CN115808482A - Method for detecting concentration of three components in evodia rutaecarpa decoction in blood plasma - Google Patents
Method for detecting concentration of three components in evodia rutaecarpa decoction in blood plasma Download PDFInfo
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Abstract
The invention discloses a method for detecting the concentration of three components in blood plasma in evodia decoction, which comprises the steps of adding internal standard working solution and acetonitrile into a blood plasma sample, centrifuging, and taking the obtained supernatant as the blood plasma sample solution to be detected; carrying out liquid chromatography separation on a plasma sample solution to be detected, and then carrying out mass spectrometry; the internal standard working solution is 450 ng/mL-550 ng/mL indometacin solution. The detection method can effectively detect the concentrations of the evodiamine, the rutaecarpine and the evodiamine in the blood plasma, has the advantages of simple operation, quick analysis, high specificity and high separation degree, can realize the simultaneous monitoring of the effective components and the toxic components in vivo after the evodiamine is taken, and has practical significance for further research and development and safety research of the evodiamine.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting the concentration of three components in evodia rutaecarpa decoction in blood plasma.
Background
Wuzhuyu decoction is named as the Chinese medicine. Is an interior-warming agent, has the effects of warming middle-jiao and tonifying deficiency, and lowering adverse qi and relieving vomiting, and mainly comprises fructus evodiae. The fructus evodiae is dry and nearly mature fruit of rutaecarpa of Rutaceae, and mainly contains chemical components including alkaloid, bitter principle, volatile oil and flavonoid, wherein the alkaloid is the main active component.
At present, the content of partial effective components of an evodia rutaecarpa decoction preparation is mainly determined by applying high performance liquid chromatography reported in the existing documents, the determination method of the content in vivo is only limited to 1-2 components, and the comprehensive research on the pharmacokinetics of the evodia rutaecarpa decoction in vivo cannot be realized due to the limitations of the instrument sensitivity and the extraction method. In addition, the pharmacopoeia records that the evodia rutaecarpa has small toxicity, and animal experiments show that the significant liver injury of experimental animals can be caused by the administration of a large dose of evodia rutaecarpa water decoction, and the mechanism of the evodia rutaecarpa water decoction can be related to peroxidation injury, inflammatory response factor mediation, mitochondrial injury, formation of drug-protein adduct and the like. Meanwhile, the detection of the situation of toxic components entering blood is also significant for guiding the clinical application of the evodia rutaecarpa decoction preparation.
Disclosure of Invention
Based on the above, one of the purposes of the present invention is to provide a method for detecting the concentration of three components in blood plasma in evodia rutaecarpa decoction, wherein the method has characteristics of simple operation, rapid analysis, high specificity and high separation degree.
The specific technical scheme for realizing the aim of the invention comprises the following steps:
a method for detecting the concentration of three components in blood plasma in evodia rutaecarpa decoction comprises the following steps:
adding an internal standard working solution and acetonitrile into a plasma sample, centrifuging, and taking obtained supernatant as a plasma sample solution to be detected; carrying out liquid chromatography separation on a plasma sample solution to be detected, and then carrying out mass spectrometry; the internal standard working solution is an indometacin solution with the concentration of 450 ng/mL-550 ng/mL.
In some embodiments, the mobile phase B used in the gradient elution in the liquid chromatography separation is acetonitrile, and the mobile phase a is aqueous solution of formic acid with a mass percentage concentration of 0.08% -0.12%.
In some of these embodiments, the gradient elution procedure is:
0.0-0.5 min, mobile phase B:50 ± 5% → 50 ± 5%;
0.5-1.0 min, mobile phase B:50 ± 5% → 95 ± 5%;
1.0-2.0 minutes, mobile phase B:95 ± 5% → 95 ± 5%;
2.0-2.2 minutes, mobile phase B:95 ± 5% → 50 ± 5%;
2.2-3.0 min, mobile phase B:50 + -5% → 50 + -5%.
In some of these embodiments, the chromatographic conditions of the liquid chromatography comprise:
a chromatographic column: a chromatographic column using octadecylsilane chemically bonded silica as a filler;
flow rate: 0.4mL/min +/-0.05 mL/min;
sample introduction volume: 3.00 mu L plus or minus 0.5 mu L;
column temperature: at 40 +/-5 ℃;
autosampler temperature: 4 +/-4 ℃;
needle washing liquid: (50 ± 5)% acetonitrile;
washing speed: 30-40 mu L/sec;
volume of needle wash: 400 mu L-600 mu L.
