Method for simultaneously detecting residual quantity of 22 acidic plant growth regulators in traditional Chinese medicinal materials

Abstract

The invention relates to the technical field of traditional Chinese medicinal material detection, in particular to a method for simultaneously detecting residual quantity of 22 acidic plant growth regulators in traditional Chinese medicinal materials. A traditional Chinese medicine raw material sample is extracted by adopting acidified acetonitrile, C18 and GCB combined purification is performed, parent ions and daughter ions are collected at the same time in a positive and negative ion mode, an external standard method is adopted for quantification, 22 acidic plant growth regulators are good in linearity in a corresponding linear range (R2 is greater than or equal to 0.999), the recovery rate of 22 acidic plant growth regulators in three different samples is 65.6%-115.4%, and the recovery rate of 22 acidic plant growth regulators in the three different samples is 65.6%-115.4%. The relative standard deviation is 0.9%-9.8%, the method detection limit of alpha-naphthylacetic acid is 120.0 [mu] g / kg, and the method detection limit of other target compounds is 0.7-7.0 [mu] g / kg. The method is simple, rapid and sensitive to operate, and is suitable for qualitative screening and quantitative detection of the acidic plant growth regulator in the traditional Chinese medicine raw materials.

Description

Technical Field

[0001] The patent of this invention relates to the technical field of Chinese medicinal materials detection, and specifically to a method for simultaneously detecting the residues of 22 acidic plant growth regulators in Chinese medicinal materials. Background Art

[0002] Plant growth regulators are synthetic chemicals that regulate plant growth and development, with chemical properties similar to plant hormones. Their use in traditional Chinese medicine production can break dormancy, promote germination, increase transplant survival rates, promote rooting of cuttings, and increase yields, ultimately improving the quality of the resulting herbs. However, while most plant growth regulator residues in traditional Chinese medicines are low-toxic, long-term, low-dose exposure to these chemicals may pose risks to human health. This is especially true for compounds that are highly lipophilic and readily bind to enzymes and proteins. These compounds can produce cumulative toxicity, affecting the normal functioning of the reproductive and endocrine systems, brain tissue, and liver, and may also increase the risk of infertility, cancer, and neurological diseases. To ensure the safety of traditional Chinese medicines, scientific testing methods are necessary for monitoring. Commonly used testing techniques include liquid chromatography, gas chromatography, and mass spectrometry. However, the current high-performance liquid chromatography-mass spectrometry detection method for plant growth regulators in traditional Chinese medicines still has problems such as complex sample matrix components and incomplete pretreatment and purification, resulting in a high final detection limit.

[0003] For example, patent application publication number CN119125358A discloses a method for simultaneously detecting the residues of 19 plant growth regulators in traditional Chinese medicines. The 19 plant growth regulators detected in the traditional Chinese medicines are gibberellins, 4-chlorophenoxyacetic acid, 6-benzyladenine, paclobutrazol, 3-indolebutyric acid, thidiazuron, sodium 2,4-dichlorophenoxyacetate, chlorfenapyr, 3-indoleacetic acid, 4-fluorophenoxyacetic acid, isopentenyl adenine, imidacloprid, N-N'-diphenylurea, mepiquat, diethylaminoethyl ester, 1-naphthylacetamide, brassinolide, abscisic acid, and 3-indolepropionic acid. The pretreatment QuEChERS cleanup step adopts the method of MgSO4+PSA+GCB, and the final detection limit is relatively high. The pretreatment cleanup method needs to be further improved. Summary of the Invention

[0004] To address the above technical issues, the present invention establishes a rapid determination method for 22 acidic plant growth regulators in traditional Chinese medicine raw materials using QuEChERS technology combined with high-performance liquid chromatography-quadrupole / electrostatic field orbitrap high-resolution mass spectrometry (HPLC-Q-Orbitrap HRMS). Samples are extracted with 0.5% acetic acid and acetonitrile, salted out with sodium chloride and anhydrous magnesium sulfate, and cleaned with a C18 and GCB mixture. The compounds are then separated on a C18 column. Full-scan and auto-triggered secondary mass spectrometry (FullMS / dd-MS2) monitoring modes are used for simultaneous positive and negative ion scanning, and quantification is performed using an external standard method. The results showed that the 22 acidic plant growth regulators exhibited good linearity within the corresponding linear range (R² ≥ 0.999). In actual sample spike recovery experiments, the recoveries of the 22 acidic plant growth regulators in three different sample types ranged from 65.6% to 115.4%, with relative standard deviations of 0.9% to 9.8%. The method detection limit was 120.0 μg / kg for α-naphthylacetic acid and 0.7-7.0 μg / kg for the other target compounds. This method is simple, rapid, and sensitive, making it suitable for the qualitative screening and quantitative detection of acidic plant growth regulators in traditional Chinese medicine raw materials.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] The present invention provides a method for simultaneously detecting the residues of 22 acidic plant growth regulators in traditional Chinese medicines, comprising the following steps:

[0007] (1) Preparation of matrix solution: The matrix raw material was crushed and soaked in deionized water. Then, a mixed solution of acetonitrile and 0.5% acetic acid was added and vortexed. Anhydrous magnesium sulfate and sodium chloride were added, shaken, and centrifuged. The supernatant was collected. The residue was extracted again with a mixed solution of acetonitrile and 0.5% acetic acid. The supernatants were combined, nitrogen was blown, and the volume was adjusted with methanol. The solution was vortexed. C18, GCB, and anhydrous magnesium sulfate were added. The solution was vortexed and centrifuged. The supernatant was filtered through a membrane to obtain the matrix solution for later use.

