Liquid mass spectrometry detection method for biogenic amine in bee tissue

Abstract

The invention discloses a liquid mass spectrometry detection method for biogenic amines in bee tissues, and relates to the technical field of compound detection. The detection method comprises the following steps: dissecting to obtain a bee brain tissue sample (with a single eye and a compound eye removed), putting the sample into a centrifugal tube, adding an extracting solution, namely a hydrochloric acid aqueous solution, crushing the tissue crushed sample by an electric grinder, carrying out ultrasonic treatment on precipitated protein, transferring supernate into a filter type sample bottle, filtering, and carrying out liquid chromatography-mass spectrometry determination. The invention establishes a method for simultaneously and rapidly detecting four biogenic amines in a bee tissue sample by HPLC-MS (High Performance Liquid Chromatography-Mass Spectrometry), the method can simultaneously analyze four chemical substances (biogenic amines) within 8 minutes, the detection time is short, the linear relation is better, the method recovery rate is high, the stability is good, and the analysis requirement is met.

Description

Technical Field

[0001] This invention relates to the field of compound detection technology, and specifically to a liquid chromatography-mass spectrometry method for detecting biogenic amines in bee tissues. Background Technology

[0002] Biogenic amines (BAs), weakly basic, low-molecular-weight nitrogen-containing compounds, are primarily formed by the decarboxylation of amino acids or the amination of aldehydes and ketones. Evolutionarily conserved neurochemicals, biogenic amines are considered to play a significant role in regulating sensory organs, influencing the state of the central nervous system, and altering initiating behaviors in both vertebrates and invertebrates. The biogenic amine group comprises five major members in both deuterostomes and protostomes. Three of these amines are shared across phyla: dopamine, histamine, and serotonin (5-hydroxytryptamine). Furthermore, some compounds appear to be preferentially utilized in deuterostomes (norepinephrine, epinephrine) or protostomes (tyramine, octopamine). However, small amounts of tyramine and octopamine have been found in vertebrates; due to their low abundance, they are classified as trace amines.

[0003] Biogenic amines play numerous roles in regulating various physiological and productive behaviors in insects. High concentrations of octopamine, dopamine, and serotonin are present in the insect nervous system; these biogenic amines are common neurotransmitters, neuromodulators, and neurohormones in invertebrates. The effects of biogenic amines on insects are not limited to the central nervous system but also involve functional regulation at the peripheral sensory level and at the level of motor output from muscles and glands. Short-term behavioral changes in insects are directly influenced by changes in biogenic amine levels.

[0004] Currently, the main methods for detecting and analyzing biogenic amines include liquid chromatography-ultraviolet (LC-UV), liquid chromatography-fluorescence (LC-FLD), thin-layer chromatography (TLC), capillary electrophoresis (CE), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS). Among these, LC-UV and LC-FLD are simple to operate, but samples usually require derivatization, leading to poor reproducibility of the results. CE has advantages such as high separation efficiency and low operating cost, but purified samples are more suitable for CE detection, which places higher demands and challenges on sample preparation techniques. TLC can rapidly separate and determine various biogenic amines; however, some biogenic amines require derivatization, which reduces the precision of sample determination. GC-MS has advantages such as high sensitivity and high separation efficiency; however, its chromatographic peaks are prone to tailing, thus affecting the accuracy and sensitivity of biogenic amines. Although LC-MS / MS is expensive, it offers high resolution and can analyze most biogenic amines without derivatization. Currently established methods for detecting biogenic amines mainly include high-performance liquid chromatography (HPLC), capillary electrophoresis, thin-layer chromatography (TLC), gas chromatography (GC), and electrochemical detection. Among these, HPLC combined with mass spectrometry (MS / MS) is currently the most suitable method for biogenic amine detection. However, existing methods are difficult to accurately determine the biogenic amine content in bee tissue samples. This is because the main challenge in determining biogenic amines in bee tissue samples lies in the small size of individual bees, the limited amount of tissue obtained, and the low biogenic amine content in the tissue samples. Summary of the Invention

[0005] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a liquid chromatography-mass spectrometry method for detecting biogenic amines in bee tissues. This invention provides a simple and rapid liquid chromatography-mass spectrometry method for detecting biogenic amines in bee tissue samples.

