CN112540138A - Combined quantitative determination method for salvianolic acid B, aspirin and salicylic acid in blood plasma - Google Patents
Combined quantitative determination method for salvianolic acid B, aspirin and salicylic acid in blood plasma Download PDFInfo
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Abstract
The invention discloses a combined quantitative determination method of salvianolic acid B, aspirin and salicylic acid in plasma, which comprises the steps of respectively drawing standard curves of the salvianolic acid B, the aspirin and the salicylic acid, and then determining chromatograms and chromatographic peak areas of the salvianolic acid B, the aspirin, the salicylic acid and an internal standard substance in the plasma to be determined by UPLC-MS/MS analysis; and calculating according to the respective standard curves to obtain the content of the salvianolic acid B, the aspirin and the salicylic acid in the plasma. The method is used for pharmacokinetics research, and when the medicines are jointly applied, the pharmacokinetic rules of all the medicines are determined, the medicine content is accurately determined, the separation degree is high, the determination method is sensitive, and the detection efficiency is high.
Description
Technical Field
The invention relates to a method for measuring the content of a drug in blood plasma, in particular to a method for jointly and quantitatively measuring the drug in a combined drug process, belonging to the field of pharmacokinetics.
Background
Before 3500 years, Sumeiren and ancient Egyptian used willow bark for treating hyperpyrexia and pain, and salicylic acid is the main effective component extracted from willow bark and having antipyretic and analgesic effects. In 1897, German chemists added an acetyl group to salicylic acid to reduce irritation of salicylic acid to gastrointestinal tract, to obtain acetylsalicylic acid (trade name aspirin), which was then gradually used in clinical treatment as antipyretic analgesic and antirheumatic. In 1971, Smith and Willis discovered that aspirin can inhibit cyclooxygenase 1(COX-1) irreversibly to produce an anti-platelet aggregation effect, and has a good application prospect in the aspect of preventing thrombosis. At present, aspirin has been widely used clinically as a primary and secondary prophylactic agent for cardiovascular and cerebrovascular diseases. The effects of secondary prevention using aspirin, particularly in myocardial infarction, stroke and vascular cause death, have been validated in the RCT test. Clinically, aspirin is often used as a basic drug in combination with other cardiovascular and cerebrovascular disease drugs.
The research shows that the salvia miltiorrhiza has very obvious effect on the cardiovascular system and is one of the most widely used traditional Chinese medicinal materials for clinically treating coronary heart disease at present. The salvia miltiorrhiza preparation and the aspirin both have the effect of resisting platelet aggregation, and clinical researches show that on the basis of conventional aspirin treatment, the tanshinone IIA sodium sulfonate injection can obviously relieve angina pectoris symptoms. The salvianolate injection is prepared from water-soluble effective components extracted from traditional Chinese medicine salvia miltiorrhiza, has the main component of salvianolic acid B (also called salvianolic acid B), has the effects of resisting platelet aggregation, resisting thrombosis, protecting myocardial cells, resisting oxidation, relieving atherosclerotic lesions and the like, is widely used for treating cardiovascular and cerebrovascular diseases such as angina pectoris, myocardial infarction and the like in clinic, and can also effectively prevent acute coronary artery events. At present, aspirin and salvianolate injection are clinically applied to treat cardiovascular and cerebrovascular diseases more commonly, but the research on the interaction relationship of the aspirin and the salvianolate injection in combined application is relatively less, and meanwhile, an in vivo quantitative method for supporting the research on the pharmacokinetics of the aspirin and the salvianolate injection is not reported.
High Performance Liquid Chromatography (HPLC) is one of the most common techniques for quantitative analysis of drugs, and is a chromatographic method for separating a sample by pumping a specified mobile phase into a chromatographic column filled with a filler by using a high-pressure infusion pump, has broad spectrum, and achieves the purpose of quantitative analysis of different types of compounds by combining different detectors, such as an ultraviolet detector, a fluorescence detector, a mass spectrometry detector and the like. Particularly, the liquid phase-mass spectrometry (LC-MS) analysis technology combined with a mass spectrometer is rapidly developed in the last decade, and shows the analysis advantages of high sensitivity, high flux and high selectivity when facing a biological sample with complex components, thereby providing a simple and rapid analysis method for analyzing and identifying metabolites in a medicament body and promoting the development of pharmacokinetics.
Ultra Performance Liquid Chromatography (UPLC) improves the system pressure and reduces the granularity of chromatographic column packing on the basic principle of a high performance liquid chromatography system, thereby improving the separation efficiency and speed. The UPLC-MS/MS is characterized in that on the basis of UPLC high-efficiency separation, the characteristic that the mass spectrometry can be used for accurately measuring the ion mass is utilized, a mother ion signal of an object to be detected is specifically collected, the selected mother ion is smashed, a daughter ion signal is specifically collected, and the interference of other ions is eliminated, is also called as a mass spectrometry multiple reaction monitoring technology (MRM), is one of the currently recognized complex sample analysis technologies with the best selectivity, and adds a plurality of assistance forces for the research of pharmacokinetics.
In the face of biological sample analysis, in addition to selecting an appropriate assay method, a pretreatment method is required to convert the sample into an easily analyzable form. The purpose of the pretreatment is to reduce a large amount of impurities in the matrix interfering with the components to be measured, and to make the sample meet the requirements of the analysis means to the maximum extent. The multiple pretreatment processes not only consume a large amount of time, but also can cause the loss of the object to be tested while removing impurities, and increase operation errors, so that the sample analysis process can be fast and stable by selecting a simple and convenient pretreatment method with high selectivity on the object to be tested, and the analysis efficiency and the repeatability are greatly improved.
Research shows that the detection method of aspirin and salicylic acid as a metabolite thereof in a biological sample mainly adopts LC-MS, and the pretreatment method mainly comprises the following steps: acidifying the sample, adding organic solvent protein precipitation or liquid-liquid extraction, or treating the sample by Solid Phase Extraction (SPE). The method for quantitatively analyzing salvianolic acid B in the biological sample mainly comprises LC-MS and also comprises HPLC-UV method. The pretreatment method mainly comprises acetonitrile precipitation, liquid-liquid extraction and SPE method. Compared with the former two methods, SPE has great advantages in detecting various target components simultaneously, is simple and convenient to operate, thoroughly treats impurities, and has relatively high cost. The liquid-liquid extraction method has high selectivity to the components to be detected, and the processed substrate is clean.
