CN110907545A - Method for simultaneously determining contents of six metabolites in small gold preparation - Google Patents
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Abstract
The invention discloses a method for simultaneously determining the content of six metabolites in a Xiaojin preparation, wherein the six metabolites comprise aconitine, songorine, lioline, 3-acetyl-11-keto- β -boswellic acid, 11-keto- β -boswellic acid and wormcast, and the determination method comprises the following steps of 1) preprocessing a plasma sample to obtain a sample solution to be detected, and 2) adding a positive ion internal standard and a negative ion internal standard into the sample solution to be detected to perform HPLC-MS/MS detection.
Description
Technical Field
The invention relates to a method for measuring the content of drug metabolites in a biological sample, in particular to a method for simultaneously measuring the content of six metabolites in a Xiaojin preparation.
Background
A traditional Chinese medicine with the effects of dissipating stagnation, relieving swelling, removing blood stasis and relieving pain is collected from the first part of the 2010 edition of Chinese pharmacopoeia, and the medicine is named as Xiaojin pill and is prepared from 10 traditional Chinese medicines of artificial musk, semen momordicae, radix aconiti agrestis, resina liquidambaris, frankincense, myrrh, trogopterus dung, angelica sinensis, earthworm, fragrant ink and the like. The formula is a classic and famous traditional Chinese medicine formula, and can be used for treating various difficult and complicated diseases such as goiter, benign prostatic hyperplasia, postherpetic neuralgia, cystic hyperplasia of breast, chronic pelvic inflammatory disease mass, nodular fasciitis and the like. At present, various preparations such as tablets, capsules and the like are on the market, for example, CN106265992A discloses Xiaojin capsules and a preparation method thereof, and CN102764311A discloses Xiaojin dispersible tablets and a preparation method thereof.
In order to find out the compatibility rule of the Chinese herbal compound and reveal the pharmacodynamic action mechanism and the material basis for treating diseases, the applicant carries out a great deal of basic research on the Xiaojin preparation on the basis of the prior research, wherein the basic research comprises the steps of extracting and separating the parts of the Chinese herbal medicine such as total polysaccharide, total saponin, total flavone and the like by adopting the modern extraction and separation technology and determining effective part groups by combining pharmacodynamic experiments; meanwhile, serum pharmacology research is carried out, and the pharmacological effects of effective components entering the body, metabolites thereof and some endogenous products generated by the body after the action of the medicament are observed. In the research process, a series of compounds possibly having physiological activity, which enter blood serum after the small gold preparation is orally taken, are found for the first time, and in order to better monitor the pharmacokinetic process of the compounds in vivo, a convenient, accurate and sensitive content measurement method is needed to be established.
Disclosure of Invention
The invention aims to provide a method for simultaneously measuring the contents of six metabolites in a small gold preparation.
The Xiaojin preparation is prepared from the following raw materials in parts by weight: 1-20 parts of artificial musk, 40-60 parts of kusnezoff monkshood root, 20-40 parts of frankincense, 40-60 parts of trogopterus dung, 40-60 parts of earthworm, 40-60 parts of cochinchina momordica seed, 40-60 parts of sweetgum resin, 20-40 parts of myrrh, 20-40 parts of Chinese angelica and 1-10 parts of Chinese ink,
the six metabolites are aconitine, songorine, riolin, 3-acetyl-11-keto- β -boswellic acid, 11-keto- β -boswellic acid, and wormcast essence, and their chemical structures are shown in figure 1.
