Method for simultaneously detecting residual quantity of 19 plant growth regulators in traditional Chinese medicinal materials

Abstract

The invention discloses a method for simultaneously detecting residual quantity of 19 plant growth regulators in traditional Chinese medicinal materials, and belongs to the technical field of traditional Chinese medicinal material detection. The 19 plant growth regulators in the detected traditional Chinese medicinal materials are gibberellins, 4-chlorophenoxyacetic acid, 6-benzyladenine, paclobutrazol, 3-indolebutyric acid, thidiazuron, 2, 4-dihydroxyacetophenone and 2, 4-dihydroxyacetophenone respectively. The pesticide is prepared from the following raw materials: sodium 4-dichlorphenoxyacetate, forchlorfenuron, 3-indoleacetic acid, 4-fluorophenoxyacetic acid, isopentenyl adenine, imazalil, N-N '-diphenylurea, mepiquat chloride, diethyl aminoethyl hexanoate, 1-naphthylacetic acid amine, brassin, abscisic acid and 3-indolepropionic acid. According to the method, the QUECHERS traditional Chinese medicinal material sample on-machine pretreatment technology is applied, the ultra-high performance liquid chromatography-tandem mass spectrometry is established to measure the residual quantity of the 19 plant growth regulators in the medicinal materials, and the detection method is simple, clear, few in operation steps, extremely easy to operate, good in linear relation, good in sensitivity and high in accuracy.

Description

Technical Field

[0001] The present invention relates to the technical field of detection of traditional Chinese medicines, and in particular to a method for simultaneously detecting the residues of 19 plant growth regulators in traditional Chinese medicines. Background Art

[0002] Plant growth regulators (PGRs) are artificially synthesized small molecule compounds used to regulate plant growth and development. They have similar effects to natural plant hormones, can enhance crop resistance, shorten crop maturity, increase yield and improve quality, and are widely used in modern crop production.

[0003] In recent years, although the development of the world pesticide market has fluctuated overall, the plant growth regulator market has maintained a steady growth trend. China is one of the countries in the world where plant growth regulators are most widely used, mainly for regulating the growth and development of crops, stabilizing and increasing production, improving quality and enhancing stress resistance. However, plant growth regulators, like other types of pesticides, have certain toxicity and residual characteristics. Blind and unreasonable use of plant growth regulators may also lead to problems such as reduced production, reduced quality and excessive residues. In recent years, due to the increasing number of food safety issues and disputes caused by the improper use of plant growth regulators, the maximum residue limit standard is particularly important for the protection of residents' health.

[0004] Plant growth regulators are now widely used in the production of Chinese herbal medicines. They play a certain role in promoting the growth and development of Chinese herbal medicines and increasing their yields. Studies have found that plant growth regulators have been used in large quantities in the cultivation of several root and rhizome herbal medicines. At the same time, the irrational use of plant growth regulators will lead to their residues in Chinese herbal medicines, which may reduce the content of active ingredients in Chinese herbal medicines and endanger the health of consumers. Chinese herbal medicines are different from general crops. The important premise for determining whether PGRs can be promoted and used in Chinese herbal medicines is to evaluate whether they have a negative impact on the effectiveness and safety of Chinese herbal medicines. my country's "Regulations on Pesticide Management" uniformly manages plant growth regulators as pesticides, but the residue limit and detection standards of plant growth regulators in Chinese herbal medicines are missing, which has led to the increasing use of plant growth regulators in the cultivation of Chinese herbal medicines, seriously affecting the quality and safety of Chinese herbal medicines and causing major hidden dangers.

[0005] At present, the main analytical detection methods for plant growth regulator residues include gas chromatography, gas chromatography-mass spectrometry, and high performance liquid chromatography. Among them, gas chromatography and gas chromatography-mass spectrometry require chemical derivatization and are relatively cumbersome to operate, while high performance liquid chromatography is completely qualitative based on retention time and has low sensitivity, a large matrix interference effect, complex pretreatment, and prone to false positives. At the same time, the above detection methods only detect a single or several plant growth regulators and are not suitable for the simultaneous detection of multi-component plant growth regulator residues in Chinese medicinal materials with different matrices. Summary of the invention

[0006] The present invention is to solve the above technical problems existing in the prior art, and provides a method for simultaneously detecting the residues of 19 plant growth regulators in Chinese medicinal materials. The present invention uses the QUECHERS Chinese medicinal material sample pretreatment technology to establish an ultra-high performance liquid chromatography-tandem mass spectrometry method to determine the residues of 19 plant growth regulators in medicinal materials. The detection method is concise and clear, with few operation steps and is very easy to use, with good linear relationship, good sensitivity and high accuracy.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A method for simultaneously detecting the residues of 19 plant growth regulators in traditional Chinese medicines comprises the following steps:

