Rapid quantitative detection method of hypoxanthine nucleosides in plant root exudates

Abstract

The application discloses a kind of rapid quantitative detection methods of plant root exudates hypoxanthine nucleoside, comprising the following steps: first, the plant root exudates powder is prepared;Second, the plant root exudates powder is dissolved in the extraction solvent obtained by mixing methanol and water with a volume ratio of 1:4, to obtain the solution to be tested;Then, the UPLC-MS / MS method is used to detect the solution to be tested, if the solution to be tested contains hypoxanthine nucleoside, the peak area corresponding to hypoxanthine nucleoside is obtained;Finally, the standard curve is combined to realize the quantitative detection of plant root exudates hypoxanthine.The detection method provided by the application can rapidly and quantitatively detect hypoxanthine nucleoside in plant root exudates, has better peak type, better accuracy and repeatability, and can be used for rapid detection of large-scale samples.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of compound detection, and particularly relates to a rapid quantitative detection method of hypoxanthine in plant root exudates. BACKGROUND

[0002] Plant root exudates contain a large number of small molecule compounds, which play an important role in plant growth and development and mutual recognition between plants. Among them, hypoxanthine is an important plant growth regulator, and the determination of its absolute content plays an important role in the study of plant growth regulation and individual recognition mechanism. However, there is no rapid quantitative detection method for hypoxanthine at present. Therefore, the rapid quantitative detection of hypoxanthine from plant root exudates has important methodological significance for the study of chemical signal recognition of plant interaction. SUMMARY

[0003] The present application relates to the technical field of compound detection, and particularly relates to a rapid quantitative detection method of hypoxanthine in plant root exudates.

[0004] In a first aspect, the present application provides a rapid quantitative detection method of hypoxanthine in plant root exudates, comprising:

[0005] S1, collecting plants in good growth condition, washing the plant roots clean and then transferring them to a light-tight sterilized culture tube containing deionized water for water culture, keeping natural light and day-night cycle, collecting the liquid in the light-tight sterilized culture tube after the water culture is completed, and obtaining a plant root exudate solution; filtering and freeze-drying the plant root exudate solution to obtain a plant root exudate powder;

[0006] S2, dissolving the plant root exudate powder in an extraction solvent to obtain a detection solution, wherein the extraction solvent is obtained by mixing methanol and water at a volume ratio of 1:4;

[0007] S3, detecting the detection solution by using UPLC-MS / MS method, and if the detection solution contains hypoxanthine, obtaining the peak area corresponding to the hypoxanthine;

[0008] S4, dissolving hypoxanthine standard into the extraction solvent to prepare a series of standard samples with gradient concentrations, detecting the standard samples by using the same UPLC-MS / MS method, and constructing a standard curve; substituting the peak area of the hypoxanthine obtained in S3 into the standard curve to obtain the concentration of the hypoxanthine in the plant root exudate, and completing the quantitative detection of the hypoxanthine in the plant root exudate.

[0009] The method as described above, in the UPLC-MS / MS method, the chromatographic column is a reversed-phase HPLC chromatographic column; the mobile phase A is a deionized water solution containing 0.1% formic acid, and the mobile phase B is acetonitrile; the flow rate is 0.3 ml / min; the column temperature is 40 DEG C; and gradient elution conditions are used for elution, and the gradient elution program comprises: 0 min, the mobile phase A is 95%, and the mobile phase B is 5%; 2 min, the mobile phase A is 95%, and the mobile phase B is 5%; 4 min, the mobile phase A is 10%, and the mobile phase B is 90%; 5.5 min, the mobile phase A is 10%, and the mobile phase B is 90%; 6 min, the mobile phase A is 95%, and the mobile phase B is 5%; and 8 min, the mobile phase A is 95%, and the mobile phase B is 5%.

[0010] The method as described above, in the UPLC-MS / MS method, the mass spectrometry conditions are as follows: an ESI source is used, data collection is carried out in a positive ion mode, the voltage is 5500 V, and the ion source temperature is 500 DEG C.