Particularly, aspirin and salicylic acid, which is a metabolite thereof, belong to high-polarity small molecules, the property is unstable, and when sample pretreatment is carried out, attention is paid to the fact that the aspirin and the metabolite thereof are degraded by biological enzymes contained in a matrix, so that it is very important to add a non-specific enzyme inhibitor or control an enzymolysis reaction by reducing the pH value and the temperature. In clinical application, aspirin is often used as a basic drug in combination with various drugs because cardiovascular and cerebrovascular diseases mostly coexist with other diseases and the clinical situation is complex. The complex physicochemical properties and unstable characteristics of aspirin and its metabolites need to be considered in developing a combined analysis method of multiple drugs, which puts high requirements on the stability and repeatability of the method development. The salvianolic acid B is easy to oxidize and has poor stability in blood plasma and tissue samples, and a proper antioxidant needs to be added during method development. In addition to the instability of salvianolic acid B, the selectivity of sample pretreatment methods for highly polar compounds needs to be considered when combined with other components for detection. At present, the joint quantitative method of salvianolic acid B, aspirin (aspirin) and salicylic acid as a metabolite in a biological sample is not reported yet.
In order to solve the problems, the invention develops a combined quantitative analysis method of salvianolic acid B, aspirin (aspirin) and salicylic acid which is a metabolite thereof in human plasma, and lays a foundation for the research of the combined medication of aspirin and a traditional Chinese medicine preparation containing salvianolic acid B.
Disclosure of Invention
The invention aims to provide a combined determination method for the content of salvianolic acid B, aspirin and salicylic acid metabolites thereof in blood plasma, which is used for pharmacokinetic research, and when drugs are combined, the pharmacokinetic rule of each drug is determined, the drug content determination is accurate, the separation degree is high, the determination method is sensitive and the detection efficiency is high, aiming at the technical problems of quantitative determination of salvianolic acid B, aspirin and salicylic acid metabolites thereof in biological samples in the combined medication process of the existing aspirin and the salvia miltiorrhiza preparations taking salvianolic acid B as a main component.
In order to achieve the purpose of the invention, the invention provides a combined quantitative determination method of salvianolic acid B, aspirin and salicylic acid in blood plasma, which comprises the following steps:
1) drawing a standard curve
1-1) mixing salvianolic acid B, aspirin and salicylic acid to prepare a mixed mother liquor, and diluting the mixed mother liquor with a methanol-water solution to obtain a series of working solutions with the concentration of 0.025-30 mu g/mL;
1-2) respectively taking 10 mu L of working solution, uniformly mixing with 40 mu L of blank plasma, 20 mu L of internal standard working solution and 300 mu L of ethyl acetate, then carrying out first centrifugation, drying the supernatant after the first centrifugation, and respectively re-dissolving the dried residues with 50 mu L of organic mixed solution; then respectively carrying out second centrifugation treatment, wherein the centrifuged supernatant is a sample solution for drawing a standard curve, and the concentrations of salvianolic acid B, aspirin and salicylic acid in the sample solution of the standard curve are respectively 5-6000 ng/mL;
1-3) respectively sampling the prepared standard curve sample solution, carrying out UPLC-MS/MS analysis, and recording chromatograms and chromatographic peak areas of salvianolic acid B, aspirin and salicylic acid in the standard curve sample solution; then respectively taking the ratio of the concentrations of the salvianolic acid B, the aspirin and the salicylic acid in the standard curve sample solution to the concentration of the internal standard as an abscissa and the ratio of the chromatographic peak areas of the salvianolic acid B, the aspirin, the salicylic acid and the internal standard as an ordinate, and performing regression operation by adopting a least square method to respectively draw standard curve equations of the salvianolic acid B, the aspirin and the salicylic acid;
2) plasma sample assay
Sampling a to-be-detected plasma sample, carrying out UPLC-MS/MS analysis, and recording chromatograms and chromatographic peak areas of salvianolic acid B, aspirin, salicylic acid and internal standard substances in the to-be-detected plasma sample; and then, respectively substituting the chromatographic peak area ratio of the aspirin, the salicylic acid and the salvianolic acid B in the plasma sample to be detected, the chromatographic peak area ratio of the internal standard substance and the concentration of the internal standard substance into a drawn standard curve equation of the aspirin, the salicylic acid and the salvianolic acid B, and calculating to obtain the concentrations of the aspirin, the salicylic acid and the salvianolic acid B in the plasma sample to be detected.
Wherein the concentrations of the salvianolic acid B, the aspirin and the salicylic acid mother liquor in the step 1-1) are 1mg/mL respectively.
Particularly, the salvianolic acid B, aspirin and salicylic acid mother liquor in the step 1-1) is prepared by the following method: accurately weighing standard substances of aspirin, salicylic acid and salvianolic acid B, dissolving the aspirin and the salicylic acid respectively by using acetonitrile, and diluting the aspirin and the salicylic acid respectively to obtain aspirin mother liquor and salicylic acid mother liquor with the concentration of 1 mg/mL; dissolving salvianolic acid B with methanol, and diluting to 1mg/mL salvianolic acid B mother liquor.
Particularly, the salvianolic acid B, aspirin and salicylic acid mother liquor in the step 1-1) are uniformly mixed according to the volume ratio of 1: 1 to prepare the mixed mother liquor.
In particular, the methanol-water solution has a methanol to water volume ratio of 80: 20.
Wherein the blank plasma in the step 1-2) is prepared by the following method: placing collected venous blood of healthy human or animal in a centrifuge for 1h after blood collection for centrifugal treatment, and marking obtained supernatant as blank plasma. The blank plasma does not contain the substance to be detected, and the blank plasma is stored in a refrigerator at the temperature of not higher than-70 ℃ for standby. Venous blood was collected from animals or humans (provided voluntarily). Usually, the anticoagulant to be used is EDTA K2The vein whole blood of healthy human or animal collected by the vacuum negative pressure tube is placed in a centrifuge for centrifugal treatment (2-8 ℃, 3000-.
In particular, the internal standard working solution is a chloramphenicol solution.
In particular, the concentration of the chloramphenicol solution as the internal standard working solution is 10 to 200ng/mL, preferably 50 ng/mL.
