Method for determining phenacetin and its metabolites in animal-derived food

Abstract

This invention discloses a method for determining phenacetin and its metabolites in animal-derived foods, belonging to the field of determination technology, including the following steps: S1, Sample pretreatment: Weigh 2g of homogenized animal-derived food sample into a 50mL plastic centrifuge tube, add 10mL of acetonitrile, shake for 1 min, add 3g of sodium chloride, shake for 1 min, and centrifuge for 10 min; take 8mL of the supernatant and add it to a dispersion solid-phase extraction purification tube, mix for 1 min, and centrifuge for 10 min; take 2mL of the supernatant, blow it to near dryness with nitrogen, redissolve it with acetonitrile-water, filter it through a 0.22 filter membrane, and then perform LC-MS / MS analysis; S2, Liquid chromatography-tandem mass spectrometry detection: Inject the test solution into LC-MS / MS, separate it using a C18 reversed-phase column, and perform gradient elution using mobile phase A and mobile phase B; S3, Quantitative analysis: Plot a standard curve using the matrix-matched external standard method, and calculate the content of phenacetin and acetaminophen in the sample based on the quantitative ion peak area of ​​the test solution. This invention discloses a determination method with low detection cost and high sensitivity.

Description

Technical Field

[0001] This invention relates to the field of assay technology, and more particularly to a method for determining phenacetin and its metabolites in animal-derived foods. Background Technology

[0002] Phenacetin (CAS No. 62-44-2) was among the first synthetically produced acetanilide antipyretic and analgesic compounds. In vivo, it is metabolized via O-deethylation to produce acetaminophen. Phenacetin has significant potential side effects, including kidney damage. The International Agency for Research on Cancer (IARC) of the World Health Organization has classified it as a Group 1 carcinogen. Due to its health risks, many countries have strictly restricted or banned its use in cosmetics.

[0003] Animal-derived foods are an important part of the public's diet. If there is illegal feeding of animals containing phenacetin (human waste drugs) or adulteration with veterinary drugs to alleviate symptoms of sick animals, or if feed and water sources are contaminated by wastewater from drug manufacturing, phenacetin and its metabolite acetaminophen residues may form in animal tissues, posing a food safety risk. Pork is one of the most consumed meat products in my country, with a complex matrix and wide distribution. Its residue levels directly affect public health; therefore, conducting simultaneous determination of phenacetin and its metabolites in pork is of significant practical importance.

[0004] Currently reported detection methods mainly include high-performance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry (HPLC-MS / MS) for determining phenacetin residues in cosmetics such as hair dyes. However, there are no reported methods specifically targeting animal-derived food matrices for simultaneous quantification using a phenacetin-acetaminophen parent-metabolite pairing. Directly applying existing cosmetic matrix detection methods suffers from problems such as significant matrix interference, unstable recovery rates, and insufficient sensitivity.

[0005] Therefore, developing a simple, highly sensitive method for detecting phenacetin and its metabolites in animal-derived foods is of great practical significance for food safety supervision. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the technical problem to be solved by the present invention is to propose a simple, highly sensitive method for detecting phenacetin and its metabolites in animal-derived foods.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] This invention provides a method for determining phenacetin and its metabolites in animal-derived foods, comprising the following steps:

[0009] S1. Sample Pretreatment: Weigh 2g of homogenized animal-derived food sample into a 50mL plastic centrifuge tube, add 10mL of acetonitrile, and vortex vigorously for 1 min. Then add 3g of sodium chloride, vortex vigorously for 1 min, and centrifuge at 4000r / min for 10 min in a benchtop low-speed centrifuge. Take 8mL of the supernatant and add it to a dispersion solid-phase extraction purification tube. Vortex and mix for 1 min, then centrifuge at 4000r / min for 10 min in a benchtop low-speed centrifuge. Take 2mL of the supernatant, purge to near dryness with nitrogen, redissolve in acetonitrile-water, and filter through a 0.22 mL filter. After filtration, the sample was analyzed by LC-MS / MS (triple quadrupole liquid chromatography-tandem mass spectrometry).