In some of these embodiments, the conditions of the mass spectrometry comprise:
ionization mode: an electrospray ion source; a positive ion mode and a negative ion mode; monitoring multiple reactions;
ion source parameters: air curtain air: 20psi; ion source gas 1:30psi; ion source gas 2:30psi;
ion source spray voltage of positive ion mode: 5500 + -10V;
ion source spray voltage of negative ion mode: -4500 ± 10V;
ion source temperature: 500 plus or minus 10 ℃;
resolution Q1/Q3: unit/Unit;
collision gas: medium;
pause time: 20 +/-1 msec;
the mass spectrum acquisition time is 3.00 +/-0.2 min.
In some of these embodiments, the mass spectrometry conditions for each component are:
evodiamine: ion pairing: 304.2 → 134.0; declustering voltage: 30.0V; inlet voltage: 10.0V; outlet voltage: 15.0V; collision energy: 35.000eV; residence time: 200.0msec;
rutaecarpine: ion pairing: 288.1 → 273.1; de-clustering voltage: 30.0V; inlet voltage: 10.0V; outlet voltage: 15.0V; collision energy: 32.000eV; residence time: 200.0msec;
evodia fruit lactone: ion pairing: 417.1 → 255.0; declustering voltage: 30.0V; inlet voltage: 10.0V; outlet voltage: 15.0V; collision energy: 32.000eV; residence time: 200.0msec;
and (3) indometacin: ion pairing: 358.0 → 139.0; de-clustering voltage: 30.0V; inlet voltage: 10.0V; outlet voltage: 15.0V; collision energy: 24.000eV; residence time: 200.0msec.
In some of these examples, liquid chromatography and mass spectrometry were performed using a Shimadzu LC 30AD liquid chromatograph in combination with a Sciex Qtrap5500 mass spectrometer using an operating system of Analyst 1.6.3; the liquid chromatographic column used was: ACQUITY
BEH C18，Waters。
In some of these embodiments, the volume ratio of the acetonitrile to the plasma sample is 3 to 6:1; and/or the volume ratio of the plasma sample to the internal standard solution is 1.5-2.5: 1; and/or the centrifugation temperature is 3-5 ℃, the centrifugation rotating speed is 2500-3500 rpm, and the centrifugation time is 8-12 min.
In some of these embodiments, the plasma sample is a human or animal plasma sample.
In some embodiments, the method further comprises the step of preparing a standard curve sample and a quality control sample, wherein the method for preparing the standard curve sample comprises the following steps: adding a mixed standard curve working solution into blank plasma; the method for preparing the quality control sample comprises the following steps: adding a mixed quality control working solution into blank plasma; the preparation of the mixed standard curve working solution and the mixed quality control working solution comprises the following steps: respectively diluting the stock solutions of the standard evodiamine, rutaecarpine and evodia fruit lactone with the concentrations of 1.00mg/mL by using 50 +/-5 percent methanol aqueous solution in a gradient manner to obtain the final product.
Compared with the prior art, the invention has the following beneficial effects:
in the invention, indomethacin is selected as an internal standard substance (the indomethacin has moderate polarity, can give consideration to the polarities of three components, can better simulate the actual condition that an analyte is influenced by a matrix effect on the three compounds, and better offset the influence caused by the matrix effect) and is added into a plasma sample to be detected, acetonitrile is used for precipitating protein, supernatant is taken for analysis through centrifugation, the sample pretreatment process is greatly simplified, and then the quantitative requirement can be met only by analyzing with extremely small sample feeding amount (the detection limit is low, only 3 mu L of sample feeding is needed); by adopting the LC-MS/MS-MRM detection method, the concentrations of three components, namely evodiamine, rutaecarpine and evodiamine in blood plasma in the evodia rutaecarpa decoction can be effectively detected, the channel of each compound only has a unique target peak, the chromatographic peak shape is excellent, the interference of other factors such as solvent effect and the like is avoided, the compound channels are mutually independent, and the separation degree is high; the sample introduction time of each sample is 3 minutes, and the analysis is rapid; no peak of the compound to be detected was found in the blank plasma, and the specificity was high. Therefore, the detection method can realize simultaneous monitoring of the effective components and toxic components in vivo after taking the evodia rutaecarpa decoction, and has practical significance for promoting further research and development and safety research of the evodia rutaecarpa decoction.
