CN110907581B - Mass spectrum detection method for concentration of seven-component blood plasma or tissue of compound salvia miltiorrhiza preparation - Google Patents
Mass spectrum detection method for concentration of seven-component blood plasma or tissue of compound salvia miltiorrhiza preparation Download PDFInfo
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Abstract
The invention relates to a mass spectrum detection method for the concentration of seven components of blood plasma or tissues in a compound salvia miltiorrhiza preparation. The method comprises the following steps: (1) preparation of stock solution: taking standard tanshinone I, tanshinone IIA, salvianolic acid B, tanshinol, and notoginsenoside R1Ginsenoside Rg1And Rb1Respectively dissolving in 80 +/-5% methanol aqueous solution to obtain standard substance stock solution; dissolving carbamazepine in 80 +/-5% methanol water solution to prepare a carbamazepine stock solution; (2) preparing a working solution; (3) preparing a sample solution to be detected; (4) performing liquid chromatography separation on the sample solution to be detected obtained in the step (3); and carrying out mass spectrum analysis on the sample subjected to the liquid chromatography separation. The chromatographic peak shape in the detection process of the method is excellent, each compound channel only has a unique target peak, the interference of other factors such as solvent effect and the like is avoided, and the method is simple to operate, rapid to analyze, high in specificity and high in separation degree.
Description
Technical Field
The invention relates to the technical field of pharmaceutical analysis, in particular to a mass spectrometry detection method for the concentration of seven-component plasma or tissue of a compound salvia miltiorrhiza preparation.
Background
The compound red sage preparation consists of 3 kinds of Chinese medicinal materials including red sage, notoginseng and borneol, and has total 9 kinds: compound Saviae Miltiorrhizae radix oral liquid, compound Saviae Miltiorrhizae radix aerosol, compound Saviae Miltiorrhizae radix buccal tablet, compound Saviae Miltiorrhizae radix capsule, compound Saviae Miltiorrhizae radix tablet, compound Saviae Miltiorrhizae radix granule, compound Saviae Miltiorrhizae radix dripping pill, compound Saviae Miltiorrhizae radix spray, and compound Saviae Miltiorrhizae radix soft capsule. The compound red sage root preparation has the functions of promoting blood circulation to disperse blood clots and regulating vital energy to alleviate pain, and is used in treating chest obstruction caused by qi stagnation and blood stasis, with the symptoms of chest distress, precordial pain, coronary atherosclerotic heart disease and angina pectoris. The incidence rate of adverse reactions of the single compound salvia miltiorrhiza preparation in clinical application is about 5-7%. The adverse reactions are complex and various in expression, and the individual difference of in vivo metabolism is large, mainly including gastrointestinal tract reaction, headache, hemorrhage and the like. Most adverse reactions are slight injury, bleeding and the like which can be tolerated by patients; a small part of adverse reactions need to be treated with stopping taking the medicines; the curative effect and adverse reaction of the compound salvia miltiorrhiza preparation are closely related to the drug concentration in blood plasma, and the drug administration scheme needs to be adjusted according to the blood drug concentration, so that the blood drug concentration needs to be monitored.
The content determination method of compound Saviae Miltiorrhizae radix preparation specified in pharmacopoeia is high performance liquid chromatographyThe spectrum method comprises content determination method of tanshinone IIA and salvianolic acid B and ginsenoside Rg under Notoginseng radix1Ginsenoside Rb1Notoginsenoside R1And a method for measuring ginsenoside Re. However, the analysis method specified in the pharmacopoeia involves a large number of chemical pretreatment steps, the operation is relatively complicated, and especially the pretreatment and calibration of the test sample for measuring the content of tanshinone IIA takes a long time, which brings inconvenience to the actual work.
At present, common methods for measuring the blood concentration of the compound salvia miltiorrhiza preparation comprise: high performance liquid chromatography and liquid chromatography-mass spectrometry are combined. The compound Saviae Miltiorrhizae radix preparation contains water soluble components (salvianolic acid B, tanshinol, and notoginsenoside R) due to large property difference of seven components1Ginsenoside Rg1Ginsenoside Rb1) And fat-soluble components (tanshinone I and tanshinone IIA), and the content is greatly different, so that the treatment and the detection by a pretreatment method are difficult. The method for detecting the main components of the compound salvia miltiorrhiza preparation generally adopts a method for separately preprocessing and detecting different components, so that the process is complex and the steps are complicated.
Disclosure of Invention
Based on the above, one of the purposes of the present invention is to provide a high performance liquid chromatography tandem mass spectrometry detection method for plasma concentration or tissue distribution of seven components of a compound salvia miltiorrhiza preparation, wherein the detection method has characteristics of simple operation, rapid analysis, high specificity and high separation degree.
