Method for determining the pentachlorophenyl sodium residue in egg

Abstract

The application discloses a method for determining the residual amount of sodium pentachlorophenol in eggs, and belongs to the technical field of detection. The method comprises the following steps: homogenizing the edible part of an egg sample 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, uniformly mixing, centrifuging, taking the upper organic phase, purifying and then deriving; preparing a series of sodium pentachlorophenol standard working solutions, adding an internal standard reagent to the sodium pentachlorophenol standard working solutions; adding a derivative reagent to the organic phase and the sodium pentachlorophenol standard working solutions, and performing derivative treatment; placing the derived organic phase and the sodium pentachlorophenol standard working solutions in a gas chromatography tandem mass spectrometry device, performing machine testing, obtaining the content of sodium pentachlorophenol in the organic phase, and determining the residual amount of sodium pentachlorophenol in the egg sample according to the content of sodium pentachlorophenol. The application realizes the technical effect of improving the detection accuracy of the residual amount of sodium pentachlorophenol in eggs.

Description

Technical Field

[0001] This application relates to the field of detection technology, and in particular to a method for determining the residual amount of sodium pentachlorophenate in eggs. Background Technology

[0002] Sodium pentachlorophenate is an organochlorine pesticide that degrades slowly in the natural environment. It can enter poultry through feed and accumulate, leading to residues in eggs and posing a significant health risk. Sodium pentachlorophenate is listed in the list of prohibited drugs and compounds for use in food animals.

[0003] Currently, methods for detecting pentachlorophenols in food include liquid chromatography-tandem mass spectrometry (LC-MS / MS), gas chromatography-mass spectrometry (GC-MS / MS), and colloidal gold immunochromatography (CIP). These methods all use external standard methods for quantification, which have poor stability and accuracy, and require highly skilled operators. LC-MS / MS is currently the mainstream detection method, but it is costly and only suitable for meat products. Furthermore, sample pretreatment often employs solid-phase extraction and gel chromatography purification methods, making the experimental procedures cumbersome and complex. Incomplete purification can easily lead to matrix effects, resulting in inaccurate quantification. Summary of the Invention

[0004] The main objective of this application is to provide a method for determining the residual amount of sodium pentachlorophenate in eggs, aiming to solve the technical problem of low accuracy in existing methods for detecting pentachlorophenol in food due to the matrix effect of eggs.

[0005] To achieve the above objectives, this application provides a method for determining the residual amount of sodium pentachlorophenate in eggs, comprising:

[0006] The edible portion of the egg sample was homogenized to obtain an egg liquid sample;

[0007] Add internal standard reagent, protein precipitant, dilute sulfuric acid and n-hexane to the egg liquid sample, mix well and centrifuge, take the upper organic phase for purification and derivatization;

[0008] Prepare a series of pentachlorophenol standard working solutions by adding the internal standard reagent to the pentachlorophenol standard working solutions;

[0009] A derivatizing reagent is added to the organic phase and the pentachlorophenol standard working solution to perform derivatization treatment;

[0010] The derivatized organic phase and pentachlorophenol standard working solution were placed in a gas chromatography-tandem mass spectrometry (GC-MS) instrument and tested to obtain the content of pentachlorophenol in the organic phase.

[0011] The residual amount of sodium pentachlorophenate in the egg sample was determined based on the content of the pentachlorophenate.

[0012] In some embodiments of this 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, in μg / kg; c represents the content of pentachlorophenol in the organic phase sample solution, in ng / mL; v represents the final volume of the organic phase sample solution, in mL; and m represents the sample amount of the egg sample, in g.

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

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

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

[0018] Take the organic phase into a centrifuge tube, add concentrated sulfuric acid, vortex to mix, and then centrifuge. The supernatant obtained is the purified organic phase.

[0019] In some embodiments of this application, the 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 this application, the derivatizing agent is an acetic anhydride-pyridine solution, wherein the volume ratio of acetic anhydride to pyridine in the derivatizing agent is 1:1.

[0021] In some embodiments of this application, the step of derivatizing the organic phase and the pentachlorophenol standard working solution includes:

[0022] Take the organic phase and pentachlorophenol standard working solution respectively, add derivatizing reagent to each, seal and react in a water bath at 60℃ for 10 min-20 min;

[0023] Remove and cool, add 0.2 mol / L potassium carbonate solution, mix thoroughly and then centrifuge.

