Detection method of 14-hydroxybrassinosterol

Abstract

The invention belongs to the technical field of analysis and detection, and particularly relates to a detection method of 14-hydroxybrassinosterol. The detection method of 14-hydroxybrassinosterol comprises the following steps: mixing a sample to be detected with a formic acid-containing organic solvent to obtain a test solution; and carrying out liquid chromatography-tandem mass spectrometry detection on the test solution, and obtaining the content of 14-hydroxybrassinosterol in the sample to be detected according to the obtained peak area and a preset standard curve. According to the method, a tedious sample pretreatment process is not needed, mass spectrometric detection can be carried out only through simple dilution, dissolution and constant volume, operation errors in the pretreatment process are avoided, the influence of variables in the derivatization reaction process on a quantitative detection result is avoided, and a test result is accurate. In addition, the method does not need to consume time, reagents and consumable costs in the derivatization process. The method is high in analysis speed, good in reproducibility and good in applicability. 527.2 is selected as a parent ion, the fundamental peak intensity is high, and the method sensitivity is high.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and specifically relates to a detection method for 14-hydroxybrassinosteroid. Background Art

[0002] Brassinosteroids (BRs), also known as brassinolide, are polyhydroxylated sterol compounds and a new class of plant growth regulators. As endogenous plant hormones, they often participate in regulating multiple growth and development processes. There are many types of BRs, including 28-homobrassinolide, brassinolide, 24-epibrassinolide, brassinosteroid, brassinosteroid and dozens of other different structural types.

[0003] 14-Hydroxybrassinosteroid is a brassinosteroid derived from natural extracts. Its chemical name is (2β,3β,5β,22R)-2,3,14,20,22,25-hexahydroxycholester-6-one. Its structural formula is as follows:

[0004]

[0005] 14-Hydroxybrassinosteroid has no ultraviolet absorption and needs to be derivatized to form special functional groups before it can be detected by liquid chromatography. The laboratory commonly uses phenylboronic acid derivatization liquid chromatography method, but the derivatization method is complicated to operate and has high time cost and reagent and consumable costs. Moreover, the derivatization reaction process of 14-hydroxybrassinosteroid with phenylboronic acid substances is affected by the derivatization time, derivatization temperature, matrix and derivatization reagent itself. There are many uncontrollable factors, which will affect the accuracy of quantitative detection results. The derivatization reaction of 14-hydroxybrassinosteroid, especially the stability of boric acid esters generated by the reaction of boric acid and vicinal alcohols, is the key to the entire detection method. The simultaneous presence of vicinal diols at positions 2, 3, 20, and 22 in 14-hydroxybrassinosteroid may competitively react with boric acid substances to form isomers, affecting the accuracy of chromatographic qualitative and quantitative detection.

[0006] 14-Hydroxybrassinosteroids are present in low levels (trace levels) in plant matrices or preparations and are susceptible to matrix interference. The phenylboronic acid derivatization liquid chromatography method has many influencing factors and is not suitable for trace quantitative analysis. Therefore, it is urgent to establish a detection method for 14-hydroxybrassinosteroids with good accuracy and high sensitivity. Summary of the invention

[0007] In view of this, the object of the present invention is to provide a method for detecting 14-hydroxybrassinosteroids. The method for detecting 14-hydroxybrassinosteroids provided by the present invention has good accuracy and high sensitivity.

[0008] The present invention provides a method for detecting 14-hydroxybrassinosteroids, comprising the following steps:

[0009] Mixing the sample to be tested and the organic solvent containing formic acid to obtain a test solution;

[0010] The test solution is subjected to liquid chromatography-tandem mass spectrometry detection, and the content of 14-hydroxybrassinosteroid in the test sample is obtained according to the obtained mass spectrum peak area of ​​14-hydroxybrassinosteroid and a predetermined standard curve; the predetermined standard curve is a linear curve of the concentration of 14-hydroxybrassinosteroid and the corresponding mass spectrum peak area;

[0011] The liquid chromatography conditions of the liquid chromatography-tandem mass spectrometry detection include: the chromatographic column is a C18 column; the mobile phase A is a 0.01% to 1% volume fraction formic acid aqueous solution, and the mobile phase B is a 0.01% to 1% volume fraction formic acid acetonitrile solution; the flow rate is 0.4 to 0.5 mL / min, gradient elution; the column temperature is 40 to 50° C.;

[0012] The elution procedure of the gradient elution includes: 0-2min, the volume fraction of mobile phase A is maintained at 95%; 2-4min, the volume fraction of mobile phase A is reduced from 95% to 85%; 4-5min, the volume fraction of mobile phase A is maintained at 85%; 5-5.5min, the volume fraction of mobile phase A is reduced from 85% to 70%; 5.5-7min, the volume fraction of mobile phase A is reduced from 70% to 60%; 7-7.5min, the volume fraction of mobile phase A is reduced from 60% to 5%; 7.5-10min, the volume fraction of mobile phase A is maintained at 5%; 10-11min, the volume fraction of mobile phase A is increased from 5% to 95%; 11-12min, the volume fraction of mobile phase A is maintained at 95%;

[0013] The mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry detection include: ionization mode: ESI - , scanning mode: MRM, nebulizer gas temperature: 240~260℃, nebulizer gas flow rate: 9~11L / min, nebulizer gas pressure: 35~45psi, sheath gas temperature: 340~360℃, sheath gas flow rate: 10~12L / min, capillary voltage: 3~4kV; parent ions include: 527.2±0.2DA, daughter ions include: 481.3±0.2DA.

[0014] Preferably, the volume fraction of formic acid in the formic acid-containing organic solvent is 0.01% to 1%.