The specific technical scheme is as follows:
a high performance liquid chromatography tandem mass spectrometry detection method for plasma concentration or tissue distribution of seven components of a compound salvia miltiorrhiza preparation comprises the following steps:
(1) preparation of stock solutions:
taking standard tanshinone I, tanshinone IIA, salvianolic acid B, tanshinol, and notoginsenoside R1Ginsenoside Rg1And Rb1Respectively dissolving in 80 +/-5% methanol aqueous solution to obtain standard substance stock solution; mixing the standard substance stock solution to obtain a mixed standard substance stock solution;
dissolving carbamazepine in 80 +/-5% methanol water solution to prepare a carbamazepine stock solution;
(2) preparation of working solution:
respectively diluting the mixed standard substance stock solution with (80 +/-5)% methanol aqueous solution to obtain a mixed standard curve sample working solution and a mixed quality control sample working solution;
diluting the carbamazepine stock solution by using (80 +/-5)% methanol aqueous solution to prepare an internal standard working solution;
(3) preparing a sample solution to be tested:
preparation of a plasma sample solution to be tested: taking a plasma sample, adding an internal standard working solution and methanol, centrifuging, and taking obtained supernatant as a plasma sample solution to be detected;
preparing a solution of a tissue sample to be detected: taking a tissue sample, homogenizing, adding an internal standard working solution and methanol into the obtained homogenate, centrifuging, and taking the obtained supernatant as a tissue sample solution to be detected;
(4) performing liquid chromatography separation on the sample solution to be detected obtained in the step (3); and carrying out mass spectrum analysis on the sample subjected to liquid chromatography separation.
Compared with the prior art, the invention has the following beneficial effects:
in the method, carbamazepine is selected as an internal standard substance to be added into a plasma sample to be detected, then methanol is used for precipitating protein, the supernatant is taken for analysis through centrifugation, then the quantitative requirement can be met only by analyzing with few micro-upgrading sample volumes, and the sample pretreatment process is greatly simplified. The chromatographic peak shape is excellent in the detection process of the method, and each compound channel only has a unique target peak, so that other interferences such as solvent effect and the like are avoided; the pretreatment adopts a protein precipitation method, and the operation is simple; the sample introduction time of each sample is 3 minutes, and the analysis is rapid; the peak of the compound to be detected is not found in blank plasma, and the specificity is high; the LC-MS/MS-MRM method is adopted, the compound channels are mutually independent, and the separation degree is high.
The method can simultaneously detect the contents of seven components of the compound salvia miltiorrhiza preparation in one method, can additionally detect the contents of tanshinone I and tanshinol besides five components specified in pharmacopoeia, can provide a faster and more efficient method for measuring the content of the compound salvia miltiorrhiza preparation, and has good practical significance.
Drawings
Fig. 1 is a liquid chromatogram of tanshinone I in a plasma sample (retention time 1.87 min):
fig. 2 is a liquid chromatogram (retention time 1.92min) of tanshinone IIA in plasma sample:
FIG. 3 is a liquid chromatogram of salvianolic acid B in a plasma sample (retention time 1.56 min):
FIG. 4 is a liquid chromatogram of tanshinol in a plasma sample (retention time 0.62 min):
FIG. 5 shows notoginsenoside R1Liquid chromatogram in plasma sample (retention time 1.63 min):
FIG. 6 shows ginsenoside Rg1Liquid chromatogram in plasma samples (retention time 1.65 min):
FIG. 7 shows ginsenoside Rb1Liquid chromatogram in plasma sample (retention time 1.74 min);
FIG. 8 is a liquid chromatogram of carbamazepine in a plasma sample (retention time 1.68 min).
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following examples, which are included to provide further understanding of the invention. 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.
The embodiment provides a high performance liquid chromatography-tandem mass spectrometry detection method for plasma concentration or tissue distribution of seven components of a compound salvia miltiorrhiza preparation, which comprises the following steps:
(1) preparation of stock solution:
taking standard tanshinone I, tanshinone IIA, salvianolic acid B, tanshinol, and notoginsenoside R1Ginsenoside Rg1And Rb1Respectively dissolving in 80 +/-5% methanol aqueous solution to obtain standard substance stock solution; mixing the standard substance stock solution to obtain a mixed standard substance stock solution;
dissolving carbamazepine in 80 +/-5% methanol water solution to prepare a carbamazepine stock solution;
(2) preparation of working solution:
respectively diluting the mixed standard substance stock solution with (80 +/-5)% methanol aqueous solution to obtain a mixed standard curve sample working solution and a mixed quality control sample working solution;
diluting the carbamazepine stock solution by using (80 +/-5)% methanol aqueous solution to prepare an internal standard working solution;
(3) preparing a sample solution to be tested:
preparation of a plasma sample solution to be tested: taking a plasma sample, adding an internal standard working solution and methanol, centrifuging, and taking obtained supernatant as a plasma sample solution to be detected;
preparing a solution of a tissue sample to be detected: taking a tissue sample, homogenizing, adding an internal standard working solution and methanol into the obtained homogenate, centrifuging, and taking the obtained supernatant as a tissue sample solution to be detected;
(4) performing liquid chromatography separation on the sample solution to be detected obtained in the step (3); and carrying out mass spectrum analysis on the sample subjected to liquid chromatography separation.