[0024] In some embodiments of this application, the analysis conditions for the on-machine test include:

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

[0026] Inlet temperature 230℃;

[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℃, hold for 2 min, increase to 290℃ at 10℃ / min, hold for 4 min;

[0031] Transmission line temperature: 280℃;

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

[0033] Ion source temperature: 230℃;

[0034] Scanning method: Multiple reaction monitoring mode.

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

[0036] The method for determining sodium pentachlorophenate residue in eggs provided in this application involves homogenizing the edible portion of an egg sample 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 thoroughly, centrifuging, and purifying the upper organic phase before derivatization; preparing a series of pentachlorophenate standard working solutions, and adding the internal standard reagent to the pentachlorophenate standard working solutions; adding a derivatization reagent to the organic phase and the pentachlorophenate standard working solutions for derivatization; placing the derivatized organic phase and the pentachlorophenate standard working solutions in a gas chromatography-tandem mass spectrometry (GC-MS) device for testing to obtain the pentachlorophenate content in the organic phase; and determining the sodium pentachlorophenate residue in the egg sample based on the pentachlorophenate content. In this embodiment, homogenized egg liquid samples are extracted with a protein precipitant, and the pH is adjusted to acidity with dilute sulfuric acid to convert sodium pentachlorophenate in the egg liquid sample into pentachlorophenol. Liquid-liquid extraction is performed using n-hexane, followed by impurity purification, derivatization with a derivatizing reagent, and the content of the derivative is determined by gas chromatography-tandem mass spectrometry. The content of pentachlorophenol is directly calculated by the instrument, and quantification is performed using the internal standard method to finally obtain the residual amount of sodium pentachlorophenate. This method solves the problems of complicated egg sample processing, high operator requirements, 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. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments or related technologies of this drawing, the drawings used in the description of the embodiments or related technologies will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this drawing. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0038] Figure 1 A flowchart illustrating a method for determining sodium pentachlorophenate residue in eggs, provided as an embodiment of this application;

[0039] Figure 2 The calibration curve provided for the embodiments of this application.

[0040] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0041] The following detailed description, with appropriate reference to the accompanying drawings, discloses embodiments of the method for determining sodium pentachlorophenate residues in eggs according to this application. However, unnecessary details may be omitted. For example, detailed descriptions of well-known matters and repetitive descriptions of essentially identical structures may be omitted. This is to avoid unnecessarily lengthy descriptions and to facilitate understanding by those skilled in the art. Furthermore, the accompanying drawings and the following description are provided for the purpose of enabling those skilled in the art to fully understand this application and are not intended to limit the subject matter of the claims.

[0042] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; 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, it is expected that ranges of 60-110 and 80-120 are also included. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In this application, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0-5" indicates that all real numbers between "0-5" have been listed in this article; "0-5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0043] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0044] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, the technical solution of this application is further described below in conjunction with the accompanying drawings and embodiments. However, this application is not limited to the listed embodiments, but should also include any other well-known modifications within the scope of the claims made in this application.

[0045] This application provides a method for determining the residual amount of sodium pentachlorophenate in eggs, referring to... Figure 1 , Figure 1 This is a flowchart illustrating a method for determining the residual amount of sodium pentachlorophenate in eggs, as provided in an embodiment of this application.

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

[0047] Step S10: Take the edible part of the egg sample and homogenize it to obtain an egg liquid sample;

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

[0049] Taking eggs as an example, eggs consist of edible and inedible parts. The inedible part is the eggshell, which is brittle and hard. The edible part consists of the yolk and albumen protected by the shell. Sodium pentachlorophenate in eggs is primarily found in the edible part. To test for sodium pentachlorophenate residue, take an egg sample, break open the shell, and mix and homogenize the edible egg white and yolk to obtain an egg liquid sample. To quantitatively determine the sodium pentachlorophenate residue in the egg sample, a certain amount of egg liquid sample can be accurately weighed into a centrifuge tube. For example, 5g (accurate to 0.001g) of egg liquid sample can be weighed into a 50mL centrifuge tube using an electronic balance.

[0050] Step S20: Add internal standard reagent, protein precipitant, dilute sulfuric acid and n-hexane to the egg liquid sample, mix well and centrifuge, take the upper organic phase for purification and derivatization.