[0008] (2) Preparation of matrix standard working solution: prepare matrix standard working solution of abscisic acid, abscisic acid, cyclohexanediol, cyclohexanediol, cyclohexanediol, salicylic acid, α-naphthylacetic acid, chloramphenic acid, gibberellic acid, gibberellic acid, gibberellin A7, 4-chlorophenoxyacetic acid, 4-fluorophenoxyacetic acid, 4-bromophenoxyacetic acid, 4-iodophenoxyacetic acid, 3-indoleacetic acid, 3-indolebutyric acid, 3-indolepropionic acid, 2-naphthylacetic acid, 2,4-dichlorophenoxyacetic acid, 2,4-dichlorpropionic acid, 2,4,5-trichlorophenoxyacetic acid and 2,3,5-triiodobenzoic acid respectively with the matrix solution in step (1) for use;

[0009] (3) Preparation of the sample solution to be tested: After the Chinese medicinal material raw materials are crushed, they are soaked in deionized water, and then a mixed solution of acetonitrile-0.5% acetic acid is added, vortexed, and then anhydrous magnesium sulfate and sodium chloride are added. The mixture is shaken and centrifuged. The supernatant is collected, and the acetonitrile-0.5% acetic acid mixed solution is added to the residue and the extraction is repeated once. The supernatants are combined, nitrogen is blown, and the volume is adjusted with methanol. The mixture is vortexed, C18, GCB and anhydrous magnesium sulfate are added, vortexed, centrifuged, and the supernatant is filtered through a membrane to obtain the sample solution to be tested, which is then set aside.

[0010] (4) Determination of standard working curve: The matrix standard working solution in step (2) is measured by high performance liquid chromatography-quadrupole / electrostatic field orbitrap high resolution mass spectrometry to draw a matrix standard curve;

[0011] (5) The sample solution to be tested in step (3) is measured by high performance liquid chromatography-quadrupole / electrostatic field orbital trap high resolution mass spectrometry, and the residual amounts of abscisic acid, abscisic acid, cyclohexane, cyclohexane, cyclohexane, salicylic acid, α-naphthylacetic acid, chloramphenic acid, gibberellic acid, gibberellin A7, 4-chlorophenoxyacetic acid, 4-fluorophenoxyacetic acid, 4-bromophenoxyacetic acid, 4-iodophenoxyacetic acid, 3-indoleacetic acid, 3-indolebutyric acid, 3-indolepropionic acid, 2-naphthylacetic acid, 2,4-dichlorophenoxyacetic acid, 2,4-dichlorpropionic acid, 2,4,5-trichlorophenoxyacetic acid, and 2,3,5-triiodobenzoic acid in the sample to be tested are calculated according to the matrix standard curve.

[0012] Furthermore, in step (1) and step (3), the mass ratio of C18, GCB and anhydrous magnesium sulfate is 2:1:4.

[0013] Further preferably, the amount of the matrix raw material added in step (1) is 1.0 g, the volume of the methanol constant volume is 2.0 mL, and the amounts of the C18, GCB and anhydrous magnesium sulfate added are 50 mg C18, 25 mg GCB and 100 mg anhydrous magnesium sulfate, respectively; the amount of the Chinese medicinal material added in step (3) is 1.0 g, the volume of the methanol constant volume is 2.0 mL, and the amounts of the C18, GCB and anhydrous magnesium sulfate added are 50 mg C18, 25 mg GCB and 100 mg anhydrous magnesium sulfate, respectively.

[0014] Furthermore, the sample to be tested in step (3) is one of Radix Ophiopogonis, Pericarpium Citri Reticulatae, Radix Astragali, Poria, Polygonatum Sibiricum, Morinda Officinalis, Schisandrae Chinensis, Semen Cassiae, Panax Notoginseng, Angelicae Sinensis, Atractylodes Macrocephalae, Ganoderma Lucidum, Codonopsis Pilosulae, Rhodiola Rosea, Fructus Amomi, and Flos Lonicerae.

[0015] Furthermore, in step (4), the linear range of the matrix standard curve is: 10-1000 μg / L of citric acid, 2-1000 μg / L of abscisic acid, 2-1000 μg / L of prohexadione, 4-1000 μg / L of citric acid, 10-1000 μg / L of citric acid, 2-1000 μg / L of salicylic acid, 200-20000 μg / L of α-naphthyl acetic acid, 3-1000 μg / L of chloramphenicol, 4-1000 μg / L of gibberellic acid, 2-1000 μg / L of gibberellin A7, 2-1000 μg / L of 4-chlorophenoxyacetic acid, 4-1000 μg / L of 4-fluorophenoxyacetic acid. Phenoxyacetic acid 4-1000μg / L, 4-bromophenoxyacetic acid 4-1000μg / L, 4-iodophenoxyacetic acid 3-1000μg / L, 3-indoleacetic acid 1-500μg / L, 3-indolebutyric acid 1-500μg / L, 3-indolepropionic acid 1-500μg / L, 2-naphthyloxyacetic acid 2-1000μg / L, 2,4-dichlorophenoxyacetic acid 5-1000μg / L, 2,4-dichlorpropionic acid 5-1000μg / L, 2,4,5-trichlorophenoxyacetic acid 5-1000μg / L, 2,3,5-triiodobenzoic acid 5-1000μg / L.

[0016] Furthermore, the conditions of the high performance liquid chromatography are as follows: the chromatographic column is ACQUITYUPLC BEH C18 (100 mm×2.1 mm, 1.7 μm); column temperature: 30°C; injection volume: 3 μL; flow rate: 0.3 mL / min; mobile phase A: 0.1% formic acid-0.5 mM ammonium formate, mobile phase B: methanol, gradient elution program: 0.0-0.5 min, 5% B; 0.5-2.0 min, 5% B-60% B; 2.0-6.0 min, 60% B-95% B; 6.0-8.0 min, 95% B; 8.0-8.1 min, 95% B-5% B; 8.1-10.0 min, 5% B.