In particular, the internal standard working solution is prepared according to the following method: dissolving and diluting a precision-weighed chloramphenicol standard substance into 1mg/mL chloramphenicol internal standard mother liquor by using acetonitrile; the working solution was then diluted with formic acid-water solution to a final concentration of 50ng/mL chloramphenicol as an internal standard working solution, wherein the formic acid-water solution had a volume ratio of formic acid to water of 50/50 (v/v).
Wherein, the conditions of the first and second centrifugal treatments are 2-8 ℃; 10000-; at least 10 min.
In particular, the organic mixed solution is a mixed solution of methanol, acetonitrile and water.
Particularly, the volume ratio of the methanol to the acetonitrile to the water is (1-80) to (20-40), and the preferred volume ratio is 40 to 20.
In particular, the drying treatment is to blow dry the supernatant after the first centrifugation treatment with nitrogen at room temperature.
Wherein, in the UPLC-MS/MS analysis process in the step 1-3), the chromatographic conditions measured by liquid chromatography (UPLC) are as follows: mobile phase: 0.5% formic acid (a) -acetonitrile (B).
In particular, during the liquid chromatographic separation, a mobile phase selective gradient elution: 0-0.3min, 95% -75% A; 0.3-1.3min, 75% -70% A; 1.3-2.1min, 70% -30% A; 2.1-2.5min, 30% A; 2.5-2.7min, 30% -95% A; 2.7-4.0min, 95% A.
In particular, the liquid chromatography separation conditions are: column temperature: 30-60 ℃, preferably 50 ℃; flow rate: 0.2-0.5mL/min, preferably 0.4 mL/min; sample introduction: 2 μ L.
In particular, the liquid chromatograph employs a reverse phase chromatography column as a separation medium; the reverse phase chromatographic column is a reverse phase C8 or C18 chromatographic column.
In particular, the reverse phase chromatography column was selected for ACQUITY UPLC BEH C18(1.7 μm, 2.1 x 100 mm).
In the UPLC-MS/MS analysis process in the step 1-3), the mass spectrometry detection adopts an electrospray ionization ion source to ionize liquid phase eluent, and adopts an anion multiple reaction ion monitoring scanning mode to detect the contents of the salvianolic acid B, aspirin, salicylic acid and internal standard in the plasma sample.
In particular, the electrospray ion source (ESI) employs a negative ion multiple reactive ion monitoring (MRM) scan mode in which the collision Gas (CAD) Is 6psi, the curtain Gas (CUR) Is 15psi, the atomizing Gas (Gas1) Is 18psi, the supplemental heating Gas (Gas2) Is 5psi, the spray voltage (Is) Is 4000V, the atomizing Temperature (TEM) Is 400 ℃, the inlet voltage (EP) Is 10V, and the collision cell outlet voltage (CXP) Is 17V.
Particularly, the standard curve equations of the salvianolic acid B and the aspirin in the step 1-3) are unary linear equations, and the linear quantitative range of the salvianolic acid B and the aspirin is 5-6000 ng/ml; the salicylic acid standard curve equation is a quadratic equation with the quantitative range of 5-6000 ng/ml.
Wherein, the blood plasma sample to be detected in the step 2) is prepared according to the following method: uniformly mixing the plasma to be detected with an internal standard working solution and an organic solvent, and then carrying out first centrifugal treatment; re-dissolving the residue of the supernatant after the first centrifugation with organic mixed solution; and then carrying out second centrifugation treatment, wherein the supernatant obtained after the second centrifugation treatment is the plasma sample to be detected.
In particular, the test plasma is prepared according to the following method: and (3) carrying out centrifugal treatment on the collected venous blood within 1h after blood collection, and taking a centrifugal treatment supernatant, namely the blood plasma to be detected.
In particular, the venous blood is venous whole blood collected using an anticoagulant vacuum negative pressure tube.
In particular, the anticoagulant is EDTAK2、EDTA K3Heparin sodium, preferably EDTA K2。
Particularly, the centrifugal processing condition in the preparation process of the blood plasma to be detected is 2-8 ℃, 3000 and 5000rpm, preferably 4500 rpm; at least 10 min; storing the blood plasma to be detected in a refrigerator at the temperature of not higher than-70 ℃ for later use.
The volume usage of the to-be-detected plasma and the internal standard working solution is that 20 mu L of the internal standard working solution is added into every 50 mu L of the to-be-detected plasma.
Particularly, the conditions of the first and second centrifugation treatments are 2-8 ℃; 10000-; at least 10 min.
In particular, the organic solvent is selected from ethyl acetate, petroleum ether, n-hexane, dichloromethane or chloroform, preferably ethyl acetate; the organic mixed solution is a mixed solution of methanol, acetonitrile and water; wherein the volume ratio of methanol to acetonitrile to water is as follows: 1-80: 20-40; preferably 40: 20 (v/v/v).
In particular, the plasma sample to be tested in the step 2) is prepared according to the following method:
2A) mixing 50 μ L of the plasma to be tested with 20 μ L of the internal standard working solution uniformly (usually by vortexing for 30 s); then 300 μ L ethyl acetate was added and mixed well (usually vortexed for 2 min); then placing the mixture in a centrifuge for first centrifugal treatment;
2B) taking all the centrifuged supernatant, drying the supernatant by using nitrogen at room temperature, re-dissolving the residue by using 50 mu L of organic mixed solution, and then placing the re-dissolved residue in a centrifuge for secondary centrifugation treatment; and the supernatant after centrifugation is the plasma sample to be detected.
The conditions of the liquid chromatogram and the mass spectrum detection of the UPLC-MS/MS analysis in the step 2) are the same as those in the step 1-3).
