Polyamine and detection method of synthetic pathway substance thereof
阅读说明：本技术 一种多胺及其合成通路物质的检测方法 (Polyamine and detection method of synthetic pathway substance thereof ) 是由 何敬全 王剑锋 刘瑜 盛正平 黄强 钟召赟 黄颖瑜 刘志鹏 于书红 秦欢欢 于 2019-12-12 设计创作，主要内容包括：本发明公开了一种多胺及其合成通路物质的检测方法,包括以下步骤：S1.取45-55mg样本于EP管中,加入500μL提取液,涡旋30秒；然后在一定的功率下进行研磨处理,将研磨后的样品在冰水浴下超声1-10min；重复研磨超声步骤3次,得混合物1；S2.将步骤S1得到的混合物1在-20摄氏度静置50-70min；然后在4摄氏度,12000rpm下离心10-20min,得上清液1；S3.取100μL上清液1于EP管中,加入50μL碳酸钠溶液和50μL 20mg/mL衍生化试剂,涡旋混匀；40摄氏度避光衍生反应1h；S4.将衍生反应后的溶液中加入1％甲酸水溶液,涡旋30s；然后在4摄氏度,12000rpm下离心10-20min；得上清液2,作为待检测液；S5.标准溶液配制；S6.将步骤S4得到的待检测液和步骤S5得到的标准溶液进行UHPLC-MS/MS检测分析,计算多胺的含量。(The invention discloses a method for detecting polyamine and a synthetic pathway substance thereof, which comprises the following steps: s1, putting a 45-55mg sample into an EP tube, adding 500 mu L of extracting solution, and vortexing for 30 seconds; then grinding under certain power, and carrying out ultrasonic treatment on the ground sample for 1-10min in ice-water bath; repeating the grinding and ultrasonic steps for 3 times to obtain a mixture 1; s2, standing the mixture 1 obtained in the step S1 for 50-70min at-20 ℃; centrifuging at 12000rpm at 4 deg.C for 10-20min to obtain supernatant 1; s3, taking 100 mu L of supernatant fluid 1, putting the supernatant fluid into an EP tube, adding 50 mu L of sodium carbonate solution and 50 mu L of 20mg/mL derivatization reagent, and uniformly mixing by vortex; carrying out light-shielding derivatization reaction for 1h at 40 ℃; s4, adding 1% formic acid water solution into the solution after the derivatization reaction, and performing vortex operation for 30 s; then centrifuging for 10-20min at 4 ℃ and 12000 rpm; obtaining supernatant 2 as a liquid to be detected; s5, preparing a standard solution; s6, carrying out UHPLC-MS/MS detection analysis on the solution to be detected obtained in the step S4 and the standard solution obtained in the step S5, and calculating the content of polyamine.)
1. A method for detecting polyamine and a synthetic pathway substance thereof is characterized by comprising the following steps:
s1, putting a 45-55mg sample into an EP tube, adding 500 mu L of extracting solution, and vortexing for 30 seconds; then grinding under certain power, and carrying out ultrasonic treatment on the ground sample for 1-10min in ice-water bath; repeating the grinding and ultrasonic steps for 3 times to obtain a mixture 1;
s2, standing the mixture 1 obtained in the step S1 for 50-70min at-20 ℃; centrifuging at 12000rpm at 4 deg.C for 10-20min to obtain supernatant 1;
s3, taking 100 mu L of supernatant fluid 1, putting the supernatant fluid into an EP tube, adding 50 mu L of sodium carbonate solution and 50 mu L of 20mg/mL derivatization reagent, and uniformly mixing by vortex; carrying out light-shielding derivatization reaction for 1h at 40 ℃;
s4, adding 1% formic acid water solution into the solution after the derivatization reaction, and performing vortex operation for 30 s; then centrifuging for 10-20min at 4 ℃ and 12000 rpm; obtaining supernatant 2 as a liquid to be detected;
s5, preparing a standard solution;
s6, carrying out UHPLC-MS/MS detection analysis on the solution to be detected obtained in the step S4 and the standard solution obtained in the step S5, and calculating the content of polyamine.
2. The detection method according to claim 1, wherein the extraction liquid in step S1 is a mixed solution of acetonitrile, a monohydric alcohol having 1 to 2 carbon atoms, and water, and the volume ratio of acetonitrile, the monohydric alcohol having 1 to 2 carbon atoms, and water is (1 to 3): 2: 1.
3. the detection method according to claim 2, wherein the volume ratio of acetonitrile, monohydric alcohol having 1-2 carbon atoms, and water is 2: 2: 1.