[0017] Furthermore, the mass spectrometry conditions are as follows: the ion source is equipped with a heated electrospray ionization (HESI) source, and positive and negative ions are scanned simultaneously; the capillary temperature is 320°C; the auxiliary gas temperature is 350°C; the spray voltage is 3.5 kV; the sheath gas and auxiliary gas are both nitrogen, with the pressure and flow rate set to 40 and 10 psi, respectively; the mass spectrometry mode is a primary mass spectrometry full scan and a data-dependent secondary mass spectrometry full scan; the scanning range is m / z 50-600; the full scan resolution is 70,000 FWHM; and the trigger threshold is 3×10 6 Secondary product ion scanning resolution: 17500FWHM; trigger threshold: 1×10 5 ; Maximum injection time: 80 ms; Separation window: m / z 3.0; Normalized collision energy set to 20%, 40%, 60%.

[0018] The beneficial effects of this application are as follows:

[0019] The present invention establishes a method for rapidly determining 22 acidic plant growth regulators in traditional Chinese medicine raw materials using a QuEChERS-HPLC-Q-Orbitrap HRMS method. The traditional Chinese medicine raw material samples are extracted with acidified acetonitrile, and C18 and GCB are jointly cleaned up. Parent ions and daughter ions are simultaneously collected in positive and negative ion modes. The results are quantified using a matrix standard solution external standard method. Abscisic acid, salicylic acid, 3-indoleacetic acid, 3-indolebutyric acid, 3-indolepropionic acid, gibberellic acid, 2,4-dichlorophenoxyacetic acid, 4-chlorophenoxyacetic acid, and furanic acid are detected in the traditional Chinese medicine raw material samples. The method has good accuracy and sensitivity and is suitable for the qualitative and quantitative analysis of acidic plant growth regulator residues in traditional Chinese medicine raw materials. The method can provide data support for standardizing the application of plant growth regulators in traditional Chinese medicine raw materials and ensuring the quality and safety of medicinal raw materials.

[0020] The results showed that the 22 acidic plant growth regulators exhibited good linearity within the corresponding linear range (R² ≥ 0.999). In actual sample spike recovery experiments, the recoveries of the 22 acidic plant growth regulators in three different sample types ranged from 65.6% to 115.4%, with relative standard deviations of 0.9% to 9.8%. The response for α-naphthylacetic acid was poor, with a method detection limit of 120.0 μg / kg. The method detection limits for the other target compounds ranged from 0.7 to 7.0 μg / kg. This method is simple, rapid, and sensitive, making it suitable for the qualitative screening and quantitative detection of acidic plant growth regulators in traditional Chinese medicine raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the extracted ion chromatogram of 22 compounds in Example 1.

[0022] Figure 2 This is a comparison chart of the extraction recovery rates of acidic growth regulators under 22 different extraction solvent conditions in Example 1, Comparative Example 1, and Comparative Example 2.

[0023] Figure 3 This is a comparison chart of the recovery rates of acidic growth regulators under different purification material conditions in Example 1, Comparative Example 3, Comparative Example 4, and Comparative Example 5, wherein 1 is 50 mg PSA + 100 mg MgSO4; 2 is 50 mg C18 + 100 mg MgSO4; 3 is 50 mg GCB + 100 mg MgSO4; and 4 is 50 mg C18 + 25 mg GCB + 100 mg MgSO4.

[0024] Figure 4 This is a comparison chart of the matrix effects of 22 compounds in Codonopsis pilosula, Panax notoginseng, and Morinda officinalis in Example 1.

[0025] Figure 5 These are the extracted ion chromatograms and secondary mass spectra of abscisic acid and salicylic acid in honeysuckle and the extracted ion chromatogram and secondary mass spectra of 3-indoleacetic acid in Schisandra chinensis in Example 1, wherein a is the extracted ion chromatogram and secondary mass spectra of abscisic acid, b is the extracted ion chromatogram and secondary mass spectra of salicylic acid, and c is the extracted ion chromatogram and secondary mass spectra of 3-indoleacetic acid in Schisandra chinensis. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0027] In the following examples and comparative examples, abscisic acid (100 mg / L), abscisic acid (1000 mg / L), salicylic acid (1000 mg / L), α-naphthylacetic acid (100 mg / L), gibberellic acid (1000 mg / L), 4-chlorophenoxyacetic acid (1000 mg / L) and 16 solid standard substances: prohexadione (99.9%), procarbamic acid (99.7%), procarbamic acid (99.9%), Chloramino cyprolic acid (99.9%), Gibberellic acid A7 (99.2%), 4-fluorophenoxyacetic acid (97.0%), 4-bromophenoxyacetic acid (98.3%), 4-iodophenoxyacetic acid (97.0%), 3-indoleacetic acid (99.2%), 3-indolebutyric acid (99.9%), 3-indolepropionic acid (98.8%), 2-naphthyloxyacetic acid (99.5%), 2,4-dichlorophenoxyacetic acid (99.4%). %), 2,4-dichlorpropionic acid (99.6%), 2,4,5-trichlorophenoxyacetic acid (96.3%), and 2,3,5-triiodobenzoic acid (99.9%) were purchased from Shanghai Anpu Experimental Technology Co., Ltd.; chromatographically pure methanol and acetonitrile (ACN) were produced by Merck, Germany; chromatographically pure formic acid (FA), acetic acid (Ac), and ammonium formate were produced by CNW, Germany; anhydrous magnesium sulfate, sodium chloride, ethylenediamine-N-propylsilane (PSA), octadecyl bonded silica gel (C18), and graphitized carbon black (GCB) were provided by Shanghai Anpu Experimental Technology Co., Ltd.; ultrapure water was prepared in-house using an ultrapure water instrument; matrix raw materials (including Codonopsis pilosula matrix raw material, Panax notoginseng matrix raw material, Morinda officinalis matrix raw material, Lonicera japonica matrix raw material, and Schisandra chinensis matrix raw material, purchased commercially); and test samples (including Codonopsis pilosula, Panax notoginseng, Morinda officinalis, Lonicera japonica, and Schisandra chinensis, purchased commercially).