[0009] (1) Preparation of standard solution: 11 plant growth regulators, including gibberellin, 4-chlorophenoxyacetic acid, 6-benzyladenine, paclobutrazol, 3-indolebutyric acid, thidiazuron, sodium 2,4-dichlorophenoxyacetate, chlorfenapyr, 3-indoleacetic acid, 4-fluorophenoxyacetic acid, and isopentenyl adenine, were dissolved in methanol to prepare standard solutions; 8 plant growth regulators, including thiazolin, N-N'-diphenylurea, mepiquat, aminoethyl hexanoate, 1-naphthylacetamide, brassinolide, abscisic acid, and 3-indolepropionic acid, were dissolved in acetonitrile to prepare standard solutions for later use;

[0010] (2) Preparation of the sample solution to be tested: Crush the Chinese herbal medicine sample into herbal medicine powder, take the herbal medicine powder and place it in a stoppered centrifuge tube, add 1% formic acid acetonitrile solution, vortex to fully infiltrate the herbal medicine powder, add acetonitrile after standing, vortex, add anhydrous magnesium sulfate and anhydrous sodium acetate, vortex, shake on an oscillator, refrigerate centrifuge, take the supernatant into a QUECHERS purification tube, vortex, refrigerate centrifuge, take all the supernatant into a stoppered centrifuge tube, blow dry with nitrogen, add methanol solution, vortex, refrigerate centrifuge, filter with a microporous filter membrane to obtain the sample solution to be tested, and set aside;

[0011] (3) using ultra performance liquid chromatography-tandem mass spectrometry to analyze the contents of 19 plant growth regulators in the sample solution, including gibberellins, 4-chlorophenoxyacetic acid, 6-benzyladenine, paclobutrazol, 3-indolebutyric acid, thidiazuron, sodium 2,4-dichlorophenoxyacetate, chlorfenapyr, 3-indoleacetic acid, 4-fluorophenoxyacetic acid, isopentenyl adenine, chlorfenapyr, N-N'-diphenylurea, mepiquat, aminoethyl hexanoate, 1-naphthylacetamide, brassinolide, abscisic acid, and 3-indolepropionic acid;

[0012] Chromatographic conditions were as follows: chromatographic column: Agilent SB-C18 RRHD, 1.8 μm, 2.1×100 mm; mobile phase: A was 0.1% formic acid, the 0.1% formic acid contained 5 mmol / L ammonium acetate, and B was methanol; injection volume: 2 μL; flow rate: 0.25 mL / min; column temperature: 35°C;

[0013] The mass spectrometry conditions were as follows: ESI source, positive and negative ion simultaneous multiple reaction monitoring mode detection, collision gas: on; gas flow: 8.0 L / min; gas temperature: 350°C; nebulizer: 45.1 psi; sheath gas flow: 10.0 L / min; sheath gas temperature: 400°C.

[0014] Furthermore, in step (1), the concentration of the 11 standard solutions prepared by dissolving in methanol is 1 mg / mL; the concentration of the 8 standard solutions prepared by dissolving in acetonitrile is 20 μg / mL.

[0015] Furthermore, the specific operation of step (2) is as follows: the Chinese medicinal material sample is crushed into medicinal material powder, 5 g of medicinal material powder is placed in a 50 mL stoppered centrifuge tube, 15 mL of 1% formic acid acetonitrile solution is added, vortexed for 1 min to fully infiltrate the medicinal material powder, and allowed to stand for 30 min. 15 mL of acetonitrile is accurately added, vortexed for 1 min, 4 g of anhydrous magnesium sulfate and 1 g of anhydrous sodium acetate are added, vortexed immediately, shaken on an oscillator for 1 min, refrigerated centrifuged for 5 min, and the centrifugal speed is 7000 r / min. 5 mL of supernatant is accurately taken and placed in a QUECHERS purification tube (900 mg MgSO4 + 150 mg PSA + 15 mg GCB, 15mL), vortex for 1min, refrigerate centrifuge for 5min, centrifuge speed 7000r / min, take all the supernatant into a 15mL stoppered centrifuge tube, blow dry with nitrogen, accurately add 1mL methanol solution, vortex for 3min, refrigerate centrifuge for 5min, centrifuge speed 7000r / min, filter into a liquid phase vial with a 0.22μm microporous filter membrane to obtain the sample solution to be tested, and set aside.