Wherein the properties of the components are as follows:
tanshinone I free molecular formula and molecular weight C18H12O/276.29, the specific structural formula is shown as formula (I).
Tanshinone IIA free molecular formula and molecular weight C19H18O3/294.34, the specific structural formula is shown as formula (II).
The salvianolic acid B is soluble in water, and has a free molecular formula and a molecular weight of C36H30O16/718.62, the specific structure is shown in formula (III).
Salvianic acid A is soluble in methanol and water, and insoluble in chloroform and diethyl ether. Free molecular formula and molecular weight C5H10O5/198.7, sodium salt formula and molecular weight C9H9O5Na/220.04, the specific structural formula is shown as formula (IV).
Notoginsenoside R1 is slightly soluble in anhydrous alcohol, and almost insoluble in water. Free molecular formula and molecular weight C47H80O18The specific structural formula of/933.13 is shown in formula (V).
Ginsenoside Rg1Dissolved in methanol, pyridine, hot acetone, slightly dissolved in ethyl acetate and chloroform. The acetylate was dissolved in methanol, pyridine, hot acetone, slightly in ethyl acetate and chloroform. Free molecular formula and molecular weight C42H72O14The specific structural formula of/801.01 is shown in formula (VI).
Ginsenoside Rb1Free molecular formula and molecular weight C54H92O23The specific structural formula of/1109.29 is shown as formula (VII).
The internal standard selected by the method is Carbamazepine (Carbamazepine), which is easily soluble in trichloromethane, slightly soluble in ethanol and hardly soluble in water and ether. Free molecular formula and molecular weight C15H12N2O/236.27, the specific structure is shown as formula (VIII).
TABLE 1
In some of these embodiments, the liquid chromatography is performed using an elution method comprising: gradient elution was carried out using (0.1. + -. 0.02)% formic acid aqueous solution as mobile phase A and methanol as mobile phase B.
In some of these embodiments, the process of gradient elution comprises: 0-0.5 min, (10 +/-5)% of mobile phase A- (90 +/-5)% of mobile phase B; 0.5-2.0 minutes, (5 +/-5)% of mobile phase A- (95 +/-5)% of mobile phase B; 2-2.2 minutes, (90 +/-5)% of mobile phase A- (10 +/-5)% of mobile phase B; 2.2-3.0 min, (90 +/-5)% of mobile phase A- (10 +/-5)% of mobile phase B; 2.4-3 minutes, (90 +/-5)% of mobile phase A- (10 +/-5)% of mobile phase B.
Preferably, the gradient elution is carried out by: 10% of mobile phase A to 90% of mobile phase B in 0-0.5 min; 5% of mobile phase A to 95% of mobile phase B in 0.5-2.0 minutes; 90% of mobile phase A-10% of mobile phase B in 2-2.2 minutes; 2.2-3.0 minutes, 90% of mobile phase A-10% of mobile phase B; 90% of mobile phase A to 10% of mobile phase B for 2.4 to 3 minutes.
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;
the flow rate is 0.3 plus or minus 0.1 mL/min;
the sample injection volume is 3.00 +/-0.5 mu L;
the column temperature is set to be 40 +/-5 ℃;
the temperature of the automatic sample injector is 15 +/-3 ℃;
needle washing liquid: (50 ± 5)% methanol;
washing speed: 30-40 μ L/sec;
volume of needle washing liquid: 400-600 μ L.
