CN112557527A - Method for measuring polypeptide content in Shuxuetong injection - Google Patents
Method for measuring polypeptide content in Shuxuetong injection Download PDFInfo
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- CN112557527A CN112557527A CN202011285739.3A CN202011285739A CN112557527A CN 112557527 A CN112557527 A CN 112557527A CN 202011285739 A CN202011285739 A CN 202011285739A CN 112557527 A CN112557527 A CN 112557527A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
Abstract
The invention relates to a method for measuring polypeptide content in Shuxuetong injection, which comprises the steps of transferring Tp-ww-1504, Tp-ww-1505, Tp-ww-1506 and Tp-ww-1511 polypeptide stock solutions, diluting the polypeptide stock solutions into polypeptide standard solutions with series concentrations step by using a diluent, adding triptorelin internal standard stock solutions, and preparing a mixed standard solution containing the internal standard triptorelin by using the diluent. Taking the mass concentration (X) of a substance to be measured as a horizontal coordinate, and taking the peak area ratio (Y) of a target analyte and an internal standard measured by liquid chromatography-mass spectrometry as a vertical coordinate to obtain a standard curve of the polypeptide; and (3) determining a test solution prepared from the Shuxuetong injection or a plasma sample containing the effective components of the Shuxuetong injection by adopting a liquid chromatography-mass spectrometry method to obtain the peak area ratio of the target analyte to the internal standard, and calculating according to the standard curve to obtain the content of the polypeptide to be detected. The determination method has the characteristics of good specificity, high sensitivity and accurate quantification.
Description
Technical Field
The invention relates to the technical field of drug analysis, in particular to polypeptides in Shuxuetong injection and a method for measuring the content of the polypeptides in blood plasma.
Background
The injection is an animal traditional Chinese medicine injection prepared from leech and earthworm extracts, contains amino acid and polypeptide substances, can be clinically used for ischemic stroke disease caused by blood stasis and collateral obstruction, has obvious effects on diabetic cerebral infarction and acute cerebral infarction, and has the effects of promoting blood circulation to remove blood stasis and clearing and activating the channels and collaterals. The Shuxuetong injection comprises four polypeptides of Tp-ww-1504 (peptide segment amino acid sequence VAPEEHPVLL), Tp-ww-1505(FAGDDAPRAVFPS), Tp-ww-1506(RVAPEEHPVLL) and Tp-ww-1511(GFAGDDAPRAVFP), the content determination of the polypeptides is not reported, and in addition, the polypeptide content is extremely low, and the determination of the polypeptides is very challenging due to the complex components, the serious matrix effect and the like of the Shuxuetong injection.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for measuring the content of polypeptide in Shuxuetong injection, which has the characteristics of good specificity, high sensitivity and accurate quantification.
In order to solve the technical problems, the invention provides a method for determining the content of polypeptide in Shuxuetong injection, which comprises the following steps:
preparing stock solution and internal standard stock solution;
weighing Tp-ww-1504 (peptide amino acid sequence VAPEEHPVLL), Tp-ww-1505(FAGDDAPRAVFPS), Tp-ww-1506(RVAPEEHPVLL) and Tp-ww-1511(GFAGDDAPRAVFP) four polypeptides, respectively adding water (v/v) containing 0.1% formic acid, and vortexing to completely dissolve the polypeptides to prepare a polypeptide stock solution; weighing triptorelin, adding water (v/v) containing 0.1% formic acid, and vortexing to dissolve triptorelin completely to obtain triptorelin internal standard stock solution;
step two, preparing a standard substance solution;
respectively transferring the polypeptide stock solutions, and gradually diluting the polypeptide stock solutions into polypeptide standard solutions with serial concentrations by using a diluent; respectively adding triptorelin internal standard stock solutions into the polypeptide standard solutions, uniformly mixing by vortex, and preparing a mixed standard solution containing the internal standard triptorelin by using a diluent;
step three, preparing a test solution
Transferring the Shuxuetong injection filtered by a 0.22 mu m microporous filter membrane and the internal standard triptorelin solution into an EP tube, diluting the injection by using a diluent, and performing vortex and uniform mixing to prepare a test solution containing the internal standard triptorelin;
or taking a plasma sample containing the effective components of the Shuxuetong injection, adding the triptorelin internal standard solution and the diluent, uniformly mixing by vortex, adding methanol, vortex, centrifuging, taking the supernatant, placing the supernatant in an EP tube, carrying out vacuum centrifugal concentration at 40 ℃, adding an appropriate amount of the diluent for redissolution, vortex, centrifuging, and taking the supernatant as a test sample solution containing the internal standard triptorelin;
taking the prepared polypeptide series concentration mixed standard solution, taking the mass concentration (X) of the substance to be detected as a horizontal coordinate, and taking the peak area ratio (Y) of the target analyte and the internal standard, which is measured by the liquid chromatography-mass spectrometry, as a vertical coordinate, so as to obtain a standard curve of the polypeptide; determining the test sample solution by adopting a liquid chromatography-mass spectrometry method to obtain the peak area ratio of the target analyte to the internal standard, and calculating according to the standard curve to obtain the content of the polypeptide to be detected;
in the fourth step, the liquid phase part chromatographic conditions are as follows: adopting a C18 chromatographic column as a separation medium, and carrying out gradient elution after sample injection, wherein the ratio of a mobile phase A: acetonitrile or methanol, mobile phase B: an organic solvent or water at a mass concentration of 10% or less or water (v/v) containing 0.1% formic acid; gradient elution conditions: 0-0.3min 5% A, 95% B; 0.6-1.2min 20% A, 80% B; 2.5-3.5min 50% A, 50% B; 5% of A and 95% of B at 3.51-4.5 min; the flow rate is 0.1-0.4 ml/min; column temperature: 20-40 ℃;
mass spectrum detection conditions: an electrospray ESI ion source positive ion detection mode; capillary voltage: 1.0-3.0 KV; taper hole voltage: 2-70V; nitrogen pressure: 5-10 psi; desolventizing gas temperature: 300 ℃ and 450 ℃; desolventizing air flow rate: 600-900 L.h-1(ii) a Taper hole air flow rate: 150 L.h-1(ii) a Collision airflow rate: 0.1-0.25 mL/min-1(ii) a The scanning mode is multi-reaction ion monitoring MRM, and the parameters are as follows:
further, in the first step, the Tp-ww-1504, Tp-ww-1505, Tp-ww-1506 and Tp-ww-1511 polypeptides are precisely weighed to be 0.44mg, 0.56mg, 0.64mg and 0.61mg respectively; adding water containing 0.1% formic acid, and vortex for 2min to dissolve completely to obtain 500 μ g/ml polypeptide stock solution; and accurately weighing 0.59mg of triptorelin, adding water (v/v) containing 0.1% formic acid, and vortexing for 2min to completely dissolve the triptorelin to prepare 500 mu g/ml triptorelin internal standard stock solution.