[0010] S2, Liquid Chromatography-Tandem Mass Spectrometry Detection: 5 The test solution was injected into a triple quadrupole liquid chromatography-tandem mass spectrometer at a flow rate of 0.3 mL / min. Separation was performed using a C18 reversed-phase column at a column temperature of 40 °C. Gradient elution was performed using a combination of mobile phases, namely mobile phase A and mobile phase B. Mass spectrometry was performed using an electrospray positive ion source (ESI+) and multiple reaction monitoring (MRM) mode.

[0011] S3. Quantitative analysis: A standard curve was plotted using the matrix-matched external standard method, and the contents of phenacetin and acetaminophen in the sample were calculated based on the quantitative ion peak area of ​​the test solution.

[0012] A preferred embodiment of the present invention is that, in step S1, the dispersed solid-phase extraction purification tube is filled with a purification material, which is one of the following: 1g anhydrous sodium sulfate + 0.2g PSA + 0.3g C18, 1g anhydrous magnesium sulfate + 0.2g PSA + 0.3g C18, 1g anhydrous magnesium sulfate + 0.2g PSA, or 1g anhydrous magnesium sulfate + 0.3g C18.

[0013] A preferred embodiment of the present invention is that, in step S1, the volume ratio of acetonitrile to water is one of 1:9, 2:8, 3:7, or 5:5.

[0014] In a preferred embodiment of the present invention, in step S2, mobile phase A is a 0.1% formic acid aqueous solution or pure water, and mobile phase B is acetonitrile.

[0015] A preferred embodiment of the present invention is that the gradient elution procedure in step S2 is as follows:

[0016] 0.0~0.5 min, mobile phase A volume fraction is 90%, mobile phase B volume fraction is 10%;

[0017] Within 0.5–6.0 min, the volume fraction of mobile phase A decreased linearly from 90% to 10%, while the volume fraction of mobile phase B increased linearly from 10% to 90%.

[0018] 6.0~7.0 min, mobile phase A volume fraction 10%, mobile phase B volume fraction 90%;

[0019] From 7.0 to 7.1 min, the volume fraction of mobile phase A increased linearly from 10% to 90%, while the volume fraction of mobile phase B decreased linearly from 90% to 10%.

[0020] 7.1~10.0 min, the volume fraction of mobile phase A is 90%, and the volume fraction of mobile phase B is 10%.

[0021] The preferred technical solution of the present invention is that the mass spectrometry conditions in step 2 are as follows: ion spray voltage 5500V, ion source temperature 550℃; curtain gas pressure 25.0psi, collision gas pressure 8psi; quantitative ion pair of phenacetin is 180.1m / z→110m / z, collision energy 27V; qualitative ion pair is 180.1m / z→138m / z, collision energy 22V; declustering voltage 79V; quantitative ion pair of acetaminophen is 152.1m / z→109.9m / z, collision energy 22V; qualitative ion pair is 152.1m / z→93.1m / z, collision energy 30V; declustering voltage 65V.

[0022] The preferred technical solution of the present invention is that, in step S3, the standard curve preparation method of matrix-matched external standard method is as follows: prepare matrix-matched standard series working solutions with concentrations of 2, 4, 8, 12, 20, and 50 ng / mL respectively, and perform linear regression of the concentration with the quantitative ion peak area to obtain the standard curve equation.

[0023] A preferred embodiment of the present invention is that the matrix-matched standard series working solutions are prepared through the following steps:

[0024] (1) Weigh 10 mg of phenacetin standard, dissolve it in methanol and dilute to 10 mL to prepare a 1000 mL solution. The standard stock solution should be stored at 4°C away from light.

[0025] (2) Accurately pipette appropriate amounts of phenacetin standard stock solution and acetaminophen standard solution, and dilute them stepwise with methanol to prepare solutions with a mass concentration of 1 for both target compounds. Mixed standard intermediate solution;

[0026] (3) Accurately pipette the mixed standard intermediate solution and dilute it stepwise with pork blank matrix extract to prepare a matrix-matched standard series of working solutions with phenacetin and acetaminophen concentrations of 2, 4, 8, 12, 20 and 50 ng / mL, which are used to plot the matrix standard curve.

[0027] The preferred embodiment of the present invention is that the animal-derived food includes pork, beef, and mutton.

