Method for simultaneously detecting arecoline and arecoline in biological sample and application thereof

Abstract

The invention discloses a method for simultaneously detecting arecoline and arecoline in a biological sample and application of the method. According to the method, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) is adopted for testing and analysis, an octadecyl bonded silica gel chromatographic column is used as a detection chromatographic column, 0.001% formic acid aqueous solution-acetonitrile is used as a mobile phase, a gradient elution procedure is adopted, and specific mass spectrometry conditions are matched, so that the arecoline and the arecoline in a biological sample can be quantitatively determined at the same time. The detection method is applied to pharmacokinetic research on arecoline and arecaine in a living body, and fills up the technical blank in the aspect of arecoline and arecaine detection at present.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical analysis and detection, and particularly to a high performance liquid chromatography tandem mass spectrometry (HPLC-MS / MS) detection method for simultaneously quantitatively detecting arecoline and arecaidine in biological samples and its application. Background Art

[0002] Areca catechu L. is an evergreen tree of the genus Areca in the Palmae family. Medicinal Arecae Semen (AS) is the dried ripe seed of the palm plant Areca catechu L. It is described in the Chinese Pharmacopoeia as the ripe fruit from late summer to early autumn, dried, with the pericarp removed and the seed taken out. Areca is one of the "four major southern medicines" in China. It enters the stomach meridian, small intestine meridian and large intestine meridian, and has the effects of killing parasites, promoting digestion and removing food retention, promoting diuresis and qi movement, and treating malaria. Jujube areca (also known as areca core, tender areca, areca kernel, green fruit areca, etc.) is the immature seed of the palm plant Areca catechu L. It has the same source and matrix as the medicinal areca in the pharmacopoeia, only with different maturity. Areca and / or jujube areca contain various chemical components such as alkaloids, polyphenols, and flavonoids. Among them, characteristic alkaloids such as arecoline and arecaidine are one of the main active substances of areca.

[0003] Pharmacokinetics is of great significance in clinical medication. It mainly studies the dynamic changes of the body's disposal of drugs, including the processes of drug absorption, distribution, biochemical transformation (or metabolism) and excretion in the body, especially the law of blood drug concentration changing with time. Through pharmacokinetic monitoring, it is possible to determine the concentration changes and apparent clearance rates of drugs and their metabolites in the body, which helps to determine the optimal dose, administration route and dosage form of drugs, and maximize the therapeutic effect of drugs in clinical trials.

[0004] Currently, there is no report on the method for simultaneously quantitatively detecting arecoline and arecaidine in biological samples, especially in chicken plasma. In order to better reveal the pharmacodynamic material basis of arecoline and arecaidine, it is necessary to develop a detection method with high specificity, good sensitivity and small matrix effect, which can simultaneously quantitatively determine the contents of arecoline and arecaidine in biological samples, especially chicken plasma, and provide a research basis for the pharmacokinetics of arecoline and arecaidine in biological samples. Summary of the Invention

[0005] In order to solve the above existing technical problems, one of the purposes of the present invention is to establish a detection method with high sensitivity, strong specificity, fast analysis speed, small matrix effect, simple operation and good repeatability, which can simultaneously quantitatively detect arecoline and arecaidine in biological samples.

[0006] The second purpose of the present invention is to apply the method for simultaneously quantitatively detecting arecoline and arecoline in biological samples to pharmacokinetic research, so as to fill the current gap in the pharmacokinetic technology of arecoline and arecoline in biological samples, especially chicken plasma.

[0007] To achieve the above object, the technical solution adopted by the present invention is: a method for simultaneously detecting arecoline and arecoline in a biological sample, using high performance liquid chromatography tandem mass spectrometry (HPLC-MS / MS), the detection method comprising the following steps:

[0008] A1: Preparation of working solution;

[0009] A2: Collect biological samples and obtain the test solution after processing;

[0010] A3: Preparation of standard curve solution;

[0011] A4: HPLC-MS / MS is used to detect and analyze the biological sample test solution and the standard curve solution, wherein: the chromatographic conditions use a C18 chromatographic column, a 0.001% volume concentration of formic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, and gradient elution; the mass spectrometry conditions use an ESI ion source, and the scanning mode is an MRM monitoring mode.