[0006] The technical solution of the present invention is as follows: This invention provides a method for detecting biogenic amines in bee tissues by liquid chromatography-mass spectrometry, comprising the following steps: S1. Dissect the bee to be tested to obtain a bee brain tissue sample; add the sample to the extraction solution, grind and sonicate the sample to obtain tissue fluid; filter the tissue fluid to obtain filtrate; S2. The filtrate is subjected to liquid chromatography-mass spectrometry analysis, wherein... Liquid chromatography conditions include: Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: methanol; Flow rate: 0.5 mL / min; Column temperature: 30℃; Injection volume: 10 μL; Column: ZORBAX high-performance column Polaris 5 C18-A 150 × 4.6 mm; Mass spectrometry conditions include: Scanning method: dual electrospray positive ion scanning; detection reaction: multiple reaction detection; gas temperature: 350℃; drying gas flow rate: 9 L / min; atomizer pressure: 45 Psi; electrospray voltage: 4000 V; sheath gas temperature: 250℃; sheath gas flow rate: 11 L / min; nozzle voltage: 500 V; collision gas: nitrogen.

[0007] Optionally, in S1, the extract is an aqueous hydrochloric acid solution with a concentration of 0.1 mol / L, and the ratio of bee brain tissue sample to extract is 500 μg / L.

[0008] Optionally, in S1, the grinding method is to grind for 1 minute using an electric grinder with a rotation speed of 11000 RPM.

[0009] Optionally, in S1, the ultrasonic treatment has a power of 200W, a frequency of 40KHz, and a duration of 0.5 min to 1 min.

[0010] Optionally, in S2, the gradient elution program for the liquid chromatography mobile phase is as follows: Run for 7 minutes afterward.

[0011] Optionally, in S2, the parameters of MS TOP include: Decomposition voltage: 90 V; Skimmer voltage: 65 V; Octopus RF voltage peak: 750 V.

[0012] Optionally, in S2, the MS scan range is 50 m / z to 250 m / z, and the scan rate is 2 spectra / s.

[0013] Optionally, in S2, the target ion is as follows:

[0014] Optionally, the detection method further includes: Using hydrochloric acid containing DHBA as a solvent, histamine, octopamine, dopamine, and 5-hydroxytryptamine were prepared into biogenic amine standard solutions of different concentrations. The biogenic amine standard solutions were then subjected to liquid chromatography-mass spectrometry detection under the same conditions as those for the bee brain tissue sample. A standard curve was plotted with the peak area of ​​the quantitative ion as the ordinate and the corresponding concentration of the biogenic amine standard solution as the abscissa. The peak area of ​​the bee brain tissue sample to be tested was substituted into the standard curve to calculate the concentration of biogenic amines in the bee brain tissue sample to be tested.

[0015] Specifically, the four biogenic amine standards are: histamine (Sigma-Aldrich, V900396); octopamine hydrochloride (Sigma-Aldrich, 68631); dopamine hydrochloride (Sigma-Aldrich, H8502); and 5-hydroxytryptamine hydrochloride (Sigma-Aldrich, H9523).

[0016] Stock solutions of four biogenic amines: Weigh appropriate amounts (accurate to 0.01 mg) of each of the four biogenic amine standards into brown volumetric flasks, and dilute to volume with 0.1 mol / L hydrochloric acid aqueous solution containing 500 ng / mL 3,4-dihydroxybenzylamine hydrobromide (DHBA, 858781, Sigma-Aldrich) to prepare 1 mg / mL stock solutions. Store at 4°C protected from light. The standard stock solution was diluted with 0.1 mol / L hydrochloric acid aqueous solution containing 500 ng / mL DHBA to prepare mixed standards of 20 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 500 ng / mL, respectively. The peak areas of each concentration of standard were statistically analyzed. The peak areas of the standard were corrected with the peak area of ​​the internal standard DHBA. A standard curve was plotted with the peak area of ​​the characteristic ion mass chromatographic peak as the ordinate and the concentration of the standard as the abscissa.

