A quantitative analysis method for intermediates in the synthesis process of brassinolide compounds

Abstract

The invention relates to a quantitative analysis method for intermediates in the synthesis process of brassinolide compounds. The analysis method is a gas chromatography method, a sterol series product is used as an internal standard, an SH-Rtx-5 (30m*0.25mm*0.1um) capillary column and an FID detector are used, and a heating program is as follows: a starting temperature of 150-200°C is maintained for 1-5min, and then the temperature is increased to 230-300°C at a heating rate of 10°C / min-20°C / min, and the temperature is maintained for 10min-20min. The invention conducts qualitative and quantitative analysis on ergosteroid-2,22-diene-6-one, an intermediate in the synthesis process of brassinolide, and solves the problems that ergosteroid-2,22-diene-6-one has low sensitivity to ultraviolet detectors and DAD detectors, and that evaporative light detectors have poor reproducibility in quantitative detection.

Description

Technical Field

[0001] The invention belongs to the technical field of gas phase analysis and detection, and specifically relates to a quantitative analysis method for intermediates in the synthesis process of brassinolide compounds. Background Art

[0002] Brassinolide is ubiquitous in higher plants, including dicotyledonous plants (rapeseed, cabbage, chestnut, tea, lentil, kidney bean, mosquito tree, morning glory), monocotyledonous plants (cattail, corn, rice), gymnosperms (black pine, spruce). However, the content is extremely low, so the products on the market are all based on synthetic brassinolide as the original drug. Different organs of plants such as roots, stems, leaves, pollen, pistil, fruit and seeds contain brassinolide, among which pollen and immature seeds have the highest content of brassinolide (1-100 micrograms / gram fresh weight), and stem and leaf tissues usually contain 0.01-0.1 micrograms / gram fresh weight of brassinolide.

[0003] Brassinolide is known as the sixth generation of plant growth regulators. It is an endogenous plant growth regulator that inhibits the activity of oxidases in plants, increases plant respiration, and regulates the growth balance of plants. Its presence can affect and effectively regulate the growth and development of plants, including a series of plant life processes from cell growth and division to rooting, germination, flowering, fruiting, maturity and shedding. It can be used at all stages of the growth of various crops. It is safe to use and has no side effects on the entire growth and development process of crops (roots, stems, leaves, flowers, fruits, seeds). Brassinolide can effectively regulate the growth process of crops to achieve the goals of stabilizing and increasing production, improving quality, and enhancing crop resistance.

[0004] Brassinolide has been promoted in China for more than 20 years and has been widely recognized. Most of the current synthesis methods of 24-epi-brassinolide are improved on the basis of the literature reported by McMorris (McMorris, TC; J.Org, Chem. 1993, 58, 2338). However, in the synthesis process of brassinolide, the intermediate ergosta-2,22-diene-6-one has almost no ultraviolet absorption, and there is no absorption peak using the ultraviolet detector and the DAD detector. The reproducibility of the evaporative light detector in quantitative detection is poor, resulting in the inability to accurately judge the progress of the reaction in the production process, and the inability to judge the accurate content of the intermediate, resulting in blindness in the production process of brassinolide. For example, Chinese patent CN112552365A discloses an improved preparation method of (22E, 24R)-5α-ergosta-2,22-dien-6-one, in which the compound (22E, 24R)-5α-ergosta-2,22-dien-6-one obtained by the improved method is detected by HPLC and a UV detector. However, the detection method is only for the detection of three compounds. The detection sensitivity and separation of multiple compounds appearing in the synthesis process of brassinolide are low and cannot meet the detection requirements. In addition, the compound (22E, 24R)-5α-ergosta-2,22-dien-6-one has no conjugated double bonds and no heteroatom electronic conjugation, and the full-band scanning characteristic absorption peak is extremely weak, making it difficult to achieve quantitative analysis and identification.

[0005] In summary, due to the lack of accurate quantitative analysis methods for the synthetic intermediates of brassinolide compounds in the current existing technology using different detection methods, it is impossible to achieve quantitative control over the synthesis process of brassinolide. Therefore, the development of an effective, convenient and fast quantitative and qualitative detection method can play a clear judgment and guidance role in the production process of brassinolide compounds. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a method for quantitative analysis of intermediates in the synthesis process of brassinolide compounds. The method can be applied to the quantitative and qualitative analysis and detection of brassinolide synthesis intermediates, as well as the component analysis of plant growth regulators containing brassinolide or brassinolide synthesis intermediates.