[0028] The beneficial effects of this invention are as follows:

[0029] 1. Excellent matrix purification effect and simple and efficient pretreatment. This invention addresses the complex matrix characteristics of animal-derived foods, which are high in protein and fat. It optimizes the pretreatment system to achieve optimal results using 3g sodium chloride single salting-out and anhydrous magnesium sulfate / PSA / C18 ternary dispersion solid-phase extraction. Sodium chloride salting-out induces a clear stratified interface between the acetonitrile and water phases, resulting in high clarity of the supernatant and good sampling stability. The ternary purification materials synergistically remove endogenous interfering substances such as fats, fatty acids, pigments, and sugars, keeping the absolute value of the matrix effect below 20%. The method eliminates the need for complex solid-phase extraction and liquid-liquid extraction operations, allowing for the simultaneous processing of dozens of samples per batch. It is simple to operate, has high throughput, and is suitable for routine large-scale supervisory sampling inspections.

[0030] 2. Excellent chromatographic and mass spectrometric response, effectively improving the detection performance of polar metabolites. This invention uses a 0.1% formic acid aqueous solution-acetonitrile mobile phase system. Formic acid can provide sufficient protons to enhance the electrospray ionization efficiency of the target compound, while suppressing the secondary adsorption of residual silanol groups on the surface of the C18 column packing. This specifically solves the technical problems of peak tailing and low response intensity of the polar metabolite acetaminophen, significantly improving the peak symmetry, signal intensity, and separation selectivity of acetaminophen, ensuring stable mass spectrometric responses for both compounds.

[0031] 3. Achieves simultaneous quantification of phenacetin parent compound and its metabolite acetaminophen, providing value for residue traceability. This invention can simultaneously and accurately quantify the residue levels of phenacetin parent compound and its metabolite acetaminophen, filling the technical gap in the simultaneous quantification method of phenacetin-acetaminophen parent compound and metabolite in animal-derived food matrices. The concentration ratio of the two compounds can help distinguish the source of residues: if only acetaminophen is detected, it may be due to endogenous animal metabolism or other drug metabolites; if phenacetin and acetaminophen are detected simultaneously in a ratio consistent with metabolic patterns, it can indicate exogenous exposure to phenacetin, providing technical support for food safety risk traceability and supervision.

[0032] 4. The methodology is stable and reliable, meeting the quality control requirements for trace detection. Systematic methodological validation showed good linearity between the two compounds in the range of 2–50 ng / mL, with a correlation coefficient r² ≥ 0.996; the limit of detection for phenacetin was 0.2. The limit of quantitation is 0.5. The detection limit for acetaminophen is 1. The limit of quantitation is 2. It can meet the detection requirements for ultra-trace residues; under three spiking levels, the average recovery rate of phenacetin is 91% to 99%, the average recovery rate of acetaminophen is 86% to 95%, and the relative standard deviation is ≤3.48%. The accuracy and precision of the method meet the requirements of GB / T 27404-2026 "Laboratory Quality Control Standard for Physicochemical Testing of Food", and the test data have legal credibility.

[0033] 5. Low detection cost and easy to promote and apply. This invention uses matrix-matched external standard method for quantification, which replaces expensive isotope internal standard reagents while ensuring quantitative accuracy and precision, significantly reducing the cost of detection reagents; the instrument used in the method is a triple quadrupole liquid chromatography-tandem mass spectrometer, which is routinely configured in food testing laboratories at all levels, requiring no special equipment, and the method parameters have strong universality, making it easy to promote and apply in grassroots testing institutions. Attached Figure Description

[0034] Figure 1 This is the MRM extraction ion chromatogram of phenacetin at 10 ng / mL when mobile phase A is 0.1% formic acid water, provided in a specific embodiment of the present invention.

[0035] Figure 2 This is an MRM extraction ion chromatogram of 10 ng / mL acetaminophen when mobile phase A is 0.1% formic acid water, provided in a specific embodiment of the present invention.

[0036] Figure 3 This is an MRM extraction ion chromatogram of phenacetin at 10 ng / mL when mobile phase A is pure water, provided in a specific embodiment of the present invention.