Drawings
FIG. 1 is a liquid-mass diagram of evodiamine in a plasma sample solution to be tested (retention time 1.58 min).
FIG. 2 is a liquid-mass diagram of rutaecarpine in a plasma sample solution to be tested (retention time 1.70 min).
FIG. 3 is a liquid-mass diagram of evodia rutaecarpa lactone in a plasma sample solution to be tested (retention time 1.22 min).
FIG. 4 is a liquid mass diagram (retention time 1.88 min) of indomethacin in a plasma sample solution to be tested.
Detailed Description
In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
In the invention, a method for detecting the concentration of three components in evodia rutaecarpa decoction in blood plasma is provided,
wherein, the properties of the 3 components are respectively as follows:
evodiamine free molecular formula and molecular weight C 19 H 17 N 3 O/303.36, and the specific structural formula is shown as a formula (I).
Rutaecarpine free molecular formula and molecular weight C 18 H 13 N 3 O/287.32, the specific structural formula is shown as the formula (II).
Evodiamine monomer free molecular formula and molecular weight C 18 H 19 NO 5 329.13, the specific structural formula is shown as a formula (III).
The internal standard adopted by the detection method is indomethacin (indomethacin), which is dissolved in acetone and slightly dissolved in ethanolDiethyl ether, chloroform and methanol, are slightly soluble in benzene, are very slightly soluble in toluene and are practically insoluble in water. Free molecular formula and molecular weight C 19 H 16 ClNO 4 /357.79, and the specific structure is shown as the formula (IV).
TABLE 1 Structure of three analytes and internal standards
The detection method specifically comprises the following steps:
(1) Preparation of stock solutions
Respectively dissolving standard evodiamine, rutaecarpine and evodiamine in DMSO with appropriate volume, and adding 3.5-4.5 times of methanol to obtain standard stock solution; mixing the standard substance stock solutions to obtain a mixed standard substance stock solution;
dissolving indometacin in DMSO with a proper volume, and adding methanol with a volume 3.5-4.5 times that of the indometacin to prepare an indometacin stock solution;
(2) Preparation of working solution
Respectively diluting the mixed standard substance stock solution in the step (1) with a (50 +/-5)% methanol aqueous solution to prepare a mixed standard curve working solution and a mixed quality control working solution;
diluting the indometacin stock solution in the step (1) by using a (50 +/-5)% methanol aqueous solution to prepare an internal standard working solution;
in the step, the mixed standard curve working solution comprises 8 gradients, wherein the concentrations of the evodiamine, the rutaecarpine and the evodiamine are respectively 10.0ng/mL, 40.0ng/mL and 40.0ng/mL;20.0ng/mL, 80.0ng/mL, and 80.0ng/mL;40ng/mL, 160ng/mL and 200ng/mL;160ng/mL, 640ng/mL and 400ng/mL;400ng/mL, 1600ng/mL and 800ng/mL;1600ng/mL, 6400ng/mL and 1600ng/mL;3200ng/mL, 12800ng/mL and 6400ng/mL;4000ng/mL, 16000ng/mL, and 8000ng/mL.
In this step, the mixed quality control working solution includes a quantitative lower-limit quality control working solution, a low-concentration quality control working solution, a medium-concentration quality control working solution, and a high-concentration quality control working solution; wherein the concentrations of the evodiamine, the rutaecarpine and the evodiamine in the quantitative lower limit quality control working solution are respectively 10.0ng/mL, 40.0ng/mL and 20.0ng/mL; the concentrations of the evodiamine, the rutaecarpine and the evodiamine in the low-concentration quality control working solution are respectively 30.0ng/mL, 120ng/mL and 50.0ng/mL; the concentrations of the evodiamine, the rutaecarpine and the evodiamine in the medium-concentration quality control working solution are respectively 300ng/mL, 1200ng/mL and 600ng/mL; the concentrations of the evodiamine, the rutaecarpine and the evodiamine in the high-concentration quality control working solution are 3000ng/mL, 12000ng/mL and 6000ng/mL respectively.