Further, in the second step, precisely transferring the stock solutions of the polypeptides Tp-ww-1504 and Tp-ww-1506, and diluting the stock solutions into polypeptide standard solutions with the serial concentrations of 3 mu g/ml, 2.5 mu g/ml, 2.0 mu g/ml, 1.5 mu g/ml and 1.0 mu g/ml by using a diluent step by step respectively; precisely transferring a proper amount of a stock solution of the polypeptide Tp-ww-1505, and gradually diluting the stock solution into polypeptide standard solutions with the serial concentrations of 500ng/ml, 200ng/ml, 150ng/ml, 100ng/ml and 50ng/ml by using a diluent; precisely transferring a proper amount of a stock solution of the polypeptide Tp-ww-1511, and gradually diluting the stock solution into polypeptide standard solutions with serial concentrations of 1 mu g/ml, 800ng/ml, 500ng/ml, 400ng/ml and 200ng/ml by using a diluent;
taking the polypeptide standard solutions, sequentially mixing according to concentration gradient, adding 100 mul of triptorelin internal standard solution with the concentration of 1 mug/ml, uniformly mixing by vortex, preparing 100ng/ml triptorelin containing internal standard by using a diluent, wherein the polypeptide Tp-ww-1504 and Tp-ww-1506 are 300ng/ml, 250ng/ml, 200ng/ml, 150ng/ml and 100 ng/ml; polypeptide Tp-ww-1505 is contained in the polypeptide, and the polypeptide is 50ng/ml, 20ng/ml, 15ng/ml, 10ng/ml and 5 ng/ml; the mixed standard solution containing the polypeptide Tp-ww-1511 is 100ng/ml, 80ng/ml, 50ng/ml, 40ng/ml and 20 ng/ml.
Further, in the third step, the injection for dredging blood circulation is taken and filtered by a microporous filter membrane of 0.22 mu m; precisely transferring 500 mu l of Shuxuetong and 100 mu l of triptorelin as an internal standard of 1 mu g/ml into a 1.5ml EP tube, diluting 2 times by using a diluent, and performing vortex mixing; preparing a test solution containing 100ng/ml of triptorelin as an internal standard.
Further, in the third step, 0.2ml of plasma sample containing the effective components of the Shuxuetong injection is taken, 50 mul of triptorelin internal standard solution and 50 mul of diluent are precisely added, vortex mixing is carried out, 0.8ml of methanol is added, vortex is carried out for 5min, 14000rpm centrifugation is carried out for 10min, 0.9ml of supernatant is taken and placed in an EP tube, vacuum concentration is carried out at 40 ℃, 0.1ml of diluent is added for redissolution, vortex is carried out for 5min, 14000rpm centrifugation is carried out for 5min, and the supernatant is taken for sample injection and determination.
Further, the diluent in the second step and the third step is selected from 60% methanol water (v/v) containing 0.01% -0.5% formic acid or 60% methanol water (v/v) containing 0.01% -0.1% trifluoroacetic acid.
The invention adopts a liquid chromatograph-mass spectrometer to establish a method capable of simultaneously determining the contents of 4 polypeptides Tp-ww-1504(VAPEEHPVLL), Tp-ww-1505(FAGDDAPRAVFPS), Tp-ww-1506(RVAPEEHPVLL) and Tp-ww-1511(GFAGDDAPRAVFP) in the Shuxuetong injection. The method has the advantages of good specificity, high sensitivity, high precision, high accuracy, high sample recovery rate, high stability and the like, and meets the requirements of drug analysis.
Drawings
FIG. 1 is a chromatogram of a methanol blank solvent;
FIG. 2 is a chromatogram of a mixed standard solution;
FIG. 3 is a chromatogram of a test solution of Shuxuetong with an internal standard;
in FIGS. 2 and 3, peak 1 IS Tp-ww-1505, peak 2 IS Tp-ww-1511, peak 3 IS Tp-ww-1506, peak 4 IS Tp-ww-1504, and peak IS IS internal triptorelin;
FIG. 4 is a chromatogram of rat blank blood;
FIG. 5 is a blank blood from a rat to which a standard substance has been added;
FIG. 6 is a chromatogram of a blood sample taken 5 or 10min after intravenous injection of Shuxuetong injection in rats.