[0011] The method as described above, the plant is sunflower.

[0012] The method as described above, in S1, the hydroponic time is at least 48 h.

[0013] The method as described above, based on per milligram of plant root exudate powder, the volume of the extraction solvent is 15 ul.

[0014] The method as described above, after the plant root exudate powder is mixed with the extraction solvent, vortex oscillation is carried out for 5 min; after the oscillation is finished, the mixture is centrifuged at a speed of 12000 rpm for 15 min, to obtain a solution to be detected.

[0015] The method as described above, the above centrifugation is carried out at 4 DEG C, so that the compounds in the plant root exudate powder are prevented from changing in a high-temperature environment caused by centrifugation.

[0016] The method as described above, in S4, the linear equation of the standard curve is y=2.26309*e 6 *x+8213.76924, x is the concentration of hypoxanthine, and y is the peak area corresponding to the hypoxanthine.

[0017] The method provided by the application can effectively and rapidly detect the hypoxanthine in the plant root exudate; the mass spectrometry peak type is better, the fitting degree is high, the quantitative accuracy is good; the total elution time is 8 min, the time consumption is short; the linear range of the standard curve for quantification is large, and the method can be suitable for samples with a large concentration fluctuation range; the repeatability is good, and the method can be used for detection of large-scale samples; the method provides a reliable and rapid quantitative method for quantitative research on the hypoxanthine excreted by the plant root system, and fills the blank of the quantitative detection method for the hypoxanthine excreted by the plant root system. Attached Figure Description

[0018] Figure 1 The mass spectrometry results of the hypoxanthine nucleoside standards obtained by dissolving them in different extraction solvents are shown. Among them, the extraction solvent used in a is a mixture of methanol and water at a volume ratio of 1:4; the extraction solvent used in b is a mixture of methanol and water at a volume ratio of 4:1; and the extraction solvent used in c is a mixture of methanol, water, and isopropanol at a volume ratio of 1:1:1. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, embodiments of this invention, and should not be construed as limiting the invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. In the description of this invention, it should be understood that the terminology used is for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0021] Example 1: Plotting the standard curve, and verifying its linearity and precision.

[0022] A hypoxanthine nucleoside (CAS No. 58-63-9) standard was dissolved in an extraction solvent (prepared by mixing methanol and water at a volume ratio of 1:4) to prepare a hypoxanthine nucleoside stock solution with a concentration of 1000 ng / mL. The hypoxanthine nucleoside stock solution was serially diluted to prepare a series of standard solutions with concentration gradients of 7.8125 ng / mL, 15.625 ng / mL, 31.25 ng / mL, 62.5 ng / mL, 125 ng / mL, 250 ng / mL, and 500 ng / mL. These standard solutions were measured using UPLC-MS / MS in multiple reaction monitoring mode. A standard curve was plotted with the peak area of ​​hypoxanthine nucleoside as the ordinate and the corresponding concentration as the abscissa.

[0023] The detection conditions for liquid chromatography and mass spectrometry are as follows:

[0024] Liquid chromatography conditions:

[0025] Chromatographic column: Phenomenex, 3 μm NX-C18, 50 x 2 mm;

[0026] Mobile phase: A phase is deionized water solution containing 0.1% formic acid; B phase is pure acetonitrile solution;

[0027] Flow rate: 0.3 ml / min, column temperature: 40℃, sample size: 1 μl;

[0028] Gradient elution procedure:

[0029] Time (min) Mobile phase A (%) Mobile phase B (%) 0 95 5 2 95 5 4 10 90 5.5 10 90 6 95 5 8 95 5

[0030] Mass spectrometry conditions:

[0031] ESI source, data collection in positive ion mode, voltage 5500V, ion source temperature 500℃. Ion detection conditions:

[0032]

[0033] Note: * is quantitative ion.

[0034] The standard curve was plotted with the measured peak area of hypoxanthine as the ordinate and the corresponding concentration as the abscissa, and the results are shown in Table 1. From the results in Table 1, it can be seen that the method has a wide detection range and good linear condition, and can be well applied to the quantitative detection of hypoxanthine.