[0028] Example 1

[0029] A method for simultaneously detecting the residues of 22 acidic plant growth regulators in traditional Chinese medicines comprises the following steps:

[0030] (1) Preparation of matrix solution: 1.0 g of ground Codonopsis pilosula matrix sample was placed in a 50 mL plastic centrifuge tube, added to 8 mL of deionized water and soaked for 20 min, added to 10 mL of acetonitrile-0.5% acetic acid, vortexed for 1 min, then added to 4 g of anhydrous magnesium sulfate and 2 g of sodium chloride, shaken for 10 min, centrifuged at 4200 r / min for 5 min, aspirated the supernatant, added 10 mL of acetonitrile-0.5% acetic acid to the residue and extracted once more, the supernatants were combined, and nitrogen was blown to about 0.5 mL at 40°C, and the volume was made up to 2.0 mL with methanol. After vortexing for 30 s, 50 mg of C18, 25 mg of GCB, and 100 mg of anhydrous magnesium sulfate were added to the fixed solution, vortexed for 1 min, centrifuged at 2000 r / min for 3 min, and the supernatant was filtered through a membrane to obtain the Codonopsis pilosula matrix solution for later use;

[0031] The chemical components in Chinese herbal medicine raw materials are rich and diverse, including alkaloids, flavonoids, glycosides and polysaccharides. These components cannot be completely removed through the purification step, so they may cause significant interference to the target analytes, resulting in a matrix effect (ME). ME = (peak area of the target in the standard solution prepared with matrix solution - peak area of the target in the matrix) / peak area of the target in the standard solution of the solvent × 100%. When ME is between 80% and 120%, it is generally believed that the matrix effect can be ignored. The present invention investigated the matrix effects of 22 acidic plant growth regulators in three different types of Chinese herbal medicine raw material samples: Codonopsis pilosula, Panax notoginseng and Morinda officinalis. The experimental results are as follows: Figure 4 As shown, eight compounds in Panax notoginseng exhibited matrix inhibition, only one compound in Codonopsis pilosula exhibited matrix inhibition, and one compound in Morinda officinalis exhibited matrix enhancement. SANTE / 11813 / 2017 stipulates that if matrix inhibition or enhancement exceeds 20%, matrix effects should be corrected. Therefore, to reduce the impact of matrix effects, the present invention uses a matrix standard curve for quantification.

[0032] (2) Preparation of matrix standard working solution: 16 kinds of solid acidic plant growth agent standard substances (hexadione (99.9%), hydroxybenzoic acid (99.7%), hydroxybenzoic acid (99.9%), chloramphenic acid (99.9%), gibberellin A7 (99.2%), 4-fluorophenoxyacetic acid (97.0%), 4-bromophenoxyacetic acid (98.3%), 4-iodophenoxyacetic acid (97.0%), 3-indoleacetic acid (99.2%), 3-indolebutyric acid (99.9%), 3-indolepropionic acid (98.8%), 2-naphthyloxyacetic acid (99.5%), 2,4-dichlorophenoxyacetic acid (99.5%), 4-chlorophenoxyacetic acid (99.2%), 4-chlorophenoxyacetic acid (99.9 ...9%), 4-chlorophenoxyacetic acid (99.9%), 4-chlorophenoxyacetic acid (99.9%), 4-chlorophenoxyacetic acid (99.9%), 4-chlorophenoxyacetic acid (99.9%), 4-chlorophenoxyacetic acid (99.9%), Acetic acid (99.4%), 2,4-dichlorpropionic acid (99.6%), 2,4,5-trichlorophenoxyacetic acid (96.3%), 2,3,5-triiodobenzoic acid (99.9%)) were dissolved in methanol to prepare a standard stock solution with a mass concentration of about 1000 mg / L; 6 liquid standard substances (bezoarcinic acid (100 mg / L), abscisic acid (1000 mg / L), salicylic acid (1000 mg / L), α-naphthylacetic acid (100 mg / L), gibberellic acid (1000 mg / L), 4-chlorophenoxyacetic acid (1000 mg / L)) were directly used as standard stock solutions, and then The standard stock solution was diluted with the Codonopsis pilosula matrix solution in step (1) to 10-1000 μg / L of citric acid, 2-1000 μg / L of abscisic acid, 2-1000 μg / L of prohexadione, 4-1000 μg / L of prohexadione, 10-1000 μg / L of prohexadione, 2-1000 μg / L of salicylic acid, 200-20000 μg / L of α-naphthylacetic acid, 3-1000 μg / L of chloramphenicol, 4-1000 μg / L of gibberellic acid, 2-1000 μg / L of gibberellin A7, 2-1000 μg / L of 4-chlorophenoxyacetic acid, 4-10 00μg / L, 4-bromophenoxyacetic acid 4-1000μg / L, 4-iodophenoxyacetic acid 3-1000μg / L, 3-indoleacetic acid 1-500μg / L, 3-indolebutyric acid 1-500μg / L, 3-indolepropionic acid 1-500μg / L, 2-naphthyloxyacetic acid 2-1000μg / L, 2,4-dichlorophenoxyacetic acid 5-1000μg / L, 2,4-dichlorpropionic acid 5-1000μg / L, 2,4,5-trichlorophenoxyacetic acid 5-1000μg / L, 2,3,5-triiodobenzoic acid 5-1000μg / L, for later use;