[0037] Figure 4 This is an MRM extraction ion chromatogram of 10 ng / mL acetaminophen when mobile phase A is pure water, provided in a specific embodiment of the present invention.

[0038] Figure 5 This describes the effect of different purification materials provided in specific embodiments of the present invention on the spiked recovery rates of phenacetin and acetaminophen.

[0039] Figure 6 This is a schematic diagram illustrating the effect of an acetonitrile-water volume ratio of 1:9 in the complex solution on the response and peak shape of the target compound, provided in a specific embodiment of the present invention.

[0040] Figure 7This is a schematic diagram illustrating the effect of an acetonitrile-water volume ratio of 2:8 in the complex solution on the response and peak shape of the target compound, provided in a specific embodiment of the present invention.

[0041] Figure 8 This is a schematic diagram illustrating the effect of an acetonitrile-water volume ratio of 3:7 in the complex solution on the response and peak shape of the target compound, provided in a specific embodiment of the present invention.

[0042] Figure 9 This is a schematic diagram illustrating the effect of an acetonitrile-water volume ratio of 5:5 in the complex solution on the response and peak shape of the target compound, provided in a specific embodiment of the present invention. Detailed Implementation

[0043] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0044] Example 1

[0045] 1. Instruments and Reagents

[0046] 1.1 Instruments

[0047] AB 3500 triple quadrupole liquid chromatography-tandem mass spectrometry system (with ESI electrospray ionization source, AB Sciex, USA; LC-30AD liquid chromatography system, Shimadzu, Japan); C18 reversed-phase column (100 mm × 2.1 mm, 1.7... Thermo Scientific); AL204 electronic balance (sensitivity 0.0001g, Sartorius, USA); M37610-33CN vortex oscillator (Thermo Fisher Scientific, USA); TDL-5-A benchtop low-speed centrifuge (Shanghai Anting Scientific Instrument Factory); HP520 nitrogen evaporator (Zhejiang Hongpu Technology Co., Ltd.); 0.22 Microporous filter membrane (nylon, Tianjin Jinteng Experimental Equipment Co., Ltd.)

[0048] 1.2 Reagents

[0049] Phenacetin standard (batch number J3023280, 25 mg, purity 99.7%, Beijing Manhag Biotechnology Co., Ltd.); acetaminophen standard solution (methanol matrix, 1000 mg / L, batch number 2433244, Shanghai Anpu Cuishi Standard Technology Service Co., Ltd.); methanol and acetonitrile were chromatographically pure (Merck, Germany); anhydrous magnesium sulfate and sodium chloride (analytical grade, Xilong Scientific Co., Ltd.); PSA (CNW, Shanghai Anpu Experimental Technology Co., Ltd.), C18 (Ron, Shanghai Yi'en Chemical Technology Co., Ltd.); ultrapure water was prepared by the Milli-Q system (resistivity ≥ 18.2 MΩ·cm).

[0050] 2. Preparation of standard solutions

[0051] Phenacetin standard stock solution: Accurately weigh 10 mg of phenacetin standard (accurate to 0.0001 g), dissolve in methanol and dilute to 10 mL to prepare a solution with a mass concentration of 1000. The standard stock solution should be stored at 4 ℃ away from light.

[0052] Mixed standard intermediate solutions: Accurately pipette appropriate amounts of phenacetin standard stock solution and acetaminophen standard solution, and dilute them stepwise with methanol to prepare solutions with a mass concentration of 1 for both target compounds. The mixed standard intermediate liquid.

[0053] Matrix-matched standard working solutions: Accurately pipette the mixed standard intermediate solution and dilute it stepwise with pork blank matrix extract through sample pretreatment to prepare matrix-matched standard working solutions with phenacetin and acetaminophen concentrations of 2, 4, 8, 12, 20, and 50 ng / mL, which are used to plot matrix standard curves.

[0054] 3. Test Methods

[0055] 3.1 Sample Pretreatment

[0056] Take 2g of homogenized pork sample into a 50mL plastic centrifuge tube, add 10mL of acetonitrile, and vortex vigorously for 1min; add 3g of sodium chloride, and vortex vigorously for 1min again to salt out the salt; centrifuge at 4000r / min for 10min to completely separate the acetonitrile phase and the aqueous phase.