(3) Preparation of a plasma sample solution to be tested
Taking a plasma sample or homogenate, adding an internal standard working solution and acetonitrile, centrifuging, and taking obtained supernatant as a plasma sample solution to be detected;
in this step, the volume ratio of the acetonitrile to the plasma sample or the homogenate is 3 to 6:1; in the sample pretreatment process, the amount of the precipitating agent is far larger than the amount of the plasma, the sample injection amount is very low, and the influence of the solvent effect can be completely ignored, so that the separation effect is excellent. The centrifugal temperature is 3-5 ℃; the centrifugal rotating speed is 2500 rpm-3500 rpm; the centrifugation time is 8-12 min. The blood plasma is human or animal blood plasma.
In one embodiment, the preparation method of the plasma sample solution to be tested comprises the following steps: precisely sucking 50 μ L of plasma sample, sequentially adding 25 μ L (50 ng/mL) of internal standard (indomethacin) and 300 μ L of acetonitrile, vortexing for 10min, mixing well, centrifuging at 4 deg.C and 3000rpm for 10min, collecting supernatant 100 μ L, adding 100 μ L of 0.1% formic acid aqueous solution, mixing well, and analyzing by sample injection.
(4) And (4) performing liquid chromatography separation on the plasma sample solution to be detected obtained in the step (3), and then performing mass spectrometry.
In the invention, the model of the adopted liquid chromatography-mass spectrometry combination instrument is Shimadzu LC 30AD liquid chromatograph combined with Sciex Qtrap5500 mass spectrometer, and the adopted operating system is Analyst 1.6.3; the Sciex Qtrap model 5500 mass spectrometer used in the invention has an order of magnitude higher sensitivity than that of the previous instrument.
The elution mode adopted by the liquid chromatography separation is as follows: gradient elution is carried out by taking (0.1 +/-0.02)% formic acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B; the gradient elution process is as follows:
0.0-0.5 min, mobile phase B:50 ± 5% → 50 ± 5%;
0.5-1.0 min, mobile phase B:50 ± 5% → 95 ± 5%;
1.0-2.0 minutes, mobile phase B:95 ± 5% → 95 ± 5%;
2.0-2.2 minutes, mobile phase B:95 ± 5% → 50 ± 5%;
2.2-3.0 min, mobile phase B:50 + -5% → 50 + -5%.
Preferably, the process of gradient elution is:
0.0-0.5 min, mobile phase B:50% → 50%;
0.5-1.0 min, mobile phase B:50% → 95%;
1.0-2.0 minutes, mobile phase B:95% → 95%;
2.0-2.2 minutes, mobile phase B:95% → 50%;
2.2-3.0 min, mobile phase B:50% → 50%.
In the present invention, the chromatographic conditions of the liquid chromatography include:
and (3) chromatographic column: a chromatographic column using octadecylsilane chemically bonded silica as a filler; in particular ACQUITY
BEH C18, waters, 1.7 μm,2.1x50mm;
flow rate: 0.4mL/min +/-0.05 mL/min;
sample introduction volume: 3.00 mu L plus or minus 0.5 mu L;
column temperature: 40 +/-5 ℃;
autosampler temperature: 4 +/-4 ℃;
needle washing liquid: (50 ± 5)% acetonitrile;
washing speed: 30-40 mu L/sec;
volume of needle washing liquid: 400 mu L-600 mu L.
In the present invention, the conditions for the mass spectrometry include:
ionization mode: an electrospray ion source; a positive ion mode and a negative ion mode; monitoring multiple reactions;
ion source parameters: air curtain air: 20psi; ion source gas 1:30psi; ion source gas 2:30psi; ion source temperature: 500 plus or minus 10 ℃; resolution Q1/Q3: unit/Unit; collision gas: medium; pause time: 20 +/-1 msec; the mass spectrum acquisition time is 3.00 +/-0.2 min; ion source spray voltage of positive ion mode: 5500V plus or minus 10V; ion source spray voltage of negative ion mode: 4500 ± 10V.
In the mass spectrometry, the quadrupole mass spectrometry conditions of each component are as follows:
evodiamine: ion pairing: 304.2 → 134.0; de-clustering voltage: 30.0V; inlet voltage: 10.0V; outlet voltage: 15.0V; collision energy: 35.000eV; residence time: 200.0msec;
rutaecarpine: ion pairing: 288.1 → 273.1; de-clustering voltage: 30.0V; inlet voltage: 10.0V; outlet voltage: 15.0V; collision energy: 32.000eV; residence time: 200.0msec;
evodia fruit lactone: ion pairing: 417.1 → 255.0; de-clustering voltage: 30.0V; inlet voltage: 10.0V; outlet voltage: 15.0V; collision energy: 32.000eV; residence time: 200.0msec;
and (3) indometacin: ion pairing: 358.0 → 139.0; declustering voltage: 30.0V; inlet voltage: 10.0V; outlet voltage: 15.0V; collision energy: 24.000eV; residence time: 200.0msec.