[0012] Preferably, the biological sample comprises an animal biological sample containing arecoline and / or arecoline-containing and an animal biological sample not containing arecoline and / or arecoline-containing.

[0013] Preferably, the biological sample is a chicken plasma sample.

[0014] Preferably, the working solution in A1 comprises a mother solution of a standard solution and a mother solution of an internal standard solution;

[0015] Preferably, the standard substance is arecoline hydrobromide or arecoline hydrobromide; the internal standard is ganciclovir;

[0016] Preferably, a method for simultaneously detecting arecoline and arecoline in a biological sample comprises the following specific steps:

[0017] A1: Preparation of working solution: The method for preparing the stock solution of the standard solution is to accurately weigh arecoline hydrobromide and arecoline hydrobromide, and use an appropriate volume of methanol to prepare arecoline hydrobromide stock solutions and arecoline hydrobromide stock solutions with a concentration of 500 μg / mL respectively; the method for preparing the stock solution of the internal standard solution is to accurately weigh an appropriate amount of ganciclovir, and add methanol to prepare a ganciclovir stock solution with a concentration of 100 μg / mL.

[0018] A2: Plasma sample treatment method: Transfer the collected biological sample into a centrifuge tube, centrifuge to separate the plasma, and then add a standard solution and / or an internal standard solution and / or an organic solvent to the plasma sample, mix evenly once, sonicate, centrifuge, concentrate the supernatant by nitrogen blowing, redissolve in the solvent, mix evenly twice, sonicate, centrifuge, and take the supernatant for filtration to obtain the biological sample to be tested.

[0019] A3: Preparation of standard curve solution: Take an appropriate amount of arecoline hydrobromide and arecaidine hydrobromide stock solutions and mix them, dilute with methanol to finally obtain a mixed standard solution of arecoline hydrobromide and arecaidine hydrobromide at different concentrations.

[0020] A4: Detect and analyze the biological sample to be tested and the standard curve solution by HPLC-MS / MS: The chromatographic conditions use a C18 chromatographic column, with an aqueous solution of 0.001% formic acid by volume as mobile phase A and acetonitrile as mobile phase B for gradient elution; the mass spectrometry conditions use an ESI ion source, and the scanning mode is the MRM monitoring mode.

[0021] Preferably, the organic solvent is formic acid and methanol.

[0022] Preferably, the plasma sample treatment method in A2 is: Take 100 μL of plasma sample and add it to a 1 mL EDTA-2K anticoagulant centrifuge tube, centrifuge at a speed of 4000 r / min for 10 min to separate the plasma, add 50 μL of ganciclovir solution (50 μg / mL), 5 μL of formic acid solution and 845 μL of methanol solution. After the mixed solution system is vigorously mixed on a vortex oscillator for 1 min, sonicate for 15 min, centrifuge for 10 min (4 °C, 10000 r / min), collect the supernatant and concentrate it by nitrogen blowing, redissolve in 1 mL of methanol, vortex twice for 1 min, sonicate for 15 min, centrifuge for 10 min (4 °C, 10000 r / min), and finally take the clear supernatant and filter it through a 0.22 μm organic microporous membrane and store it in the dark at 4 °C for testing.

[0023] Preferably, an animal biological sample (blank plasma sample) in A2 that does not contain arecoline and / or arecaidine can also be spiked: Take 100 μL of plasma sample and add it to a 1.5 mL EDTA-2K anticoagulant centrifuge tube. Then, add 50 μL of the alkaloid mixed standard solution (arecoline hydrobromide 2 μg / mL, arecaidine hydrobromide 4 μg / mL), 50 μL of ganciclovir solution (internal standard, 50 μg / mL), 5 μL of formic acid solution, and 795 μL of methanol solution in sequence. After the mixed solution system is vigorously mixed on a vortex oscillator for 1 min, it is ultrasonicated for 15 min, centrifuged for 10 min (4 °C, 10000 r / min). The supernatant is collected and concentrated by nitrogen blowing, redissolved in 1 mL of methanol, vortexed for 1 min for the second time, ultrasonicated for 15 min, and centrifuged for 10 min (4 °C, 10000 r / min). Finally, the clarified supernatant is filtered through a 0.22 μm organic microporous membrane and stored in the dark at 4 °C for testing.