In some of these embodiments, the conditions of the mass spectrometry comprise:
ionization mode: an electrospray ion source; a positive ion mode; monitoring multiple reactions;
ion source parameters: air curtain air: 25 psi; ion source gas 1: 50 psi; ion source gas 2: 50 psi; ion source spray voltage: 5500 + -10V; ion source temperature: 550 +/-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:
tanshinone I: ion pairing: 277 → 249; de-clustering voltage: 120.0V; inlet voltage: 10.00V; outlet voltage: 25.00V; collision energy: 28.00 eV; residence time: 100.0 msec;
tanshinone IIA: ion pairing: 295 → 277; de-clustering voltage: 120.0V; inlet voltage: 10.00V; outlet voltage: 25.00V; collision energy: 21.00 eV; residence time: 100.0 msec;
salvianolic acid B: ion pairing: 717.3 → 519.2; de-clustering voltage: -50.0V; inlet voltage: -10.00V; outlet voltage: -16.00V; collision energy: -25.00 eV; residence time: 100.0 msec;
danshensu: ion pairing: 197 → 135; de-clustering voltage: -50.0V; inlet voltage: -10.00V; outlet voltage: -16.00V; collision energy: -23.00 eV; residence time: 100.0 msec;
notoginseng radix saponin R1: ion pairing: 935.3 → 755.5; de-clustering voltage: 120.0V; inlet voltage: 10.00V; outlet voltage: 25.00V; collision energy: 28.00 eV; residence time: 100.0 msec;
ginsenoside Rg1: ion pairing: 823.2 → 643.3; de-clustering voltage: 120.0V; inlet voltage: 10.00V; outlet voltage: 25.00V; collision energy: 45.00 eV; residence time: 100.0 msec;
ginsenoside Rb1: ion pairing: 1131.5 → 365.1; de-clustering voltage: 120.0V; inlet voltage: 10.00V; outlet voltage: 25.00V; collision energy: 53.00 eV; residence time: 100.0 msec;
carbamazepine: ion pairing: 237 → 1954; de-clustering voltage: 120.0V; inlet voltage: 10.00V; outlet voltage: 25.00V; collision energy: 25.00 eV; residence time: 100.0 msec.
In some of these embodiments, in step (3), the volume ratio of the methanol to the plasma sample or the homogenate is from 4 to 6: 1; in the sample pretreatment process, the amount of the precipitant is far larger than the amount of the plasma, the proportion of the sample injection solution system to the initial mobile phase is close, 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.
And/or, in the step (3), the temperature of the centrifugation is 3-5 ℃; the rotation speed of the centrifugation is 8000-12000 rpm; the centrifugation time is 8-12 min.
In some of these examples, the liquid chromatography mass spectrometer used was a Shimadzu LC30AD 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: xbridge BEH C18 UPLC Column. The specification is 1.7 μm, 2.1x100 mm; the Sciex Qtrap model 5500 mass spectrometer used in the invention has an order of magnitude higher sensitivity than that of the previous instrument.
In one embodiment, in step (2), in the mixed standard curve sample working solution, tanshinone I, tanshinone IIA, salvianolic acid B, tanshinol, and notoginsenoside R1Ginsenoside Rg1And Rb1Are 20.0ng/mL, 40.0ng/mL, 80.0ng/mL, 160ng/mL, 400ng/mL, 1600ng/mL, 4000ng/mL, 16000ng/mL, 32000ng/mL, and 40000ng/mL, respectively.
In one embodiment, in step (2), in the mixed quality control sample working solution, tanshinone I, tanshinone IIA, salvianolic acid B, tanshinol, and notoginsenoside R1Ginsenoside Rg1And Rb1The concentrations of (A) are respectively: the lower limit quality control of quantification is 20.0ng/mL, the low concentration quality control is 50.0ng/mL, the concentration quality control in geometric mean is 1000ng/mL, the concentration quality control in arithmetic mean is 10000ng/mL, and the high concentration quality control is 15000 ng/mL.
In some embodiments, the preparation method of the standard curve sample and the quality control sample in the high performance liquid chromatography tandem mass spectrometry detection method comprises the following steps:
adding the mixed standard curve sample working solution into blank plasma to prepare a standard curve sample; and adding the mixed quality control sample working solution into blank plasma to prepare a quality control sample.
In one embodiment, in the standard curve sample, tanshinone I, tanshinone IIA, salvianolic acid B, tanshinol, and notoginsenoside R1Ginsenoside Rg1And Rb1Are 1.00ng/mL, 2.00ng/mL, 4.00ng/mL, 8.00ng/mL, 20.0ng/mL, 80.0ng/mL, 200ng/mL, 800ng/mL, 1600ng/mL, and 2000ng/mL, respectively.
In one embodiment, the tanshinone I, the tanshinone IIA, the salvianolic acid B, the danshensu and the notoginsenoside R in the quality control sample1Ginsenoside Rg1And Rb1The concentrations of the two types of the test solution are respectively 1.00ng/mL of quantitative lower limit quality control, 2.50ng/mL of low concentration quality control, 50.0ng/mL of geometric mean medium concentration quality control, 500ng/mL of arithmetic mean medium concentration quality control and 750ng/mL of high concentration quality control.
