Quantitative detection method of phenolic acids in rape root exudates based on UPLC-MS / MS

Abstract

The present application relates to the technical field of detection of phenolic acid, and discloses a quantitative detection method of phenolic acid components in rape root exudates based on UPLC-MS / MS, wherein the phenolic acid in the rape root exudates is p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin. The method comprises the following steps: (1) mixing freeze-dried rape root exudate powder with an extraction solvent, fully shaking and mixing, centrifuging the obtained mixture to obtain root exudate extract; (2) preparing a mixed mother liquor of p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin standard substances, and drawing a standard curve; (3) determining the root exudate extract prepared in step (1) in a UPLC-MS / MS multiple reaction monitoring mode according to the operation method and conditions of step (2). The method can be used for rapid quantitative detection of phenolic acid components in rape root exudates.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of Chinese Patent Application No. 202411702488.2, filed on November 26, 2024, the contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of phenolic acid detection technology, specifically to a quantitative detection method for phenolic acid components in rapeseed root exudates based on UPLC-MS / MS. Background Technology

[0004] Plant root exudates are important efflux substances in plants, playing a crucial role in plant-plant interactions and plant-environment interactions. Root exudates have a complex composition, including small molecules such as flavonoids, hormones, and phenolic acids, as well as large molecules such as polypeptides and proteins. They play a vital role in signal transduction between plants and between plants and the environment.

[0005] Currently, the development of methods for detecting phenolic acids mainly focuses on the food and traditional Chinese medicine fields, while methods for extracting and detecting phenolic acids from plant root exudates are still lacking. Technically, the detection of phenolic acids is primarily achieved through high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). HPLC-based methods are relatively inexpensive, but their sensitivity is low and they cannot be used for detecting trace biological samples. Compared to HPLC-based methods, LC-MS methods offer significantly improved sensitivity. LC-MS typically utilizes either a high-performance liquid chromatograph (HPLC) or an ultra-high-performance liquid chromatograph (UPLC). Compared to conventional HPLC, UPLC offers advantages such as shorter detection time, higher efficiency, and better stability. Summary of the Invention

[0006] The purpose of this invention is to fill the gap in the detection technology of phenolic acids in rapeseed root exudates and overcome the problems of low sensitivity and poor repeatability of HPLC and HPLC-MS / MS detection. This invention provides a quantitative detection method for phenolic acids in rapeseed root exudates based on UPLC-MS / MS. The method described in this invention can achieve rapid quantitative detection of p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid, and hesperidin in rapeseed root exudates.

[0007] To achieve the above objectives, this invention provides a method for the quantitative detection of phenolic acid components in rapeseed root exudates based on UPLC-MS / MS, the method comprising the following steps:

[0008] (1) mixing the freeze-dried rape root exudate powder with an extraction solvent, fully shaking and mixing, and centrifuging the obtained mixture to obtain a root exudate extract, wherein the extraction solvent is a mixture of methanol and water;

[0009] (2) preparing a mixed mother liquor of p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin standard substance, and preparing a series of standard solutions with gradient concentrations by gradient dilution, and determining the standard solutions in UPLC-MS / MS multiple reaction monitoring mode, taking the peak area of the target substance as the ordinate and the corresponding concentration as the abscissa to draw a standard curve;

[0010] (3) according to the operation method and conditions of step (2), determining the root exudate extract prepared in step (1) in UPLC-MS / MS multiple reaction monitoring mode, comparing the multiple reaction monitoring chromatogram of the root exudate extract with the multiple reaction monitoring chromatogram of the standard solution obtained in step (2), identifying p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin according to the retention time and characteristic ions obtained by UPLC-MS / MS, and determining the content of p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin in the root exudate extract according to the chromatographic peak area of the root exudate extract combined with the standard curve.

[0011] Preferably, in the extraction solvent, the volume ratio of methanol to water is (2.5-5.5):1.

[0012] Preferably, in step (1), the mass-volume ratio of the rape root exudate powder to the extraction solvent is (15-25) mg:300 μL.

[0013] Preferably, in step (1), the centrifugation conditions include a temperature of 2-6℃ and a time of 10-20 min.

[0014] Preferably, in steps (2) and (3), the gradient elution is carried out with a mixed solution of methanol and acetonitrile as the mobile phase during chromatographic detection, wherein in the aqueous formic acid solution, the volume ratio of formic acid to water is (0.05-0.2):100; in the mixed solution of methanol and acetonitrile, the volume ratio of methanol to acetonitrile is 1:(0.5-2).

