Method for detecting arecoline residues in biological sample and application of method
The detection of arecoline in edible chicken tissues using high performance liquid chromatography-mass spectrometry (HPLC-MS) fills the gap in the detection of arecoline residues, achieving high sensitivity and high accuracy.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- 湖南九安禾生物科技有限公司
- Filing Date
- 2026-04-16
- Publication Date
- 2026-05-15
AI Technical Summary
There is a lack of rapid detection methods for arecoline residues in biological samples, especially in edible chicken tissues, in the current technology.
Arecoline was detected using high performance liquid chromatography-mass spectrometry. Biological sample pretreatment was performed by preparing standards and internal standard solutions, including homogenization, solid-phase extraction, and gradient elution. A matrix standard curve was established to achieve quantitative detection of arecoline.
This method provides a highly sensitive, precise, and accurate method for detecting arecoline residues. It is suitable for edible chicken tissues, has a low detection limit, and meets the requirements for biological sample testing.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of analytical detection technology, specifically to a method for detecting arecoline residues in biological samples and its application. Background Technology
[0002] Areca catechu (Areca catechum μ L.) is an evergreen tree belonging to the Areca genus of the palm family. Medicinal areca catechu (Arecae Semen, AS) refers to the dried, mature seeds of the Areca catechu plant. The Chinese Pharmacopoeia describes it as the mature fruit from late summer to early autumn, dried, with the pericarp removed, and the seeds extracted. Areca catechu is one of the "Four Great Southern Chinese Medicines," and it enters the stomach, small intestine, and large intestine meridians, possessing insecticidal, digestive, diuretic, and antimalarial effects. Jujube areca catechu (also known as areca kernel, tender areca catechu, areca seed, green areca catechu, etc.) is the immature seed of the Areca catechu plant, sharing the same source and matrix as the medicinal areca catechu in the Pharmacopoeia, differing only in maturity. Areca catechu and / or jujube areca catechu contain various chemical components such as alkaloids, polyphenols, and flavonoids. Characteristic alkaloids, such as arecoline and arecaine, are among the main active substances in areca catechu.
[0003] Bioresidue detection is a scientific method that analyzes residual microorganisms, antibiotics, pesticides, and other substances in samples to assess their impact on food safety, environmental health, or pharmaceutical quality. Its core objective is to ensure products meet safety standards and reduce harm to humans and the environment. It is widely used in food processing, environmental monitoring, and pharmaceutical production. Currently, there are no reported methods for detecting arecoline residues in biological samples, particularly in edible tissues of chicken (including muscle, liver, kidneys, and subcutaneous fat). Summary of the Invention
[0004] To address the aforementioned technical problems, the present invention aims to provide a rapid detection method for arecoline residues in biological samples and its application.
[0005] The specific technical solution is as follows:
[0006] A method for detecting arecoline residues in biological samples, using high-performance liquid chromatography-mass spectrometry, characterized by comprising the following steps:
[0007] A1: Preparation of working solutions: Preparation of standard stock solution, internal standard stock solution, standard working solution and internal standard working solution; wherein the standard is arecoline hydrobromide and the internal standard is pilocarpine;
[0008] A2: Biological sample pretreatment: Take biological sample tissue, add water and grinding beads to homogenize, add acidic acetonitrile solution, vortex centrifuge, take the supernatant for solid phase extraction and / or dispersion solid phase extraction purification, collect the eluent, filter and wait for analysis;
[0009] A3: Preparation of matrix standard curve solution: Prepare blank biological sample matrix solution according to step A2, add standard working solution and internal standard working solution, and prepare matrix standard curve solutions of a series of concentrations;
[0010] A4: The processed biological sample solution and the matrix standard curve solution of A3 were analyzed by high performance liquid chromatography-mass spectrometry to obtain the matrix standard curve of arecoline. The content of arecoline in the biological sample to be tested was calculated based on the standard curve.
[0011] The liquid chromatography conditions used an octadecylsilane-bonded silica gel column, with mobile phase A being 0.1% formic acid-10 mmol / L ammonium formate aqueous solution and mobile phase B being methanol, for gradient elution.
[0012] Preferably, the biological sample is edible chicken tissue.
[0013] Preferably, the edible chicken tissue is selected from at least one of muscle, liver, kidney, and subcutaneous fat.
[0014] Preferably, in A1, the working solution concentration is diluted using 0.1% formic acid solution - 10 mmol / L ammonium formate solution.
