Determination of brucine and strychnine in Nux vomica

Abstract

The present invention belongs to the field of testing or analytical methods, and specifically relates to a method for determining strychnine and strychnine in Strychnos nux vomica medicinal materials. The method uses an extraction method using ultrasonic treatment of an acid-water solution, followed by purification using a magnetic solid-phase extraction material, which greatly shortens the pre-treatment time and reduces environmental pollution. UPLC-MS is then used for determination, without using a chromatographic column for separation, but with direct injection. The mobile phase is 0.5% formic acid in water; the flow rate is 0.2 mL·min –1 Injection volume: 1 μL; The mass spectrometry ion source is an electrospray ionization source, positive ion, multiple reaction monitoring mode, capillary voltage 3.5 kV, ion source temperature 150 ℃, and desolvation temperature 500 ℃. This method has a fast determination speed, and the entire process can be completed in 30-40 minutes. It does not use a chromatographic column, which can save detection costs. In addition, it can complete the detection with a small amount of reagents, which is green and environmentally friendly.

Description

Technical Field

[0001] The invention belongs to the field of testing or analysis methods, and relates to the determination of certain chemical components in traditional Chinese medicines, and in particular to a method for determining brucine and strychnine in Strychnos nux vomica medicinal materials. Background Art

[0002] Strychnos nux vomica Strychnos nux-vomica The dried mature seeds of Strychnos nux vomica L. have the properties of dredging the meridians, relieving pain, dispersing nodules, and reducing swelling. They are used to treat traumatic injuries, fractures, swelling and pain, rheumatic arthritis, numbness and paralysis, carbuncles, sores, and sore throats. It is listed in the 2025 edition of the Chinese Pharmacopoeia, Volume 1. It is primarily produced in Yunnan, Guangdong, Guangxi, Fujian, and Hainan. Strychnos nux vomica contains dozens of chemical components, including alkaloids, glycosides, acids, and alcohols. Strychnine and strychnine are representative alkaloids in Strychnos nux vomica, with pronounced neurostimulant and analgesic effects, and are the primary active ingredients in the drug. Furthermore, the therapeutic and toxic doses of strychnine and strychnine are very close. A single dose of 5-10 mg of strychnine can cause poisoning in adults, while an oral dose of 15-100 mg can cause poisoning, and overdose can even be fatal. Strychnine and strychnine are both active and toxic components, and are important quality control indicators for Strychnos nux vomica medicinal materials and their preparations.

[0003] In the 2025 edition of the Chinese Pharmacopoeia, the sample pretreatment for the determination of Strychnine content is to add sodium hydroxide test solution, let it stand for 30 minutes, and then treat it in a chloroform water bath with reflux for 2 hours. 3mL of chloroform solution is diluted to 10mL with methanol as the test solution, and then the content of strychnine and strychnine is determined by ion-pair high-performance liquid chromatography. There are three main problems with this operation: first, it takes a long time, at least about 3 hours; second, the use of chloroform is strictly regulated and highly toxic, and it is included in the "List of New Pollutants Under Key Control (2023 Edition)", posing a major health risk to laboratory workers and the environment; third, the use of ion-pair reagents and potassium dihydrogen phosphate salts in the chromatographic conditions is very damaging to the chromatographic column, requiring high column maintenance costs.

[0004] Chinese patent CN 1945309 A discloses a method for determining the content of strychnine and strychnine in refined Haima Zhuifeng ointment. The method involves preparing a test solution and a reference solution. The determination is then performed using an octadecylsilane bonded silica gel column, a methanol-acetonitrile-water mobile phase, and an ion-pairing reagent. This method involves complex sample pretreatment steps, is time-consuming, and utilizes traditional high-performance liquid chromatography, which is slow.