The reagents used in the following examples are all commercially available.
The invention is described in further detail below with reference to the figures and specific examples.
Example 1 method for detecting the concentration of three components in Evodia rutaecarpa decoction in blood plasma
1. Preparation of the solution
(1) Preparation of stock solutions
Weighing a proper amount of standard substances of evodiamine, rutaecarpine and evodia fruit lactone, respectively dissolving the standard substances in a proper amount of DMSO, and then adding acetonitrile with the volume of 4 times of that of the DMSO to prepare standard substance stock solutions with the concentrations of 1.00mg/mL respectively.
Taking a proper amount of indometacin standard substance, dissolving the indometacin standard substance in DMSO, and adding acetonitrile with the volume 4 times that of the indometacin standard substance to prepare an indometacin stock solution with the concentration of 1.00 mg/mL.
(2) Preparation of standard curve working solution
Diluting the evodiamine stock solution, the rutaecarpine stock solution and the evodia fruit lactone stock solution by using 50% methanol aqueous solution to obtain:
(1) mixed standard curve working solutions with the concentrations of the evodiamine, the rutaecarpine and the evodiamine being respectively 10.0ng/mL, 40.0ng/mL and 40.0ng/mL;
(2) mixed standard curve working solutions with the concentrations of the evodiamine, the rutaecarpine and the evodiamine being respectively 20.0ng/mL, 80.0ng/mL and 80.0ng/mL;
(3) mixed standard curve sample working solutions with the concentrations of the evodiamine, the rutaecarpine and the evodiamine being 40ng/mL, 160ng/mL and 200ng/mL respectively;
(4) mixed standard curve working solutions with the concentrations of the evodiamine, the rutaecarpine and the evodiamine being respectively 160ng/mL, 640ng/mL and 400ng/mL;
(5) mixed standard curve working solutions with the concentrations of the evodiamine, the rutaecarpine and the evodiamine being respectively 400ng/mL, 1600ng/mL and 800ng/mL;
(6) mixed standard curve working solutions with the concentrations of the evodiamine, the rutaecarpine and the evodiamine being 1600ng/mL, 6400ng/mL and 1600ng/mL respectively;
(7) mixed standard curve working solutions with the concentrations of the evodiamine, the rutaecarpine and the evodiamine being 3200ng/mL, 12800ng/mL and 6400ng/mL respectively;
(8) the concentrations of the evodiamine, the rutaecarpine and the evodiamine are 4000ng/mL, 16000ng/mL and 8000ng/mL of mixed standard curve working solution respectively.
(3) Preparation of standard curve plasma samples
Adding 20.0 μ L of the above mixed standard curve working solution into 380 μ L of blank plasma, and mixing well to obtain evodiamine, rutaecarpine and evodiamine lactone with concentrations of: (1) 0.50ng/mL, 2.00ng/mL, and 2.00ng/mL; (2) 1.00ng/mL, 4.00ng/mL, and 4.00ng/mL; (3) 2.00ng/mL, 8.00ng/mL, and 10.0ng/mL; (4) 8.00ng/mL, 32.0ng/mL, and 20.0ng/mL; (5) 20.0ng/mL, 80.0ng/mL, and 40.0ng/mL; (6) 80.0ng/mL, 320ng/mL, and 80.0ng/mL; (7) 160ng/mL, 640ng/mL and 320ng/mL; (8) standard curve plasma samples at 200ng/mL, 800ng/mL, and 400 ng/mL.
(4) Preparation of quality control working solution
The indomethacin stock solution was diluted with 50% aqueous methanol to make an internal standard working solution at a concentration of 500 ng/mL.