[0024] Preferably, the preparation of the A3 standard curve solution is as follows: Take an appropriate amount of the mother liquor of arecoline hydrobromide and arecaidine hydrobromide and mix them, then dilute with methanol. Finally, the concentrations of arecoline hydrobromide and arecaidine hydrobromide are (1 μg / mL, 2 μg / mL), (0.5 μg / mL, 1 μg / mL), (0.25 μg / mL, 0.5 μg / mL), (0.125 μg / mL, 0.25 μg / mL), (0.0625 μg / mL, 0.125 μg / mL), (0.03125 μg / mL, 0.0625 μg / mL), (0.015625 μg / mL, 0.03125 μg / mL), (0.0078125 μg / mL, 0.015625 μg / mL), and a total of 8 mixed standard solutions are prepared for standby. Replace the alkaloid mixed standard solution in A2 with the mixed standard solution to prepare the standard curve solution for testing, which is used for the determination of the standard curve.

[0025] Preferably, the chromatographic conditions and mass spectrometry conditions in A4 are as follows:

[0026] Chromatographic conditions: Chromatographic column: Agilent StableBond Analytical 4.6×250 mm 5-Micron; Mobile phase: 0.001% formic acid in water (A) - acetonitrile (B); Column temperature: 35 °C; Flow rate: 1 mL / min; Injection volume: 2 μL; Needle washing solution: 50% methanol in water; Gradient elution program is shown in Table 1:

[0027] Table 1 Gradient elution program

[0028]

[0029] Mass spectrometry conditions: The electrospray ionization source (ESI) was used in the positive ion multiple reaction monitoring (MRM) mode; the capillary voltage was 4000 V; the drying gas temperature was 350 °C; the drying gas flow rate was 13 L / min; the nebulizer pressure was 30 psi. The drying gas was N2 (purity 99.9%), and the collision gas was N2 (purity 99.999%). The mass spectrometry parameters are shown in Table 2:

[0030] Table 2 Mass spectrometry parameters of arecoline, arecaidine, and ganciclovir

[0031]

[0032] Advantages of the present invention:

[0033] (1) In the prior art, there is no literature report on the detection method for simultaneously quantifying arecoline and arecaidine in chicken plasma.

[0034] (2) The detection method provided by the present invention can simultaneously quantify arecoline and arecaidine in biological samples, especially chicken plasma. The specificity, precision, accuracy, stability, etc. of this detection method are all good, meeting the requirements for biological sample detection. In addition, it is also convenient to monitor the pharmacokinetic processes of arecoline and arecaidine in vivo.

[0035] (3) In the detection method provided by the present invention, the quantification range of arecoline is within 0.975 - 249.6 ng / mL, and the quantification range of arecaidine is within 1.99 - 499.2 ng / mL. It has high sensitivity and is suitable for the detection of arecoline and arecaidine in vivo. Description of the drawings

[0036] Figure 1 Chromatogram of blank matrix; Arecoline (upper left), Arecaidine (upper right), Ganciclovir (lower)

[0037] Figure 2 Chromatogram of blank matrix spiked with standards; Arecoline (upper left), Arecaidine (upper right), Ganciclovir (lower)

[0038] Figure 3 betel nut Standard curve graphs of arecoline (left) and arecaidine (right)

[0039] Figure 4 betel nut Average drug-time curve graphs of arecoline (left) and arecaidine (right) Detailed implementation manners

[0040] Preparation of working solutions:

[0041] 1. Preparation of standard solutions: Accurately weigh hydrobromide arecoline and hydrobromide arecaidine, and use an appropriate volume of methanol to prepare mother solutions of hydrobromide arecoline and hydrobromide arecaidine with a concentration of 500 μg / mL each;

[0042] 2. Preparation of internal standard solution: Weigh an appropriate amount of ganciclovir accurately, and dissolve it in methanol to prepare a ganciclovir stock solution with a concentration of 100 μg / mL.