[0007] In a first aspect, the present invention provides a method for quantitative analysis of intermediates in the synthesis process of brassinolide compounds, wherein the analysis method uses gas chromatography, and the gas chromatography conditions are:

[0008] The injection port temperature is 200℃-280℃;

[0009] The detector temperature is 200℃-280℃;

[0010] The carrier gas is nitrogen;

[0011] Column flow rate is 1mL / min-2.5mL / min;

[0012] The split ratio is (15-25):1;

[0013] The hydrogen flow rate is 25mL / min-35mL / min;

[0014] The air flow rate is 250mL / min-350mL / min;

[0015] The tail gas flow rate is 20mL / min-50mL / min.

[0016] Preferably, the chromatographic analysis conditions are:

[0017] The injection port temperature was 280°C;

[0018] The detector temperature is 280°C;

[0019] The carrier gas is nitrogen;

[0020] The column flow rate was 2.5 mL / min;

[0021] The split ratio is 20:1;

[0022] The hydrogen flow rate was 30 mL / min;

[0023] The air flow rate is 300 mL / min;

[0024] The tail gas flow rate was 50 mL / min.

[0025] Furthermore, the temperature rise program in the gas chromatography conditions is:

[0026] The starting temperature is 150°C-200°C, maintained for 1 min-5 min, then increased to 230°C-300°C at a heating rate of 10°C / min-20°C / min, and maintained for 10 min-20 min.

[0027] Preferably, the temperature rise program in the chromatographic analysis conditions is:

[0028] The initial temperature was 200 °C, maintained for 1 min, and then increased to 280 °C at a heating rate of 10 °C / min and maintained for 13 min.

[0029] Furthermore, the analysis method of the present invention also includes the following processing steps before gas chromatography injection:

[0030] S1. Preparation of internal standard solution

[0031] Accurately weigh the internal standard, dissolve it in the diluent, make up to volume, mix well, and prepare an internal standard solution with a concentration of 0.1-10 mg / ml for later use;

[0032] S2. Preparation of standard sample solution

[0033] Accurately weigh the standard sample of the synthetic intermediate of brassinolide compounds, place it in a volumetric flask, use a pipette to accurately absorb the internal standard solution of the quantitative step S1 to dissolve it, and prepare a standard sample solution with a concentration of 0.1-10 mg / ml. After the standard sample solution is cooled, inject 1 ul of the standard sample solution into a gas chromatograph with set chromatographic analysis conditions for analysis.

[0034] Furthermore, the present invention also provides a result analysis of the analysis method, which specifically includes the following method:

[0035] (1) Calculation of correction factor f

[0036] The calculation formula of the correction coefficient f is shown in formula (1):

[0037]

[0038] Where: A S is the peak area of ​​the internal standard; W B is the mass of the standard, mg; A B is the peak area of ​​the standard; W S is the mass of the internal standard, mg; C is the mass fraction of the standard, %;

[0039] (2) Determination of the content of intermediate samples in the synthesis process of brassinolide

[0040] The calculation formula of the intermediate sample content in the synthesis process of brassinolide is shown in formula (2):

[0041]

[0042] Where: A 甾 A is the peak area of ​​the intermediate in the synthesis process of brassinolide in the sample; s is the peak area of ​​the internal standard; M s is the mass of the internal standard, mg; M is the mass of the sample, mg; f is the correction factor.

[0043] Further, the internal standard in step S1 is one of cholesterol, stigmasterol, ergosterol, sitosterol, campesterol, β-sitosterol, campesterol, dihydrocholesterol, alginosterol, and α-sitosterol;

[0044] Preferably, the internal standard in step S1 is cholesterol or stigmasterol.

[0045] Furthermore, the diluent in step S1 is one of methanol, ethanol, ethyl acetate, acetonitrile, tetrahydrofuran, or a mixed solvent;

[0046] Preferably, the diluent in step S1 is tetrahydrofuran.

[0047] Furthermore, the chromatographic column used in the gas chromatography in step S2 is a SH-Rtx-5 capillary column.

[0048] Preferably, the concentration of the internal standard solution in step S1 is 0.8-2 mg / ml;

[0049] Preferably, the concentration of the standard sample solution in step S2 is 0.5-2 mg / ml.

[0050] Furthermore, the brassinolide compound comprises 24-epi-brassinolide, 28-homo-brassinolide, 28-epi-homo-brassinolide, mixed-epi-brassinolide or propionyl-brassinolide.

