Method for determining residual quantity of sodium pentachlorophenate in eggs

Abstract

The invention discloses a method for measuring the residual quantity of sodium pentachlorophenate in eggs, and belongs to the technical field of detection.The method comprises the steps that edible parts of egg samples are taken and subjected to homogenization treatment, and egg liquid samples are obtained; the method comprises the following steps: adding an internal standard reagent, a protein precipitant, dilute sulphuric acid and normal hexane into an egg liquid sample, uniformly mixing, centrifuging, taking an upper organic phase, and purifying for derivation; preparing a series of pentachlorophenol standard working solutions, and adding an internal standard reagent into the pentachlorophenol standard working solutions; adding a derivatization reagent into the organic phase and the pentachlorophenol standard working solution, and carrying out derivatization treatment; putting the derived organic phase and a pentachlorophenol standard working solution into gas chromatography-tandem mass spectrometry equipment, and testing on a machine to obtain the content of pentachlorophenol in the organic phase; and determining the residual quantity of the sodium pentachlorophenate in the egg sample according to the content of the pentachlorophenate. The technical effect of improving the detection accuracy of the residual quantity of the sodium pentachlorophenate in the eggs is achieved.

Description

Technical Field

[0001] This application relates to the field of detection technology, and particularly to a method for determining the residue amount of sodium pentachlorophenolate in eggs. Background Art

[0002] Sodium pentachlorophenolate is an organochlorine pesticide that degrades slowly in the natural environment. It can enter the body of poultry through feed and accumulate, resulting in residue in egg foods, which is extremely harmful to the human body. Sodium pentachlorophenolate has been included in the list of drugs and their compounds prohibited from use in food animals.

[0003] Currently, the testing methods for pentachlorophenol substances in food include liquid chromatography-tandem mass spectrometry, gas chromatography-mass spectrometry, colloidal gold immunochromatography, etc. These methods all use the external standard method for quantification, with poor stability and accuracy, and high requirements for personnel operation. Among them, liquid chromatography-tandem mass spectrometry is the current mainstream detection method, with high costs and only applicable to meat foods. At the same time, the pretreatment process of samples mostly uses solid-phase extraction and gel chromatography purification methods. The experimental steps are relatively cumbersome and complex, and when the purification is incomplete, it is extremely easy to cause matrix effects, resulting in inaccurate quantification. Summary of the Invention

[0004] The main purpose of this application is to provide a method for determining the residue amount of sodium pentachlorophenolate in eggs, aiming to solve the technical problem that the existing detection methods for pentachlorophenol substances in food have low test accuracy due to the matrix effect of eggs.

[0005] To achieve the above purpose, this application provides a method for determining the residue amount of sodium pentachlorophenolate in eggs, including:

[0006] Take the edible part of the egg sample for homogenization treatment to obtain an egg liquid sample;

[0007] Add an internal standard reagent, a protein precipitant, dilute sulfuric acid, and n-hexane to the egg liquid sample. After mixing evenly, perform centrifugation treatment, and take the upper organic phase for purification and then wait for derivatization;

[0008] Prepare a series of pentachlorophenol standard working solutions, and add the internal standard reagent to the pentachlorophenol standard working solutions;

[0009] Add a derivatization reagent to the organic phase and the pentachlorophenol standard working solutions for derivatization treatment;

[0010] Place the derivatized organic phase and pentachlorophenol standard working solutions in a gas chromatography-tandem mass spectrometry device for on-machine testing to obtain the content of pentachlorophenol in the organic phase;

[0011] Determine the residue amount of sodium pentachlorophenolate in the egg sample according to the content of pentachlorophenol.

[0012] In some embodiments of the present application, the residual amount of sodium pentachlorophenate is calculated according to the following expression:

[0013] C = c × v × 1.08 / m;

[0014] Wherein, C represents the residual amount of sodium pentachlorophenate in the egg sample, with the unit of μg / kg; c represents the content of pentachlorophenol in the sample solution of the organic phase, with the unit of ng / mL; v represents the constant volume of the sample solution of the organic phase, with the unit of mL; m represents the sampling amount of the egg sample, with the unit of g.

[0015] In some embodiments of the present application, the internal standard reagent is 2,4,6-tribromophenol.

[0016] In some embodiments of the present application, the protein precipitant is trichloroacetic acid.