[0016] Furthermore, in step (3), the gradient elution procedure in the chromatographic conditions is as follows:

[0017] Time, min A，％ B，％ 0.00 0.00 100.00 2.00 75.00 25.00 8.00 25.00 75.00 16.50 20.00 80.00 18.00 75.00 25.00 20.00 75.00 25.00。

[0018] Furthermore, in step (3), the mass spectrometry parameters of the 19 plant growth regulators are as follows:

[0019]

[0020]

[0021] The 2,4-D is sodium 2,4-dichlorophenoxyacetate, the 2-IP is isopentenyladenine, the 3-IBA is 3-indolebutyric acid, the 3-IPA is 3-indolepropionic acid, the 4-CPA is 4-chlorophenoxyacetic acid, the 4-FPA is 4-fluorophenoxyacetic acid, the 6-BA is 6-benzyladenine, the ABA is abscisic acid, the BAS is mepiquat, the BR is brassinolide, the BSI is chlorfenapyr, the CPPU is chlorfenapyr, the DA-6 is ampicillin, the GA is gibberellin, the IAA is 3-indoleacetic acid, the NAAAmide is 1-naphthylacetamide, the NND is N-N'-diphenylurea, the PP333 is paclobutrazol, and the TDZ is thidiazuron.

[0022] Furthermore, the Chinese medicinal materials include Pueraria root, Chinese yam, Asparagus cochinchinensis, Curcuma aromatica, ginger, and Curcuma zedoaria.

[0023] Beneficial effects:

[0024] The present invention uses QUECHERS Chinese herbal medicine sample pretreatment technology to establish an ultra-high performance liquid chromatography-tandem mass spectrometry method to simultaneously determine the residual amounts of 19 plant growth regulators in medicinal materials. By optimizing the separation degree and conditions of the 19 plant growth regulators in ultra-high performance liquid chromatography and determining the pretreatment method of the Chinese herbal medicine sample, the 19 plant growth regulators have good linear relationships within their respective concentration ranges, and the linear correlation coefficients (r) are all greater than 0.990. Using asparagus as a representative matrix, three levels of three samples were used for spiked recovery experiments, and a good recovery rate was obtained, with a relative standard deviation (RSD) of 0.19%-12.77%. The detection method of the present invention is concise and clear, has few operating steps and is extremely easy to use, has a good linear relationship, good sensitivity, and high accuracy, and can provide a reference for the determination of the residual amounts of various plant growth regulators in Chinese herbal medicines such as kudzu root, Chinese yam, asparagus, turmeric, ginger, and zedoaria. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the total ion current of the detected sample;

[0026] Figure 2It is the MRM chromatogram of plant growth regulators, in which A is 2,4-D, B is 2-IP, C is 3-IBA, D is 3-IPA, E is 4-CPA, and F is 4-FPA;

[0027] Figure 3 This is the MRM chromatogram of plant growth regulators, where G is 6-BA, H is ABA, I is BAS, J is BR, K is BSI, and L is CPPU;

[0028] Figure 4 This is the MRM chromatogram of plant growth regulators, where M is DA-6, N is GA, O is IAA, P is NAAAmide, Q is NND, and R is PP333;

[0029] Figure 5 This is the MRM chromatogram of plant growth regulators, where S is TDZ. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0031] Example

[0032] A method for simultaneously detecting the residues of 19 plant growth regulators in traditional Chinese medicines comprises the following steps:

[0033] 1 Materials and methods

[0034] 1.1 Drug testing

[0035] 2,4-dichlorophenoxyacetic acid sodium (2,4-D, mass fraction 99.40%, batch number G146114), isopentenyl adenine (2-IP, mass fraction 99.83%, batch number YF190825-17), 3-indolebutyric acid (3-IBA, mass fraction 99.20%, batch number G162446), 3-indolepropionic acid (3-IPA, 99.8μg / mL, batch number S041555), 4-chlorophenoxyacetic acid (4-CPA, mass fraction 98.5%, batch number C0005132), 4-fluorophenoxyacetic acid (4-FPA, mass fraction 99.8%, batch number AF170802-11), 6-benzyladenine (6-BA, mass fraction 98.51%, batch number C15696 00), abscisic acid (ABA, 99.7μg / mL, batch number S012301), bacitracin (BAS, 100.3μg / mL, batch number S030861), brassinolide (BR, 1002.5μg / mL, batch number S028519), bromide imidazole (BSI, 100.0μg / mL, batch number S040133), chlorfenapyr (CPPU, mass fraction 99.60%, batch number G163580), aminoethyl amine (DA-6, 100.3μg / mL, batch number S035007), gibberellin (GA, mass fraction 97.9%, batch number G124928), 3-indoleacetic acid (IAA, mass fraction 99.60%, batch number G737768), 1-naphthylacetic acid amide (NAA Amide, 100.6μg / mL, S029695), N-N'-diphenylurea (NND, 100.1μg / mL, S037335), paclobutrazol (PP333, mass fraction 98.80%, G145460), thidiazuron (TDZ, mass fraction 99.30%, G687087), wherein, sodium 2,4-dichlorophenoxyacetic acid, 3-indolebutyric acid, 6-benzyladenine, chlorfenapyr, gibberellin, 3-indoleacetic acid, paclobutrazol, thidiazuron were purchased from Dr. Ehrenstorfer Gmbh, 3-indolepropionic acid, abscisic acid, mepiquat, brassinolide, thiazolin, aminoethyl hexanoate, 1-naphthylacetamide, N-N'-diphenylurea were purchased from Fint Standang, isopentenyl adenine and 4-fluorophenoxyacetic acid were purchased from Standford Ghemical Company, and 4-chlorophenoxyacetic acid was purchased from BePure Company.Glacial acetic acid (Guangdong Guanghua Technology Co., Ltd., analytical grade), methanol (Lichrosolve, chromatographic grade), acetonitrile (Lichrosolve, chromatographic grade), formic acid (Tianjin Komiou Chemical Reagent Co., Ltd., chromatographic grade), QUECHERS Clean-up Kit (StanQuik), CNWBOND GCB / NH2 SPE Cartridge (StanQuik), anhydrous sodium acetate (Tianjin Guangfu Fine Chemical Research Institute, analytical grade), anhydrous magnesium sulfate (Guangdong Guanghua Technology Co., Ltd., analytical grade), LC-C18 (ANPELLaboratory Technologies Shanghai Inc., analytical grade). Asparagus, Pueraria root, Chinese yam, Curcuma, ginger, and Curcuma are all commercially available products in Yulin Chinese herbal medicine market.

[0036] 1.2 Instruments

[0037] Liquid spectrometer-mass spectrometer Agilent 1290-6460A (with automatic sample injector, Agilent, USA), ultrapure water machine (Synergy), nitrogen blowdown instrument (Degent), XS-205DU electronic balance (Mettler-Toledo, USA), BSA 822-CW electronic balance (Sartorius, Germany), 3K15 high-speed refrigerated centrifuge (Sigma, Germany), HYQ-3110 vortex mixer (Seiki, USA), KQ-500GDV constant temperature CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0038] 1.3 Methods

[0039] 1.3.1 Chromatographic conditions

[0040] The present invention optimizes the chromatographic column during the research process. For the analysis of multi-component test compounds, selecting a suitable chromatographic column is the premise and the key. The present invention investigates Agilent SB-C18 RRHD (1.8 μm, 2.1×100 mm), ACQUITY BEH C18 (1.8μm, 2.1×100mm), ACQUITY HSS T3 (1.8μm, 2.1×100mm), Agilent Eclip seplusC18 RRHD (1.8μm, 2.1×50mm), XSE LECT CSH TM C18(3.5μm, 4.6×150mm), ACQUITY The separation effects of 6 BEH HLIC (1.7μm, 2.1×100mm) columns with different performance on the analytes. After comprehensive consideration of the peak shape, response and other factors of each analyte, the Agilent SB-C18RRHD column was finally selected to analyze the analytes.

[0041] The present invention optimizes the mobile phase during the research process. The present invention compares the effects of different mobile phase compositions on the peak shape of the test compound when methanol and acetonitrile are used as the organic phase and 0.1% formic acid solution (5mmol / L ammonium acetate solution) and 0.1% formic acid (5mmol / L ammonium formate) solution are used as the aqueous phase. The results show that when methanol is used as the mobile phase, each test compound responds well and has a higher sensitivity than acetonitrile, so methanol is used as the organic phase. When 0.1% formic acid (5mmol / L ammonium acetate solution) is used as the aqueous phase, the retention and separation effects of the test compound are better, and the peak shape is good. Therefore, the present invention uses methanol-0.1% formic acid (5mmol / L ammonium acetate solution) as the mobile phase.

[0042] The chromatographic conditions of the present invention are finally determined as follows:

[0043] Chromatographic column: Agilent SB-C18 RRHD (1.8 μm, 2.1×100 mm); mobile phase: A is 0.1% formic acid (5 mmol / L ammonium acetate), B is methanol; injection volume: 2 μL; flow rate: 0.25 mL / min; column temperature: 35°C; gradient elution conditions are shown in Table 1.