In particular, it also comprises a step 1A): and QC quality inspection, wherein the QC quality inspection comprises the following steps:
1A-1) mixing mother liquor of salvianolic acid B, aspirin and salicylic acid to prepare mixed mother liquor, and diluting with methanol-water solution to obtain QC working solution with concentration of 0.075-22.5 μ g/mL;
1A-2) respectively taking 10 mu L of QC working solution, respectively uniformly mixing with 40 mu L of blank plasma, 20 mu L of internal standard working solution and 300 mu L of ethyl acetate, performing first centrifugation, drying supernatant after the first centrifugation, and respectively re-dissolving dried residues with 50 mu L of organic mixed liquor; then respectively carrying out second centrifugation treatment, wherein the centrifuged supernatant is a QC sample solution, and the concentrations of salvianolic acid B, aspirin and salicylic acid in the QC sample solution are respectively 15-4500 ng/mL;
1A-3) respectively sampling QC sample solutions with different concentrations, carrying out UPLC-MS/MS analysis, and recording chromatograms and chromatographic peak areas of salvianolic acid B, aspirin and salicylic acid in the QC sample solutions; then, respectively substituting the ratio of the chromatographic peak areas of the aspirin, the salicylic acid and the salvianolic acid B in the QC sample solution to the chromatographic peak area of the internal standard and the concentration of the internal standard substance into the standard curve equation of the aspirin, the salicylic acid and the salvianolic acid B drawn in the step 2), and calculating to obtain the concentrations of the aspirin, the salicylic acid and the salvianolic acid B in the QC sample solution;
1A-4) comparing the calculated concentration of aspirin, salicylic acid and salvianolic acid B in the QC sample solution with the real concentration of aspirin, salicylic acid and salvianolic acid B in the QC sample solution, and calculating the accuracy of the QC sample; the accuracy of the components aspirin, salicylic acid and salvianolic acid B in the QC sample solution is 85-115% of a standard curve for determining the concentration of the plasma sample.
Wherein, the salvianolic acid B, the aspirin and the salicylic acid mother liquor in the step 1A-1) are uniformly mixed according to the volume ratio of 1: 1.
In particular, the methanol-water solution of step 1A-1) has a methanol to water volume ratio of 80: 20.
Particularly, the concentrations of the salvianolic acid B, the aspirin and the salicylic acid mother liquor are respectively 1 mg/mL.
Wherein, the concentrations of the salvianolic acid B, the aspirin and the salicylic acid in the QC sample solution in the step 1A-3) are respectively 15ng/mL, 3000ng/mL and 4500 ng/mL.
Particularly, the accuracy of the QC sample in the step 1A-4) is the ratio of the concentrations of aspirin, salicylic acid, and salvianolic acid B in the QC sample solution to the actual concentrations of aspirin, salicylic acid, and salvianolic acid B in the QC sample solution, which are calculated according to the standard curve equation.
The invention develops a combined quantitative analysis method of salvianolic acid B, aspirin and salicylic acid as a metabolite in plasma based on UPLC-MS/MS technology, and fills the blank of the existing 3-component in-vivo quantitative method.
Compared with the prior art, the invention has the following advantages:
the method of the invention has accurate measurement results of the contents of the salvianolic acid B, the aspirin and the salicylic acid in the plasma, provides technical and theoretical supports for researching the combined medication of the aspirin and the salvianolic acid B as the main component, provides a technical method for researching the pharmacokinetics of the combined medicine, clearly knows the blood concentration of the metabolism of the medicine in the body and guides the combined medication.
The method has high practicability and wide measurement range, and the blood concentration in vivo measured by the method has wide range, wherein the concentration ranges of the salvianolic acid B, the aspirin and the salicylic acid are respectively as follows: 5-6000ng/mL salvianolic acid B; 5-6000ng/mL aspirin; 5-6000ng/mL of salicylic acid.
In the method, the liquid-liquid extraction method is used in the pretreatment process of the plasma, the operation is simple and rapid, the operation error is reduced, and the treated sample has good parallelism and stability; the UPLC-MS/MS analysis method has high sensitivity and high speed, and is suitable for the detection and analysis of a large number of samples supporting pharmacokinetics.
The method is a combined quantitative analysis method for salvianolic acid B, aspirin (acetylsalicylic acid) and salicylic acid as a metabolite in vivo plasma, wherein the preparation of a working solution, the pretreatment of a sample, UPLC chromatographic conditions and mass spectrum conditions are important characteristic parameters. The method carries out methodology verification by a method of 'biological sample quantitative analysis method verification guiding principle' in 2015 edition of Chinese pharmacopoeia, and the verification result meets the requirements of the guiding principle, so that the method can be used for measuring actual samples.
Drawings
FIG. 1A is a MRM chromatogram of aspirin, salicylic acid, salvianolic acid B, and chloramphenicol (internal standard) in blank plasma;
FIG. 1B is a chromatogram of plasma MRM of aspirin, salicylic acid, salvianolic acid B, and chloramphenicol (internal standard) containing 15ng/mL of each drug component and 20ng/mL of internal standard, wherein in FIGS. 1A and 1B, a is aspirin; b is salicylic acid; c is salvianolic acid B; d is chloramphenicol (internal standard).
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
Test example 1
1. Preparation of working solution:
1.1 preparation of mother liquor:
accurately weighing standard substances of aspirin, salicylic acid and salvianolic acid B by using an analytical balance, dissolving the standard substances by using organic solvent solutions respectively, and diluting the standard substances to obtain aspirin mother liquor, salicylic acid mother liquor and salvianolic acid B mother liquor with final concentration of 1mg/mL, wherein the aspirin and the salicylic acid are dissolved and diluted by using acetonitrile; dissolving salvianolic acid B in methanol, and diluting.
1.2 preparation of series working solution and QC (Quality Control) working solution:
mixing salvianolic acid B, aspirin and salicylic acid mother liquor according to the volume ratio of 1: 1 to prepare mixed mother liquor, and then respectively diluting the mixed mother liquor into working solutions with a series of concentrations and QC working solutions with 3 concentrations by using methanol to water (80/20, v/v) solutions.
Mixing 10 μ L serial working solution with 40 μ L blank plasma to prepare serial standard curve sample solutions, wherein the concentrations of salvianolic acid B, aspirin, and salicylic acid in the standard curve sample solutions are shown in Table 1;
mixing 10 μ L of QC working solution with 40 μ L of blank plasma to prepare QC sample solution, wherein the concentrations of salvianolic acid B, aspirin and salicylic acid in the QC sample solution are shown in Table 1, and 15ng/mL is low concentration of the QC sample solution (expressed as LQC); 3000ng/mL expressed as MQC; 4500ng/mL is expressed as HQC.