[0051] Furthermore, the quantitative analysis method of intermediates in the synthesis process of brassinolide compounds can also be used for the analysis of intermediates including ergosta-2,22-dien-6-one, 3α-5-cyclo-5α-ergosta-7,22-dien-6-ol, 3α-5-cyclo-5α-ergosta-7,22-dien-6-one, and 3α-5-cyclo-5α-ergosta-22-ene-6-one.

[0052] In a second aspect, the present invention also provides a method for quantitative analysis of intermediates in the synthesis process of brassinolide compounds, wherein the internal standard detection method used in the pretreatment can also be used for external standard detection, and the internal standard can also be used as a reference substance for external standard detection.

[0053] In a third aspect, the present invention also provides the quantitative analysis method of intermediates in the synthesis process of brassinolide compounds and its application in the preparation of plant growth regulators containing brassinolide or brassinolide synthesis intermediates.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] 1. The present invention provides a quantitative analysis method for intermediates in the synthesis process of brassinolide compounds, which solves the detection problem of intermediates in the synthesis process of brassinolide compounds in the prior art, that is, UV detectors and DAD detectors have no absorption, and evaporative light detectors have poor reproducibility.

[0056] 2. The detection method using gas chromatography plus internal standard of the present invention solves the problem of quantitative analysis of the intermediate ergosta-2,22-dien-6-one, and the detection method is convenient and fast. It can not only achieve quantitative control during the reaction process, but also quantitatively analyze the finished product of the intermediate ergosta-2,22-dien-6-one, which provides clear judgment and guidance for the production process of brassinolide. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is the synthetic route of 24-epibrassinolide.

[0058] Figure 2 This is the gas chromatogram of ergosterol.

[0059] Figure 3 This is the gas chromatogram of compound 2.

[0060] Figure 4 This is the gas chromatogram of compound 3.

[0061] Figure 5 Gas chromatogram of compound 4

[0062] Figure 6 This is the gas chromatogram of ergosta-2,22-dien-6-one (compound 5).

[0063] Figure 7 Cholesterol gas chromatogram.

[0064] Figure 8 This is the gas chromatogram of stigmasterol.

[0065] Fig. 9 This is a gas chromatogram of ergosta-2,22-dien-6-one (Compound 5) using stigmasterol as the internal standard.

[0066] Fig.10 This is the linear relationship between ergosta-2,22-dien-6-one concentration and peak area.

[0067] Fig.11 This is the liquid chromatogram of 24-epi-brassinolide.

[0068] Fig.12 It is the gas chromatogram of Comparative Example 2. DETAILED DESCRIPTION

[0069] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0070] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0071] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0072] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that these descriptions are exemplary only and are not intended to limit the scope of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0073] The present invention is further described in the following examples, but the examples are not intended to limit the scope of protection of the present invention.

[0074] Example 1

[0075] The synthetic route of 24-epi-brassinolide of the present invention is as follows:

[0076]

[0077] In the embodiments and comparative examples of the present invention, unless otherwise specified, tetrahydrofuran, ethyl acetate, acetonitrile, methanol, cholesterol, stigmasterol, ergosterol, dichloromethane, chloroform, trifluoroacetic anhydride, etc. are all commercially available products;

[0078] The intermediate ergosta-2,22-diene-6-one (compound 5) in the synthesis process of brassinolide of the present invention is prepared by the method reported by McMorris (McMorris, TC; J. Org, Chem. 1993, 58, 2338).

[0079] The specific synthesis steps are as follows:

[0080] (1) Add 39.6 g (0.1 mol) of ergosterol and 400 ml of pyridine to a four-necked flask, lower the temperature to 0-5°C, slowly drop 127 g (0.11 mol) of methylsulfonyl chloride, react at below 10°C for 0.5 hour, add 1000 ml of water, stir for 10 minutes, and filter out compound 1;

[0081] (2) Add 43 g of compound 1 to 150 ml of acetone, add 100 g of an aqueous solution containing 10% potassium bicarbonate (10 g of potassium bicarbonate dissolved in 90 ml of water) under reflux, heat under reflux for 5 hours, distill off 120 ml of acetone, add 200 ml of water, and filter to obtain compound 2;

[0082] (3) 18 g of chromium trioxide was added to 180 ml of pyridine in three portions, and the mixture was stirred at room temperature for 1 hour. Then 38 g of compound 2 was added at 0-5° C., and the mixture was stirred at room temperature for 3 hours. The mixture was filtered, and the filtrate was evaporated to dryness. The mixture was then passed through a chromatography column to obtain 20 g of 3α-5-cyclo-5α-ergosta-7,22-diene-6-one (compound 3).