[0051] Internal standard reagents are reagents used for the quantitative determination of sodium pentachlorophenate residues using the internal standard method. Internal standard reagents should meet the following conditions: similar properties to pentachlorophenate, no chemical reaction with pentachlorophenate, and similar retention time and response value to pentachlorophenate during chromatographic analysis. For example, 2,4,6-tribromophenol can be chosen as the internal standard reagent at a concentration of 1.0 μg / ml.

[0052] Protein precipitants are reagents used to denature and precipitate proteins. Egg yolks are rich in fats and phospholipids, while egg whites are even richer in protein. During chromatographic analysis, proteins may interact with pentachlorophenol derivatives, affecting retention time and response values. Using a protein precipitant to remove protein precipitate can reduce substrate interference. Optionally, the protein precipitant is trichloroacetic acid at a concentration of 5%.

[0053] Dilute sulfuric acid is a strong acid, and hydrogen ions in the solution can be completely ionized. Adding dilute sulfuric acid adjusts the pH of the egg liquid sample system to acidity, causing sodium pentachlorophenate to convert to pentachlorophenol. In this example, 10% dilute sulfuric acid can be used. Hydrochloric acid and nitric acid are also strong acids; however, hydrochloric acid contains chlorine, which may affect the chromatographic analysis results. Nitric acid is a strong oxidizing agent, and its addition to the egg liquid sample may react with other substances in the matrix, generating impurities and making the composition of the egg liquid sample more complex. Using dilute sulfuric acid avoids these problems and improves the accuracy of the determination.

[0054] Hexane, as an organic solvent, can dissolve pentachlorophenol, separating it from other impurities. Its structure is relatively stable and it does not react with pentachlorophenol. After adding dilute sulfuric acid and a protein precipitant, sodium pentachlorophenol is converted to pentachlorophenol. Hexane is then added, mixed thoroughly, and centrifuged for liquid-liquid extraction. Pentachlorophenol is present in the upper organic phase. Further purification of the organic phase can remove impurities.

[0055] In some embodiments of this application, the purification of the organic phase includes the following steps: taking the organic phase into a centrifuge tube, adding concentrated sulfuric acid, vortexing to mix, centrifuging, and obtaining the supernatant as the purified organic phase. The concentrated sulfuric acid used in this embodiment can be 98% concentrated sulfuric acid. Using concentrated sulfuric acid as the purification reagent has two advantages: firstly, concentrated sulfuric acid has strong oxidizing properties, which can remove impurities such as phospholipids; secondly, it has the same composition as dilute sulfuric acid used to adjust the pH of the system, thus avoiding the introduction of more impurities into the system.

[0056] In some embodiments of this application, 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 are added to a 50 mL centrifuge tube containing 5 g of egg liquid sample. The mixture is vigorously shaken for 1 min, centrifuged at 8000 rpm for 3 min, and about 2 mL of the upper organic phase is transferred to another 15 mL centrifuge tube. 0.5 mL of concentrated sulfuric acid is added, the mixture is vortexed for 1 min, centrifuged at 8000 rpm for 3 min, and 1 mL of the n-hexane supernatant is collected for further processing.

[0057] Step S30: Prepare a series of pentachlorophenol standard working solutions by adding an internal standard reagent to the pentachlorophenol standard working solutions;

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

[0059] In some embodiments of this application, the series of concentrations of the pentachlorophenol standard working solution includes 0.1 ng / mL, 0.5 ng / mL, 1.0 ng / mL, 2.0 ng / mL, and 5 ng / mL. This series of concentrations includes five concentration points, which, together with the origin, form six concentration points. The solution corresponding to the origin does not contain pentachlorophenol. For example, a series of 10 mL volumetric flasks can be used to take 10 μL of 100 ng / mL pentachlorophenol standard working solution, 5 μL of 1 μg / mL pentachlorophenol standard working solution, 10 μL of 1 μg / mL pentachlorophenol standard working solution, 20 μL of 1 μg / mL pentachlorophenol standard working solution, and 50 μL of 1 μg / mL pentachlorophenol standard working solution to obtain pentachlorophenol standard working solutions of different concentrations. Then, 100 μL of 1.0 μg / mL 2,4,6-tribromophenol internal standard reagent is added to each solution, and the solution is diluted to 10 mL with n-hexane and mixed well. 1 mL of each solution is then used for derivatization.

