Quantitative detection method for phenolic acid components in rape root exudates based on UPLC-MS / MS

Abstract

The invention relates to the technical field of phenolic acid substance detection, and discloses a quantitative detection method for phenolic acid components in rape root exudates based on UPLC-MS / MS. The phenolic acids in the rape root exudates are 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 oscillating and uniformly mixing, and centrifuging the obtained mixture to obtain a root exudate extracting solution; (2) preparing a mixed mother solution of p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin standard substances, and drawing a standard curve; and (3) determining the root exudate extracting solution prepared in the step (1) in a UPLC-MS / MS (Ultra Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) multi-reaction monitoring mode according to the operation method and conditions in the step (2). The method disclosed by the invention can be used for rapidly and quantitatively detecting the phenolic acid components in the rape root exudates.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of Chinese patent application 202411702488.2 filed on November 26, 2024, the contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the technical field of detection of phenolic acid substances, and in particular to a quantitative detection method of phenolic acid components in rapeseed root exudates based on UPLC-MS / MS. Background Art

[0004] Plant root secretions are important excretion substances of plants, playing an important role in the process of plant interaction and plant-environment interaction. The composition of root secretions is complex, including small molecules such as flavonoids, hormones and phenolic acids, as well as macromolecules such as peptides and proteins. They play an important role in the process of signal transmission between plants and between plants and the environment.

[0005] At present, the development of detection methods for phenolic acids is mainly concentrated in the fields of food and traditional Chinese medicine. There is still a blank in the extraction and detection methods of phenolic acids in plant root secretions. From the perspective of technical means, the detection of phenolic acids is currently mainly achieved through liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) technology. The detection method based solely on liquid chromatography (HPLC) has a relatively low cost, but its sensitivity is low and cannot be used for the detection of trace biological samples. Compared with the method based solely on liquid chromatography (HPLC), the detection method of liquid chromatography-mass spectrometry (LC-MS) has significantly improved sensitivity. In liquid chromatography-mass spectrometry technology, a high performance liquid chromatograph (HPLC) or an ultra-high performance liquid chromatograph (UPLC) is usually equipped. Compared with conventional high performance liquid chromatographs (HPLC), ultra-high performance liquid chromatographs (UPLC) have the advantages of short detection time, high efficiency and good stability. Summary of the invention

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

[0007] In order to achieve the above object, the present invention provides a method for quantitatively detecting 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, shaking and mixing thoroughly, and centrifuging the resulting mixture to obtain a root exudate extract, wherein the extraction solvent is a mixture of methanol and water;

[0009] (2) preparing a mixed stock solution of p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin standard substances, and preparing a series of standard solutions with concentration gradients by gradient dilution, measuring the standard solutions under UPLC-MS / MS multiple reaction monitoring mode, and plotting a standard curve with the peak area of ​​the measured target substance as the ordinate and the corresponding concentration as the abscissa;

[0010] (3) According to the operation method and conditions of step (2), the root exudate extract prepared in step (1) is measured under UPLC-MS / MS multiple reaction monitoring mode, and the obtained multiple reaction monitoring chromatogram of the root exudate extract is compared with the multiple reaction monitoring chromatogram of the standard solution obtained in step (2), and p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin are identified according to the retention time and characteristic ions obtained by UPLC-MS / MS, and the content of p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin in the root exudate extract is determined 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 rapeseed root exudate powder to the extraction solvent is (15-25) mg:300 μL.

[0013] Preferably, in step (1), the conditions for the centrifugal treatment include: a temperature of 2-6°C and a time of 10-20 min.

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

[0015] Preferably, based on the total volume of the mobile phase as 100%, the elution time is t, and the elution conditions include:

[0016] 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%;

[0017] 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%;

[0018] 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%;

[0019] 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%;

[0020] 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%.

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

[0022] Preferably, the conditions for the chromatographic detection also include: a column temperature of 30-50°C and an injection volume of 0.5-1.5 μL.

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

[0024] Preferably, the detection conditions of the mass spectrometer include: 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°C.

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

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

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

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

[0029] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0030] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0031] The method for quantitatively detecting phenolic acid components in rapeseed root exudates based on UPLC-MS / MS of the present invention comprises the following steps:

[0032] (1) mixing the freeze-dried rape root exudate powder with an extraction solvent, shaking and mixing thoroughly, and centrifuging the resulting 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 preparing a series of standard solutions with concentration gradients by gradient dilution, measuring the standard solutions under UPLC-MS / MS multiple reaction monitoring mode, and plotting a standard curve with the peak area of ​​the measured target substance as the ordinate and the corresponding concentration as the abscissa;

[0034] (3) According to the operation method and conditions of step (2), the root exudate extract prepared in step (1) is measured under UPLC-MS / MS multiple reaction monitoring mode, and the obtained multiple reaction monitoring chromatogram of the root exudate extract is compared with the multiple reaction monitoring chromatogram of the standard solution obtained in step (2), and p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin are identified according to the retention time and characteristic ions obtained by UPLC-MS / MS, and the content of p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin in the root exudate extract is determined according to the chromatographic peak area of ​​the root exudate extract combined with the standard curve.