[0005] Chinese patent CN 119086735 A discloses a "mass spectrometry rapid determination kit for strychnine and strychnine." The detection method of the mass spectrometry rapid determination kit is as follows: adding the sample to be tested to a porous plate filled with a mixed-mode cation exchange (MCX) adsorbent and a pyrolidone-styrene polymer (PLS) adsorbent, then adding an acetonitrile-water solution, mixing evenly, and eluting with the methanol-ammonia solution to obtain an eluate; the eluate is then subjected to liquid chromatography-tandem mass spectrometry to obtain the peak area of ​​strychnine or strychnine, and finally, the concentration of strychnine or strychnine in the sample to be tested is calculated using a standard curve of strychnine or strychnine. Although this method can achieve rapid, accurate and highly sensitive detection of strychnine and strychnine, it has three major problems: first, the kit is relatively complex, including reagent A, reagent B and reagent C, and reagent A includes three reagents; reagent B includes multiple standard solutions, and reagent C includes a 96-well extraction cleanup plate and a 96-well sample injection plate. The 96-well extraction cleanup plate is loaded with MCX cation exchange resin and 5mgPLS (pyrrolidone-styrene polymer) filler; second, it requires the use of expensive UPLC chromatographic columns; and third, it requires more reagents, including toxic reagents such as acetonitrile and methanol. Summary of the Invention

[0006] In view of the shortcomings and deficiencies of the prior art, the object of the present invention is to provide a method for the determination of strychnine and strychnine in the medicinal material Strychnine. The method adopts an extraction method using ultrasonic treatment of an acid-water solution, followed by purification using a magnetic solid-phase extraction material, which greatly shortens the pretreatment time (shortening the time for preparing the test solution to within 30 minutes) and reduces environmental pollution. In addition, UPLC-MS is used for determination, without the use of a chromatographic column for separation, and direct sample injection is used, thereby achieving rapid analysis and determination while reducing chromatographic column maintenance costs.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for determining brucine and strychnine in a Nux vomica medicinal material comprises the following steps:

[0009] Step 1. Prepare a control extract solution;

[0010] Step 2. Prepare the test sample extract:

[0011] Take the test sample, place it in a conical flask, add 0.5% formic acid aqueous solution, weigh it, treat it ultrasonically, make up the weight loss with 0.5% formic acid aqueous solution, filter it, take the filtrate, place it in a volumetric flask, and dilute it to the scale with 0.5% formic acid aqueous solution;

[0012] Step 3. Purification process:

[0013] Preparation of magnetic extractant: Weigh magnetic extractant particles, add 0.5% formic acid aqueous solution to prepare extractant particle dispersion, and fully shake the extractant particle dispersion to keep the extractant particle dispersion in a uniform suspension state;

[0014] Balance: Pipette the extractant particle dispersion into a test tube, add 0.5% formic acid aqueous solution, vortex, place the test tube on a magnetic rack, and let it stand until the magnetic extractant particles are completely adsorbed on one side of the inner wall of the test tube; aspirate and discard the residual liquid;

[0015] Sample loading: Pipette the sample extract from step 2 into a test tube, vortex, place the test tube on a magnetic rack, and let it stand until the magnetic extractant particles are completely adsorbed on one side of the inner wall of the test tube; aspirate and discard the residual liquid;

[0016] Elution: Pipette methanol into a test tube, vortex, place the test tube on a magnetic rack, and let it stand until the magnetic extractant particles are completely adsorbed on one side of the inner wall of the test tube; aspirate and discard the residual liquid;

[0017] Elution: Pipette 8% ammonia solution into a test tube, vortex, place the test tube on a magnetic rack, and let it stand until the magnetic extractant particles are completely adsorbed on one side of the inner wall of the test tube. Collect the eluate, concentrate to dryness under reduced pressure, and dilute to 1 mL with 0.5% formic acid aqueous solution to use as the test solution;

[0018] Step 4. Chromatography-mass spectrometry conditions:

[0019] No chromatographic column was used, and the chromatographic column was replaced by a two-way connector; mobile phase: 0.5% formic acid aqueous solution; flow rate: 0.2 mL·min –1 The injection volume was 1 μL. The mass spectrometry ion source was an electrospray ionization source with positive ionization, multiple reaction monitoring mode, capillary voltage of 3.5 kV, ion source temperature of 150 °C, and desolvation temperature of 500 °C.

[0020] As a preferred embodiment of the present invention, the specific steps of step 1 are: weighing the total alkaloid extract of Strychnos nux vomica and dissolving it in 0.5% formic acid aqueous solution to prepare a solution containing 0.923 μg·mL of strychnine. –1 , strychnine 1.009 μg·mL –1 Take 1 mL of the control extract stock solution, add 0.5% formic acid aqueous solution to dissolve it, and dilute to 25 mL.