[0044] Table 1 Gradient elution conditions

[0045]

[0046] 1.3.2 Mass spectrometry conditions

[0047] The present invention optimizes the mass spectrometry conditions during the research process. 19 plant growth regulators are respectively prepared into standard solutions with a concentration of 1 μg / mL, and the test compounds are analyzed using a single needle automatic injection, and full scans are performed in positive and negative ion modes to determine the molecular ions of the 19 plant growth regulators, and the molecular ions are used as parent ions, a certain collision energy is given, and the secondary ions are fully scanned, and the daughter ions with higher abundance are selected as quantitative and qualitative ions, respectively. Among them, gibberellins, 4-chlorophenoxyacetic acid, 6-benzyladenine, thiadiazole, 2,4-dichlorophenoxyacetic acid sodium, chlorfenapyr, 4-fluorophenoxyacetic acid, and isopentenyl adenine respond in the negative ion mode, and the negative ion mode is selected for scanning; paclobutrazol, 3-indolebutyric acid, 3-indoleacetic acid, thiazolin, N-N'-diphenylurea, mepiquat, aminoethyl hexanoate, 1-naphthylacetamide, brassinolide, abscisic acid, and 3-indolepropionic acid respond in the positive ion mode, and the positive ion mode is selected for scanning.

[0048] The mass spectrometry conditions of the present invention are finally determined as follows:

[0049] ESI source, positive ion and negative ion simultaneous multiple reaction monitoring mode (MRM) detection, collision gas: on; gas flow: 8.0 L / mim; gas temperature: 350°C; nebulizer: 45.1 psi; sheath gas flow: 10.0 L / min; sheath gas temperature: 400°C; the mass spectrometry parameters of 19 plant growth regulators are shown in Table 2.

[0050] Table 2 Mass spectrometry parameters of 19 plant growth regulators

[0051]

[0052]

[0053] Note: “*” indicates quantitative ion

[0054] 1.3.3 Preparation of standard solutions: 11 plant growth regulators including gibberellin, 4-chlorophenoxyacetic acid, 6-benzyladenine, paclobutrazol, 3-indolebutyric acid, thidiazuron, sodium 2,4-dichlorophenoxyacetate, chlorforuzonium, 3-indoleacetic acid, 4-fluorophenoxyacetic acid and isopentenyl adenine were dissolved in methanol to prepare 1 mg / mL reference stock solutions, and 8 plant growth regulators including thiazolin, N-N'-diphenylurea, mepiquat, aminoethyl hexadecene, 1-naphthylacetamide, brassinolide, abscisic acid and 3-indolepropionic acid were dissolved in acetonitrile to prepare 20 μg / mL reference stock solutions, and the above reference stock solutions were stored in a -4°C refrigerator.

[0055] 1.3.4 Preparation of test solution The Chinese herbal medicine sample was crushed into powder, and about 5 g of sample powder was placed in a 50 mL stoppered centrifuge tube. 15 mL of 1% formic acid acetonitrile solution was added, and vortexed for 1 min to fully infiltrate the sample. After standing for 30 min, 15 mL of acetonitrile was accurately added, and vortexed for 1 min. 4 g of anhydrous magnesium sulfate + 1 g of anhydrous sodium acetate were added, and vortexed immediately. The solution was shaken on an oscillator for 1 min, and refrigerated centrifuged (7000 r / min) for 5 min. 5 mL of supernatant was accurately taken and placed in a QUECHERS purification tube (900 mg MgSO4 + 150 mg PSA + 15 mg GCB, 15 mL), vortex for 1 min, refrigerate centrifuge (7000 r / min) for 5 min, take all the supernatant into a 15 mL stoppered centrifuge tube, blow dry with nitrogen, accurately add 1 mL of methanol solution, vortex for 3 min, refrigerate centrifuge (7000 r / min) for 5 min, filter into a liquid vial with a microporous filter membrane (0.22 μm) for ultra performance liquid chromatography-tandem mass spectrometry analysis.

[0056] In the process of pre-treatment of Chinese medicinal materials, the present invention screens the extraction solvent. Chinese medicinal materials are low-water samples. In order to fully extract the target compound and make it fully free, the present invention investigates the extraction rate of 19 plant growth regulators by three extraction methods: 1% formic acid acetonitrile solution, 1% glacial acetic acid solution, and pure acetonitrile solution, which are soaked and left to stand for 30 minutes and then added with acetonitrile for extraction. The results show that the extraction efficiency of the target compound is higher when the target compound is extracted after soaking and standing for 30 minutes with 1% formic acid acetonitrile solution.

