CN111435128A - Method for measuring 4 diester-type aconite alkaloids in blood - Google Patents
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Abstract
The invention relates to the technical field of drug detection, and provides a method for determining 4 diester-type aconite alkaloids in blood, which aims to solve the problems of more interferents, long detection time and low accuracy in the detection method of aconite alkaloids.
Description
Technical Field
The invention relates to the technical field of drug detection, in particular to a method for determining 4 diester-type aconite alkaloids in blood.
Background
Aconitum plant has effects of dispelling pathogenic wind, removing dampness, warming channels and relieving pain, and can be used for treating arthralgia due to wind-cold-warm pathogen, arthralgia, psychroalgia of heart and abdomen, cold hernia pain, and pain caused by anesthesia, and can be widely used in clinical treatment. The main component of the traditional Chinese medicine is diester-type aconite alkaloids, and the total weight of the traditional Chinese medicine is about 7-10%. Diester-type aconite alkaloids are toxic components of aconitum Chinese medicine and are the material basis of the efficacy of the aconitum Chinese medicine. Because the aconite traditional Chinese medicines are not processed uniformly, poisoning or death is caused by excessive dosage, insufficient decoction, raw preparation, misuse or toxic administration.
At present, the aconite alkaloid poisoning cases are mostly identified by sampling and detecting 3-7 days or more after the poisoning occurs, but the aconite alkaloid is low in content and rapid in metabolism in a sample in vivo, so that the forensic toxicological identification and clinical poisoning inspection are difficult. Therefore, it is necessary to establish a highly sensitive detection method for detecting aconite alkaloids in a biological sample.
The detection method of alkaloids in aconite plants mainly comprises high performance liquid chromatography (HP L C), derivatization gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (L C-MS/MS), wherein detection of various aconite alkaloids in a sample mainly focuses on diester alkaloids such as aconitine, hypaconitine, mesaconine and aconitine, and the method has the problems of a large number of interferents, long detection time, low accuracy and incapability of real-time monitoring.
Disclosure of Invention
The invention provides a method for determining 4 diester-type aconitine alkaloids in blood, which is rapid and accurate, has objective and easy-to-analyze result and can realize real-time monitoring, and aims to overcome the problems of more interferents, long detection time, low accuracy and incapability of real-time monitoring in the existing detection method of aconitine alkaloids.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for measuring 4 diester aconitine alkaloids in blood comprises the following steps:
(1) adding a stabilizing agent into the plasma sample, and storing at a low temperature for later use; the plasma sample is obtained by centrifuging a whole blood sample for 5min under the centrifugal force of 3000g and sucking supernatant; after the plasma sample is collected, because the plasma contains various ions, carbohydrates, lipids, proteins, various enzymes, hormones and various metabolites of the body, the existence of the chemicals and the enzymes can promote the further metabolism of alkaloids (such as aconitine, hypaconitine, mesaconine, aconitine and the like). The stabilizer has the function of keeping the content of 4 diester aconite alkaloids in the plasma sample stable, and improving the detection accuracy; adding a stabilizer from blood sample collection to perform quality control of the whole process;
(2) adding an extraction solvent into the plasma sample treated in the step (1) for extraction to obtain an extracting solution;
(3) adding a magnetic graphene-based adsorbent into the extracting solution for purification treatment to obtain a purified solution; purifying the extracting solution by using a magnetic graphene-based adsorbent, thereby eliminating the matrix effect and improving the recovery rate;
(4) the purified solution is filtered and then analyzed by HP L C-MS/MS detection on machine, and the filtration is preferably performed by a 0.22 μm filter membrane.
The invention realizes the purpose of accurately detecting 4 diester aconitine alkaloids in blood samples by applying HP L C-MS/MS technology, ensures the specificity of a detected object by respectively carrying out quantification and qualitative operation by two pairs of ions, reduces the influence of interferents, has simple, convenient and quick operation and low flux and high cost, effectively detects the levels of the 4 aconitine alkaloids in clinical poisoned patients, has important guiding significance for clinical diagnosis and treatment of the poisoning of the aconitine alkaloids, and is easy to popularize and popularize clinically.
