Method for detecting primary metabolites in plants

Abstract

The invention relates to the technical field of analytical chemistry and metabonomics, in particular to a method for detecting primary metabolites in plants. According to the present invention, the primary substituted writing product comprises saccharides and organic acids, and the detection method comprises single sample extraction and derivatization treatment to obtain the to-be-detected substance synchronously adapted to the GC-FID system and the GC-MS system so as to achieve the synchronous detection of the GC-FID system and the GC-MS system. Compared with the prior art, the method provided by the invention has the advantages that the detection efficiency is remarkably improved, the detection stability is remarkably optimized, the pretreatment compatibility is outstanding, and the application universality is high.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical chemistry and metabolomics, and in particular to a method for synchronously detecting primary metabolites such as sugars and organic acids in plants. Background Art

[0002] Primary metabolites, including sugars, organic acids, amino acids, polyamines, and sugar alcohols, are not only important components of fruit nutrition but also participate in plant growth and development and stress response through pathways such as glycolysis, the TCA cycle, amino acid metabolism, and the GABA pathway. Citrus, the world's largest fruit, has a flavor quality determined by the dynamic balance between soluble sugars (primarily sucrose, fructose, and glucose) and organic acids (primarily citric acid, accounting for over 60% of the total acid). Therefore, accurate detection of primary metabolites such as sugars and organic acids is crucial for research on fruit and vegetable quality and plant growth and development.

[0003] Current detection technologies for primary metabolites of fruits and vegetables such as citrus have significant deficiencies: when GC-FID is used to detect sugars and organic acids, it is necessary to undergo multi-step derivatization with hydroxylamine hydrochloride, hexamethyldisilazane (HMDS) and trimethylchlorosilane (TMCS). The steps are cumbersome and the product stability is poor. Although separation can be completed within 20 minutes using a DB-5 column, the pretreatment has low compatibility with different metabolites. GC-MS metabolomics detection uses methoxyamine hydrochloride / N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) derivatization combined with DB-5MS column analysis. A single detection takes 55 minutes and has low detection throughput. In addition, the pretreatment processes (extraction solvents, derivatization reagents) of the two methods are independent, and simultaneous analysis requires repeated sampling, resulting in increased time, reagent and sample consumption, which restricts the efficiency of metabolomics research.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a method for the simultaneous detection of primary metabolites such as plant sugars and organic acids based on a single sample pretreatment coupled with a GC-FID / GC-MS dual system. The method is particularly suitable for the efficient synergistic analysis of primary metabolites such as high-abundance sugars and organic acids and low-abundance amino acids and secondary organic acids in fruits and vegetables, and can also be expanded to the primary metabolomics research of various plant tissues.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] The present invention provides a method for the simultaneous detection of primary metabolites such as sugars and organic acids in plants. Through single sample extraction and derivatization treatment, analytes that are simultaneously adapted to the GC-FID and GC-MS dual systems are obtained, thereby realizing simultaneous detection by the GC-FID and GC-MS dual systems.

[0008] Preferably, the sample extraction step comprises: ball-milling the sample to powder using liquid nitrogen; using pre-cooled extraction buffer and ice bath sonication; extracting at 60-80°C; centrifuging to obtain the supernatant; adding pre-cooled chloroform and pre-cooled ultrapure water; centrifuging to collect the aqueous phase; and vacuum rotary evaporation to dryness.

[0009] Preferably, the derivatization step comprises: adding methoxyamine hydrochloride solution for oximation; adding MSTFA for reaction and then filtering with a filter membrane.

[0010] Preferably, the GC-FID uses a DB-5 chromatographic column with a flow rate of 3±1 mL / min.

[0011] Preferably, the temperature program of GC-FID is: 130±5°C→8±1°C / min→152±5°C→12±1°C / min→176±5°C→16±2°C / min→198±5°C→20±2°C / min→238±5°C→24±3°C / min→280±5°C, hold for 5±2 min.

[0012] Preferably, the detection conditions of GC-FID are: hydrogen 40±5 mL / min, air 450±10 mL / min, nitrogen tail gas 45±5 mL / min, and detector temperature 300±10°C.