[0057] Take 8 mL of the supernatant and add it to a dispersion solid-phase extraction purification tube pre-loaded with 1 g anhydrous magnesium sulfate, 0.2 g PSA, and 0.3 g C18 adsorbent. Vortex mix for 1 min for purification, then centrifuge at 4000 r / min for 10 min. Take 2 mL of the supernatant, purge with nitrogen until nearly dry, and redissolve in 1 mL of acetonitrile-water solution (1:9 v / v). Filter through a 0.22 mL filter. The microporous membrane was then used for LC-MS / MS analysis.

[0058] 3.2 Chromatographic and Mass Spectrometric Conditions

[0059] Chromatographic conditions: C18 reversed-phase column, column temperature 40℃, injection volume 5 μL / mL. The flow rate was 0.3 mL / min; mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was acetonitrile; the gradient elution program was as follows:

[0060] 0.0~0.5min, 90% A + 10% B;

[0061] From 0.5 to 6.0 min, phase A linearly decreased to 10%, while phase B linearly increased to 90%.

[0062] 6.0~7.0 min, 10% A + 90% B;

[0063] From 7.0 to 7.1 min, phase A linearly increased to 90%, while phase B linearly decreased to 10%.

[0064] 7.1~10.0min, 90% A + 10% B.

[0065] Mass spectrometry conditions: Ion source was electrospray positive ion source (ESI+); monitoring mode was multiple reaction monitoring (MRM); ion spray voltage was 5500 V; ion source temperature was 550 °C; curtain gas pressure was 25.0 psi; collision gas pressure was 8 psi. MRM parameters for each compound are shown in Table 1. Experimental results are as follows: Figure 1-2 As shown.

[0066] Table 1. MRM parameters for phenacetin and acetaminophen

[0067]

[0068] 3.3 Quantitative Calculation

[0069] The matrix-matched standard series working solutions were injected sequentially from low to high concentration for analysis. A standard curve equation was established by performing linear regression with the peak area of ​​the quantitative ion as the ordinate and the corresponding mass concentration as the abscissa. The test solution was then injected for analysis, and the measured peak area of ​​the quantitative ion was substituted into the standard curve equation to calculate the concentration of the target analyte in the test solution. This concentration was then converted to the residual amount in the sample based on the pretreatment dilution factor.

[0070] 4. Methodological Validation

[0071] 4.1 Linear range, detection limit and quantitation limit

[0072] A series of matrix-matched standard solutions with concentrations of 2, 4, 8, 12, 20, and 50 ng / mL were prepared using pork blank matrix extract. Linear regression was performed on the concentration using quantitative ion peak area. Blank pork samples were spiked and fully pretreated before analysis. The limits of detection (LOD) and quantitation (LOQ) were determined based on the signal-to-noise ratio (S / N): the sample concentration corresponding to S / N ≈ 3 was used as the LOD, and the sample concentration corresponding to S / N ≈ 10 was used as the LOD. The results are shown in Table 2. The two compounds showed good linearity in the range of 2–50 ng / mL, with a correlation coefficient r² ≥ 0.996; the LOD for phenacetin was 0.2. The LOQ is 0.5. The LOD of acetaminophen is 1. LOQ is 2 Its sensitivity meets the needs of food safety monitoring for trace residue detection.

[0073] Table 2. Matrix-matched standard curve, linear range, correlation coefficient, limit of detection, and limit of quantitation.

[0074]

[0075] 4.2 Matrix effect

[0076] Mixed standard solutions of the same concentration were added to both the pork blank matrix extract and pure solvent (acetonitrile-water volume ratio 1:9), setting three concentration levels: 2, 5, and 20 ng / mL, with three replicates for each level. The matrix effect was calculated using the following formula:

[0077] ME% = (Matrix spiking response ÷ Pure solvent spiking response - 1) × 100%

[0078] The results are shown in Table 3. Generally, |ME%|≤20% is considered to be a controllable matrix effect, and there is no need to use isotope internal standards or deeper purification.