The method of the invention respectively selects a pair of qualitative ions (aconitine: 646.3>105.0, mesaconine: 632.3>105.0, hypaconitine 616.3>105.0 and mesaconine 660.3>135.0) and a pair of quantitative ions (aconitine: 646.3>586.3, mesaconine: 632.3>572.3, hypaconitine 616.3>556.3 and mesaconine 660.3>600.3), and takes the relative retention time and the qualitative ion pair thereof as qualitative basis and takes the standard substance to make a standard curve for quantification. Meanwhile, the method applies three levels of quality control quality inspection methods to examine the accuracy and effectiveness of the method and avoids the distortion of the detection result.
Further, the preparation method of the standard working solution comprises the steps of preparing 1.0mg/m L standard stock solution by using acetonitrile, preparing 7 gradients of 0.1, 0.5,1.0,5.0,10.0,50.0,100.0 and 150.0 mu g/L by using acetonitrile solution, and subpackaging the standard solution in 1.5m L brown bottles for storage at-20 ℃ for later use.
Further, the method comprises the following steps of: the quality control product is prepared by adding 4 diester-type aconitine (aconitine, neoaconitine, hypaconitine and yunaconitine) standard substances into artificial plasma, the concentrations of the substances are respectively consistent with the concentrations of the first, fourth and seventh gradients in eight gradients of standard solution, and the target value is determined by detection.
Preferably, in the step (1), the stabilizer is 10% by volume of formic acid aqueous solution, the addition amount of the stabilizer in the plasma sample is 2-3 mu L/m L, and the temperature for low-temperature storage is 3-5 ℃.
In the research process, the diester-type aconitine alkaloid is found to have poor stability in a blood sample and is easy to degrade, and the stability of aconitine in the plasma sample can be ensured by adding a certain amount of formic acid into the plasma sample.
Preferably, in the step (2), the extraction solvent is a formic acid/acetonitrile mixture solution, and the volume fraction of formic acid in the formic acid/acetonitrile mixture solution is 0.5%.
When pure acetonitrile is used as an extraction solvent, the recovery rate of 4 alkaloids is within 42.4-58.4%, the recovery rate of 4 alkaloids is greatly improved along with the increase of the content of formic acid in the acetonitrile, and when the volume content of the formic acid in the acetonitrile reaches 0.5%, the recovery rate of 4 alkaloids is within 91.9-94.5%. However, when the formic acid content was further increased, the recovery of all the target products was not improved significantly. A satisfactory recovery rate can be obtained with 0.5% formic acid/acetonitrile as the extraction solvent, so 0.5% formic acid/acetonitrile is selected as the extraction solvent.
Preferably, in the step (3), the adding amount of the magnetic graphene-based adsorbent in the extracting solution is 20mg/m L, and the time of the purification treatment is 2 min.
When the addition amount of the magnetic graphene-based adsorbent in the extracting solution is 20-50 mg/m L, impurities can be effectively removed, and meanwhile, a satisfactory recovery rate (91.5-95.9%) can be obtained.
The MSPE process is based on adsorption-desorption balance, and the interaction time of the magnetic graphene-based adsorbent and the extracting solution to be purified has direct influence on the purification effect. When the interaction time between the adsorbent and the extract to be purified is 0.5min, the recovery rate of the 4 alkaloids is not ideal and is 52.1-72.3%. The recovery rate of most alkaloids is obviously improved along with the increase of the interaction time, and when the interaction time reaches 2min, the recovery rate of 4 alkaloids is between 85.9 and 96.4 percent. The time of purification treatment is 2min, and 4 kinds of alkaloids can obtain ideal recovery rate. The extraction time is so short that the impurities such as grease and pigment can be effectively adsorbed due to the ultra-large surface area of the magnetic graphene-based adsorbent.
Preferably, in the step (3), the magnetic graphene-based adsorbent is magnetic graphene-based poly (amide amine) (PAA @ Mag-G). The adsorbent has good reusability and mechanical stability.
Preferably, the preparation method of the magnetic graphene-based polyamide comprises the following steps:
(a) preparation of a polyimide polyamine dendrimer:
adding methyl acrylate into a methanol solution of tris (2-aminoethyl) amine, and continuously reacting for 18-24 h at the temperature of 30-35 ℃; adding triethylene diamine, continuously reacting for 18-24 h at 30-35 ℃, and performing rotary evaporation to obtain a crude product; dissolving the crude product in a methanol solution, repeating the step of adding methyl acrylate and triethylene diamine, and synthesizing the polyamide polyamine dendrimer taking N as a core through two Michael addition reactions and two amidation reactions;
(b) preparation of magnetic graphene-based polyamine:
FeCl is added3·6H2O，Ni(NO3)2·6H2Sequentially adding O, NaAc and NaOH into ethylene glycol for ultrasonic dissolution until the solution is transparent; then adding the polyimide dendritic macromolecule prepared in the step (a) and dissolving by ultrasonic; and adding carboxylated graphene, strongly stirring for 30-45 min at 50-60 ℃, transferring to a high-pressure reaction kettle, reacting for 48h at 180-200 ℃, separating a product by means of an external magnetic field, and washing to be neutral to obtain the magnetic graphene-based polyamide.
