A method for detecting plant free polyamines by HPLC

Abstract

The application discloses a method for detecting plant free polyamines by HPLC, which can clearly separate the target substances putrescine (Put), spermidine (Spd), thermospermine (T-Spm) and spermine (Spm) in a plant sample. The method has stable retention time of the separated target substances, and the peaks corresponding to the target substances separated from different samples and the standard sample can be well overlapped, data is clear, and the result is reliable. The separation time is shortened by half, the use amount of the mobile phase is halved, the operation cost is greatly reduced, the waste liquid is halved, the recovery cost of the waste liquid is also halved, and the potential pollution to the environment is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection of free polyamines, in particular, to a method for detecting free polyamines in plants by HPLC. BACKGROUND

[0002] Polyamines (PAs) are a class of highly active small molecule aliphatic compounds containing two or more amino groups, which are widely distributed in prokaryotes and eukaryotes. Putrescine (Put), spermidine (Spd) and spermine (Spm) are the most common and early discovered polyamines in plants.

[0003] According to the number of amino groups in the molecular structure, common polyamines can be divided into diamines (i.e. Put), triamines (i.e. Spd) and tetramines (i.e. Spm and T-Spm). Thermospermine (T-Spm) is a structural isomer of Spm, which was first isolated in Thermus thermophilus, and later found to be ubiquitous in higher plants.

[0004] Studies have shown that PAs widely participate in plant life activities in the form of free or small molecules such as cinnamic acid, such as DNA synthesis, cell division, vascular development, pollen development, seed size development, fruit ripening, senescence, stress response, etc. Therefore, accurate determination of polyamine content in plants is crucial in the study of plant growth and development.

[0005] Currently, there are related reports on the determination method of free polyamine content in Arabidopsis thaliana. The HPLC procedure for determining the polyamine content in Arabidopsis thaliana is shown in Table 1 (PMID: 24906355):

[0006] Table 1. Running program for detecting polyamines in Arabidopsis thaliana by HPLC

[0007]

[0008] Currently, acetonitrile and water are mostly used as mobile phases for separating free polyamines in Arabidopsis thaliana, but this method has the disadvantages of long separation time, high cost of mobile phase, large amount of organic waste liquid due to the use of acetonitrile, increased waste liquid recovery cost and environmental pollution. SUMMARY

[0009] The purpose of the present application is to overcome the above-mentioned shortcomings of the prior art and provide a method for detecting free polyamines in plants by HPLC.

[0010] The first object of the present application is to provide a method for sample pretreatment of detecting free polyamines in plants by HPLC.

[0011] A second object of the present application is to provide a method for detecting free plant polyamines by HPLC.

[0012] To achieve the above object, the present application is implemented by the following scheme:

[0013] The new method uses methanol and water as mobile phase, and by constantly adjusting the running program of HPLC, the following optimal methanol and water ratio and the retention time of the target are obtained. The new method shortens the separation time by nearly half, reduces the use of mobile phase by half, greatly reduces the running cost, and reduces the waste liquid recovery cost and the possibility of environmental pollution. The specific running program is shown in Table 2.

[0014] Table 2: Running program for detecting polyamines by HPLC:

[0015]

[0016] Therefore, the present application claims to protect a method for sample pretreatment for detecting free plant polyamines by HPLC, comprising the following steps:

[0017] S1. Grinding the sample to be tested into powder in liquid nitrogen, mixing with perchloric acid aqueous solution, homogenizing, then separating the solid and liquid, taking the supernatant, and removing impurities;

[0018] S2. Mixing the supernatant obtained in step S1 with NaOH solution, then mixing with benzoyl chloride, then mixing with NaCl solution, then mixing with diethyl ether, then separating the solid and liquid, and taking the supernatant;

[0019] S3. Taking the supernatant obtained in step S2, removing the solvent, drying, mixing with methanol, removing impurities, and obtaining sample extraction solution.

[0020] Preferably, in step S1, 500 μL of 5%-10% (v / v) perchloric acid (PCA) aqueous solution is added per gram of sample.

[0021] More preferably, in step S1, 500 μL of 5% (v / v) PCA is added per gram of sample.

[0022] Preferably, in step S2, the concentration of NaOH solution is 2-3 M, the concentration of NaCl solution is 2-3 M, and the volume ratio of NaOH solution, benzoyl chloride, NaCl and diethyl ether is 1-1.5 mL: 10-15 μL: 1-1.5 mL: 2-3 mL.

