Method for rapidly and quantitatively detecting inosine in plant root exudates

Abstract

The invention discloses a method for rapidly and quantitatively detecting inosine in plant root exudates. The method comprises the following steps: firstly, preparing plant root exudates powder; secondly, dissolving the plant root exudate powder in an extraction solvent obtained by mixing methanol and water according to a volume ratio of 1: 4, so as to obtain a solution to be detected; detecting the solution to be detected by adopting a UPLC-MS / MS (Ultra Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) method, and if the solution to be detected contains hypoxanthine nucleoside, obtaining a peak area corresponding to the hypoxanthine nucleoside; and finally, quantitatively detecting the hypoxanthine in the plant root exudates in combination with the standard curve. The detection method provided by the invention can be used for rapidly and quantitatively detecting the hypoxanthine nucleoside in the plant root exudates, is relatively good in peak pattern, has relatively good accuracy and repeatability, and can be used for rapidly detecting large-scale samples.

Description

Technical Field

[0001] The present invention relates to the technical field of compound detection, and particularly relates to a rapid quantitative detection method for inosine in plant root exudates. Background Art

[0002] Plant root exudates contain a large number of small molecule compounds, which play important roles in plant growth and development and plant-plant recognition. Among them, inosine is an important plant growth regulator, and the determination of its absolute content is of great significance for studying plant growth regulation and individual recognition mechanisms. However, there is currently no rapid quantitative detection method for inosine. Therefore, the rapid quantitative detection of inosine from plant root exudates has important methodological significance for the study of chemical signal recognition in plant interactions. Summary of the Invention

[0003] The purpose of the present invention is to provide a rapid quantitative detection method for inosine in plant root exudates.

[0004] In the first aspect, the present invention provides a rapid quantitative detection method for inosine in plant root exudates, including:

[0005] S1. Collect plants with good growth conditions, rinse the plant roots thoroughly and transfer them to an opaque sterilized culture tube containing deionized water for hydroponics, maintain natural light and the day-night rhythm, and collect the liquid in the opaque sterilized culture tube after hydroponics to obtain a plant root exudate solution; filter and freeze-dry the plant root exudate solution to obtain a plant root exudate powder;

[0006] S2. Dissolve the plant root exudate powder in an extraction solvent to obtain a solution to be detected, and the extraction solvent is obtained by mixing methanol and water with a volume ratio of 1:4;

[0007] S3. Detect the solution to be detected by UPLC-MS / MS method. If inosine is contained in the solution to be detected, obtain the peak area corresponding to inosine;

[0008] S4. Dissolve the inosine standard product in the extraction solvent to prepare a series of standard products with concentration gradients, detect the standard products by the same UPLC-MS / MS method, and construct a standard curve; substitute the peak area of inosine obtained in S3 into the standard curve to obtain the concentration of inosine in the plant root exudate, and complete the quantitative detection of inosine in the plant root exudate.

[0009] The method described above. In the UPLC-MS / MS method, the chromatographic column is a reverse HPLC column; mobile phase A is an aqueous deionized solution containing 0.1% formic acid, and 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 includes: at 0 min, mobile phase A is 95% and mobile phase B is 5%; at 2 min, mobile phase A is 95% and mobile phase B is 5%; at 4 min, mobile phase A is 10% and mobile phase B is 90%; at 5.5 min, mobile phase A is 10% and mobile phase B is 90%; at 6 min, mobile phase A is 95% and mobile phase B is 5%; at 8 min, mobile phase A is 95% and mobile phase B is 5%.

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

[0011] The method described above. The plant is sunflower.

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

[0013] The method described above. Based on each milligram of the plant root exudate powder, the volume of the extraction solvent is 15 μl.

[0014] The method described above. After mixing the plant root exudate powder with the extraction solvent, vortex and oscillate for 5 minutes; after the oscillation ends, centrifuge the mixture at a speed of 12000 rpm for 15 min to obtain the solution to be detected.

[0015] The method described above. The above centrifugation is carried out at 4 °C to prevent the compounds in the plant root exudate powder from changing in the high-temperature environment generated by centrifugation.