The determination method comprises the following steps:
1) pretreating a plasma sample to obtain a sample solution to be detected;
2) adding a positive ion internal standard and a negative ion internal standard into a sample solution to be detected, and carrying out HPLC-MS/MS detection, wherein the stationary phase of the high-performance liquid phase is a C18 bonded silica gel chromatographic column, the mobile phase A is 0.05-0.5% formic acid aqueous solution, the mobile phase B is acetonitrile, gradient elution is adopted, the flow rate is 0.1-0.5mL/min, the column temperature is 25-40 ℃, the mass spectrum adopts a positive ion mode and a negative ion mode, an electrospray ionization ion source is adopted, the scanning mode is a multi-reaction ion monitoring mode, under the positive ion mode, aconitine, pseudosongorine, riolin, 3-acetyl-11-keto- β -boswellic acid, 11-keto- β -boswellic acid and positive ion internal standard protopine are detected, and under the negative ion mode, imperial and negative ion internal standard isofraxidine are detected.
Preferably, the positive ion internal standard is protopine, and the negative ion internal standard is isofraxidin.
Preferably, the mobile phase a is 0.1% aqueous formic acid.
Preferably, the flow rate is 0.3 mL/min.
Preferably, the column temperature is 35 ℃.
Preferably, the gradient elution is in positive ion mode, 0.01-2.5min, 10-80% acetonitrile; 2.5-3.5min, 80-100% acetonitrile; 3.5-6min, 100% acetonitrile; 6-7min, 100-10% acetonitrile; 7-10min, 10% acetonitrile, 10min of single running time; under the negative ion mode, 0.01-0.5min, 30% acetonitrile; 0.5-2min, 30-80% acetonitrile; 2-2.1min, 80-30% acetonitrile; 2.1-5min, 30% acetonitrile, 5min single run time.
Preferably, the drying gas temperature of the mass spectrum is 550 ℃; the air curtain air is 40 psi; the collision gas is medium; the ion spray voltage is 5500(+)/-4500(-) V; the atomizing gas was 50psi and the auxiliary heating gas was 55 psi.
Preferably, the pretreatment method of the plasma sample is that the plasma sample is vortexed and shaken for 2min, centrifuged at 13000rpm at 4 ℃ for 10min, supernatant solution is removed, the solvent is volatilized by nitrogen, residues are redissolved by methanol solution, centrifuged at 13000rpm at 4 ℃ for 10min, and the supernatant solution is absorbed.
The invention establishes a convenient, accurate, sensitive and reliable LC-MS content determination method for six metabolites of the Xiaojin preparation in a living body for the first time, and the method can be applied to serum pharmacology research of the Xiaojin preparation and better monitor the pharmacokinetic process of a medicament in the body.
Drawings
FIG. 1 shows the structural formulae of six test compounds and 2 internal standard compounds.
FIG. 2 HPLC-MS/MS chromatogram of 6 test compounds and 2 internal standard compounds in female SD rat plasma. A: a blank plasma chromatogram of a rat; b: 6 chromatograms of the compounds to be detected and the internal standard mixed solution; c: adding 6 to-be-detected compounds and an internal standard mixed solution chromatogram map to blank plasma; d: the chromatogram of the plasma and the internal standard compound of the rat 45min after the oral administration of the small gold capsule. (1. aconitine, 2. songorine, 3. rioline, 4.AKBA, 5.KBA, 6. protopine (IS +), 7. imperial, 8. isofraxidin (IS-))
Detailed Description
The invention establishes and develops an HPLC-MS/MS quantitative analysis method of 6 biological metabolites in Chinese medicinal compound small gold capsules produced by Jianmin pharmaceutical industry group member company in rat plasma, and carries out methodology investigation of biological samples, including verification on aspects of specificity, linear range, lower limit of quantitation, precision, accuracy, matrix effect, stability and the like. Therefore, an accurate, rapid, stable and reliable analysis means is provided for the research of the pharmacokinetics of the multicomponent small gold capsules.