[0043] 3. Preparation of quality control solutions: Pipette an appropriate amount of the reference stock solution, dilute it with methanol to prepare three quality control solutions with different concentrations: low (arecoline hydrobromide 0.015625 μg / mL, arecaidine hydrobromide 0.03125 μg / mL), medium (arecoline hydrobromide 0.0625 μg / mL, arecaidine hydrobromide 0.125 μg / mL), and high (arecoline hydrobromide 1 μg / mL, arecaidine hydrobromide 2 μg / mL).

[0044] 4. Sample pretreatment: The mixed solution system is vigorously mixed on a vortex oscillator for 1 min, sonicated for 15 min, centrifuged for 10 min (4 °C, 10000 r / min). Collect the supernatant, concentrate it by nitrogen blowing, redissolve it in 1 mL of methanol, vortex for 1 min again, sonicated for 15 min, centrifuged for 10 min (4 °C, 10000 r / min). Take the supernatant and filter it through a 0.22 μm organic microporous membrane. The filtrate is reserved for use.

[0045] The prepared working solution is used in the following examples.

[0046] Example 1: Detection method for arecoline and arecaidine in biological samples - Investigation of methodological specificity

[0047] (1) The method steps are as follows:

[0048] S1 Pretreatment of blank plasma samples: Take 100 μL of blank plasma sample and place it in a 1.5 mL EDTA-2K anticoagulant centrifuge tube, add 5 μL of formic acid solution and 845 μL of methanol solution to make a 1000 μL solution system. The subsequent steps are carried out according to 4 in the preparation of the working solution.

[0049] S2 Pretreatment of blank plasma samples with added standard: Take 100 μL of blank plasma sample and place it in a 1.5 mL EDTA-2K anticoagulant centrifuge tube, and successively add 50 μL of alkaloid mixed standard solution (arecoline hydrobromide 2 μg / mL, arecaidine hydrobromide 4 μg / mL), 50 μL of ganciclovir solution (internal standard, 50 μg / mL), 5 μL of formic acid solution and 795 μL of methanol solution. The subsequent steps are carried out according to 4 in the preparation of the working solution.

[0050] S3 Standard curve preparation: Take an appropriate amount of arecoline hydrobromide and arecaidine hydrobromide stock solutions and mix them to prepare a mixed standard solution. Finally, obtain eight mixed standard solutions with the concentrations of arecoline hydrobromide and arecaidine hydrobromide being (1 μg / mL, 2 μg / mL), (0.5 μg / mL, 1 μg / mL), (0.25 μg / mL, 0.5 μg / mL), (0.125 μg / mL, 0.25 μg / mL), (0.0625 μg / mL, 0.125 μg / mL), (0.03125 μg / mL, 0.0625 μg / mL), (0.015625 μg / mL, 0.03125 μg / mL), and (0.0078125 μg / mL, 0.015625 μg / mL) for standby. Replace the alkaloid mixed standard solution in S2 with the mixed standard solution to prepare samples for standard curve determination.

[0051] S4 Test and analysis: Use HPLC-MS / MS to test and analyze the test solutions obtained in S1, S2, and S3. The chromatographic and mass spectrometric conditions are as follows:

[0052] 1. Chromatographic conditions: Chromatographic column: Agilent StableBond Analytical 4.6×250 mm 5-Micron; Mobile phase: 0.001% formic acid in water (A) - acetonitrile (B); Column temperature: 35°C; Flow rate: 1 mL / min; Injection volume: 2 μL; Needle wash solution: 50% methanol in water; Gradient elution program is shown in Table 3:

[0053] Table 3 Gradient elution program

[0054]