[0017] In some embodiments of the present application, the purification treatment of the organic phase includes the following steps:

[0018] Take the organic phase in a centrifuge tube, add concentrated sulfuric acid, vortex and mix evenly, and then perform centrifugation treatment. The supernatant obtained is the purified organic phase.

[0019] In some embodiments of the present application, the series of concentrations of the pentachlorophenol standard working solution include 0.1 ng / mL, 0.5 ng / mL, 1.0 ng / mL, 2.0 ng / mL, and 5 ng / mL.

[0020] In some embodiments of the present application, the derivatization reagent is an acetic anhydride-pyridine solution, and the volume ratio of acetic anhydride to pyridine in the derivatization reagent is 1:1.

[0021] In some embodiments of the present application, the steps for performing derivatization treatment on the organic phase and the pentachlorophenol standard working solution include:

[0022] Respectively take the organic phase and the pentachlorophenol standard working solution, add the derivatization reagent to each and seal, and react in a water bath at 60 °C for 10 min - 20 min;

[0023] Take out and cool, add 0.2 mol / L potassium carbonate solution, mix well and then perform centrifugation treatment.

[0024] In some embodiments of the present application, the analysis conditions for the machine testing include:

[0025] Chromatographic column: HP-5MS capillary chromatographic column, column length 30 m, inner diameter 0.25 mm, film thickness 0.25 μm;

[0026] Injection port temperature 230 °C;

[0027] Carrier gas: helium, purity ≥ 99.999%;

[0028] Splitless injection mode, injection volume: 1 μL;

[0029] Constant flow mode, flow rate 1.0 mL / min;

[0030] Temperature program: initial temperature 80°C, hold for 2 min, increase to 290°C at 10°C / min, hold for 4 min;

[0031] Transfer line temperature: 280°C;

[0032] Ionization mode: EI, energy 70 eV;

[0033] Ion source temperature: 230°C;

[0034] Scanning mode: multiple reaction monitoring mode.

[0035] In some embodiments of the present application, the quantification limit of the residual amount of sodium pentachlorophenate is 0.2 μg / kg.

[0036] The method for determining the residual amount of sodium pentachlorophenate in eggs provided by the embodiments of the present application comprises taking the edible part of the egg sample for homogenization to obtain an egg liquid sample; adding an internal standard reagent, a protein precipitant, dilute sulfuric acid and n-hexane to the egg liquid sample, mixing evenly and then centrifuging, taking the upper organic phase for purification and then waiting for derivatization; preparing a series of pentachlorophenol standard working solutions, and adding the internal standard reagent to the pentachlorophenol standard working solutions; adding a derivatization reagent to the organic phase and the pentachlorophenol standard working solutions for derivatization treatment; placing the derivatized organic phase and pentachlorophenol standard working solutions in a gas chromatography-tandem mass spectrometry device for on-machine testing to obtain the content of pentachlorophenol in the organic phase; and determining the residual amount of sodium pentachlorophenate in the egg sample according to the content of pentachlorophenol. In the embodiments of the present application, the homogenized egg liquid sample is extracted with a protein precipitant, the pH is adjusted to acidic with dilute sulfuric acid to convert sodium pentachlorophenate in the egg liquid sample into pentachlorophenol, liquid-liquid extraction is carried out with n-hexane, then impurity purification is carried out, derivatization is carried out with a derivatization reagent, the content of the derivative is determined by gas chromatography-tandem mass spectrometry, the content of pentachlorophenol is directly calculated by the instrument, quantification is carried out by the internal standard method, and finally the residual amount of sodium pentachlorophenate is obtained, solving the problems of complicated processing of egg samples, high requirements for personnel operation and the influence of high background on the determination of sodium pentachlorophenate. This method has high accuracy, good precision, low detection cost, good stability and good safety. Description of the Drawings

[0037] To more clearly illustrate the technical solutions in the embodiments of the present drawings or in the related art, the following will briefly introduce the drawings required for use in the description of the embodiments or the related art. Obviously, the drawings in the following description are only some embodiments of the present drawings. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on the structures shown in these drawings.

[0038] Figure 1 It is a schematic flowchart of a method for determining the residue amount of sodium pentachlorophenol in eggs provided by an embodiment of the present application;

[0039] Figure 2 It is a calibration curve provided by an embodiment of the present application.