[0035] Table 1 Standard curve

[0036]

[0037] According to the above method, the standard sample was detected 6 times within 24h to calculate the intra-day precision, and the detection was performed for 3 consecutive days to calculate the inter-day precision, and the precision was evaluated by RSD value. The precision verification results are shown in Table 2.

[0038] Table 2 Precision

[0039]

[0040] From the results in Table 2, it can be seen that the inter-day precision RSD of hypoxanthine detection is 3.44%, and the intra-day precision RSD is 0.73%, which shows that the method of the present application has high precision and excellent quantitative detection effect.

[0041] Example 2, quantitative detection of hypoxanthine in sunflower root exudates

[0042] Step 1, the aboveground and underground parts of sunflower in good growing condition were completely taken out from the soil, the root system was kept intact and the plant roots were washed clean with tap water and sterilized deionized water respectively. Then, the whole plant was transferred to a light-tight sterilized culture tube containing deionized water for hydroponic culture, natural light and day-night cycle were maintained, and the liquid in the light-tight sterilized culture tube was collected after 48 h of culture to obtain a plant root exudate solution; the collected plant root exudate solution was filtered and freeze-dried to obtain a plant root exudate powder.

[0043] Step 2, 20 mg of sunflower root exudate powder was accurately weighed, 300 μl of extraction solvent (obtained by mixing methanol and water at a volume ratio of 1:4) was added, vortexed for 5 minutes, and centrifuged at 4°C for 15 min (12000 rpm); the supernatant was taken to obtain a sample to be detected.

[0044] Step 3, the sample to be detected obtained in step 2 was detected by the same method as in Example 1. The multiple reaction monitoring chromatogram of the obtained sample to be detected was compared with the multiple reaction monitoring chromatogram of the standard sample obtained in step 1, hypoxanthine was identified according to the retention time and characteristic ions obtained by high performance liquid chromatography-mass spectrometry, and the sample to be detected was quantified by combining the chromatographic peak area with the standard curve.

[0045] Step 4, standard addition recovery rate verification: a known concentration of hypoxanthine standard was added to the sample to be detected in step S3, and the same method as in Example 1 was used for detection to calculate the recovery rate of hypoxanthine. The results are shown in Table 3. From the data in Table 3, it can be seen that the detection accuracy of the method of the present application for the target substance is high, and the detection result is more accurate.

[0046] Table 3 extraction effect

[0047] Target substance Spiked recovery Inosine 115.00％

[0048] Step 5, repeatability verification: a known concentration of hypoxanthine standard was added to the same sample to be detected, mixed and divided into 6 parallel samples for repeatability verification. The results are shown in Table 4. The results show that the method has good repeatability.

[0049] Table 4 repeatability

[0050] Target substance Repeatability (RSD %, N=6) Inosine 7.41％

[0051] Comparative Example:

[0052] 1, different extraction solvents were prepared, specifically, comparative extraction solvent 1 was obtained by mixing methanol and water at a volume ratio of 4:1; comparative extraction solvent 2 was obtained by mixing methanol, water and isopropanol at a volume ratio of 1:1:1.

[0053] The hypoxanthine standard was dissolved in comparative extraction solvent 1 and comparative extraction solvent 2 respectively to obtain comparative standard 1 and comparative standard 2; the mass spectrometry detection of comparative standard 1 and comparative standard 2 was carried out according to the same method as in Example 1, and the detection results are shown in Table 4. Figure 1 as shown in b-c of Table 4.

[0054] According to Figure 1 It can be seen that when comparative extraction solvent 1 is used, the peak of the standard is not single (as shown in b of Table 4), there is false positive, which seriously affects the qualitative and quantitative results. Figure 1

[0055] 2. Using the same method as in Example 2, the sunflower root exudate powder was dissolved in comparative extraction solvent 1 and comparative extraction solvent 2 respectively to obtain comparative detection solution 1 and comparative detection solution 2; the detection of comparative detection solution 1 and comparative detection solution 2 was carried out using the same method, and the concentration of hypoxanthine was calculated. The known concentration of hypoxanthine standard was added to the comparative detection solution 1 and comparative detection solution 2, and the recovery rate under different extraction solvents was calculated, and the results are shown in Table 5.