[0023] More preferably, in step S1, the concentration of NaOH solution is 2 M, the NaCl solution is saturated NaCl solution, and the volume ratio of NaOH solution, benzoyl chloride, saturated NaCl and diethyl ether is 1 mL: 10 μL: 2 mL: 2 mL.

[0024] Preferably, the volume ratio of the supernatant to methanol in step S3 is 5-7:1.

[0025] More preferably, the volume ratio of the supernatant to methanol in step S3 is 6:1.

[0026] The present application also claims a method for detecting free polyamines in plants by HPLC, wherein the sample is pretreated by any of the above methods to obtain a sample extract; and the content of free polyamines in the sample extract is detected by HPLC.

[0027] Preferably, an SB-C18 reverse-phase chromatographic column is used.

[0028] More preferably, the SB-C18 reverse-phase chromatographic column is a ZORBAX SB-C18 reverse-phase chromatographic column.

[0029] Preferably, the HPLC detection uses gradient elution, and the gradient elution is performed according to the following procedure:

[0030] From 0 to 2 min, the volume ratio of water in the mobile phase decreases from 60% to 40%, and the volume ratio of methanol increases from 40% to 60%.

[0031] From 2 to 22 min, the volume ratio of water in the mobile phase decreases from 40% to 30%, and the volume ratio of methanol increases from 60% to 70%.

[0032] From 22 to 24.5 min, the volume ratio of water in the mobile phase decreases from 30% to 0%, and the volume ratio of methanol increases from 70% to 100%.

[0033] From 24.5 to 28 min, the volume ratio of water in the mobile phase increases from 0% to 40%, and the volume ratio of methanol decreases from 100% to 60%.

[0034] From 28 to 35 min, the volume ratio of water in the mobile phase is 40%, and the volume ratio of methanol is 60%.

[0035] Preferably, the column temperature is 25-30°C.

[0036] More preferably, the column temperature is 30°C.

[0037] Preferably, the polyamines are one or more of putrescine, spermidine, thermospermine, and / or spermine.

[0038] Preferably, the plants are tomatoes or rice.

[0039] Compared with the prior art, the present application has the following beneficial effects:

[0040] 1, strong separation ability: can clearly separate the target substances putrescine (Put), spermidine (Spd), thermospermine (T-Spm) and spermine (Spm) in plant samples. Among them, since T-Spm and Spm belong to tetramine isomers, their molecular structures are very similar, and it is also difficult to separate them successfully. Using acetonitrile and water as mobile phase can barely separate the two tetramines, but the two target peaks are still connected to each other, indicating that the separation effect is not ideal. The new method of the present patent can separate T-Spm and Spm more clearly than the currently commonly used acetonitrile and water as mobile phase, and can clearly separate T-Spm and Spm.

[0041] 2, low experimental cost: reduces experimental cost, shortens separation time by half, reduces the amount of mobile phase by half, and greatly reduces operating cost (both the purchase cost of mobile phase and the use cost of HPLC are halved).

[0042] 3, short experimental period: experimental time is shortened by half, saving valuable time for researchers.

[0043] 4, stable operation and reliable data: the retention time of the target substance separated by the method of the present patent is stable, and the target substance separated from different samples can well overlap with the corresponding peak of the standard sample, the data is clear, and the result is reliable.

[0044] 5, environmental protection: more environmentally friendly, operating time is halved, mobile phase usage is halved, waste liquid is halved, waste liquid recovery cost is halved, and potential environmental pollution is reduced. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 The results of the method for detecting different tomato free polyamines by HPLC in Example 2; A is a polyamine standard (containing Put, Spd, T-Spm and Spm four standard substances); B-F are respectively five biological repeat samples of wild type tomato (S. lycopersium L. cv. Micro-Tom).

[0046] Figure 2 The free polyamine content in different tomato leaf samples; A, putrescine (Put); B, spermidine (Spd); C, thermospermine (T-Spm); D, spermine (Spm); WT: wild type tomato, 1-1 and 1-4: two polyamine oxidase mutant tomato lines.

[0047] Figure 3 The detection of free polyamine content in different rice leaf samples; A, standard; B-E, different rice leaf samples. DETAILED DESCRIPTION

[0048] The application will be further described in conjunction with the accompanying drawings and specific embodiments, which are only used to explain the application and are not used to limit the scope of the application. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0049] Example 1: Establishment of a standard curve for HPLC detection of plant free polyamines

[0050] I. Experimental methods

[0051] Prepare a 25 mM putrescine solution and dilute it into a series of gradient standard putrescine solutions with concentrations of 25, 25 / 3, 25 / 9, 25 / 27, and 25 / 81 mM. Perform sample pretreatment and HPLC detection, and take the peak area of the target putrescine at each concentration. Establish a standard curve equation with the peak area as the Y-axis and the concentration as the X-axis.

