Detection method suitable for three aconitine in multiple compound traditional Chinese medicine preparations

Abstract

The invention relates to a method suitable for detecting three aconitine in various compound traditional Chinese medicine preparations, which comprises the following steps: preparing a reference extract solution and a test solution, introducing a magnetic solid-phase extraction technology when the test solution is prepared, and accurately'capturing 'and separating target aconitine from a complex chemical component background; a multi-reaction monitoring mode of triple quadrupole mass spectrometry is adopted, and a Shim-pack Scept C18-120 chromatographic column is adopted as a chromatographic column; the column temperature is 40 DEG C; a mobile phase is 0.14% formic acid aqueous solution (A)-acetonitrile (B), gradient elution is carried out for 0-5 min, 95% A > 5% A; the flow rate is 0.3 mL * min <-1 >; the sample size is 1 microliter; a mass spectrum ion source is an electrospray ionization source, a positive ion multi-reaction monitoring mode is adopted, the capillary voltage is 3.5 kV, the ion source temperature is 150 DEG C, and the solvent removal temperature is 500 DEG C. The high-efficiency purification of MCX and the high-specificity detection of LC-MS / MS are combined to form a set of powerful method system, the method system is successfully applied to various dosage forms with different formulas, and the industry dilemma of'one preparation and one method 'is solved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of test or analysis method, relates to the determination of certain chemical components in Chinese patent medicine, and in particular to a detection method suitable for three aconitine in various compound Chinese medicine preparations. BACKGROUND

[0002] Compound Chinese medicine preparation is the main form of clinical medicine of traditional Chinese medicine, and achieves the treatment purpose through the synergistic effect of multiple components and multiple targets. With the promotion and application of traditional Chinese medicine in the world, the standardization control of its safety and effectiveness has become the core issue of the industry development. Among them, the compound preparations containing aconite medicinal materials (such as aconite, Sichuan aconite and caowu) (such as rheumatism and pain drugs) are widely used in clinical practice, but the aconite alkaloids (especially double-ester aconitine) contained in them have a narrow therapeutic window and strong toxicity, which are not only the key effective substances, but also the main toxic components. Therefore, establishing an accurate, sensitive and reliable detection method for aconitine is the key technical prerequisite for ensuring the safety of clinical use of such compound preparations and realizing quality control.

[0003] At present, there are many reports on the detection of aconitine in single aconite medicinal materials or preparations with relatively simple components (such as HPLC, LC-MS, etc.). However, when facing compound Chinese medicine preparations with extremely complex components, the existing conventional methods face severe challenges: Severe matrix interference: A large number of coexisting components (such as flavones, saponins, pigments, sugars, etc.) in compound preparations will seriously interfere with the separation and detection of target aconitine, resulting in poor chromatographic peak separation, high baseline noise and inaccurate quantification.

[0004] Extremely low content of target components: The most toxic double-ester aconitine (such as aconitine, new aconitine and hypaconitine) usually has trace or ultra-trace content in preparations, which puts high requirements on the sensitivity (LOD / LOQ) and selectivity of the detection method.

[0005] Complicated pretreatment process and unstable recovery rate: The existing methods mostly use complex liquid-liquid extraction or solid-phase extraction for purification, which is tedious, time-consuming and labor-intensive, and the recovery rate under different preparation matrixes fluctuates greatly, affecting the accuracy and universality of the method.

[0006] Lack of universal method: The compound preparations on the market have different formulas and various dosage forms (such as pills, powders, tablets and injection solutions). At present, there is a lack of a standardized detection method that can be applied across different varieties and dosage forms, which leads to poor comparability of data from different laboratories and different products, and is not conducive to supervision and enterprise quality control.

[0007] In summary, the current urgent need to develop a high selectivity, high sensitivity, high throughput, and can effectively overcome the interference of complex matrix detection method, used for a variety of compound Chinese medicine preparation in 3 key aconitine (aconitine, new aconitine, hypaconitine) synchronous quantitative analysis. SUMMARY

[0008] In view of the shortcomings and deficiencies of the prior art, the purpose of the present application is to provide a method for detecting three aconitines in various compound Chinese medicine preparations, which has strong anti-interference ability, can accurately "capture" and separate the target aconitine from the complex chemical composition background, and has excellent sensitivity, accuracy, good universality and operability, can be applied to different formula compound preparations, realizes the synchronous, rapid and accurate detection of three aconitines (aconitine, new aconitine, hypaconitine) in various compound preparations, and solves the industry dilemma of "one preparation one method".