[0015] Preferably, the organic solvent in the formic acid-containing organic solvent includes methanol or ethyl acetate.

[0016] Preferably, the types of samples to be tested include: liquid, powder, granule, emulsifiable concentrate, parent drug or original drug.

[0017] Preferably, the dosage ratio of the sample to be tested and the formic acid-containing organic solvent is (0.001-5) g:10 mL.

[0018] Preferably, the predetermined standard curve uses a matrix-matched standard solution, and the solvent of the matrix-matched standard solution includes a blank matrix solution and a 0.01% to 1% volume fraction formic acid methanol solution.

[0019] Preferably, the linear range of the predetermined standard curve is 50 ng / mL to 21900 ng / mL.

[0020] Preferably, the chromatographic column comprises an AgilentZobax RRHD Eclipse Plus C18 chromatographic column.

[0021] Preferably, the injection volume of the liquid chromatography is 1 to 20 μL.

[0022] Preferably, the daughter ions further include: 387.1±0.2DA and 347.1±0.2DA.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides a method for detecting 14-hydroxybrassinosteroids, comprising the following steps:

[0025] Mixing the sample to be tested and the organic solvent containing formic acid to obtain a test solution;

[0026] The test solution is subjected to liquid chromatography-tandem mass spectrometry detection, and the content of 14-hydroxybrassinosteroid in the test sample is obtained according to the obtained mass spectrum peak area of ​​14-hydroxybrassinosteroid and a predetermined standard curve; the predetermined standard curve is a linear curve of the concentration of 14-hydroxybrassinosteroid and the corresponding mass spectrum peak area;

[0027] The liquid chromatography conditions of the liquid chromatography-tandem mass spectrometry detection include: the chromatographic column is a C18 column; the mobile phase A is a 0.01% to 1% volume fraction formic acid aqueous solution, and the mobile phase B is a 0.01% to 1% volume fraction formic acid acetonitrile solution; the flow rate is 0.4 to 0.5 mL / min, gradient elution; the column temperature is 40 to 50° C.;

[0028] The elution procedure of the gradient elution includes: 0-2min, the volume fraction of mobile phase A is maintained at 95%; 2-4min, the volume fraction of mobile phase A is reduced from 95% to 85%; 4-5min, the volume fraction of mobile phase A is maintained at 85%; 5-5.5min, the volume fraction of mobile phase A is reduced from 85% to 70%; 5.5-7min, the volume fraction of mobile phase A is reduced from 70% to 60%; 7-7.5min, the volume fraction of mobile phase A is reduced from 60% to 5%; 7.5-10min, the volume fraction of mobile phase A is maintained at 5%; 10-11min, the volume fraction of mobile phase A is increased from 5% to 95%; 11-12min, the volume fraction of mobile phase A is maintained at 95%;

[0029] The mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry detection include: ionization mode: ESI - , scanning mode: MRM, nebulizer gas temperature: 240~260℃, nebulizer gas flow rate: 9~11L / min, nebulizer gas pressure: 35~45psi, sheath gas temperature: 340~360℃, sheath gas flow rate: 10~12L / min, capillary voltage: 3~4kV; parent ions include: 527.2±0.2DA, daughter ions include: 481.3±0.2DA.

[0030] The present invention establishes a direct, rapid, efficient and sensitive quantitative detection method for 14-hydroxybrassinosteroids. Compared with the derivatization liquid chromatography method, the present invention does not require a cumbersome sample pretreatment process, and only requires simple dilution, dissolution and volume determination for mass spectrometry detection, thereby avoiding sample handling errors in the pretreatment process, and avoiding the influence of variables in the derivatization reaction process on the quantitative detection results, and the test results are accurate. Moreover, the present invention does not require the time, reagents and consumables costs in the derivatization process. The present invention has fast analysis speed, good reproducibility and good applicability. The present invention selects 527.2 as the parent ion, has a high base peak intensity, high method sensitivity, and a quantitative limit of 0.00000278%.

[0031] Furthermore, the formulations of 14-hydroxybrassinosteroid preparations are complex and diverse. The present invention adopts a matrix matching standard curve method, which can directly reduce the influence of most matrix substances on the quantitative detection of low-content 14-hydroxybrassinosteroids, thereby enhancing the sensitivity and specificity of the detection method.

[0032] The data from the examples show that the detection method of the present invention has the advantages of high accuracy, high sensitivity, high specificity, simplicity and rapidity, small sample amount required, simple sample pretreatment and good tolerance, etc. It can be used for the quality control of 14-hydroxybrassinosteroids, and is suitable for low-content, trace or trace level detection, such as 0.0075% 14-hydroxybrassinosteroid aqueous solution, 0.01% 14-hydroxybrassinosteroid soluble powder, 0.0036% 14-hydroxybrassinosteroid soluble solution, and 0.004% 14-hydroxybrassinosteroid soluble solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 This is the standard mass spectrum of 14-hydroxybrassinosteroid;

[0035] Figure 2 This is the total ion chromatogram of the extracted ions of 45 μg / mL 14-hydroxybrassinosteroid standard (adduct molecular ion peak 527.2);

[0036] Figure 3 This is the total ion chromatogram of extracted ions of 45 μg / mL 14-hydroxybrassinosteroid standard (quasi-molecular ion peak 481.3);

[0037] Figure 4 It is the multiple reaction monitoring chromatogram and mass spectrum (MRM detection mode) of 21.9 μg / mL 14-hydroxybrassinosteroid standard;