[0033] (3) Preparation of the sample solution: 1.0 g of the crushed Codonopsis pilosula was placed in a 50 mL plastic centrifuge tube, and 8 mL of deionized water was added to soak for 20 min. 10 mL of acetonitrile-0.5% acetic acid was added and vortexed for 1 min. 4 g of anhydrous magnesium sulfate and 2 g of sodium chloride were added, and the mixture was shaken for 10 min. The mixture was centrifuged at 4200 r / min for 5 min. The supernatant was aspirated, and 10 mL of acetonitrile-0.5% acetic acid was added to the residue and the extraction was repeated once. The supernatants were combined and nitrogen was blown to about 0.5 mL at 40°C. The mixture was diluted to 2.0 mL with methanol and vortexed for 30 s. 50 mg of C18, 25 mg of GCB, and 100 mg of anhydrous magnesium sulfate were added to the diluted solution. The mixture was vortexed for 1 min and centrifuged at 2000 r / min for 3 min. The supernatant was filtered through a membrane to obtain the Codonopsis pilosula sample solution for later use.

[0034] (4) Determination of standard working curve: The matrix standard working solution in step (2) was determined by high performance liquid chromatography-quadrupole / electrostatic field orbitrap high resolution mass spectrometry to draw a matrix standard curve; wherein, the conditions of the high performance liquid chromatography: the chromatographic column is ACQUITY UPLC BEH C18 (100 mm × 2.1 mm, 1.7 μm) (the effects of three chromatographic columns with different fillers including ACQUITY UPLC HSS T3 (100 mm × 2.1 mm, 1.8 μm), ACQUITY UPLC BEH C18 (100 mm × 2.1 mm, 1.7 μm), and ACQUITY UPLC BEHAmide (100 mm × 2.1 mm, 1.7 μm) on the peak shape and retention time of 22 acidic plant growth regulators were compared. The results showed that under the same mobile phase conditions, when the Amide column was used, the peak shape of some plant growth regulators was poor and the response was lower than that of the other two chromatographic columns; while when the HSS column was used, the peak shape of some plant growth regulators was poor and the response was lower than that of the other two chromatographic columns; When using the T3 and C18 columns, the peak shapes of all targets were good, and the responses of most targets were significantly better than those of the Amide column. When using the C18 column, the responses of all compounds were the best). Column temperature: 30°C (column temperature can affect the retention time and response value of the compound. The retention time and response of acidic plant growth regulators were studied at column temperatures of 30°C, 35°C, and 40°C. The results showed that with the increase of column temperature, the retention time and response of the compound gradually decreased. Therefore, the final column temperature was selected as 30°C. ; Injection volume 3 μL; Flow rate: 0.3 mL / min; Mobile phase A: 0.1% formic acid-0.5 mM ammonium formate, Mobile phase B: methanol, Gradient elution program: 0.0-0.5 min, 5% B; 0.5-2.0 min, 5% B-60% B; 2.0-6.0 min, 60% B-95% B; 6.0-8.0 min, 95% B; 8.0-8.1 min, 95% B-5% B; 8.1-10.0 min, 5% B.(The organic phase compared the effects of methanol and acetonitrile on the peak shape and response intensity of 22 acidic plant growth agents. The results showed that when acetonitrile was used as the organic phase, some plant growth agents such as 3-indoleacetic acid, 3-indolebutyric acid, and 3-indolepropionic acid responded very poorly to the standard solutions of the same concentration, and procyclic acid showed a split peak. When methanol was used as the organic phase, the responses of most targets were better than acetonitrile and the peak shape was better. Methanol was used as the organic phase; the aqueous phase mainly compared the effects of formic acid and ammonium formate concentrations on the peak shape and response intensity of plant growth agents. Pure water, 0.1% formic acid, 0.3% formic acid, 1mM ammonium formate, 0.1% formic acid-0.5mM ammonium formate, 0.1% formic acid-1mM ammonium formate, 0.1% formic acid- 5mM ammonium formate is used as the aqueous phase of the mobile phase. When pure water is used as the aqueous phase, most acidic plant growth agents respond best, but the peak width is very wide and the peak shape is poor, and tailing is prone to occur. When formic acid is added, formic acid will inhibit the ionization of acidic plant growth agents in negative ion mode, resulting in a decrease in response, but the peak shape of the compound is significantly improved. However, some compounds still have a wide peak width, and with the increase of formic acid concentration, the response of each compound gradually decreases. When ammonium formate is added, the response of acidic plant growth agents will also decrease, but the peak shape will become better. The higher the ammonium formate concentration, the worse the response. Therefore, for acidic plant growth agents, after comprehensively considering the peak shape and response of the target compound, 0.1% formic acid-0.5mM ammonium formate is selected as the aqueous phase). The mass spectrometry conditions were as follows: the ion source was equipped with a heated electrospray ionization (HESI) source, with simultaneous scanning of positive and negative ions; the capillary temperature was 320°C; the auxiliary gas temperature was 350°C; the spray voltage was 3.5 kV; nitrogen was used for both sheath gas and auxiliary gas, with the pressure and flow rate set to 40 and 10 psi, respectively; the mass spectrometry modes were primary mass spectrometry full scan and data-dependent secondary mass spectrometry full scan; the scan range was m / z 50-600; the full scan resolution was 70,000 FWHM; and the trigger threshold was 3 × 10. 6 Secondary product ion scanning resolution: 17500FWHM; trigger threshold: 1×10 5 Maximum injection time: 80 ms; separation window: m / z 3.0; normalized collision energy set to 20%, 40%, 60%. The chromatographic mass spectrometric information of the 22 acidic plant growth regulators obtained is shown in Table 1 below:

[0035] Table 1 Chromatographic and mass spectrometric information of 22 acidic plant growth regulators

[0036]

[0037]

[0038] The extracted ion chromatograms of the 22 compounds were obtained as follows: Figure 1 As shown;

[0039] At the same time, the peak area of the parent ion (Y) was plotted against the corresponding mass concentration (X, μg / L) to obtain the linear equation and correlation coefficient (R 2 The standard solution was gradually diluted until the concentration corresponding to the target substance signal-to-noise ratio S / N ≥ 3 was the instrument detection limit (ILOD), and the concentration corresponding to S / N ≥ 10 was the instrument quantification limit (ILOQ). The ILOD and ILOQ were combined with the sample dilution factor (2 times) and the signal-to-noise ratio method to estimate the method detection limit (MLOD) and method quantification limit (MLOQ). The experimental results are shown in Table 2. The correlation coefficients (R 2 ) were all greater than 0.999; the response of α-naphthylacetic acid was poor, with its MLOD of 120.0 μg / kg and MLOQ of 400.0 μg / kg. The MLOD of the other 21 acidic plant growth regulators were between 0.7 and 7.0 μg / kg, and their MLOQs were between 2.0 and 20.0 μg / kg.

[0040] Table 2 Linear range and linear equation, correlation coefficient, detection limit and quantification limit of 22 acidic plant growth regulators

[0041]

[0042]

[0043] (5) The Codonopsis pilosula sample solution to be tested in step (3) is measured by high performance liquid chromatography-quadrupole / electrostatic field orbital trap high resolution mass spectrometry, and the residual amounts of abscisic acid, abscisic acid, cyclohexane, cyclohexane, cyclohexane, salicylic acid, α-naphthylacetic acid, chloramphenic acid, gibberellic acid, gibberellin A7, 4-chlorophenoxyacetic acid, 4-fluorophenoxyacetic acid, 4-bromophenoxyacetic acid, 4-iodophenoxyacetic acid, 3-indoleacetic acid, 3-indolebutyric acid, 3-indolepropionic acid, 2-naphthylacetic acid, 2,4-dichlorophenoxyacetic acid, 2,4-dichlorpropionic acid, 2,4,5-trichlorophenoxyacetic acid, and 2,3,5-triiodobenzoic acid in the sample to be tested are calculated according to the matrix standard curve. At the same time, this embodiment also uses the same method to detect the residual amount of 22 kinds of acidic plant growth regulators in multiple batches of Radix Ophiopogonis, Tangerine Peel, Radix Astragali, Poria, Polygonatum, Morinda Officinalis, Schisandrae Chinensis, Cassiae Seed, Panax Notoginseng, Radix Angelicae Sinensis, Atractylodes Macrocephalae, Ganoderma Lucidum, Codonopsis Pilosula, Rhodiola Rosea, Fructus Amomi, and Flos Lonicerae. It is found that the Chinese medicine raw material samples tested all contain salicylic acid, with a content of 13.4-16996.4μg / kg; most samples contain compounds such as abscisic acid and 3-indoleacetic acid, with a detection rate of 91.4% and a content of 6.0-16798.1μg / kg; the rest include 3-indoleacetic acid, 3-indolepropionic acid, gibberellic acid, 3-indolebutyric acid, and furanic acid, with contents ranging from a few μg / kg to several hundred μg / kg. The extracted ion chromatograms and secondary mass spectra of abscisic acid and salicylic acid in Flos Lonicerae and the extracted ion chromatograms and secondary mass spectra of 3-indoleacetic acid in Schisandrae Chinensis are shown in Figure 2. Figure 5 As shown, a is the extracted ion chromatogram and secondary mass spectrum of abscisic acid, b is the extracted ion chromatogram and secondary mass spectrum of salicylic acid, and c is the extracted ion chromatogram and secondary mass spectrum of 3-indoleacetic acid in Schisandra chinensis. The retention times, primary parent ions and secondary product ions are consistent with the standard substance information in Table 1.

[0044] Since no blank matrix was found that was completely free of the 22 target compounds, the spike concentrations for the undetected compounds in the samples were set at the LOQ, 5×LOQ, and 10×LOQ, respectively. For the detected target compounds, the spike concentrations were approximately 0.5×, 1×, and 2× the detection value. Each concentration was repeated three times, and the average recovery was calculated. The results are shown in Table 3. The average recovery of the 22 acidic plant growth regulators in Codonopsis pilosula ranged from 70.9% to 115.4%, with relative standard deviations of 1.2% to 8.6%; the average recovery of the 22 acidic plant growth regulators in Panax notoginseng ranged from 65.6% to 105.6%, with relative standard deviations of 1.1% to 9.8%; and the average recovery of the 22 acidic plant growth regulators in Morinda officinalis ranged from 70.0% to 111.6%, with relative standard deviations of 0.9% to 9.5%. All recoveries met the national standard requirements, indicating that the method has good accuracy.

[0045] Table 3 Recoveries and relative standard deviations of 22 acidic plant growth regulators in Codonopsis pilosula, Panax notoginseng, and Morinda officinalis (n=3)

[0046]

[0047] Comparative Example 1

[0048] The difference from Example 1 is that, during the pretreatment process, acetonitrile is used as the extraction solvent instead of acetonitrile-0.5% acetic acid.