[0021] As a preferred embodiment of the present invention, the specific steps of step 2 are: after the medicinal material is crushed, it is passed through a No. 4 sieve, 0.2 g of powder is taken, accurately weighed, and placed in a stoppered conical flask, 50 mL of 0.5% formic acid aqueous solution is added, the weight is weighed, and ultrasonic treatment is performed for 10 minutes to make up for the weight loss, and filtered. 1 mL of the filtrate is taken and placed in a 100 mL volumetric flask, and diluted to the scale with 0.5% formic acid aqueous solution to serve as the test sample extract.

[0022] As the preferred embodiment of the present invention, the specific steps of step 3 are:

[0023] Preparation of magnetic extractant: Weigh 0.1 g of magnetic extractant particles and place them in a 1.5 mL centrifuge tube. Add 1 mL of 0.5% formic acid solution to prepare a dispersion of the extractant particles. Thoroughly shake the dispersion to keep it in a uniform suspension.

[0024] Balance: Pipette 100 μl of the extractant particle dispersion into a test tube, add 1 mL of 0.5% formic acid aqueous solution, vortex for 1 minute, place the test tube on a magnetic rack, and let it stand for 30 seconds to allow the magnetic extractant particles to be completely adsorbed on one side of the inner wall of the test tube; aspirate and discard the residual liquid;

[0025] Sample loading: Pipette 100 μl of the sample extract from step 2 into a test tube, vortex for 1 minute, place the test tube on a magnetic rack, and let it stand for 30 seconds to allow the magnetic extractant particles to be completely adsorbed to one side of the inner wall of the test tube; aspirate and discard the residual liquid;

[0026] Washing: Pipette 1 mL of methanol into a test tube, vortex for 1 minute, place the test tube on a magnetic rack, and let it stand for 30 seconds to allow the magnetic extractant particles to be completely adsorbed on one side of the inner wall of the test tube; aspirate and discard the residual liquid;

[0027] Elution: Pipette 1 mL of 8% ammonia solution into a test tube, vortex for 1 minute, place the test tube on a magnetic rack, and let it stand for 30 seconds to allow the magnetic extractant particles to be completely adsorbed on one side of the inner wall of the test tube. Collect the eluate, concentrate to dryness under reduced pressure, and dilute to 1 mL with 0.5% formic acid aqueous solution to use as the test solution.

[0028] As the preferred embodiment of the present invention, the mass spectrometry parameters of strychnine are: parent ion m / z is 395.0, the fragmentor voltage is 195V, and the quantitative ion m / z 244.1, qualitative ion m / z is 324.1, and the collision energies of the quantitative ion and the qualitative ion are 44eV and 37eV, respectively;

[0029] The mass spectrometric parameters of strychnine are: parent ion m / z is 336.0, the fragmentor voltage is 190V, and the quantitative ion m / z 185.1, qualitative ion m / zThe collision energy of the quantitative ion and the qualitative ion is 46eV and 62eV respectively.

[0030] As a further preference of the present invention, the power of the ultrasonic treatment in step 2 is 300 W and the frequency is 40 kHz.

[0031] As a further preferred embodiment of the present invention, mixed cation exchange MCX magnetic extractant particles are used in step 3.

[0032] Advantages and positive effects of the present invention:

[0033] (1) The present invention establishes a method for determining brucine and strychnine in Strychnos nux vomica medicinal material. In the method, when preparing the test solution, an extraction method of ultrasonic treatment of an acid aqueous solution is adopted, and then purification is performed using a cationic magnetic purification material, so that the time for preparing the test solution is shortened to within 30 minutes, thereby solving the problem of long test sample preparation time. At the same time, this method can also reduce the use of organic reagents, saving sample pretreatment time, reducing testing costs, and protecting the environment.

[0034] (2) The present invention adopts UPLC-MS for determination, without using chromatographic columns for separation, and directly injects samples (using mobile phase to promote sample injection), and then uses the extracted ions of mass spectrometry for quantitative analysis; this method has three major advantages: first, it saves time and cost, and the determination speed is fast. The determination time can be completed within 1-2 minutes. Including the pre-treatment time, the whole process can be completed in 30-40 minutes; second, no chromatographic column is used, saving detection costs; third, it is green and environmentally friendly, and the detection can be completed with a small amount of reagents.