[0017] Optionally, the linear range, standard curve equation, correlation coefficient, limit of detection (LOD), and limit of quantitation (LOQ) of the detection method for the four biogenic amines—histamine, octopamine, dopamine, and serotonin—are as follows:

[0018] This invention has at least one of the following beneficial effects: This invention establishes a rapid HPLC-MS method for the simultaneous detection of four biogenic amines in bee tissue samples. On one hand, due to the small size of bees and the limited amount of tissue obtained, coupled with the low content of biogenic amines in the tissue samples, the sensitivity of the detection method is determined by the extraction method of biogenic amines. Therefore, this invention explored different extraction methods and optimized the extraction conditions to extract biogenic amines from brain tissue samples. On the other hand, this invention utilizes the advantages of HPLC-MS, optimizing HPLC and MS conditions, selecting appropriate ionization modes, and optimizing MS parameters. Qualitative and quantitative ions were selected for the four biogenic amines, ultimately achieving the separation, extraction, and confirmation of the four chemical substances, and realizing their quantitative analysis. This method can simultaneously analyze four chemical substances within 8 minutes, exhibiting short detection time, good linearity, high recovery rate, and good stability, meeting the analytical requirements. Attached Figure Description

[0019] Figure 1 This is the MBR chromatogram of the standard solution (400 ng / ML) in Example 1 of the present invention.

[0020] Figure 2 This is the chromatogram of the 5HT precursor ion in Comparative Example 4 of the present invention. Detailed Implementation

[0021] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0022] Example 1 A liquid chromatography-mass spectrometry method for detecting biogenic amines in bee tissues includes the following steps: 1. Materials and Methods 1.1 Materials: Western honeybee ( Apis mellifera ) Instruments: Liquid chromatography-mass spectrometry-TOF system (G-7120A, G6546A, Agilent Technologies); homogenizer (MP FastPrep®-24); ultrasonic cleaner (Bangjie, BG-04C); high-speed centrifuge (Eppendorf, 5424R); handheld electric grinder (TIANGEN, OSE-Y30).

[0023] Mass spectrometry grade methanol, mass spectrometry grade formic acid, ultrapure water. Hydrochloric acid (Xilong Scientific); 3,4-dihydroxybenzylamine-hydrobromide (DHBA; Sigma-Aldrich, 858781); histamine (Sigma-Aldrich, V900396); octopamine hydrochloride (Sigma-Aldrich, 68631); dopamine hydrochloride (Sigma-Aldrich, H8502); 5-hydroxytryptamine hydrochloride (Sigma-Aldrich, H9523); formic acid (mass spectrometry grade, Maclean's).

[0024] 2.0 mL screw-cap microtubes (Thermo Sciencefic, 3470s and 3471TS); 0.1 mm diameter glass beads (BioSpec, 11079101); 0.22 μm hydrophilic PTFE filter vials (CNW Technologies, 2.FV6102.2001); 200 μL inner tubes (5183-2090, Agilent Technologies).

[0025] 1.2 Test Methods 1.2.1 Chromatographic conditions Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: methanol; Flow rate: 0.5 mL / min; Column temperature: 30℃; Injection volume: 10 μL; Column: ZORBAX high performance column (Polaris 5 C18-A 150 × 4.6 mm, Agilent Technologies) To ensure the reproducibility of the experiment, column rinsing and equilibration were added. The specific mobile phase system procedure is as follows: Table 1 Mobile Phase Procedures for Liquid Chromatography Run for 7 minutes afterward.

[0026] 1.2.2 Mass Spectrometry Conditions Dual AJS ESI electro-injection ion source, Gas Temp 350℃, Gas Flow 9 L / min, Nebulizer 45 Psi, Capillary positive 4000 V, Sheath Gas Temp 250℃, Sheath Gas Flow 11 L / min, Nozzle Voltage 500 V. MS TOP: Fragmentor 90 V, Skimmer 65 V, Oct 1 RF Vpp 750 V. Positive ion Scan mode: MS range: 50-250 m / z, Rate 2 spectra / s; Target ions are shown in the table below: Table 2 Target Ions 1.3 Preparation of standard solutions 1.3.1 Preparation of Standard Solution Stock Solution Histamine, octopamine hydrochloride, dopamine hydrochloride, and 5-hydroxytryptamine hydrochloride standards were accurately weighed using a high-precision electronic balance. A 1 mg / mL standard stock solution was prepared using 0.1 mol / L hydrochloric acid containing 500 ng / mL DHBA as the solvent.