[0038] Figure 5 This is the multiple reaction monitoring chromatogram and mass spectrum (MRM detection mode) of 0.0075% 14-hydroxybrassinosteroid aqueous solution;

[0039] Figure 6 This is the total ion chromatogram of the 45 μg / mL 14-hydroxybrassinosteroid standard Scan;

[0040] Figure 7 It is the multiple reaction monitoring chromatogram and mass spectrum (MRM detection mode) of chromatographically pure methanol;

[0041] Figure 8 It is the multiple reaction monitoring chromatogram and mass spectrum (MRM detection mode) of chromatographically pure acetonitrile;

[0042] Fig. 9It is the multiple reaction monitoring chromatogram and mass spectrum of blank matrix solution (MRM detection mode);

[0043] Fig.10 This is the linear relationship diagram of the standard curve in Test Example 2. DETAILED DESCRIPTION

[0044] The present invention provides a method for detecting 14-hydroxybrassinosteroids, comprising the following steps:

[0045] Mixing the sample to be tested and the organic solvent containing formic acid to obtain a test solution;

[0046] The test solution is subjected to liquid chromatography-tandem mass spectrometry detection, and the content of 14-hydroxybrassinosteroid in the test sample is obtained according to the obtained mass spectrum peak area of ​​14-hydroxybrassinosteroid and a predetermined standard curve; the predetermined standard curve is a linear curve of the concentration of 14-hydroxybrassinosteroid and the corresponding mass spectrum peak area;

[0047] The liquid chromatography conditions of the liquid chromatography-tandem mass spectrometry detection include: the chromatographic column is a C18 column; the mobile phase A is a 0.01% to 1% volume fraction formic acid aqueous solution, and the mobile phase B is a 0.01% to 1% volume fraction formic acid acetonitrile solution; the flow rate is 0.4 to 0.5 mL / min, gradient elution; the column temperature is 40 to 50° C.;

[0048] The elution procedure of the gradient elution includes: 0-2min, the volume fraction of mobile phase A is maintained at 95%; 2-4min, the volume fraction of mobile phase A is reduced from 95% to 85%; 4-5min, the volume fraction of mobile phase A is maintained at 85%; 5-5.5min, the volume fraction of mobile phase A is reduced from 85% to 70%; 5.5-7min, the volume fraction of mobile phase A is reduced from 70% to 60%; 7-7.5min, the volume fraction of mobile phase A is reduced from 60% to 5%; 7.5-10min, the volume fraction of mobile phase A is maintained at 5%; 10-11min, the volume fraction of mobile phase A is increased from 5% to 95%; 11-12min, the volume fraction of mobile phase A is maintained at 95%;

[0049] The mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry detection include: ionization mode: ESI - , scanning mode: MRM, nebulizer gas temperature: 240~260℃, nebulizer gas flow rate: 9~11L / min, nebulizer gas pressure: 35~45psi, sheath gas temperature: 340~360℃, sheath gas flow rate: 10~12L / min, capillary voltage: 3~4kV; parent ions include: 527.2±0.2DA, daughter ions include: 481.3±0.2DA.

[0050] In the present invention, unless otherwise specified, the materials and equipment used are commercially available products in the art.

[0051] The present invention mixes a sample to be tested with an organic solvent containing formic acid to obtain a test solution.

[0052] In the present invention, the volume fraction of formic acid in the formic acid-containing organic solvent is preferably 0.01% to 1%, more preferably 0.1%, and the organic solvent in the formic acid-containing organic solvent preferably includes methanol or ethyl acetate.

[0053] In the present invention, the types of the sample to be tested preferably include: liquid, powder, granule, emulsifiable concentrate, parent drug or original drug; the liquid preferably includes an aqueous solution, the liquid is preferably a soluble liquid, the powder is preferably a soluble powder, and the granule is preferably a soluble granule. The mass content of 14-hydroxybrassinosteroid in the sample to be tested is preferably 0.0001% to 95%, specifically 0.0075%, 0.01% or 0.002%.

[0054] When the sample to be tested is a liquid, the mass content of 14-hydroxybrassinosteroid in the sample to be tested is preferably 0.0001% to 0.01%; the dosage ratio of the sample to be tested and the formic acid-containing organic solvent is preferably (0.05 to 5) g:10 mL, specifically 1 g:10 mL or 2 g:10 mL, and the specific sample concentration detection range is 50 ng / mL to 21900 ng / mL.

[0055] When the sample to be tested is a powder or granule, the mass content of 14-hydroxybrassinosteroid in the sample to be tested is preferably 0.0001% to 0.01%; the dosage ratio of the sample to be tested and the organic solvent containing formic acid is preferably (0.05 to 5) g:10 mL, specifically 1 g:10 mL, and the specific sample concentration detection range is 50 ng / mL to 21900 ng / mL. The mixing preferably includes: adding the sample to be tested to the organic solvent containing formic acid for ultrasonic dissolution, centrifugation, and filtering the obtained supernatant. The time for ultrasonic dissolution is preferably 5 min; the speed of the centrifugation is preferably 5000 r / min, and the time is preferably 5 min; the filtration preferably uses a 0.22 μm filter membrane.

[0056] When the sample to be tested is emulsifiable concentrate, the mass content of 14-hydroxybrassinosteroid in the sample to be tested is preferably 0.0001% to 0.01%; the dosage ratio of the sample to be tested and the formic acid-containing organic solvent is preferably (0.05 to 5) g:10 mL, specifically 1 g:10 mL, and the specific sample concentration detection range is 50 ng / mL to 21900 ng / mL. The organic solvent in the formic acid-containing organic solvent is preferably ethyl acetate.