Preferably, in the step (b), the addition amount of the carboxylated graphene is 1-1.5 mg/m L.
Preferably, in step (4), the chromatographic conditions for the HP L C-MS/MS on-machine detection analysis are mobile phase A of HP L C grade water containing 0.05% formic acid at pH 3 and mobile phase B of acetonitrile containing 0.05% formic acid at pH 3.
Preferably, the gradient elution conditions are 30% B, 0.00min → 4.00min, 30% → 90% B, 4.00min → 7.00min, 90% B, 7.00min → 10.00min, 300. mu. L/min for the flow rate, and 5. mu. L for the sample introduction.
Preferably, in the step (4), the mass spectrum conditions of HP L C-MS/MS on-machine detection and analysis are that a positive ion electrospray ionization multi-ion reaction monitoring mode is adopted, the atomization gas is 60kPa, the heating gas is 50kPa, the air curtain gas is 25kPa, the spraying voltage is 4.5kV, the desolvation temperature is 425 ℃, and the mass spectrum reference conditions of aconitine, mesaconine, hypaconitine and mesaconine are shown in Table 1.
TABLE 1 Aconitine, mesaconine, hypaconitine and mesaconine analysis Mass Spectrometry reference conditions (ESI Source Positive ion mode)
Therefore, the invention has the following beneficial effects:
(1) establishing a method for detecting 4 diester aconite alkaloids in a blood sample by pretreating the blood sample and optimizing HP L C-MS/MS detection conditions;
(2) the sample is stored, and 10% formic acid is adopted to ensure that the content of 4 diester-type aconite alkaloids in the sample to be detected is more stable. The method adopts 0.5 percent formic acid/acetonitrile solution to extract 4 diester aconite alkaloids, so that the sample treatment is more efficient, the operation is simple and convenient, the protein can be better precipitated, and the matrix effect can be basically and completely eliminated by adopting magnetic graphene-based polyamide for purification;
(3) the ultra-high performance liquid chromatography-tandem mass spectrometry is rapid and accurate, the result is objective and easy to analyze, the content change of 4 diester-type aconitine alkaloids in the poisoning patients can be monitored in real time, and scientific basis can be provided for clinical diagnosis and treatment of aconitine alkaloid poisoning.
Detailed Description
The technical solution of the present invention is further specifically described below by way of specific examples.
In the present invention, all the equipment and materials are commercially available or commonly used in the art, and the methods in the following examples are conventional in the art unless otherwise specified.
Reagent: formic acid, acetic acid, concentrated ammonia (chromatographical purity, Shanghai 'an spectrum), acetonitrile (chromatographical purity, Beijing Bailingwei science and technology Co., Ltd.), sodium chloride (analytically pure, Nanjing chemical reagents Co., Ltd.), and bottled distilled water (Chen's, China) and ferric chloride hexahydrate (FeCl)3·6H2O), nickel nitrate hexahydrate [ Ni (NO)3)2·6H2O]Sodium hydroxide, sodium acetate, Ethylene Glycol (EG), tris (2-aminoethyl) amine, triethylenediamine and Methyl Acrylate (MA) (analytical grade, nanjing chemicals ltd), carboxylated graphene (nanjing piont nanomaterials science ltd).
Aconitine (aconitine), hypaconine (hypaconine), mesaconine (mesaconine), mesaconine (yunaconitine), Chengduiensis Biotech, Inc.
The solutions used in the following examples of the invention were prepared as follows:
standard stock solution 1000 mg/L, weighing 4 alkaloids 5.0mg each in a brown bottle, adding 5.0m L acetonitrile respectively, vortex dissolving, and storing at-4 deg.C for use.
Mixing standard solutions, transferring a certain amount of 4 standard stock solutions, diluting with acetonitrile, and concocting in brown bottle, wherein the alkaloid concentrations are aconitine, hypaconitine, mesaconine, and mesaconine (5 mg/L), storing at-4 deg.C, and diluting with acetonitrile to desired concentration when using.
