Process for detecting biogenic amine of plant group by liquid chromatography-mass spectrometry

Abstract

The invention relates to the field of biological detection, and discloses a process for detecting plant group biogenic amine by liquid chromatography-mass spectrometry, which comprises the following detection steps: mobile phase configuration, biogenic amine extraction and chromatographic instrument and mass spectrometer reading calculation, the mobile phase is divided into an organic phase and an inorganic phase, the organic phase is 100% acetonitrile, the inorganic phase is 100% acetonitrile, and the inorganic phase is 100% acetonitrile. The preparation of the inorganic phase comprises the following specific steps: taking a 0.1% formic acid aqueous solution, taking 900mL of ultrapure water, adding into a 1L volumetric flask, adding 1mL of formic acid, performing ultrapure constant volume to 1L, and reversing and uniformly mixing. According to the process for detecting the biogenic amine of the plant group by using the liquid chromatography-mass spectrometry, a sample extraction method and loading conditions are optimized, so that a large batch of plant samples can be quickly treated, the biogenic amine of the plant group can be simultaneously detected, and the plant samples are quickly treated through a relatively simple plant pretreatment method; and the contents of spermine, spermidine, putrescine and cadaverine in a sample are rapidly and accurately detected by a liquid chromatography-mass spectrometry method, so that the detection efficiency is greatly improved.

Description

Technical Field

[0001] The invention relates to the field of biological detection, and in particular to a process for detecting plant biogenic amines by liquid chromatography-mass spectrometry. Background Art

[0002] Biogenic amines are a class of biologically active nitrogen-containing low molecular weight organic compounds that are commonly found in animals, plants and food.

[0003] Biogenic amine compounds themselves lack chromophores and have neither ultraviolet absorption nor fluorescence and electrochemical activity, making conventional analysis and determination difficult. There are many extraction and detection methods for plant biogenic amines, but each of the existing methods has its own shortcomings, such as cumbersome pre-treatment cycles, the need for derivatization, low sensitivity, low recovery rate and other technical difficulties.

[0004] Therefore, there is an urgent need for a process that is simple and can be carried out in batches to detect biogenic amines in plant tissues to solve the above problems. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] In view of the deficiencies in the prior art, the present invention provides a process for detecting biogenic amines in plant tissues using liquid chromatography-mass spectrometry, which has the advantages of being able to quickly process large quantities of plant samples and solves the problems in the above-mentioned background technology.

[0007] (II) Technical solution

[0008] In order to achieve the above-mentioned purpose of being able to quickly process large quantities of plant samples, the present invention provides the following technical solution: a process for detecting biogenic amines in plant groups using liquid chromatography-mass spectrometry, comprising the following detection steps: mobile phase configuration, biogenic amine extraction, and chromatograph and mass spectrometer reading calculation.

[0009] Preferably, the mobile phase is divided into an organic phase and an inorganic phase, and the organic phase is 100% acetonitrile.

[0010] Preferably, the inorganic phase configuration comprises the following specific steps:

[0011] Take 0.1% formic acid aqueous solution, take 900mL of ultrapure water and add it to a 1L volumetric flask, add 1mL of formic acid, make up to 1L with ultrapure solution, and mix by inversion.

[0012] Preferably, the biogenic amine extraction comprises the following specific steps:

[0013] Step a1: Grind the plant sample into powder in liquid nitrogen, weigh 0.5 g of the crushed sample into a test tube, and then add 4 mL of 20% acetonitrile solution;

[0014] Step a2: Ultrasonic extraction in an ice water bath for 30 min;

[0015] Step a3: Centrifuge at 10,000 g for 3 min at a controlled temperature of 4°C, take the supernatant, and extract the precipitate again with 4 mL of 20% acetonitrile;

[0016] Step a4: Combine the two supernatants obtained in step a3 and dilute to 10 mL with 100% acetonitrile solution;

[0017] Step a5: Mix the fixed volume extract, take 1 mL of the supernatant, add 100 μL of formic acid, filter through a 0.22 μm organic phase filter, and test on an analyzer.

[0018] Preferably, the chromatograph liquid phase conditions are:

[0019] Chromatographic liquid phase conditions:

[0020] Chromatographic column: Waters UPLC HSS T3 3.0×100mm 1.8μm;

[0021] Column temperature: 35°C;

[0022] Mobile phase: A:B = (water / 0.1% formic acid): (acetonitrile);

[0023] Flow rate: 0.3 mL / min;

[0024] Injection volume: 2 μl.