[0057] In the process of pre-treatment of Chinese medicinal materials, the present invention optimizes the purification method. This invention investigates the purification effects of three different extraction material combinations: 300mg anhydrous MgSO4+100mgC18, QUECHERS purification tube (900mgMgSO4+150mgPSA+15mgGCB, 15mL), and CNWBOND GCB / NH2 SPE Cartridge (500mg / 500mg, 6mL). The test results show that the QUECHERS purification tube has a better purification effect, so the QUECHERS purification tube is selected for purification.

[0058] In addition, for matrix effect, the present invention makes relevant selections in the optimization of conditions for Chinese herbal medicine pretreatment. Matrix refers to the components in the sample except the analyte. Since the matrix often has significant interference in the analysis process of the analyte and affects the accuracy of the analysis result, these influences and interferences are called matrix effect. The present invention selects asparagus as a matrix curve, and compares it with the standard curve of the reference substance, and finds that its matrix effect is stronger. In order to reduce the influence of the matrix effect on the result, it is necessary to use matrix matching standard solution for calibration and quantification.

[0059] 2 Results and analysis

[0060] 2.1 Linear range, detection limit and quantification limit

[0061] The mixed standard solution was diluted into a series of mass concentrations using blank asparagus matrix extract, and analyzed according to the detection conditions of 1.3.1 and 1.3.2. The detection limit (LOD) and quantification limit (LOQ) of each compound were calculated using a 3-fold signal-to-noise ratio and a 10-fold signal-to-noise ratio. The obtained correlation coefficients were all greater than 0.990, and the linear relationship was good, as shown in Table 3. The total ion current of the test sample is shown in Figure 1 , MRM chromatograms of 19 plant growth regulators, the results are shown in Figure 2-4 .

[0062] Table 3 Linear regression equations and detection limits and quantification limits of 19 PGRs

[0063]

[0064]

[0065] 2.2 Precision and recovery experiments

[0066] Taking Asparagus cochinchinensis as a representative matrix, the addition recovery and precision experiments were carried out. Accurately weigh 5g (accurate to 0.01g) of blank sample, add 19 kinds of plant growth regulator standard solutions, and make addition levels of 40μg / kg, 120μg / kg, and 240μg / kg (3 copies for each sample, and carry out spiked recovery experiment according to 1.3.4 method, calculate the average recovery rate and relative standard deviation, and examine the accuracy and precision of the method. As shown in Table 4, the average recovery rate of each addition level is between 71.46% and 118.60%, and the precision is between 0.19% and 12.77%. The accuracy and precision of the method meet the requirements of residue analysis. In order to further verify the precision of the method, the middle and high points in the matrix curve were taken and repeated 6 times, as shown in Table 5, and the RSD was 0.51%-5.86%, further indicating that this method meets the regulations.

[0067] Table 4 Recovery and relative standard deviation (RSD) of 19 PGRs (n=3)

[0068]

[0069]

[0070]

[0071] Table 5 Average concentrations and relative standard deviations (RSD) of the high points in the matrix curve (n=6)

[0072]

[0073]

[0074] 2.3 Sample determination

[0075] Applying the detection and analysis method established above, 18 batches of samples of Pueraria lobata, Chinese yam, Asparagus cochinchinensis, Curcuma aromatica, ginger, and Curcuma zedoaria purchased on the market were measured. The results showed that among the 18 batches of samples, except for one batch of Curcuma aromatica samples with an abscisic acid content of 53.1 μg / kg, the rest of the samples had no detection content of abscisic acid, which may be related to the limited number of samples collected and the higher quality of the purchased samples.

[0076] The method of the present invention for simultaneously detecting the residues of 19 plant growth regulators in Chinese medicinal materials is widely used after obtaining these data, especially in quality control and evaluation. These data can not only be used to ensure the safety of Chinese medicinal materials, but also promote the standardized development of the Chinese medicinal materials industry. The following are some specific application scenarios and evaluation methods:

[0077] 1. Quality Control

[0078] 1. Establish personalized residue standards:

[0079] (1) Based on the test data, personalized plant growth regulator residue standards can be established for different types of Chinese medicinal materials, origins, growth cycles, etc. These standards can more accurately reflect the actual quality status of Chinese medicinal materials and provide a scientific basis for the quality control of Chinese medicinal materials.