Example 1:
a method for measuring 4 diester aconitine alkaloids in blood comprises the following steps:
(1) taking 2ml of a whole blood sample in a centrifuge tube, centrifuging for 5min under the centrifugal force of 3000g, sucking supernatant into another test tube to obtain a plasma sample, adding 5 mu l of 10% formic acid aqueous solution in volume ratio into the plasma sample, and storing the mixture in a refrigerator at 4 ℃ for later use;
(2) sucking 200 μ l of the plasma sample treated in step (1), adding 1m of L0.5.5% formic acid/acetonitrile solution as extraction solvent, centrifuging at 3000g for 2min, and sucking supernatant to obtain extractive solution;
(3) transferring 0.5m L extractive solution to another 2m L polypropylene centrifuge tube, adding 10.0mg of magnetic graphene-based polyamide (PAA @ Mag-G), vortex oscillating for 5min, centrifuging at centrifugal force of 3000G for 5min, and collecting supernatant to obtain purified solution;
(4) filtering the purified solution with 0.22 μm filter membrane, injecting sample, analyzing by HP L C-MS/MS detection on machine:
the chromatographic conditions comprise mobile phase A of HP L C grade water containing 0.05% formic acid and having pH value of 3, mobile phase B of acetonitrile containing 0.05% formic acid and having pH value of 3, gradient elution conditions of 30% B, 0.00min → 4.00min, 30% → 90% B, 4.00min → 7.00min, 90% B, 7.00min → 10.00min, flow rate of 300 mu L/min and sample introduction amount of 5 mu L.
The mass spectrum conditions are as follows: adopting a multi-ion reaction monitoring mode of positive ion electric spray ionization, namely atomizing: 60kPa, heating gas: 50kPa, air curtain air: 25kPa, spray voltage: 4.5kV, desolvation temperature: 425 ℃. The reference conditions of aconitine, mesaconine, hypaconitine and mesaconine analysis mass spectrometry are shown in table 1.
In the step (3), the preparation method of the magnetic graphene-based polyamide (PAA @ Mag-G) comprises the following steps:
(a) preparation of polyimide polyamine dendrimer tris (2-aminoethyl) amine (0.5g, 0.51m L) was weighed into a round-bottomed flask, 100m L of methanol was added to dissolve, Methyl Acrylate (MA) (2.4g, 2.51m L) was added to the round-bottomed flask, and reaction was continued at 30 ℃ for 24 hours, then triethylenediamine (2.0g, 2.22m L) was added to the reaction system, and reaction was continued at 30 ℃ for 24 hours, and when the reaction was completed, rotary evaporation was performed to obtain a crude product, the obtained crude product was added to 100m L of methanol and dissolved in 250m L of round-bottomed flask, and the above-mentioned steps of adding MA and triethylenediamine were repeated, and through two michael addition reactions and two amidation reactions, a polyimide Polyamine (PAA) dendrimer having N as a core was finally synthesized;
(b) preparation of amino functionalized magnetic graphene
On the basis of a solvothermal method, amino-functionalized magnetic graphene is synthesized by a one-pot method. In particular toThe synthesis method is as follows.A round-bottom flask with 250m L is selected, 100m L ethylene glycol is added, and FeCl is added3·6H2O(2.0g)，Ni(NO3)2·6H2O (2.0g), NaAc (6.0g) and NaOH (0.8g) were added thereto in this order, and the mixture was dissolved by ultrasonic waves until the solution became transparent; then adding the PAA (200mg) crude product prepared in the step (a) into a reaction system, and dissolving for 10min by ultrasonic; finally, 100mg of carboxylated graphene was added to a round bottom flask and vigorously stirred at 60 ℃ for 40 min. Transferring the reaction liquid into a stainless steel high-pressure reaction kettle with polytetrafluoroethylene as a lining, and continuously reacting for 48 hours at 200 ℃. The amino functionalized magnetic graphene carbon (PAA @ Mag-G) synthesized above is separated from the reaction system by means of a magnetic field formed by an external magnet. The resultant material was then washed repeatedly with deionized water and ethanol in sequence until the pH of the wash solution was near neutral. The magnetic graphene-based polyamide is prepared and dried for later use.