[0013] Preferably, the GC-MS uses a DB-35MS column with a flow rate of 1.5±0.5 mL / min.

[0014] Preferably, the temperature program of GC-MS is: 85±5°C, hold for 2±1 min, → 15±2°C / min → 300±10°C, hold for 6±2 min.

[0015] Preferably, the mass spectrum acquisition of GC-MS is: full scan mode m / z 45-650, ion source temperature 250±10°C.

[0016] Preferably, the time taken for simultaneous detection by the GC-FID and GC-MS dual systems does not exceed 25 minutes.

[0017] Beneficial effects:

[0018] The present invention provides a method for synchronous detection of primary metabolites such as sugars and organic acids in plants. Through single sample extraction and derivatization treatment, an analyte that is synchronously adapted to the GC-FID and GC-MS dual systems is obtained, thereby achieving synchronous detection of the GC-FID and GC-MS dual systems. Compared with the prior art, the advantages of the present invention include: significantly improved detection efficiency, with the dual-system synchronous detection taking only 22 minutes per sample, which is 71% shorter than the traditional step-by-step detection method; significantly optimized stability, with the GC-FID system repeatability RSD <3%, and the GC-MS system repeatability RSD <10%, and detection stability superior to traditional methods; outstanding pretreatment compatibility, with a unified pretreatment process adapted to the dual detection system, reducing sample consumption by 92%, and reducing reagent and consumable costs by 50%; strong universal application, the method can be applied to the analysis of primary metabolites such as sugars and organic acids in various plant materials, and has wide applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0020] Figure 1 This is the GC-FID chromatogram obtained by the method of Example 1 of the present invention.

[0021] Figure 2 This is the GC-MS chromatogram obtained by the method of Example 1 of the present invention. DETAILED DESCRIPTION

[0022] The present invention discloses a method for the simultaneous detection of primary metabolites, such as sugars and organic acids, in plants. Through single sample extraction and derivatization, combined with a gas chromatography-flame detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS) dual system, the method achieves absolute quantification of high-abundance sugars (fructose, glucose, sucrose) and organic acids (citric acid, malic acid, quinic acid), as well as relative quantification of all primary metabolites. The GC-FID system achieves baseline separation and precise quantification of sugar and organic acid metabolites within 15 minutes, while the GC-MS system achieves highly sensitive detection of low-abundance primary metabolites (such as amino acids, sugar alcohols, and secondary organic acids) within 22 minutes.

[0023] By optimizing the pretreatment process, chromatographic configuration, and combining two systems, this method overcomes the complex extraction and derivatization processes and poor system compatibility of traditional methods. This method improves detection efficiency by 71%, reduces sample consumption by 92%, and reduces reagent and consumable costs by 50%. It also exhibits excellent stability (GC-FID repeatability RSD <3%, GC-MS <10%). This method is suitable for the complementary analysis of high- and low-abundance primary metabolites in citrus fruits and can be expanded to the analysis of primary metabolites in various fruits, vegetables, and plants.

[0024] The specific technical solutions provided by the present invention include the following:

[0025] (1) Experimental materials and equipment

[0026] Reagents: chloroform (pre-cooled at -20°C), methanol (chromatographic grade), pyridine (chromatographic grade), ribitol (internal standard, purity ≥98%), methoxyamine hydrochloride (purity ≥95%), N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA, purity ≥98%).

[0027] Consumables: 2 mL centrifuge tubes, 25 mm stainless steel grinding beads.

[0028] Pre-treatment equipment: ball mill, constant temperature oscillator, high-speed centrifuge, vacuum rotary evaporator, ultrasonic cleaning instrument.

[0029] Detection equipment: GC-MS (with DB-35MS column, 30m×0.32mm×0.25μm), GC-FID (with DB-5 column, 30m×0.32mm×0.25μm).

[0030] (2) Solution preparation

[0031] Internal standard stock solution: ribitol 4 mg / mL, dissolved in ultrapure water, stored at 4°C.