According to the principles of 'representative components in monarch drugs and rare drugs, components with larger in vitro content, drug effect components reported in documents, representative components of single drugs, representative components of different compounds' and the like, the research results of earlier-stage medicinal chemistry of the small gold capsule serum of the applicant are synthesized, 6 components of aconitine, pseudosongorine, riolin, 3-acetyl-11-keto- β -boswellic acid (AKBA), 11-keto- β -boswellic acid (KBA) and imperial element are selected for methodological research, the components mainly comprise aconitine alkaloids represented by aconitine, pseudosongorine and riolin, AKBA and KBA are triterpenic acid compounds, the wormcapine is a furan sulfonic acid compound newly found in earthworms, the metabolic rule in vivo of the compound is not reported, and a theoretical basis can be provided for the subsequent research of the chemical structures of the 6 compounds, and the chemical structures of the 6 compounds are shown in figure 1.
1. Experimental Material
1.1 instruments
1.2 drugs and reagents
| Drugs and reagents | Production company | State of the country |
| Aconitine | China institute for testing and testing food and medicine | China (China) |
| Erpartum songaricum alkali | Shanghai leaf Biotech Co., Ltd | China (China) |
| Liaolin | Shanghai leaf Biotech Co., Ltd | China (China) |
| 11-keto- β -boswellic acid | Shanghai leaf Biotech Co., Ltd | China (China) |
| 3-acetyl-11-keto- β -boswellic acid | Shanghai leaf Biotech Co., Ltd | China (China) |
| Protopine (IS) | China institute for testing and testing food and medicine | China (China) |
| Isofraxidin (IS) | China institute for testing and testing food and medicine | China (China) |
| Xiaojin capsule (batch number: 20170231) | Wuhan Jianmin group stocks Co Ltd | China (China) |
| Chloral hydrate | Chemical reagents of national drug group Co Ltd | China (China) |
| Heparin sodium | Chemical reagents of national drug group Co Ltd | China (China) |
| Methanol (quality spectrum level) | Merck | Germany |
| Acetonitrile (quality spectrum level) | Merck | Germany |
| Formic acid (quality spectrum level) | Sigma-Aldrich | United states of America |
| Leucine enkephalin correction fluid | Sigma-Aldrich | United states of America |
| Ultrapure water (18.2 omega) | MilliQ 50SP pure water system | United states of America |
| Ethyl acetate | Shanghai Rujie chemical Co., Ltd | China (China) |
The earthworm extract is obtained by separating and purifying single medicine earthworm in the laboratory, and the structure of the earthworm extract is processed by nuclear magnetic resonance mass spectrum (1HNMR,13C NMR), UPLC-Q-TOF-MS to verify that its structure is 2-hexyl-5-ethyl-furan-3-sulfonic acid. The purity of all standard compounds was higher than 98% by HPLC.
1.3 Experimental animals
Healthy, adult female SD rats, weighing 200 ± 20g, provided by shanghai sipel-bikeka laboratory animals limited company, animal quality certification No.: SCXK (Shanghai) 2018 and 0006. The animals are conventionally bred in the experimental animal center of the medical institute of medicine and pharmacology of the naval military for 7 days, and are fed with standard feed and drinking water, the room temperature is kept at 24 +/-2 ℃, the relative humidity is kept at 55 +/-5%, and the dark illumination period is 12 h. Fasted for 12h before the experiment, but drinking water was still given.
2. Experimental methods and results
2.1 preparation of the solution
Respectively and precisely weighing 5.03mg, 4.95mg, 5.04mg, 4.98mg, 4.93mg and 4.90mg of aconitine, isosongorine, lyoxaline, AKBA, KBA and wormcast element standard substances, putting the weighed materials into a 5mL brown volumetric flask, firstly adding a proper amount of methanol solution for dissolving, then adding methanol to scale for constant volume, and uniformly mixing and shaking to obtain standard substance stock solutions with mass concentrations of 1.006mg/mL, 0.990mg/mL, 1.008mg/mL, 0.996mg/mL, 0.986mg/mL and 0.998 mg/mL.
Respectively and precisely weighing 5.0mg of internal standard protopine and isofraxidin standard, preparing a mixed internal standard stock solution with the mass concentration of 400ng/mL by using a methanol-acetonitrile (1:3, v/v) mixed solution, and storing in a refrigerator at 4 ℃.