4. the detection method according to claim 3, wherein the extraction solution is a mixed solution of acetonitrile, methanol and water, and the volume ratio of the acetonitrile, the methanol and the water is 2: 2: 1.
5. the detection method of claim 1, wherein the derivatizing reagent in step S3 is dansyl chloride or benzoyl chloride.
6. The detection method of claim 5, wherein the derivatizing agent in step S3 is dansyl chloride.
7. The detection method according to claim 1, wherein in step S6, the liquid chromatography mobile phase a is an ammonium formate/formic acid solution, and the mobile phase B is acetonitrile.
8. The assay of claim 7, wherein the liquid chromatography mobile phase A is 9-11mM ammonium formate/formic acid solution and the mobile phase B is acetonitrile.
9. The detection method according to claim 8, wherein the gradient elution conditions of the liquid chromatography are:
0-0.5 min: mobile phase a 75% → 75%, mobile phase B25% → 25%;
0.5-5.9 min: mobile phase a 75% → 2%, mobile phase B25% → 98%;
5.9-9.4 min: mobile phase a is 2% → 2%, mobile phase B is 98% → 98%;
9.4-9.5 min: mobile phase a is 2% → 75%, mobile phase B is 98% → 25%;
9.5-12 min: mobile phase a was 75% → 75%, and mobile phase B was 25% → 25%.
10. The detection method according to claim 1, wherein in step S6, the ion source parameters are as follows: capillary voltage +4000/-3500V, nzzle voltage +500/-500V, gas (N)2)temperature＝300℃，gas(N2)flow＝5L/min，sheath gas(N2)temperature＝250℃，sheath gas flow＝11L/min，nebulizer＝45psi。
Technical Field
The application belongs to the technical field of polyamine detection, and particularly relates to a method for detecting polyamine and a synthetic pathway substance thereof.
Background
Polyamines are compounds containing two or more amino groups, the synthetic raw materials of the polyamines are mainly ornithine and arginine, and the key enzymes are ornithine decarboxylase and arginine decarboxylase. Polyamines, which are also most commonly important physiological functions, are putrescine, cadaverine, spermidine, spermine, etc. polyamines have the effect of promoting the growth of certain tissues and also play an important role in the normal maintenance of membranes.
The polyamine has polycation property, can be combined with membrane phospholipid with negative charge and enzyme protein, is an important regulating substance for plant growth and development, participates in important physiological processes of plant growth and development, fruit maturation and aging, sex differentiation, delayed aging, adversity stress and the like, and also participates in biochemical processes, including DNA replication and transcription, membrane stabilization, RNA and protein translation and the like. Under the environmental stress, a large amount of polyamine substances are accumulated in plants, and have important effect on stabilizing the conformation of macromolecular substances such as cell membranes, nucleic acids, proteins and the like, so that the polyamine substances are considered to be substances which have close relation with plant adversity physiology; regarding the action mechanism of polyamines, it is generally considered that they act as a mediator of plant hormones, acting like cAMP as a "second messenger", and therefore it is very interesting to detect polyamine contents in plants.
Disclosure of Invention
In order to solve the above technical problems, a first aspect of the present invention provides a method for detecting polyamine and its synthetic pathway material, comprising the steps of:
s1, putting a 45-55mg sample into an EP tube, adding 500 mu L of extracting solution, and vortexing for 30 seconds; then grinding under certain power, and carrying out ultrasonic treatment on the ground sample for 1-10min in ice-water bath; repeating the grinding and ultrasonic steps for 3 times to obtain a
s2, standing the
s3, taking 100 mu L of
s4, adding 1% formic acid water solution into the solution after the derivatization reaction, and performing vortex operation for 30 s; then centrifuging for 10-20min at 4 ℃ and 12000 rpm; obtaining
s5, preparing a standard solution;
s6, carrying out UHPLC-MS/MS detection analysis on the solution to be detected obtained in the step S4 and the standard solution obtained in the step S5, and calculating the content of polyamine.
In a preferred embodiment, the extraction liquid in step S1 is a mixed solution of acetonitrile, a monohydric alcohol having 1 to 2 carbon atoms, and water, wherein the volume ratio of acetonitrile to monohydric alcohol having 1 to 2 carbon atoms to water is (1 to 3): 2: 1.
in a preferred embodiment, the volume ratio of acetonitrile to a monohydric alcohol having 1 to 2 carbon atoms to water is 2: 2: 1.
as a preferable technical scheme, the extracting solution is a mixed solution of acetonitrile, methanol and water, and the volume ratio of the acetonitrile to the methanol to the water is 2: 2: 1.
as a preferred technical solution, in the step S3, the derivatization reagent is dansyl chloride or benzoyl chloride.