[0057] When the sample to be tested is a parent drug, the mass content of 14-hydroxybrassinosteroid in the sample to be tested is preferably 0.01% to 90%; the dosage ratio of the sample to be tested and the formic acid-containing organic solvent is preferably (0.001 to 0.1) g:10 mL, and the specific sample concentration detection range is 50 ng / mL to 21900 ng / mL.

[0058] When the sample to be tested is the original drug, the mass content of 14-hydroxybrassinosteroid in the sample to be tested is preferably 90% to 95%; the dosage ratio of the sample to be tested and the organic solvent containing formic acid is preferably (0.001 to 0.1) g:10 mL, and the specific sample concentration detection range is 50 ng / mL to 21900 ng / mL. The organic solvent in the organic solvent containing formic acid is preferably methanol.

[0059] In the present invention, the mixing preferably further comprises filtering, and the filtration preferably uses a 0.22 μm filter membrane.

[0060] After obtaining the test solution, the present invention performs liquid chromatography-tandem mass spectrometry detection on the test solution, and obtains the content of 14-hydroxybrassinosteroid in the test sample according to the obtained mass spectrum peak area of ​​14-hydroxybrassinosteroid and a predetermined standard curve; the predetermined standard curve is a linear curve of the concentration of 14-hydroxybrassinosteroid and the corresponding mass spectrum peak area;

[0061] The liquid chromatography conditions of the liquid chromatography-tandem mass spectrometry detection include: the chromatographic column is a C18 column; the mobile phase A is a 0.01% to 1% volume fraction formic acid aqueous solution, and the mobile phase B is a 0.01% to 1% volume fraction formic acid acetonitrile solution; the flow rate is 0.4 to 0.5 mL / min, gradient elution; the column temperature is 40 to 50° C.;

[0062] The elution procedure of the gradient elution includes: 0-2min, the volume fraction of mobile phase A is maintained at 95%; 2-4min, the volume fraction of mobile phase A is reduced from 95% to 85%; 4-5min, the volume fraction of mobile phase A is maintained at 85%; 5-5.5min, the volume fraction of mobile phase A is reduced from 85% to 70%; 5.5-7min, the volume fraction of mobile phase A is reduced from 70% to 60%; 7-7.5min, the volume fraction of mobile phase A is reduced from 60% to 5%; 7.5-10min, the volume fraction of mobile phase A is maintained at 5%; 10-11min, the volume fraction of mobile phase A is increased from 5% to 95%; 11-12min, the volume fraction of mobile phase A is maintained at 95%;

[0063] The mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry detection include: ionization mode: ESI -, scanning mode: MRM, nebulizer gas temperature: 240~260℃, nebulizer gas flow rate: 9~11L / min, nebulizer gas pressure: 35~45psi, sheath gas temperature: 340~360℃, sheath gas flow rate: 10~12L / min, capillary voltage: 3~4kV; parent ions include: 527.2±0.2DA, daughter ions include: 481.3±0.2DA.

[0064] In the present invention, the chromatographic column preferably comprises an AgilentZobax RRHD Eclipse Plus C18 chromatographic column, and the specification is preferably 2.1×50 mm, 1.8 μm.

[0065] In the present invention, the mobile phase A is preferably a 0.1% by volume formic acid aqueous solution, and the mobile phase B is preferably a 0.1% by volume formic acid acetonitrile solution.

[0066] In the present invention, the flow rate is preferably 0.45 mL / min.

[0067] In the present invention, the injection volume of the liquid chromatography is preferably 1 to 20 μL.

[0068] In the present invention, the column temperature is preferably 45°C.

[0069] In the present invention, the nebulizing gas temperature is preferably 250°C, the nebulizing gas flow rate is preferably 10 L / min, the nebulizing gas pressure is preferably 40 psi, the sheath gas temperature is preferably 350°C, the sheath gas flow rate is preferably 11 L / min, and the capillary voltage is preferably 3.5 kV.

[0070] In the present invention, the daughter ions preferably also include: 387.1±0.2DA and 347.1±0.2DA. The present invention selects 527.2 as the quantitative detection adduct molecular ion peak of 14-hydroxybrassinosteroid, and the method has high sensitivity. The present invention preferably uses 481.3 as the quantitative ion, that is, the quantitative ion pair is 527.2>481.3; the qualitative ion pair is 527.2>387.1 and 527.2>347.1.

[0071] In the present invention, the linear range of the predetermined standard curve is preferably 50 ng / mL to 21900 ng / mL, specifically 219 ng / mL to 21900 ng / mL, the predetermined standard curve preferably uses a matrix-matched standard solution, the solvent of the matrix-matched standard solution preferably includes a blank matrix solution and a 0.01% to 1% formic acid methanol solution, the volume ratio of the blank matrix solution to the 0.01% to 1% formic acid methanol solution is preferably (0.1 to 1): (9 to 9.9), and the solvent can specifically be a mixture of a blank matrix solution and a 0.1% formic acid methanol solution in a volume ratio of 1:9.

[0072] In the present invention, the predetermined standard curve is preferably: y=5.830573*x+104.1467, R 2 =0.996658, where x is the concentration and y is the response peak area.

[0073] In the present invention, the detection method of 14-hydroxybrassinosteroids is preferably quantitatively performed by external standard method.

[0074] Brassinolide substances have great similarities in structure, but the differentiation of different structures, different substituents, and different substitution sites can lead to huge differences in their functionality and effects. The detection method is not applicable to different brassinolide substances. The data of the embodiment show that the detection method of 14-hydroxybrassinolide provided by the present invention has the advantages of high accuracy, high sensitivity, strong specificity, simplicity and rapidity, small sample amount required, simple sample pretreatment and good tolerance, and is suitable for trace or constant detection of 14-hydroxybrassinolide.