[0083] (4) The temperature in a 500 mL single-layer glass reactor was lowered to below -55°C and 40 g of liquid ammonia was introduced into the reaction mixture. 0.7 g of metallic lithium was added in three portions. After the addition of metallic lithium, the mixture was stirred for 30 minutes. A solution of 18 g of compound 3 dissolved in 100 ml of tetrahydrofuran was added dropwise to the reaction mixture. After stirring for 1 hour, 20 g of ammonium chloride was added. The mixture was slowly heated to room temperature and then 100 ml of water and 100 ml of ethyl acetate were added for extraction. The ethyl acetate layer was concentrated and passed through a column to obtain 15 g of compound 4.

[0084] (5) 15 g of 3α-5-cyclo-5α-ergosta-22-dien-6-one, 100 ml of N,N-dimethylformamide, 4 g of p-toluenesulfonic acid, and 2.5 g of sodium bromide were added to a three-necked flask, heated to 150° C. and refluxed for 3 hours, cooled to room temperature, added with 500 ml of water, stirred for 5 minutes, filtered, and the solid was recrystallized from methanol to obtain 11 g of compound 5;

[0085] (6) Add 10 g of ergosta-2,22-dien-6-one (Compound 5), 500 ml of tert-butyl alcohol, 45 g of potassium ferrocyanide, 20 g of anhydrous potassium carbonate, 2 g of methanesulfonamide, 0.1 g of potassium osmate (and 2 g of ligand DHQD-CLB) to a flask, stir at room temperature for 4 days, add 20 g of sodium sulfite and stir for 1 hour, separate the liquid and release the aqueous layer, concentrate the organic layer, dissolve it in ethyl acetate, wash it once with 10% sulfuric acid water, wash it once with brine, and then concentrate the ethyl acetate to obtain a solid, which is then recrystallized from ethyl acetate to obtain 8 g of Compound 6;

[0086] (7) Add 150 ml of chloroform and 25 ml of trifluoroacetic anhydride to a four-necked flask, cool to below 5°C, add 6 ml of 30% hydrogen peroxide dropwise, and stir for 30 minutes. Add 8 g of compound 6 and 150 ml of chloroform solution at below 10°C. After reacting at room temperature for 3 hours, add 250 ml of water to separate. Wash the chloroform layer with water, sodium sulfite and brine in sequence, dry and concentrate, and pass through a column to obtain 7 g of 24-epi-brassinolide.

[0087] Example 2

[0088] Part I: Analytical Methods

[0089] (1) Quantitative analysis of ergosta-2,22-diene-6-one (Compound 5), an intermediate in the synthesis of 24-epi-brassinolide, was performed. The specific analysis steps are as follows:

[0090] S1. Preparation of internal standard solution

[0091] Accurately weigh 1g of cholesterol (accurate to 0.0002g), dissolve it in tetrahydrofuran, dilute to 1000ml, mix well, and set aside;

[0092] S2. Preparation of standard sample solution

[0093] Accurately weigh the ergosta-2,22-dien-6-one standard sample (accurate to 0.0002 g), place it in a 10 ml volumetric flask, and use a pipette to accurately pipette 10 ml of the internal standard solution (1 mg / ml) in step S1 to dissolve it to prepare a standard sample solution. After the standard sample solution is cooled, inject 1 ul of the standard sample solution into a gas chromatograph set with chromatographic analysis conditions for analysis;

[0094] The gas chromatography analysis conditions set in step S2 of this embodiment are specifically shown in Table 1:

[0095] Table 1: Gas chromatography analysis conditions

[0096]

[0097] (2) The raw material ergosterol, the intermediates (Compound 2, Compound 3, Compound 4), the internal standard cholesterol, and the solvent tetrahydrofuran in the synthesis process of 24-epi-brassinolide were qualitatively detected using the gas phase detection method described in Table 1. The sample processing steps before gas chromatography injection are as follows:

[0098] 5 mg of sample ergosterol, intermediates (Compound 2, Compound 3, Compound 4) and internal standard cholesterol were weighed into a 10 ml volumetric flask, dissolved with tetrahydrofuran and fixed to volume, and then used for detection.