[0040] The realization of the purpose of the present application, functional features and advantages will be further described with reference to the embodiments and the drawings. Detailed implementation manners

[0041] Hereinafter, the embodiments of the method for determining the residue amount of sodium pentachlorophenol in eggs of the present application will be specifically disclosed in detail with reference to the drawings as appropriate. However, there may be cases where unnecessary detailed descriptions are omitted. For example, there are cases where the detailed descriptions of well-known matters and the repeated descriptions of actually identical structures are omitted. This is to avoid the following descriptions from becoming unnecessarily long and to facilitate the understanding of those skilled in the art. In addition, the drawings and the following descriptions are provided for those skilled in the art to fully understand the present application, and are not intended to limit the subject matter recited in the claims.

[0042] The "range" disclosed in the present application is defined in the form of a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of a particular range. The range defined in this way can include or not include the end values, and can be combined arbitrarily, that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, ranges of 60-110 and 80-120 are also contemplated. In addition, if the minimum range values 1 and 2 are listed, and if the maximum range values 3, 4, and 5 are listed, the following ranges are all contemplated: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In the present application, unless otherwise stated, the numerical range "a-b" represents an abbreviated representation of any real number combination between a and b, where a and b are both real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" have been fully listed herein, and "0-5" is only an abbreviated representation of these numerical combinations. In addition, when it is stated that a certain parameter is an integer ≥2, it is equivalent to disclosing that the parameter is, for example, the integer 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0043] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form new technical solutions.

[0044] To make the above objects, features, and advantages of the present application more obvious and understandable, the technical solutions of the present application will be further described below with reference to the accompanying drawings and embodiments. However, the present application is not limited to the listed embodiments, and should also include any other well-known changes within the scope of the rights required by the present application.

[0045] The embodiment of the present application provides a method for determining the residue amount of sodium pentachlorophenol in eggs, with reference to Figure 1 , Figure 1 which is a schematic flow chart of a method for determining the residue amount of sodium pentachlorophenol in eggs provided by the embodiment of the present application.

[0046] In this embodiment, the method for determining the residue amount of sodium pentachlorophenol in eggs includes:

[0047] Step S10, taking the edible part of the egg sample for homogenization to obtain an egg liquid sample;

[0048] Eggs are a type of food rich in protein. Poultry such as chickens and ducks can produce egg foods such as eggs and duck eggs. Sodium pentachlorophenol is a highly toxic organochlorine pesticide with good water solubility. It can be widely spread through water carriers, accumulate in poultry bodies, and remain in eggs.

[0049] Taking eggs as an example, an egg includes an edible part and an inedible part. The inedible part is the eggshell, which is brittle and hard. The edible part is the egg yolk and egg white protected inside the eggshell. Sodium pentachlorophenol basically remains in the edible part of the egg. Take an egg sample to be detected for the residue amount of sodium pentachlorophenol, break its outer shell, take the egg white and egg yolk of the edible part and mix them for homogenization to obtain an egg liquid sample. In order to quantitatively determine the residue amount of sodium pentachlorophenol in the egg sample, a certain amount of the egg liquid sample can be accurately weighed and placed in a centrifuge tube for standby. For example, 5 g (accurate to 0.001 g) of the egg liquid sample can be weighed with an electronic balance and placed in a 50 mL centrifuge tube.

[0050] Step S20, adding an internal standard reagent, a protein precipitant, dilute sulfuric acid, and n-hexane to the egg liquid sample, mixing evenly and then performing centrifugation, and taking the upper organic phase for purification and then waiting for derivatization;

[0051] The internal standard reagent refers to a reagent used for quantitatively determining the residue amount of sodium pentachlorophenol by the internal standard method. The internal standard reagent should meet the conditions such as being similar in properties to pentachlorophenol and not reacting chemically with pentachlorophenol, and having a retention time and response value similar to those of pentachlorophenol during chromatographic analysis. For example, the internal standard reagent can be selected as 2,4,6-tribromophenol, and its concentration is 1.0 μg / ml.

[0052] Protein precipitants are reagents used to precipitate proteins after denaturation. Egg yolks are rich in substances such as fat and phospholipids, while egg whites are richer in protein content. During chromatographic analysis, proteins may interact with derivatives of pentachlorophenol, affecting the retention time and response value. Removing the precipitated proteins using a protein precipitant can reduce matrix interference. Optionally, the protein precipitant is trichloroacetic acid, and the concentration of trichloroacetic acid is 5%.