[0075] In order to further illustrate the present invention, the detection method of 14-hydroxybrassinosteroid provided by the present invention is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0076] In the embodiments of the present invention, the 0.1% formic acid methanol solution, 0.1% formic acid aqueous solution or 0.1% formic acid acetonitrile solution used are all formic acid in volume concentration.

[0077] Example 1

[0078] The high-sensitivity quantitative detection method of 14-hydroxybrassinosteroid by high performance liquid chromatography tandem mass spectrometry comprises the following steps:

[0079] Sample preparation: Accurately weigh 1.0 g (accurate to 0.0002 g) of 0.0075% 14-hydroxybrassinosteroid aqueous solution into a 10 mL volumetric flask, dilute to volume with 0.1% formic acid methanol solution, shake well and filter (0.22 μm filter membrane) before testing on an instrument.

[0080] Standard solution: accurately weigh 0.01 g (accurate to 0.00002 g, actual weight 10.98 mg) of 14-hydroxybrassinosteroid standard product on an analytical balance and dissolve it in chromatographic methanol to make up to 25 mL. Take 1.0 mL and transfer it to a 10 mL volumetric flask, make up to volume, and filter it through a 0.22 μm filter membrane for sample analysis.

[0081] High performance liquid chromatography tandem triple quadrupole mass spectrometry (LC-MS / MS) detection parameters:

[0082] Liquid phase conditions: Agilent Zobax RRHD Eclipse Plus C18 chromatographic column, 2.1×50 mm, 1.8 μm; injection volume 1 μL; mobile phase A is 0.1% formic acid in water, and mobile phase B is 0.1% formic acid in acetonitrile; flow rate is 0.45 mL / min; column temperature is 45°C; peak retention time is about 6 min, running time is 12 min; gradient elution, elution conditions are shown in Table 1:

[0083] Table 1 Liquid phase gradient elution conditions

[0084]

[0085] Mass spectrometry conditions: The MRM mass spectrometry parameters were established based on the molecular weight information obtained in Scan mode. The strongest response signal was obtained by scanning in negative ion mode. The ion source parameters are shown in Table 2 below:

[0086] Table 2 Ion source parameters of mass spectrometry

[0087] Ionization method <![CDATA[ESI - ]]> Atomizing gas temperature 250℃ Nebulizer gas flow rate 10L / min Atomizing gas pressure 40psi Sheath gas temperature 350℃ Sheath gas flow rate 11L / min Capillary voltage 3.5kV

[0088] The multiple reaction monitoring ion pairs and mass spectrometry related parameters of 14-hydroxybrassinosteroids are shown in Table 3 below:

[0089] Table 31 Multiple reaction monitoring ion pairs and mass spectrometry related parameters of 4-hydroxybrassinosteroids

[0090]

[0091] Note: * indicates quantitative ion.

[0092] 14-Hydroxybrassinosteroid Molecular formula: C 27 H 46 O7, molecular weight: 482.7 g / mol, CAS number: 457603-63-3, its (ESI - The theoretical quasi-molecular ion peak in the )Scan scanning mode is 481.7, and the molecular weight scan is performed using a 45 μg / mL 14-hydroxybrassinosteroid standard solution, and the quasi-molecular ion peak is 481.3.

[0093] Figure 1 is the standard mass spectrum of 14-hydroxybrassinosteroid; the mass spectrum represents the characteristic spectrum of a compound, which can reflect the characteristic quasi-molecular ion peak mass spectrum, fragment ion mass spectrum, and adduct ion mass spectrum of the substance; in ESI - In scanning mode, the quasi-molecular ion peak should be 481.3, but due to the particularity of the substance, the formate ion is added, which increases its response in the mass spectrum by dozens of times. The response intensity of the base peak ion is 100%, which can cover the quasi-molecular ion peak response.

[0094] Figure 2 This is the total ion chromatogram of the extracted ions of 45 μg / mL 14-hydroxybrassinosteroid standard (adduct molecular ion peak 527.2); Figure 3 This is the total ion chromatogram of the extracted ions of the 45 μg / mL 14-hydroxybrassinosteroid standard (quasi-molecular ion peak 481.3). It can be seen that at the same concentration of 45 μg / mL, the quantitative detection parameters were established with 527.2 as the adducted molecular ion peak, and the method sensitivity was 161.8 times higher than that with 481.3 as the quasi-molecular ion peak (8401008.8 / 51918.52=161.8). The adducted molecular mass of 527.2 is [M+HCOOH-H] - , 14-hydroxybrassinosteroid standard product has a molecular weight of 482.7 and a formate group (HCOO - ) loses another hydrogen. Since the ESI ion source ionization in mass spectrometry follows the "law of non-conservation of mass energy", the molecular weight addition will lose part of the mass. Therefore, the present invention selects 527.2 as the 14-hydroxybrassinosteroid quantitative detection adduct molecular ion peak, takes 527.2 as the base peak intensity of 100%, and selects [M+HCOOH-H] - The MRM (multiple reaction ion monitoring) quantitative detection parameters were established based on the molecular mass of 527.2.