Diluting the evodiamine stock solution, the rutaecarpine stock solution and the evodia fruit lactone stock solution by using 50% methanol aqueous solution to obtain:
(1) the concentrations of the evodiamine, the rutaecarpine and the evodiamine are respectively 10.0ng/mL, 40.0ng/mL and 20.0ng/mL (quantitative lower limit quality control);
(2) the concentrations of the evodiamine, the rutaecarpine and the evodiamine are respectively 30.0ng/mL, 120ng/mL and 50.0ng/mL (low concentration quality control);
(3) the concentrations of the evodiamine, the rutaecarpine and the evodiamine are respectively 300ng/mL, 1200ng/mL and 600ng/mL (medium concentration quality control);
(4) the concentrations of the evodiamine, the rutaecarpine and the evodiamine are 3000ng/mL, 12000ng/mL and 6000ng/mL respectively (high-concentration quality control);
(5) Quality control plasma sample preparation
20.0. Mu.L of the mixed quality control working solution is added to 380. Mu.L of blank plasma and mixed uniformly. The concentrations of the obtained evodiamine, rutaecarpine and evodiamine lactone are respectively as follows: (1) the lower limit of quantification quality control is 0.500ng/mL, 2.00ng/mL and 1.00ng/mL; (2) the low concentration quality control is 1.50ng/mL, 6.00ng/mL and 2.50ng/mL; (3) medium concentration quality control is 15.0ng/mL, 60.0ng/mL and 30.0ng/mL; (4) high concentration quality control 150ng/mL, 600ng/mL and 300ng/mL quality control plasma samples.
2. Sample pretreatment
After 30 minutes of oral administration of the evodia rutaecarpa decoction to rats, 50 mu L of rat plasma samples are precisely sucked, 25 mu L (500 ng/mL) of internal standard (indometacin) and 300 mu L of acetonitrile are sequentially added, vortex is carried out for 10min, the mixture is fully mixed, centrifugation is carried out at 3000rpm at 4 ℃ for 10min, 100 mu L of supernate is taken, 100 mu L of 0.1% formic acid aqueous solution is added, and the mixture is fully mixed.
3. Liquid chromatography-mass spectrometry detection
And (3) carrying out liquid chromatography-mass spectrometry detection on the pretreated rat plasma sample.
(1) Liquid chromatography conditions
The instrument equipment comprises: shimadzu LC-30AD liquid chromatography system.
And (3) chromatographic column: ACQUITY
BEH C181.7 μm 2.1X50mm, waters; the column temperature was set to 40 ℃ and the autosampler temperature was set to 4 ℃.
A mobile phase A:0.1% aqueous formic acid; mobile phase B:100% acetonitrile.
Total flow rate: 0.40mL/min.
The mobile phase elution procedure was:
0.0-0.5 min, mobile phase B:50% → 50%;
0.5-1.0 min, mobile phase B:50% → 95%;
1.0-2.0 minutes, mobile phase B:95% → 95%;
2.0-2.2 minutes, mobile phase B:95% → 50%;
2.2-3.0 min, mobile phase B:50% → 50%.
Needle washing liquid: 50% acetonitrile. The needle washing mode is external flushing; and (3) washing speed: 35 μ L/sec; flushing the port with liquid: r1; volume of flushing fluid: 500 mu L of the solution; a flushing mode: flushing before and after needle insertion; washing and soaking time: 0 second; the washing method comprises the following steps: port only flush; washing time: 2 seconds;
the injection volume was 5.00. Mu.L.
The standard chromatograms of the three compound standards and the internal standard are shown in fig. 1-4. The retention times of evodiamine, rutaecarpine, evodiamine and IS (indomethacin) are 1.58min, 1.70min, 1.22min and 1.88min respectively.
(2) Mass spectrum conditions
An instrument device: sciex Qtrap5500 Mass Spectrometry System.
Ionization mode: electrospray ion source (ESI); positive ion mode (Positive) and Negative ion mode (Negative); multiple Reaction Monitoring (MRM).
Ion source parameters: air curtain air: 20psi; ion source gas 1:30psi; ion source gas 2:30psi; ion source temperature: 500 plus or minus 10 ℃; resolution Q1/Q3: unit/Unit; collision gas: medium; pause time: 20 +/-1 msec; the mass spectrum acquisition time is 3.00 +/-0.2 min; ion source spray voltage of positive ion mode: 5500 + -10V; ion source spray voltage in negative ion mode: 4500 ± 10V.