[0053] Dilute sulfuric acid has strong acidity, and hydrogen ions in the solution can be completely ionized. After adding dilute sulfuric acid, the pH value of the egg liquid sample system is adjusted to acidic, converting sodium pentachlorophenol into pentachlorophenol. The dilute sulfuric acid used in this example can be 10% dilute sulfuric acid. Hydrochloric acid and nitric acid also have strong acidity, but hydrochloric acid contains chlorine elements, which may affect the chromatographic analysis results, and nitric acid has strong oxidizing properties. Adding it to the egg liquid sample may react with other substances in the matrix to generate impurities, making the composition of the egg liquid sample more complex. Using dilute sulfuric acid can avoid these situations and improve the measurement accuracy.

[0054] As an organic solvent, n-hexane can dissolve pentachlorophenol and separate pentachlorophenol from other impurities. Its structure is relatively stable and will not react with pentachlorophenol. After adding dilute sulfuric acid and protein precipitant, sodium pentachlorophenol is converted into pentachlorophenol. After adding n-hexane and mixing evenly, centrifugation is carried out for liquid-liquid extraction, and pentachlorophenol is present in the upper organic phase. Then, the organic phase is purified to further remove impurities.

[0055] In some embodiments of the present application, the purification treatment of the organic phase includes the following steps: Take the organic phase in a centrifuge tube, add concentrated sulfuric acid, vortex and mix evenly, then centrifuge to obtain the supernatant, which is the purified organic phase. The concentrated sulfuric acid used in this example can be 98% concentrated sulfuric acid. Using concentrated sulfuric acid as the purification reagent, on the one hand, concentrated sulfuric acid has strong oxidizing properties and can remove impurities such as phospholipids; on the other hand, it has the same composition as the dilute sulfuric acid that adjusts the system pH, avoiding introducing more impurities into the system.

[0056] In some embodiments of the present application, add 100 μL of 1.0 μg / ml 2,4,6-tribromophenol internal standard reagent, 10 mL of 5% trichloroacetic acid protein precipitant, 0.5 mL of 10% dilute sulfuric acid, and 10 mL of n-hexane to a 50 mL centrifuge tube containing 5 g of egg liquid sample. Vigorously shake and mix evenly for 1 min, centrifuge at 8000 rpm for 3 min. Take about 2 mL of the upper organic phase and transfer it to another 15 mL centrifuge tube. Add 0.5 mL of concentrated sulfuric acid, vortex and mix evenly for 1 min, centrifuge at 8000 rpm for 3 min, and take 1 mL of the n-hexane supernatant for derivatization.

[0057] Step S30: Prepare a series of pentachlorophenol standard working solutions, and add an internal standard reagent to the pentachlorophenol standard working solutions.

[0058] The concentration of pentachlorophenol in the pentachlorophenol standard working solutions is known and can be used to establish a calibration curve. The series of pentachlorophenol standard working solutions contain multiple different concentrations. The higher the correlation between the concentration points, the higher the measurement accuracy. The same internal standard reagent as that in the egg liquid sample is added to the pentachlorophenol standard working solutions.

[0059] In some embodiments of the present application, the series of concentrations of the pentachlorophenol standard working solutions include 0.1 ng / mL, 0.5 ng / mL, 1.0 ng / mL, 2.0 ng / mL, and 5 ng / mL. The above series of concentrations include 5 concentration points, and together with the coordinate origin, 6 concentration points can be formed. No pentachlorophenol is added to the solution corresponding to the coordinate origin. For example, a series of 10 mL volumetric flasks can be taken, and 10 μL of 100 ng / mL pentachlorophenol standard stock solution, 5 μL of 1 μg / mL pentachlorophenol standard stock solution, 10 μL of 1 μg / mL pentachlorophenol standard stock solution, 20 μL of 1 μg / mL pentachlorophenol standard stock solution, and 50 μL of 1 μg / mL pentachlorophenol standard stock solution are taken respectively to obtain the above different concentrations of pentachlorophenol standard working solutions. Then, 100 μL of 1.0 μg / ml 2,4,6-tribromophenol internal standard reagent is added to each, and the volume is fixed to 10 mL with n-hexane and mixed evenly. 1 mL of each is taken for derivatization.

[0060] Step S40: Add a derivatization reagent to the organic phase and the pentachlorophenol standard working solutions for derivatization treatment.

[0061] Under chromatographic analysis conditions, the structural stability of pentachlorophenol is limited. Higher-stability pentachlorophenol derivatives can be obtained through derivatization treatment to improve the quantitative accuracy.