[0095] Figure 4 It is the multiple reaction monitoring chromatogram and mass spectrum (MRM detection mode) of 21.9 μg / mL 14-hydroxybrassinosteroid standard; Figure 5 This is the multiple reaction monitoring chromatogram and mass spectrum (MRM detection mode) of 0.0075% 14-hydroxybrassinosteroid aqueous solution;

[0096] Figure 6 This is the total ion chromatogram of the 45 μg / mL 14-hydroxybrassinosteroid standard Scan;

[0097] Figure 7 It is the multiple reaction monitoring chromatogram and mass spectrum (MRM detection mode) of chromatographically pure methanol;

[0098] Figure 8 It is the multiple reaction monitoring chromatogram and mass spectrum (MRM detection mode) of chromatographically pure acetonitrile;

[0099] Fig. 9 It is the multiple reaction monitoring chromatogram and mass spectrum of blank matrix solution (MRM detection mode);

[0100] The above figures show that no interfering ions and impurities were introduced into the chromatographic methanol, chromatographic acetonitrile, formic acid and sample pretreatment process; the reagents and consumables used did not contain the substances to be detected.

[0101] Example 2

[0102] 1. Determination of linear relationship:

[0103] Accurately weigh about 0.01g (accurate to 0.00002g) of 14-hydroxybrassinosteroid standard with a purity of 99%. Prepare a matrix matching working solution with a blank matrix solution (i.e., a matrix solution without the target substance of 14-hydroxybrassinosteroid) and a 0.1% formic acid methanol solution in a volume ratio of 1:9. Prepare a standard curve with the matrix matching working solution to make its concentration range from 219ng / mL to 21900ng / mL, which are 219, 876, 2190, 4380, 8760, and 21900ng / mL respectively; quantitative ion pair: 527.2>481.3; qualitative ion pairs 527.2>387.1 and 527.2>347.1 are used as characteristic fragment ions of quantitative ions to exclude the influence of other fragment ions on the quantitative detection results. Establish a standard curve with concentration as the horizontal axis and peak area as the vertical axis, and calculate the regression equation and correlation coefficient.

[0104] Fig.10 The linear relationship diagram of the standard curve. The regression equation of the matrix standard curve of this method is y = 5.830573*x + 104.1467, R 2 =0.996658 (y is the response peak area, x is the concentration), and the linear relationship is good.

[0105] 2. Recovery rate of detection method:

[0106] The standard was added to the blank matrix solution of 14-hydroxybrassinosteroid aqueous solution. The prepared standard solution concentration was 0.438 mg / mL. The spiked amounts were 20 μL, 50 μL, and 200 μL, respectively. Three blank matrix solutions were weighed at each spiked concentration point. A total of 9 spiked samples were used to complete the spiked recovery test. The spiked recovery and relative standard deviation (RSD) of 14-hydroxybrassinosteroid were obtained. The specific data are shown in Table 4 below:

[0107] Table 4 Recovery and relative standard deviation of 14-hydroxybrassinosteroids

[0108]

[0109]

[0110] From the data in the table, it can be seen that the average recoveries of the three added levels of 14-hydroxybrassinosteroid aqueous solution blank matrix solution were 96.79%, 101.76% and 101.92%, respectively, and the relative standard deviations were 0.905%, 0.546% and 2.382%, respectively, which met the requirements, indicating that the detection method has high accuracy.

[0111] 3. Precision of the detection method: Under the fixed determination conditions of the method, the closeness between the results obtained by multiple sampling determinations. Accurately weigh 6 portions of 0.0075% 14-hydroxybrassinosteroid aqueous solution and process according to the sample pretreatment process of Example 1, inject the sample for detection and analysis, and process the data with the established standard curve. The precision test results are shown in Table 5 below:

[0112] Table 5 Precision test results

[0113]

[0114] The detection result of 0.0075% 14-hydroxybrassinosteroid aqueous solution was 0.00786%, and the relative standard deviation was 0.805%, which met the requirements, indicating that this method has good detection precision and accurate test results.

[0115] 4. Limit of Quantitation

[0116] The 14-hydroxybrassinosteroid standard was gradually diluted to an instrument signal-to-noise ratio of S / N=10 as the minimum quantitative detection concentration, and the quantification limit of HPLC tandem mass spectrometry was 0.00000278%.

[0117] The present invention provides a high-sensitivity quantitative detection method for 14-hydroxybrassinosteroids by high-performance liquid chromatography tandem mass spectrometry, with a chromatographic peak retention time of 6 minutes, a total running time of 12 minutes, and a sample preparation time of approximately 10 minutes. The total running time of a sample from preparation to result detection does not exceed 30 minutes. Compared with the derivatization liquid chromatography method, the total time consumed for the sample from weighing to concentration and then to derivatization reaction is approximately 120 minutes; the present invention can fully save time costs. At the same time, the present invention does not require derivatization, and the sample can be tested on the machine after simple treatment, which fully saves reagents (derivative reagents such as phenylboronic acid or boric acid reagents) and reduces energy consumption in the concentration and derivatization processes. The present invention can be completed at room temperature, and does not require an additional derivatization heating environment, which not only saves energy and reduces emissions, but also reduces costs and increases efficiency; and the sample dosage of the present invention is small, only 0.05 to 5g (derivation requires at least 10g, HG / T4924-2016 brassinolide aqueous solution, weighing about 0.001g of brassinolide), or even lower, the mobile phase is simple to prepare, only simple chromatographic acetonitrile and formic acid water are needed, and multi-batch and multi-sample detection can be completed with high throughput and high efficiency, comprehensively improving work efficiency and reducing time, reagents, and consumables costs. The comparison data of the two methods are shown in Table 6 below:

[0118] Table 6 Comparison between the detection method of the present invention and the derivatization method

[0119]

[0120] Example 3

[0121] 1. Determination of 14-hydroxybrassinosteroid soluble powder:

[0122] Sample preparation: Under the conditions of Example 1, when the sample to be tested is a 0.01% 14-hydroxybrassinosteroid soluble powder, accurately weigh 1.0 g (accurate to 0.0002 g) of 0.01% 14-hydroxybrassinosteroid soluble powder, accurately add 10 mL of formic acid-containing organic solvent (0.1% formic acid methanol solution) to the sample to be tested, ultrasonically dissolve for 5 min, centrifuge at 5000 r / min for 5 min to obtain the supernatant, filter (0.22 μm filter membrane) and then test on the machine. The specific sample concentration detection range is 50 ng / mL to 21900 ng / mL.