Wherein the blank plasma is plasma without analyte, and the anticoagulant is EDTA K2Placing the collected vein whole blood of healthy human or animal in a centrifuge for centrifugation (2-8 ℃, 3000-; storing blank plasma in refrigerator at-70 deg.C or below for use. Venous blood was collected from animals or humans (provided voluntarily).
Anticoagulant except EDTAK2In addition, other anticoagulants useful in anticoagulation are suitable for use in the present invention.
TABLE 1 Standard Curve sample and quality control sample concentrations of the ingredients
1.3 preparation of internal standard working solution:
precisely weighing a chloramphenicol standard substance, dissolving acetonitrile, and diluting to obtain 1mg/mL chloramphenicol internal standard mother liquor; the working solution was then diluted with formic acid/water (50/50, v/v) to a final concentration of 50ng/mL of chloramphenicol as an internal standard for use.
2. Sample measuring method
2.1 sample plasma separation:
the anticoagulant is EDTA K2Storing venous blood collected by vacuum negative pressure tube in ice bath, centrifuging in centrifuge at 2-8 deg.C (4500 rpm for 10min) for 1 hr, collecting supernatant (blood plasma), and storing in refrigerator at no higher than-70 deg.C. Venous blood was collected from animals or humans (provided voluntarily).
2.2 plasma sample pretreatment:
taking 50 mu L of supernatant plasma after centrifugation according to the method of separating 2.1 sample plasma, adding 20 mu L of internal standard working solution (50ng/mL of chloramphenicol internal standard working solution), and vortexing for 30 s; add 300. mu.L ethyl acetate, vortex for 2 min; centrifuging in a centrifuge (2-8 deg.C, 12000rpm, 10 min);
drying all the upper layer solution with nitrogen at room temperature, re-dissolving the residue with 50 μ L of mixed solution of methanol, acetonitrile and water (40/40/20, v/v/v), vortexing for 2min, and centrifuging in a centrifuge (2-8 deg.C, 12000rpm, 10 min); centrifuging the supernatant to obtain a pretreated plasma sample, wherein the content of chloramphenicol (internal standard) in the pretreated plasma sample is 20 ng/mL.
2.3UPLC (Ultra Performance Liquid Chromatography) chromatographic conditions:
the type of the chromatograph: acquity I-class (waters)
The type of the chromatographic column: ACQUITY UPLC BEH C18(1.7 μm, 2.1 × 100 mm);
mobile phase: 0.5% formic acid (a) -acetonitrile (B);
gradient elution: 0-0.3min, 95% -75% A; 0.3-1.3min, 75% -70% A; 1.3-2.1min, 70% -30% A; 2.1-2.5min, 30% A; 2.5-2.7min, 30% -95% A; 2.7-4.0min, 95% A;
column temperature: 50 ℃; flow rate: 0.4 mL/min; sample introduction: 2 μ L.
2.4 Mass Spectrometry conditions:
the mass spectrometer model: triple QuadTM5500(AB Sciex)
Using electrospray ion source (ESI), using negative ion multiple reactive ion monitoring (MRM) scan mode, 6psi collision Gas (CAD), 15psi air curtain Gas (CUR), 18psi atomizing Gas (Gas1), 5psi auxiliary heating Gas (Gas2), 4000V atomizing voltage (Is), 400 ℃ atomizing Temperature (TEM), 10V inlet voltage (EP), 17V collision cell outlet voltage (CXP), ion pair, collision voltage (CE), and declustering voltage (DP) information for each component as in table 2 below:
TABLE 2 Mass Spectrometry conditions for Aspirin, salicylic acid, Salvianolic acid B and Chloramphenicol (internal standards)
3. And (3) verification of methodology:
the methodology verification is carried out by adopting a method of 'biological sample quantitative analysis method verification guiding principle' of the year version of Chinese pharmacopoeia 2015, and the result proves that the analysis method is stable and reliable and can be used for measuring actual samples.
3.1 specificity
3.1.1 blank plasma is plasma without the substance to be detected, namely, the whole blood of a healthy person is collected by adopting an anticoagulant blood collection tube, and a blank plasma sample is obtained according to the operation of '2.1 sample plasma separation'.
3.1.2 mix 10 μ L of aqueous solution with 40 μ L of blank plasma sample, replace the internal standard solution with formic acid aqueous solution (50/50, v/v) without internal standard, then proceed according to "2.2 plasma sample pretreatment", then take 2 μ L of supernatant after pretreatment centrifugation (pretreated plasma sample), sample injection, UPLC-MS/MS analysis, get blank plasma aspirin, salicylic acid, salvianolic acid B, chloramphenicol (internal standard) chromatogram, as shown in FIG. 1A.
3.1.3 mixing 10 μ L of QC working solution with 40 μ L of blank plasma sample to prepare QC sample solution (50 μ L, wherein the concentration of the drug to be detected is 15ng/mL respectively); then, the operation is carried out according to the '2.2 plasma sample pretreatment'; then 2 μ L of the supernatant (pretreated plasma sample) after pretreatment and centrifugation is taken, sample introduction is carried out, UPLC-MS/MS analysis is carried out, and chromatograms of aspirin, salicylic acid, salvianolic acid B and chloramphenicol (internal standard) in the plasma are obtained, as shown in figure 1B.
The result shows that endogenous substances in human plasma do not interfere with the detection of the target component under the UPLC-MS/MS detection condition. The retention times were respectively: aspirin 2.08min, salicylic acid 2.40min, salvianolic acid B1.93min, and chloramphenicol 2.32 min. Test results show that under the experimental conditions of the invention, aspirin, salicylic acid, salvianolic acid B and chloramphenicol can be completely separated, endogenous substances in plasma cannot interfere with the detection of substances to be detected (aspirin, salicylic acid and salvianolic acid B) and internal standard substances (chloramphenicol), and the specificity of the method is good.