The service life of the adsorbent is an important index for evaluating the performance of a magnetic material. In order to evaluate the reusability of the synthesized amino-functionalized magnetic graphene, the amino-functionalized magnetic graphene adsorbent is regenerated after extraction is completed each time, and is put into use again next time. Before the adsorbent is put into next use, the used adsorbent is washed with 5.0% ammonia/methanol (v/v) solution and deionized water in sequence, and the washing is repeated three times. When the reusability experiment is carried out, the adding standard concentration of the sample is respectively aconitine, hypaconitine, mesaconine and 0.4ng/g of aconitine. As a result, as shown in Table 2, the purification ability of the adsorbent was not significantly reduced when the adsorbent was used in cycles of 6 times. Therefore, the synthesized adsorption material PAA @ Mag-G has good reusability and mechanical stability.
TABLE 2 reusability measurement results
Evaluation of matrix Effect
Taking 3 blank blood plasmas from different sources respectively, 6 parts of each blood plasma, and 2 parts of each of the 3 blank blood plasmas from different sources to be 1 group, totally 3 groups, treating according to a test method, drying by nitrogen, re-dissolving residues by using 4 aconite alkaloid mixed standard solutions (diluted by methanol) with high, medium and low concentration levels respectively to be 1m L, measuring the chromatographic peak area (S1), taking the same 4 aconite alkaloid mixed standard solutions (diluted by methanol), measuring the chromatographic peak area (S2), and taking the ratio of S1 to S2 as a matrix effect, wherein the result is shown in Table 3.
Table 3 evaluation of matrix effect (n ═ 3)
Standard Curve, detection Limit and quantitation Limit of the method
Adding a series of mixed standard solutions with concentration into blank plasma, and preparing a series of matrix matching standard working solutions, so that the concentration ranges of aconitine, hypaconitine, neoaconitine and aconitine are 0.5-200 mug/L (0.5,1.0,5.0,10.0,50.0,100.0 and 200.0 mug/L). In an optimized instrument condition, carrying out sample injection detection, drawing a standard working curve by taking the sample concentration as an abscissa and the instrument response value as an ordinate, carrying out quantification by a matrix marking curve method, calculating the detection limit by a signal-to-noise ratio S/N ═ 3 calculation method, calculating the quantification limit by an S/N inner line ═ 10, and for plasma samples, the results are shown in Table 3, wherein the linear ranges of the 4 alkaloids are good in relation (r is not less than 0.9995), and the method detection limit of the 4 alkaloids obtained by calculating the lowest concentration marking sample is 0.16-0.26 mug/L, and the quantification limit is 0.054-0.085 mug/L.
TABLE 3.4 Linear equation, correlation coefficient, Linear Range, detection Limit and quantitation Limit for alkaloid
Precision and accuracy of the method
Adding different amounts of aconitine, mesaconine, hypaconitine and aconitine standard stock solutions into blank plasma to prepare a whole blood Quality Control (QC) sample with low, medium and high concentrations of 3 (respectively 1.0, 20.0 and 100.0 mug/L), repeatedly extracting and measuring 6 times per day, calculating the recovery rate and the day precision, selecting 8 days in 2 weeks, repeatedly extracting and measuring the labeled sample 2 times per day, calculating the day precision, and obtaining the experiment result, wherein the recovery rate of 4 alkaloids in the plasma is 84.0-103.0%, the day precision is 3.2-6.9%, and the day precision is 3.3-8.8%.
TABLE 4 recovery of mesaconine, hypaconitine and mesaconine spiked in plasma samples (n ═ 3)
Example 2:
a method for measuring 4 diester aconitine alkaloids in blood comprises the following steps:
(1) taking 2ml of a whole blood sample in a centrifuge tube, centrifuging for 5min under the centrifugal force of 3000g, sucking supernatant into another test tube to obtain a plasma sample, adding 6 mu l of 10% formic acid aqueous solution with volume fraction into the plasma sample, and storing the mixture in a refrigerator at 3 ℃ for later use;
(2) sucking 200 μ l of the plasma sample treated in step (1), adding 1m of L0.5.5% formic acid/acetonitrile solution as extraction solvent, centrifuging at 3000g for 2min, and sucking supernatant to obtain extractive solution;
(3) transferring 0.5m L extractive solution to another 2m L polypropylene centrifuge tube, adding 10.0mg of magnetic graphene-based polyamide (PAA @ Mag-G), vortex oscillating for 5min, centrifuging at centrifugal force of 3000G for 5min, and collecting supernatant to obtain purified solution;
(4) the purified solution was filtered through a 0.22 μm filter and analyzed by sample injection, and the conditions of chromatography and mass spectrometry were the same as in example 1, using HP L C-MS/MS machine for detection and analysis.