[0032] Extraction buffer: methanol-ultrapure water-internal standard stock solution (80:19:1, v / v / v), pre-cooled at -20°C.

[0033] Standard buffer: extraction buffer-ultrapure water (1:1, v / v).

[0034] Methoxyamine hydrochloride derivative solution: 20 mg / mL pyridine solution (prepare immediately before use).

[0035] (III) Sample pretreatment and derivatization

[0036] The first step is the metabolite extraction, which includes:

[0037] 1. The fresh sample was ball-milled to powder using liquid nitrogen, and 100 mg was weighed into a 2 mL centrifuge tube (error ≤ 10%);

[0038] 2. Add 1 mL of pre-chilled extraction buffer and sonicate on ice for 15 minutes;

[0039] 3. Extract at 70℃ constant temperature with shaking for 30 min;

[0040] Centrifuge at 12,000 rpm for 15 min and collect 700 μL of supernatant.

[0041] 5. Add 350 μL of pre-cooled chloroform and 700 μL of pre-cooled ultrapure water and mix well;

[0042] 6. Centrifuge at 3,000 rpm for 10 min and collect the aqueous phase;

[0043] 7. Take 200 μL of the extract and evaporate to dryness under vacuum;

[0044] Next comes the derivatization step, which includes:

[0045] 8. Add 40 μL of methoxyamine hydrochloride solution and incubate at 37°C and 950 rpm for 2 h;

[0046] 9. Add 70 μL MSTFA, react at 37°C, 300 rpm for 30 min, and filter through a 0.22 μm filter membrane.

[0047] (IV) Chromatographic detection conditions

[0048] GC-MS parameters are as follows:

[0049] Chromatographic column: DB-35MS (30m×0.32mm×0.25μm) with high-purity helium carrier gas at a flow rate of 1.5mL / min;

[0050] Heating program: 85°C (hold for 2 min) → 15°C / min → 300°C (hold for 6 min);

[0051] Mass spectrometry acquisition: full scan mode (m / z 45-650), ion source temperature 250 °C.

[0052] The GC-FID parameters are as follows:

[0053] Chromatographic column: DB-5 (30m×0.32mm×0.25μm), high-purity nitrogen carrier gas, flow rate 3mL / min;

[0054] Heating program:

[0055] 130℃→8℃ / min→152℃→12℃ / min→176℃→16℃ / min→198℃→20℃ / min→238℃→24℃ / min→280℃(hold for 5min);

[0056] Detector: hydrogen 40 mL / min, air 450 mL / min, nitrogen tail gas 45 mL / min, detection temperature 300 °C.

[0057] (V) Quantitative analysis

[0058] Standard curve establishment: Prepare gradient concentration standard solutions and establish GC-FID sugar acid absolute quantitative curve using ribitol internal standard method;

[0059] Metabolite identification: GC-MS detection is qualitatively determined by matching with the NIST mass spectral library and comparing retention indices;

[0060] Relative quantification: calculated based on the normalization of the ribitol internal standard peak area and the fresh weight of the sample.

[0061] Based on the above technical solution, the method was verified by using typical citrus pulp samples, and the results showed that this method can achieve efficient and simultaneous detection of sugars, acids and primary metabolites.

[0062] The present invention provides a simultaneous detection method for citrus sugar acids and primary metabolites based on a dual GC-FID / GC-MS system. This method enables the coordinated analysis of high- and low-abundance metabolites through a single pretreatment step, shortening the total detection time to 22 minutes per sample. This method improves detection efficiency by 71% and reduces sample consumption by 92% compared to existing conventional techniques. Furthermore, the method exhibits excellent stability (GC-FID repeatability RSD <3%, GC-MS <10%), significantly improving analytical efficiency and stability. For detailed data, please refer to the following examples and accompanying tables.

[0063] To make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below. Obviously, the embodiments described are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, ordinary technicians in this field can optimize parameters according to actual needs. All other embodiments obtained without creative work are within the scope of protection of the present invention.

[0064] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the exact 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, and these numerical ranges should be considered to be specifically disclosed herein.