3.2 Standard Curve
Mixing 10 mu L of series working solution with 40 mu L of blank plasma to prepare a series of standard curve sample solutions, wherein the concentration of each drug to be detected in the series of standard curve sample solutions is shown in Table 1; then, the operation is carried out according to the '2.2 plasma sample pretreatment'; then sucking the supernatant (2 mu L) after the pretreatment and centrifugation, injecting a sample, and carrying out UPLC-MS/MS analysis;
taking the ratio of the concentration of the drug to be detected (salvianolic acid B, aspirin and salicylic acid) to the concentration of the internal standard (chloramphenicol) in the plasma as a horizontal coordinate, taking the ratio of the chromatographic peak area of the drug to be detected to the internal standard as a vertical coordinate, performing regression operation by adopting a least square method, and calculating to obtain a regression equation which is a standard curve. The regression equation is shown in table 3, the r values of the regression equation of the drugs to be tested in table 3 are all greater than 0.99, the verification guiding principle of the quantitative analysis method of the biological sample is met, and the regression equation can be used for calculating the content of the components to be tested in a linear range, wherein: the linear range of standard curves of salvianolic acid B, aspirin and salicylic acid is 5-6000ng/mL, and the lower limit of the quantification is 5 ng/mL.
TABLE 3 regression equation and Linear Range for Aspirin, salicylic acid, Salvianolic acid B
3.3 precision and accuracy
Mixing blank plasma with quantitative lower limit working solution and low, medium and high 3 concentration QC working solution respectively to prepare quantitative lower limit sample solution (LLOQ: 5ng/ml) and low, medium and high QC sample solution (LQC, MQC and HQC), preparing 6 parts of each concentration in parallel, performing 3 batches in total, and performing operation according to '2.2 plasma sample pretreatment'; then, sample injection, UPLC-MS/MS analysis and determination are carried out.
The lower limit of quantitation and the concentration of the analyte in the QC sample were calculated from the standard curve, and then the accuracy, defined as (measured/true) x 100%, and the precision, defined as the relative standard deviation (RSD%) of the measured values, were calculated. The results of the precision measurement of the contents of the drugs to be measured in the plasma are shown in tables 4, 5 and 6.
The measurement results show that: the precision of the aspirin, the salicylic acid and the salvianolic acid B at each concentration point in batch and between batches is less than 10 percent, the accuracy is between 85 and 115 percent and less than +/-15 percent, and the method conforms to the verification guide principle of the quantitative analysis method of the biological sample.
3.4 extraction recovery
3.4.1 mixing the blank plasma with QC working solution, preparing QC sample solution with low, medium and high concentration points (as shown in Table 1), preparing 6 parts of each concentration in parallel, and operating according to '2.2 plasma sample pretreatment'; then sample introduction is carried out, UPLC analysis is carried out, and the peak area (A) of the drug to be detected is determined;
3.4.2 taking another group of blank plasma, performing operation according to '2.2 plasma sample pretreatment', then respectively and correspondingly adding low, medium and high concentration QC working solutions, then injecting a sample, and performing UPLC-MS/MS analysis to obtain the peak area (B) of the drug to be detected;
3.4.3 calculate the extraction recovery, wherein: the extraction recovery rate is a/B × 100%.
The extraction recoveries for each concentration were averaged and the average extraction recovery measurements are shown in table 7, where: the average extraction recovery rate of aspirin is 93.2-115.3%, the average extraction recovery rate of salicylic acid is 94.6-117.5%, and the average extraction recovery rate of salvianolic acid B is 68.3-88.2%.
TABLE 7 measurement results of average extraction recovery rates of aspirin, salicylic acid, and salvianolic acid B
3.5 matrix Effect
Taking 6 blank plasma samples from different sources, operating according to '2.2 plasma sample pretreatment', then respectively and correspondingly adding low, medium and high concentration QC working solutions to prepare low, medium and high QC sample solutions, wherein the concentrations of the low, medium and high QC sample solutions are respectively 15, 3000 and 4500ng/mL, then carrying out sample injection analysis, and respectively determining peak areas (C) of the drug to be detected and the interior label chloramphenicol;
mixing the water solution instead of blank plasma with low, medium and high concentration QC working solutions, then referring to '2.2 plasma sample pretreatment', then carrying out sample injection analysis, respectively determining peak areas (D) of a drug to be detected and an internal standard, and calculating a matrix effect factor and an internal standard normalization matrix effect factor, wherein:
the matrix effect factor is C/D multiplied by 100%, and the internal standard normalization matrix effect factor is the matrix effect factor of the substance to be detected/the internal standard matrix effect factor multiplied by 100%.
The matrix effect measurement results of aspirin, salicylic acid and salvianolic acid B are shown in Table 8, and the internal standard normalized matrix effect variation coefficients of the aspirin, the salicylic acid and the salvianolic acid B are all less than 10%, so that the method conforms to the verification guide principle of the quantitative analysis method of the biological sample.
TABLE 8 determination of aspirin, salicylic acid, and salvianolic acid B matrix effect
3.6 stability
This verification investigates the stability of 3 storage conditions:
(1) long-term stability for 60 days: mixing blank plasma with low, medium and high concentration QC working solution respectively, storing at-70 deg.C for 60 days, performing plasma sample pretreatment according to 2.2, and analyzing by sample injection;
(2)4h room temperature stability: mixing blank plasma with low, medium and high concentration QC working solutions respectively, standing at room temperature for 4h, performing pretreatment according to a 2.2 plasma sample, and then performing sample injection analysis;
(3) and (3) placing in a sample injector at 4 ℃ for 24h for stabilization: the blank plasma was mixed with low, medium and high concentration QC working solutions, respectively, and after reference to "2.2 plasma sample pretreatment", the samples were placed in a sample injector for 24h at 4 ℃ and then analyzed by sample injection.
After the above QC samples were determined, the content calculation was performed using a freshly prepared standard curve. The measurements were averaged over 6 replicates per storage condition. The determination results are shown in tables 9, 10 and 11, wherein the stability accuracy of aspirin, salicylic acid and salvianolic acid B is between 85 and 115 percent and less than +/-15 percent, and the determination results accord with the verification guide principle of the quantitative analysis method of the biological sample.
TABLE 9 Long-term stability results of 60 days for Aspirin, salicylic acid, Salvianolic acid B
TABLE 10 results of stability of aspirin, salicylic acid, salvianolic acid B4 h at room temperature
TABLE 11 results of stability of aspirin, salicylic acid, salvianolic acid B in a sample injector at 4 deg.C for 24h
Example 1
1. Preparation of plasma
Healthy adult Beagle dog, male, body weight 10-14kg, after gavage aspirin enteric-coated tablet (100 mg/tablet, Bayer medicine health promotion Co., Ltd.), injecting salvianolate injection (200 mg/tablet, Shanghai green grain pharmacy Co., Ltd.) into vein, respectively before administration, 15min, 30min, 45min, 1h, 2h, 4h, 8h, 12h, 24h before administration, taking 2mL of venous blood, placing in EDTA K2Storing in ice bath in anticoagulation tube, centrifuging in centrifuge (2-8 deg.C, 4500rpm, 10min) in 1h, collecting supernatant (blood plasma), and storing in refrigerator at no higher than-70 deg.C.