[0009] To achieve the above object, the technical scheme adopted by the present application is as follows: A method for detecting three aconitines in various compound Chinese medicine preparations, the three aconitines are new aconitine, hypaconitine and aconitine, and the method specifically comprises the following steps: Step 1. Preparation of control extract solution; Step 2. Preparation of test sample solution: Step 2.1. Sample solution extraction: take the test sample and place it in a conical flask, add a mixed solution of hydrochloric acid-methanol=1:100, weigh, ultrasonic treatment, cool, re-weigh, make up the weight loss with a mixed solution of hydrochloric acid-methanol=1:100, filter, take the filtrate, reduce the pressure to recover the solvent to dryness, add 0.1% formic acid solution to the residue, shake to dissolve, centrifuge, and take the supernatant as the sample solution; Step 2.2. Purification of sample solution: Magnetic extractant preparation: weigh the magnetic extractant particles and place them in a centrifuge tube, add 5% methanol solution, prepare the extractant particle dispersion, shake the extractant particle dispersion well, and keep the extractant particle dispersion in uniform suspension; wherein the magnetic extractant particles are mixed cation exchange magnetic extractant particles loaded with benzene sulfonic acid groups on superparamagnetic Fe3O4 particles; Equilibrium: take the extractant particle dispersion into a test tube, add 0.1% formic acid solution, vortex, place the test tube on a magnetic stand, and stand still to make the magnetic extractant particles completely adsorbed on one side of the inner wall of the test tube; discard the residual liquid; Loading: take the sample solution of step 2.1 into the test tube, and perform the same operation as "equilibrium", and discard the residual liquid; Elution: take methanol into the test tube, perform the same operation as "equilibrium", and discard the residual liquid; Elution: Take 8% ammonia methanol solution in a test tube, same as "equilibrium" operation, and collect the eluent, concentrate to dryness under reduced pressure, dilute to 1 mL with 0.1% formic acid aqueous solution as the test sample solution; Step 3. Chromatography-mass spectrometry conditions: Chromatographic column: Shim-pack Scepter C18-120 chromatographic column; column temperature: 40 ℃; mobile phase: 0.14% formic acid aqueous solution (A) - acetonitrile (B), gradient elution: 95% A→5% A for 0-5 min; flow rate: 0.3 mL·min -1 ; injection volume: 1 μL; mass spectrometry ion source is an electrospray ionization source, positive ion multiple reaction monitoring mode, capillary voltage 3.5 kV, ion source temperature 150 ℃, desolvation temperature 500 ℃.

[0010] As a preferred embodiment of the present application, the specific steps of step 1 are as follows: weigh the aconitum bifurcatum ester-type alkaloid control extract, dissolve it with 0.1% formic acid aqueous solution to prepare a control extract stock solution; then take the control extract stock solution, dilute to the mark with 0.1% formic acid aqueous solution as the control extract solution.

[0011] As a preferred embodiment of the present application, the concentration of hydrochloric acid in step 2.1 is 36%-38%, and the concentration of ammonia water in step 2.2 is 25%-28%.

[0012] As a preferred embodiment of the present application, in step 2.1, the ultrasonic treatment is performed for 30 minutes, the power is 250 W, and the frequency is 33 kHz; the centrifugation is performed for 5 minutes at a speed of 8000 revolutions per minute.

[0013] As a preferred embodiment of the present application, the specific steps of step 2.2 are as follows: Magnetic extractant preparation: weigh 0.1 g of magnetic extractant particles, put them in a 1.5 mL centrifuge tube, add 1 mL of 5% methanol aqueous solution to prepare an extractant particle dispersion, shake the extractant particle dispersion thoroughly, and keep the dispersion in a uniform suspension state; Equilibrium: take 100 μl of the extractant particle dispersion in a test tube, add 1 mL of 0.1% formic acid aqueous solution, vortex for 1 minute, place the test tube on a magnetic stand, and stand for 30 seconds to make the magnetic extractant particles completely adsorbed on one side of the inner wall of the test tube; then suck and discard the residual liquid; Loading: take 100 μl of the sample solution of step 2.1 in a test tube, vortex for 1 minute, place the test tube on a magnetic stand, and stand for 30 seconds to make the magnetic extractant particles completely adsorbed on one side of the inner wall of the test tube; then suck and discard the residual liquid; Elution: take 1 mL of methanol in a test tube, vortex for 1 minute, place the test tube on a magnetic stand, and stand for 30 seconds to make the magnetic extractant particles completely adsorbed on one side of the inner wall of the test tube; then suck and discard the residual liquid; Elution: 1 ml of 8% ammonia methanol solution was taken in a test tube, vortexed for 1 minute, and the test tube was placed on a magnetic stand and allowed to stand for 30 seconds, so that the magnetic extractant particles were completely adsorbed on one side of the inner wall of the test tube. The eluent was collected, concentrated under reduced pressure to dryness, diluted with 0.1% formic acid solution to 1 mL as the test sample solution.