[0060] Step S40: Add a derivatizing reagent to the organic phase and the pentachlorophenol standard working solution to perform derivatization treatment;

[0061] Under chromatographic conditions, pentachlorophenol has limited structural stability. Derivatization can be used to obtain more stable pentachlorophenol derivatives, thereby improving quantitative accuracy.

[0062] In some embodiments of this application, the derivatizing agent used is an acetic anhydride-pyridine solution, wherein the volume ratio of acetic anhydride to pyridine in the derivatizing agent is 1:1. The acetic anhydride-pyridine solution is an acetylation agent that can react with pentachlorophenol. During the reaction, pyridine acts as a catalyst, and the phenolic hydroxyl group of pentachlorophenol undergoes an esterification reaction with acetic anhydride to generate pentachlorophenylacetic acid ester.

[0063] In some embodiments of this application, the derivatization process of the organic phase and the pentachlorophenol standard working solution includes: taking the organic phase and the pentachlorophenol standard working solution separately, adding derivatizing reagents to each, sealing, and reacting in a 60°C water bath for 10-20 min; removing and cooling, adding 0.2 mol / L potassium carbonate solution, mixing thoroughly, and centrifuging. After adding derivatizing reagents to the organic phase and the pentachlorophenol standard working solution, derivatization is carried out at a temperature of 60°C to obtain pentachlorophenylacetic acid ester. After cooling, potassium carbonate solution is added to remove excess derivatizing reagents, with carbon dioxide generated during the process. Pentachlorophenylacetic acid ester has higher structural stability. For example, adding 0.2 ml of derivatizing reagent (volume ratio of acetic anhydride:pyridine = 1:1) to both the organic phase and the pentachlorophenol standard working solution, sealing, reacting in a 60°C water bath for 15 min, removing and cooling, adding 2 ml of 0.2 mol / L potassium carbonate solution, mixing thoroughly, and centrifuging at 8000 rpm for 2 min.

[0064] The embodiments of this application have a relatively short processing time for egg samples, 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 and test them to obtain the content of pentachlorophenol in the organic phase;

[0066] Gas chromatography-tandem mass spectrometry directly measures the content of pentachlorophenol phenylacetate. A conversion calculation mode can be set in the equipment, and the equipment will directly output the content of pentachlorophenol.

[0067] In some embodiments of this application, the analysis conditions for the on-machine test include the following conditions, as shown in Table 1 below:

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

[0069] Inlet temperature 230℃;

[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 program: Initial temperature 80℃, hold for 2 min, increase to 290℃ at 10℃ / min, hold for 4 min;

[0074] Transmission line temperature: 280℃;

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

[0076] Ion source temperature: 230℃;

[0077] Scanning method: Multiple reaction monitoring mode.

[0078] Table 1

[0079]

[0080] Step S60: Determine the residual amount of sodium pentachlorophenate in the egg sample based on the content of pentachlorophenate.

[0081] Gas chromatography-tandem mass spectrometry (GC-MS) equipment can also be used to set the conversion relationship between pentachlorophenylacetate and sodium pentachlorophenate in egg samples, and the residual amount can be directly output by the equipment. Alternatively, the content of pentachlorophenol output by the equipment can be used as a basis to further calculate the residual amount of sodium pentachlorophenate.

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

[0083] C = c × v × 1.08 / m;

[0084] Where C represents the residual amount of sodium pentachlorophenate in the egg sample, in μg / kg; c represents the content of pentachlorophenol in the organic phase sample solution, in ng / mL; v represents the final volume of the organic phase sample solution, in mL; and m represents the sample weight of the egg sample, in g. 1.08 in the formula is the conversion factor between pentachlorophenol and sodium pentachlorophenate.

[0085] This application uses the internal standard method for calibration quantification, which effectively reduces human error in the complex pretreatment process and eliminates interference from matrix effects caused by the matrix sample, resulting in a lower limit of quantitation and greater accuracy. Furthermore, the use of multi-reaction monitoring mode in gas chromatography-tandem mass spectrometry effectively reduces testing interference beyond that introduced during pretreatment purification.