[0055] 2. Mass spectrometric conditions: Use an electrospray ionization source (ESI) in the positive ion multiple reaction monitoring (MRM) mode; Capillary voltage is 4000 V; Dry gas temperature is 350°C; Dry gas flow rate is 13 L / min; Nebulizer pressure is 30 psi. The dry gas is N2 (purity 99.9%), and the collision gas is N2 (purity 99.999%). The mass spectrometric parameters are shown in Table 4:

[0056] Table 4 Mass spectrometric parameters of arecoline, arecaidine, and ganciclovir

[0057]

[0058] (II) Results

[0059] According to Table 5 and Figures 1-2 The results show that no interference from endogenous substances in plasma on the analytes arecoline, arecaidine, and the internal standard ganciclovir was found.

[0060] Table 5. Response values of standards and internal standards

[0061]

[0062] Example 2: Detection method of arecoline and arecaidine in biological samples - Methodological linear investigation:

[0063] (1) Method:

[0064] A1: Pretreatment of blank plasma samples before spiking: The same as in Example S2.

[0065] A2: Preparation of standard curve: The same as in Example S3.

[0066] A3: Test and analysis using HPLC-MS / MS method: The same as in Example S4.

[0067] (2) Results of linear investigation:

[0068] As shown in Table 6, the quantitative range of arecoline is within 0.641 - 164.1 ng / mL, and the quantitative range of arecaidine is within 1.26 - 317.32 ng / mL. The calibration curve has good linearity, with R 2 > 0.999. The standard curves of arecoline and arecaidine are shown in Figure 3.

[0069] Arecoline = weight of arecoline hydrobromide / 1.5214

[0070] Arecaidine = weight of arecaidine hydrobromide / 1.5732

[0071] Table 6. Standard curves, linear ranges, and correlation coefficients (R 2 )

[0072]

[0073] Example 3: Detection method of arecoline and arecaidine in biological samples - Investigation of methodological accuracy and precision

[0074] (1) Method

[0075] Add three different concentrations of quality control solutions (i.e., low, medium, and high) to blank plasma samples, and prepare quality control samples according to the sample pretreatment method described in Example S2 (replace the alkaloid mixed standard solution with the quality control solution). Conduct six repeated determinations for all concentrations of samples. Repeat the determinations for samples within one day and for three consecutive days. The detection method is the same as in Example 1.

[0076] (2) Results

[0077] The precision and accuracy of arecoline and arecaidine are shown in Table 7. The within-batch accuracy recovery rate of arecoline is 100.39%-103.63%, and the within-batch precision RSD is 4.56%-13.74%; the between-batch accuracy recovery rate is 100.45%-103.89%, and the between-batch precision RSD is 5.01%-14.62%. The within-batch accuracy recovery rate of arecaidine is 101.66%-104.50%, and the within-batch precision RSD is 5.74%-10.39%; the between-batch accuracy recovery rate is 102.57%-106.12%, and the between-batch precision RSD is 4.26%-12.81%. All meet the test requirements.

[0078] Table 7. Accuracy and Precision of Arecoline and Arecaidine in Plasma (n = 6)

[0079]

[0080] Example 4: Investigation on Matrix Effect and Extraction Recovery Rate

[0081] (1) Method

[0082] Group A: Take 900 μL of methanol solution, add 50 μL of quality control solution (low, medium, high) and 50 μL of internal standard solution and mix, and then proceed according to the preparation of working solution 4 (no nitrogen blowing for reconstitution); (n = 6)

[0083] Group B: Take 100 μL of blank plasma, add 50 μL of quality control solution (low, medium, high) and 50 μL of internal standard solution and mix, add 5 μL of formic acid solution and 795 μL of methanol solution to make a 1000 μL solution system. Then proceed according to the preparation of working solution 4; (n = 6)

[0084] Group C: Take 100 μL of blank plasma, add 795 μL of methanol and 5 μL of formic acid, and then proceed according to the preparation of working solution 4. Add 50 μL of quality control solution (low, medium, high) and 50 μL of internal standard solution to the obtained supernatant. (n = 6)

[0085] The detection method is the same as that in Example 1. The matrix effect is qualified when it reaches 80%-120%, and the recovery rate should be greater than 70%.