[0014] As a preferred embodiment of the present application, the mass spectrometry parameters of aconitine are as follows: parent ion m / z 646.3 ， Fragmentation voltage is 200V ， Quantitative ion m / z 586.3 ， Qualitative ion m / z 554.3, the collision energies of the quantitative ion and the qualitative ion are 41eV and 45eV respectively; The mass spectrometry parameters of hypaconitine are as follows: parent ion m / z 616.3 ， Fragmentation voltage is 155V, quantitative ion m / z 556.3 ， Qualitative ion m / z 524.3 ， The collision energies of the quantitative ion and the qualitative ion are 37eV and 45eV respectively; The mass spectrometry parameters of mesaconitine are as follows: parent ion m / z 632.3 ， Fragmentation voltage is 155V, quantitative ion m / z 572.3 ， Qualitative ion m / z 540.3 ， The collision energies of the quantitative ion and the qualitative ion are 37eV and 45eV respectively.

[0015] The advantages and beneficial effects of the present application are as follows: (1) Revolutionary advantage of pretreatment technology: introduction of magnetic solid phase extraction Excellent anti-matrix interference ability: the present application first applies the magnetic solid phase extraction technology to the purification of aconitine in complex traditional Chinese medicine. Through the specific adsorbent-coated magnetic nanoparticles, the target molecules can be captured with high selectivity and high efficiency from the complex sample solution, while most of the sugar, pigment, protein and non-target polar components are left in the supernatant. This "adsorption-magnetic separation" mode fundamentally solves the core problem of incomplete matrix purification in traditional methods.

[0016] Easy operation, fast and efficient: MCX purification process does not require the column packing, activation, sample loading, elution, and other cumbersome steps required by traditional solid phase extraction. Only need to add the sample solution to the magnetic adsorbent material, vortex adsorption, and use the external magnet to realize the liquid-solid rapid separation in a few seconds. The whole pretreatment time can be shortened to less than one third of the traditional method, greatly improving the analysis throughput.

[0017] Save solvent and cost, environmentally friendly: MCX purification requires less adsorbent (usually a few tens of milligrams), and the elution solvent consumption is very small (usually only 1-2 mL). Compared with traditional liquid-liquid extraction (consumes hundreds of milliliters of organic solvent) or conventional SPE, it significantly reduces the consumption of organic solvents and waste liquid production, in line with the development trend of green analytical chemistry.

[0018] Stable and high recovery rate: The large specific surface area of the magnetic nanomaterial provides abundant adsorption sites, combined with optimized adsorption / elution conditions, which can achieve nearly quantitative adsorption and release of trace aconitine. This method shows high and stable recovery rate (such as 91.1%-96.1%) in various different matrices, ensuring the accuracy and reliability of quantitative results.

[0019] (2) Core advantages of detection technology: LC-MS / MS precise quantification High sensitivity and specificity: The multiple reaction monitoring mode of triple quadrupole mass spectrometry can detect the characteristic parent ion-daughter ion pair of aconitine. This method completely avoids chromatographic co-elution interference, even if the target peak does not achieve complete baseline chromatographic separation, it can also be accurately quantified through the mass spectrometry channel. Its detection limit for 3 aconitines can reach ng / mL level, which fully meets the monitoring needs of trace toxic ingredients, and ensures the accurate quantification of trace toxic ingredients, providing a reliable basis for safety threshold setting.

[0020] Simultaneous and accurate quantitative analysis: LC-MS / MS method can complete the simultaneous separation and quantification of aconitine, hypaconitine, and mesaconitine in 5 minutes with one injection. Mass spectrometry detection has a wide linear range and good linear relationship, providing accurate quantitative basis for samples with different content levels.