[0015] Preferably, the elution conditions include that, taking the total volume of the mobile phase as 100%, the elution time is t.

[0016] 0≤t<2min, the content of the aqueous formic acid solution is 70-90%, and the content of the mixed solution of methanol and acetonitrile is 10-30%;

[0017] 2≤t<6.5min, the content of the aqueous formic acid solution is 0-85%, and the content of the mixed solution of methanol and acetonitrile is 15-100%;

[0018] 6.5≤t<7min, the content of the aqueous formic acid solution is 0-10%, and the content of the mixed solution of methanol and acetonitrile is 90-100%;

[0019] 7≤t<8min, the content of the aqueous formic acid solution is 0-85%, and the content of the mixed solution of methanol and acetonitrile is 15-100%;

[0020] 8≤t<9min, the content of the aqueous formic acid solution is 70-90%, and the content of the mixed solution of methanol and acetonitrile is 10-30%.

[0021] Preferably, the flow rate of the gradient elution is 0.2-0.6mL / min.

[0022] Preferably, the conditions of the chromatographic detection further comprise that the column temperature is 30-50℃, and the injection volume is 0.5-1.5μL.

[0023] Preferably, the chromatographic column of the ultra-high performance liquid chromatograph of the UPLC-MS / MS is a chromatographic column (Phenomenex).

[0024] Preferably, the detection conditions of the mass spectrometer comprise that the ion source is ESI, the scanning mode is positive ion detection, the voltage is 5300-5800V, and the ion source temperature is 400-500℃.

[0025] In the present application, the analysis and detection are carried out by the ultra-high performance liquid chromatograph-mass spectrometer (UPLC-MS / MS) technology, so that the method has high sensitivity, large quantitative range and good repeatability; through the design and optimization of the chromatographic conditions, the rapid quantitative detection of p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin in the root exudates of Brassica napus can be realized. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 the extracted ion chromatogram (XIC) of the standard sample p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin;

[0027] Figure 2 the extracted ion chromatogram (XIC) of the sample of Comparative Example 1 based on UPLC-MS / MS detection;

[0028] Figure 3Extraction ion chromatogram (XIC) based on HPLC-MS / MS detection of the sample of Comparative Example 1. DETAILED DESCRIPTION

[0029] The specific embodiments of the present application are described in detail below. It should be understood that the detailed description and specific examples, while indicating preferred embodiments of the application, are given by way of illustration only, and are not by way of limitation.

[0030] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The endpoints of the ranges are provided as a separate value from but are included in the range. For values which are less than or equal to the lower limit of the range, this is indicated by the low end of the range, and for values which are greater than or equal to the upper limit of the range, this is indicated by the upper end of the range. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. For ranges including an upper and lower limit, the range includes each individual value subsumed therein. For ranges excluding the endpoints, the range is intended to include the endpoints as used in the description and the claims.

[0031] The method for quantitatively detecting the phenolic acid components in the rape root exudates based on UPLC-MS / MS according to the present application comprises the following steps:

[0032] (1) mixing the freeze-dried rape root exudate powder with an extraction solvent, fully shaking and mixing, and centrifuging the obtained mixture to obtain a root exudate extract, wherein the extraction solvent is a mixture of methanol and water;

[0033] (2) preparing a mixed stock solution of p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin standard substances, and gradient dilution to prepare a series of standard solutions with concentration gradient, determining the standard solution under UPLC-MS / MS multiple reaction monitoring mode, taking the peak area of the target substance as the ordinate and the corresponding concentration as the abscissa, and drawing a standard curve;

[0034] (3) determining the root exudate extract prepared according to the operation method and conditions of step (2) under UPLC-MS / MS multiple reaction monitoring mode, comparing the multiple reaction monitoring chromatogram of the root exudate extract with the multiple reaction monitoring chromatogram of the standard solution obtained in step (2), identifying p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin according to the retention time and characteristic ions obtained by UPLC-MS / MS, and determining the content of p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin in the root exudate extract according to the chromatographic peak area of the root exudate extract combined with the standard curve.

[0035] In the method according to the present application, the volume ratio of methanol to water in the extraction solvent can be (2.5-5.5):1, preferably (3-5):1.

[0036] In step (1), the mass-volume ratio of the rape root exudate powder to the extraction solvent can be (15-25) mg:300 μL, preferably (16-24) mg:300 μL.