[0035] (3) The determination method provided by the present invention is simple and quick, has a very fast determination speed, good repeatability, high detection sensitivity, and accurate and reliable detection results. It can be used to detect the content of strychnine and strychnine in Strychnos nux vomica medicinal materials. Moreover, since no chromatographic column is used, the maintenance cost of the chromatographic column can be reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is an MRM (mass spectrometry multiple reaction monitoring) chromatogram of a control extract solution; 1 in the figure represents strychnine, and 2 in the figure represents strychnine;

[0037] Figure 2 This is the MRM (mass spectrometry multiple reaction monitoring) chromatogram of the test solution; 1 in the figure represents strychnine, and 2 in the figure represents strychnine. DETAILED DESCRIPTION

[0038] The present invention is further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the technical solutions of the present invention, but the present invention is not limited thereto.

[0039] 1. Instruments and Materials:

[0040] Agilent 1290 Infinity II chromatograph (Agilent); Agilent 6470LC / TQ triple quadrupole tandem mass spectrometer (Agilent); KQ-500DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); XPE205DR 1 / 100,000 electronic analytical balance (METTLER TOLEDO); and Milli-Q ultrapure water system (Millipore).

[0041] Strychnine total alkaloid control extract (batch number 112030-202202, content calculated as strychnine 45.3%, strychnine 49.5%, origin: China Food and Drug Administration), acetonitrile of chromatography grade (Fisher), ammonia of analytical grade (Sinopharm Chemical Reagent Co., Ltd.), MCX magnetic extraction material (Putun Laboratory Equipment (Shanghai) Co., Ltd.). Six batches of Strychnine medicinal materials, sourced as shown in Table 1, were identified as Strychnine (a plant of the Logarithaceae family) by Associate Researcher Zan Ke of the Institute of Traditional Chinese Medicine, China Food and Drug Administration. Strychnos nux-vomica The dried mature seeds of L.

[0042] Table 1 Source information of Nux vomica

[0043] batch number Origin serial number 220601 Fujian MQZ-1 230801 Hainan MQZ-2 20230501 Guangxi MQZ-3 230601 Hainan MQZ-4 240301 Fujian MQZ-5 20230901 Fujian MQZ-6

[0044] 2. Methods and Results

[0045] Solution preparation

[0046] 2.1.1. Control extract solution:

[0047] Accurately weigh an appropriate amount of Strychnine total alkaloid control extract, add an appropriate amount of 0.5% formic acid aqueous solution to dissolve it, and prepare a solution containing 0.923 μg·mL of strychnine. –1 , containing strychnine 1.009μg·mL –1 1 mL of the control extract stock solution was taken, dissolved in 0.5% formic acid aqueous solution, and diluted to 25 mL as the control extract solution.

[0048] 2.1.2. Test sample extract:

[0049] After the medicinal material is crushed, it is passed through a No. 4 sieve. 0.2 g of powder is taken and placed in a stoppered conical flask. 50 mL of 0.5% formic acid aqueous solution is accurately added and weighed. The mixture is ultrasonically treated (300 W, 40 kHz) for 10 minutes and weighed again. The lost weight is supplemented with 0.5% formic acid aqueous solution, shaken and filtered. 1 mL of the filtrate is accurately drawn and placed in a 100 mL volumetric flask. The mixture is diluted to the mark with 0.5% formic acid aqueous solution to serve as the test sample extract.

[0050] 2.1.3.MCX purification process:

[0051] Preparation of magnetic extractant: Weigh 0.1 g of magnetic extractant particles (mixed cation exchange MCX magnetic extraction material) into a 1.5 ml centrifuge tube. Add 1 mL of 0.5% formic acid aqueous solution to prepare a dispersion of extractant particles (equivalent to 10 mg of extractant particles per 100 μl of dispersion). Vortex the dispersion thoroughly to maintain a uniform suspension.

[0052] Balance: Pipette 100 μl of the above-mentioned extractant particle dispersion into a test tube, add 1 mL of 0.5% formic acid aqueous solution, vortex for 1 minute, place the test tube on a magnetic rack, and let it stand for 30 seconds to allow the magnetic extractant particles (MCX magnetic extraction material) to be completely adsorbed on one side of the inner wall of the test tube; aspirate and discard the residual liquid to avoid carrying out the magnetic extractant particles.