2.2 plasma sample Pre-treatment
Precisely measuring 180 mu L of blank plasma of normal rats, placing the blank plasma in a 1.5mL EP tube, and respectively adding 20 mu L of mixed solution of 6 standard substances with various series concentrations and 800 mu L of mixed internal standard solution of protopine and isofraxidin with the solubility of 400 ng/mL. Vortex for 2min, centrifuge at 13000rpm for 10min at 4 ℃ and remove the supernatant solution in another clean 1.5mL EP tube. The solvent was evaporated at 35 ℃ with a nitrogen instrument, the residue was redissolved in 200. mu.L of methanol solution, centrifuged at 13000rpm for 10min at 4 ℃ and the supernatant solution was aspirated and 2. mu.L of sample was injected.
2.3 conditions of analysis
2.3.1 liquid phase conditions
An Agilent Eclipse Plus C18 (2.1X 100mm, 1.8 μm) chromatographic column was used, and the mobile phase consisted of 0.1% (v/v) aqueous formic acid (A) and acetonitrile (B), with a flow rate of 0.3mL/min, a column temperature of 35 ℃ and a sample injection of 2 μ L. The 6 compounds are collected in a positive and negative ion mode, and in order to shorten the collection time and improve the analysis efficiency, the elution gradient is respectively optimized in the positive and negative modes. In positive ion mode, acetonitrile 10-80% (v/v) for 0.01-2.5 min; 2.5-3.5min, 80-100% acetonitrile; 3.5-6.0min, 100% acetonitrile; 6.0-7.0min, 100-10% acetonitrile; 7.0-10.0min, 10% acetonitrile, 10.0min of single running time. Under the negative ion mode, 0.01-0.5min, 30% acetonitrile; 0.5-2.0min, 30-80% acetonitrile; 2.0-2.1min, 80-30% acetonitrile; 2.1-5.0min, 30% acetonitrile, 5.0min of single running time.
2.3.2 Mass Spectrometry conditions
AB SCIEX 4500QTRAPTMMass spectrometry (AB SCIEX, usa) using an electrospray ionization (ESI) ion source, scanning in multiple reaction ion monitoring (MRM) mode. The atomizing gas, the drying gas and the collision gas are all nitrogen; temperature of the drying gas: 550 ℃, Curtain Gas (Curtain Gas): 40 psi; collisional gas (CollisionGas): medium; spray Voltage (Ionspray Voltage): 5500(+)/-4500(-) V; nebulized Gas (Gas 1): 50psi, auxiliary heating Gas (Gas 2): 55 psi. Using AnalystTM1.6.3 the workstation performs the acquisition of mass spectral data. Detecting aconitine, songorine, riolin, AKBA, KBA and positive ion internal standard protopine in a positive ion mode; and detecting the earthworm element and the anion internal standard isofraxidin in the anion mode. The optimal MRM parameters for each component to be tested are shown in Table 1.
TABLE 1 Primary and secondary ion pairs and MRM parameters of six compounds to be tested and two internal standard compounds
2.4 methodological considerations
2.4.1 specificity Studies
The method comprises the following steps of carrying out pretreatment on a biological sample according to a method under the item 2.2 on a rat blank plasma, a mixed solution of 6 compounds to be detected and an internal standard, a mixed solution of 6 compounds to be detected and the internal standard in the blank plasma, and a sample of the plasma of the rat and the internal standard after the mixed solution of the 6 compounds to be detected and the internal standard in the small gold capsule are orally administered for 45min, carrying out sample injection analysis and detection according to the conditions of liquid phase and mass spectrum under the item 2.3, and obtaining a chromatogram of the result shown in. The result shows that endogenous substances in rat plasma do not interfere with the detection of the 6 compounds to be detected and the internal standard, and the chromatographic behaviors of the 6 compounds to be detected and the internal standard solution added into the rat blank plasma are consistent with those of the rat plasma and the internal standard solution after the administration of the Xiaojin capsule.