Detailed Description
The method can be used for accurately determining the content of the polypeptides in the Shuxuetong injection to provide reference for product quality control in later period of enterprises.
The invention provides a method for measuring the content of polypeptide in Shuxuetong injection, which comprises the following steps:
preparing stock solution and internal standard stock solution;
weighing four polypeptides of Tp-ww-1504, Tp-ww-1505, Tp-ww-1506 and Tp-ww-1511, respectively adding water (v/v) containing 0.1% formic acid, and vortexing to completely dissolve the polypeptides to prepare polypeptide stock solution; triptorelin is weighed, water (v/v) containing 0.1% formic acid is added, and vortex is carried out to completely dissolve the triptorelin to prepare triptorelin internal standard stock solution.
Preferably, in the first step, the Tp-ww-1504, Tp-ww-1505, Tp-ww-1506 and Tp-ww-1511 polypeptides are precisely weighed to be 0.44mg, 0.56mg, 0.64mg and 0.61mg respectively; adding water containing 0.1% formic acid, and vortex for 2min to dissolve completely to obtain 500 μ g/ml polypeptide stock solution; and accurately weighing 0.59mg of triptorelin, adding water (v/v) containing 0.1% formic acid, and vortexing for 2min to completely dissolve the triptorelin to prepare 500 mu g/ml triptorelin internal standard stock solution.
And step two, preparing a standard solution.
Respectively transferring the four polypeptide stock solutions, and gradually diluting the four polypeptide stock solutions into polypeptide standard solutions with serial concentrations by using a diluent; and respectively adding triptorelin internal standard stock solutions into the polypeptide standard solutions, uniformly mixing by vortex, and preparing a mixed standard solution containing the internal standard triptorelin by using a diluent.
Preferably, in the second step, the polypeptide Tp-ww-1504 and Tp-ww-1506 stock solutions are precisely transferred and diluted by a diluent into polypeptide standard solutions with the serial concentrations of 3 mug/ml, 2.5 mug/ml, 2.0 mug/ml, 1.5 mug/ml and 1.0 mug/ml respectively; precisely transferring a proper amount of a stock solution of the polypeptide Tp-ww-1505, and gradually diluting the stock solution into polypeptide standard solutions with the serial concentrations of 500ng/ml, 200ng/ml, 150ng/ml, 100ng/ml and 50ng/ml by using a diluent; precisely transferring a proper amount of a stock solution of the polypeptide Tp-ww-1511, and gradually diluting the stock solution into polypeptide standard solutions with serial concentrations of 1 mu g/ml, 800ng/ml, 500ng/ml, 400ng/ml and 200ng/ml by using a diluent;
taking the four polypeptide standard solutions, mixing the four polypeptide standard solutions in sequence according to concentration gradient, respectively adding 100 mul of triptorelin internal standard solution with the concentration of 1 mug/ml, uniformly mixing the solutions in a vortex mode, preparing 100ng/ml triptorelin containing the internal standard by using a diluent, wherein the polypeptide Tp-ww-1504 and Tp-ww-1506 are 300ng/ml, 250ng/ml, 200ng/ml, 150ng/ml and 100 ng/ml; polypeptide Tp-ww-1505 is contained in the polypeptide, and the polypeptide is 50ng/ml, 20ng/ml, 15ng/ml, 10ng/ml and 5 ng/ml; the mixed standard solution containing the polypeptide Tp-ww-1511 is 100ng/ml, 80ng/ml, 50ng/ml, 40ng/ml and 20 ng/ml.
The diluent is selected from 60% methanol water (v/v) containing 0.01% -0.5% formic acid or 60% methanol water (v/v) containing 0.01% -0.1% trifluoroacetic acid.
Step three, preparing a test solution
The present embodiment is directed to two sample solutions.
One is Shuxuetong injection which is not injected into human bodies. And transferring the Shuxuetong injection filtered by a 0.22 mu m microporous filter membrane and the internal standard triptorelin solution into an EP tube, diluting the injection by using a diluent, and performing vortex and uniform mixing to prepare a test solution containing the internal standard triptorelin. Preferably, the injection for dredging blood is taken and filtered by a 0.22 mu m microporous filter membrane; precisely transferring 500 mu l of Shuxuetong and 100 mu l of 1 mu g/ml internal standard triptorelin solution into a 1.5ml EP tube, diluting 2 times by using a diluent, and performing vortex mixing and uniform mixing; preparing a test solution containing 100ng/ml of triptorelin as an internal standard.
The other is a plasma sample containing the effective components of the Shuxuetong injection obtained after the injection is injected into human bodies or animals. Adding triptorelin internal standard solution and diluent into a plasma sample containing the Shuxuetong injection, uniformly mixing by vortex, adding methanol, vortex, centrifuging, taking supernate, placing the supernate into an EP (ethylene propylene glycol) tube, carrying out vacuum centrifugal concentration at 40 ℃, adding the diluent for redissolving, vortex, centrifuging, and taking the supernate as the mixed standard solution of the internal standard triptorelin. Preferably, 0.2ml of plasma sample containing the Shuxuetong injection is taken, 50 mul of triptorelin internal standard solution and 50 mul of diluent are precisely added, vortex mixing is carried out, 0.8ml of methanol is added, vortex is carried out for 5min, centrifugation is carried out at 14000rpm for 10min, 0.9ml of supernatant is taken and placed in an EP tube, vacuum centrifugal concentration is carried out at 40 ℃, 0.1ml of diluent is added for redissolution, vortex is carried out for 5min, centrifugation is carried out at 14000rpm for 5min, and the supernatant is taken for sampling and determination. For plasma samples, the amount of one or more polypeptides therein may be determined.