[0025] Preferably, the mass spectrometer mass spectrometry parameters are:

[0026] Ionization mode: ESI positive ion mode;

[0027] Scan type: MRM;

[0028] Curtain gas: 45psi;

[0029] Spray voltage: +4000v;

[0030] Atomizing gas pressure: 65psi;

[0031] Auxiliary gas pressure: 70psi;

[0032] Atomization temperature: 350℃.

[0033] Preferably, the sample solution is injected into a liquid chromatography-mass spectrometer to obtain a corresponding chromatographic peak response value, the concentration of polyamine in the test solution is obtained according to a standard curve, and the polyamine content is calculated using the following formula:

[0034] Xii=pi*VV / m

[0035] Where:

[0036] Xi is the polyamine content in the sample, in nanograms per gram (ng / g);

[0037] ρ i is the concentration of the polyamine solution obtained from the standard curve, in nanograms per milliliter (ng / mL);

[0038] V is the final volume of the sample solution, in milliliters (mL);

[0039] m is the mass of the sample weighed, in grams (g).

[0040] (III) Beneficial effects

[0041] Compared with the prior art, the present invention provides a process for detecting biogenic amines in plant tissues by liquid chromatography-mass spectrometry, which has the following beneficial effects:

[0042] The process of detecting plant biogenic amines by liquid chromatography-mass spectrometry can rapidly process large quantities of plant samples and simultaneously detect plant biogenic amines by optimizing sample extraction methods and machine conditions. Plant samples can be rapidly processed by a relatively simple plant pretreatment method, and the contents of spermine, spermidine, putrescine, and cadaverine in the samples can be rapidly and accurately detected by liquid chromatography-mass spectrometry. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] Example 1

[0045] A preferred embodiment of a process for detecting biogenic amines in plant groups using liquid chromatography-mass spectrometry provided by the present invention is as follows: A process for detecting biogenic amines in plant groups using liquid chromatography-mass spectrometry includes the following detection steps: mobile phase configuration, biogenic amine extraction, and chromatograph and mass spectrometer reading calculation.

[0046] In this embodiment, the mobile phase is divided into an organic phase and an inorganic phase, and the organic phase is 100% acetonitrile.

[0047] Example 2

[0048] Based on Example 1, a preferred embodiment of a process for detecting biogenic amines in plant tissues by liquid chromatography-mass spectrometry provided by the present invention is as follows: the inorganic phase configuration includes the following specific steps:

[0049] Take 0.1% formic acid aqueous solution, take 900mL of ultrapure water and add it to a 1L volumetric flask, add 1mL of formic acid, make up to 1L with ultrapure solution, and mix by inversion.

[0050] In this embodiment, the biogenic amine extraction comprises the following specific steps:

[0051] Step a1: Grind the plant sample into powder in liquid nitrogen, weigh 0.5 g of the crushed sample into a test tube, and then add 4 mL of 20% acetonitrile solution;

[0052] The sample specimen obtained by grinding the sample under liquid nitrogen can terminate all biological reactions inside and outside the cell, such as enzyme degradation reactions, etc. At the same time, it can also increase the overall brittleness of the sample, make the grinding faster, and liquid nitrogen grinding can reduce the heat loss on the sample surface, thereby better maintaining the authenticity of the sample.

[0053] Step a2: Ultrasonic extraction in an ice water bath for 30 min;

[0054] Step a3: Centrifuge at 10,000 g for 3 min at a controlled temperature of 4°C, take the supernatant, and extract the precipitate again with 4 mL of 20% acetonitrile;

[0055] Step a4: Combine the two supernatants obtained in step a3 and dilute to 10 mL with 100% acetonitrile solution;

[0056] Step a5: Mix the fixed volume extract, take 1 mL of the supernatant, add 100 μL of formic acid, filter through a 0.22 μm organic phase filter, and test on an analyzer.

[0057] Further, the chromatograph liquid phase conditions are:

[0058] Chromatographic liquid phase conditions:

[0059] Chromatographic column: Waters UPLC HSS T3 3.0×100mm 1.8μm;

[0060] Column temperature: 35°C;

[0061] Mobile phase: A:B = (water / 0.1% formic acid): (acetonitrile);

[0062] Flow rate: 0.3 mL / min;

[0063] Injection volume: 2 μl;

[0064] The liquid phase gradient parameters are as follows:

[0065]

[0066] Furthermore, the mass spectrometer parameters are:

[0067] Ionization mode: ESI positive ion mode;

[0068] Scan type: MRM;

[0069] Curtain gas: 45psi;

[0070] Spray voltage: +4000v;

[0071] Atomizing gas pressure: 65psi;

[0072] Auxiliary gas pressure: 70psi;