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

[0036] In step (1), the mass volume ratio of the rapeseed 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 conditions may include: temperature of 2-6°C, time of 10-20 min. In a preferred case, the centrifugal treatment conditions include: temperature of 3-5°C, time of 12-18 min.

[0038] In step (1), in a preferred case, the shaking time is 5-10 min.

[0039] In steps (2) and (3), in a preferred case, gradient elution is performed using a formic acid aqueous solution and a mixed solution of methanol and acetonitrile as mobile phases during chromatographic detection, wherein in the formic acid aqueous 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).

[0040] In the method described in the present invention, taking the total volume of the mobile phase as 100%, the elution time is t, and the elution conditions include:

[0041] 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%;

[0042] 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%;

[0043] 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%;

[0044] 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%;

[0045] 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%.

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

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

[0048] 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%;

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

[0050] 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%;

[0051] When 8≤t<9min, the content of the formic acid aqueous 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 chromatographic detection gradient program is set as shown in the following table:

[0053]

[0054]

[0055] In the method described in the present invention, in a preferred case, the flow rate of the gradient elution is 0.2-0.6 mL / min.

[0056] In the method of the present invention, the chromatographic detection conditions may include: column temperature of 30-50°C, injection volume of 0.5-1.5 μL. In a preferred case, the chromatographic detection conditions include: column temperature of 33-48°C, injection volume of 0.6-1.3 μL.

[0057] In the method described in the present invention, the chromatographic column of the ultra-high performance liquid chromatograph of the UPLC-MS / MS can be In a preferred embodiment, the chromatographic column of the ultra-high performance liquid chromatograph of the UPLC-MS / MS is Chromatographic column (Phenomenex, 2.6 μm×50 mm×3 mm).

[0058] In the method described in the present invention, in a preferred case, the detection conditions of the mass spectrometer include: 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°C.

[0059] The following examples further illustrate the quantitative detection method of phenolic acid components in rapeseed root exudates based on UPLC-MS / MS of the present invention. The examples are implemented based on the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following examples.

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

[0061] Example 1

[0062] (1) Extraction of root exudates: 20 mg of freeze-dried rapeseed root exudate powder was added to a 2 mL centrifuge tube. Then, 300 μL of a mixed solution of methanol and water (the volume ratio of methanol to water was 4:1) was added to the centrifuge tube and shaken for 10 min. The tube was then centrifuged at 4 °C for 15 min to obtain a rapeseed root exudate extract.

[0063] (2) Preparation of standard curve: Prepare a mixed stock solution of 10000 ng / ml of p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin standard substances, and prepare a series of standard solutions with concentration gradients by gradient dilution. The concentrations are 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 measured in UPLC-MS / MS multiple reaction monitoring mode. The liquid chromatography conditions are as follows: the model of the chromatographic column is Chromatographic column (Phenomenex, 2.6 μm × 50 mm × 3 mm); mobile phase A was formic acid solution (the volume ratio of formic acid to water was 0.1:100), and mobile phase B was a mixed solution of methanol and acetonitrile (the volume ratio of methanol to acetonitrile was 1:1); the flow rate was 0.4 mL / min, the column temperature was 40 °C, and the injection volume was 1 μL; 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 scan mode was positive ion detection, the voltage was 5500 V, and the ion source temperature was 450° C. The detected ion conditions are shown in Table 2 below.

[0067] Table 2

[0068]

[0069]

[0070] Note: * indicates quantitative ion.

[0071] The standard solution was taken for UPLC-MS / MS detection. The extracted ion current chromatograms (XIC) of p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin of the standard sample were as shown in FIG. Figure 1 shown.

[0072] The standard curve was drawn with the peak area of ​​the target substance measured as the ordinate and the corresponding concentration as the abscissa, and the results are shown in Table 3. From the results in Table 3, it can be seen that this method has a wide detection range and good linear conditions, 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 tested 6 times within 24 hours to calculate the intra-day precision, and the test was performed for 3 consecutive days to calculate the inter-day precision. The precision was evaluated by the RSD value. The precision verification results are shown in Table 4 below. From the results in Table 4, it can be seen that the inter-day precision and intra-day precision RSD of the five phenolic acids are all less than 6%, indicating that the method of the present invention has high precision and excellent quantitative detection effect.