2.4.2 Linear investigation
Precisely absorbing 180 mu L of blank plasma by a pipette, transferring the blank plasma into a blank 1.5mL EP tube, respectively adding 20 mu L of mixed standard substance solution of 6 compounds to be detected with different concentrations, and preparing the mixed standard substance solution with the mass concentration of respectively: aconitine 0.10, 0.20, 1.00, 2.00, 5.00, 10.00, 20.00, 40.00 ng/mL; 0.20, 0.40, 2.00, 4.00, 10.00, 20.00, 40.00 and 80.00ng/mL of isosongorine; riolin 0.50, 1.00, 5.00, 10.00, 25.00, 50.00, 100.00, 200.00 ng/mL; AKBA7.50, 15.00, 30.00, 75.00, 150.00, 375.00, 750.00, 1500.00 ng/mL; KBA7.50, 15.00, 30.00, 75.00, 150.00, 375.00, 750.00, 1500.00 ng/mL; taking 25.00, 62.50, 125.00, 250.00, 625.00, 1250.00, 2500.00 and 5000.00ng/mL plasma samples, carrying out pretreatment on biological samples according to the method under the item '2.2', carrying out sample injection analysis and detection according to the conditions of liquid phase and mass spectrum under the item '2.3', recording data, drawing a standard curve, and calculating the linear regression equation of each compound, wherein the result is shown in Table 2. As a result, the compounds aconitine, songorine, riolin, AKBA, KBA and wormcast element have good linear relations in the ranges of 0.10-40.00ng/mL, 0.20-80.00 ng/mL, 0.50-200.00ng/mL, 7.50-1500.00ng/mL, 7.50-1500.00ng/mL and 25.00-5000.00ng/mL respectively.
TABLE 2 regression equation and regression coefficient for six test compounds in rat plasma
| Compound (I) | Linear range/(ng/mL) | Regression equation | R |
| Aconitine | 0.10～40.00 | y＝0.00322x+0.00119 | 0.9982 |
| Erpartum songaricum alkali | 0.20～80.00 | y＝0.0146x+0.00709 | 0.9910 |
| Liaolin | 0.50～200.00 | y＝0.0202x+0.0118 | 0.9946 |
| AKBA | 7.50～1500.00 | y＝0.1104x+0.0967 | 0.9927 |
| KBA | 7.50～1500.00 | y＝0.0284x-0.3621 | 0.9912 |
| Earthworm element | 25.00～5000.00 | y＝0.0065x+0.0477 | 0.9974 |
2.4.3 quantitative lower bound investigation
Precisely absorbing 180 mu L of blank plasma by a pipette, adding 20 mu L of 6 mixed solutions of compounds to be detected into a blank 1.5mL EP tube, preparing 5 parts of plasma sample solutions of 6 compounds to be detected with mass concentrations of 0.10, 0.20, 0.50, 7.50 and 25.00ng/mL respectively, carrying out pretreatment on a biological sample according to a method under the item '2.2', carrying out sample injection analysis and detection according to liquid phase and mass spectrum conditions under the item '2.3', recording data, analyzing the actual solubility of the sample, and calculating the RSD value. The results are shown in Table 3. As can be seen from the data in the table, the RSD value is in the range of 5.11-11.02%, which is less than 15%, and meets the analysis requirement of the plasma sample.