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

[0017] The method provided by the present invention can effectively and rapidly quantify inosine in plant root exudates; moreover, the mass spectrometry peak shape is good, the fitting degree is high, and the quantification accuracy is good; the total elution time is 8 minutes, which is time-consuming; the linear range of the standard curve for quantification is large, and it can be applied to samples with a large concentration fluctuation range; the repeatability is good, and it can be used for the detection of a large number of samples; it provides a reliable rapid quantification method for the quantitative research of inosine secreted by plant roots, filling the blank of the quantitative detection method for inosine secreted by plant roots. Description of the Drawings

[0018] Figure 1 Mass spectrometry detection results of the standard product obtained after dissolving the inosine standard product with different extraction solvents; among them, the extraction solvent used in a is a mixture of methanol and water in a volume ratio of 1:4; the extraction solvent used in b is a mixture of methanol and water in a volume ratio of 4:1; the extraction solvent used in c is a mixture of methanol, water and isopropanol in a volume ratio of 1:1:1. Detailed Description of the Invention

[0019] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention, and they should not be construed as limitations to the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the scope of protection of the present invention. In the description of the present invention, it should be understood that the terms used are only for the purpose of description and cannot be construed as indicating or implying relative importance.

[0020] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0021] Example 1: Drawing of the standard curve, linearity and precision test:

[0022] Dissolve the inosine (CAS No. 58-63-9) standard product in the extraction solvent (a mixture of methanol and water in a volume ratio of 1:4) to prepare an inosine stock solution with a concentration of 1000 ng / mL. Gradient dilute the inosine stock solution 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. Measure these standard solutions in the multiple reaction monitoring mode of UPLC-MS / MS, use the peak area of the measured inosine as the ordinate, and the corresponding concentration as the abscissa to draw the standard curve;

[0023] Among them, the detection conditions of liquid chromatography and mass spectrometry are as follows:

[0024] Liquid chromatography conditions:

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

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

[0027] Flow rate: 0.3ml / min, column temperature: 40°C, injection volume: 1μl;

[0028] Gradient elution program:

[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 was carried out in positive ion mode, voltage 5500V, ion source temperature was 500°C. Conditions for detected ions:

[0032]

[0033] Note: * is the quantitative ion.

[0034] Taking the peak area of inosine measured as the ordinate and the corresponding concentration as the abscissa, a standard curve was plotted, and the results are shown in Table 1 below. From the results in Table 1, it can be seen that the detection range of this method is wide, the linear conditions are good, and it can be better applied to the quantitative detection of inosine.

[0035] Table 1 Standard curve

[0036]

[0037] According to the above method, the standard sample was continuously detected 6 times within 24h to calculate the within-day precision, and detected continuously for 3 days to calculate the between-day precision, and the precision was evaluated by the 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 between-day precision RSD for inosine detection is 3.44% and the within-day precision RSD is 0.73%, indicating that the method of the present invention has high precision and excellent quantitative detection effect.

[0041] Example 2, Quantitative detection of inosine in sunflower root exudates

[0042] Step 1: Completely remove the above-ground and underground parts of sunflowers with good growth conditions from the soil, keep the root systems intact, and rinse the plant roots clean with tap water and sterilized deionized water respectively. Then, transfer the whole plant to an opaque sterilized culture tube containing deionized water for hydroponic cultivation, maintain natural light and the day-night rhythm, collect the liquid in the opaque sterilized culture tube after 48 hours of cultivation to obtain the plant root exudate solution; filter and freeze-dry the collected plant root exudate solution to obtain the plant root exudate powder.

[0043] Step 2: Accurately weigh 20 mg of sunflower root exudate powder, add 300 μl of extraction solvent (obtained by mixing methanol and water in a volume ratio of 1:4), vortex for 5 minutes, and centrifuge at 4 °C for 15 min (12,000 rpm); take the supernatant to obtain the sample to be detected.

[0044] Step 3: Detect the sample to be detected obtained in Step 2 using the same method as in Example 1. Compare the multi-reaction monitoring chromatogram of the sample to be detected with the multi-reaction monitoring chromatogram of the standard sample obtained in Step 1, identify inosine riboside based on the retention time and characteristic ion pairs obtained by high-performance liquid chromatography-mass spectrometry, and quantify it by combining the chromatographic peak area of the sample to be detected with the standard curve.