[0086] Calculation method: Matrix effect = C / A × 100%; Recovery rate = B / A × 100%

[0087] (2) Results

[0088] The results of the extraction recovery rate and matrix effect of arecoline and arecaidine measured by the research institute are shown in Table 8. The matrix effect and recovery rate of arecoline are respectively in the range of 100.23%-104.10% and 98.119%-99.869%; the matrix effect and recovery rate of arecaidine are respectively in the range of 88.49%-93.12% and 97.656%-99.350%. It shows that the extraction method is stable, not interfered by the matrix, and basically meets the requirements of the biological sample analysis method.

[0089] Table 8. Matrix effect and recovery rate of arecoline and arecaidine in plasma (n = 6)

[0090]

[0091] Example 5: Stability methodology investigation

[0092] (1) Method

[0093] By measuring quality control samples with low, medium, and high concentrations under different conditions, the stability of arecoline and arecaidine components in the samples was determined. For example: (1) Store at room temperature for 24 h; (2) Freeze-thaw repeatedly at -80 °C for 3 times; (3) Store in a -80 °C refrigerator to detect long-term stability. The detection method is the same as that in Example 1.

[0094] (2) Results

[0095] The results of the stability investigation are shown in Table 9. The accuracy (RSD) under the following three conditions: short-term stability (24 h at room temperature), freeze-thaw stability (3 freeze-thaw cycles), and long-term stability (stored at -80 °C for 1 month) is within 15%, and the precision (RE) is between 100.02% and 104.13%. The results show that the stability of arecoline and arecaidine in biological samples under various conditions is good, and the stability meets the detection requirements.

[0096] Table 9. Stability of arecoline and arecaidine in plasma under different environments (n = 6)

[0097]

[0098] Example 6: Application of the detection method of arecoline and arecaidine in biological samples in pharmacokinetic studies

[0099] White - feather broilers in each dose group were bled from the wing - vein at 0 min, 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 4 h, 8 h, 16 h, and 24 h after intragastric administration of chickens (11381.11 mg / kg areca extract, arecoline content 1.073%, arecaidine content 0.618%, provided by Hunan Jiu'anhe Biotechnology Co., Ltd.). The collected blood samples were transferred into anticoagulant centrifuge tubes containing EDTA - 2K and centrifuged at a speed of 4000 r / min for 10 min to separate the plasma. Subsequently, a formic acid solution with a volume of 5% of the plasma volume was added to the plasma samples, and the treated plasma samples were stored in a - 80 °C refrigerator for subsequent pretreatment and detection analysis.

[0100] (I) Method

[0101] Y1: Pretreatment of plasma samples: Take 105 μL of plasma samples and place them in a 1.5 mL EDTA - 2K anticoagulant centrifuge tube, add 50 μL of internal standard solution and 845 μL of methanol solution to make a 1000 μL solution system. The subsequent steps are carried out according to the preparation of working solutions 4.

[0102] Y2: Preparation of standard curve: Carry out according to Example 1 S2.

[0103] Y3: Test and analysis using HPLC - MS / MS method: Carry out according to Example 1 S3.

[0104] (II) Drawing of the standard curve (same as Example 2)

[0105] (III) Pharmacokinetic parameters of arecoline and arecaidine in plasma samples at different time points after administration

[0106] The concentration values of arecoline and arecaidine in biological samples at different administration time points (within the standard curve range) were deduced by the standard curve method. The pharmacokinetic parameters of arecoline and arecaidine were calculated using the non - compartmental pharmacokinetic model in the PKSolver 2.0 plug - in program. The drug concentration - time curve was drawn using GraphPad Prism 8.0 software.