[0053] Sample loading: Pipette 100 μl of the sample extract from step 2.1.2 into a test tube, vortex for 1 minute, place the test tube on a magnetic rack, and let it stand for 30 seconds to allow the magnetic extractant particles to be completely adsorbed to one side of the inner wall of the test tube; aspirate and discard the residual liquid;

[0054] Washing: Pipette 1 mL of methanol into a test tube, vortex for 1 minute, place the test tube on a magnetic rack, and let it stand for 30 seconds to allow the magnetic extractant particles to be completely adsorbed on one side of the inner wall of the test tube; aspirate and discard the residual liquid;

[0055] Elution: Pipette 1 mL of 8% ammonia solution (concentrated ammonia solution is diluted with water to contain 8% ammonia) into a test tube, vortex for 1 minute, place the test tube on a magnetic rack, and let it stand for 30 seconds to allow the magnetic extractant particles to be completely adsorbed on one side of the inner wall of the test tube. Collect the eluate, concentrate it to dryness under reduced pressure, and dilute it to 1 mL with 0.5% formic acid aqueous solution to use as the test solution.

[0056] 2.2. Chromatography-mass spectrometry conditions:

[0057] Mobile phase: 0.5% formic acid in water; flow rate: 0.2 mL·min –1The injection volume was 1 μL. The mass spectrometry ion source was electrospray ionization (ESI), positive ion, multiple reaction monitoring (MRM) mode, capillary voltage 3.5 kV, ion source temperature 150 °C, and desolvation temperature 500 °C.

[0058] In this embodiment, the test solution is purified, leaving almost only two components, strychnine and strychnine, with very few other interfering components; when performing chromatography-mass spectrometry detection, a chromatographic column is not used, and the chromatographic column portion is replaced with a two-way connector; the MRM technology in the triple quadrupole mass spectrometer selectively selects data for mass spectrometry signal acquisition, records signals for ions that meet the rules, removes interference from ion signals that do not meet the rules, and extracts the peak area of ​​the quantitative ion for content calculation; for parent ions or daughter ions, different compounds can be directly extracted for quantification without separation. Specifically, in this embodiment, the mass spectrometry conditions of strychnine and strychnine are shown in Table 2, and the MRM chromatograms of the control extract and sample are shown in Table 2. Figure 1 and Figure 2 .

[0059] Table 2 MS parameters of target components

[0060]

[0061] 2.3. Methodological Investigation:

[0062] 2.3.1. Linear relationship investigation: Accurately pipette 0.05, 0.10, 0.25, 0.50, 1.0, and 2.0 mL of the reference extract stock solution under 2.1.1, and place them in 6 10 mL volumetric flasks, add 0.5% formic acid aqueous solution to the scale, shake well, and obtain a reference substance mixed solution of each concentration series. Filter with a 0.22 μm microporous membrane, take the filtrate, and inject the sample. Perform the determination according to the conditions under "2.2". Take the peak area Y of the reference substance as the ordinate, and the corresponding concentration (ng·mL -1 )X is the horizontal axis, and linear regression is performed. The results are shown in Table 3.

[0063] Table 3 Linear equation and linear range

[0064] Element Linear equations r <![CDATA[Linear range / (ng·mL -1 )]]> Strychnine Y = 431.7X + 5830.0 0.9996 4.62～184.6 Strychnine Y = 116.9X + 1127.6 0.9991 5.04～201.8

[0065] 2.3.2. Precision test: Accurately pipette 1 μL of the control extract solution in item “2.1.1” and inject it 6 times in succession. Record the peak area. n =6) were 1.39% and 1.64% respectively, indicating that the instrument had good precision.

[0066] 2.3.3. Stability test: Take the same sample solution (MQZ-6) and inject it at 0, 2, 4, 8, 12 and 24 hours. n =6) were 1.15% and 1.73%, respectively, indicating that the test solution was stable within 24 h.

[0067] 2.3.4. Repeatability Test: Six aliquots of the test solution (MQZ-6) were prepared according to the method described in 2.1.2-2.1.3 and injected separately for analysis. The average contents of strychnine and strychnine were 1.95% and 2.69%, respectively, with RSDs less than 3.0%, indicating good repeatability of the method.