[0021] (3) Innovation and application value of the whole method Strong method universality and reliability: The combination of MCX's efficient purification and LC-MS / MS's high specificity detection forms a powerful method system. Through verification, this method has been successfully applied to various dosage forms (such as pills, capsules, etc.) of different formulations, and satisfactory analysis results have been obtained. It solves the industry dilemma of "one formulation one method".

[0022] This method provides a universal solution for the quality control and standard upgrading of traditional Chinese medicine (TCM): It integrates high sensitivity, high selectivity, high accuracy, high speed, low cost, and environmental friendliness. It is not only suitable for enterprises' daily quality control and process monitoring, but also provides a powerful technical tool for drug regulatory authorities' sampling inspections, anti-counterfeiting efforts, and adverse reaction tracing. Its establishment and implementation are expected to promote the unification and improvement of quality standards for compound preparations of toxic TCM containing aconite, thus ensuring public medication safety from a technical perspective.

[0023] It has good prospects for promotion: the pretreatment steps of this method are simple and easy to master, and the detection process is highly automated. It is very suitable for promotion and application in the routine laboratories of drug testing institutes, enterprise quality inspection centers and research institutions, and has important industrial application value. Attached Figure Description

[0024] Figure 1 This is a quantitative ion diagram of aconitine in the control extract; Figure 2 Quantitative ion chromatogram of aconitine in control extract; Figure 3 Quantitative ion chromatogram of neoaconitine in the control extract; Figure 4 Quantitative ion chromatogram of aconitine in Papaya Pills (batch number: 2305020); Figure 5 Quantitative ion chromatogram of aconitine in Papaya Pills (batch number: 2305020); Figure 6 Quantitative ion chromatogram of neoaconitine in Papaya Pills (batch number: 2305020); Figure 7 Quantitative ion chromatogram of aconitine in Fugui Gutong Granules (batch number: 2203003); Figure 8 Quantitative ion chromatogram of aconitine in Fugui Gutong Granules (batch number: 2203003); Figure 9 Quantitative ion chromatogram of neoaconitine in Fugui Gutong Granules (batch number: 2203003); Figure 10 The liquid chromatogram of Papaya Pill (batch number: 2305020) was determined using the pharmacopoeia method; Figure 11 The liquid chromatogram of Fugui Gutong Granules (batch number: 2203003) was determined using the pharmacopoeia method. Detailed Implementation

[0025] The present invention will be 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 described herein, but this does not limit the present invention.

[0026] 1. Instruments and Materials 1.1 Instruments Agilent 1290 Infinity II chromatograph (Agilent Technologies); Agilent 6470LC / TQ triple quadrupole tandem mass spectrometer detector (Agilent Technologies); KQ-500DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); XPE205DR 0.0001 g electronic analytical balance (METTLER TOLEDO); Milli-Q ultrapure water system (Millipore).

[0027] 1.2 Reagents used in the experiment Acetonitrile, methanol, and formic acid were chromatographically pure; hydrochloric acid, sulfuric acid, dichloromethane, ammonia, and diethyl ether were analytically pure products of Sinopharm Chemical Reagent Co., Ltd. The aconitine diester-type alkaloid reference extract (batch number: 112029-202302, content calculated as neoaconitine 32.9%, hypoaconitine 30.9%, aconitine 32.7%), hypoaconitine (batch number: 110798-202010, content calculated as 99.2%), and neoaconitine (batch number: 110799-202409, content calculated as 98.6%) were products of the China National Institutes for Food and Drug Control. Aconitine (batch number: DSTDW000602) was from Lemeitian Pharmaceutical | Desite Biotechnology. The MCX magnetic extraction material was superparamagnetic Fe3O4 particles + benzenesulfonic acid groups (strong cation exchange) from Putton Laboratory Equipment (Shanghai) Co., Ltd.

[0028] 1.3 Medicines The traditional Chinese medicines, Papaya Pills and Cinnamon Bone Pain Granules, were purchased from pharmacies. The manufacturers and batch numbers are shown in Table 1.