[0086] In this embodiment, the edible portion of an egg sample is homogenized 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, mixed thoroughly, and centrifuged. The upper organic phase is purified and then derivatized. A series of pentachlorophenol standard working solutions are prepared, and the internal standard reagent is added to the pentachlorophenol standard working solutions. A derivatizing reagent is added to the organic phase and the pentachlorophenol standard working solutions for derivatization. The derivatized organic phase and the pentachlorophenol standard working solutions are placed in a gas chromatography-tandem mass spectrometry (GC-MS) instrument for testing to obtain the pentachlorophenol content in the organic phase. The residual amount of sodium pentachlorophenolate in the egg sample is determined based on the pentachlorophenol content. In this embodiment, homogenized egg liquid samples are extracted with a protein precipitant, and the pH is adjusted to acidity with dilute sulfuric acid to convert sodium pentachlorophenate in the egg liquid sample into pentachlorophenol. Liquid-liquid extraction is performed using n-hexane, followed by impurity purification, derivatization with a derivatizing reagent, and the content of the derivative is determined by gas chromatography-tandem mass spectrometry. The content of pentachlorophenol is directly calculated by the instrument, and quantification is performed using the internal standard method to finally obtain the residual amount of sodium pentachlorophenate. This method solves the problems of complicated egg sample processing, high operator requirements, 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.

[0087] The following detailed description of the present application solution is provided in conjunction with specific embodiments.

[0088] The instruments used in the examples are: 8890-700D triple quadrupole tandem mass spectrometer (Agilent Technologies, USA); Sartorius electronic balance (0.1 g / L, Beijing Sartorius Balance Co., Ltd.); JW-3024HR high-speed refrigerated centrifuge (Anhui Jiawen Instrument Equipment Co., Ltd.); and 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 test.

[0091] Accurately weigh 5 grams (accurate to 0.001 g) of 20 homogenized negative fresh egg samples into centrifuge tubes. Among them, there are 2 background samples and 6 spiked samples in each of the 3 groups of spiked samples: low concentration spiked sample, medium concentration spiked sample, and high concentration spiked sample. Add 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, and then prepare for the next step of the experiment.

[0092] 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 centrifuge tubes containing each sample, respectively. Shake vigorously 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 for 1 min, and centrifuge at 8000 rpm for 3 min. Take 1 mL of the n-hexane supernatant to be used together with the standard working solution for derivatization.

[0093] Preparation of standard working solutions:

[0094] Take a series of six 10 mL volumetric flasks and transfer 10 μL of 100 ng / mL pentachlorophenol standard working solution, 5 μL of 1 μg / mL pentachlorophenol standard working solution, 10 μL of 1 μg / mL pentachlorophenol standard working solution, 20 μL of 1 μg / mL pentachlorophenol standard working solution, and 50 μL of 1 μg / mL pentachlorophenol standard working solution to each flask. Then add 100 μL of 1.0 μg / mL 2,4,6-tribromophenol internal standard reagent to each volumetric flask, dilute to volume with n-hexane and mix well. Take 1 mL of each solution for derivatization.

[0095] Derivatization: Add 0.2 ml of derivatizing reagent (acetic anhydride:pyridine = 1:1 by volume) to the above sample extract and standard working solution, seal, react in a 60℃ water bath for 15 min, remove and cool, add 2 ml of 0.2 mol / L potassium carbonate solution, mix thoroughly and extract, note that carbon dioxide will be generated, centrifuge at 8000 rpm for 2 min, take the organic phase and use gas chromatography-tandem mass spectrometry for analysis.

[0096] Two background samples were negative, which can be considered as no detection of sodium pentachlorophenate. The test results of the remaining 18 spiked samples are shown in Table 2 below.

[0097] Table 2

[0098]

[0099] The calibration curve is plotted using the internal standard method, as shown in the figure. Figure 2 As shown, the correlation coefficient R² of the regression equation of its linear standard working curve is 0.9999. Using this calibration curve to calibrate the sample solution, the corresponding recovery rate and precision were calculated. The relative standard deviation of precision (RSD) was 3.3-8.3%, and the recovery rate was 81.8-118.5%.

[0100] Combining Table 2 above and Figure 2 It can be concluded that the above data shows that the detection method of this application embodiment is reliable in testing the stability of sodium pentachlorophenate in egg samples.

[0101] Example 2: Limit of Quantitation Test.

[0102] Accurately weigh 5 grams (accurate to 0.001 g) of the homogenized negative fresh egg sample into six centrifuge tubes, add 1 ng of pentachlorophenol to each tube, and prepare for the next 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 tube containing each sample. Shake vigorously for 1 min, centrifuge at 8000 rpm for 3 min, take 2 mL of the upper organic phase into another 15 mL centrifuge tube, add 0.5 mL of concentrated sulfuric acid, vortex for 1 min, centrifuge at 8000 rpm for 3 min, and take 1 mL of the n-hexane supernatant to be used together with the standard working solution for derivatization.