[0037] In step (1), the centrifugal treatment can be performed at a temperature of 2-6°C for 10-20 min. In a preferred embodiment, the centrifugal treatment is performed at a temperature of 3-5°C for 12-18 min.

[0038] In step (1), the time for the oscillation is preferably 5-10 min.

[0039] In steps (2) and (3), the gradient elution is preferably performed using a mixture of a formic acid aqueous solution and a mixture of methanol and acetonitrile as the mobile phase in the chromatographic detection process, wherein the volume ratio of formic acid to water in the formic acid aqueous solution is (0.05-0.2):100, and the volume ratio of methanol to acetonitrile in the mixture of methanol and acetonitrile is 1:(0.5-2).

[0040] In the method of the present application, the elution conditions include:

[0041] when 0≤t<2 min, the content of the formic acid aqueous solution is 70-90%, and the content of the mixture of methanol and acetonitrile is 10-30%;

[0042] when 2≤t<6.5 min, the content of the formic acid aqueous solution is 0-85%, and the content of the mixture of methanol and acetonitrile is 15-100%;

[0043] when 6.5≤t<7 min, the content of the formic acid aqueous solution is 0-10%, and the content of the mixture of methanol and acetonitrile is 90-100%;

[0044] when 7≤t<8 min, the content of the formic acid aqueous solution is 0-85%, and the content of the mixture of methanol and acetonitrile is 15-100%;

[0045] when 8≤t<9 min, the content of the formic acid aqueous solution is 70-90%, and the content of the mixture of methanol and acetonitrile is 10-30%.

[0046] In a preferred embodiment, the elution conditions include:

[0047] when 0≤t<2 min, the content of the formic acid aqueous solution is 80-90%, and the content of the mixture of methanol and acetonitrile is 10-20%;

[0048] 2≤t<6.5min, the content of the aqueous formic acid solution is 0-85%, and the content of the mixed solution of methanol and acetonitrile is 15-100%;

[0049] 6.5≤t<7min, the content of the aqueous formic acid solution is 0-5%, and the content of the mixed solution of methanol and acetonitrile is 95-100%;

[0050] 7≤t<8min, the content of the aqueous formic acid solution is 0-85%, and the content of the mixed solution of methanol and acetonitrile is 15-100%;

[0051] 8≤t<9min, the content of the aqueous formic acid solution is 80-90%, and the content of the mixed solution of methanol and acetonitrile is 10-20%.

[0052] In the most preferred embodiment, the gradient program of the chromatographic detection is set as shown in the following table:

[0053]

[0054]

[0055] In the method of the present application, the flow rate of the gradient elution is preferably 0.2-0.6 mL / min.

[0056] In the method of the present application, the conditions of the chromatographic detection can include a column temperature of 30-50℃ and an injection volume of 0.5-1.5 μL. Preferably, the conditions of the chromatographic detection include a column temperature of 33-48℃ and an injection volume of 0.6-1.3 μL.

[0057] In the method of the present application, the chromatographic column of the UPLC-MS / MS ultra performance liquid chromatograph can be a chromatographic column (Phenomenex). Preferably, the chromatographic column of the UPLC-MS / MS ultra performance liquid chromatograph is a chromatographic column (Phenomenex, 2.6 μm x 50 mm x 3 mm).

[0058] In the method of the present application, the detection conditions of the mass spectrometer preferably include an ion source of ESI, a positive ion detection scanning mode, a voltage of 5300-5800 V, and an ion source temperature of 400-500℃.

[0059] The UPLC-MS / MS-based method for quantitatively detecting phenolic acid components in oilseed rape root exudates according to the present application is further illustrated by the following examples. The examples are implemented on the premise of the technical solutions of the present application, and detailed implementation modes and specific operation processes are given, but the protection scope of the present application is not limited to the following examples.

[0060] In the following examples, the experimental methods are all conventional methods in the art unless otherwise specified. The experimental materials used in the following examples are all commercially available unless otherwise specified.

[0061] Example 1

[0062] (1) Extraction of root exudates: 20 mg of freeze-dried oilseed rape root exudate powder was added to a 2 mL centrifuge tube, then 300 μL of a mixed solution of methanol and water (volume ratio of methanol to water was 4:1) was added to the centrifuge tube, and then shaken for 10 min, followed by centrifugal treatment at 4℃ for 15 min to obtain the oilseed rape root exudate extract.