The conditions of the quadrupole mass spectrum of each component are as follows:
evodiamine: ion pairing: 304.2 → 134.0; declustering voltage: 30.0V; inlet voltage: 10.0V; outlet voltage: 15.0V; collision energy: 35.000eV; residence time: 200.0msec;
rutaecarpine: ion pairing: 288.1 → 273.1; declustering voltage: 30.0V; inlet voltage: 10.0V; outlet voltage: 15.0V; collision energy: 32.000eV; residence time: 200.0msec;
evodia fruit lactone: ion pairing: 417.1 → 255.0; declustering voltage: 30.0V; inlet voltage: 10.0V; outlet voltage: 15.0V; collision energy: 32.000eV; residence time: 200.0msec;
indomethacin: ion pairing: 358.0 → 139.0; de-clustering voltage: 30.0V; inlet voltage: 10.0V; outlet voltage: 15.0V; collision energy: 24.000eV; residence time: 200.0msec.
4. The result of the detection
The concentrations of evodiamine, rutaecarpine and evodiamine in the plasma samples of rats are shown in Table 2 after the rats are orally administered with the evodiamine decoction for 30 minutes.
TABLE 2 measurement results of 3-component concentration in rat plasma
| Test object | Plasma sample (ng/mL) |
| Evodiamine | 50.2 |
| Rutaecarpine | 2.69 |
| Evodiamine derivative | 0.00 |
Example 2 methodological validation of the detection method of the invention
1. Specificity property
The liquid chromatography-mass spectrometry detection conditions of example 1 are adopted to detect rat blank plasma, and the result shows that endogenous impurities in the rat blank plasma do not interfere with the determination of the evodiamine, the rutaecarpine and the evodiamine, and the rat blank plasma has no chromatographic peak at three components.
2. Lowest quantitative limit and linear range
In rat plasma:
the lowest quantitative limit of the evodiamine is 0.500ng/mL, and the linear range of the evodiamine is 0.500-200ng/mL
The lowest quantitative limit of the rutaecarpine is 2.00ng/mL, and the linear range is 2.00-800ng/mL;
the lowest quantitative limit of the evodia rutaecarpa lactone is 2.00ng/mL, and the linear range is 2.00-400ng/mL;
r of three compounds 2 Between 0.9957 and 0.9992.
3. Accuracy and precision
For evodiamine, preparing a quality control plasma sample with low concentration quality control of 1.50ng/mL, medium concentration quality control of 15.0ng/mL and high concentration quality control of 150 ng/mL;
for the rutaecarpine, the concentrations of prepared quality control plasma samples are respectively 6.00ng/mL, 60.0ng/mL and 600ng/mL;
for the evodia rutaecarpa lactone, the concentrations of the prepared quality control plasma samples are respectively 2.50ng/mL, 30.0ng/mL and 300ng/mL.
Each 6-concentration sample was analyzed and continuously measured for 3 days, and the QC (quality control) sample concentration was calculated from the standard curve on the day to obtain the RE value and RSD value for each concentration, as shown in Table 3.
TABLE 3 daily precision results for the ingredients
Table 3 the results show that: the precision of the 3 component quality control samples is between-15.0 percent and 15.0 percent in the daytime and the RSD is within 15.0 percent, and the requirement of the verification of the biological sample quantitative analysis method in Chinese pharmacopoeia-9012 is met.
4. Recovery rate
Preparing 6 samples from the low-concentration, medium-concentration and high-concentration quality control samples respectively, and simultaneously extracting 18 control samples (mixed matrixes) which do not contain the substance to be detected and contain the internal standard. After the control sample is extracted, a compound is added into the extracting solution to ensure that the theoretical concentration of the extracting solution is consistent with that of the low, medium and high extracting samples. Calculating the extraction recovery rate of the object to be detected: r (%) =100% xc/S, where C is the peak area of the analyte in the QC sample and S is the peak area of the analyte in the control sample, as shown in table 4.
TABLE 4 results for recovery of the ingredients
Table 4 the results show that: the recovery rate of the 3 component quality control samples is between 79.8 and 112.8, and the RSD is within 15.0 percent, which shows that the method is stable and reliable.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent should be subject to the appended claims.
Claims (10)
1. A method for detecting the concentration of three components in blood plasma in evodia rutaecarpa decoction is characterized by comprising the following steps:
adding an internal standard working solution and acetonitrile into a plasma sample, centrifuging, and taking obtained supernatant as a plasma sample solution to be detected; performing liquid chromatography separation on a plasma sample solution to be detected, and then performing mass spectrometry; the internal standard working solution is an indometacin solution with the concentration of 450 ng/mL-550 ng/mL.
2. The detection method according to claim 1, wherein a mobile phase B adopted by gradient elution in the liquid chromatography separation is acetonitrile, and a mobile phase A is aqueous solution of formic acid with the mass percentage concentration of 0.08-0.12%.