[0015] Preferably, the preparation method of the stock solution in A1 is as follows: 5 mg of arecoline hydrobromide reference standard is placed in a 10 mL volumetric flask and diluted with pure water to obtain a 500 μg / mL arecoline hydrobromide stock solution, which is then stored at 4 °C; 50 mg of pilocarpine reference standard is placed in a 50 mL volumetric flask and diluted with methanol / water (1:1) to obtain a 1 mg / mL pilocarpine stock solution; the preparation method of the standard working solution is as follows: an appropriate amount of arecoline stock solution is diluted with 0.1% formic acid water-10 mmol / L ammonium formate water to prepare standard working solutions of 1 μg / mL and 100 ng / mL; the pilocarpine stock solution is diluted with 0.1% formic acid water-10 mmol / L ammonium formate water to prepare internal standard working solutions of 1 μg / mL and 100 ng / mL.
[0016] Preferably, the solid-phase extraction in A2 is purified using at least one of HLB solid-phase extraction columns and MCX solid-phase extraction columns; the dispersion solid-phase extraction uses a dSPE extraction package.
[0017] Preferably, in A2, for muscle or subcutaneous tissue, the solid-phase extraction is performed sequentially through an HLB solid-phase extraction column and an MCX solid-phase extraction column; for liver or kidney tissue, the solid-phase extraction is performed first through a dSPE extraction pack and then through an MCX solid-phase extraction column; the acidic acetonitrile solution is 0.5% formic acid acetonitrile.
[0018] Preferably, 3.2mm grinding beads are used in A2.
[0019] Preferably, the specific operation of step A2 is as follows: (1) Muscle and subcutaneous tissue: Take 2 grams of tissue and place it in a 50 ml round-bottom centrifuge tube. Add 1 mL of pure water and 3 grinding beads for homogenization. Add 8 mL of 0.5% formic acid acetonitrile and 1 mL of pure water. Vortex for 5 min, centrifuge at 8000 rpm for 5 min, take the supernatant and pass it through an HLB solid phase extraction column, then through an MCX solid phase extraction column. Collect the eluent, take 1 ml and filter it through a 0.22 μm organic filter membrane before testing. (2) Liver and kidney tissues: Take 1 gram of tissue and place it in a 50 ml round-bottom centrifuge tube. Add 1 ml of 0.5% formic acid water and 2 grinding beads for homogenization. Add 8 mL of 0.5% formic acid acetonitrile and 1 mL of pure water. Vortex for 5 min and centrifuge at 10,000 rpm for 5 min. Take the supernatant to another 50 ml centrifuge tube, add 2 g of dSPE extraction packet, vortex for 5 min and centrifuge at 10,000 rpm for 5 min. Take the supernatant and pass it through an MCX solid phase extraction column. Collect the eluent and filter 1 ml through a 0.22 μm organic filter membrane before testing.
[0020] Preferably, the lower limit of quantification for muscle tissue detection is 2 ng / mL; and the lower limit of quantification for subcutaneous fat, liver, and kidney tissue detection is 4 ng / mL.
[0021] Preferably, step A3 is specifically performed as follows: Following step A2, a blank muscle matrix solution is obtained. Appropriate amounts of arecoline standard solutions (1 μg / mL and 100 ng / mL) and 0.5 mL of internal standard working solution (100 ng / mL) are added to a 5 mL volumetric flask and diluted to volume to obtain a series of matrix standard curve solutions with concentrations of 100, 75, 50, 20, 10, 5, and 2 ng / mL. Following step A2, blank sebum, liver, and kidney matrix solutions are obtained. Appropriate amounts of arecoline standard solutions (1 μg / mL and 100 ng / mL) and 0.5 mL of internal standard working solution (100 ng / mL) are added to a 5 mL volumetric flask and diluted to volume to obtain a series of matrix standard curve solutions with concentrations of 100, 75, 50, 40, 25, 16, 8, and 4 ng / mL.
[0022] Preferably, the gradient elution procedure described in A4 is as follows:
[0023]
[0024] Preferably, the chromatographic conditions in A4 are as follows: column: Endeavorsil C18-A (2.1×150mm, 1.8μm), column temperature 40℃, flow rate 0.3mL / min, injection volume 2µL; mass spectrometry conditions are as follows: ion source: electrospray ionization (ESI+); scanning mode: positive ion multiple reaction monitoring (MRM) mode; positive and negative capillary voltages: 4500V and 3500V; nebulizer pressure: 35psi; drying gas temperature: 280℃; drying gas flow rate: 13L / min; Delta EMV: 500; drying gas: N2 (purity 99.9%); collision gas: N2 (purity 99.9%); the quantitative ion pair of arecoline is m / z 156.00 → 44, and the quantitative ion pair of the internal standard pilocarpine is m / z 209.20 → 96.1.