The diluent is selected from 60% methanol water (v/v) containing 0.01% -0.5% formic acid or 60% methanol water (v/v) containing 0.01% -0.1% trifluoroacetic acid.
Taking the prepared polypeptide series concentration mixed standard solution, taking the mass concentration (X) of the substance to be detected as a horizontal coordinate, and taking the peak area ratio (Y) of the target analyte and the internal standard, which is measured by the liquid chromatography-mass spectrometry, as a vertical coordinate, so as to obtain a standard curve of the polypeptide; determining the sample solution by adopting a liquid chromatography-mass spectrometry method to obtain the peak area ratio of the target analyte to the internal standard, and calculating according to the standard curve to obtain the content of the polypeptide to be detected in the Shuxuetong injection;
in the fourth step, the liquid phase part chromatographic conditions are as follows: adopting a C18 chromatographic column as a separation medium, and carrying out gradient elution after sample injection, wherein the ratio of a mobile phase A: acetonitrile or methanol, mobile phase B: organic solvent or water with mass concentration of less than 10% or water containing 0.1% formic acid; gradient elution conditions: 0-0.3min 5% A, 95% B; 0.6-1.2min 20% A, 80% B; 2.5-3.5min 50% A, 50% B; 5% of A and 95% of B at 3.51-4.5 min; the flow rate is 0.1-0.4 ml/min; column temperature: 20-40 ℃;
mass spectrum detection conditions: an electrospray ESI ion source positive ion detection mode; capillary voltage: 1.0-3.0 KV; taper hole voltage: 2-70V; nitrogen pressure: 5-10 psi; desolventizing gas temperature: 300 ℃ and 450 ℃; desolventizing air flow rate: 600-900 L.h-1(ii) a Taper hole air flow rate: 150 L.h-1(ii) a Collision airflow rate: 0.1-0.25 mL/min-1(ii) a Scanning mode (multiple reactive ion monitoring MRM):
the detection method provided by the invention is a liquid chromatography-mass spectrometry quantitative method so as to meet the data requirement of content determination of 4 polypeptides in the Shuxuetong injection. The effects of the method in terms of specificity, sensitivity, precision, accuracy, extraction recovery rate, stability, and the like will be described below through experiments.
Method for establishing polypeptide concentration determination in Shuxuetong injection
Purpose of the experiment: a content determination method of 4 polypeptides with good specificity and high sensitivity is established by a liquid chromatography-mass spectrometry quantitative method so as to meet the data requirements of content determination of the 4 polypeptides in the Shuxuetong injection.
Materials and methods
1. Experimental Material
The polypeptide is purchased from Shanghai Tanpai Biotechnology limited, and the purity is 99 percent; triptorelin is purchased from SINOPEP pharmaceutical Co., Ltd, and has the purity of 98%; methanol from Sigma, chromatographically pure; formic acid was purchased from ROE corporation and chromatographically pure; the water is ultrapure water, and the trifluoroacetic acid is purchased from Beijing Bailingwei science and technology Limited and has the purity of 99.9 percent; shuxuetong injection (provided by peony Jiangyoubo pharmaceutical Co., Ltd.).
Waters ACQUITY UPLC ultra high performance liquid chromatograph; a Waters Xevo TQ-S triple quadrupole mass spectrometer; one-tenth-ten-thousandth analytical balance (METTLER TOLEDO corporation); vortex mixer (lebert, usa).
2. Experimental methods
2.1 preparation of test solution:
taking 1ml of Shuxuetong injection, filtering the injection by a 0.22 mu m filter membrane, precisely transferring 500 mu l of Shuxuetong and 100 mu l of 1 mu g/ml internal standard triptorelin into a 1.5ml EP tube, diluting 2 times by using 60% methanol water (containing 0.05% trifluoroacetic acid), and vortexing and uniformly mixing; and (6) sample injection.
2.2 methodological validation
2.2.1 specificity
Under the test condition, methanol, a standard solution and a test solution are respectively sampled and analyzed to obtain chromatograms of the polypeptide and the internal standard in the Shuxuetong injection (see figure 2 and figure 3). As can be seen from the figure, the chromatographic peak shapes of 4 polypeptides (Tp-ww-1504, Tp-ww-1505, Tp-ww-1506 and Tp-ww-1511) and the internal standard triptorelin are good, the measurement is free from interference, and the specificity of the method is good. The parameters for the LC/MS measurements were the same as in example 1.
2.2.2 Standard Curve and Linear Range
Precisely weighing polypeptides Tp-ww-1504(0.44mg), Tp-ww-1505(0.56mg), Tp-ww-1506(0.64mg) and Tp-ww-1511(0.61mg), placing in a 1ml EP tube, adding a proper amount of formic acid water, and uniformly mixing by vortex to obtain a stock solution. The stock solution was stored in a refrigerator at 4 ℃ in the dark. It was diluted to the desired concentration with 60% aqueous methanol (containing 0.05% trifluoroacetic acid) immediately before use. Taking 4 polypeptide series mixed standard solutions (containing 100ng/ml of internal standard), injecting 2 mul of sample for determination, drawing a standard curve by taking the mass concentration (X) of the substance to be detected as a horizontal coordinate and the peak area ratio (Y) of the target analyte and the internal standard as a vertical coordinate, wherein the correlation coefficient meets the requirement.