[0080] (2) Regularly test Chinese medicinal materials, compare the test data with the standard values, evaluate the quality stability of Chinese medicinal materials, and ensure that the quality of Chinese medicinal materials meets relevant requirements.

[0081] 2. Risk assessment and early warning:

[0082] (1) Use the test data to conduct risk assessment on plant growth regulator residues in Chinese medicinal materials and predict their potential harm to human health.

[0083] (2) Establish an early warning mechanism to promptly issue an early warning signal when the test data exceeds the safety threshold, reminding relevant departments and consumers to pay attention to the safety of Chinese medicinal materials.

[0084] 2. Innovation in Evaluation Methods

[0085] 1. Comprehensive evaluation from multiple dimensions:

[0086] (1) In addition to the single residue value, other quality indicators of Chinese medicinal materials (such as active ingredient content, microbial contamination, etc.) can also be combined for multi-dimensional comprehensive evaluation. This method can more comprehensively reflect the quality characteristics of Chinese medicinal materials and improve the accuracy and scientificity of the evaluation.

[0087] (2) Using statistical analysis and data mining techniques, a quality evaluation model for Chinese medicinal materials is established, and a comprehensive analysis of the test data is conducted to derive the overall quality score or grade of the Chinese medicinal materials.

[0088] 2. Dynamic monitoring and tracing:

[0089] (1) Establish a dynamic monitoring system for the quality of Chinese medicinal materials to monitor the entire process of growth, processing, and storage of Chinese medicinal materials, and record relevant data and information in real time.

[0090] (2) Realize the quality traceability of Chinese medicinal materials. Through the test data and traceability information, the source of the problem can be quickly located and corresponding measures can be taken to rectify and optimize.

[0091] 3. Differentiated evaluation:

[0092] (1) Differentiated evaluation criteria and methods should be developed for Chinese herbal medicines with different uses (such as medicinal, edible, and health care). For example, for medicinal Chinese herbal medicines, more emphasis can be placed on the balance between their active ingredients and residues; for edible Chinese herbal medicines, more emphasis can be placed on their safety and taste.

[0093] (2) Introduce third-party testing agencies and expert review panels to independently judge and review the quality of Chinese medicinal materials to improve the fairness and authority of the evaluation.

[0094] 3. Practical Application Scenarios

[0095] 1. Market supervision of Chinese medicinal materials:

[0096] Market supervision departments can use testing data to supervise the Chinese medicinal materials market, crack down on counterfeit and shoddy products and illegal additives, and maintain market order and consumer rights.

[0097] 2. Export trade of Chinese herbal medicines:

[0098] In the export trade of Chinese medicinal materials, test data can serve as an important basis for product quality, helping Chinese medicinal materials companies to successfully pass international quality certification and testing standards and improve the international competitiveness of their products.

[0099] 3. Standardization of Chinese herbal medicine industry:

[0100] By continuously improving and promoting testing data and evaluation methods, we can promote the standardized development of the Chinese medicinal materials industry and improve the overall quality and market competitiveness of Chinese medicinal materials.

[0101] Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make some modifications and improvements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be based on the definition of the claims.

Claims

1. A method for simultaneously detecting the residues of 19 plant growth regulators in traditional Chinese medicines, characterized in that: The steps include: (1) Preparation of standard solution: 11 plant growth regulators, including gibberellin, 4-chlorophenoxyacetic acid, 6-benzyladenine, paclobutrazol, 3-indolebutyric acid, thidiazuron, sodium 2,4-dichlorophenoxyacetate, chlorfenapyr, 3-indoleacetic acid, 4-fluorophenoxyacetic acid, and isopentenyl adenine, were dissolved in methanol to prepare standard solutions; 8 plant growth regulators, including thiazolin, N-N'-diphenylurea, mepiquat, ampicillin, 1-naphthylacetamide, brassinolide, abscisic acid, and 3-indolepropionic acid, were dissolved in acetonitrile to prepare standard solutions for later use; (2) Preparation of the sample solution to be tested: Crush the Chinese herbal medicine sample into herbal medicine powder, take the herbal medicine powder and place it in a stoppered centrifuge tube, add 1% formic acid acetonitrile solution, vortex to fully infiltrate the herbal medicine powder, add acetonitrile after standing, vortex, add anhydrous magnesium sulfate and anhydrous sodium acetate, vortex, shake on an oscillator, refrigerate centrifuge, take the supernatant into a QUECHERS purification tube, vortex, refrigerate centrifuge, take all the supernatant into a stoppered centrifuge tube, blow dry with nitrogen, add methanol solution, vortex, refrigerate centrifuge, filter with a microporous filter membrane to obtain the sample solution to be tested, and set aside; (3) using ultra performance liquid chromatography-tandem mass spectrometry to analyze the contents of 19 plant growth regulators in the sample solution, including gibberellins, 4-chlorophenoxyacetic acid, 6-benzyladenine, paclobutrazol, 3-indolebutyric acid, thidiazuron, sodium 2,4-dichlorophenoxyacetate, chlorfenapyr, 3-indoleacetic acid, 4-fluorophenoxyacetic acid, isopentenyl adenine, chlorfenapyr, N-N'-diphenylurea, mepiquat, aminoethyl hexanoate, 1-naphthylacetamide, brassinolide, abscisic acid, and 3-indolepropionic acid; Chromatographic conditions were as follows: chromatographic column: Agilent SB-C18 RRHD, 1.8 μm, 2.1×100 mm; mobile phase: A was 0.1% formic acid, the 0.1% formic acid contained 5 mmol / L ammonium acetate, and B was methanol; injection volume: 2 μL; flow rate: 0.25 mL / min; column temperature: 35°C; The mass spectrometry conditions were as follows: ESI source, positive and negative ion simultaneous multiple reaction monitoring mode detection, collision gas: on; gas flow: 8.0 L / min; gas temperature: 350 °C; Atomizer : 45.1psi; sheath gas flow rate: 10.0L / min; sheath gas temperature: 400℃.