In one embodiment, the plasma sample solution to be tested is prepared by: precisely sucking 50 μ L of plasma sample, sequentially adding 20 μ L (50ng/mL) of internal standard (carbamazepine) and 230 μ L of methanol, vortexing for 5min, mixing, centrifuging at 4 ℃ for 10min at 10000rpm, and sampling 200 μ L of supernatant for analysis; the preparation of the tissue sample solution to be detected comprises the following steps: thawing frozen viscera naturally at room temperature, cutting in ice bath, adding physiological saline according to a ratio of 1:2(w/v), homogenizing, precisely sucking 100 μ L of homogenate, sequentially adding 20 μ L (50ng/mL) of internal standard (carbamazepine) and 460 μ L of methanol, vortexing for 5min, mixing completely, centrifuging at 10000rpm and 4 ℃ for 10min, and sampling 200 μ L of supernatant for analysis.
In some embodiments, the compound red sage root preparation is prepared from raw materials including red sage root, notoginseng and borneol; the compound Saviae Miltiorrhizae radix preparation comprises compound Saviae Miltiorrhizae radix oral liquid, compound Saviae Miltiorrhizae radix aerosol, compound Saviae Miltiorrhizae radix buccal tablet, compound Saviae Miltiorrhizae radix capsule, compound Saviae Miltiorrhizae radix tablet, compound Saviae Miltiorrhizae radix granule, compound Saviae Miltiorrhizae radix dripping pill, compound Saviae Miltiorrhizae radix spray or compound Saviae Miltiorrhizae radix soft capsule.
In some of these embodiments, the plasma is human or animal plasma.
The present invention will be described in further detail with reference to specific examples.
Example 1
(1) Preparation of the solution
Preparation of stock solution:
weighing appropriate amount of Saviae Miltiorrhizae radix preparation, and making into pillTanshinone I, tanshinone IIA, salvianolic acid B, tanshinol, and notoginsenoside R1Ginsenoside Rg1Ginsenoside Rb and ginsenoside Rb1The standard substances of (1) are respectively dissolved in a proper amount of 80% methanol aqueous solution to prepare standard substance stock solutions with the concentration of 1.00mg/mL respectively. Seven standard substance stock solutions with the concentration of 1.00mg/mL are mixed in equal volume, and a proper amount of 80% methanol aqueous solution is added to prepare seven mixed standard substance stock solutions with the component concentration of 0.100 mg/mL.
Taking a proper amount of carbamazepine standard substance, and dissolving the standard substance in 80% methanol aqueous solution to prepare a carbamazepine stock solution with the concentration of 1.00 mg/mL.
Preparation of standard curve working solution:
diluting the seven mixed standard stock solutions with the concentrations of 0.100mg/mL by using 80% methanol aqueous solution to obtain mixed standard curve sample working solutions with the concentrations of 20.0ng/mL, 40.0ng/mL, 80.0ng/mL, 160ng/mL, 400ng/mL, 1600ng/mL, 4000ng/mL, 16000ng/mL, 32000ng/mL and 40000ng/mL respectively;
preparing a quality control working solution:
and diluting the seven mixed standard substance stock solutions with the concentrations of 0.100mg/mL by using 80% methanol aqueous solution to obtain mixed quality control sample working solutions with the concentrations of 20.0ng/mL of quantitative lower limit quality control, 50.0ng/mL of low concentration quality control, 1000ng/mL of geometric mean medium concentration quality control, 10000ng/mL of arithmetic mean medium concentration quality control and 15000ng/mL of high concentration quality control respectively.
The stock solution of carbamazepine was diluted with 80% aqueous methanol to make an internal standard working solution with a concentration of 50.0 ng/mL.
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 each component (tanshinone I, tanshinone IIA, salvianolic acid B, tanshinol, notoginsenoside R) of compound Saviae Miltiorrhizae radix preparation1Ginsenoside Rg1And Rb1) The concentrations of (A) are respectively: standard curves of 1.00ng/mL, 2.00ng/mL, 4.00ng/mL, 8.00ng/mL, 20.0ng/mL, 80.0ng/mL, 200ng/mL, 800ng/mL, 1600ng/mL and 2000ng/mLLine plasma samples.
Preparation of quality control plasma sample:
20.0. mu.L of the mixed quality control working solution is added to 380. mu.L of blank plasma and mixed uniformly. The obtained compound Saviae Miltiorrhizae radix preparation contains tanshinone I, tanshinone IIA, salvianolic acid B, tanshinol, and notoginsenoside R1Ginsenoside Rg1And Rb1) The concentrations of (A) are respectively: and (3) quantifying a quality control plasma sample with the lower limit quality control of 1.00ng/mL, the low concentration quality control of 2.50ng/mL, the concentration quality control in a geometric mean of 50.0ng/mL, the concentration quality control in an arithmetic mean of 500ng/mL and the high concentration quality control of 750 ng/mL.