[0027] 1.3.2 Plotting the Standard Curve The standard stock solution was diluted to prepare standard samples of 500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, and 20 ng / mL, and analyzed by instrument. Each sample was analyzed three times. The peak areas of the standards at each concentration were statistically analyzed. The peak areas of the standards were corrected by the peak area of ​​the internal standard DHBA, and the average value was calculated to plot a standard curve.

[0028] 1.4 Sample Pretreatment Extraction: Dissect the bees, remove the ocelli and compound eyes, and obtain 5 brains. Place them in centrifuge tubes, add 300 μL of mixed internal standard extraction solution (500 μg / L 0.1 mol / L hydrochloric acid aqueous solution), and manually grind the tissue for 1 min using an RPM11000 electric grinder. Transfer to a centrifuge and centrifuge at 4°C, 10000 rpm for 2 min. Then transfer to an ultrasonicator containing an ice-water mixture and sonicate for 1 min at a power of 200 W and a frequency of 40 kHz. Finally, transfer to a centrifuge and centrifuge at 4°C, 10000 rpm for 1 min.

[0029] 1.5 Determination of recovery rate and precision Due to individual differences among bees, to ensure the reproducibility of the experiment, a mixed standard of 50 ng / mL, 80 ng / mL, and 100 ng / mL was used to conduct the recovery experiment using the experimental treatment method.

[0030] Comparative Example 1 A liquid chromatography-mass spectrometry method for detecting biogenic amines in bee tissues differs from Example 1 only in that "manual grinding with an electric grinder for 1 min" is replaced with "breaking with a tissue homogenizer at a rate of 6.5 m / s for 1 min", while the other steps are the same as in Example 1.

[0031] Comparative Example 2 A liquid chromatography-mass spectrometry method for detecting biogenic amines in bee tissues differs from Example 1 only in that the ultrasonic treatment time is replaced by "30s, 2min, 3min" instead of "1min". The other steps are the same as in Example 1.

[0032] Comparative Example 3 A liquid chromatography-mass spectrometry method for detecting biogenic amines in bee tissues differs from Example 1 only in that the "0.1 mol / L hydrochloric acid aqueous solution" in the sample pretreatment is replaced with "1 mol / L hydrochloric acid aqueous solution, 0.05 mol / L hydrochloric acid aqueous solution, and 0.1 mol / L trichloroacetic acid aqueous solution", while the other steps are the same as in Example 1.

[0033] Comparative Example 4 A liquid chromatography-mass spectrometry method for detecting biogenic amines in bee tissues differs from Example 1 only in that the mobile phase B is replaced with "0.1% formic acid methanol" instead of "methanol". All other steps are the same as in Example 1.

[0034] Comparative Example 5 A liquid chromatography-mass spectrometry method for detecting biogenic amines in bee tissues differs from Example 1 only in that the liquid chromatography mobile phase program is replaced with "Table 3 Liquid Chromatography Mobile Phase Program", while the other steps are the same as in Example 1.

[0035] Table 3 Mobile Phase Procedures for Liquid Chromatography Run for 5 minutes afterward.

[0036] Comparative Example 6 A liquid chromatography-mass spectrometry method for detecting biogenic amines in bee tissues differs from Example 1 only in that "Fragmentor 90 V" in the mass spectrometry conditions is replaced with "50 V, 60 V, 70 V, 80 V, 100 V, 110 V, 120 V, 130 V, 140 V", while the other steps are the same as in Example 1.

[0037] Results and Analysis The results of Example 1 and Comparative Examples 1-6 were analyzed, and the results are as follows: 1. Optimization of sample pretreatment The main challenge in the detection and analysis of biogenic amines is that the actual concentration in biological samples is low, and the sensitivity of the detection method is determined by how biogenic amines are extracted from tissue samples.

[0038] This invention compares the extraction effects of manual grinding for 1 min using an electric grinder (Example 1) and tissue homogenization at a rate of 6.5 m / s for 1 min using a tissue homogenizer (Comparative Example 1). The experiment found that the tissue homogenizer had a poor extraction effect on histamine and 5-hydroxytryptamine.