In the step (3), the preparation method of the magnetic graphene-based polyamide (PAA @ Mag-G) comprises the following steps:
(a) preparation of polyimide polyamine dendrimer tris (2-aminoethyl) amine (0.5g, 0.51m L) was weighed into a round-bottomed flask, 100m L of methanol was added to dissolve, Methyl Acrylate (MA) (2.4g, 2.51m L) was added to the round-bottomed flask, and continuous reaction was carried out at 35 ℃ for 18 hours, followed by addition of triethylenediamine (2.0g, 2.22m L) to the reaction system, and reaction was continued at 35 ℃ for 18 hours, and after completion of the reaction, rotary evaporation was carried out to obtain a crude product, which was added to 100m L of methanol and dissolved in 250m L of round-bottomed flask, and the above-mentioned steps of adding MA and triethylenediamine were repeated, and by two michael addition reactions and two amidation reactions, a polyimide Polyamine (PAA) dendrimer having N as a core was finally synthesized;
(b) preparation of amino functionalized magnetic graphene
On the basis of a solvothermal method, amino functionalized magnetic graphene is synthesized by a one-pot method, the specific synthesis method is as follows, a round-bottom flask with the length of 250m L is selected, 100m L ethylene glycol is added, and FeCl is added3·6H2O(2.0g)，Ni(NO3)2·6H2O (2.0g), NaAc (6.0g) and NaOH (0.8g) were added thereto in this order, and the mixture was dissolved by ultrasonic waves until the solution became transparent; then adding the PAA (2.0G, 200mg) crude product prepared in the step (a) into a reaction system, and dissolving for 10min by ultrasonic waves; finally, 100mg of carboxylated graphene was added to a round bottom flask and vigorously stirred at 50 ℃ for 40 min. Transferring the reaction liquid into a stainless steel high-pressure reaction kettle with polytetrafluoroethylene as a lining, and continuously reacting for 48 hours at 180 ℃. The amino functionalized magnetic graphene carbon (PAA @ Mag-G) synthesized above is separated from the reaction system by means of a magnetic field formed by an external magnet. The resultant material was then washed repeatedly with deionized water and ethanol in sequence until the pH of the wash solution was near neutral. The magnetic graphene-based polyamide is prepared and dried for later use.
Comparative example 1 (without addition of stabilizer)
Comparative example 1 differs from example 1 in that in step (1), no stabilizer is added for pretreatment and the remaining steps are identical.
The stability of 10.0 mg/L of aconitine was measured in the plasma samples treated in example 1 and comparative example 1 after step (1), and the results are shown in Table 5:
TABLE 5 stability of aconitine in plasma samples of example 1 and comparative example 1
Comparative examples 2 to 7 (different amounts of stabilizer added)
Comparative examples 2 to 7 are different from example 1 in that the amount of the stabilizer added in step (1) is different and the rest of the steps are identical.
The stability of 10.0 mg/L of aconitine after standing for 36 hours in the plasma samples treated in example 1 and comparative examples 2 to 7 by the step (1) was measured, and the results are shown in Table 6:
TABLE 6 stability of aconitine in plasma samples of example 1 and comparative examples 2-7
As can be seen from Table 6, the addition amount of the stabilizer in the plasma sample needs to be strictly controlled, and the stability of aconitine in the plasma sample can be affected by the addition amount which is too low (lower than 2 μ L/m L, comparative examples 2 and 3) or too high (higher than 3 μ L/m L, comparative examples 4 to 7).
Comparative examples 8 to 11 (different extraction solvents)
Comparative examples 8 to 11 are different from example 1 in that the formic acid content in the extraction solvent in step (2) is different and the rest of the steps are identical.