[0079] Table 3. Matrix effects of phenacetin and acetaminophen (n = 3)

[0080]

[0081] The results showed that the absolute values ​​of the matrix effects of phenacetin and acetaminophen at all three concentrations were less than 20%, which were weak matrix effects. They could be effectively corrected by matrix-matched external standard method without the need for isotope internal standard.

[0082] 4.3 Spike Recovery and Precision

[0083] Select pork blank matrix, and apply at concentrations of 2, 5, and 20 respectively. Three spiking levels were achieved by adding a mixed standard solution of phenacetin and acetaminophen. Six parallel samples were prepared for each level, and the samples were pretreated and injected according to the sample pretreatment method in 3.1. The results are shown in Table 4.

[0084] Table 4. Spike recoveries and precision of phenacetin and acetaminophen in pork (n = 6)

[0085]

[0086] The results showed that the average recovery rate of phenacetin at the three spiking levels was 91%–99%, with a relative standard deviation (RSD) of 1.87%–2.74%; the average recovery rate of acetaminophen was 86%–95%, with an RSD of 0.98%–3.48%. The accuracy and precision of the method meet the requirements for quality control of food physicochemical testing in GB / T 27404—2026 "Laboratory Quality Control Standard for Food Physicochemical Testing" and are suitable for routine laboratory testing and analysis.

[0087] Example 2

[0088] In this embodiment, pure water is used as the mobile phase A, and other conditions are the same as in Embodiment 1.

[0089] 1. Experimental Methods

[0090] Sample pretreatment, purification materials, reconstitution ratio, mass spectrometry parameters, and standard solution preparation were completely consistent with Example 1, except that mobile phase A in the chromatographic conditions was replaced with ultrapure water, and the gradient elution program remained unchanged. The 10 ng / mL mixed standard solution was then mixed with 5... Spiked pork samples were tested, with three parallel samples set up for each level.

[0091] 2. Experimental Results

[0092] The test results are attached. Figure 3-4 As shown, in conjunction with the appendix Figure 1-2 It can be seen that the two compositions have almost no effect on phenacetin, but the metabolite acetaminophen differs significantly. When using 0.1% formic acid aqueous solution as mobile phase A, the compound has a symmetrical peak shape, narrower half-peak width, higher signal intensity, and better separation selectivity. When using pure water as mobile phase A, acetaminophen shows obvious peak tailing, poor symmetry, weaker signal intensity, and a larger half-peak width than when using 0.1% formic acid aqueous solution as mobile phase A.

[0093] Example 3

[0094] This embodiment only changes the composition of the purification material in the dispersed solid-phase extraction purification tube; the other conditions are the same as in Embodiment 1.

[0095] 1. Experimental Methods

[0096] The sample pretreatment process, mobile phase composition, reconstitution ratio, chromatographic and mass spectrometric parameters, and standard solution preparation were completely consistent with Example 1. The only difference was that the purification material in the dispersed solid-phase extraction purification tube was replaced with one of the following three combinations. Three parallel experiments were conducted on 10 samples. Spiked pork samples were tested, with three parallel samples set up for each level:

[0097] Combination a: 1g anhydrous sodium sulfate + 0.2g PSA + 0.3g C18;

[0098] Combination b: 1g anhydrous magnesium sulfate + 0.2g PSA;

[0099] Combination c: 1g anhydrous magnesium sulfate + 0.3g C18;

[0100] In Example 1, 1g of anhydrous magnesium sulfate + 0.2g of PSA + 0.3g of C18 was used as a performance control benchmark.

[0101] 2. Experimental Results

[0102] The effects of different purification materials on the recovery rates of phenacetin and acetaminophen are shown in the attached figure. Figure 5 As shown, in conjunction with the appendix Figure 5 It can be seen that using 1g anhydrous magnesium sulfate + 0.2g PSA + 0.3g C18 as the evolution material in Example 1 resulted in the highest peak area of ​​the target compound, the lowest background of the blank matrix, and the best response. Anhydrous magnesium sulfate, as a dehydrating agent, can reduce the non-specific adsorption of the target compound by PSA and C18 adsorbents. PSA mainly removes weakly basic interferences such as fatty acids, organic acids, sugars, and pigments, while C18 mainly removes non-polar aliphatic interferences. The three work together to achieve high matrix purification efficiency.