[0076] Table 4

[0077]

[0078]

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

[0080] Verification of spiked recovery rate: Accurately weigh 20 mg of rapeseed root exudate dry powder, add a mixed standard of known concentration, extract according to the method of step (1), detect and calculate the concentration according to the methods of steps (2) and (3), and then calculate the recovery rates of the five phenolic acids. The recovery rate verification results are shown in Table 5. From the results in Table 5, it can be seen that the extraction recovery rate of phenolic acids in rapeseed root exudates based on this method is as high as 90-110%, indicating that this method can be well applied to the quantitative detection of phenolic acids in rapeseed root exudates.

[0081] Table 5

[0082]

[0083] Repeatability verification: From the same sample of rapeseed root exudates, 6 portions were weighed, each with 20 mg, as 6 parallel samples, and the phenolic acid components were determined according to the extraction and detection methods of steps (1), (2) and (3), and the repeatability of the method was evaluated by RSD value. The results are shown in Table 6. From the results in Table 6, it can be seen that the detection stability of phenolic acids in rapeseed root exudates based on this method is good (RSD is less than 5%), which once again shows that this method can be well applied to the quantitative detection of phenolic acids in rapeseed 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 tested 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 current chromatogram (XIC) of the mixed standard solution based on UPLC-MS / MS detection is shown in FIG. Figure 2 As shown, the extracted ion current chromatogram (XIC) of the mixed standard solution based on HPLC-MS / MS detection is as follows Figure 3 shown.

[0088] Depend on Figure 2 and Figure 3 From the comparison, it can be seen that at low concentrations, compared with the detection method based on UPLC-MS / MS, the detection method based on HPLC-MS / MS has a worse chromatographic peak shape, a wider peak width, a significantly lower signal-to-noise ratio S / N, and a lower response. This shows that the detection method based on UPLC-MS / MS has a stronger advantage and can effectively solve the problem of low sensitivity of HPLC and HPLC-MS / MS detection.

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

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

Claims

1. A method for quantitatively detecting phenolic acid components in rapeseed root exudates based on UPLC-MS / MS, wherein the phenolic acids in the rapeseed 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, shaking and mixing thoroughly, and centrifuging the resulting mixture to obtain a root exudate extract, wherein the extraction solvent is a mixture of methanol and water; (2) preparing a mixed stock solution of p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin standard substances, and preparing a series of standard solutions with concentration gradients by gradient dilution, measuring the standard solutions under UPLC-MS / MS multiple reaction monitoring mode, and plotting a standard curve with the peak area of ​​the measured target substance as the ordinate and the corresponding concentration as the abscissa; (3) According to the operation method and conditions of step (2), the root exudate extract prepared in step (1) is measured under UPLC-MS / MS multiple reaction monitoring mode, and the obtained multiple reaction monitoring chromatogram of the root exudate extract is compared with the multiple reaction monitoring chromatogram of the standard solution obtained in step (2), and p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin are identified according to the retention time and characteristic ions obtained by UPLC-MS / MS, and the content of p-hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and hesperidin in the root exudate extract is determined according to the chromatographic peak area of ​​the root exudate extract combined with the standard curve.

2. The method according to claim 1, characterized in that In the extraction solvent, the volume ratio of methanol to water is (2.5-5.5):

1.

3. The method according to claim 1 or 2, characterized in that: In step (1), the mass volume ratio of the rapeseed root exudate powder to the extraction solvent is (15-25) mg:300 μL.

4. The method according to any one of claims 1 to 3, characterized in that: In step (1), the conditions of the centrifugal treatment include: temperature of 2-6°C and time of 10-20 min.

5. The method according to claim 1, characterized in that: In steps (2) and (3), gradient elution is performed using a formic acid aqueous solution and a mixed solution of methanol and acetonitrile as mobile phases during chromatographic detection, wherein in the formic acid aqueous 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).

6. The method according to claim 5, characterized in that Taking the total volume of the mobile phase as 100%, the elution time is t, and the elution conditions include: 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 formic acid aqueous solution is 0-10%, and the content of methanol and acetonitrile mixed solution 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%.

7. The method according to claim 5 or 6, characterized in that: The flow rate of the gradient elution was 0.2-0.6 mL / min.

8. The method according to any one of claims 5 to 7, wherein the conditions for chromatographic detection further include: The column temperature was 30-50°C and the injection volume was 0.5-1.5 μL.

9. The method according to any one of claims 1 to 8, characterized in that: The chromatographic column of the ultra-high performance liquid chromatograph of the UPLC-MS / MS is Chromatographic column (Phenomenex).

10. The method according to any one of claims 1 to 9, characterized in that: The detection conditions of the mass spectrometer include: 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°C.

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