Wherein, the supernatant obtained by centrifuging venous blood before administration is plasma without medicine and blank plasma; the supernatant after the venous blood is centrifuged after administration is the drug-containing plasma.
2. Drawing a standard curve
2.1 preparation of mother liquor:
the same procedure as in "preparation of 1.1 mother liquor" in test example 1 was repeated.
2.2 preparation of internal standard working solution:
the procedure was as in test example 1 "preparation of 1.3 internal standard working solution".
2.3 preparation of Standard Curve sample solution and QC (Quality Control) sample solution
The procedure was the same as in test example 1, except that "preparation of 1.2 standard curve sample solution and QC (Quality Control) sample solution" was carried out.
Respectively mixing 10 mu L of working solution with serial concentration with 40 mu L of blank plasma, respectively adding 20 mu L of internal standard working solution, and vortexing for 30 s; add 300. mu.L ethyl acetate, vortex for 2 min; centrifuging in a centrifuge (2-8 deg.C, 12000rpm, 10 min); drying all the upper layer solution with nitrogen at room temperature, re-dissolving the residue with 50 μ L of mixed solution of methanol, acetonitrile and water (40/40/20, v/v/v), vortexing for 2min, and centrifuging in a centrifuge (2-8 deg.C, 12000rpm, 10 min); the centrifuged supernatant was the standard curve sample solution, and the concentration of the standard curve sample solution was as shown in table 2.
And mixing 10 mu L of low, medium and high concentration QC working solutions with 40 mu L of blank plasma respectively, processing according to the processing method of the standard curve sample solution, centrifuging to obtain QC sample solution, wherein the concentration of the QC sample solution is shown in the table 2, and preparing 2 parts of QC sample solution with each concentration in parallel.
2.4 preparation of Standard Curve sample solutions
2.4.1, 2 μ L of the prepared standard curve sample solution is sampled and subjected to UPLC-MS/MS analysis under the same analysis conditions as "2.3 UPLC chromatographic conditions" and "2.4 mass spectrometric conditions" in test example 1, and the chromatogram and the chromatographic peak area of each component in the standard curve sample solution are recorded.
2.4.2 taking the ratio of the concentration of the drug to be detected (salvianolic acid B, aspirin and salicylic acid) to the concentration of the internal standard (chloramphenicol) in the standard curve sample solution as the abscissa and the ratio of the chromatographic peak area of the drug to be detected to the internal standard as the ordinate, performing regression operation by adopting a least square method, and calculating to obtain a regression equation which is a standard curve, wherein the standard curves of all components are shown in Table 12.
TABLE 12 regression equation for aspirin, salicylic acid, salvianolic acid B
3. QC detection
And (3) respectively taking 2 mu L of the prepared QC sample solution for sample injection, carrying out UPLC-MS/MS analysis under the same analysis conditions as the 2.3UPLC chromatographic conditions and the 2.4 mass spectrum conditions in the test example 1, and recording the chromatogram and the chromatographic peak area of each component in the QC sample solution.
And (3) respectively substituting the ratio of the chromatographic peak area of each measured component to the chromatographic peak area of the internal standard into a drawn standard curve equation (namely the regression equation in the table 12) of aspirin, salicylic acid and salvianolic acid B, calculating the concentration of each component in the QC sample solution, comparing the concentration with the real concentration value of the QC sample solution, and calculating the accuracy of the QC sample, wherein the measurement result is shown in the table 13.
TABLE 13 plasma sample concentrations and QC sample concentrations
From the measurement results in table 13, it is found that: the accuracy of the QC sample is between 85 and 115 percent, which shows that the drawn standard curve has accurate determination result and stable analysis method.
4. Sample assay
Accurately measuring 50 mu L of plasma samples prepared in the step 1 and the step for preparing plasma respectively, adding 20 mu L of internal standard working solution (50ng/mL of chloramphenicol internal standard working solution) respectively, and vortexing for 30 s; add 300. mu.L ethyl acetate, vortex for 2 min; centrifuging in a centrifuge (2-8 deg.C, 12000rpm, 10 min);
drying all the upper layer solution with nitrogen at room temperature, re-dissolving the residue with 50 μ L of mixed solution of methanol, acetonitrile and water (40/40/20, v/v/v), vortexing for 2min, and centrifuging in a centrifuge (2-8 deg.C, 12000rpm, 10 min); and (4) centrifuging to obtain supernatant as a plasma sample to be detected.
2 mu L of the prepared plasma sample to be detected is sampled and subjected to UPLC-MS/MS analysis, the analysis conditions of the sample are the same as the '2.3 UPLC chromatographic conditions' and '2.4 mass spectrum conditions' of the test example 1, and the chromatogram and the chromatographic peak area of each component in the plasma sample to be detected are recorded.
And (3) respectively substituting the ratio of the chromatographic peak area of each measured component to the chromatographic peak area of the internal standard into the standard curve equation (namely the regression equation in the table 12) of the drawn aspirin, the salicylic acid and the salvianolic acid B, and calculating to obtain the concentration of each component in the plasma sample to be measured, wherein the measurement result is shown in the table 14.
And (3) alternating the measurement steps of preparing the concentration of the QC sample in the standard curve sample solution and the QC sample solution in the sample measurement process, namely uniformly distributing the QC sample solution in the measurement process of the plasma sample, measuring the concentration of aspirin, salicylic acid and salvianolic acid B in the QC sample solution, detecting the accuracy of the QC sample, and ensuring the accuracy of the measurement result of the sample.
TABLE 14 concentration of each component in plasma samples
BLQ indicates that the detection value is below the lower limit of quantitation.