[0065] In the description of this specification, the reference terms "one embodiment", "some embodiments", "specific implementation methods", or "some specific implementation methods" and the like mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiments of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0066] Unless otherwise specified, the materials and reagents used in the following examples are all commercially available. Experimental methods without specific conditions specified in the examples are generally performed under conventional conditions or the conditions recommended by the manufacturer.

[0067] In the following examples, the reagents and instruments used include: methanol (chromatographic grade); pyridine (chromatographic grade, Aladdin P111513); ribitol (Sigma A5502); methoxyamine hydrochloride (Sigma89803, stored dry); MSTFA (Sigma69479 / Aladdin M106662, stored at 4°C); LAVIBE 2mL centrifuge tube; ball mill (with 25mm steel balls); constant temperature shaker; high-speed centrifuge; vacuum rotary evaporator; gas chromatography-mass spectrometry (GC-MS, Thermo ISQ II); gas chromatograph (GC-FID, Agilent 8860).

[0068] Example 1

[0069] This example provides a method for simultaneous detection of primary metabolites such as citrus sugars and organic acids based on a GC-FID / GC-MS dual system, as follows:

[0070] 1. Instrument and reagent configuration

[0071] Detection system: gas chromatography-mass spectrometry system (equipped with DB-35MS chromatographic column), gas chromatography-hydrogen flame detection system (equipped with DB-5 chromatographic column).

[0072] Auxiliary equipment: ultrasonic cleaner, constant temperature shaking centrifuge, vacuum rotary concentrator.

[0073] Reagents and consumables: chloroform (pre-cooled at -20°C), methanol (chromatographic grade), pyridine (chromatographic grade), ribitol (internal standard, purity ≥98%), methoxyamine hydrochloride (purity ≥95%), N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA, purity ≥98%), 2 mL centrifuge tube, 25 mm stainless steel grinding beads.

[0074] 2. Sample pretreatment and testing

[0075] 2.1 Standard curve establishment

[0076] Six sugar and acid standard gradient solutions, including citric acid, malic acid, and sucrose, were prepared according to Table 1, with ribitol (final concentration 20 ng / μL) as the internal standard.

[0077] Table 1 GC-FID standard curve gradient solution preparation method

[0078]

[0079] A 200 μL sample was rotary evaporated to dryness, followed by oximation (40 μL methoxyamine hydrochloride solution, 37°C for 2 hours) and silylation (70 μL MSTFA, 37°C for 30 minutes). The sample was filtered and analyzed by GC-FID. A calibration curve was constructed using the peak area ratio of the target compound to the internal standard as the ordinate and the concentration ratio as the abscissa (see Table 2).

[0080] Table 2 Linear range, regression equation and correlation coefficient of GC-FID standard curve

[0081]

[0082] 2.2 Actual sample testing

[0083] A 100 mg sample of citrus pulp ground with liquid nitrogen (accurate to ±0.1 mg) was added to 1 mL of pre-chilled extraction buffer (methanol:water:internal standard stock solution = 80:19:1, v / v / v). The mixture was sonicated on ice for 15 minutes, followed by constant temperature extraction at 70°C for 30 minutes. The supernatant was centrifuged at 12,000 rpm for 15 minutes and purified by chloroform-water biphasic separation. A 200 μL sample of the aqueous phase was evaporated to dryness and derivatized for simultaneous analysis by GC-FID (absolute quantification of sugars and acids) and GC-MS (relative quantification of primary metabolites).

[0084] 3. Analysis of test results

[0085] 3.1 Verification of chromatographic system complementarity

[0086] GC-FID Detection Performance: The GC-FID system achieved baseline separation of the six target sugar acids within 15 minutes, with retention times of: malic acid 4.027 minutes, citric acid 6.821 minutes, quinic acid 7.144 minutes, fructose (7.245 / 7.305 minutes), glucose (7.410 / 7.515 minutes), and sucrose 10.913 minutes. The peak areas of fructose and glucose were combined to calculate the total amount due to their isomer separation characteristics. The standard curve fit of each target compound was R 2 >0.999, with linear ranges as follows: sugars: fructose and glucose 100–3200 μg / mL, sucrose 200–6400 μg / mL; organic acids: citric and malic acid 30–960 μg / mL, quinic acid 10–320 μg / mL.