The recovery rates of the 4 alkaloids in example 1 and comparative examples 8-11 were measured and are shown in table 7:
TABLE 7 recovery of 4 alkaloids in example 1 and comparative examples 8-11
| Numbering | Comparative example 8 | Comparative example 9 | Example 1 | Comparative example 10 | Comparative example 11 |
| Formic acid content | 0％ | 0.1％ | 0.5％ | 1.0％ | 2.0％ |
| Aconitine | 42.7 | 73.4 | 91.9 | 88.6 | 86.9 |
| Novel aconitine | 43.5 | 72.9 | 93.4 | 88.9 | 87.4 |
| Hypaconitine | 42.4 | 83.1 | 93.5 | 99.5 | 95.8 |
| Aconitine from Yunnan aconitine | 58.4 | 78.4 | 94.5 | 93.2 | 90.2 |
As can be seen from Table 7, the effect of the formulation of the extraction solvent on the recovery is critical, when pure acetonitrile (comparative example 8) is used as the extraction solvent, the recovery of 4 alkaloids is in the range of 42.4-58.4%, the recovery of 4 alkaloids is greatly improved with the increase of formic acid content in acetonitrile, and when the formic acid content in acetonitrile reaches 0.5% (example 1), the recovery of 4 alkaloids is in the range of 91.9% -94.5%. However, when the formic acid content was further increased (comparative examples 10 and 11), the recovery of all the targets was not significantly improved. Since a satisfactory recovery rate can be obtained when 0.5% formic acid/acetonitrile is used as the extraction solvent, 0.5% formic acid/acetonitrile is used as the extraction solvent.
Comparative examples 12 to 15 (different amounts of adsorbent)
Comparative examples 12 to 15 are different from example 1 in that the amount of magnetic graphene-based poly (amine) (PAA @ Mag-G) added to the 0.5m L extract in step (3) was 7.5mg, and the remaining steps were completely the same.
The recovery rates of the 4 alkaloids in example 1 and comparative examples 12-15 were measured and are shown in table 8:
TABLE 8 recovery of 4 alkaloids in example 1 and comparative examples 12-15
| Numbering | Comparative example 12 | Comparative example 13 | Example 1 | Comparative example 14 | Comparative example 15 |
| 5m of L extract adsorbent dosage | 5.0mg | 7.5mg | 10.0mg | 12.5mg | 15.0mg |
| Aconitine | 52.4 | 70.3 | 95.9 | 98.6 | 96.9 |
| Novel aconitine | 76.9 | 80.5 | 93.8 | 97.9 | 97.4 |
| Hypaconitine | 53.1 | 71.8 | 92.5 | 95.5 | 97.8 |
| Aconitine from Yunnan aconitine | 58.4 | 72.2 | 91.5 | 93.8 | 96.2 |
As can be seen from Table 8, the influence of the amount of the adsorbent on the recovery rate is critical, when the amount of the adsorbent is too low (comparative examples 11 and 12), the recovery rate is low, and a minimum of 10.0mg of the adsorbent is required for every 0.5m L extract to effectively remove impurities, and a satisfactory recovery rate (91.5 to 95.9%) can be obtained at the same time.
Comparative examples 16 to 20 (different time of purification treatment)
Comparative examples 16 to 20 are different from example 1 in that the time of the purging treatment is different in step (3), and the remaining steps are completely the same.
The recovery rates of the 4 alkaloids in example 1 and comparative examples 16-20 were measured and are shown in table 9:
TABLE 9 recovery of 4 alkaloids in example 1 and comparative examples 16-20
| Numbering | Comparative example 16 | Comparative example 17 | Comparative example 18 | Example 1 | Comparative example 19 | Comparative example 20 |
| Time of purification treatment | 0.5min | 1.0min | 1.5min | 2.0min | 2.5min | 3.0min |
| Aconitine | 54.7 | 72.5 | 83.3 | 85.9 | 92.6 | 95.6 |
| Novel aconitine | 72.3 | 79.8 | 82.5 | 92.8 | 94.6 | 95.6 |
| Hypaconitine | 47.3 | 53.9 | 79.4 | 91.3 | 95.2 | 97.3 |
| Aconitine from Yunnan aconitine | 52.1 | 78.6 | 92.5 | 96.4 | 97.5 | 96.1 |
As can be seen from Table 9, the MSPE process is based on an adsorption-desorption equilibrium, the interaction time of the adsorbent and the extract to be purified having a direct influence on the purification effect. Thus, the interaction time between the adsorbent and the extraction liquid to be purified is optimized. When the interaction time between the adsorbent and the extract to be purified is 0.5min, the recovery rate of 4 alkaloids is not ideal, and is between 52.1-72.3%. The recovery rate of most alkaloids is obviously improved along with the increase of the interaction time, and when the interaction time reaches 2min, the recovery rate of 4 alkaloids is between 85.9 and 96.4 percent. The experimental result shows that the interaction time is 2min, and the ideal recovery rate can be obtained by 4 kinds of alkaloids. The extraction time is so short that the PAA @ Mag-G adsorbent can effectively adsorb impurities such as grease, pigment and the like due to the ultra-large surface area. Finally the interaction time was set to 2 min.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.