[0025] The beneficial effects of this invention are:
[0026] (1) In the prior art, there are no literature reports on the detection method of arecoline residue in biological tissues, especially edible chicken tissues.
[0027] (2) The detection method provided by the present invention can quantitatively detect arecoline in biological samples, especially edible chicken tissues. The specificity, precision and accuracy, and stability of the detection method are all good and meet the requirements for biological sample detection.
[0028] (3) The detection method provided by this invention has been validated. The range of arecoline calibration curves in muscle tissue is 2 ng / mL to 100 ng / mL; the range of arecoline calibration curves in sebum, liver, and kidney tissue is 4 ng / mL to 100 ng / mL. This method has high sensitivity and low detection limit, and is suitable for the detection of arecoline residues in biological tissues, especially in edible chicken tissues. Attached Figure Description
[0029] Figure 1 MRM chromatograms of blank muscle sample (A), blank muscle sample with spiked (LOQ) (B), and arecoline-containing muscle tissue sample (C).
[0030] Figure 2 MRM chromatograms of blank sebum sample (A), blank sebum sample with spiked (LOQ) (B), and arecoline-residual sebum sample (C).
[0031] Figure 3 MRM chromatograms of blank liver sample (A), blank liver sample with added spike (LOQ) (B), and arecoline residue liver sample (C).
[0032] Figure 4MRM chromatograms of blank kidney sample (A), blank kidney sample with spiked (LOQ) (B), and arecoline residue kidney sample (C).
[0033] Figure 5 Chromatogram of the lower limit of quantification of arecoline in muscle matrix
[0034] Figure 6 Chromatogram of the lower limit of quantification of arecoline sebum matrix
[0035] Figure 7 Chromatogram of lower limit of quantification of arecoline in liver matrix
[0036] Figure 8 Chromatogram of lower limit of quantification of arecoline in the kidney matrix Detailed Implementation
[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0038] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.
[0039] All raw materials, reagents, instruments, and equipment used in the examples are commercially available or can be prepared using existing methods. Unless otherwise specified, all reagents used in this invention are of analytical grade.
[0040] Example 1: Methodology Construction and Validation
[0041] 1. Chromatographic conditions:
[0042] Chromatographic column: Endeavorsil C18-A (2.1 × 150 mm, 1.8 μm); mobile phase: 0.1% formic acid aqueous solution - 10 mmol / L ammonium formate aqueous solution (A) - methanol (B); column temperature: 40℃; flow rate: 0.3 mL / min; injection volume: 2 µL; gradient elution program as follows:
[0043]
[0044] 2. Mass spectrometry conditions:
[0045] Ion source: Electrospray ionization (ESI+); Detection method: MRM; Positive and negative capillary voltages: 4500V and 3500V; Nebulizer pressure: 35psi; Drying gas temperature: 280℃; Drying gas flow rate: 13L / min; Delta EMV in positive ion mode: 500; Drying gas: N2 (99.9% purity); Collision gas: N2 (99.9% purity), positive ion mode. The target compound was used for quantitative monitoring of ion pairs, fragmentation voltage, and collision energy as follows:
[0046]
[0047] Note: * indicates quantitative ions.
[0048] 3. Test methods
[0049] 3.1 Preparation of standard stock solution:
[0050] Take 5 mg of arecoline hydrobromide reference standard and place it in a 10 mL volumetric flask. Dilute to volume with pure water to obtain a 500 μg / mL arecoline hydrobromide stock solution, which is stored at 4 °C.
[0051] Take 50 mg of pilocarpine reference standard and place it in a 50 mL volumetric flask. Dilute to volume with methanol / water (1:1) to obtain a 1 mg / mL pilocarpine stock solution, and store at 4 °C.
[0052] 3.2 Preparation of working solution:
[0053] Internal standard working solutions: Dilute pilocarpine stock solution with 0.1% formic acid water-10mmol / L ammonium formate water to prepare internal standard working solutions of 1μg / mL and 100ng / mL.
[0054] Standard working solutions: Take an appropriate amount of arecoline stock solution and dilute it with 0.1% formic acid water-10mmol / L ammonium formate water to prepare standard solutions of 1μg / mL and 100ng / mL.