[0104] Preparation of standard working solutions:

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

[0106] derivative:

[0107] Add 0.2 ml of derivatizing reagent (acetic anhydride:pyridine = 1:1, v / v) to the above sample extract and standard working solution, seal, and react in a 60℃ water bath for 15 min. Remove and cool, add 2 ml of 0.2 mol / L potassium carbonate solution, mix thoroughly for extraction (note that carbon dioxide will be generated), centrifuge at 8000 rpm for 2 min, collect the organic phase, and perform gas chromatography-tandem mass spectrometry (GC-MS). Use the internal standard method to plot a calibration curve, correct the sample solution, and calculate the signal-to-noise ratio (SNR) of the target peak. The test results are shown in Table 3 below, with SNR ranging from 11.01 to 29.47, all greater than or equal to 10.

[0108] Table 3

[0109] 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] Table 3 shows that substituting the concentration of 0.1 ng / ml into 'c' in the above expression yields a limit of quantitation of 0.2 μg / kg. This data indicates that the detection method of this application can achieve a limit of quantitation of 0.2 μg / kg for sodium pentachlorophenate in egg samples. This is lower than the limit of 1.0 μg / kg specified in the liquid chromatography-mass spectrometry section of GB23200.92-2016 National Food Safety Standard for the determination of sodium pentachlorophenate residues in animal-derived foods, and thus meets the daily requirements for detecting sodium pentachlorophenate residues in eggs.

[0111] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A method for determining the residual amount of sodium pentachlorophenate in eggs, characterized in that, Includes the following steps: The edible portion of the egg sample was homogenized to obtain an egg liquid sample; Add internal standard reagent, protein precipitant, dilute sulfuric acid and n-hexane to the egg liquid sample, mix well and centrifuge, take the upper organic phase into a centrifuge tube, add concentrated sulfuric acid, vortex mix well and centrifuge, the supernatant obtained is the purified organic phase, wherein the protein precipitant is trichloroacetic acid; Prepare a series of pentachlorophenol standard working solutions by adding the internal standard reagent to the pentachlorophenol standard working solutions; Take the purified organic phase and the pentachlorophenol standard working solution respectively, add derivatizing reagent to each, seal and react in a water bath at 60°C for 10 min-20 min. The derivatizing reagent is an acetic anhydride-pyridine solution, and the volume ratio of acetic anhydride to pyridine in the derivatizing reagent is 1:

1. Remove and cool, add 0.2 mol / L potassium carbonate solution, mix thoroughly and centrifuge to obtain the derivatized organic phase and the derivatized pentachlorophenol standard working solution; The derivatized organic phase and the derivatized pentachlorophenol standard working solution were placed in a gas chromatography-tandem mass spectrometry (GC-MS) instrument for analysis to obtain the content of pentachlorophenol in the organic phase. The analytical conditions for the analysis included: Chromatographic column: HP-5MS capillary column, 30m in length, 0.25mm in inner diameter, and 0.25μm in film thickness; Inlet temperature 230℃; Carrier gas: Helium, purity ≥ 99.999%; Splitless injection mode, injection volume: 1 μL; Constant flow mode, flow rate 1.0 mL / min; Temperature program: Initial temperature 80℃, hold for 2 min, increase to 290℃ at 10℃ / min, hold for 4 min; Transmission line temperature: 280℃; Ionization mode: EI, energy 70 eV; Ion source temperature: 230℃; Scanning method: Multiple reaction monitoring mode; The residual amount of sodium pentachlorophenate in the egg sample was determined based on the content of the pentachlorophenate.

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

3. The method for determining the residual amount of sodium pentachlorophenate in eggs as described in claim 1, characterized in that, The internal standard reagent is 2,4,6-tribromophenol.

4. The method for determining the residual amount of sodium pentachlorophenate in eggs as described in claim 1, characterized in that, 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.

5. The method for determining the residual amount of sodium pentachlorophenate in eggs according to any one of claims 1 to 4, characterized in that, The limit of quantification for the residual amount of sodium pentachlorophenate is 0.2 μg / kg.

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