[0103] Example 4

[0104] This embodiment only changes the volume ratio of acetonitrile to water in the reconstitution solution; all other conditions are the same as in Example 1.

[0105] 1. Experimental Methods

[0106] The sample pretreatment process, dehydration salts, mobile phase composition, purification materials, and chromatographic and mass spectrometric parameters were all consistent with those in Example 1. The only difference was that the reconstituted solution after nitrogen blowing was replaced with three systems with acetonitrile-water volume ratios of 2:8, 3:7, and 5:5, respectively, and the chromatographic peak shapes and response intensities under different ratios were compared.

[0107] 2. Experimental Results

[0108] All four ratios of the complex solution can achieve the detection of two target compounds. The effects of the four complex solutions on the target compound response and peak shape are shown in the appendix. Figure 6-9 As shown, when the acetonitrile-water volume ratio is 1:9, the chromatographic peak symmetry, half-peak width, and response of the two target compounds are optimal. As the acetonitrile ratio increases, solvent front broadening gradually occurs, peak symmetry decreases, and response intensity gradually declines. When the acetonitrile ratio exceeds 30%, the solvent effect becomes quite significant, and peak shape deterioration is pronounced.

[0109] This invention establishes a method for the simultaneous determination of phenacetin and its metabolite acetaminophen in pork by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Through systematic optimization of pretreatment conditions (extraction solvent, dehydration salt, purification package formulation) and chromatographic-mass spectrometry conditions (mobile phase composition, reconstitution ratio, column temperature), the final method employs acetonitrile extraction, single salting out with 3 g sodium chloride, purification with anhydrous magnesium sulfate / PSA / C18 three-component QuEChERS, separation on a C18 column using 0.1% formic acid-water-acetonitrile as the mobile phase, detection by ESI+MRM, and quantification by matrix-matched external standard method.

[0110] This study fills the gap in the simultaneous quantitative method of phenacetin-acetaminophen parent compound and metabolite in animal-derived food matrices, and can distinguish between two sources of residues: exogenous illegal addition and in vivo metabolic transformation, by comparing the concentration ratio of the two. In ESI+ mode, the effects of pure water and 0.1% formic acid water mobile phase on this specific compound system were systematically compared, revealing the advantages of 0.1% formic acid water mobile phase in peak shape and response. The matrix-matched external standard method was used to replace the isotope internal standard method, which significantly reduced the detection cost while ensuring accuracy and precision, and is suitable for routine supervision and sampling inspection.

[0111] This invention has been described through preferred embodiments. Those skilled in the art will understand that various changes or equivalent substitutions can be made to these features and embodiments without departing from the spirit and scope of the invention. This invention is not limited to the specific embodiments disclosed herein; other embodiments falling within the scope of the claims are also within the protection scope of this invention.

Claims

1. A method for determining phenacetin and its metabolites in animal-derived foods, characterized in that: Includes the following steps: S1. Sample pretreatment: Weigh 2g of homogenized animal-derived food sample into a 50mL plastic centrifuge tube, add 10mL of acetonitrile, place it in a vortex shaker and shake vigorously for 1 min, then add 3g of sodium chloride, shake vigorously in a vortex shaker for 1 min, and place it in a benchtop low-speed centrifuge and centrifuge at 4000r / min for 10 min. Add 8 mL of the supernatant to a dispersion solid-phase extraction purification tube, mix thoroughly in a vortex mixer for 1 min, then centrifuge at 4000 r / min for 10 min in a benchtop low-speed centrifuge; take 2 mL of the supernatant, purge to near dryness with nitrogen, redissolve in acetonitrile-water, and dilute through a 0.22 mL filter. After filtration, the sample was analyzed by LC-MS / MS (triple quadrupole liquid chromatography-tandem mass spectrometry). S2, Liquid Chromatography-Tandem Mass Spectrometry Detection: 5 The test solution was injected into a triple quadrupole liquid chromatography-tandem mass spectrometer at a flow rate of 0.3 mL / min. Separation was performed using a C18 reversed-phase column at a column temperature of 40 °C. Gradient elution was performed using a combination of mobile phases, namely mobile phase A and mobile phase B. Mass spectrometry was performed using an electrospray positive ion source (ESI+) and multiple reaction monitoring (MRM) mode. S3. Quantitative analysis: A standard curve was plotted using the matrix-matched external standard method, and the contents of phenacetin and acetaminophen in the sample were calculated based on the quantitative ion peak area of ​​the test solution.