As a preferred technical solution, in the step S3, the derivatizing agent is dansyl chloride.
In a preferred embodiment, in step S6, the liquid chromatography mobile phase a is an ammonium formate/formic acid solution, and the mobile phase B is acetonitrile.
As a preferable technical scheme, the mobile phase A of the liquid chromatogram is 9-11mM ammonium formate/formic acid solution, and the mobile phase B is acetonitrile.
As a preferred technical solution, the gradient elution conditions of the liquid chromatography are as follows:
0-0.5 min: mobile phase a 75% → 75%, mobile phase B25% → 25%;
0.5-5.9 min: mobile phase a 75% → 2%, mobile phase B25% → 98%;
5.9-9.4 min: mobile phase a is 2% → 2%, mobile phase B is 98% → 98%;
9.4-9.5 min: mobile phase a is 2% → 75%, mobile phase B is 98% → 25%;
9.5-12 min: mobile phase a was 75% → 75%, and mobile phase B was 25% → 25%.
As a preferred technical solution, in step S6, the ion source parameters of the mass spectrum are as follows: capillaryvoltage +4000/-3500V, nzzle voltage +500/-500V, gas (N)2)temperature＝300℃，gas(N2)flow＝5L/min，sheath gas(N2)temperature＝250℃，sheath gas flow＝11L/min，nebulizer＝45psi。
Has the advantages that: the experiment optimizes the pretreatment conditions of the sample, and adopts UHPLC-MS/MS for the first time to establish a method for extracting 9 kinds of biogenic amines in citrus root systems and a method for accurately and quantitatively detecting the biogenic amines. Under the established chromatographic conditions, the sample can be detected within 9 minutes. The method has good reproducibility, simplicity, convenience, rapidness, accuracy and reliability, and is suitable for rapid detection of large-batch samples.
Drawings
FIG. 1 is an Extracted Ion Chromatogram (EICs) of citrus root system according to example 1 of the present invention.
FIG. 2 is an Extracted Ion Chromatogram (EICs) of a standard solution of example 1 of the present invention.
Description of the symbols: 1-herring spermine; 2-arginine; 3-putrescine; 4-cadaverine; 5-ornithine; 6-spermidine; 7-S-adenosylmethionine; 8-spermine.
Detailed Description
In order to solve the above problems, the present invention provides a method for detecting polyamine and its synthetic pathway material, comprising the steps of:
s1, putting a 45-55mg sample into an EP tube, adding 500 mu L of extracting solution, and vortexing for 30 seconds; then grinding under certain power, and carrying out ultrasonic treatment on the ground sample for 1-10min in ice-water bath; repeating the grinding and ultrasonic steps for 3 times to obtain a
s2, standing the
s3, taking 100 mu L of
s4, adding 1% formic acid water solution into the solution after the derivatization reaction, and performing vortex operation for 30 s; then centrifuging for 10-20min at 4 ℃ and 12000 rpm; obtaining
s5, preparing a standard solution;
s6, carrying out UHPLC-MS/MS detection analysis on the solution to be detected obtained in the step S4 and the standard solution obtained in the step S5, and calculating the content of polyamine.
Wherein the polyamine and its synthetic pathway substance are extracted from root system of Mandarin orange, such as putrescine, spermidine, spermine, cadaverine, ornithine, arginine, S-adenosylmethionine, and herring spermine.
The Citrus (Citrus reticulata Blanco) belongs to the genus Rutaceae.
In a preferred embodiment, the extraction liquid in step S1 is a mixed solution of acetonitrile, a monohydric alcohol having 1 to 2 carbon atoms, and water, and the volume ratio of acetonitrile to monohydric alcohol having 1 to 2 carbon atoms to water is (1 to 3): 2: 1.
preferably, the volume ratio of the acetonitrile to the monohydric alcohol having 1-2 carbon atoms to the water is 2: 2: 1.
preferably, the extracting solution is a mixed solution of acetonitrile, methanol and water, and the volume ratio of the acetonitrile to the methanol to the water is (1-3): 2: 1.
more preferably, the extracting solution is a mixed solution of acetonitrile, methanol and water, and the volume ratio of the acetonitrile to the methanol to the water is 2: 2: 1.
the pretreatment process of the sample is the whole process of extracting the target compound. The extraction is a method for separating the target compound with high purity by using physical, chemical and biochemical principles and methods under the condition of not damaging the structure and the function of the target compound, and is a strict, delicate and complex process.