[0062] In some embodiments of the present application, the derivatization reagent used is acetic anhydride-pyridine solution, and the volume ratio of acetic anhydride to pyridine in the derivatization reagent is 1:1. The acetic anhydride-pyridine solution is an acetylation reagent and can react with pentachlorophenol. During the reaction, pyridine plays a catalytic role, and the phenolic hydroxyl group of pentachlorophenol undergoes an esterification reaction with acetic anhydride to form pentachlorophenyl acetate.

[0063] In some embodiments of the present application, the steps of derivatizing the organic phase and the pentachlorophenol standard working solution include: separately taking the organic phase and the pentachlorophenol standard working solution, adding a derivatizing reagent to each, sealing, and reacting in a water bath at 60°C for 10 min - 20 min; taking out and cooling, adding 0.2 mol / L potassium carbonate solution, fully mixing, and then performing centrifugation. After adding the derivatizing reagent to the organic phase and the pentachlorophenol standard working solution, derivatization is carried out at a reaction temperature of 60°C to obtain pentachlorophenyl acetate. After cooling, potassium carbonate solution is added to remove the excess derivatizing reagent, and carbon dioxide is generated during the process. Pentachlorophenyl acetate has higher structural stability. For example, add 0.2 ml of a derivatizing reagent (volume ratio acetic anhydride:pyridine = 1:1) to the organic phase and the pentachlorophenol standard working solution respectively, seal, react in a water bath at 60°C for 15 min, take out and cool, add 2 ml of 0.2 mol / L potassium carbonate solution, fully mix and extract, and centrifuge at 8000 rpm for 2 min.

[0064] The processing time of egg samples in the examples of the present application is relatively short, which is beneficial for batch processing.

[0065] Step S50: Place the derivatized organic phase and pentachlorophenol standard working solution in a gas chromatography-tandem mass spectrometry device for on-machine testing to obtain the content of pentachlorophenol in the organic phase;

[0066] The gas chromatography-tandem mass spectrometry device directly measures the content of pentachlorophenol phenyl acetate. A conversion calculation method can be set in the device, and then the device directly outputs the content of pentachlorophenol.

[0067] In some embodiments of the present application, the analysis conditions for on-machine testing include the following conditions and refer to Table 1 below:

[0068] Chromatographic column: HP-5MS capillary chromatographic column, column length 30 m, inner diameter 0.25 mm, film thickness 0.25 μm;

[0069] Injector temperature 230°C;

[0070] Carrier gas: helium, purity ≥ 99.999%;

[0071] Splitless injection mode, injection volume: 1 μL;

[0072] Constant flow mode, flow rate 1.0 mL / min;

[0073] Temperature programming: initial temperature 80°C, hold for 2 min, increase to 290°C at 10°C / min, hold for 4 min;

[0074] Transfer line temperature: 280°C;

[0075] Ionization mode: EI, energy 70 ev;

[0076] Ion source temperature: 230 °C;

[0077] Scanning mode: Multiple reaction monitoring mode.

[0078] Table 1

[0079]

[0080] Step S60, determine the residue amount of sodium pentachlorophenate in the egg sample according to the content of pentachlorophenol.

[0081] In the gas chromatography tandem mass spectrometry equipment, the conversion relationship between pentachlorophenyl acetate and sodium pentachlorophenate in the egg sample can also be set, and the residue amount can be directly output by the equipment, or the content of pentachlorophenol output by the equipment can be used as a basis to further calculate the residue amount of sodium pentachlorophenate.

[0082] In some embodiments of the present application, the residue amount of sodium pentachlorophenate is calculated according to the following expression:

[0083] C = c × v × 1.08 / m;

[0084] Wherein, C represents the residue amount of sodium pentachlorophenate in the egg sample, with the unit of μg / kg; c represents the content of pentachlorophenol in the organic phase sample solution, with the unit of ng / mL; v represents the fixed volume of the organic phase sample solution, with the unit of mL; m represents the sampling amount of the egg sample, with the unit of g. The 1.08 in the formula is the calculation conversion coefficient between pentachlorophenol and sodium pentachlorophenate.

[0085] The embodiments of the present application use the internal standard method for calibration and quantification, which can effectively reduce the human operation error in the complex pretreatment process, eliminate the interference caused by the matrix effect of the matrix sample, and has a lower quantification limit and higher accuracy. In addition, using the multiple reaction monitoring mode of the gas chromatography tandem mass spectrometer for determination can effectively reduce the test interference brought about except after the purification of the pretreatment process.