[0045] Step 4: Verification of spike recovery rate: Add a known concentration of inosine riboside standard to the sample to be detected in Step S3, detect it using the same method as in Example 1 above, and calculate the recovery rate of inosine riboside. The results are shown in Table 3. From the data in Table 3, it can be seen that the method of the present invention has a high detection accuracy for the target substance, and the detection results are more accurate.

[0046] Table 3 Extraction effect

[0047] Target substance Spiked recovery rate Inosine 115.00％

[0048] Step 5: Verification of repeatability: Add a known concentration of inosine riboside standard to the same sample to be detected, mix well, and divide it into 6 parallel samples for repeatability verification. The results are shown in Table 4. The results show that this method has good repeatability.

[0049] Table 4 Repeatability

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

[0051] Comparative example:

[0052] 1. Prepare different extraction solvents. Specifically, mix methanol and water in a volume ratio of 4:1 to obtain comparative extraction solvent 1; mix methanol, water, and isopropanol in a volume ratio of 1:1:1 to obtain comparative extraction solvent 2.

[0053] Dissolve the inosine standard in extraction solvent 1 and extraction solvent 2 respectively to obtain reference standard 1 and reference standard 2; perform mass spectrometry detection on reference standard 1 and reference standard 2 in the same method as in Example 1, and the detection results are as shown in Figure 1 shown in b-c of

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

[0055] 2. Using the same method as in Example 2, dissolve the sunflower root exudate powder in extraction solvent 1 and extraction solvent 2 respectively to prepare reference test solutions 1 and 2; perform detection on reference test solutions 1 and 2 in the same method and calculate the concentration of inosine. Add the inosine standard with a known concentration to reference test solutions 1 and 2, calculate the recovery rates under different extraction solvents, and the results are shown in Table 5.

[0056] Table 5 Recovery Rates

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

[0058] According to Table 5, it can be seen that different extraction solvents have a great influence on the recovery rate. When using extraction solvent 1, the peak of the standard is not single, the recovery rate is on the high side, and there is a false positive result, which seriously affects the qualitative and quantitative results; when using extraction solvent 2, the qualitative peak pattern is normal, but the recovery rate is low. Therefore, choose the quantitative detection method provided by the present invention, that is, when methanol: water = 1:4, it takes into account both the recovery rate and the accuracy, and can effectively assist the quantitative detection of inosine in root exudates.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; 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 various embodiments of the present invention.

Claims

1. A method for quantitatively detecting inosine in plant root exudates, characterized in that, Including: S1. Collect plants with good growth conditions, rinse the plant roots thoroughly and transfer them to an opaque sterilized culture tube containing deionized water for hydroponics. Keep natural light and day-night rhythm. After hydroponics, collect the liquid in the opaque sterilized culture tube to obtain a plant root exudate solution; filter and lyophilize the plant root exudate solution to obtain a plant root exudate powder; S2. Dissolve the plant root exudate powder in an extraction solvent to obtain a solution to be detected. The extraction solvent is obtained by mixing methanol and water with a volume ratio of 1:4; S3. Detect the solution to be detected by UPLC-MS / MS method. If inosine is contained in the solution to be detected, obtain the peak area corresponding to inosine; S4. Dissolve inosine standard in the extraction solvent to prepare a series of standard products with concentration gradients. Detect the standard products by the same UPLC-MS / MS method and construct a standard curve; substitute the peak area of inosine obtained in S3 into the standard curve to obtain the concentration of inosine in the plant root exudate, and complete the quantitative detection of inosine in the plant root exudate.

2. The method according to claim 1, wherein In the UPLC-MS / MS method, the chromatographic column is a reverse HPLC chromatographic column; mobile phase A is an aqueous deionized solution containing 0.1% formic acid, and 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 includes: 0 min, mobile phase A is 95%, mobile phase B is 5%; 2 min, mobile phase A is 95%, mobile phase B is 5%; 4 min, mobile phase A is 10%, mobile phase B is 90%; 5.5 min, mobile phase A is 10%, mobile phase B is 90%; 6 min, mobile phase A is 95%, mobile phase B is 5%; 8 min, mobile phase A is 95%, mobile phase B is 5%.

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

4. The method according to claim 1, wherein The plant is sunflower.

5. The method according to claim 1, characterized in that, In S1, the hydroponics time is at least 48 h.

6. The method according to claim 1, wherein Based on each milligram of the plant root exudate powder, the volume of the extraction solvent is 15 μl.

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

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