In the measurement of the content of polyamine in citrus root systems, the polyamine needs to be extracted because citrus root systems contain various substances such as saccharides, proteins, amino acids and the like, and the currently used extracting agent is generally water and an acidic solution (such as trichloroacetic acid, hydrochloric acid and the like), but the results of extraction detection using an acidic solution in the present application are not ideal, and the applicant found that the extracting solution is a mixed solution of acetonitrile, a monohydric alcohol having 1 to 2 carbon atoms and water, and the volume ratio of the acetonitrile, the monohydric alcohol having 1 to 3 carbon atoms and water is 2: 2: 1, the detection accuracy can be improved, and the detection accuracy is improved presumably because different solvents form extracting solutions with proper polarity, carbon atoms and nitrogen atoms in acetonitrile are connected through triple bonds, and hydroxyl groups, methyl groups and water in methanol cooperate to promote the precipitation of putrescine, spermidine, spermine, cadaverine, ornithine, arginine, S-adenosylmethionine and herring spermine and prevent the precipitation of other amino acids, uronic acid and the like.
In step S1, the grinding power is 45 Hz.
As a preferred embodiment, the step S1 is specifically: taking 50mg of a sample in an EP tube, adding 500 mu L of the extracting solution, and vortexing for 30 seconds; then grinding for 4min at 45Hz, and carrying out ultrasonic treatment on the ground sample for 5min in an ice water bath; repeating the grinding and ultrasonic steps for 3 times to obtain a
as a preferred embodiment, the step S2 is specifically: standing the
As a preferred embodiment, the derivatizing agent in step S3 is dansyl chloride or benzoyl chloride.
The derivatization reagent in the step S3 is dansyl chloride or benzoyl chloride.
Preferably, the derivatizing agent in step S3 is dansyl chloride.
The sodium carbonate solution in the step S3 is a saturated sodium carbonate solution.
The 1% aqueous formic acid solution in step S4 is referred to as a volume percentage.
The structure differences of putrescine, cadaverine, spermidine, spermine and the like are few, and the differences are mainly reflected on one or more aminopropyl groups or aminoethyl groups, dansyl chloride is used as a derivatization reagent, the dansyl chloride mainly reacts with primary amine groups under the condition of sodium carbonate, and the existence of the residual secondary amine groups enables dansyl sulfonate products to generate different degrees of binding force with 1.8-micron high-strength silica gel particles on a Waters ACQUITY UHPLC HSS T3 chromatographic column, so that good separation can be realized, and the accuracy and the sensitivity of detection are improved. And we choose BEH-C18When the method is used for a column, the retention time of putrescine, spermidine and cadaverine is 5.01 min, 16.91 min and 9.03min respectively, dansyl chloride is selected to be combined with an ACQUITY UHPLC HSS T3 chromatographic column under the same chromatographic condition, the retention time of putrescine, spermidine and cadaverine is 6.20 min, 7.35 min and 6.71min respectively, the separation of polyamine can be realized within 10min, and the separation degree is good. Benzoyl chloride is selected as a derivatization reagent, so that the detection effect is poor, probably because benzoyl chloride and polyamine react to connect benzoyl groups on primary and secondary amino groups, and the benzoyl groups cannot be well separated by ultraviolet detection.
S5, preparing standard solution
The method comprises the following specific steps:
(1) accurately weighing a corresponding amount of standard substance into a 10mL volumetric flask, adding a solvent (acetonitrile: methanol: water: 2: 1) into the volumetric flask, and preparing into 10mmol/L standard substance stock solutions respectively.
(2) Then, a corresponding amount of the standard substance stock solution is taken and placed in a 10mL volumetric flask to prepare a mixed standard solution. And adding a solvent (acetonitrile: methanol: water: 2: 1) to sequentially dilute the mixed standard solution to obtain a series of standard solutions, wherein the concentration of the series of polyamine standard solutions is shown in table 1 and the unit is nmol/L.
(3) And performing derivatization treatment on the obtained series of standard solutions according to the methods in the steps S3 and S4, and performing on-machine detection after the derivatization treatment to obtain a standard curve equation.
TABLE 1 concentration of polyamine series of standards
S6, detecting the machine
The conditions of the UHPLC are as follows: the target compound was chromatographed using an Agilent 1290Infinity IIseries (Agilent technologies) ultra performance liquid chromatograph on a Waters ACQUITY UHPLC HSS T3 (100X 2.1mm,1.8 μm, Waters) liquid chromatography column.