[0086] In this embodiment, the edible part of the egg sample is taken for homogenization treatment to obtain an egg liquid sample; an internal standard reagent, a protein precipitant, dilute sulfuric acid, and n-hexane are added to the egg liquid sample, and after mixing evenly, centrifugation treatment is carried out. The upper organic phase is taken for purification and then waiting for derivatization; a series of pentachlorophenol standard working solutions are prepared, and the internal standard reagent is added to the pentachlorophenol standard working solutions; a derivatization reagent is added to the organic phase and the pentachlorophenol standard working solutions for derivatization treatment; the derivatized organic phase and pentachlorophenol standard working solutions are placed in a gas chromatography-tandem mass spectrometry device for on-machine testing to obtain the content of pentachlorophenol in the organic phase; the residual amount of sodium pentachlorophenol in the egg sample is determined according to the content of pentachlorophenol. In the embodiment of the present application, the homogenized egg liquid sample is extracted with a protein precipitant, and the pH is adjusted to acidic with dilute sulfuric acid to convert sodium pentachlorophenol in the egg liquid sample into pentachlorophenol. Liquid-liquid extraction is carried out with n-hexane, followed by impurity purification. A derivatization reagent is used for derivatization, and the content of the derivative is determined by gas chromatography-tandem mass spectrometry. The instrument directly calculates the content of pentachlorophenol, and quantitative analysis is carried out by the internal standard method. Finally, the residual amount of sodium pentachlorophenol is obtained, which solves the problems of complicated processing procedures for egg samples, high requirements for personnel operation, and the influence of high background on the determination of sodium pentachlorophenol. This method has high accuracy, good precision, low detection cost, good stability, and good safety.

[0087] The following elaborates on the solution of the present application in combination with specific embodiments.

[0088] Instruments used in the embodiment: 8890-700D triple quadrupole tandem mass spectrometer (Agilent Technologies, USA); Sartorius electronic balance (one-thousandth, Sartorius Beijing Co., Ltd.); JW-3024HR high-speed refrigerated centrifuge (Anhui Jiawen Instrument and Equipment Co., Ltd.); XW-80A vortex mixer (Shanghai Jingke Industrial Co., Ltd.).

[0089] Standard products: Purchased from Shanghai Anpu Experimental Technology Co., Ltd.

[0090] Example 1: Accuracy and repeatability tests.

[0091] Accurately weigh 20 portions of 5 grams (accurate to 0.001 g) of homogenized negative fresh egg samples into centrifuge tubes, including 2 portions of background samples and 6 portions of each of the three groups of spiked samples, namely low-concentration spiked samples, medium-concentration spiked samples, and high-concentration spiked samples. After adding 1 ng of pentachlorophenol to the low-concentration spiked sample group, 2 ng of pentachlorophenol to the medium-concentration spiked sample group, and 10 ng of pentachlorophenol to the high-concentration spiked sample group, prepare for the next test.

[0092] To each centrifuge tube containing each sample, 100 μL of 1.0 μg / ml 2,4,6-tribromophenol internal standard reagent, 10 mL of 5% trichloroacetic acid solution, 0.5 mL of 10% sulfuric acid solution, and 10 mL of n-hexane solution were successively added. First, shake vigorously and mix well for 1 min, then centrifuge at 8000 rpm for 3 min. Take 2 mL of the upper organic phase and transfer it to another 15 mL centrifuge tube. Add 0.5 mL of concentrated sulfuric acid, vortex and mix well for 1 min, then centrifuge at 8000 rpm for 3 min. Take 1 mL of the n-hexane supernatant for derivatization operation together with the standard working solution.

[0093] Preparation of the standard working solution:

[0094] Take a series of six 10 mL volumetric flasks. First, respectively pipette 10 μL of 100 ng / mL pentachlorophenol standard stock solution, 5 μL of 1 μg / mL pentachlorophenol standard stock solution, 10 μL of 1 μg / mL pentachlorophenol standard stock solution, 20 μL of 1 μg / mL pentachlorophenol standard stock solution, and 50 μL of 1 μg / mL pentachlorophenol standard stock solution. Then, add 100 μL of 1.0 μg / ml 2,4,6-tribromophenol internal standard reagent to each volumetric flask, make up the volume to the mark with n-hexane and mix well. Take 1 mL of each solution for derivatization operation.