[0073] Atomization temperature: 350℃;

[0074] Among them, the protonation selective reaction detection conditions are as follows:

[0075] Substance name polarity Precursor ion (m / z) Product ion (m / z) Declustering voltage(V) Collision energy (V) Putrescine + 89 72.1* / 30.1 30 8 / 20 Spermine + 203.2 112.1* / 84.1 90 18 / 34 Spermidine + 146.1 112.1* / 72.1 80 14 / 14 Cadaverine + 103.1 86.1* / 41.1 70 8 / 24

[0076] In addition, the sample solution is injected into a liquid chromatography-mass spectrometer to obtain the corresponding chromatographic peak response value, the concentration of the polyamine in the test solution is obtained according to the standard curve, and the polyamine content is calculated using the following formula:

[0077] Xi=pi*V / m

[0078] Where:

[0079] Xi is the polyamine content in the sample, in nanograms per gram (ng / g);

[0080] ρ i is the concentration of the polyamine solution obtained from the standard curve, in nanograms per milliliter (ng / mL);

[0081] V is the final volume of the sample solution, in milliliters (mL);

[0082] m is the mass of the sample weighed, in grams (g).

[0083] In summary, this method can quickly process large quantities of plant samples and simultaneously detect biogenic amines in plant groups by optimizing the sample extraction method and machine conditions.

[0084] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0085] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A process for detecting biogenic amines in plants using liquid chromatography-mass spectrometry, characterized in that: The following detection steps are included: mobile phase preparation, biogenic amine extraction, and calculation of chromatograph and mass spectrometer readings.

2. A process for detecting biogenic amines in plants by liquid chromatography-mass spectrometry according to claim 1, characterized in that: The mobile phase is divided into an organic phase and an inorganic phase, and the organic phase is 100% acetonitrile.

3. The process for detecting biogenic amines in plants by liquid chromatography-mass spectrometry according to claim 1, characterized in that: The inorganic phase configuration comprises the following specific steps: Take 0.1% formic acid aqueous solution, take 900mL of ultrapure water and add it to a 1L volumetric flask, add 1mL of formic acid, make up to 1L with ultrapure solution, and mix by inversion.

4. The process for detecting biogenic amines in plants by liquid chromatography-mass spectrometry according to claim 1, characterized in that: The biogenic amine extraction comprises the following specific steps: Step a1: Grind the plant sample into powder in liquid nitrogen, weigh 0.5 g of the crushed sample into a test tube, and then add 4 mL of 20% acetonitrile solution; Step a2: Ultrasonic extraction in an ice water bath for 30 min; Step a3: Centrifuge at 10,000 g for 3 min at a controlled temperature of 4°C, take the supernatant, and extract the precipitate again with 4 mL of 20% acetonitrile; Step a4: Combine the two supernatants obtained in step a3 and dilute to 10 mL with 100% acetonitrile solution; Step a5: Mix the fixed volume extract, take 1 mL of the supernatant, add 100 μL of formic acid, filter through a 0.22 μm organic phase filter, and test on an analyzer.

5. The process for detecting biogenic amines in plants by liquid chromatography-mass spectrometry according to claim 1, characterized in that: The chromatograph liquid phase conditions are: Chromatographic liquid phase conditions: Chromatographic column: Waters UPLC HSS T3 3.0×100mm 1.8μm; Column temperature: 35 °C; Mobile phase: A:B = (water / 0.1% formic acid): (acetonitrile); Flow rate: 0.3 mL / min; Injection volume: 2 μ l.

6. The process for detecting biogenic amines in plants by liquid chromatography-mass spectrometry according to claim 1, characterized in that: The mass spectrometer mass spectrometry parameters are: Ionization mode: ESI positive ion mode; Scan type: MRM; Curtain gas: 45psi; Spray voltage: +4000v; Atomizing gas pressure: 65psi; Auxiliary gas pressure: 70psi; Atomization temperature: 350℃.

7. The process for detecting biogenic amines in plants by liquid chromatography-mass spectrometry according to claim 1, characterized in that: The sample solution is injected into a liquid chromatography-mass spectrometer to obtain the corresponding chromatographic peak response value, the concentration of polyamine in the test solution is obtained according to the standard curve, and the polyamine content is calculated using the following formula: Xi=pi*V / m Where: Xi is the polyamine content in the sample, in nanograms per gram (ng / g); ρ iThe concentration of the polyamine solution obtained from the standard curve, in nanograms per milliliter (ng / mL); V is the final volume of the sample solution, in milliliters (mL); m is the mass of the sample weighed, in grams (g).

Images