Table 3 lower limit of aconitine quantitation in rat plasma (n ═ 5)
2.4.4 precision investigation
Precisely absorbing 180 mu L of blank plasma by a pipette into a blank 1.5mL EP tube, adding 20 mu L of a mixed solution of 6 compounds to be detected, and preparing four Quality Control (QC) samples with quantitative lower limit, low concentration, medium concentration and high concentration of the 6 compounds to be detected, wherein 5 parts of aconitine is prepared in parallel at each concentration, namely the concentration of aconitine is 0.10, 0.30, 5.00 and 32.00 ng/mL; the concentrations of the songorine are 0.20, 0.60, 10.00 and 64.00 ng/mL; the concentration of the lioline is 0.50, 1.50, 25.00 and 160.00 ng/mL; the concentration of AKBA is 7.50, 22.50, 150.00 and 1200.00 ng/mL; KBA concentration is 7.50, 22.50, 150.00, 1200.00 ng/mL; plasma samples with the concentrations of the earthworm element of 25.00, 75.00, 625.00 and 4000.00 ng/mL. The pretreatment of the biological sample is carried out according to the method under the item 2.2, the injection analysis and detection are carried out according to the conditions of liquid phase and mass spectrum under the item 2.3, the actual solubility of the biological sample is analyzed by recording data, and the precision in the day are calculated. The results are shown in tables 4-1 to 4-6, and show that the RSD is between 2.11 and 10.48 percent and less than 15 percent, which meets the analysis requirement of the plasma sample.
Table 4-1 precision measurement of aconitine in rat plasma (n ═ 5)
Table 4-2 precision determination of songorine in rat plasma (n ═ 5)
Table 4-3 determination of lioline precision in rat plasma (n ═ 5)
Table 4-4 AKBA precision determination in rat plasma (n ═ 5)
Table 4-5 results of KBA precision measurement in rat plasma (n ═ 5)
Table 4-6 precision measurement of wormcast in rat plasma (n ═ 5)
2.4.5 accuracy survey
In the experiment, SD rat blank plasma is used for accuracy (namely relative recovery rate) inspection, a liquid transfer gun precisely absorbs 180 mu L of blank plasma into a blank 1.5mL EP tube, 20 mu L of mixed solution of 6 compounds to be detected is added to prepare Quality Control (QC) samples with four levels of concentration, namely, lower limit of quantitation, low concentration, medium concentration and high concentration of the 6 compounds to be detected, 5 parts of aconitine is prepared in parallel for each concentration, namely, the concentration of aconitine is 0.10, 0.30, 5.00 and 32.00 ng/mL; the concentrations of the songorine are 0.20, 0.60, 10.00 and 64.00 ng/mL; the concentration of Rioprolin is 0.50, 1.50, 25.00, 160.00 ng/mL; the concentration of AKBA is 7.50, 22.50, 150.00 and 1200.00 ng/mL; KBA concentration is 7.50, 22.50, 150.00, 1200.00 ng/mL; plasma samples with the concentrations of the earthworm element of 25.00, 75.00, 625.00 and 4000.00 ng/mL. The method of '2.2' is adopted to carry out pretreatment of biological samples, the conditions of liquid phase and mass spectrum under '2.3' are adopted to carry out injection analysis and detection, the recorded data is used to analyze the actual solubility, and the ratio of the actual measured concentration to the labeled concentration is used to calculate the accuracy (namely the relative recovery rate), and the specific results are shown in tables 5-1 to 5-2, the relative recovery rate of 6 compounds is between 85.580% and 108.250%, and the RSD is between 0.75% and 11.72% and is less than 15%. Namely, the accuracy of the 6 compounds in the method is good, and the method meets the analysis requirement of biological samples.