TABLE 1 Linear Range and regression equation for Standard Curve
2.2.3 precision test
Precisely sucking mixed reference substance solutions (containing 100ng/mL of triptorelin as an internal standard) with polypeptide Tp-1504 and Tp-1506 concentrations of 200ng/mL and polypeptide Tp-1505 and Tp-1511 concentrations of 15ng/mL and 50ng/mL respectively, repeatedly sampling for 6 times, recording peak areas of polypeptide analyte and triptorelin as internal standards, calculating peak area ratio(AAnalyte/AInternal standard) And RSD. The results show that the precision of the instrument is good.
TABLE 2 results of precision test
2.2.4 stability test
At room temperature, processing the Shuxuetong injection with the batch number of 2017042121 according to the preparation method of the test sample to prepare the test sample solution, injecting samples for analysis at 0, 2, 4, 8, 12, 24 and 48h respectively, recording the peak areas of the polypeptide analyte and the internal standard, and calculating the peak area ratio (A)Analyte/AInternal standard) And RSD. The results show that the sample solution is stable within 48h at room temperature.
TABLE 3 stability test results
2.2.5 sample recovery test
24 parts of Shuxuetong injection (batch number is 2017051312) with known polypeptide content are transferred and divided into 4 groups, each group is separately added with 6 parts of a certain amount of reference substance (the addition amounts of polypeptide Tp-1504 and polypeptide Tp-1506 are both 150ng, the addition amount of polypeptide Tp-1505 is 7.50ng, and the addition amount of polypeptide Tp-1511 is 25ng), sample treatment is carried out according to the preparation method of the test sample, 2 muL of sample injection is determined, and the recovery rate is calculated according to (the polypeptide measurement amount-the polypeptide content of the test sample)/the polypeptide addition amount multiplied by 100 percent, and the result shows that the recovery rate meets the analysis requirement.
TABLE 4 polypeptide Tp-1504 recovery test results
TABLE 5 polypeptide Tp-1505 recovery test results
TABLE 6 test results on the recovery ratio of the polypeptide Tp-1506
TABLE 7 polypeptide Tp-1511 recovery test results
2.2.6 reproducibility test
6 parts of test solution is prepared from 2017042121 batch injection for dredging blood, and is injected for determination.
TABLE 8 results of reproducibility test
3. Results of the experiment
The methodological and methodological verification result shows that the method has good specificity; precision RSD is less than 1.5%; the RSD of the stability experiment is less than 5.5 percent; the sample adding recovery rate meets the requirements; the repeatability RSD is less than 2.5 percent, and the results meet the requirement of content determination of the traditional Chinese medicine injection.
(II) measurement of content
In the experiment, 20 batches of Shuxuetong injection are taken to prepare a test solution, and the content of 4 polypeptides in the injection is determined by sample injection.
TABLE 9 content of 4 Polypeptides in Shuxuetong injection
4. Conclusion
The experiment adopts a liquid chromatograph-mass spectrometer, establishes a method for simultaneously measuring the content of 4 polypeptides in the Shuxuetong injection, and the method has the advantages of good specificity, high sensitivity, precision, accuracy, extraction recovery rate, stability and the like which all meet the requirements of pharmaceutical analysis.
Establishing a method for measuring the blood concentration of the polypeptide for dredging blood in blood plasma, and determining the polypeptide of a rat after intravenous injection of the polypeptide for dredging blood
Exposure of the ingredients to the body.
1. Experimental methods
Preparation of plasma samples: taking 0.2mL of rat plasma, adding 50 μ L of internal standard solution (200ng/mL triptorelin) and 50 μ L of 60% methanol water (containing 0.05% TFA), uniformly mixing by vortex, adding 0.8mL of methanol reagent, vortex for 5min, carrying out 10min centrifugal treatment under 14000r/min, taking 0.9mL of supernatant into an EP tube, carrying out vacuum centrifugal concentration at 40 ℃, adding 0.1mL of 60% methanol water (containing 0.05% TFA) for redissolution, vortex for 5min, continuing to centrifuge for 5min under 14000r/min, and taking 60 μ L of supernatant, injecting 2 μ L for determination.
2. Methodology investigation
2.1 specificity
And (4) taking blank plasma, blank plasma + a reference substance, and 10min plasma samples after administration, and processing according to the preparation method of the plasma samples. The polypeptide Tp-ww-1504, the polypeptide Tp-ww-1506 and the internal standard triptorelin have good chromatographic peak shapes and no interference in measurement when the sample is injected by 2 muL respectively for measurement, and the results are shown in figures 4, 5 and 6.
2.2 Standard Curve and quality control
2.2.1 Standard Curve
Taking 0.2mL of blank plasma, precisely adding 50 mu L of internal standard solution (200ng/mL triptorelin) to the blank plasmaThe control solution was mixed at 50. mu.L per column concentration, and the concentration was measured by injecting 2. mu.L of the mixture according to the method for preparing the plasma sample. Taking the ratio (Y) of peak areas of the polypeptides Tp-ww-1504 and Tp-ww-1506 to the peak area of the internal standard as the ordinate, taking the concentration (X) of the polypeptide as the abscissa and weighting 1/X2Linear regression was performed, and the mean regression equation for Tp-ww-1504 was 4.18 × 10-2X+1.93×10-2The correlation coefficient r is 0.9972; the average regression equation of Tp-ww-1506 is Y ═ 8.26 × 10-3X+2.45×10-3The correlation coefficient r is 0.9971.