[0123] 2. Determination of the recovery rate of 14-hydroxybrassinosteroid soluble powder

[0124] The standard substance was added to the blank matrix of 14-hydroxybrassinosteroid soluble powder, and the prepared standard solution concentration was 0.438 mg / mL. The spiked amounts were 20 μL, 50 μL, and 200 μL, respectively. Three blank matrices were weighed at each spiked concentration point, and a total of 9 spiked samples were used to complete the spiked recovery test. The spiked recovery and relative standard deviation (RSD) of 14-hydroxybrassinosteroid were obtained. The specific data are shown in Table 7 below:

[0125] Table 7 Recovery and relative standard deviation of 14-hydroxybrassinosteroid soluble powder

[0126]

[0127] From the data in the table, it can be seen that the average recoveries of the three added levels of 14-hydroxybrassinosteroid soluble powder blank matrix were 98.07%, 99.42% and 101.37%, respectively, and the relative standard deviations were 1.319%, 0.572% and 1.155%, respectively, which met the relevant requirements, indicating that the detection method has high accuracy.

[0128] 3. Determination of the precision of 14-hydroxybrassinosteroid soluble powder

[0129] Under the determination conditions of the method, the closeness between the results obtained by multiple sampling determinations. Accurately weigh 6 portions of 0.01% 14-hydroxybrassinosteroid soluble powder and process according to the sample pretreatment process of Example 3, inject the sample for detection and analysis, and process the data with the established standard curve. The precision test results are shown in Table 8 below:

[0130] Table 8 Precision test results

[0131]

[0132]

[0133] The detection result of 0.01% 14-hydroxybrassinosteroid soluble powder was 0.01009%, and the relative standard deviation was 1.073%, which met the requirements, indicating that this method has good detection precision and accurate test results.

[0134] Example 4

[0135] 1. Determination of 14-hydroxybrassinosteroid soluble granules:

[0136] Sample preparation: Under the conditions of Example 1, when the sample to be tested is 40% 14-hydroxybrassinosteroid·gibberellic acid soluble granules, accurately weigh 1.0g (accurate to 0.0002g) of 40% 14-hydroxybrassinosteroid·gibberellic acid soluble granules (14-hydroxybrassinosteroid content is 0.002%), accurately add 10mL of formic acid-containing organic solvent (0.1% formic acid methanol solution) to the sample to be tested, ultrasonically dissolve for 5min, centrifuge at 5000r / min for 5min to obtain the supernatant, filter (0.22μm filter membrane) and then test on the machine. The specific sample concentration detection range is 50ng / mL to 21900ng / mL.

[0137] 2. Determination of the recovery rate of 40% 14-hydroxybrassinosteroid·gibberellic acid soluble granules

[0138] The standard substance was added to the blank matrix of 40% 14-hydroxybrassinosteroid·gibberellic acid soluble granules. The prepared standard solution concentration was 0.438 mg / mL. The spiked amounts were 20 μL, 50 μL, and 200 μL, respectively. Three blank matrices were weighed at each spiked concentration point. A total of 9 spiked samples were used to complete the spiked recovery test. The spiked recovery and relative standard deviation (RSD) of 14-hydroxybrassinosteroid were obtained. The specific data are shown in Table 9 below:

[0139] Table 9 Addition recovery and relative standard deviation of 40% 14-hydroxybrassinosteroid·gibberellic acid soluble granules

[0140]

[0141] From the data in the table, it can be seen that the average recoveries of the three addition levels in the blank matrix of 40% 14-hydroxybrassinosteroid·gibberellic acid soluble granules were 98.10%, 99.19% and 100.86%, respectively, and the relative standard deviations were 1.277%, 0.810% and 1.220%, respectively, which met the relevant requirements, indicating that the detection method has high accuracy.

[0142] 3. Determination of the precision of 40% 14-hydroxybrassinosteroid·gibberellic acid soluble granules

[0143] Under the determination conditions fixed by the method, the closeness between the results obtained by multiple sampling determinations. Accurately weigh 6 portions of 40% 14-hydroxybrassinosteroid·gibberellic acid soluble granules and process them according to the sample pretreatment process of Example 4, inject the samples for detection and analysis, and process the data with the established standard curve. The precision test results are shown in Table 10 below:

[0144] Table 10 Precision test results

[0145]

[0146] The detection result of 40% 14-hydroxybrassinosteroid·gibberellic acid soluble granules was 0.00200%, and the relative standard deviation was 0.949%, which met the requirements, indicating that this method has good detection precision and accurate test results.

[0147] Example 5

[0148] 1. Determination of 0.01% 14-hydroxybrassinosteroid emulsifiable concentrate:

[0149] Sample preparation: Accurately weigh 1.0g (accurate to 0.0002g) of 0.01% 14-hydroxybrassinosteroid emulsifiable concentrate into a 10mL volumetric flask, dilute to scale with 0.1% formic acid ethyl acetate solution, shake well and filter (0.22μm filter membrane) before testing on the machine. The specific sample concentration detection range is 50ng / mL to 21900ng / mL.