(2) Sample pretreatment
Plasma sample pretreatment (preparation of test plasma sample solution): precisely sucking 50 mu L of rat plasma sample, sequentially adding 20 mu L (50ng/mL) of internal standard (carbamazepine) and 230 mu L of methanol, vortexing for 5min, fully and uniformly mixing, centrifuging at 4 ℃ for 10min at 10000rpm, taking 200 mu L of supernatant, and analyzing by sample injection.
Tissue distribution sample pretreatment (preparation of a tissue sample solution to be tested): organs (heart, liver, spleen, lung, kidney, brain) of rats were minced in ice bath and the ratio of 1: adding physiological saline at a ratio of 2(w/v), homogenizing, precisely sucking 100 μ L of homogenate, sequentially adding 20 μ L (50ng/mL) of internal standard (carbamazepine) and 460 μ L of methanol, vortexing for 5min, mixing, centrifuging at 10000rpm and 4 ℃ for 10min, collecting 200 μ L of supernatant, and analyzing by sample injection.
(3) Conditions of liquid chromatography
The instrument equipment comprises: shimadzu LC-30AD liquid chromatography system.
A chromatographic column: UPLC XBridge BEH C18 ultra performance chromatography column (1.7 μm, 2.1x100mm), Waters; the column temperature was set to 40 ℃ and the autosampler temperature was set to 15 ℃.
The mobile phase A is 0.1 percent formic acid aqueous solution; mobile phase B was 100% methanol. The total flow rate was 0.30 mL/min.
The mobile phase elution procedure was: 10% of mobile phase A-90% of mobile phase B in 0-0.5 min; 5% of mobile phase A to 95% of mobile phase B in 0.5-2.0 minutes; 90% of mobile phase A-10% of mobile phase B in 2-2.2 minutes; 2.2-3.0 minutes, 90% of mobile phase A-10% of mobile phase B; 90% of mobile phase A to 10% of mobile phase B for 2.4 to 3 minutes.
The mass spectrum switching valve program is: 0 to 1 minute, valve B (into waste); 1 to 2 minutes, a valve (into mass spectrum); 2 to 3 minutes, valve B (into waste).
Needle washing liquid: 50% methanol. The needle washing mode is external flushing; 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;
sample injection volume of 3.00 μ L, tanshinone I, tanshinone IIA, salvianolic acid B, tanshinol, and notoginsenoside R1Ginsenoside Rg1Ginsenoside Rb1And the retention time of IS (carbamazepine) IS 1.87, 1.92, 1.56, 0.62, 1.63, 1.65, 1.74 and 1.68min respectively.
(4) Conditions of Mass Spectrometry
The instrument equipment comprises: sciex Qtrap5500 Mass Spectrometry System.
Ionization mode: electrospray ion source (ESI); positive ion mode (Positive); multiple Reaction Monitoring (MRM).
Ion source parameters: air curtain gas (CUR): 25 psi; ion source Gas 1(Gas 1): 50 psi; ion source Gas 2(Gas 2): 50 psi; ion source spray voltage (IS): 5500V; ion source Temperature (TEM): 550 ℃; resolution Q1/Q3(Resolution Q1/Q3): Unit/Unit; collision gas (CAD): medium; pause time (MR Pause): 20 msec; the mass spectrum acquisition time was 3.00 min.
Monitoring ion pair quadrupole rod parameters:
tanshinone I: ion pairing: 277 → 249; de-clustering voltage: 120.0V; inlet voltage: 10.00V; outlet voltage: 25.00V; collision energy: 28.00 eV; residence time: 100.0 msec;
tanshinone IIA: ion pairing: 295 → 277; de-clustering voltage: 120.0V; inlet voltage: 10.00V; outlet voltage: 25.00V; collision energy: 21.00 eV; residence time: 100.0 msec;
salvianolic acid B: ion pairing: 717.3 → 519.2; de-clustering voltage: -50.0V; inlet voltage: -10.00V; outlet voltage: -16.00V; collision energy: -25.00 eV; residence time: 100.0 msec;
danshensu: ion pairing: 197 → 135; de-clustering voltage: -50.0V; inlet voltage: -10.00V; outlet voltage: -16.00V; collision energy: -23.00 eV; residence time: 100.0 msec;
notoginseng radix saponin R1: ion pairing: 935.3 → 755.5; de-clustering voltage: 120.0V; inlet voltage: 10.00V; outlet voltage: 25.00V; collision energy: 28.00 eV; residence time: 100.0 msec;
ginsenoside Rg1: ion pairing: 823.2 → 643.3; de-clustering voltage: 120.0V; inlet voltage: 10.00V; outlet voltage: 25.00V; collision energy: 45.00 eV; residence time: 100.0 msec;
ginsenoside Rb1: ion pairing: 1131.5 → 365.1; de-clustering voltage: 120.0V; inlet voltage: 10.00V; outlet voltage: 25.00V; collision energy: 53.00 eV; residence time: 100.0 msec;
carbamazepine: ion pairing: 237 → 1954; de-clustering voltage: 120.0V; inlet voltage: 10.00V; outlet voltage: 25.00V; collision energy: 25.00 eV; residence time: 100.0 msec.