2.2.2 quality control
Each batch of test samples follows the standard curve and is subjected to a quality control test. Quality control measurements were performed on three samples at different concentrations, 2.00, 10.0 and 37.50ng/mL, as measured for polypeptides in plasma. The relative error (RSD) of the quality control samples is less than 15 percent, and the quality control requirements of the technical guidance principle of the clinical pharmacokinetics research of the chemical drugs are met.
2.3 precision and accuracy investigation
Taking blank plasma, preparing 6 parts of polypeptide plasma samples with the concentrations of 1.00ng/mL, 2.00ng/mL, 10.00ng/mL and 37.50ng/mL, operating according to the item of 'plasma sample preparation', measuring, recording the peak area of the polypeptide and the peak area of an internal standard, calculating the ratio of the peak area of the polypeptide and the peak area of the internal standard, and substituting the ratio into a following standard curve to calculate the concentration. Precision is the variation of the measured concentration expressed as RSD and accuracy is expressed as a percentage of the ratio of the measured concentration to the true concentration. 3 batches of plasma samples were processed and measured, and the precision and accuracy within and between batches were calculated, respectively.
TABLE 10 determination of the precision and accuracy of the polypeptide Tp-ww-1504 within and between batches
TABLE 11 determination of the precision and accuracy of the polypeptide Tp-ww-1506 within and between batches
2.4 extraction recovery
Taking blank plasma, preparing 6 parts of low, medium and high plasma samples with the concentrations of 2.00, 10.0 and 37.5ng/mL, operating according to the item of a plasma sample preparation method, carrying out sample injection of 2 mu L for determination, and recording the peak area of the short peptide As; taking blank plasma, adding 100 mu L of 60% methanol water (containing 0.05% trifluoroacetic acid), uniformly mixing by vortex, operating from 'adding methanol 0.8 mL', carrying out vacuum centrifugal concentration and volatilizing to obtain blank matrix residue, adding 50 mu L of reference substance solution and 50 mu L of internal standard solution for redissolution, preparing a composite solution containing the blank plasma matrix, and recording the peak area to be Asd, wherein the calculation formula of the extraction recovery rate is R ═ As/Asd × 100%.
TABLE 12 recovery of Tp-ww-1504 extraction of the polypeptide
TABLE 13 recovery of the polypeptide Tp-ww-1506 extraction
2.5 matrix Effect
Taking blank plasma, adding 100 mu L of 60% methanol water (containing 0.05% trifluoroacetic acid), uniformly mixing by vortex, operating from the point of adding 0.8mL of methanol under the term of a plasma treatment method, carrying out vacuum centrifugal concentration and volatilizing to obtain blank matrix residue, adding 50 mu L of reference substance solution and 50 mu L of internal standard solution for redissolution, and preparing the composite solution containing the blank plasma matrix. Comparing the peak areas of the composite matrix solution with high, medium and low concentrations with the pure control solution shows that the method has no obvious matrix effect.
TABLE 14 polypeptide Tp-ww-1504 matrix Effect
TABLE 15 polypeptide Tp-ww-1506 matrix Effect
2.6 stability Studies
Blank plasma is taken to prepare plasma samples with three concentrations, the stability of the plasma samples at room temperature for 8 hours and 24 hours, repeated freeze thawing for 1 time and 3 times, freezing for 1 month and standing of the processed samples for 24 hours are respectively inspected, the RSD values are all less than 15 percent, and the RSD values are all less than 10 percent.
TABLE 16 polypeptide Tp-ww-1504 stability Studies
TABLE 17 polypeptide Tp-ww-1506 stability Studies
2.7 lower limit of quantitation
Taking 0.2mL and 6 parts of blank plasma, respectively adding 50 μ L of internal standard solution and 50 μ L of polypeptide reference solution with the concentration of 2ng/mL, operating according to the item of 'plasma sample preparation method', and respectively setting RSD values to be 5.3% and 7.5%, so that the lowest limit of the Tp-ww-1504 and Tp-ww-1506 in the plasma is 1 ng/mL.
TABLE 18 lower limit of quantitation for Tp-ww-1504
TABLE 19 lower limit of quantitation for Tp-ww-1506
3 results of the experiment
Plasma samples of rats at a certain time point after intravenous injection of the Shuxuetong injection are taken, and the plasma concentration of 2 polypeptides is determined by sample injection according to the operation under the item of 'plasma sample preparation'. The result shows that the polypeptide Tp-ww-1504 can be detected from a blood sample, and the blood concentration is basically about 1-2 ng/mL; the polypeptide Tp-ww-1506 could not be detected from the blood sample.
The technical solution claimed by the present invention will be further clearly and completely explained below with reference to some embodiments. The sources of the experimental materials used in the examples are the same as those described in the above section, and are not described again.
Example 1
Firstly, preparing stock solution and internal standard solution
Precisely weighing Tp-ww-1504, Tp-ww-1505, Tp-ww-1506 and Tp-ww-1511 into 0.44mg, 0.56mg, 0.64mg and 0.61mg respectively; adding water containing 0.1% formic acid, and vortex for 2min to dissolve completely to obtain 500 μ g/ml polypeptide stock solution; another 0.59mg triptorelin was precisely weighed, added with water (v/v) containing 0.1% formic acid, vortexed for 2min to completely dissolve it, and made into 500. mu.g/ml triptorelin internal standard solution.