TABLE 5-1 accuracy of aconitine in rat plasma (n ═ 5)
Table 5-2 rat plasma songorine accuracy determinations (n ═ 5)
Table 5-3 determination of lioalin accuracy in rat plasma (n ═ 5)
Table 5-4 AKBA accuracy in rat plasma (n ═ 5)
Table 5-5 results of KBA accuracy determination in rat plasma (n ═ 5)
Table 5-6 accuracy measurement of wormcast in rat plasma (n ═ 5)
2.4.6 matrix Effect investigation
Precisely absorbing 180 mu L of blank plasma by a liquid-transferring gun into a blank 1.5mL EP tube, adding 20 mu L of a mixed solution of 6 compounds to be detected to prepare a quality control sample containing the 6 compounds to be detected in three concentration levels of low concentration, medium concentration and high concentration, preparing 6 parts of each concentration in parallel, carrying out pretreatment on a biological sample according to a method under the item '2.2', carrying out sample injection analysis and detection according to the conditions of a liquid phase and a mass spectrum under the item '2.3', and recording the peak areas of aconitine, pseudosongorine, liolin, AKBA, KBA and wormcrin as A1; in addition, 180 μ L of purified water is used to replace 180 μ L of blank plasma, and the other operations are the same as the above, and the peak area of the aconitine, the isosbaconine, the riolin, the AKBA, the KBA and the chrysopharin is recorded as A2, so that the matrix effect of the aconitine, the isosbaconine, the riolin, the AKBA, the KBA and the chrysopharin is as follows: (A1/A2). times.100%. No matrix effect was observed if the A1/A2 obtained from each biological sample was in the range of 85% to 115%. The experimental results are shown in table 6, the matrix effect of the 6 compounds is between 93.45% and 100.64%, and the RSD is between 1.27% and 5.13%, which meets the quantitative requirements of biological samples.
Table 6 rat plasma 6 test compounds and internal standard matrix effect (n ═ 6)
2.4.7 stability examination
Precisely absorbing 180 mu L of blank plasma by a pipette into a blank 1.5mL EP tube, adding 20 mu L of mixed solution of 6 compounds to be detected, and preparing Quality Control (QC) samples with three grades of low concentration, medium concentration and high concentration of the 6 compounds to be detected, wherein 5 parts of aconitine is prepared in parallel at each concentration, namely the concentration of aconitine is 0.30, 5.00 and 32.00 ng/mL; the concentrations of the songorine are 0.60, 10.00 and 64.00 ng/mL; the concentration of the lyoline is 1.50, 25.00 and 160.00 ng/mL; the concentration of AKBA is 22.50, 150.00, 1200.00 ng/mL; KBA concentration is 22.50, 150.00, 1200.00 ng/mL; plasma samples with concentrations of wormcast 75.00, 625.00, and 4000.00ng/mL were examined for short-term and long-term stability of 6 compounds tested in plasma. Wherein the short-term stability investigation mainly comprises investigating room temperature standing stability, automatic sampler stability and repeated freeze-thaw stability; the long-term stability was evaluated by examining the stability of the plasma samples after 20 days of storage in a refrigerator at-80 ℃ and the results are shown in tables 7-1 to 7-4. According to the results, the RSD is between 1.27% and 8.49% and less than 15%, namely, the quality control samples with 3 concentrations of low concentration, medium concentration and high concentration of aconitine, isosbonoconine, riolin, AKBA, KBA and wormcast element show good stability in short-term and long-term investigation, and meet the analysis conditions of biological samples.
Table 7-1 stability of 6 test compounds in rat plasma at room temperature (n ═ 5)
Table 7-2 rat plasma 6 compounds tested autosampler stability (n ═ 5)
Table 7-3 repeated freeze-thaw stability of 6 compounds tested in rat plasma (n ═ 5)
Table 7-4 long term stability of 6 compounds tested in rat plasma (n ═ 5)
3. Small knot
The invention establishes a sensitive, rapid, simple, convenient and stable in vivo content determination method for simultaneously determining 6 small gold capsule metabolites, namely, aconine, isosongorine, riolin, AKBA, KBA and earthworm element, and internal standard protopine and isooxazine picoline in a biological sample based on HPLC-MS/MS. Collecting aconitine, songorine, lionoline, AKBA, KBA and internal standard protopine in positive ion mode for 10 min; the earthworm element and the internal standard isofraxidin are collected in a negative ion mode, and the analysis time is 5 minutes. And the methodology investigation of biological samples is carried out, the established LC-MS/MS determination method is examined in the aspects of specificity, linear range, lower limit of quantification, precision, accuracy, matrix effect, stability and the like, all the investigation results conform to the analysis requirements of the biological samples, and the established 6 metabolic products are simple, stable and reliable in analysis method and can be used for determining the blood concentration of the 6 metabolic products in the rat body.