[0107] Using the drug half - life (T 1 / 2 ), peak concentration (C max ), peak time (T max ) and the area under the time - concentration curve (AuC) and other parameters to characterize the pharmacokinetic characteristics of the drug, as shown in Table 10. The average drug - time curve of the compound in chicken plasma is shown in

[0108] Figure 4. ​

[0109] Table 10. Pharmacokinetic parameters of arecoline and arecaidine in chicken plasma after administration

[0110]

[0111] Figure 4 can It is known that arecoline shows a multi-peak phenomenon, suggesting that its absorption may have non-linear kinetic characteristics (such as absorption saturation / or enterohepatic circulation); the exposure (AuC) and time to peak concentration (Tmax) of arecaidine deviate significantly from the expected values of simple exogenous administration, which may be due to the rapid hydrolysis of arecoline to arecaidine catalyzed by carboxylesterase (CES), resulting in the blood concentration of arecaidine being composed of dual sources.

[0112] The above specific embodiments shall not be construed as limiting the scope of the present invention. For those skilled in the art of the present technology, any alternative improvement or transformation made to the embodiments of the present invention shall fall within the scope of the present invention. Where the present invention is not described in detail, it is all well-known technology in the technical field of the present technology.

Claims

1. A method for simultaneously detecting arecoline and arecaidine in biological samples, using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), the detection method comprising the following steps: A1: Preparation of working solutions; A2: Collection of biological samples, and after treatment, obtaining a test solution of the biological samples; A3: Preparation of standard curve solutions; A4: Detect and analyze the test solution of biological samples and the standard curve solution by HPLC-MS / MS, where: The chromatographic conditions use a C18 chromatographic column, with an aqueous solution of formic acid with a volume concentration of 0.001% as mobile phase A, and acetonitrile as mobile phase B, with gradient elution; the mass spectrometry conditions use an ESI ion source, and the scanning mode is the MRM monitoring mode.

2. According to claim 1, it is characterized in that The biological samples include animal biological samples containing arecoline and / or arecaidine and animal biological samples not containing arecoline and / or arecaidine.

3. According to claim 2, it is characterized in that The biological sample is a chicken plasma sample.

4. According to claim 1, it is characterized in that The working solutions in A1 include a standard solution and an internal standard solution. The standards are hydrobromide arecoline and hydrobromide arecaidine; the internal standard is ganciclovir.

5. According to claim 1, it is characterized in that A method for simultaneously detecting arecoline and arecaidine in biological samples, the specific steps are as follows: A1: Preparation of working solutions: The method for preparing the mother liquor of the standard solution is to accurately weigh hydrobromide arecoline and hydrobromide arecaidine, and use appropriate volumes of methanol to respectively prepare mother liquors of hydrobromide arecoline and hydrobromide arecaidine with a concentration of 500 μg / mL each; the method for preparing the mother liquor of the internal standard solution is to accurately weigh an appropriate amount of ganciclovir, and add methanol to prepare a mother liquor of ganciclovir with a concentration of 100 μg / mL. A2: Plasma sample treatment method: Transfer the collected biological sample into a centrifuge tube, centrifuge to obtain plasma, then add the standard solution and / or internal standard solution and / or organic solvent to the plasma sample, mix evenly once, ultrasonicate, centrifuge, concentrate the supernatant by nitrogen blowing, re-dissolve in the solvent, mix evenly twice, ultrasonicate, centrifuge, and take the supernatant for filtration to obtain a test solution of the biological sample. A3: Preparation of standard curve solutions: Take appropriate amounts of the mother liquors of hydrobromide arecoline and hydrobromide arecaidine and mix them, dilute with methanol to finally obtain a mixed standard solution of hydrobromide arecoline and hydrobromide arecaidine with different concentrations. A4: Use HPLC-MS / MS to detect and analyze the test solution of the biological sample and the standard curve solution: Among them, the chromatographic conditions use a C18 chromatographic column, with an aqueous solution of formic acid with a volume concentration of 0.001% as mobile phase A, and acetonitrile as mobile phase B, with gradient elution; the mass spectrometry conditions use an ESI ion source, and the scanning mode is the MRM monitoring mode.