[0056] Table 5 Recovery rate

[0057] Extraction reagent (volume ratio) Spiked recovery Methanol: water = 4: 1 142.80％ Methanol: water: isopropanol = 1: 1: 1 72.36％

[0058] According to Table 5, the use of different extraction solvents has a great influence on the recovery rate. When comparative extraction solvent 1 is used, the peak of the standard is not single, the recovery rate is high, there is false positive result, which seriously affects the qualitative and quantitative results; when comparative extraction solvent 2 is used, the qualitative peak chart is normal, but the recovery rate is low. Therefore, the quantitative detection method provided by the present application, i.e. methanol: water = 1:4, takes into account the recovery rate and accuracy, which can effectively assist the quantitative detection of hypoxanthine in root exudates.

[0059] 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 them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions described in the foregoing examples can be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.​

Claims

1. A method for quantitative detection of a hypoxanthine nucleoside in plant root exudates, characterized in that, The method comprises the following steps: S1, collecting a plant with good growth conditions, washing the root system of the plant, and then transferring the plant to a light-proof sterilized culture tube containing deionized water for hydroponics, keeping natural light and day-night cycle, collecting the liquid in the light-proof sterilized culture tube after the hydroponics, and obtaining a plant root exudate solution; filtering and freeze-drying the plant root exudate solution to obtain a plant root exudate powder; S2, dissolving the plant root exudate powder in an extraction solvent to obtain a to-be-tested solution, wherein the extraction solvent is obtained by mixing methanol and water at a volume ratio of 1:4; S3, detecting the to-be-tested solution by using an UPLC-MS / MS method, and if the to-be-tested solution contains hypoxanthine, the peak area corresponding to the hypoxanthine is obtained; S4, dissolving a hypoxanthine standard in the extraction solvent to prepare a series of standard solutions with different concentrations, detecting the standard solutions by using the same UPLC-MS / MS method, and constructing a standard curve; substituting the peak area of the hypoxanthine obtained in S3 into the standard curve to obtain the concentration of the hypoxanthine in the plant root exudate, and completing the quantitative detection of the hypoxanthine in the plant root exudate. In the UPLC-MS / MS method, the chromatographic column is a C18 HPLC chromatographic column; the mobile phase A is a deionized water solution containing 0.1% formic acid, and the mobile phase B is acetonitrile; the flow rate is 0.3 ml / min; the column temperature is 40°C; gradient elution conditions are used for elution, and the gradient elution program comprises: 0 min, the mobile phase A is 95%, and the mobile phase B is 5%; 2 min, the mobile phase A is 95%, and the mobile phase B is 5%; 4 min, the mobile phase A is 10%, and the mobile phase B is 90%; 5.5 min, the mobile phase A is 10%, and the mobile phase B is 90%; 6 min, the mobile phase A is 95%, and the mobile phase B is 5%; 8 min, the mobile phase A is 95%, and the mobile phase B is 5%.

2. The method of claim 1, wherein, In the UPLC-MS / MS method, the mass spectrometry conditions are as follows: ESI source, data collection is performed in a positive ion mode, the voltage is 5500V, and the ion source temperature is 500°C.

3. The method of claim 1, wherein, The plant is a sunflower.

4. The method of claim 1, wherein, In S1, the hydroponics time is at least 48 h.

5. The method of claim 1, wherein, Based on 1 mg of the plant root exudate powder, the volume of the extraction solvent is 15 μl.

6. The method of claim 1, wherein, In S4, the linear equation of the standard curve is y = 2.26309 e 6 x + 8213.76924, x is the concentration of hypoxanthine nucleoside, and y is the peak area corresponding to the hypoxanthine nucleoside.

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