[0039] The present invention also compared the extraction effect of ultrasonic treatment for 1 min (Example 1) and ultrasonic treatment for 30 s, 2 min, and 3 min (Comparative Example 2) after tissue grinding and crushing. The experiment found that the extraction effect was improved by ultrasonic treatment for 30 s to 1 min, while 5-HT was partially degraded when ultrasonicated for 2 min to 3 min. The peak areas of 100 ng / ML 5-HT during ultrasonic treatment for 30 s, 1 min, 2 min, and 3 min were 169194, 226546, 225551, and 202378, respectively.

[0040] This invention also compared the extraction effects of hydrochloric acid aqueous solutions with concentrations of 0.1 mol / L, 1 mol / L, and 0.05 mol / L during sample pretreatment. The experiments showed that 0.1 mol / L hydrochloric acid aqueous solution had the best extraction effect, 1 mol / L hydrochloric acid aqueous solution led to partial degradation of 5-HT, and the concentration of 0.05 mol / L hydrochloric acid aqueous solution was too low to completely extract the biogenic amines. This invention also compared the extraction effects of different extractants, 0.1 mol / L hydrochloric acid aqueous solution and 0.1 mol / L trichloroacetic acid aqueous solution. The experiments showed that 0.1 mol / L hydrochloric acid aqueous solution had a better extraction effect, and samples extracted with 0.1 mol / L trichloroacetic acid aqueous solution degraded faster after storage.

[0041] 2. Determination of chromatographic and mass spectrometric conditions (1) Determination of chromatographic conditions: To simultaneously detect histamine, octopamine, dopamine, and serotonin, the inventors experimented with different chromatographic conditions to separate the four chemicals. The experiments revealed that when mobile phase A was a 0.1% formic acid aqueous solution, mobile phase B was methanol, the flow rate was 0.5 mL / min, the column temperature was 30℃, the injection volume was 10 μL, and the chromatographic column was a ZORBAX high-performance column (Polaris 5 C18-A150 × 4.6 mm, Agilent Technologies), and the mobile phase system program was followed according to Table 1, the separation of the four chemicals could be achieved.

[0042] In Comparative Example 4, replacing mobile phase B with "0.1% formic acid methanol" resulted in changes in the peak shape of the ion chromatogram, an increase in the window time, and the appearance of peak splitting, such as... Figure 2 As shown in Table 4. In Comparative Example 5, due to changes in the mobile phase system program, the elution time of the target peak was delayed, and the window time was lengthened. The elution times of each substance are shown in Table 4.

[0043] Table 4 Target Ions (2) Determination of mass spectrometry conditions: Four biogenic amines were analyzed by first-order mass spectrometry (Q1 scan) in electrospray positive ion mode to obtain molecular ion information, i.e., parent ion and fragment ion information, i.e., daughter ions. Then, by optimizing the fragmentor voltage to achieve the maximum ion intensity, qualitative and quantitative ion pairs for the four biogenic amines were selected. Their multiple reaction detection spectra are shown below. Figure 1 .

[0044] In Comparative Example 6, as the fragmentor voltage gradually increases from 50V, the precursor ion response gradually increases, reaching a maximum at 90V. When the fragmentor voltage continues to increase to 140V, the precursor ion response begins to decrease, while the fragment ion response increases. Therefore, the optimal fragmentor voltage is 90V.

[0045] 3. Linearity and sensitivity of the method Linearity tests were performed using a mixed solution of biogenic amine standards. The determinations were conducted according to the method established in this study. A standard curve was then plotted with the peak area of ​​the quantitative ion as the ordinate (Y) and the corresponding standard solution concentration as the abscissa (X). The linear regression equation and correlation coefficient of the obtained standard curve were calculated. The results showed that the squared values ​​of the correlation coefficients R for the four organisms were all greater than 0.99, indicating that the method has a good linear relationship within the concentration range of 20 ng / mL to 500 ng / mL (see Table 3). The limit of detection (LOD) was defined as the content of the target analyte corresponding to a signal-to-noise ratio of 3 (S / N=3), and the limit of quantitation (LOQ) was defined as the content of the target analyte corresponding to a signal-to-noise ratio of 10 (S / N=10) (results are shown in Table 5).