2. The method for simultaneously detecting the residues of 19 plant growth regulators in Chinese medicinal materials according to claim 1, characterized in that: In step (1), the concentration of the 11 standard solutions prepared by dissolving in methanol is 1 mg / mL; the concentration of the 8 standard solutions prepared by dissolving in acetonitrile is 20 μg / mL.

3. The method for simultaneously detecting the residues of 19 plant growth regulators in traditional Chinese medicine according to claim 1, characterized in that: The specific operation of step (2) is as follows: grind the Chinese medicinal material sample into medicinal material powder, take 5g of medicinal material powder and place it in a 50mL stoppered centrifuge tube, add 15mL of 1% formic acid acetonitrile solution, vortex for 1min to fully infiltrate the medicinal material powder, let it stand for 30min, accurately add 15mL of acetonitrile, vortex for 1min, add 4g of anhydrous magnesium sulfate and 1g of anhydrous sodium acetate, vortex immediately, shake on an oscillator for 1min, refrigerate centrifuge for 5min, centrifuge at a speed of 7000r / min, accurately take 5mL of supernatant and place it in a QUECHERS purification tube (900mgMgSO4+150mgPSA+15mg GCB, 15mL), vortex for 1min, refrigerate centrifuge for 5min, centrifuge speed 7000r / min, take all the supernatant into a 15mL stoppered centrifuge tube, blow dry with nitrogen, accurately add 1mL methanol solution, vortex for 3min, refrigerate centrifuge for 5min, centrifuge speed 7000r / min, filter into a liquid phase vial with a 0.22μm microporous filter membrane to obtain the sample solution to be tested, and set aside.

4. The method for simultaneously detecting the residues of 19 plant growth regulators in Chinese medicinal materials according to claim 1, characterized in that: In step (3), the gradient elution procedure in the chromatographic conditions is as follows:

5. The method for simultaneously detecting the residues of 19 plant growth regulators in Chinese medicinal materials according to claim 1, characterized in that: In step (3), the mass spectrometry parameters of 19 plant growth regulators are as follows: The 2,4-D is sodium 2,4-dichlorophenoxyacetate, the 2-IP is isopentenyladenine, the 3-IBA is 3-indolebutyric acid, the 3-IPA is 3-indolepropionic acid, the 4-CPA is 4-chlorophenoxyacetic acid, the 4-FPA is 4-fluorophenoxyacetic acid, the 6-BA is 6-benzyladenine, the ABA is abscisic acid, the BAS is mepiquat, the BR is brassinolide, the BSI is chlorfenapyr, the CPPU is chlorfenapyr, the DA-6 is ampicillin, the GA is gibberellin, the IAA is 3-indoleacetic acid, the NAAAmide is 1-naphthylacetamide, the NND is N-N'-diphenylurea, the PP333 is paclobutrazol, and the TDZ is thidiazuron.

6. The method for simultaneously detecting the residues of 19 plant growth regulators in Chinese medicinal materials according to claim 1, characterized in that: The Chinese medicinal materials include kudzu root, Chinese yam, asparagus cochinchinensis, turmeric, ginger and zedoaria.

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