[0029] Table 1 List of Drugs No. Name Manufacturing enterprise Batch No. 1 Mugua Pill Fusheng Pharmaceutical Co. 2305020 2 Mugua Pill Taifu Pharmaceutical Co. 230701 3 Mugua Pill Shandong Xin Dalu Pharmaceutical Co. 2305213 4 Mugua Pill Shandong Xin Dalu Pharmaceutical Co. 23052531 5 Mugua Pill Beijing Tong Ren Tang 22073509 6 Fugui Osteodynia Granules Dongguan Jinmei Pharmaceutical Co. 2203003 7 Fugui Osteodynia Granules Dongguan Jinmei Pharmaceutical Co. 2110007 8 Fugui Osteodynia Granules Dongguan Jinmei Pharmaceutical Co. 2111022 2. Methods and Results 2.1 Preparation of control solution 2.1.1 Preparation of the reference extract stock solution: Take about 10 mg of aconitine diester type alkaloid reference extract (content calculated as neoaconitine 32.9%, hypoaconitine 30.9%, aconitine 32.7%), accurately weigh it, place it in a 50 ml volumetric flask, add 0.1% formic acid aqueous solution to dissolve and dilute to the mark, and use it as the reference extract stock solution.

[0030] 2.1.2 Preparation of control extract solution: Take 0.1 ml of the solution in 2.1.1 above, place it in a 100 ml volumetric flask, and dilute to the mark with 0.1% formic acid aqueous solution to obtain the control extract solution.

[0031] 2.2 Preparation of Sample Solution 2.2.1 Sample solution extraction: Weigh approximately 0.5g of sample powder accurately and place it in a stoppered conical flask. Accurately add 50ml of a mixed solution of hydrochloric acid (concentration 36%–38%) and methanol (1:100), weigh the solution, sonicate (power 250W, frequency 33kHz) for 30 minutes, cool, weigh the solution again, replenish the lost weight with the mixed solution of hydrochloric acid (36%–38%) and methanol (1:100), shake well, filter, accurately measure 20ml of the filtrate, and recover the solvent under reduced pressure below 40℃ until dry. Add 2ml of 0.1% formic acid solution to the residue, shake to dissolve, centrifuge (speed 8000 rpm) for 5 minutes, and take the supernatant as the sample solution.

[0032] 2.2.2 Sample solution purification Preparation of magnetic extractant: Weigh approximately 0.1g of extractant (magnetic extractant particles) into a 1.5mL centrifuge tube, add 1mL of 5% methanol aqueous solution to obtain an extractant particle dispersion. This is equivalent to 10mg of extractant per 100μl dispersion. Shake the extractant particle dispersion thoroughly to maintain a uniform suspension.

[0033] Equilibrium: Transfer 100 μl of the above extractant particle dispersion into a test tube, add 1 ml of 0.1% 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 onto one side of the inner wall of the test tube; remove and discard the residual liquid to avoid carrying out the magnetic extractant particles.

[0034] Sample loading: Transfer 100 μl of the sample solution from section 2.2.1 into a test tube, perform the same "equilibration" procedure, and discard the residual liquid (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 onto one side of the inner wall of the test tube; aspirate and discard the residual liquid). Rinse: Transfer 1 ml of methanol into a test tube, perform the same procedure as "equilibration", and discard the residual liquid; Elution: Transfer 1 ml of 8% ammonia water (concentration of 25% to 28%) methanol solution (V / V), perform the same "equilibrium" operation, collect the eluent, concentrate to dryness under reduced pressure, and dilute to 1 ml with 0.1% formic acid aqueous solution to obtain the test solution.

[0035] 2.3 Chromatographic-Mass Spectrometry Conditions Chromatographic column: Shim-pack Scepter C18-120 column (2.1 mm × 150 mm, 1.9 μm); column temperature: 40 ℃; mobile phase: 0.14% formic acid aqueous solution (A) - acetonitrile (B), gradient elution (0–5 min, 95% A → 5% A); flow rate: 0.3 mL·min -1Injection volume: 1 μL; Mass spectrometry ion source: electrospray ionization (ESI), positive ion multiple reaction monitoring (MRM) mode, capillary voltage: 3.5 kV, ion source temperature: 150 ℃, desolventization temperature: 500 ℃; Mass spectrometry conditions for aconitine, hypoaconitine and neoaconitine are shown in Table 2.