[0068] 2.3.5. Sample recovery test: Take 6 portions of the test sample (MQZ-6) powder with a known content, each of about 0.1g, accurately weigh, and place them in stoppered conical flasks. Accurately add 1mL of Strychnine control extract solution (take 25.15mg of Strychnine total alkaloid control extract, place it in a 10mL volumetric flask, add 0.5% formic acid aqueous solution to dissolve and dilute to the scale, that is, 1mL contains 1.1393mg of brucine and 1.2449mg of strychnine). Then accurately add 50mL of 0.5% formic acid aqueous solution. The remaining steps are in parallel to prepare the test solution according to the method of "2.1.2-2.1.3", sample analysis, and calculate the recovery rate. Results The average sample recovery rate of brucine and strychnine ( n =6) were 97.62% and 97.79% respectively, with RSDs of 1.64% and 2.17% respectively. The results are shown in Table 4.

[0069] Table 4 Recovery results of two components (n=6)

[0070]

[0071] 2.4. Content determination:

[0072] Take 0.2 g of Strychnine powder and prepare the test solution according to the methods under "2.1.2-2.1.3". Determine the sample according to the method under "2.2". Calculate the contents of strychnine and strychnine according to the external standard method. The results are shown in Table 5.

[0073] Table 5 Sample content determination results (%)

[0074] No. Strychnine Strychnine MQZ-1 0.99 1.61 MQZ-2 1.04 1.83 MQZ-3 0.94 1.74 MQZ-4 0.96 1.78 MQZ-5 0.98 1.81 MQZ-6 0.92 1.39

[0075] The present invention selected magnetic solid-phase extraction materials produced by Pudun Laboratory Equipment (Shanghai) Co., Ltd., Shimadzu (Shanghai) Laboratory Equipment Co., Ltd., and Ajie Boya Bioelectronic Technology Co., Ltd. for comparison. The solid-phase extraction material solution has good stability, fast separation speed, and the recovery rate results can meet the requirements.

[0076] The present invention establishes a method for determining the contents of brucine and strychnine in strychnine seeds. The method is simple to operate, has a short pretreatment time, and consumes less organic reagents. Methodological verification shows that all indicators meet the requirements of the guidance principles for validation of analytical methods of drug quality standards, confirming that the method is accurate and reliable.

[0077] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Non-essential technical features can be added or removed according to actual needs to meet the requirements of different situations.

Claims

1. A method for determining brucine and strychnine in Strychnos nux vomica medicinal material, characterized in that: The method comprises the following steps: Step 1. Prepare a control extract solution; Step 2. Prepare the test sample extract: Take the test sample, place it in a conical flask, add 0.5% formic acid aqueous solution, weigh it, treat it ultrasonically, make up the weight loss with 0.5% formic acid aqueous solution, filter it, take the filtrate, place it in a volumetric flask, and dilute it to the scale with 0.5% formic acid aqueous solution; Step 3. Purification process: Preparation of magnetic extractant: Weigh magnetic extractant particles, add 0.5% formic acid aqueous solution to prepare extractant particle dispersion, and fully shake the extractant particle dispersion to keep the extractant particle dispersion in a uniform suspension state; Balance: Pipette the extractant particle dispersion into a test tube, add 0.5% formic acid aqueous solution, vortex, place the test tube on a magnetic rack, and let it stand until the magnetic extractant particles are completely adsorbed on one side of the inner wall of the test tube; aspirate and discard the residual liquid; Sample loading: Pipette the sample extract from step 2 into a test tube, vortex, place the test tube on a magnetic rack, and let it stand until the magnetic extractant particles are completely adsorbed on one side of the inner wall of the test tube; aspirate and discard the residual liquid; Elution: Pipette methanol into a test tube, vortex, place the test tube on a magnetic rack, and let it stand until the magnetic extractant particles are completely adsorbed on one side of the inner wall of the test tube; aspirate and discard the residual liquid; Elution: Pipette 8% ammonia solution into a test tube, vortex, place the test tube on a magnetic rack, and let it stand until the magnetic extractant particles are completely adsorbed on one side of the inner wall of the test tube. Collect the eluate, concentrate to dryness under reduced pressure, and dilute to 1 mL with 0.5% formic acid aqueous solution to use as the test solution; Step 4. Chromatography-mass spectrometry conditions: No chromatographic column was used, and the column was replaced by a two-way connector; mobile phase: 0.5% formic acid in water; flow rate: 0.2 mL·min –1 The injection volume was 1 μL. The mass spectrometry ion source was an electrospray ionization source with positive ionization, multiple reaction monitoring mode, capillary voltage of 3.5 kV, ion source temperature of 150 °C, and desolvation temperature of 500 °C.