[0150] 2. Determination of the recovery rate of 0.01% 14-hydroxybrassinosteroid emulsifiable concentrate

[0151] The standard was added to the blank matrix of 0.01% 14-hydroxybrassinosteroid emulsifiable concentrate, and the prepared standard solution concentration was 0.438 mg / mL. The spiked amounts were 20 μL, 50 μL, and 200 μL, respectively. Three blank matrices were weighed at each spiked concentration point, and a total of 9 spiked samples were used to complete the spiked recovery test, and the spiked recovery and relative standard deviation (RSD) of 14-hydroxybrassinosteroid were obtained. The specific data are shown in Table 11 below:

[0152] Table 11 Recovery and relative standard deviation of 0.01% 14-hydroxybrassinosteroid emulsifiable concentrate

[0153]

[0154]

[0155] From the data in the table, it can be seen that the average recoveries of the three added levels in the blank matrix of 0.01% 14-hydroxybrassinosteroid emulsifiable concentrate were 99.39%, 99.49% and 101.43%, respectively, and the relative standard deviations were 1.461%, 0.330% and 1.066%, respectively, which met the relevant requirements, indicating that the detection method has high accuracy.

[0156] 3. Determination of the precision of 0.01% 14-hydroxybrassinosteroid emulsifiable concentrate

[0157] Under the determination conditions of the method, the closeness between the results obtained by multiple sampling determinations was measured. 6 portions of 0.01% 14-hydroxybrassinosteroid emulsifiable concentrate were accurately weighed and processed according to the sample pretreatment process of Example 5, and the samples were injected for detection and analysis. The data were processed with the established standard curve. The precision test results are shown in Table 12 below:

[0158] Table 12 Precision test results

[0159]

[0160] The detection result of 0.01% 14-hydroxybrassinosteroid emulsifiable concentrate was 0.01010%, and the relative standard deviation was 1.327%, which met the requirements, indicating that this method has good detection precision and accurate test results.

[0161] Although the above-mentioned embodiments have made a detailed description of the present invention, they are only some embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on the embodiments of the present invention without creative work, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for detecting 14-hydroxybrassinosteroids, characterized in that: The following steps are involved: Mixing the sample to be tested and the organic solvent containing formic acid to obtain a test solution; The test solution is subjected to liquid chromatography-tandem mass spectrometry detection, and the content of 14-hydroxybrassinosteroid in the test sample is obtained according to the obtained mass spectrum peak area of ​​14-hydroxybrassinosteroid and a predetermined standard curve; the predetermined standard curve is a linear curve of the concentration of 14-hydroxybrassinosteroid and the corresponding mass spectrum peak area; The liquid chromatography conditions of the liquid chromatography-tandem mass spectrometry detection include: the chromatographic column is a C18 column; the mobile phase A is a 0.01% to 1% volume fraction formic acid aqueous solution, and the mobile phase B is a 0.01% to 1% volume fraction formic acid acetonitrile solution; the flow rate is 0.4 to 0.5 mL / min, gradient elution; the column temperature is 40 to 50° C.; The elution procedure of the gradient elution includes: 0-2min, the volume fraction of mobile phase A is maintained at 95%; 2-4min, the volume fraction of mobile phase A is reduced from 95% to 85%; 4-5min, the volume fraction of mobile phase A is maintained at 85%; 5-5.5min, the volume fraction of mobile phase A is reduced from 85% to 70%; 5.5-7min, the volume fraction of mobile phase A is reduced from 70% to 60%; 7-7.5min, the volume fraction of mobile phase A is reduced from 60% to 5%; 7.5-10min, the volume fraction of mobile phase A is maintained at 5%; 10-11min, the volume fraction of mobile phase A is increased from 5% to 95%; 11-12min, the volume fraction of mobile phase A is maintained at 95%; The mass spectrometry conditions of the liquid chromatography-tandem mass spectrometry detection include: ionization mode: ESI - , scanning mode: MRM, nebulizer gas temperature: 240~260℃, nebulizer gas flow rate: 9~11L / min, nebulizer gas pressure: 35~45psi, sheath gas temperature: 340~360℃, sheath gas flow rate: 10~12L / min, capillary voltage: 3~4kV; parent ions include: 527.2±0.2DA, daughter ions include: 481.3±0.2DA.

2. The detection method according to claim 1, characterized in that: The volume fraction of formic acid in the formic acid-containing organic solvent is 0.01% to 1%.

3. The detection method according to claim 1 or 2, characterized in that: The organic solvent in the formic acid-containing organic solvent includes methanol or ethyl acetate.

4. The detection method according to claim 1, characterized in that: The types of samples to be tested include: liquid, powder, granule, emulsifiable concentrate, parent drug or original drug.

5. The detection method according to claim 2 or 4, characterized in that: The dosage ratio of the sample to be tested and the formic acid-containing organic solvent is (0.001-5) g:10 mL.

6. The detection method according to claim 1, characterized in that: The predetermined standard curve uses a matrix matching standard solution, and the solvent of the matrix matching standard solution includes a blank matrix solution and a 0.01% to 1% volume fraction formic acid methanol solution.

7. The detection method according to claim 1 or 6, characterized in that: The linear range of the predetermined standard curve is 50 ng / mL to 21900 ng / mL.

8. The detection method according to claim 1, characterized in that: The chromatographic column comprises an Agilent Zobax RRHDEclipse Plus C18 chromatographic column.

9. The detection method according to claim 1, characterized in that: The injection volume of the liquid chromatography is 1 to 20 μL.

10. The detection method according to claim 1 or 9, characterized in that: The daughter ions also include: 387.1±0.2DA and 347.1±0.2DA.

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