[0052] 1. Sample pretreatment

[0053] (1) Centrifuge the series of gradient standard putrescine solutions at low temperature (15000g, 30min), carefully pipette the supernatant into a new 2mL centrifuge tube, filter the impurities with a 0.22μm filter membrane to obtain the filtrate, and immediately proceed to the next step or store overnight at -20℃.

[0054] (2) Transfer 1.2mL of the prepared filtrate from the previous step into a 15mL centrifuge tube, add 1mL of NaOH solution (2M) and mix well by inverting several times; then, add 10μL of benzoyl chloride (in a fume hood), vortex for 2min, and stand at room temperature for 20min; add 2mL of saturated NaCl, shake for 15s, then add 2mL of diethyl ether, vortex for 2min; then, centrifuge at low temperature (1500g, 5min), carefully pipette an equal amount of the upper liquid, and record the volume.

[0055] (3) Volatilize the diethyl ether in a metal bath at 50℃, completely dry, then dissolve in 200μL of HPLC-grade methanol, and filter the impurities with a 0.22μm filter membrane.

[0056] (4) Vortex for 5min in a vortex shaker, centrifuge briefly, store temporarily at 4℃, or transfer to the inner cannula of a small brown bottle, and clearly label the sample name.

[0057] 2. HPLC detection

[0058] Use HPLC to detect the content of free polyamines in the sample, perform gradient elution with the running program shown in Table 3. The column temperature is 30℃, and the reverse-phase chromatographic column from Agilent is used, with a column model of (ZORBAX SB-C18 4.6×250mm, PACKING LOT#: B17225, US).

[0059] Table 3: HPLC detection of polyamines running program:

[0060]

[0061] The standard curve equation of spermidine, thermospermine and spermine was made in the same way as the putrescine standard curve equation.

[0062] II. Experimental results

[0063] The standard curve equation obtained is as follows:

[0064] Putrescine: X = (Y peak area + 58.7) / 16.65 (R 2 = 0.9876);

[0065] Spermidine: X = (Y + 66.946) / 18.737 (R 2 = 0.9883);

[0066] Thermospermine: X = (Y + 101.12) / 21.438 (R 2 = 0.9835);

[0067] Spermine: X = (Y + 82.313) / 19.877 (R 2 = 0.9855);

[0068] Where X represents the concentration, Y represents the peak area, and the sample content (μmol·g -1 FW).

[0069] Example 2: A method for detecting free polyamines in tomatoes by HPLC

[0070] I. Experimental method

[0071] 1. Sample collection

[0072] Take the mature leaves of tomatoes grown for 2 months, 0.4 g per sample, 5 replicates, quickly frozen in liquid nitrogen and stored at -80℃.

[0073] 2. Sample pretreatment

[0074] (1) Prepare two sets of clean mortar and a medicine spoon for each sample; sterilized centrifuge tubes 1.5 mL, 2 mL, 15 mL; prepare 5% (v / v) PCA (perchloric acid) aqueous solution, pre-cooled on ice.

[0075] (2) A set of mortar plus liquid nitrogen pre-cooled after putting the sample, quickly grinding into powder, with a spoon (liquid nitrogen pre-cooled) to transfer the powder to the ice pre-cooled mortar (each 0.1 g sample plus 500 μL 5% (v / v) PCA aqueous solution) added 5% (v / v) PCA aqueous solution, grinding into homogenate and then using a pipette (the tip of the gun is cut) to suck into a 2 mL centrifuge tube, and then standing on ice for 1 h.

[0076] (3) Low temperature centrifugation (15000g, 30 min), carefully sucking the supernatant into a new 2 mL centrifuge tube, 0.22 μm filter membrane to remove impurities to obtain the filtrate, and then immediately proceeding to the next step or storing overnight at -20℃.

[0077] (4) Taking 1.2 mL of the sample prepared in the previous step into a 15 mL centrifuge tube, adding 1 mL of NaOH solution (2M), and mixing several times; then, adding 10 μL of benzoyl chloride (in a fume hood), vortexing for 2 min, and standing at room temperature for 20 min; adding 2 mL of saturated NaCl, shaking for 15 s, and then adding 2 mL of diethyl ether, vortexing for 2 min; then, low temperature centrifugation (1500g, 5 min), carefully sucking 1.2 mL of the upper liquid, and recording the volume.