[0036] Table 2 MS parameters of the target components

[0037] In this embodiment, the mobile phase also included: 0.1% formic acid aqueous solution (A) - acetonitrile (B), 0.1% ammonium acetate solution - acetonitrile, and acetonitrile - ammonium bicarbonate solution. However, it was found that 0.14% formic acid aqueous solution (A) - acetonitrile (B) was the most effective, achieving efficient separation. Furthermore, compared to the fluidity of 0.1% formic acid aqueous solution (A) - acetonitrile (B), 0.14% formic acid aqueous solution (A) - acetonitrile (B) effectively prevented tailing. In addition, other gradient elution methods were tried at the fluidity of 0.14% formic acid aqueous solution (A) - acetonitrile (B), but the results were not ideal. The above-mentioned sample pretreatment method combined with the above-mentioned chromatographic conditions is necessary to achieve effective detection of three aconitines in different compound traditional Chinese medicine preparations. Moreover, by comparing the chromatograms of the samples measured in this application with the high-performance liquid chromatography (HPLC) of the pharmacopoeia, it can be seen that the sample pretreatment method provided in this application is not only fast and uses less reagent, but also has a better purification effect, with only a small amount of alkaloid impurities remaining, and these impurities can be well separated under the above-mentioned chromatographic conditions.

[0038] 2.4. Methodological Examination: 2.4.1 Linearity Assessment: Accurately pipette 20, 50, 100, 200, 500, and 1000 μL of the reference extract stock solution from section 2.1.1 into six 100 mL volumetric flasks. Dilute to the mark with 0.1% formic acid solution, shake well, and the resulting series of reference standard mixed solutions are obtained. Inject the solutions according to the conditions described in section 2.3. Plot the peak area Y of the reference standard on the ordinate, corresponding to the concentration (ng·mL⁻¹). -1 X is the horizontal axis. Linear regression was performed, and the results are shown in Table 3.

[0039] Table 3 Linear Equations and Linear Ranges Component Linear equation Linear range / (ng mL -1 ) Aconitine =125.32722.7 0.9992 13.54～676.9 Hypaconitine =94.12161.6 0.9997 12.79～639.6 Mesaconitine = 98.4+ 1553.8 0.9994 13.62～681.0 2.4.2 Limit of Detection and Quantitative Test: Accurately pipette the reference extract solution from section “2.1.2”, and use the dilution method to determine the limit of detection and limit of quantitation. Quantify the signal-to-noise ratio of the ion chromatography peak. S / NThe lowest concentration corresponding to ≥3 was defined as the limit of detection (LOD), and the lowest concentration corresponding to a quantitative ion chromatography peak signal-to-noise ratio ≥10 was defined as the limit of quantitation (LOQ). Peak areas were recorded. The results showed that the LOD for aconitine was 0.68 ng / ml and the LOD for hypoaconitine was 1.35 ng / ml; the LOD for hypoaconitine was 0.64 ng / ml and the LOD for hypoaconitine was 1.28 ng / ml; and the LOD for neoaconitine was 0.68 ng / ml and the LOD for neoaconitine was 1.36 ng / ml.

[0040] 2.4.3 Precision Test: Accurately pipette 1 μL of the control extract solution from section "2.1.2", inject it 6 times consecutively, and record the peak area. The RSD of the aconitine peak area is calculated as follows: n =6) respectively is 0.61%; the RSD of the peak area of ​​aconitine is ( n =6) respectively is 0.72%; the RSD of the peak area of ​​neoacin is ( n =6) is 0.55%, which indicates that the instrument has good precision.

[0041] 2.4.4. Stability Test: The same sample solution (Papaya Pill, batch number 2305020) was injected and measured at 0, 2, 4, 8, 12, and 24 h. The RSD of the aconitine peak area was determined. n =6) is 1.06%; the RSD of the peak area of ​​hypoaconitine ( n =6) is 0.98%; the RSD of the peak area of ​​neoaconitine ( n =6) is 1.15%, indicating that the test solution is stable within 24 h.

[0042] 2.4.5. Repeatability Test: Six test solutions were prepared according to the method in section “2.2”, and each solution was injected for determination. The results showed that the average content of aconitine was 0.329 μg / g; the average content of hypoaconitine was 1.352 μg / g; and the average content of neoaconitine was 0.990 μg / pill. The RSDs of all solutions were less than 3.0%, indicating that the method had good repeatability.