3. The detection method according to claim 2, wherein the gradient elution procedure is:
0.0-0.5 min, mobile phase B:50 ± 5% → 50 ± 5%;
0.5-1.0 min, mobile phase B:50 ± 5% → 95 ± 5%;
1.0-2.0 minutes, mobile phase B:95 ± 5% → 95 ± 5%;
2.0-2.2 minutes, mobile phase B:95 ± 5% → 50 ± 5%;
2.2-3.0 min, mobile phase B:50 + -5% → 50 + -5%.
4. The detection method according to claim 1, wherein the chromatographic conditions of the liquid chromatography comprise:
a chromatographic column: a chromatographic column using octadecylsilane chemically bonded silica as a filler;
flow rate: 0.4mL/min +/-0.05 mL/min;
sample introduction volume: 3.00 mu L plus or minus 0.5 mu L;
column temperature: at 40 +/-5 ℃;
autosampler temperature: 4 +/-4 ℃;
needle washing liquid: (50 ± 5)% acetonitrile;
washing speed: 30 mu L/sec-40 mu L/sec;
volume of needle washing liquid: 400 mu L-600 mu L.
5. The detection method according to claim 1, wherein the conditions of mass spectrometry comprise:
ionization mode: an electrospray ion source; a positive ion mode and a negative ion mode; monitoring multiple reactions;
ion source parameters: air curtain air: 20psi; ion source gas 1:30psi; ion source gas 2:30psi;
ion source spray voltage of positive ion mode: 5500 + -10V;
ion source spray voltage of negative ion mode: -4500 ± 10V;
ion source temperature: 500 plus or minus 10 ℃;
resolution Q1/Q3: unit/Unit;
collision gas: medium;
pause time: 20 +/-1 msec;
the mass spectrum acquisition time is 3.00 +/-0.2 min.
6. The detection method according to claim 5, wherein in the mass spectrometry, the quadrupole mass spectrometry conditions of each component are as follows:
evodiamine: ion pairing: 304.2 → 134.0; de-clustering voltage: 30.0V; inlet voltage: 10.0V; outlet voltage: 15.0V; collision energy: 35.000eV; residence time: 200.0msec;
rutaecarpine: ion pairing: 288.1 → 273.1; de-clustering voltage: 30.0V; inlet voltage: 10.0V; outlet voltage: 15.0V; collision energy: 32.000eV; residence time: 200.0msec;
evodia fruit lactone: ion pairing: 417.1 → 255.0; de-clustering voltage: 30.0V; inlet voltage: 10.0V; outlet voltage: 15.0V; collision energy: 32.000eV; residence time: 200.0msec;
indomethacin: ion pairing: 358.0 → 139.0; declustering voltage: 30.0V; inlet voltage: 10.0V; outlet voltage: 15.0V; collision energy: 24.000eV; residence time: 200.0msec.
7. The detection method according to any one of claims 1 to 6, characterized in that liquid chromatography and mass spectrometry are carried out by using Shimadzu LC 30AD liquid chromatograph in combination with Sciex Qtrap5500 mass spectrometer, and the operating system used is Analyst 1.6.3; the liquid chromatographic column used was: ACQUITY
BEH C18，Waters。
8. The assay of any one of claims 1 to 6, wherein the volume ratio of acetonitrile to the plasma sample is from 3 to 6:1; and/or the volume ratio of the plasma sample to the internal standard working solution is 1.5-2.5: 1; and/or the centrifugation temperature is 3-5 ℃, the centrifugation rotating speed is 2500-3500 rpm, and the centrifugation time is 8-12 min.
9. The assay of any one of claims 1 to 6, wherein the plasma sample is a human or animal plasma sample.
10. The detection method according to any one of claims 1 to 6, further comprising a step of preparing a standard curve sample and a quality control sample,
the method for preparing the standard curve sample comprises the following steps: adding a mixed standard curve working solution into blank plasma;
the method for preparing the quality control sample comprises the following steps: adding a mixed quality control working solution into blank plasma;
the preparation of the mixed standard curve working solution and the mixed quality control working solution comprises the following steps: respectively diluting the stock solutions of the standard evodiamine, rutaecarpine and evodia fruit lactone with the concentrations of 1.00mg/mL by using 50 +/-5 percent methanol aqueous solution in a gradient manner to obtain the final product.
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