[0055] 3.3 Sample Pretreatment:
[0056] (1) Muscle and subcutaneous fat: Take 2 grams of tissue and place it in a 50 ml round-bottom centrifuge tube. Add 1 ml of pure water and 3 grinding beads for homogenization. Add 8 mL of 0.5% formic acid acetonitrile and 1 mL of pure water. Vortex for 5 min and centrifuge at 8000 rpm for 5 min. Take the supernatant and pass it through an HLB solid-phase extraction column, then through an MCX solid-phase extraction column. Collect the eluent and take 1 ml to filter through a 0.22 μm organic filter membrane for LC-MS / MS analysis.
[0057] (2) Liver and kidney: Take 1 gram of tissue and place it in a 50 ml round-bottom centrifuge tube. Add 1 ml of 0.5% formic acid water and 2 grinding beads for homogenization. Add 8 mL of 0.5% formic acid acetonitrile and 1 mL of pure water. Vortex for 5 min and centrifuge at 10,000 rpm for 5 min. Take the supernatant to another 50 ml centrifuge tube, add 2 g of dSPE extraction packet, vortex for 5 min and centrifuge at 10,000 rpm for 5 min. Take the supernatant and pass it through an MCX solid phase extraction column. Collect the eluent and take 1 ml and filter it through a 0.22 μm organic filter membrane for LC-MS / MS analysis.
[0058] 3.4 Preparation of matrix standard curve:
[0059] Following the steps in “3.3 Sample Pretreatment”, a blank muscle matrix solution was obtained. Appropriate amounts of arecoline standard solutions of 1 μg / mL and 100 ng / mL, and 0.5 mL of internal standard working solution of 100 ng / mL were taken and diluted to volume in a 5 mL volumetric flask to obtain a series of matrix standard curve solutions of 100, 75, 50, 20, 10, 5, and 2 ng / mL.
[0060] Following the steps in “3.3 Sample Pretreatment”, blank sebum, liver, and kidney matrix solutions were obtained. Appropriate amounts of arecoline standard solutions of 1 μg / mL and 100 ng / mL, and 0.5 mL of internal standard working solution of 100 ng / mL were taken and diluted to volume in a 5 mL volumetric flask to obtain a series of matrix standard curve solutions of 100, 75, 50, 40, 25, 16, 8, and 4 ng / mL.
[0061] 3.5 Quality control sample preparation:
[0062] Muscle: Take an appropriate volume of arecoline stock solution and dilute it with 0.1% formic acid-10mmol / L ammonium formate water to prepare high, medium, and low concentrations and quantitation limit concentrations of 7.5, 5, 0.6, and 0.2 μg / mL, respectively.
[0063] Sebum, liver, and kidneys: Take an appropriate volume of arecoline stock solution and dilute it with 0.1% formic acid water-10mmol / L ammonium formate water to prepare high, medium, and low concentrations with quantitation limits of 7.5, 5, 1.2, and 0.4 μg / mL, respectively.
[0064] 4. Methodological Validation
[0065] Referring to the "Guiding Principles for Veterinary Drug Residue Elimination Tests" and the "Guiding Principles for Validation of Quantitative Analysis Methods for Biological Samples", the main contents of method validation include: specificity, limit of quantitation, standard curve, matrix effect, precision, accuracy, and stability.
[0066] 5. Test Results
[0067] 5.1 Specificity
[0068] from Figures 1-4 It can be seen that arecoline and internal standard were not detected in the blank tissue sample. The chromatographic peak responses of the standard at the limit of quantitation concentration added to the blank tissue, as well as the target compound and internal standard in the drug sample, were good. The results indicate that the endogenous substances in the blank tissue did not significantly interfere with the chromatographic peak responses of arecoline and internal standard. No interfering peaks were present in the MRM monitoring mode. The chromatographic mass spectrometry conditions are suitable for the analysis of the analytes.
[0069] 5.2 Lower limit of quantification
[0070] Muscle: The signal-to-noise ratio (S / N) of the arecoline muscle matrix standard solution at 2 ng / mL is 12.3, and that at 1 ng / mL is 5.0. Therefore, the limit of quantification is 2 ng / mL. (See [link to relevant documentation]). Figure 5 .
[0071] Sebum: The signal-to-noise ratio (S / N) of the arecoline sebum matrix standard solution at 4 ng / mL is 30.3, and that at 2 ng / mL is 7.7. Therefore, the limit of quantification is 4 ng / mL. (See [link to relevant documentation]). Figure 6 .