[0043] 2.4.6. Spiking Recovery Test: Take approximately 0.25 g of the test sample with known content (Papaya Pills, batch number 2305020, after removing the coating, average pill weight 0.1864 g / pill; Fugui Gutong Granules, batch number 2203003, take the contents, average fill weight 5.0200 g / bag), accurately weigh it, place it in a stoppered conical flask, and accurately add an appropriate amount of reference standard (Papaya Pills plus 0.10 ml of aconitine reference solution (concentration 0.922 μg / ml), and 0.40 ml of hypoaconitine reference solution (concentration 0.9...). 0.25 ml of neoaconitine reference solution (concentration 1.001 μg / ml) was added to Fugui Gutong granules; 0.05 ml of aconitine reference solution (concentration 0.922 μg / ml), 1.00 ml of hypoaconitine reference solution (concentration 0.930 μg / ml), and 0.10 ml of neoaconitine reference solution (concentration 1.001 μg / ml) were added to the other steps in parallel with the preparation of the test solution in section "2.2 Preparation of Test Solution". The samples were injected for analysis, and the recovery rate was calculated. The results are shown in Tables 4 to 9.

[0044] Table 4. Recovery rate of aconitine in papaya pills (n=6)

[0045] Table 5. Recovery rate of aconitine in papaya pills (n=6)

[0046] Table 6. Recovery rate of neoaconitine in papaya pills (n=6)

[0047] Table 7. Recovery rate of aconitine in Fugui Gutong Granules (n=6)

[0048] Table 8. Recovery rate of aconitine in Fugui Gutong Granules (n=6)

[0049] Table 9. Recovery rate of neoaconitine in Fugui Gutong Granules (n=6)

[0050] 2.5. Content determination: Two different types of preparations, Papaya Pills and Fugui Gutong Granules, were taken in appropriate amounts and test solutions were prepared according to the method described in section "2.2". The solutions were then injected and analyzed according to the method described in section "2.3". The contents of the three aconitines were calculated, and the contents were determined according to the method specified in Part I of the 2025 edition of the Chinese Pharmacopoeia. The results are shown in Table 10. Quantitative ion chromatograms of the reference standard and the test samples are shown in [Table 10].Figures 1-9 .

[0051] Table 10 Results of Sample Content Determination Name Batch No. Content determined by the method (μg / g) Content determined by pharmacopoeia method (μg / g) Relative average deviation (%) Mugua Pill 2305020 2.671 2.695 0.89 Mugua Pill 230701 14.121 14.062 0.42 Mugua Pill 2305213 8.247 8.193 0.66 Mugua Pill 23052531 12.675 12.715 0.32 Mugua Pill 22073509 21.880 21.623 1.18 Fugui Osteodynia Granules 2203003 4.019 4.036 0.42 Fugui Osteodynia Granules 2110007 3.823 3.811 0.31 Fugui Osteodynia Granules 2111022 8.362 8.426 0.76 After verification, the results of the method of this invention are consistent with those of the pharmacopoeia method, therefore the method is considered feasible. This method has high specificity, high purification level, wide applicability, and fast detection speed, completing sample extraction, purification, and detection within one hour.

[0052] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Unnecessary technical features can be added or removed according to actual needs to meet the needs of different situations.

Claims

1. A method for detecting three aconitines in various compound traditional Chinese medicine preparations, wherein the three aconitines are neoaconitine, hypoaconitine, and aconitine, characterized in that, The method includes the following steps: Step 1. Prepare the control extract solution; Step 2. Prepare the test solution: Step 2.

1. Sample solution extraction: Take the test sample, place it in an Erlenmeyer flask, add a mixed solution of hydrochloric acid and methanol = 1:100, weigh it, sonicate it, cool it, weigh it again, make up the weight loss, filter it, measure the filtrate, recover the solvent under reduced pressure until dry, add 0.1% formic acid solution to the residue, shake to dissolve it, centrifuge it, and take the supernatant as the sample solution; Step 2.