Secondly, preparing a standard substance solution
Precisely transferring the stock solutions of the polypeptides Tp-ww-1504 and Tp-ww-1506, and diluting the stock solutions into polypeptide solutions with the serial concentrations of 3 mu g/ml, 2.5 mu g/ml, 2.0 mu g/ml, 1.5 mu g/ml and 1.0 mu g/ml by using a diluent respectively; precisely transferring a proper amount of polypeptide Tp-ww-1505 stock solution, and gradually diluting the stock solution into polypeptide solutions with the serial concentrations of 500ng/ml, 200ng/ml, 150ng/ml, 100ng/ml and 50ng/ml by using a diluent; precisely transferring a proper amount of a stock solution of the polypeptide Tp-ww-1511, and gradually diluting the stock solution into polypeptide solutions with serial concentrations of 1 mug/ml, 800ng/ml, 500ng/ml, 400ng/ml and 200ng/ml by using a diluent;
taking the four polypeptide standard solutions, respectively adding 100 mu l of triptorelin internal standard solution with the concentration of 1 mu g/ml, uniformly mixing by vortex, and preparing 100ng/ml triptorelin containing the internal standard by using a diluent, wherein the Tp-ww-1504 or Tp-ww-1506 containing the polypeptide is 300ng/ml, 250ng/ml, 200ng/ml, 150ng/ml or 100 ng/ml; polypeptide Tp-ww-1505 is contained in the polypeptide, and the polypeptide is 50ng/ml, 20ng/ml, 15ng/ml, 10ng/ml and 5 ng/ml; the mixed standard solution containing the polypeptide Tp-ww-1511 is 100ng/ml, 80ng/ml, 50ng/ml, 40ng/ml and 20 ng/ml.
Thirdly, preparing a test solution
And transferring the Shuxuetong injection filtered by a filter membrane of 0.22 mu m and the internal standard triptorelin into an EP tube, diluting the mixture by using a diluent, and performing vortex and uniform mixing to prepare a test solution containing the internal standard triptorelin. Preferably, the injection for dredging blood is taken and filtered by a 0.22 mu m filter membrane; precisely transferring 500 mu l of Shuxuetong and 100 mu l of 1 mu g/ml internal standard triptorelin into a 1.5ml EP tube, diluting 2 times by using a diluent, and performing vortex and uniform mixing; preparing a test solution containing 100ng/ml of triptorelin as an internal standard.
Fourthly, adopting a liquid chromatography-mass spectrometer for measurement
Chromatographic conditions are as follows:
waters ACQUITY UPLC ultra high performance liquid chromatograph system: comprises a binary pump, an automatic sample injector and a column incubator. And (3) analyzing the column: waters Acquity UPLC BEH C18(100 mm. times.2.1 mm, 1.7 μm); protection of the column: WatersAcquity UPLC BEH C18(5 mm. times.2.1 mm, 1.7 μm). Adopting a C18 chromatographic column as a separation medium, and carrying out gradient elution after sample injection, wherein the ratio of a mobile phase A: acetonitrile, mobile phase B: water containing 0.1% formic acid; gradient elution conditions:
flow rate: 0.4ml/min, column temperature: 40 ℃; temperature of a sample injection tray: 25 ℃; sample introduction amount: 2 μ l.
Mass spectrum conditions:
waters Xevo TQ-S triple quadrupole mass spectrometry; ESI ion source: a positive ion analysis mode; MRM scan mode. Setting parameters: capillary voltage: 1.9 KV; taper hole voltage: 30V; nitrogen pressure: 7 psi; desolventizing gas temperature: 400 ℃; desolventizing air flow rate: 800 L.h-1(ii) a Taper hole air flow rate: 150 L.h-1(ii) a Collision airflow rate: 0.15 mL/min-1；
Multiple reaction detection mode (MRM):
taking the prepared mixed standard solution with the concentration of the four polypeptide series, taking the mass concentration (X) of a substance to be detected as a horizontal coordinate, determining by adopting a liquid chromatography-mass spectrometry method, and taking the peak area ratio (Y) of a target analyte and an internal standard as a vertical coordinate to obtain standard curves of the four polypeptides; and obtaining the contents of the four polypeptides in the Shuxuetong injection according to a standard curve.
Example 2
The difference from example 1 is that: in the fourth step, the flow rate is 0.1 ml/min; column temperature: 20 ℃; temperature of a sample injection tray: 25 ℃; sample introduction amount: 2 μ l.
Mass spectrum conditions: capillary voltage: 1.0 KV; taper hole voltage: 2V; nitrogen pressure: 5 psi; desolventizing gas temperature: 300 ℃; desolventizing air flow rate: 600 L.h-1(ii) a Taper hole air flow rate: 150 L.h-1(ii) a Collision airflow rate: 0.1mL/min-1；
Multiple reaction detection mode (MRM):
example 3
The difference from example 1 is that: in step four, mobile phase a: methanol, mobile phase B: 0.1% formic acid in water at a flow rate of 0.4 ml/min; column temperature: 40 ℃; temperature of a sample injection tray: 25 ℃; sample introduction amount: 2 μ l.
Mass spectrum conditions: capillary voltage: 3.0 KV; taper hole voltage: 50V; nitrogen pressure: 10 psi; desolventizing gas temperature: at 450 ℃; desolventizing air flow rate: 900 L.h-1(ii) a Taper hole air flow rate: 150 L.h-1(ii) a Collision airflow rate: 0.25 mL/min-1；
Multiple reaction detection mode (MRM):
example 4
Firstly, preparing stock solution and internal standard solution
Precisely weighing Tp-ww-1504, Tp-ww-1505, Tp-ww-1506 and Tp-ww-1511 into 0.44mg, 0.56mg, 0.64mg and 0.61mg respectively; adding water (v/v) containing 0.1% formic acid, and vortexing for 2min to dissolve completely to obtain 500 μ g/ml polypeptide stock solution; another 0.59mg triptorelin was precisely weighed, added with water (v/v) containing 0.1% formic acid, vortexed for 2min to completely dissolve it, and made into 500. mu.g/ml triptorelin internal standard solution.