The above-described embodiments of the present invention are merely exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (9)
1. A combined quantitative determination method for salvianolic acid B, aspirin and salicylic acid in blood plasma is characterized by comprising the following steps:
1) drawing a standard curve
1-1) mixing mother liquor of salvianolic acid B, aspirin and salicylic acid to prepare mixed mother liquor, and diluting with methanol-water solution to obtain a series of working solutions with the concentration of 0.025-30 mug/mL;
1-2) respectively and uniformly mixing 10 mu L of working solution with 40 mu L of blank plasma, 20 mu L of internal standard working solution and 300 mu L of ethyl acetate in sequence, then carrying out first centrifugation treatment, drying the supernatant after the first centrifugation treatment, and respectively re-dissolving the dried residues with 50 mu L of organic mixed solution; then respectively carrying out second centrifugation treatment, wherein the centrifuged supernatant is a sample solution for drawing a standard curve, and the concentrations of salvianolic acid B, aspirin and salicylic acid in the sample solution of the standard curve are respectively 5-6000 ng/mL;
1-3) respectively sampling the prepared standard curve sample solution, carrying out UPLC-MS/MS analysis, and recording chromatograms and chromatographic peak areas of salvianolic acid B, aspirin and salicylic acid in the standard curve sample solution; then respectively taking the ratio of the concentrations of the salvianolic acid B, the aspirin and the salicylic acid in the standard curve sample solution to the concentration of the internal standard as an abscissa and the ratio of the chromatographic peak areas of the salvianolic acid B, the aspirin, the salicylic acid and the internal standard as an ordinate, and performing regression operation by adopting a least square method to respectively draw standard curve equations of the salvianolic acid B, the aspirin and the salicylic acid;
2) plasma sample assay
Sampling a to-be-detected plasma sample, carrying out UPLC-MS/MS analysis, and recording chromatograms and chromatographic peak areas of salvianolic acid B, aspirin, salicylic acid and internal standard substances in the to-be-detected plasma sample; and then respectively substituting the chromatographic peak area ratio of the aspirin, the salicylic acid and the salvianolic acid B in the plasma sample to be detected, the chromatographic peak area ratio of the internal standard substance and the concentration of the internal standard substance into the standard curve equation of the aspirin, the salicylic acid and the salvianolic acid B to be drawn, and calculating to obtain the concentrations of the aspirin, the salicylic acid and the salvianolic acid B in the plasma sample to be detected.
2. The method of claim 1, wherein during the UPLC-MS/MS analysis in step 1-3), the chromatographic conditions determined by liquid chromatography (UPLC) are as follows: mobile phase: 0.5% formic acid (a) -acetonitrile (B).
3. The method of claim 2, wherein during the liquid chromatography separation, a mobile phase selective gradient elution: 0-0.3min, 95% -75% A; 0.3-1.3min, 75% -70% A; 1.3-2.1min, 70% -30% A; 2.1-2.5min, 30% A; 2.5-2.7min, 30% -95% A; 2.7-4.0min, 95% A.
4. The method as claimed in claim 1, wherein the mass spectrometric detection uses an electrospray ionization ion source to ionize a liquid phase eluate during UPLC-MS/MS analysis in step 1-3), and uses a negative ion multiple reaction ion monitoring scan mode to detect the content of salvianolic acid B, aspirin, salicylic acid and internal standard in a plasma sample.
5. The method according to any one of claims 1 to 4, wherein the test plasma sample in step 2) is prepared as follows: uniformly mixing the plasma to be detected with an internal standard working solution and an organic solvent, and then carrying out first centrifugal treatment; drying the supernatant after the first centrifugation treatment, and redissolving the supernatant by using an organic mixed solution; and then carrying out second centrifugation treatment, wherein the supernatant obtained after the second centrifugation treatment is the plasma sample to be detected.
6. The process according to claim 5, wherein the organic solvent is selected from ethyl acetate, petroleum ether, n-hexane, dichloromethane or chloroform, preferably ethyl acetate.
7. A method according to any one of claims 1 to 6, further comprising the step of 1A) QC quality inspection, wherein said QC quality inspection comprises the steps of:
1A-1) mixing mother liquor of salvianolic acid B, aspirin and salicylic acid to prepare mixed mother liquor, and diluting with methanol-water solution to obtain QC working solution with concentration of 0.075-22.5 μ g/mL;
1A-2) respectively carrying out QC working solutions of 10 mu L, respectively mixing with blank plasma of 40 mu L, internal standard working solution of 20 mu L and ethyl acetate of 300 mu L uniformly, carrying out first centrifugation treatment, drying supernate obtained after the first centrifugation treatment, and respectively re-dissolving dried residues with organic mixed liquor of 50 mu L; then respectively carrying out second centrifugation treatment, wherein the centrifuged supernatant is a QC sample solution, and the concentrations of salvianolic acid B, aspirin and salicylic acid in the QC sample solution are respectively 15-4500 ng/mL;
1A-3) respectively sampling QC sample solutions with different concentrations, carrying out UPLC-MS/MS analysis, and recording chromatograms and chromatographic peak areas of salvianolic acid B, aspirin and salicylic acid in the QC sample solutions; then, respectively substituting the ratio of the chromatographic peak areas of the aspirin, the salicylic acid and the salvianolic acid B in the QC sample solution to the chromatographic peak area of the internal standard and the concentration of the internal standard substance into the standard curve equation of the aspirin, the salicylic acid and the salvianolic acid B drawn in the step 2), and calculating to obtain the concentrations of the aspirin, the salicylic acid and the salvianolic acid B in the QC sample solution;
1A-4) comparing the calculated concentration of aspirin, salicylic acid and salvianolic acid B in the QC sample solution with the real concentration of aspirin, salicylic acid and salvianolic acid B in the QC sample solution, and calculating the accuracy of the QC sample; the accuracy of the components aspirin, salicylic acid and salvianolic acid B in the QC sample solution is 85-115% of a standard curve for determining the concentration of the plasma sample.
8. The method as claimed in claim 7, wherein the concentrations of salvianolic acid B, aspirin, and salicylic acid in the QC sample solutions of steps 1A-3) are 15, 3000, and 4500ng/mL, respectively.
9. The method according to claim 7, wherein the QC sample accuracy in steps 1A-4) is the ratio of the concentration of aspirin, salicylic acid, salvianolic acid B in the QC sample solution to the real concentration of aspirin, salicylic acid, salvianolic acid B in the QC sample solution calculated according to the standard curve equation, respectively.
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