[0072] Liver: The signal-to-noise ratio (S / N) of the arecoline liver matrix standard solution at 4 ng / mL was 50.8, and that at 2 ng / mL was 10.1. However, the peak difference significantly interfered with quantification; therefore, the lower limit of quantification was set at 4 ng / mL. (See [link to relevant documentation]). Figure 7 .
[0073] Kidney: The signal-to-noise ratio (S / N) of the arecoline kidney matrix standard solution at 4 ng / mL is 45.4, and that at 2 ng / mL is 9.4. Therefore, the limit of quantification is 4 ng / mL. (See [link to relevant documentation]). Figure 8 .
[0074] 5.3 Standard Curve
[0075] Arecoline exhibits good linearity in different tissues, R 2 ≥0.999, the regression equation and other parameters are shown in Table 1.
[0076]
[0077] 5.4 Accuracy and Precision
[0078] Table 2 shows the intra-batch and inter-batch accuracy and precision results of arecoline in different tissues. The data shows that arecoline has good accuracy and precision. The accuracy meets the requirements at all four concentration levels, and the precision RSD is less than 15%.
[0079]
[0080] 5.5 Matrix effect
[0081] The matrix effect verification results are shown in Table 3. The results show that the average matrix effect of arecoline at high and low quality control concentrations is between 88.6% and 106.26% (the matrix effect coefficient of variation satisfies RSD ≤15%), and the matrix effect has little impact on the response of arecoline.
[0082]
[0083] 5.6 Stability
[0084] The stability results of arecoline in different tissues under different environments are shown in Table 4. As can be seen from the table, after being placed in the injector environment (8℃) and room temperature (22℃) for 24 hours, the mean of each concentration level deviated from the labeled concentration within ±15%. The sample still showed good stability after repeated freeze-thaw cycles at -40℃ and after being stored at -40℃ for 30 days.
[0085]
[0086] VI. Conclusion
[0087] The assay method was systematically validated with reference to the "Guiding Principles for Validation of Quantitative Analysis Methods for Biological Samples" under the 2020 edition of the Chinese Pharmacopoeia and the "Guiding Principles for Veterinary Drug Residue Elimination Tests". The results showed that all indicators met the requirements and the method was feasible.
[0088] Example 2: Detection of arecoline residue in edible tissues of broiler chickens
[0089] I. Animal grouping and administration
[0090] The experiment used 480 healthy 1-day-old Kobo broiler roosters, randomly divided into 4 treatments, with 8 replicates per treatment and 15 chickens per replicate. The experiment lasted for 42 days. The 4 treatment groups were supplemented with 0, 1000, 5000, and 10000 mg / kg ASE in their basal diet, respectively, and were designated A0, A1, A2, and A3.
[0091] II. Sample Collection
[0092] 2.1 Sampling time point setting and number of animals
[0093] According to the relevant provisions of the "Guidelines for Veterinary Drug Residue Elimination Tests", the experimental group should have a sampling point on the day of drug withdrawal (2-6 hours after withdrawal in poultry tissue), and the residue level at the last sampling point should be below the limit. Four sampling points were set up, namely 0 (6 h), 1, 2 and 5 days after drug withdrawal, with at least 6 chickens at each point. The control group only sampled on day 0.
[0094] 2.2 Sample Collection
[0095] Collect 100g-200g of broiler muscle (breast muscle) and 20g of skin fat at each sampling point. Collect whole liver and whole kidney. Do not wash or process the collected samples. Immediately divide the collected samples into several equal portions, package and label them, and store them on dry ice immediately. Transfer them to a freezer at -20℃ or -80℃.
[0096] III. Test Results
[0097] The method of this invention is used to detect arecoline residues in biological tissues.
[0098] 3.1 Results of the low-dose group
[0099] Table 6 shows the residual arecoline levels in tissues from the low-dose group (1000 mg / kg). Experimental data indicate that no arecoline residues were detected in any of the four tissue samples from the low-dose group.
[0100]
[0101] 3.2 Results of the medium-dose group
[0102] Table 7 shows the residual arecoline levels in the tissues of the medium-dose group (5000 mg / kg). Experimental data indicate that the residual arecoline level in the muscle of the medium-dose group was 4.79 ± 0.21 μg / kg at day 0 after drug withdrawal. No arecoline residues were detected in the remaining tissues of the medium-dose group in subsequent tests.