1. Methodology validation
Specific attribute
The result shows that endogenous impurities in the rat blank plasma do not interfere the determination of the seven effective components, and the rat blank plasma has no chromatographic peak at the seven effective components.
② minimum quantitative limit and linear range
The lowest limit of quantification of seven compounds contained in the compound salvia miltiorrhiza preparation in rat plasma is 1.00ng/mL, the linear range is 1.00-2000ng/mL, and the R of the seven compounds2Between 0.9912 and 0.9961.
Accuracy and precision
Preparing quality control plasma samples with the quantitative lower limit quality control of 1.00ng/mL, the low concentration quality control of 2.50ng/mL, the geometric mean medium concentration quality control of 50.0ng/mL, the arithmetic mean medium concentration quality control of 500ng/mL and the high concentration quality control of 750ng/mL, analyzing each concentration of 6 samples, continuously measuring for 3 days, calculating the concentration of QC (quality control) samples according to the standard curve of the day, and obtaining the RE value and the RSD value of each concentration, wherein the specific values are shown in Table 2.
TABLE 2
Recovery rate
6 samples are prepared from low-concentration, medium-concentration and high-concentration quality control samples respectively, and 18 control samples (mixed matrixes) which do not contain the substance to be detected and contain the internal standard are extracted simultaneously. 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 substance to be detected: r (%). times.c/S, 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 3.
TABLE 3
2. The result of the detection
Measuring tanshinone I, tanshinone IIA, salvianolic acid B, tanshinol, and notoginsenoside R in rat plasma sample and tissue sample after rat oral gavage administration for 1 hr1Ginsenoside Rg1、Rb1The concentrations of (a) and (b) are shown in table 4, and the standard chromatograms of the seven compound standards and the carbamazepine standard are shown in fig. 1-8.
TABLE 4
| Plasma sample (ng/mL) | Tissue distribution samples (liver, ng/g) | |
| Tanshinone I | 6.98 | 23.7 |
| Tanshinone IIA | 32.3 | 193 |
| Salvianolic acid B | 673 | 0.00 |
| Salvianic acid A | 283 | 25.1 |
| Notoginseng radix saponin R1 | 88.1 | 86.2 |
| Ginsenoside Rg1 | 156 | 311 |
| Ginsenoside Rb1 | 3050 | 151 |
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, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (7)
1. A high performance liquid chromatography tandem mass spectrometry detection method of seven ingredient plasma or tissue concentrations of a compound radix salviae miltiorrhizae preparation is characterized in that the compound radix salviae miltiorrhizae preparation is prepared from raw materials including radix salviae miltiorrhizae, radix notoginseng and borneol; the method comprises the following steps:
(1) preparation of stock solutions:
taking standard tanshinone I, tanshinone IIA, salvianolic acid B, tanshinol, and notoginsenoside R1Ginsenoside Rg1And Rb1Respectively dissolving in 80 + -5% methanol water solution to obtain standard stock solution; mixing the standard substance stock solution to obtain a mixed standard substance stock solution;
dissolving carbamazepine in 80 +/-5% methanol water solution to prepare a carbamazepine stock solution;
(2) preparation of working solution:
diluting the mixed standard substance stock solution with 80 +/-5% methanol aqueous solution respectively to prepare a mixed standard curve sample working solution and a mixed quality control sample working solution;
diluting the carbamazepine stock solution by using 80 +/-5% methanol aqueous solution to prepare an internal standard working solution;
(3) preparing a sample solution to be tested:
preparation of a plasma sample solution to be tested: taking a plasma sample, adding an internal standard working solution and methanol, centrifuging, and taking obtained supernatant as a plasma sample solution to be detected;
preparing a solution of a tissue sample to be detected: taking a tissue sample, homogenizing, adding an internal standard working solution and methanol into the obtained homogenate, centrifuging, and taking the obtained supernatant as a tissue sample solution to be detected;
(4) performing liquid chromatography separation on the sample solution to be detected obtained in the step (3); performing mass spectrometry on the sample subjected to liquid chromatography separation;
the elution mode adopted by the liquid chromatographic separation is as follows: performing gradient elution by using 0.1 +/-0.02% formic acid aqueous solution as a mobile phase A and methanol as a mobile phase B; the liquid chromatographic column used was: XBridge BEH C18 UPLC Column;
the process of gradient elution includes:
0-0.5 min, (10 +/-5)% of mobile phase A- (90 +/-5) mobile phase B;
0.5-2.0 minutes, (5 +/-5)% of mobile phase A- (95 +/-5) of mobile phase B;
2-2.2 minutes, (90 +/-5)% of mobile phase A- (10 +/-5) mobile phase B;
2.2-3.0 min, (90 +/-5)% of mobile phase A- (10 +/-5) mobile phase B;
wherein the conditions of the mass spectrometry comprise:
ionization mode: an electrospray ion source; a positive ion mode; monitoring multiple reactions;
ion source parameters: air curtain air: 25 psi; ion source gas 1: 50 psi; ion source gas 2: 50 psi;
ion source spray voltage: 5500 + -10V;
ion source temperature: 550 +/-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.
