acetonitrile to acetic acid is 99:

1. The amount of acetonitrile-acetic acid solution added should simultaneously meet the requirements of completely extracting the five nitrophenol compounds in the sample and reaching the detection limit.

3. The method for simultaneously determining multiple nitrophenolic compounds in different food matrices according to claim 2, characterized in that... The purification process for the food matrix being tested is as follows: (1) The food matrix being tested is tea and its products. The extract of tea leaves and their products is added to a container containing a dehydrating agent and a purifying agent and mixed evenly. Then, the mixture is centrifuged to obtain the supernatant. The supernatant is then filtered through an organic filter membrane, and the resulting filtrate is the sample. The dehydrating agent is anhydrous magnesium sulfate, and the purifying agent is C18 and PSA. The mass ratio of anhydrous magnesium sulfate, C18, and PSA is 3:1:

1. The mass ratio of the extract of tea leaves and their products to the total mass of the dehydrating agent and purifying agent is 4:0.8~1.

2. (2) The food matrix being tested is other foods besides tea and its products. The enhanced lipid removal purifier is activated with deionized water, wherein the mass ratio of the purifier to the deionized water is 1:5-8. The extracts from other food samples and the purified solution obtained from activation are measured at a volume ratio of 1.3 to 2:1, and mixed evenly. Then, the mixture is centrifuged to obtain the first supernatant. The first supernatant is then added to a container containing sodium chloride and anhydrous sodium sulfate, mixed evenly, and centrifuged again to obtain the second supernatant. The second supernatant is filtered through an organic filter membrane, and the resulting filtrate is the sample. The mass ratio of sodium chloride to other food samples is 1 to 2:1, the mass ratio of anhydrous sodium sulfate to other food samples is 1 to 2:1, and the mass ratio of the first supernatant to the total mass of sodium chloride and anhydrous sodium sulfate is 1:0.2 to 0.

3.

4. The method for simultaneously determining multiple nitrophenolic compounds in different food matrices according to any one of claims 1 to 3, characterized in that... The procedure for detecting samples and standards using high-performance liquid chromatography-tandem mass spectrometry is as follows: (1) Qualitative detection The samples and a series of standards were determined according to the high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) conditions. The chromatographic retention times of each nitrophenol compound in the samples and standards were recorded. The relative ionic abundance of the qualitative ion pair was taken as the percentage relative to the abundance of the strongest ion. The relative ionic abundance of the corresponding nitrophenol compounds in the samples and standards with equivalent concentrations was also recorded. When a chromatographic peak was detected in the sample whose chromatographic retention time varied within ±2.5% of that of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in a certain standard, and the allowable deviation of the relative ionic abundance did not exceed the specified range, it was determined that the sample contained the corresponding nitrophenol compound. Otherwise, the sample did not contain the corresponding nitrophenol compound. (2) Quantitative detection ① Construction of standard curve According to the high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) conditions, a series of standard samples were determined to obtain the quantitative ion chromatographic peak areas of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in each standard sample. The mass concentration of nitrophenol compounds in the standard samples was plotted on the x-axis, and the peak area of ​​the quantitative ion chromatographic peak was plotted on the y-axis. ② Detection of the sample The sample was analyzed under high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) conditions to obtain the quantitative chromatographic peak areas of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in the sample. Then, based on the standard curves of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol, the concentrations of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in the sample were obtained.

5. The method for simultaneously determining multiple nitrophenolic compounds in different food matrices according to claim 4, characterized in that... The content of each of the above-mentioned nitrophenol compounds in the tested food matrix was calculated by the following formula based on the concentration of 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol, 3,4-dinitrophenol, and 3,5-dinitrophenol in the sample: ； In the formula, X represents the content of nitrophenolic compounds in the tested food matrix. C, The concentration of nitrophenol compounds in the obtained sample was detected. V, the volume of acetonitrile-acetic acid solution added during the extraction and processing of the food matrix being tested. m is the sample size of the food matrix sample being tested.

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