The liquid chromatography mobile phase A is 10mM ammonium formate/formic acid solution, and the mobile phase B is acetonitrile;
the gradient elution conditions of the liquid chromatogram are as follows:
0-0.5 min: mobile phase a 75% → 75%, mobile phase B25% → 25%;
0.5-5.9 min: mobile phase a 75% → 2%, mobile phase B25% → 98%;
5.9-9.4 min: mobile phase a is 2% → 2%, mobile phase B is 98% → 98%;
9.4-9.5 min: mobile phase a is 2% → 75%, mobile phase B is 98% → 25%;
9.5-12 min: mobile phase a was 75% → 75%, and mobile phase B was 25% → 25%.
The 10mM ammonium formate/formic acid solution shows a concentration of ammonium formate in formic acid of 10 mM.
The temperature of the column oven was 35 degrees Celsius, the sample tray was set to 4 degrees Celsius, the sample introduction volume was 1 μ L, and the flow rate was 0.4 mL/min.
The structure differences of putrescine, cadaverine, spermidine, spermine and the like are few, and the differences are mainly reflected on one or more aminopropyl groups or aminoethyl groups, dansyl chloride is used as a derivatization reagent, the dansyl chloride mainly reacts with primary amine groups under the condition of sodium carbonate, and the existence of the residual secondary amine groups enables dansyl sulfonate products to generate different degrees of binding force with 1.8-micron high-strength silica gel particles on a Waters ACQUITY UHPLC HSS T3 chromatographic column, so that good separation can be realized, and the accuracy and the sensitivity of detection are improved. And we choose BEH-C18When the method is used for a column, the retention time of putrescine, spermidine and cadaverine is 5.01 min, 16.91 min and 9.03min respectively, dansyl chloride is selected to be combined with an ACQUITY UHPLC HSS T3 chromatographic column under the same chromatographic condition, the retention time of putrescine, spermidine and cadaverine is 6.20 min, 7.35 min and 6.71min respectively, the separation of polyamine can be realized within 9min, and the separation degree is good. Benzoyl chloride is selected as a derivatization reagent, so that the detection effect is poor, probably because benzoyl chloride and polyamine react to connect benzoyl groups on primary and secondary amino groups, and good separation cannot be realized.
The applicant has also found that the accuracy, sensitivity, etc. of detection can be improved when the mobile phase a of the liquid chromatography is 10mM ammonium formate/formic acid solution and the mobile phase B is acetonitrile, and elution is carried out in different ratios. The guess is that the mixed solution of ammonium formate and formic acid can adjust the contact time and the combination degree of acetonitrile and the packed particles of the chromatographic column in different time periods, and the peak emergence of part of target compounds in different time periods can be realized by adjusting the retention time through the acetonitrile; achieving good separation effect. When the content of acetonitrile is high, the separation time can be shortened, but the peak is not completely generated, and the components cannot be completely separated.
The mass spectrum conditions are as follows: mass spectrometry was performed in the Multiple Reaction Monitoring (MRM) mode using an Agilent 6460 triple quadrupole mass spectrometer equipped with an AJS-ESI ion source.
The ion source parameters were as follows: capillary voltage +4000/-3500V, nzzle voltage +500/-500V, gas (N)2)temperature＝300℃，gas(N2)flow＝5L/min，sheath gas(N2)temperature＝250℃，sheath gas flow＝11L/min，nebulizer＝45psi。
Before UHPLC-MS/MS analysis is carried out, a target compound standard stock solution is introduced into a mass spectrum, a plurality of parent ion-daughter ion pairs (transitions) with the highest signal intensity are selected for each target compound, the MRM parameters of the parent ion-daughter ion pairs are optimized, the ion pair with the best response is selected for quantitative analysis, other ion pairs are used for qualitative analysis of the target compound, and the specific parameters are shown in Table 2.
In this project, all mass spectrometric data acquisition and target compound quantification work was done by AgilentMassHunterWork Station Software (B.08.00, Agilent Technologies).
TABLE 2
The applicant finds that the selection of the mobile phase also considers the ionization of the compound besides the separation effect of the chromatography, and because the electrospray ionization, positive and negative ion modes are selected in the application, the addition of the mixed solution of ammonium formate and formic acid in the mobile phase is beneficial to the ionization of the target compound and the improvement of the chromatographic peak.
The present invention will be specifically described below by way of examples. It should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and that the insubstantial modifications and adaptations of the present invention by those skilled in the art based on the above disclosure are still within the scope of the present invention.
In addition, the starting materials used are all commercially available, unless otherwise specified.