[0099] Part II: Analysis Results

[0100] The gas chromatography results obtained by the first part of the analysis method in this embodiment 2 are as shown in the attached Figures 2 to 7 As shown in Table 2;

[0101] Table 2: Analysis and test results

[0102] Serial number sample Retention time (min) 1 Ergosterol 9.6 2 Compound 2 5.198 3 Compound 3 10.323 4 Compound 4 8.215 5 Ergosta-2,22-dien-6-one (Compound 5) 8.545 6 cholesterol 8.070 7 Tetrahydrofuran 1.543

[0103] By the attached Figures 2 to 7As shown in Table 2, the intermediate control method provided in this embodiment can effectively separate the peaks of each raw material, intermediate product and internal standard, and the peaks can be well separated. However, the interval time between the separation of internal standard cholesterol and compound 4 is only about 0.14 minutes, which sometimes has an impact on the intermediate control of compound 5.

[0104] Example 3

[0105] (1) Quantitative analysis of ergosta-2,22-diene-6-one (Compound 5), an intermediate in the synthesis of 24-epi-brassinolide, was performed. The specific analysis steps are as follows:

[0106] S1. Preparation of internal standard solution

[0107] Accurately weigh 1 g of stigmasterol (accurate to 0.0002 g), dissolve it in tetrahydrofuran, dilute to 1000 ml, mix well, and set aside;

[0108] S2. Preparation of standard sample solution

[0109] Accurately weigh the ergosta-2,22-dien-6-one standard sample (accurate to 0.0002g), place it in a 10ml volumetric flask, use a pipette to accurately pipette 10ml of internal standard solution (1mg / ml) to dissolve, and prepare the standard sample solution. After the standard sample solution is cooled, inject 1ul of the standard sample solution into the gas chromatograph set with the chromatographic analysis conditions for analysis.

[0110] The gas chromatography analysis conditions set in step S2 of this embodiment are specifically shown in Table 3:

[0111] Table 3: Gas chromatography analysis conditions

[0112]

[0113] (3) The raw material ergosterol, intermediates (Compound 2, Compound 3, Compound 4), internal standard stigmasterol, and solvent tetrahydrofuran in the synthesis process of 24-epi-brassinolide were qualitatively detected using the gas phase detection method described in Table 2. The sample processing steps before gas chromatography injection are as follows:

[0114] 5 mg of sample ergosterol, intermediates (Compound 2, Compound 3, Compound 4) and internal standard stigmasterol were weighed into a 10 ml volumetric flask, dissolved with tetrahydrofuran and fixed to volume, and then used for detection.

[0115] Part II: Analysis Results

[0116] The gas chromatography results obtained by the first part of the analysis method in this embodiment 3 are as shown in the attached Figures 8-9 and as shown in Table 4;

[0117] Table 4: Analysis and test results

[0118] Serial number sample Retention time 1 Ergosterol 9.65 2 Compound 2 5.23 3 Compound 3 10.413 4 Compound 4 8.268 5 Ergosta-2,22-dien-6-one (Compound 5) 8.549 6 Stigmasterol 10.574 7 Tetrahydrofuran 1.581

[0119] The HG / T 4922-2016 standard was used to analyze 24-epi-brassinolide, and the retention time was 19.051 min. Fig.11 ;

[0120] As attached Figures 8-9 As shown in the results of Table 4, the in-process control method provided in this embodiment can effectively separate the peaks of each raw material, intermediate product, internal standard, and solvent, and the peaks can be well separated without overlapping, so that the compounds can be accurately qualitatively analyzed.

[0121] Example 4

[0122] The analytical method of Example 3 was used to quantitatively analyze ergosta-2,22-diene-6-one samples with concentrations of 0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, and 1.0 mg / ml, respectively, and a linear regression curve was made for the content and peak area. The analytical method is as follows:

[0123] (1) Calculation of correction factor f

[0124]

[0125] Where: A S is the peak area of ​​the internal standard; W B is the mass of the standard substance (ergosta-2,22-dien-6-one), mg; A B is the peak area of ​​the standard substance (ergosta-2,22-dien-6-one); W S is the mass of the internal standard, mg; C is the mass fraction of the standard (ergosta-2,22-dien-6-one), %;

[0126] (2) Determination of the content of ergosta-2,22-dien-6-one sample

[0127] The calculation formula for the content of ergosta-2,22-dien-6-one sample is:

[0128]

[0129] Where: A 甾 A is the peak area of ​​ergosta-2,22-dien-6-one in the sample; s is the peak area of ​​the internal standard; M s is the mass of the internal standard, mg; M is the mass of the sample, mg; f is the correction factor.