2. The method for determining phenacetin and its metabolites in animal-derived foods according to claim 1, characterized in that: In step S1, the dispersed solid-phase extraction purification tube is filled with a purification material, which is one of the following: 1g anhydrous sodium sulfate + 0.2g PSA + 0.3g C18, 1g anhydrous magnesium sulfate + 0.2g PSA + 0.3g C18, 1g anhydrous magnesium sulfate + 0.2g PSA, or 1g anhydrous magnesium sulfate + 0.3g C18.

3. The method for determining phenacetin and its metabolites in animal-derived foods according to claim 1, characterized in that: In step S1, the acetonitrile-water volume ratio is one of 1:9, 2:8, 3:7, or 5:

5.

4. The method for determining phenacetin and its metabolites in animal-derived foods according to claim 1, characterized in that: In step S2, mobile phase A is a 0.1% formic acid aqueous solution or pure water, and mobile phase B is acetonitrile.

5. The method for determining phenacetin and its metabolites in animal-derived foods according to claim 1, characterized in that: The gradient elution procedure in step S2 is as follows: 0.0~0.5 min, mobile phase A volume fraction is 90%, mobile phase B volume fraction is 10%; Within 0.5–6.0 min, the volume fraction of mobile phase A decreased linearly from 90% to 10%, while the volume fraction of mobile phase B increased linearly from 10% to 90%. 6.0~7.0 min, mobile phase A volume fraction 10%, mobile phase B volume fraction 90%; From 7.0 to 7.1 min, the volume fraction of mobile phase A increased linearly from 10% to 90%, while the volume fraction of mobile phase B decreased linearly from 90% to 10%. 7.1~10.0 min, the volume fraction of mobile phase A is 90%, and the volume fraction of mobile phase B is 10%.

6. The method for determining phenacetin and its metabolites in animal-derived foods according to claim 1, characterized in that: The mass spectrometry conditions in step 2 are as follows: ion spray voltage 5500V, ion source temperature 550℃; curtain gas pressure 25.0psi, collision gas pressure 8psi; quantitative ion pair for phenacetin: 180.1m / z→110m / z, collision energy 27V; qualitative ion pair: 180.1m / z→138m / z, collision energy 22V; declustering voltage 79V; quantitative ion pair for acetaminophen: 152.1m / z→109.9m / z, collision energy 22V; qualitative ion pair: 152.1m / z→93.1m / z, collision energy 30V; declustering voltage 65V.

7. The method for determining phenacetin and its metabolites in animal-derived foods according to claim 1, characterized in that: In step S3, the standard curve preparation method of matrix-matched external standard method is as follows: prepare matrix-matched standard series working solutions with concentrations of 2, 4, 8, 12, 20, and 50 ng / mL respectively, and perform linear regression of the concentration with the quantitative ion peak area to obtain the standard curve equation.

8. The method for determining phenacetin and its metabolites in animal-derived foods according to claim 7, characterized in that: The matrix-matched standard series of working solutions are prepared through the following steps: (1) Weigh 10 mg of phenacetin standard, dissolve it in methanol and dilute to 10 mL to prepare a 1000 mL solution. The standard stock solution should be stored at 4°C away from light. (2) Accurately pipette appropriate amounts of phenacetin standard stock solution and acetaminophen standard solution, and dilute them stepwise with methanol to prepare solutions with a mass concentration of 1 for both target compounds. Mixed standard intermediate solution; (3) Accurately pipette the mixed standard intermediate solution and dilute it stepwise with pork blank matrix extract to prepare a matrix-matched standard series of working solutions with phenacetin and acetaminophen concentrations of 2, 4, 8, 12, 20 and 50 ng / mL, which are used to plot the matrix standard curve.

9. The method for determining phenacetin and its metabolites in animal-derived foods according to claim 1, characterized in that: The animal-derived foods include pork, beef, and mutton.

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