Claims (10)
1. A method for measuring 4 diester aconitine alkaloids in blood is characterized by comprising the following steps:
(1) adding a stabilizing agent into the plasma sample, and storing at a low temperature for later use;
(2) adding an extraction solvent into the plasma sample treated in the step (1) for extraction to obtain an extracting solution;
(3) adding a magnetic graphene-based adsorbent into the extracting solution for purification treatment to obtain a purified solution;
(4) the purified solution was filtered and analyzed by HP L C-MS/MS machine detection.
2. The method for detecting 4 diester-type aconitine alkaloids in blood as claimed in claim 1, wherein in step (1), the stabilizer is 10% by volume aqueous solution of formic acid, the amount of the stabilizer added to the plasma sample is 2-3 μ L/m L, and the temperature for storing at low temperature is 3-5 ℃.
3. The method according to claim 1, wherein in the step (2), the extraction solvent is a formic acid/acetonitrile mixture, and the volume fraction of formic acid in the formic acid/acetonitrile mixture is 0.5%.
4. The method for detecting 4 diester-type aconitine alkaloids in blood as claimed in claim 1, wherein in step (3), the amount of said magnetic graphene-based adsorbent added to the extract is 20mg/m L, and the time of purification treatment is 2 min.
5. The method according to claim 1, wherein in the step (3), the magnetic graphene-based adsorbent is magnetic graphene-based polyimide.
6. The method for detecting 4 diester-type aconitine alkaloids in blood as claimed in claim 5, wherein the preparation method of magnetic graphene-based poly-amine comprises the following steps:
(a) preparation of a polyimide polyamine dendrimer:
adding methyl acrylate into a methanol solution of tris (2-aminoethyl) amine, and continuously reacting for 18-24 h at the temperature of 30-35 ℃; adding triethylene diamine, continuously reacting for 18-24 h at 30-35 ℃, and performing rotary evaporation to obtain a crude product; dissolving the crude product in a methanol solution, repeating the step of adding methyl acrylate and triethylene diamine, and synthesizing the polyamide polyamine dendrimer taking N as a core through two Michael addition reactions and two amidation reactions;
(b) preparation of magnetic graphene-based polyamine:
FeCl is added3·6H2O，Ni(NO3)2·6H2Sequentially adding O, NaAc and NaOH into ethylene glycol for ultrasonic dissolution until the solution is transparent; then adding the polyimide dendritic macromolecule prepared in the step (a) and dissolving by ultrasonic; and adding carboxylated graphene, strongly stirring for 30-45 min at 50-60 ℃, transferring to a high-pressure reaction kettle, reacting for 48h at 180-200 ℃, separating a product by means of an external magnetic field, and washing to be neutral to obtain the magnetic graphene-based polyamide.
7. The method for detecting 4 diester-type aconitine alkaloids in blood as claimed in claim 6, wherein the amount of the carboxylated graphene added in step (b) is 1-1.5 mg/m L.
8. The method for detecting 4 diester-type aconitine alkaloids in blood as claimed in claim 1, wherein the chromatographic conditions of HP L C-MS/MS in step (4) are mobile phase A of HP L C grade water containing 0.05% formic acid and having pH of 3, and mobile phase B of acetonitrile containing 0.05% formic acid and having pH of 3.
9. The method according to claim 1, wherein the gradient elution conditions comprise 30% B, 0.00min → 4.00min, 30% → 90% B, 4.00min → 7.00min, 90% B, 7.00min → 10.00min, 300 μ L/min for the flow rate, and 5 μ L for the sample amount.
10. The method for detecting 4 diester-type aconitine alkaloids in blood as claimed in claim 1, wherein the mass spectrometric conditions of HP L C-MS/MS on-machine detection and analysis in step (4) are multi-ion reaction monitoring mode using positive ion electro-spray ionization, atomizing gas 60kPa, heating gas 50kPa, curtain gas 25kPa, spray voltage 4.5kV, and desolvation temperature 425 ℃.
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