[0063] (2) Preparation of a standard curve: a mixed stock solution of p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin standard substances was prepared at 10000 ng / ml, and a series of standard solutions with concentration gradients were prepared by gradient dilution, with the concentrations being 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL and 10000 ng / mL from low to high. The standard solution was determined under the UPLC-MS / MS multiple reaction monitoring mode, and the conditions of liquid chromatography were as follows: the type of chromatographic column was the chromatographic column (Phenomenex, 2.6 μm x 50 mm x 3 mm); the mobile phase A was formic acid solution (volume ratio of formic acid to water was 0.1:100), and the mobile phase B was a mixed solution of methanol and acetonitrile (volume ratio of methanol to acetonitrile was 1:1); the flow rate was 0.4 mL / min, the column temperature was 40℃, the injection volume was 1 μL; and the gradient elution program was as shown in Table 1.

[0064] Table 1

[0065]

[0066] The mass spectrometry conditions were as follows: the ion source was ESI, the scanning mode was positive ion detection, the voltage was 5500 V, and the ion source temperature was 450℃. The detected ion conditions were as shown in Table 2.

[0067] Table 2

[0068]

[0069]

[0070] Note: * is quantitative ion.

[0071] The standard solution was subjected to UPLC-MS / MS detection, and the extracted ion chromatogram (XIC) of p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin of the standard sample is shown in Figure 1. Figure 1

[0072] The standard curve was plotted with the measured peak area of the target substance as the vertical coordinate and the corresponding concentration as the horizontal coordinate, and the results are shown in Table 3. From the results in Table 3, 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 p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin.

[0073] Table 3

[0074]

[0075] According to the above method, the standard sample was detected 6 times within 24 hours to calculate the intra-day precision, and the detection was performed for 3 consecutive days to calculate the inter-day precision. The precision was evaluated by RSD value. The precision verification results are shown in Table 4. From the results in Table 4, it can be seen that the inter-day precision and intra-day precision RSD of the 5 kinds of phenolic acids are less than 6%, which indicates that the method of the present application has high precision and excellent quantitative detection effect.

[0076] Table 4

[0077]

[0078]

[0079] (3) Extraction and detection of rape root exudates: according to the operation method and conditions of step (2), the rape root exudate extract prepared in step (1) was detected under the multiple reaction monitoring mode of UPLC-MS / MS, and the obtained multiple reaction monitoring chromatogram of the rape root exudate extract was compared with the multiple reaction monitoring chromatogram of the standard solution obtained in step (2). According to the retention time and characteristic ions obtained by UPLC-MS / MS, p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin were identified, and according to the chromatographic peak area of the rape root exudate extract combined with the standard curve, the content of p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin in the rape root exudate extract was determined.

[0080] ​Spiked recovery verification: 20 mg of dry powder of rape root exudates was accurately weighed, and a mixed standard with a known concentration was added. Extraction was performed according to step (1), and detection and concentration calculation were performed according to steps (2) and (3), and then the recovery rates of the five phenolic acids were calculated respectively. The recovery rate verification results are shown in Table 5. As can be seen from the results in Table 5, the extraction recovery rate of phenolic acids in rape root exudates based on the method is as high as 90-110%, which shows that the method can be well applied to the quantitative detection of phenolic acids in rape root exudates.

[0081] Table 5

[0082]

[0083] Repeatability verification: 6 portions, each weighing 20 mg, were weighed from the same sample of rape root exudates as 6 parallel samples, and the determination of each phenolic acid component was performed according to the extraction and detection methods of steps (1), (2) and (3), and the repeatability was evaluated by RSD value. The results are shown in Table 6. As can be seen from the results in Table 6, the detection stability of phenolic acids in rape root exudates based on the method is good (RSD is less than 5%), which again shows that the method can be well applied to the quantitative detection of phenolic acids in rape root exudates.

[0084] Table 6

[0085]

[0086] Comparative Example 1

[0087] The mixed standard solution (50 ng / ml) containing p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin was detected according to the method of Example 1, except that in step (2), ultra-high performance liquid chromatography (UPLC) was replaced by high performance liquid chromatography (HPLC). The extracted ion chromatogram (XIC) of the mixed standard solution based on UPLC-MS / MS detection is shown in Figure 2 , and the extracted ion chromatogram (XIC) of the mixed standard solution based on HPLC-MS / MS detection is shown in Figure 3 .