[0087] GC-MS detection characteristics: The GC-MS system completed the relative quantitative analysis of the target primary metabolites within 22 minutes. However, the high sugar content of citrus (>80 mg / g FW) caused the glucose, fructose and sucrose chromatographic peak signals to be overloaded (peak height>10 8At the same time, quinic acid-fructose and glucose-citric acid coeluted on the DB-35MS column (Δt < 0.1 min), leading to deviations in peak area calculations and significantly affecting quantitative accuracy.

[0088] System Synergy: The GC-FID system leverages the wide dynamic range of the FID detector and the highly selective separation of sugars and acids by the DB-5 column to accurately capture and independently quantify high-concentration sugar and acid signals. The GC-MS system, with its high sensitivity, focuses on the relative quantification of low-abundance primary metabolites such as amino acids and secondary organic acids, minimizing interference from sugar overload. The two systems operate simultaneously (total time: 22 minutes per sample), forming a complementary detection system for high-concentration sugar and acid components and low-abundance metabolites, fully covering the full abundance analysis needs of citrus primary metabolites.

[0089] 3.2 Sample test results and stability verification

[0090] GC-FID sugar and acid data analysis: Based on the quantitative analysis of the GC-FID standard curve, the detection results of each component in the citrus pulp sample are shown in Table 3.

[0091] Table 3 Results of relative quantitative analysis of sugar and acid in citrus pulp samples by GC-FID

[0092]

[0093] As shown in Table 3:

[0094] Organic acid content (μg / g FW): malic acid 240.10±5.54 (RSD=2.31%), citric acid 9382.31±249.72 (RSD=2.66%), quinic acid 42.94±1.25 (RSD=2.91%), total acid 9665.35±249.78 (RSD=2.58%), of which citric acid accounted for 97.0%.

[0095] Carbohydrate content (μg / g FW): fructose 20664.64±549.40 (RSD=2.66%), glucose 21864.46±589.03 (RSD=2.69%), sucrose 55215.89±1606.83 (RSD=2.91%), total sugar 97744.99±1797.39 (RSD=1.84%), of which sucrose accounted for 56.5%.

[0096] Method stability: The RSD of the repeatability of each component was less than 3% (n=5), indicating that the extraction, derivatization process and GC-FID system were excellently stable.

[0097] Data reliability: The acid content (0.97% of the fresh weight of the pulp) and sugar content (9.8% of the fresh weight of the pulp) were both within the range reported in the literature (sugar content 8%–15%, acid content 0.3%–2%), verifying the validity of the data.

[0098] GC-MS primary metabolic data analysis: Primary metabolites were identified by NIST mass spectral library matching, retention index and characteristic ion peak comparison, and the relative content was normalized by ribitol internal standard peak area and sample fresh weight. A total of 58 metabolites were identified (excluding ribitol internal standard, isomers and derivatized products were combined), mainly including sugars, sugar alcohols, organic acids, secondary organic acids, amino acids, amino acid derivatives, polyamines, etc., as shown in Table 4. The GC-MS system successfully identified μg-level metabolites such as pyruvate, succinate, fumarate, methionine, tyrosine, spermidine, etc. These metabolites are involved in the TCA cycle, amino acid metabolism and polyamine metabolism. Their accurate analysis is of great significance for analyzing the metabolic laws of citrus. In terms of sample stability, the repeatability RSD of all metabolites was <10% (n=5), indicating that the system meets the requirements for fruit and vegetable metabolomics analysis.