[0095] Derivatization: Add 0.2 ml of the derivatization reagent (volume ratio acetic anhydride:pyridine = 1:1) to the above sample extract and standard working solution, seal, react in a water bath at 60 °C for 15 min, take out and cool, add 2 ml of 0.2 mol / L potassium carbonate solution, mix well and extract. Note that carbon dioxide will be generated. Centrifuge at 8000 rpm for 2 min, take the organic phase for determination by gas chromatography-tandem mass spectrometry.

[0096] 2 background samples were negative, and pentachlorophenol sodium could be regarded as not detected. The test results of the remaining 18 spiked samples are shown in Table 2 below.

[0097] Table 2

[0098]

[0099] The internal standard method was used to draw the calibration curve. The calibration curve is as Figure 2 shown. It can be found that the correlation coefficient R2 of the linear standard working curve is 0.9999; use this calibration curve to correct the sample solution, calculate the corresponding recovery rate and precision. The relative standard deviation RSD of the precision is 3.3 - 8.3%; the recovery rate is 81.8 - 118.5%.

[0100] Combined with Table 2 above and Figure 2 it can be concluded that: The above data indicate that the detection method of the embodiment of the present application for testing pentachlorophenol sodium in egg samples is stable and reliable.

[0101] Example 2: Limit of Quantification Test

[0102] Accurately weigh 6 portions of 5 g (accurate to 0.001 g) of homogenized negative fresh egg samples into centrifuge tubes, and add 1 ng of pentachlorophenol to each for the next step of the test.

[0103] Add 100 μL of 1.0 μg / ml 2,4,6-tribromophenol internal standard reagent, 10 mL of 5% trichloroacetic acid solution, 0.5 mL of 10% sulfuric acid solution, and 10 mL of n-hexane solution to the centrifuge tubes containing each sample. Vigorously shake and mix well for 1 min, centrifuge at 8000 rpm for 3 min. Take 2 mL of the upper organic phase and transfer it to another 15 mL centrifuge tube. Add 0.5 mL of concentrated sulfuric acid, vortex and mix well for 1 min, centrifuge at 8000 rpm for 3 min. Take 1 mL of the n-hexane supernatant for derivatization operation together with the standard working solution.

[0104] Preparation of standard working solution:

[0105] Take a series of six 10 mL volumetric flasks. First, transfer 10 μL of 100 ng / mL pentachlorophenol standard stock solution, 5 μL of 1 μg / mL pentachlorophenol standard stock solution, 10 μL of 1 μg / mL pentachlorophenol standard stock solution, 20 μL of 1 μg / mL pentachlorophenol standard stock solution, and 50 μL of 1 μg / mL pentachlorophenol standard stock solution into each volumetric flask respectively. Then add 100 μL of 1.0 μg / ml 2,4,6-tribromophenol internal standard reagent to each volumetric flask, make up the volume with n-hexane and mix well. Take 1 mL of each solution for derivatization operation.

[0106] Derivatization:

[0107] Add 0.2 ml of derivatization reagent (volume ratio acetic anhydride:pyridine = 1:1) to the above sample extract and standard working solution, seal, react in a water bath at 60 °C for 15 min, take out and cool. Add 2 ml of 0.2 mol / L potassium carbonate solution, mix well for extraction. Note that carbon dioxide will be generated. Centrifuge at 8000 rpm for 2 min, take the organic phase for determination by gas chromatography-tandem mass spectrometry. Use the internal standard method to draw a calibration curve, correct the sample solution, and calculate the signal-to-noise ratio of the target peak. The test results are shown in Table 3 below. The signal-to-noise ratio is 11.01 - 29.47, all of which are greater than or equal to 10.

[0108] Table 3

[0109] Concentration Signal-to-noise ratio 0.1 ng / ml 21.99 0.1 ng / ml 11.01 0.1 ng / ml 11.62 0.1 ng / ml 21.49 0.1 ng / ml 13.12 0.1 ng / ml 29.47

[0110] It can be concluded from Table 3 that by substituting the above concentration of 0.1 ng / ml into c in the above expression, the quantification limit is calculated to be 0.2 μg / kg. The above data indicate that the quantification limit of the detection method in the examples of the present application for testing the content of sodium pentachlorophenol in egg samples can reach 0.2 μg / kg; compared with the determination section of the residue of sodium pentachlorophenol in animal-derived foods in the national food safety standard GB 23200.92-2016 regarding the liquid chromatography-mass spectrometry method, the specified quantification limit of 1.0 μg / kg is lower, which can meet the detection requirements for the daily residue of sodium pentachlorophenol in eggs.