[0130] The results obtained according to the above method are shown in Table 5:

[0131] Table 5

[0132]

[0133] A linear regression curve was made based on the data in Table 5, y = 578858x-5246.72. Fig.10 It can be seen that the linear relationship R 2 =0.9986, indicating that this method can meet the requirements of quantitative analysis.

[0134] Comparative Example 1

[0135] This comparative example conducts a qualitative analysis on the intermediate ergosta-2,22-diene-6-one (Compound 5) in the synthesis process of 24-epi-brassinolide. The specific analysis steps are the same as those in Example 3, except that the temperature increase program of the column temperature is different. The gas chromatography analysis conditions set in step S2 of this comparative example are specifically shown in Table 6:

[0136] Table 6: Gas chromatography analysis condition settings

[0137]

[0138]

[0139] Using the gas chromatography analysis conditions set in Table 6 above, it was found that compound 5 did not have a peak in the detection results, so the column temperature was too low and it was not feasible to achieve qualitative and quantitative analysis of the compound.

[0140] Comparative Example 2

[0141] In this comparative example, ergosta-2,22-diene-6-one (Compound 5), an intermediate of 24-epi-brassinolide, was quantitatively analyzed. The specific analysis steps were the same as those in Example 3, except that the temperature increase program of the column temperature was different. The gas chromatography analysis conditions set in step S2 of this comparative example are specifically shown in Table 7:

[0142] Table 7: Gas chromatography analysis condition settings

[0143]

[0144] The test results obtained according to the gas chromatography analysis conditions set in Table 7 above are shown in the attached Fig.12 From the figure, we can see that compound 5 and the internal standard have obvious tailing, especially during the in-process control, the impurities will be wrapped in the tailing and affect the effect of quantitative detection. The detection effect under this condition is not ideal, which in turn affects the qualitative and quantitative analysis of the compound.

[0145] It should be noted that the specific features, structures, materials or characteristics described in this specification can be combined arbitrarily. In order to make the description concise, not all possible combinations of the various technical features in the above embodiments are described. If there is no contradiction, technicians in this field can combine and combine the different embodiments and features of different embodiments described in this specification.

[0146] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A quantitative analysis method for intermediates in the synthesis process of brassinolide compounds, characterized in that: The intermediate in the synthesis process of the brassinolide compound is ergosta-2,22-diene-6-one, and the analysis method uses gas chromatography, the internal standard is cholesterol or stigmasterol, and the gas chromatography conditions are: The chromatographic column was a SH-Rtx-5 capillary column; The injection port temperature is 200℃-280℃; The heating program was as follows: starting temperature 200 °C, held for 1 min, then raised to 280 °C at a heating rate of 10 °C / min, held for 13 min; The detector temperature is 200℃-280℃; The carrier gas is nitrogen; Column flow rate is 1mL / min-2.5mL / min; The split ratio is (15-25):1; The hydrogen flow rate is 25mL / min-35mL / min; The air flow rate is 250mL / min-350mL / min; The tail gas flow rate is 20mL / min-50mL / min.

2. The quantitative analysis method of intermediates in the synthesis process of brassinolide compounds according to claim 1, characterized in that: The analytical method also includes the following processing steps before gas chromatography injection: S1. Preparation of internal standard solution Accurately weigh the internal standard, dissolve it in the diluent, make up to volume, mix well, and prepare an internal standard solution with a concentration of 0.1-10 mg / ml for later use; S2. Preparation of standard sample solution Accurately weigh the standard sample of the synthetic intermediate of brassinolide compounds, place it in a volumetric flask, use a pipette to accurately absorb the internal standard solution of the quantitative step S1 to dissolve it, and prepare a standard sample solution with a concentration of 0.1~10mg / mL. After the standard sample solution is cooled, inject 1uL of the standard sample solution into a gas chromatograph with set chromatographic analysis conditions for analysis.

3. The quantitative analysis method of intermediates in the synthesis process of brassinolide compounds according to claim 2, characterized in that: The diluent in step S1 is one of methanol, ethanol, ethyl acetate, acetonitrile, tetrahydrofuran, or a mixed solvent.

4. The method for quantitative analysis of intermediates in the synthesis process of brassinolide compounds according to claim 2, characterized in that: The concentration of the internal standard solution in step S1 is 0.8-2 mg / mL, and the concentration of the standard sample solution in step S2 is 0.5-2 mg / mL.

5. The quantitative analysis method of intermediates in the synthesis process of brassinolide compounds according to any one of claims 1 to 4 is used in the preparation of plant growth regulators containing brassinolide or brassinolide synthetic intermediates.

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