6. According to claim 5, wherein The organic solvents used in A2 are formic acid and methanol.

7. According to claim 5, characterized in that, The plasma sample treatment method in A2 is as follows: Take 100 μL of plasma sample and add it to a 1.5 mL EDTA-2K anticoagulant centrifuge tube. Centrifuge at a speed of 4000 r / min for 10 min to separate the plasma. Then add 50 μL of ganciclovir solution (50 μg / mL), 5 μL of formic acid solution, and 845 μL of methanol solution. After the mixed solution system is vigorously mixed on a vortex oscillator for 1 min, ultrasonicate for 15 min, centrifuge for 10 min (4°C, 10000 r / min), collect the supernatant, concentrate it by nitrogen blowing, redissolve it in 1 mL of methanol, vortex for 1 min for the second time, ultrasonicate for 15 min, centrifuge for 10 min (4°C, 10000 r / min). Finally, take the clear supernatant and filter it through a 0.22 μm organic microporous membrane, and store it in the dark at 4°C for testing.

8. According to claim 5, characterized in that, The animal biological samples without arecoline and / or arecaidine in A2 can also be spiked: Take 100 μL of plasma sample and add it to a 1.5 mL EDTA-2K anticoagulant centrifuge tube. Then add 50 μL of alkaloid mixed standard solution (arecoline hydrobromide 2 μg / mL, arecaidine hydrobromide 4 μg / mL), 50 μL of ganciclovir solution (internal standard, 50 μg / mL), 5 μL of formic acid solution, and 795 μL of methanol solution in sequence. After the mixed solution system is vigorously mixed on a vortex oscillator for 1 min, ultrasonicate for 15 min, centrifuge for 10 min (4°C, 10000 r / min), collect the supernatant, concentrate it by nitrogen blowing, redissolve it in 1 mL of methanol, vortex for 1 min for the second time, ultrasonicate for 15 min, centrifuge for 10 min (4°C, 10000 r / min). Finally, take the clear supernatant and filter it through a 0.22 μm organic microporous membrane, and store it in the dark at 4°C for testing.

9. According to claim 5, it is characterized in that The preparation of the A3 standard curve solution is as follows: Take an appropriate amount of the mother liquor of arecoline hydrobromide and arecaidine hydrobromide and mix them, then dilute with methanol. Finally, obtain mixed standard solutions with concentrations of arecoline hydrobromide and arecaidine hydrobromide as (1 μg / mL, 2 μg / mL), (0.5 μg / mL, 1 μg / mL), (0.25 μg / mL, 0.5 μg / mL), (0.125 μg / mL, 0.25 μg / mL), (0.0625 μg / mL, 0.125 μg / mL), (0.03125 μg / mL, 0.0625 μg / mL), (0.015625 μg / mL, 0.03125 μg / mL), (0.0078125 μg / mL, 0.015625 μg / mL), a total of 8 mixed standard solutions for standby. Replace the alkaloid mixed standard solution in A2 with the mixed standard solution to prepare the standard curve solution test solution for standard curve determination.

10. As described in claim 5, characterized in that The chromatographic conditions in A4 are as follows: Chromatographic column: Agilent StableBond Analytical 4.6×250mm 5-Micron; Mobile phase: 0.001% formic acid in water (A) - acetonitrile (B); Column temperature: 35°C; Flow rate: 1 mL / min; Injection volume: 2 μL; Needle wash solution: 50% methanol in water; Gradient elution conditions: 0 - 3 min, 50% mobile phase A, 50% mobile phase B; 3 - 3.1 min, 50% → 10% mobile phase A, 50% → 90% mobile phase B; 3.1 - 9 min, 10% → 10% mobile phase A, 90% → 90% mobile phase B; 9 - 9.1 min, 10% → 50% mobile phase A, 90% → 50% mobile phase B; 9.1 - 15 min, 50% mobile phase A, 50% mobile phase B. The mass spectrometry conditions are as follows: Using an electrospray ionization source (ESI) in the positive ion multiple reaction monitoring (MRM) mode; Capillary voltage is 4000 V; Drying gas temperature is 350°C; Drying gas flow rate is 13 L / min; Nebulizer pressure is 30 psi; Drying gas is N2 (purity 99.9%); Collision gas is N2 (purity 99.999%).

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