2. Sample solution purification: Preparation of magnetic extractant: Weigh the magnetic extractant particles, place them in a centrifuge tube, add 5% methanol aqueous solution to obtain the extractant particle dispersion, shake thoroughly to keep the extractant particle dispersion in a uniform suspension state; wherein, the magnetic extractant particles are mixed cation exchange magnetic extractant particles of superparamagnetic Fe3O4 particles loaded with benzenesulfonic acid groups. Equilibrium: Transfer the extractant particle dispersion into a test tube, add 0.1% formic acid aqueous solution, vortex, place the test tube on a magnetic rack, and let it stand to allow the magnetic extractant particles to be completely adsorbed onto one side of the inner wall of the test tube; remove and discard the residual liquid. Sample loading: Transfer the sample solution from step 2.1 into a test tube, perform the same "equilibration" operation, and discard the residual liquid; Rinsing: Transfer methanol into a test tube, perform the same procedure as "equilibration", and discard the residual liquid; Elution: Transfer 8% ammonia-methanol solution into a test tube, perform the same "equilibrium" procedure, collect the eluent, concentrate to dryness under reduced pressure, and dilute to 1 mL with 0.1% formic acid aqueous solution to obtain the test solution; Step 3. Chromatographic-mass spectrometry conditions: Chromatographic column: Shim-pack Scepter C18-120 column; column temperature: 40 ℃; mobile phase: 0.14% formic acid aqueous solution (A) - acetonitrile (B), gradient elution: 0–5 min, 95% A → 5% A; flow rate: 0.3 mL·min -1 Injection volume: 1 μL; Mass spectrometry ion source is electrospray ionization source, positive ion multiple reaction monitoring mode, capillary voltage 3.5 kV, ion source temperature 150 ℃, desolventization temperature 500 ℃.

2. The method for detecting three aconitines in various compound traditional Chinese medicine preparations according to claim 1, characterized in that, The specific steps of step 1 are as follows: Weigh aconite diester-type alkaloid reference extract, add 0.1% formic acid aqueous solution to dissolve it, and prepare reference extract stock solution; then take the reference extract stock solution, add 0.1% formic acid aqueous solution to dilute to the mark, and use it as reference extract solution.

3. The method for detecting three aconitines in various compound traditional Chinese medicine preparations according to claim 1, characterized in that, In step 2.1, the concentration of hydrochloric acid is 36%–38%, and in step 2.2, the concentration of ammonia is 25%–28%.

4. The method for detecting three aconitines in various compound traditional Chinese medicine preparations according to claim 1, characterized in that, In step 2.1, ultrasonic treatment is performed for 30 minutes at a power of 250W and a frequency of 33kHz; centrifugation is performed for 5 minutes at a speed of 8000 revolutions per minute.

5. The method for detecting three aconitines in various compound traditional Chinese medicine preparations according to claim 1, characterized in that, The specific steps of step 2.2 are as follows: Preparation of magnetic extractant: Weigh 0.1g of magnetic extractant particles, place them in a 1.5mL centrifuge tube, add 1mL of 5% methanol aqueous solution to obtain extractant particle dispersion, and shake the extractant particle dispersion thoroughly to keep the dispersion in a uniform suspension state. Equilibration: Transfer 100 μl of the extractant particle dispersion into a test tube, add 1 mL of 0.1% 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 onto one side of the inner wall of the test tube; remove and discard the residual liquid; Sample loading: Transfer 100 μl of the sample solution from step 2.1 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 onto one side of the inner wall of the test tube; remove and discard the residual liquid. Washing: Transfer 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 onto one side of the inner wall of the test tube; remove and discard the residual liquid. Elution: Transfer 1 ml of 8% ammonia-methanol 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 onto one side of the inner wall of the test tube. Collect the eluent, concentrate it to dryness under reduced pressure, and dilute it to 1 mL with 0.1% formic acid aqueous solution to obtain the test solution.

6. The method for detecting three aconitines in various compound traditional Chinese medicine preparations according to claim 1, characterized in that, The mass spectrometry parameters of aconitine are: precursor ion m / z It is 646.3 ， The breaking voltage is 200V ， Quantitative ions m / z It is 586.3 ， Qualitative ions m / z The value is 554.3, and the collision energies of quantitative and qualitative ions are 41 eV and 45 eV, respectively. The mass spectrometry parameters of aconitine are: parent ion m / z It is 616.3 ， The fragmentation voltage is 155V, and the quantitative ion... m / z It is 556.3 ， Qualitative ions m / z It is 524.

3. ， The collision energies of quantitative and qualitative ions are 37 eV and 45 eV, respectively. The mass spectrometry parameters of neoaconitine are: parent ion m / z It is 632.3 ， The fragmentation voltage is 155V, and the quantitative ion... m / z It is 572.

3. ， Qualitative ions m / z It is 540.3 ， The collision energies of quantitative and qualitative ions are 37 eV and 45 eV, respectively.

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