2. The method for determining brucine and strychnine in the Nux vomica medicinal material according to claim 1, characterized in that: The specific steps of step 1 are as follows: weigh the total alkaloid extract of Strychnos nux vomica and dissolve it in 0.5% formic acid aqueous solution to prepare a solution containing 0.923 μg·mL of strychnine. –1 , strychnine 1.009 μg·mL –1 Take 1 mL of the control extract stock solution, add 0.5% formic acid aqueous solution to dissolve it, and dilute to 25 mL.

3. The method for determining brucine and strychnine in the Nux vomica medicinal material according to claim 1, characterized in that: The specific steps of step 2 are as follows: after the medicinal material is crushed, it is passed through a No. 4 sieve, 0.2 g of powder is taken, accurately weighed, and placed in a stoppered conical flask, 50 mL of 0.5% formic acid aqueous solution is added, the weight is weighed, ultrasonic treatment is performed for 10 minutes, the weight loss is compensated, and the solution is filtered. 1 mL of the filtrate is taken and placed in a 100 mL volumetric flask, and diluted to the scale with 0.5% formic acid aqueous solution to serve as the test sample extract.

4. The method for determining brucine and strychnine in the Nux vomica medicinal material according to claim 1, characterized in that: The specific steps of step 3 are: Preparation of magnetic extractant: Weigh 0.1 g of magnetic extractant particles and place them in a 1.5 mL centrifuge tube. Add 1 mL of 0.5% formic acid solution to prepare a dispersion of the extractant particles. Thoroughly shake the dispersion to keep it in a uniform suspension. Balance: Pipette 100 μl of the extractant particle dispersion into a test tube, add 1 mL of 0.5% formic acid aqueous solution, vortex for 1 minute, place the test tube on a magnetic rack, and let it stand for 30 seconds to allow the magnetic extractant particles to be completely adsorbed on one side of the inner wall of the test tube; aspirate and discard the residual liquid; Sample loading: Pipette 100 μl of the sample extract from step 2 into a test tube, vortex for 1 minute, place the test tube on a magnetic rack, and let it stand for 30 seconds to allow the magnetic extractant particles to be completely adsorbed to one side of the inner wall of the test tube; aspirate and discard the residual liquid; Washing: Pipette 1 mL of methanol into a test tube, vortex for 1 minute, place the test tube on a magnetic rack, and let it stand for 30 seconds to allow the magnetic extractant particles to be completely adsorbed on one side of the inner wall of the test tube; aspirate and discard the residual liquid; Elution: Pipette 1 mL of 8% ammonia solution into a test tube, vortex for 1 minute, place the test tube on a magnetic rack, and let it stand for 30 seconds to allow the magnetic extractant particles to be completely adsorbed on one side of the inner wall of the test tube. Collect the eluate, concentrate to dryness under reduced pressure, and dilute to 1 mL with 0.5% formic acid aqueous solution to use as the test solution.

5. The method for determining brucine and strychnine in the Nux vomica medicinal material according to claim 1, characterized in that: The mass spectrometric parameters of strychnine are: parent ion m / z is 395.0, the fragmentor voltage is 195V, and the quantitative ion m / z 244.1, qualitative ion m / z is 324.1, and the collision energies of the quantitative ion and the qualitative ion are 44eV and 37eV, respectively; The mass spectrometric parameters of strychnine are: parent ion m / z is 336.0, the fragmentor voltage is 190V, and the quantitative ion m / z 185.1, qualitative ion m / z The collision energy of the quantitative ion and the qualitative ion is 46eV and 62eV respectively.

6. The method for determining brucine and strychnine in the Nux vomica medicinal material according to claim 3, characterized in that: The power of the ultrasonic treatment in step 2 was 300 W and the frequency was 40 kHz.

7. The method for determining brucine and strychnine in the Nux vomica medicinal material according to claim 4, characterized in that: Mixed cation exchange MCX magnetic extractant particles are used in step 3.

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