[0099] Table 4 Absolute quantitative analysis results of primary metabolites of citrus pulp samples by GC-MS

[0100]

[0101]

[0102] In summary, the method provided by the present invention supports GC-FID and GC-MS dual system detection simultaneously through single extraction and derivatization, with a total time of 22 min / sample, which is faster than the traditional step-by-step detection method (Bartolozzi, F., Bertazza, G., Bassi, D., Cristoferi, G. (1997). Simultaneous determination of soluble sugars and organic acids as their trimethylsilyl derivatives in apricot fruits by gas-liquid chromatography. Journal of Chromatography A, 758 (1), 99-107.)(Sun, X., Zhu, A., Liu, S., Sheng, L., Ma, Q., Zhang, L., Nishawy, E. M., Zeng, Y., Xu, J., Ma, Z., Cheng, Y., Deng, X. (2013). Integration of metabolomics and subcellular organelle expression microarray to increase understanding the organic acid changes in The post-harvest citrus fruit. J Integr Plant Biol, 55(11), 1038-1053.) was shortened by 71% (the traditional method, GC-FID takes 20 minutes, GC-MS takes 55 minutes, the present invention only takes 22 minutes for simultaneous detection, shortening by 71%), the sample consumption was reduced by 92% (the traditional method: 1g sugar and acid sample, 0.3g primary metabolite sample, see Sun et al., 2013, the present invention only requires 0.1g, reducing by 92%), and the cost of reagents and consumables was reduced by 50%. The stability of the GC-FID and GC-MS systems met RSD < 3% and RSD < 10%, respectively. Through the complementary detection of high- and low-abundance components, the quantitative analysis requirements of citrus primary metabolites are fully covered, and can be expanded to the primary metabolome analysis of other fruit, vegetable and plant samples.

[0103] Finally, it should be noted that the above embodiments are only preferred embodiments of the present invention, which are used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention. Therefore, any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for detecting primary metabolites in plants, characterized in that: The primary metabolites include sugars and organic acids. The method obtains analytes that are synchronously adapted to the GC-FID and GC-MS dual systems through single sample extraction and derivatization treatment, thereby realizing synchronous detection of the GC-FID and GC-MS dual systems.

2. The method for detecting primary metabolites in plants according to claim 1, characterized in that: The sample extraction steps include: ball-milling the sample to powder using liquid nitrogen; using a pre-cooled extraction buffer solution for ice bath ultrasonication; extracting at 60-80° C.; centrifuging to obtain the supernatant; adding pre-cooled chloroform and pre-cooled ultrapure water; centrifuging to collect the aqueous phase; and vacuum rotary evaporation to dryness.

3. The method for detecting primary metabolites in plants according to claim 2, characterized in that: The derivatization treatment steps include: adding methoxyamine hydrochloride solution for oximation; adding MSTFA for reaction and then filtering with a filter membrane.

4. The method for detecting primary metabolites in plants according to any one of claims 1 to 3, characterized in that The GC-FID used a DB-5 chromatographic column with a flow rate of 3±1 mL / min.

5. The method for detecting primary metabolites in plants according to claim 4, characterized in that: The heating program of GC-FID was as follows: 130±5℃→8±1℃ / min→152±5℃→12±1℃ / min→176±5℃→16±2℃ / min→198±5℃→20±2℃ / min→238±5℃→24±3℃ / min→280±5℃, and held for 5±2min.

6. The method for detecting primary metabolites in plants according to claim 5, characterized in that: The detection conditions of GC-FID were as follows: hydrogen 40±5 mL / min, air 450±10 mL / min, nitrogen makeup gas 45±5 mL / min, and detector temperature 300±10°C.

7. The method for detecting primary metabolites in plants according to any one of claims 1 to 3, characterized in that: The GC-MS used a DB-35MS column with a flow rate of 1.5±0.5 mL / min.

8. The method for detecting primary metabolites in plants according to claim 7, characterized in that: The heating program of GC-MS was as follows: 85 ± 5 °C, hold for 2 ± 1 min, → 15 ± 2 °C / min → 300 ± 10 °C, hold for 6 ± 2 min.

9. The method for detecting primary metabolites in plants according to claim 7, characterized in that: The mass spectra of GC-MS were collected in full scan mode with m / z 45-650 and an ion source temperature of 250±10°C.

10. The method for detecting primary metabolites in plants according to any one of claims 1 to 3, characterized in that: The simultaneous detection of GC-FID and GC-MS dual systems takes no more than 25 minutes.

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