[0103]
[0104] 3.3 Results of the high-dose group
[0105] Table 8 shows the residual arecoline levels in tissues of the high-dose group (10000 mg / kg). Experimental data indicate that the residual arecoline level in muscle tissue of the high-dose group was 5.64 ± 0.32 μg / kg at day 0 after drug withdrawal and 4.45 ± 0.27 μg / kg at day 1 after drug withdrawal; the residual arecoline level in subcutaneous fat tissue was 10.50 ± 0.68 μg / kg at day 0 after drug withdrawal; and no arecoline residues were detected in other tissues of the high-dose group.
[0106]
[0107] IV. Conclusion
[0108] The experimental results demonstrate that this method has high sensitivity and low detection limit, making it suitable for the detection of arecoline residues in biological tissues, especially in edible chicken tissues.
Claims
1. A method for detecting arecoline residues in biological samples and its application, comprising detection using high performance liquid chromatography-mass spectrometry, characterized in that, Includes the following steps: A1: Preparation of working solutions: Preparation of standard stock solution, internal standard stock solution, standard working solution and internal standard working solution; wherein the standard is arecoline hydrobromide and the internal standard is pilocarpine; A2: Biological sample pretreatment: Take biological sample tissue, add water and grinding beads to homogenize, add acidic acetonitrile solution, vortex centrifuge, take the supernatant for solid phase extraction and / or dispersion solid phase extraction purification, collect the eluent, filter and wait for analysis; A3: Preparation of matrix standard curve solution: Prepare blank biological sample matrix solution according to step A2, add standard working solution and internal standard working solution, and prepare matrix standard curve solutions of a series of concentrations; A4: The processed biological sample solution and the matrix standard curve solution of A3 were analyzed by high performance liquid chromatography-mass spectrometry to obtain the matrix standard curve of arecoline. The content of arecoline in the biological sample to be tested was calculated based on the standard curve. The liquid chromatography conditions used an octadecylsilane-bonded silica gel column, with mobile phase A being 0.1% formic acid-10 mmol / L ammonium formate aqueous solution and mobile phase B being methanol, for gradient elution.
2. The detection method according to claim 1, characterized in that, The biological sample was edible chicken tissue.
3. The detection method according to claim 2, characterized in that, The edible chicken tissue is selected from at least one of muscle, liver, kidney, and subcutaneous fat.
4. The detection method according to claim 1, characterized in that, In A1, the working solution concentration was diluted using 0.1% formic acid solution and 10 mmol / L ammonium formate solution.
5. The detection method according to claim 1, characterized in that, The solid-phase extraction described in A2 uses at least one of HLB solid-phase extraction columns and MCX solid-phase extraction columns for purification; the dispersion solid-phase extraction uses a dSPE extraction package.
6. The detection method according to claim 5, characterized in that, In A2, for muscle or subcutaneous tissue, the solid-phase extraction is performed sequentially through an HLB solid-phase extraction column and an MCX solid-phase extraction column; for liver or kidney tissue, the solid-phase extraction is performed first through a dSPE extraction pack and then through an MCX solid-phase extraction column; the acidic acetonitrile solution is 0.5% formic acid acetonitrile.
7. The detection method according to claim 1, characterized in that, The lower limit of quantification for muscle tissue detection is 2 ng / mL; the lower limit of quantification for subcutaneous fat, liver, and kidney tissue detection is 4 ng / mL.
8. The detection method according to claim 1, characterized in that, The gradient elution procedure is as follows:
9. The detection method according to claim 1, characterized in that, The chromatographic conditions in A4 were as follows: column: Endeavorsil C18-A (2.1×150mm, 1.8μm), column temperature: 40℃, flow rate: 0.3mL / min, injection volume: 2µL; mass spectrometry conditions were as follows: ion source: electrospray ionization (ESI+); scanning mode: positive ion multiple reaction monitoring (MRM) mode; positive and negative capillary voltages: 4500V and 3500V; nebulizer pressure: 35psi; drying gas temperature: 280℃; drying gas flow rate: 13L / min; Delta EMV: 500; drying gas: N2 (purity 99.9%); collision gas: N2 (purity 99.9%); the quantitative ion pair for arecoline was m / z 156.00 → 44, and the quantitative ion pair for the internal standard pilocarpine was m / z 209.20 → 96.
1.
10. The application of the method for detecting arecoline residues in biological samples according to any one of claims 1-9 in the detection of arecoline residues in edible chicken tissues.