[0111] It should be noted that the present application is not limited to the above embodiments. The above embodiments are only examples, and embodiments with the same composition and the same effect as the technical idea within the scope of the technical solution of the present application are included in the technical scope of the present application. In addition, within the scope of not departing from the gist of the present application, various modifications that can be conceived by those skilled in the art to the embodiments and other ways constructed by combining some of the constituent elements of the embodiments are also included in the scope of the present application.

Claims

1. A method for determining the residual amount of sodium pentachlorophenol in eggs, characterized in that: The following steps are involved: Taking the edible part of the egg sample and homogenizing it to obtain an egg liquid sample; Adding an internal standard reagent, a protein precipitant, dilute sulfuric acid and n-hexane to the egg liquid sample, mixing well and then centrifuging, taking an upper organic phase for purification and then derivatization; A series of pentachlorophenol standard working solutions are prepared, and the internal standard reagent is added to the pentachlorophenol standard working solutions; Adding a derivatization reagent to the organic phase and the pentachlorophenol standard working solution to perform a derivatization treatment; The derivatized organic phase and the pentachlorophenol standard working solution are placed in a gas chromatography tandem mass spectrometry device for testing to obtain the content of pentachlorophenol in the organic phase; The residual amount of sodium pentachlorophenol in the egg sample is determined according to the content of pentachlorophenol.

2. The method for determining the residual amount of sodium pentachlorophenol in eggs according to claim 1, wherein: The residual amount of sodium pentachlorophenol is calculated according to the following expression: C = c × v × 1.08 / m; Wherein, C represents the residual amount of sodium pentachlorophenol in the egg sample, in μg / kg; c represents the content of pentachlorophenol in the sample solution of the organic phase, in ng / mL; v represents the fixed volume of the sample solution of the organic phase, in mL; m represents the sampling amount of the egg sample, in g.

3. The method for determining the residual amount of sodium pentachlorophenol in eggs according to claim 1, wherein: The internal standard reagent is 2,4,6-tribromophenol.

4. The method for determining the residual amount of sodium pentachlorophenol in eggs according to claim 1, wherein: The protein precipitant is trichloroacetic acid.

5. The method for determining the residual amount of sodium pentachlorophenol in eggs according to claim 1, characterized in that: The purification of the organic phase comprises the following steps: The organic phase is placed in a centrifuge tube, concentrated sulfuric acid is added, vortexed and mixed, and then centrifuged to obtain the supernatant, which is the purified organic phase.

6. The method for determining the residual amount of sodium pentachlorophenol in eggs according to claim 1, characterized in that: The concentration series of the pentachlorophenol standard working solution include 0.1 ng / mL, 0.5 ng / mL, 1.0 ng / mL, 2.0 ng / mL, and 5 ng / mL.

7. The method for determining the residual amount of sodium pentachlorophenol in eggs according to claim 1, characterized in that: The derivatization reagent is an acetic anhydride-pyridine solution, and the volume ratio of acetic anhydride to pyridine in the derivatization reagent is 1:

1.

8. The method for determining the residual amount of sodium pentachlorophenol in eggs according to claim 7, characterized in that: The steps of derivatizing the organic phase and the pentachlorophenol standard working solution include: Separately take the organic phase and the pentachlorophenol standard working solution, add the derivatization reagent to each, seal the mixture, and react in a water bath at 60° C. for 10 min-20 min; Take out and cool, add 0.2 mol / L potassium carbonate solution, mix thoroughly and centrifuge.

9. The method for determining the residual amount of sodium pentachlorophenol in eggs according to claim 1, characterized in that: The analysis conditions of the on-machine test include: Chromatographic column: HP-5MS capillary column, column length 30m, inner diameter 0.25mm, film thickness 0.25μm; Inlet temperature 230°C; Carrier gas: helium, purity ≥ 99.999%; Injection in splitless mode, injection volume: 1 μL; Constant flow mode, flow rate 1.0 mL / min; Heating program: initial temperature 80℃, hold for 2min, increase to 290℃ at 10℃ / min, hold for 4min; Transmission line temperature: 280°C; Ionization mode: EI, energy 70 eV; Ion source temperature: 230°C; Scanning mode: multiple reaction monitoring mode.

10. The method for determining the residual amount of sodium pentachlorophenol in eggs according to any one of claims 1 to 9, characterized in that: The quantitative limit of the residual amount of sodium pentachlorophenol is 0.2 μg / kg.

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