[0088] As can be seen from the comparison of Figure 2 and Figure 3 , in the low concentration state, compared with the detection method based on UPLC-MS / MS, the detection result chromatographic peak type of the detection method based on HPLC-MS / MS is worse, the peak width is wider, the signal-to-noise ratio S / N is significantly lower, and the response is lower. It shows that the detection method based on UPLC-MS / MS has stronger superiority, which can effectively solve the problem of low detection sensitivity of HPLC and HPLC-MS / MS.

[0089] It can be seen from the detection results of Example 1 and Comparative Example 1 that the UPLC-MS / MS-based quantitative detection method for phenolic acid components in rape root exudates has the advantages of high sensitivity, wide linear range, high recovery rate, good stability and small sample loading amount, and can realize rapid quantitative detection of p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin in rape root exudates.

[0090] The preferred embodiments of the present application are described in detail above, but the present application is not limited thereto. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be considered as disclosed by the present application and belong to the protection scope of the present application.

Claims

1. A method for quantitatively detecting phenolic acids in Brassica root exudates based on UPLC-MS / MS, wherein the phenolic acids in the Brassica root exudates are p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin, characterized in that, The method comprises the following steps: (1) mixing the freeze-dried rape root exudate powder with an extraction solvent, fully shaking and mixing, and centrifuging the obtained mixture to obtain a root exudate extract, wherein the extraction solvent is a mixture of methanol and water; (2) preparing a mixed mother liquor of p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin standard substance, and gradient dilution to prepare a series of standard solutions with concentration gradient, determining the standard solutions in UPLC-MS / MS multiple reaction monitoring mode, taking the peak area of the measured target as the ordinate and the corresponding concentration as the abscissa to draw a standard curve; (3) determining the root exudate extract prepared in step (1) in UPLC-MS / MS multiple reaction monitoring mode according to the operation method and conditions of step (2), comparing the multiple reaction monitoring chromatogram of the root exudate extract with the multiple reaction monitoring chromatogram of the standard solution obtained in step (2), identifying p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin according to the retention time and characteristic ions obtained by UPLC-MS / MS, and determining the content of p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin in the root exudate extract according to the chromatographic peak area of the root exudate extract combined with the standard curve; In steps (2) and (3), the gradient elution is carried out with a mixed solution of formic acid aqueous solution and methanol and acetonitrile as the mobile phase, wherein the volume ratio of formic acid to water in the formic acid aqueous solution is (0.05-0.2):100, and the volume ratio of methanol to acetonitrile in the mixed solution of methanol and acetonitrile is 1:(0.5-2); The elution conditions include that, taking the total volume of the mobile phase as 100%, the elution time as t, when 0≤t<2min, the content of the formic acid aqueous solution is 70-90% and the content of the mixed solution of methanol and acetonitrile is 10-30%; When 2≤t<6.5min, the content of the formic acid aqueous solution is 0-85% and the content of the mixed solution of methanol and acetonitrile is 15-100%; When 6.5≤t<7min, the content of the formic acid aqueous solution is 0-10% and the content of the mixed solution of methanol and acetonitrile is 90-100%; When 7≤t<8min, the content of the formic acid aqueous solution is 0-85% and the content of the mixed solution of methanol and acetonitrile is 15-100%; When 8≤t<9min, the content of the formic acid aqueous solution is 70-90% and the content of the mixed solution of methanol and acetonitrile is 10-30%; In the extraction solvent, the volume ratio of methanol to water is (2.5-5.5):

1. The column of the UPLC-MS / MS is Kinetex ® Column.

2. The method of claim 1, wherein, In step (1), the mass-volume ratio of the rape root exudate powder to the extraction solvent is (15-25)mg:300μL.

3. The method according to claim 1 or 2, characterized in that, In step (1), the centrifuging conditions include that the temperature is 2-6℃ and the time is 10-20min.

4. The method according to claim 1 or 2, characterized in that, The flow rate of the gradient elution is 0.2-0.6mL / min.

5. The method of claim 1, wherein, ​ 6. The method of claim 1 or 5, the conditions for chromatographic detection further comprising: The column temperature is 30-50℃, and the injection volume is 0.5-1.5 μL.

7. The method of claim 1, wherein, The mass spectrometer detection conditions include: the ion source is ESI, the scanning mode is positive ion detection, the voltage is 5300-5800 V, and the ion source temperature is 400-500℃.

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