[0049] Comparative Example 2

[0050] The difference from Example 1 is that, during the pretreatment process, the extraction solvent is acetonitrile-1% acetic acid instead of acetonitrile-0.5% acetic acid.

[0051] Comparative Example 3

[0052] The difference from Example 1 is that, in the pretreatment process, the purification material uses 50 mg PSA + 100 mg MgSO 4 instead of 50 mg C18 + 25 mg GCB + 100 mg MgSO 4 .

[0053] Comparative Example 4

[0054] The difference from Example 1 is that, in the pretreatment process, the purification material uses 50 mg C18 + 100 mg MgSO4 instead of 50 mg C18 + 25 mg GCB + 100 mg MgSO4.

[0055] Comparative Example 5

[0056] The difference from Example 1 is that, in the pretreatment process, the purification material uses 50 mg GCB + 100 mg MgSO 4 instead of 50 mg C18 + 25 mg GCB + 100 mg MgSO 4 .

[0057] The comparison of the extraction efficiency of acetonitrile-0.5% acetic acid, acetonitrile, and acetonitrile-1% acetic acid for 22 acidic plant growth regulators in Example 1, Comparative Example 1, and Comparative Example 2 is shown in the figure below: Figure 2 As shown in the figure, the extraction efficiency of the three extraction solvents for most acidic plant growth regulators is not much different. When pure acetonitrile is used as the extraction solvent, only the extraction recoveries of furanic acid and naphthylacetic acid are poor (48.9% and 68.0%). After adding 0.5% acetic acid, the pH is lowered and the acidic conditions are more conducive to the extraction of acidic plant growth regulators. Therefore, the extraction recovery of these two compounds can be increased to more than 70%, and the recovery rates of other compounds are also greater than 70%. When the volume of acetic acid is increased to 1%, the recovery rates of some compounds such as gibberellic acid and 4-fluorophenoxyacetic acid decrease slightly.

[0058] The recovery rate comparison of the acidic growth regulator under the four purification material conditions in Example 1, Comparative Example 3, Comparative Example 4 and Comparative Example 5 is shown in the figure below: Figure 3 As shown, because PSA contains -NH2 bonds, it can combine with carboxyl-containing compounds. Therefore, when PSA is used as a purification material, the recovery rates of various acidic plant growth regulators such as abscisic acid, prohexadione, and salicylic acid are very poor; when C18 is used as a purification material, the recoveries of 22 target substances are all good; when 50 mg of GCB is used as a purification material, the recovery rates of substances containing planar structures such as 3-indoleacetic acid and gibberellic acid are poor; and when the amount of GCB used is reduced to 25 mg and combined with 50 mg of C18 as a purification material (50 mg C18 + 25 mg GCB + 100 mg MgSO4), the recoveries of 3-indoleacetic acid and gibberellic acid increase. At this time, the recoveries of 22 acidic plant growth regulators are 73.5% to 109.3%, and the recoveries all meet the requirements.

[0059] The above is a specific description of the preferred implementation of the present invention, but the invention is not limited to the embodiments. Those skilled in the art can make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A method for simultaneously detecting the residual amounts of 22 acidic plant growth regulators in Chinese medicinal materials, characterized in that: The following steps are involved: (1) Preparation of matrix solution: The matrix raw material was crushed and soaked in deionized water. Then, a mixed solution of acetonitrile and 0.5% acetic acid was added and vortexed. Anhydrous magnesium sulfate and sodium chloride were added, shaken, and centrifuged. The supernatant was collected. The residue was extracted again with a mixed solution of acetonitrile and 0.5% acetic acid. The supernatants were combined, nitrogen was blown, and the volume was adjusted with methanol. The solution was vortexed. C18, GCB, and anhydrous magnesium sulfate were added. The solution was vortexed and centrifuged. The supernatant was filtered through a membrane to obtain the matrix solution for later use. (2) Preparation of matrix standard working solution: prepare matrix standard working solution of abscisic acid, abscisic acid, cyclohexanediol, cyclohexanediol, cyclohexanediol, salicylic acid, α-naphthylacetic acid, chloramphenic acid, gibberellic acid, gibberellic acid, gibberellin A7, 4-chlorophenoxyacetic acid, 4-fluorophenoxyacetic acid, 4-bromophenoxyacetic acid, 4-iodophenoxyacetic acid, 3-indoleacetic acid, 3-indolebutyric acid, 3-indolepropionic acid, 2-naphthylacetic acid, 2,4-dichlorophenoxyacetic acid, 2,4-dichlorpropionic acid, 2,4,5-trichlorophenoxyacetic acid and 2,3,5-triiodobenzoic acid respectively with the matrix solution in step (1) for use; (3) Preparation of the sample solution to be tested: After the Chinese medicinal material raw materials are crushed, they are soaked in deionized water, and then a mixed solution of acetonitrile-0.5% acetic acid is added, vortexed, and then anhydrous magnesium sulfate and sodium chloride are added. The mixture is shaken and centrifuged. The supernatant is collected, and the acetonitrile-0.5% acetic acid mixed solution is added to the residue and the extraction is repeated once. The supernatants are combined, nitrogen is blown, and the volume is adjusted with methanol. The mixture is vortexed, C18, GCB and anhydrous magnesium sulfate are added, vortexed, centrifuged, and the supernatant is filtered through a membrane to obtain the sample solution to be tested, which is then set aside. (4) Determination of standard working curve: The matrix standard working solution in step (2) is measured by high performance liquid chromatography-quadrupole / electrostatic field orbitrap high resolution mass spectrometry to draw a matrix standard curve; (5) The sample solution to be tested in step (3) is measured by high performance liquid chromatography-quadrupole / electrostatic field orbital trap high resolution mass spectrometry, and the residual amounts of abscisic acid, abscisic acid, cyclohexane, cyclohexane, cyclohexane, salicylic acid, α-naphthylacetic acid, chloramphenic acid, gibberellic acid, gibberellin A7, 4-chlorophenoxyacetic acid, 4-fluorophenoxyacetic acid, 4-bromophenoxyacetic acid, 4-iodophenoxyacetic acid, 3-indoleacetic acid, 3-indolebutyric acid, 3-indolepropionic acid, 2-naphthylacetic acid, 2,4-dichlorophenoxyacetic acid, 2,4-dichlorpropionic acid, 2,4,5-trichlorophenoxyacetic acid, and 2,3,5-triiodobenzoic acid in the sample to be tested are calculated according to the matrix standard curve.