[0046] Table 5. Linearity, Limit of Detection (LODs), and Limit of Quantification (LOQs) 4. Recovery rate and precision of the method Four biogenic amines were prepared at three concentration levels: 50 ng / mL, 80 ng / mL, and 100 ng / mL. Recovery and precision tests were performed at these three concentration levels (n=6) according to this experimental method. The results are shown in Table 6. It can be seen that the average recoveries of the four analytes ranged from 89.56% to 111.23%, and the relative standard deviations (RSDs) ranged from 3.63% to 9.67%, indicating that the method has good recovery and precision.

[0047] Table 6 Spike recoveries and relative standard deviations (n=6) In summary, the method of the present invention can detect biogenic amines in bee tissue samples and can simultaneously and rapidly detect four biogenic amines. This method can complete the simultaneous analysis of four chemical substances (biogenic amines) within 8 minutes, with short detection time, good linearity, high method recovery rate, good stability, and meets the analytical requirements.

[0048] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for detecting biogenic amines in honeybee tissues by liquid chromatography-mass spectrometry, characterized by, The method comprises the following steps: S1, dissecting the honeybee to be tested to obtain a honeybee brain tissue sample; adding the sample to an extraction solution, grinding and ultrasonic treating the sample to obtain a tissue liquid; filtering the tissue liquid to obtain a filtrate; S2, performing liquid chromatography-mass spectrometry on the filtrate, wherein the liquid chromatography conditions comprise: mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: methanol; flow rate 0.5 mL / min; column temperature 30 DEG C; sample injection amount 10 ul; chromatographic column: ZORBAX high-performance chromatographic column Polaris 5 C18-A 150*4.6mm; the mass spectrometry conditions comprise: scanning mode: double-electrospray positive ion scanning; detection reaction: multiple reaction detection; gas temperature: 350 DEG C; drying gas flow rate: 9 L / min; atomizer pressure 45 Psi; electrospray voltage: 4000 V; sheath gas temperature: 250 DEG C; sheath gas flow rate: 11 L / min; nozzle voltage: 500 V, collision gas: nitrogen.

2. The detection method according to claim 1, characterized in that, characterized in that, In S1, the extraction solution is an aqueous hydrochloric acid solution, the concentration of the aqueous hydrochloric acid solution is 0.1 mol / L, and the addition ratio of the honeybee brain tissue sample to the extraction solution is 500 ug / L.

3. The method of claim 1, wherein, In S1, the grinding method is to use an electric grinder with a rotation speed of 11000 RPM to grind for 1 min.

4. The method of claim 1, wherein In S1, the ultrasonic treatment power is 200 W, the frequency is 40 KHz, and the time is 0.5 min-1 min.

5. The method of claim 1, wherein In S2, the liquid chromatography mobile phase gradient elution program is as follows: running for 7 min.

6. The method of claim 1, wherein In S2, the parameters of MS TOP comprise: fragmentation voltage: 90 V; skimmer voltage: 65 V; octupole radio frequency voltage peak value: 750 V.

7. The method of claim 1, wherein, In S2, the MS scanning range is 50 m / z-250 m / z, and the scanning rate is 2 spectra / s.

8. The method of claim 1, wherein, In S2, the target ions are as follows: 。 9. The method of claim 1, wherein, The detection method further comprises: using an aqueous hydrochloric acid containing DHBA as a solvent, preparing histamine, octopamine, dopamine and 5-hydroxytryptamine into different concentrations of biological amine standard solution respectively; performing liquid chromatography-mass spectrometry on the biological amine standard solution, the detection conditions being the same as those of the honeybee brain tissue sample to be tested, taking the peak area of the quantitative ion as the vertical coordinate and the concentration of the corresponding biological amine standard solution as the horizontal coordinate to draw a standard curve; substituting the peak area of the honeybee brain tissue sample to be tested into the standard curve to calculate the concentration of the biological amine in the honeybee brain tissue sample to be tested.

10. The detection method according to claim 9, characterized in that, The linear range, standard curve equation, correlation coefficient, detection limit LOD and quantitative limit LOQ of the detection method for the four biological amines of histamine, octopamine, dopamine and 5-hydroxytryptamine are as follows: 。

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