[0078] (5) Metal bath 50℃ volatilize diethyl ether, completely dry, then add HPLC grade methanol 200 μL to dissolve, and use 0.22 μm filter membrane to remove impurities.

[0079] (6) Vortex for 5 min, centrifuge briefly, and store temporarily at 4℃ or transfer to the inner cannula of a small brown bottle, and mark the sample name clearly.

[0080] 3. HPLC detection

[0081] The same as example 1.

[0082] The polyamine content is calculated according to the polyamine standard curve formula established in example 1.

[0083] II. Experimental results

[0084] The results are shown in Figure 1 The method can clearly separate the four free polyamines, putrescine (Put), spermidine (Spd), thermospermine (T-Spm) and spermine (Spm), and the retention time is stable, the peak time of the target substance remains basically unchanged, the data is reliable, and the detection accuracy using the new method for sample detection can reach 1 nmol·g -1 FW (see Figure 2 ).

[0085] Example 3. Influence of HPLC running program on detection of free polyamines in rice

[0086] I. Experimental method

[0087] 1. Sample collection

[0088] The aerial part of the rice seedlings grown for 12 days was taken, 0.4 g per sample, 5 replicates, and stored at -80°C after quick freezing in liquid nitrogen.

[0089] 2. Sample pretreatment

[0090] The same as in Example 2.

[0091] 3. HPLC detection

[0092] The same as in Example 2.

[0093] II. Experimental results

[0094] The results are shown in Table 1. Figure 3 It can be seen that the method can clearly separate the four free polyamines, putrescine (Put), spermidine (Spd), thermospermine (T-Spm) and spermine (Spm), and the retention time is stable, the peak time of the target substance remains basically unchanged, the data is reliable, and the detection accuracy can reach 1 nmol·g -1 FW.

[0095] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. For those skilled in the art, on the basis of the above description and ideas, other different forms of changes or variations can also be made, which do not need or cannot be exhaustively listed here. Any modification, equivalent replacement and improvement made within the spirit and principles of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. A method for detecting free polyamines in plants by HPLC, characterized in that, The sample is pretreated to obtain a sample extract; the content of free polyamines in the sample extract is detected by HPLC; The sample pretreatment method comprises the following steps: S1. grinding the sample to be tested into powder in liquid nitrogen, mixing the powder with an aqueous perchloric acid solution, homogenizing, and then separating the solid and the liquid, taking the supernatant, and removing impurities; S2. mixing the supernatant obtained in step S1 with a NaOH solution, then mixing with benzoyl chloride, then mixing with a NaCl solution, and then mixing with diethyl ether; then separating the solid and the liquid, and taking the supernatant; S3. taking the supernatant obtained in step S2, removing the solvent, mixing with methanol after drying, removing impurities, and obtaining a sample extract; The HPLC detection is performed by gradient elution, and the gradient elution is performed according to the following procedure: from 0 to 2 min, the volume fraction of water in the mobile phase decreases from 60% to 40%, and the volume fraction of methanol increases from 40% to 60%; from 2 to 22 min, the volume fraction of water in the mobile phase decreases from 40% to 30%, and the volume fraction of methanol increases from 60% to 70%; from 22 to 24.5 min, the volume fraction of water in the mobile phase decreases from 30% to 0%, and the volume fraction of methanol increases from 70% to 100%; from 24.5 to 28 min, the volume fraction of water in the mobile phase increases from 0% to 40%, and the volume fraction of methanol decreases from 100% to 60%; and from 28 to 35 min, the volume fraction of water in the mobile phase is 40%, and the volume fraction of methanol is 60%; The polyamines are putrescine, spermidine, thermospermine and spermine; An SB-C18 reverse-phase chromatographic column is used.

2. The method of claim 1, wherein, In step S1, 500 μL of a 5%-10% (v / v) aqueous perchloric acid solution is added per gram of sample.

3. The method of claim 1, wherein, In step S2, the concentration of the NaOH solution is 2-3 M, the concentration of the NaCl solution is 2-3 M, and the volume ratio of the NaOH solution, benzoyl chloride, NaCl and diethyl ether is 1-1.5 mL: 10-15 μL: 1-1.5 mL: 2-3 mL.

4. The method of claim 1, wherein, In step S3, the volume ratio of the supernatant to methanol is 5-7:

1.

5. The method of claim 1, wherein, The SB-C18 reverse-phase chromatographic column is a ZORBAX SB-C18 reverse-phase chromatographic column.

6. The method of claim 1, wherein, The column temperature is 25-30℃.

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