2. The method for simultaneously detecting the residues of 22 acidic plant growth regulators in Chinese medicinal materials according to claim 1, characterized in that: In step (1) and step (3), the mass ratio of C18, GCB and anhydrous magnesium sulfate is 2:1:

4.

3. The method for simultaneously detecting the residues of 22 acidic plant growth regulators in Chinese medicinal materials according to claim 1, characterized in that: The amount of the matrix raw material added in step (1) is 1.0 g, the volume of the methanol constant volume is 2.0 mL, and the amounts of the C18, GCB, and anhydrous magnesium sulfate added are 50 mg C18, 25 mg GCB, and 100 mg anhydrous magnesium sulfate, respectively; the amount of the Chinese medicinal material added in step (3) is 1.0 g, the volume of the methanol constant volume is 2.0 mL, and the amounts of the C18, GCB, and anhydrous magnesium sulfate added are 50 mg C18, 25 mg GCB, and 100 mg anhydrous magnesium sulfate, respectively.

4. The method for simultaneously detecting the residues of 22 acidic plant growth regulators in Chinese medicinal materials according to claim 1, characterized in that: The sample to be tested in step (3) is one of Radix Ophiopogonis, Pericarpium Citri Reticulatae, Radix Astragali, Poria, Polygonatum Sibiricum, Morinda Officinalis, Fructus Schisandrae Chinensis, Fructus Cassiae, Panax Notoginseng, Radix Angelicae Sinensis, Atractylodes Macrocephalae, Ganoderma Lucidum, Codonopsis Pilosulae, Rhodiola Rosea, Fructus Amomi, and Flos Lonicerae.

5. The method for simultaneously detecting the residues of 22 acidic plant growth regulators in Chinese medicinal materials according to claim 1, characterized in that: In step (4), the linear range of the matrix standard curve is: 10-1000 μg / L of citric acid, 2-1000 μg / L of abscisic acid, 2-1000 μg / L of prohexadione, 4-1000 μg / L of citric acid, 10-1000 μg / L of citric acid, 2-1000 μg / L of salicylic acid, 200-20000 μg / L of α-naphthyl acetic acid, 3-1000 μg / L of chloramphenicol, 4-1000 μg / L of gibberellic acid, 2-1000 μg / L of gibberellin A7, 2-1000 μg / L of 4-chlorophenoxyacetic acid, 4-fluorophenoxyacetic acid, Acid 4-1000μg / L, 4-bromophenoxyacetic acid 4-1000μg / L, 4-iodophenoxyacetic acid 3-1000μg / L, 3-indoleacetic acid 1-500μg / L, 3-indolebutyric acid 1-500μg / L, 3-indolepropionic acid 1-500μg / L, 2-naphthyloxyacetic acid 2-1000μg / L, 2,4-dichlorophenoxyacetic acid 5-1000μg / L, 2,4-dichlorpropionic acid 5-1000μg / L, 2,4,5-trichlorophenoxyacetic acid 5-1000μg / L, 2,3,5-triiodobenzoic acid 5-1000μg / L.

6. The method for simultaneously detecting the residues of 22 acidic plant growth regulators in Chinese medicinal materials according to claim 1, characterized in that: The HPLC conditions were as follows: ACQUITY UPLC BEH C18 column; column temperature: 30°C; injection volume: 3 μL; flow rate: 0.3 mL / min; mobile phase A: 0.1% formic acid-0.5 mM ammonium formate, mobile phase B: methanol, gradient elution program: 0.0-0.5 min, 5% B; 0.5-2.0 min, 5% B-60% B. 2.0~6.0min, 60%B~95%B; 6.0~8.0min, 95%B; 8.0~8.1min, 95%B~5%B; 8.1~10.0min, 5%B.

7. The method for simultaneously detecting the residues of 22 acidic plant growth regulators in Chinese medicinal materials according to claim 1, characterized in that: The mass spectrometry conditions were as follows: the ion source was equipped with a heated electrospray ion source, with simultaneous scanning of positive and negative ions; the capillary temperature was 320°C; the auxiliary gas temperature was 350°C; the spray voltage was 3.5 kV; both the sheath gas and the auxiliary gas were nitrogen, with the pressure and flow rate set to 40 and 10 psi, respectively; the mass spectrometry modes were primary mass spectrometry full scan and data-dependent secondary mass spectrometry full scan; the scan range was m / z 50-600; the full scan resolution was 70,000 FWHM; and the trigger threshold was 3 × 10 6 Secondary product ion scanning resolution: 17500FWHM; trigger threshold: 1×10 5 ; Maximum injection time: 80 ms; Separation window: m / z 3.0; Normalized collision energy set to 20%, 40%, 60%.

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