Claims (8)
1. The method for simultaneously determining the contents of six metabolites in a Xiaojin preparation comprises the following raw materials, by weight, 1-20 parts of artificial musk, 40-60 parts of radix aconiti agrestis, 20-40 parts of frankincense, 40-60 parts of trogopterus dung, 40-60 parts of earthworm, 40-60 parts of cochinchina momordica seed, 40-60 parts of liquidambar formosana, 20-40 parts of myrrh, 20-40 parts of angelica sinensis and 1-10 parts of balsam, and is characterized in that the six metabolites are aconitine, isokuramine, riodenine, 3-acetyl-11-keto- β -boswellic acid, 11-keto- β -boswellic acid and earthworm extract, and the determination method comprises the following steps:
1) pretreating a plasma sample to obtain a sample solution to be detected;
2) adding a positive ion internal standard and a negative ion internal standard into a sample solution to be detected, and carrying out HPLC-MS/MS detection, wherein the stationary phase of the high-performance liquid phase is a C18 bonded silica gel chromatographic column, the mobile phase A is a 0.05-0.5% formic acid aqueous solution, the mobile phase B is acetonitrile, gradient elution is adopted, the flow rate is 0.1-0.5mL/min, the column temperature is 25-40 ℃, the mass spectrum adopts a positive ion mode and a negative ion mode, an electrospray ionization ion source is adopted, the scanning mode is a multi-reaction ion monitoring mode, and under the positive ion mode, protopine, isosbane, lioline, 3-acetyl-11-keto- β -boswellic acid, 11-keto- β -boswellic acid and positive ion internal standard protopine are detected, and under the negative ion mode, aconitine and negative ion internal standard isofraxidine are detected.
2. The method for simultaneously determining the contents of six metabolites in a Xiaojin preparation as claimed in claim 1, wherein: the positive ion internal standard is protopine, and the negative ion internal standard is isofraxidin.
3. The method for simultaneously determining the contents of six metabolites in a Xiaojin preparation as claimed in claim 1, wherein: the mobile phase a was 0.1% aqueous formic acid.
4. The method for simultaneously determining the contents of six metabolites in a Xiaojin preparation as claimed in claim 1, wherein: the flow rate was 0.3 mL/min.
5. The method for simultaneously determining the contents of six metabolites in a Xiaojin preparation as claimed in claim 1, wherein: the column temperature was 35 ℃.
6. The method for simultaneously determining the contents of six metabolites in a Xiaojin preparation as claimed in claim 1, wherein: the gradient elution is carried out in a positive ion mode for 0.01-2.5min and 10-80% of acetonitrile; 2.5-3.5min, 80-100% acetonitrile; 3.5-6min, 100% acetonitrile; 6-7min, 100-10% acetonitrile; 7-10min, 10% acetonitrile, 10min of single running time; under the negative ion mode, 0.01-0.5min, 30% acetonitrile; 0.5-2min, 30-80% acetonitrile; 2-2.1min, 80-30% acetonitrile; 2.1-5min, 30% acetonitrile, 5min single run time.
7. The method for simultaneously determining the contents of six metabolites in a Xiaojin preparation as claimed in claim 1, wherein: the drying gas temperature of the mass spectrum is 550 ℃; the air curtain air is 40 psi; the collision gas is medium; the ion spray voltage is 5500(+)/-4500(-) V; the atomizing gas was 50psi and the auxiliary heating gas was 55 psi.
8. The method for simultaneously determining the contents of six metabolites in a Xiaojin preparation as claimed in claim 1, wherein: the pretreatment method of the plasma sample comprises the steps of carrying out vortex oscillation for 2min, centrifuging at 13000rpm at 4 ℃ for 10min, transferring a supernatant solution, volatilizing the solvent by nitrogen, redissolving residues by using a methanol solution, centrifuging at 13000rpm at 4 ℃ for 10min, and sucking the supernatant solution.
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