2. The high performance liquid chromatography-tandem mass spectrometry detection method according to claim 1, wherein the chromatographic conditions of the liquid chromatography comprise:
flow rate: 0.3 plus or minus 0.1 mL/min;
sample introduction volume: 3.00 +/-0.5 mu L;
column temperature: 40 +/-5 ℃;
autosampler temperature: 15 +/-3 ℃;
needle washing liquid: 50 plus or minus 5% methanol water solution;
washing speed: 30-40 μ L/sec;
volume of needle washing liquid: 400-600 μ L.
3. The HPLC-MS/MS detection method of claim 1, wherein in said mass spectrometry, the quadrupole mass spectrometry conditions of each component are:
tanshinone I: ion pairing: 277 → 249; de-clustering voltage: 120.0V; inlet voltage: 10.00V; outlet voltage: 25.00V; collision energy: 28.00 eV; residence time: 100.0 msec;
tanshinone IIA: ion pairing: 295 → 277; de-clustering voltage: 120.0V; inlet voltage: 10.00V; outlet voltage: 25.00V; collision energy: 21.00 eV; residence time: 100.0 msec;
salvianolic acid B: ion pairing: 717.3 → 519.2; de-clustering voltage: -50.0V; inlet voltage: -10.00V; outlet voltage: -16.00V; collision energy: -25.00 eV; residence time: 100.0 msec;
danshensu: ion pairing: 197 → 135; declustering voltage: -50.0V; inlet voltage: -10.00V; outlet voltage: -16.00V; collision energy: -23.00 eV; residence time: 100.0 msec;
notoginseng radix saponin R1: ion pairing: 935.3 → 755.5; de-clustering voltage: 120.0V; inlet voltage: 10.00V; outlet voltage: 25.00V; collision energy: 28.00 eV; residence time: 100.0 msec;
ginsenoside Rg1: ion pairing: 823.2 → 643.3; de-clustering voltage: 120.0V; inlet voltage: 10.00V; outlet voltage: 25.00V; collision energy: 45.00 eV; residence time: 100.0 msec;
ginsenoside Rb1: ion pairing: 1131.5 → 365.1; de-clustering voltage: 120.0V; inlet voltage: 10.00V; outlet voltage: 25.00V; collision energy: 53.00 eV; stayTime: 100.0 msec;
carbamazepine: ion pairing: 237 → 194; de-clustering voltage: 120.0V; inlet voltage: 10.00V; outlet voltage: 25.00V; collision energy: 25.00 eV; residence time: 100.0 msec.
4. The method according to any one of claims 1 to 3, wherein in step (3), the volume ratio of the methanol to the plasma sample or the homogenate is from 4 to 6: 1;
and/or, in the step (3), the temperature of the centrifugation is 3-5 ℃; the rotating speed of the centrifugation is 8000-12000 rpm; the centrifugation time is 8-12 min.
5. The HPLC-MS/MS detection method of any one of claims 1-3, wherein the LC-MS/MS used is a Shimadzu LC30AD LC MS/MS combined with a Sciex Qtrap5500 mass spectrometer and the operating system used is Analyst 1.6.3.
6. The HPLC-MS/MS detection method of any one of claims 1-3, wherein said red sage root preparation is FUFANGDANSHEN oral liquid, FUFANGDANSHEN Aerosol, FUFANGDANSHEN Capsule, FUFANGDANSHEN tablet, FUFANGDANSHEN granule, FUFANGDANSHEN dripping pill, or FUFANGDANSHEN spray.
7. The high performance liquid chromatography-tandem mass spectrometry detection method of any one of claims 1 to 3, wherein the plasma is human or animal plasma; and/or the preparation method of the standard curve sample and the quality control sample in the high performance liquid chromatography tandem mass spectrometry detection method comprises the following steps:
adding the mixed standard curve sample working solution into blank plasma to prepare a standard curve sample; and adding the mixed quality control sample working solution into blank plasma to prepare a quality control sample.
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