Secondly, preparing a standard substance solution
Precisely transferring the stock solutions of the polypeptides Tp-ww-1504 and Tp-ww-1506, and diluting the stock solutions into polypeptide solutions with the serial concentrations of 3 mu g/ml, 2.5 mu g/ml, 2.0 mu g/ml, 1.5 mu g/ml and 1.0 mu g/ml by using a diluent respectively; precisely transferring a proper amount of polypeptide Tp-ww-1505 stock solution, and gradually diluting the stock solution into polypeptide solutions with the serial concentrations of 500ng/ml, 200ng/ml, 150ng/ml, 100ng/ml and 50ng/ml by using a diluent; precisely transferring a proper amount of a stock solution of the polypeptide Tp-ww-1511, and gradually diluting the stock solution into polypeptide solutions with serial concentrations of 1 mug/ml, 800ng/ml, 500ng/ml, 400ng/ml and 200ng/ml by using a diluent;
taking the four polypeptide standard solutions, respectively adding 100 mu l of triptorelin internal standard solution with the concentration of 1 mu g/ml, uniformly mixing by vortex, and preparing 100ng/ml triptorelin containing the internal standard by using a diluent, wherein the Tp-ww-1504 or Tp-ww-1506 containing the polypeptide is 300ng/ml, 250ng/ml, 200ng/ml, 150ng/ml or 100 ng/ml; polypeptide Tp-ww-1505 is contained in the polypeptide, and the polypeptide is 50ng/ml, 20ng/ml, 15ng/ml, 10ng/ml and 5 ng/ml; the mixed standard solution containing the polypeptide Tp-ww-1511 is 100ng/ml, 80ng/ml, 50ng/ml, 40ng/ml and 20 ng/ml.
Thirdly, preparing a test solution
Taking 0.2ml of a plasma sample containing Shuxuetong injection, precisely adding 50 mul of triptorelin internal standard solution and 50 mul of diluent, uniformly mixing by vortex, adding 0.8ml of methanol, centrifuging for 5min by vortex at 14000rpm for 10min, taking 0.9ml of supernatant, placing the supernatant in an EP tube, vacuum concentrating at 40 ℃, adding 0.1ml of diluent for redissolving, centrifuging for 5min by vortex at 14000rpm, taking the supernatant, injecting and measuring.
Fourthly, adopting a liquid chromatography-mass spectrometer for measurement
Chromatographic conditions are as follows:
waters ACQUITY UPLC ultra high performance liquid chromatograph system: comprises a binary pump, an automatic sample injector and a column incubator. And (3) analyzing the column: waters Acquity UPLC BEH C18(100 mm. times.2.1 mm, 1.7 μm); protection of the column: waters Acquity UPLC BEH C18(5 mm. times.2.1 mm, 1.7 μm). Adopting a C18 chromatographic column as a separation medium, and carrying out gradient elution after sample injection, wherein the ratio of a mobile phase A: acetonitrile, mobile phase B:
water containing 0.1% formic acid; gradient elution conditions:
the flow rate is 0.4 ml/min; column temperature: 40 ℃; temperature of a sample injection tray: 25 ℃; sample introduction amount: 2 μ l.
Mass spectrum conditions:
waters Xevo TQ-S triple quadrupole mass spectrometry; ESI ion source: a positive ion analysis mode; MRM scan mode.
Setting parameters: capillary voltage: 1.9 KV; taper hole voltage: 30V; nitrogen pressure: 7 psi; desolventizing gas temperature: 400 ℃; desolventizing air flow rate: 800 L.h-1(ii) a Taper hole air flow rate: 150 L.h-1(ii) a Collision airflow rate: 0.15 mL/min-1；
Multiple reaction detection mode (MRM):
taking the prepared polypeptide series concentration mixed standard solution, taking the mass concentration (X) of a substance to be detected as a horizontal coordinate, determining by adopting a liquid chromatography-mass spectrometry method, and taking the peak area ratio (Y) of a target analyte and an internal standard as a vertical coordinate to obtain a standard curve of the polypeptide; and obtaining the content of the polypeptide in the effective components of the Shuxuetong injection in the blood sample according to the standard curve.
Claims (6)
1. A method for measuring the content of polypeptide in Shuxuetong injection is characterized by comprising the following steps:
preparing stock solution and internal standard stock solution;
weighing four polypeptides of Tp-ww-1504, Tp-ww-1505, Tp-ww-1506 and Tp-ww-1511, respectively adding water (v/v) containing 0.1% formic acid, and vortexing to completely dissolve the polypeptides to prepare polypeptide stock solution; weighing triptorelin, adding water (v/v) containing 0.1% formic acid, and vortexing to dissolve triptorelin completely to obtain triptorelin internal standard stock solution;
step two, preparing a standard substance solution;
respectively transferring the stock solutions, and gradually diluting the stock solutions into polypeptide standard solutions with serial concentrations by using a diluent; respectively adding triptorelin internal standard solutions into the polypeptide standard solutions, uniformly mixing by vortex, and preparing a mixed standard solution containing the internal standard triptorelin by using a diluent;
step three, preparing a test solution
Transferring the Shuxuetong injection filtered by a 0.22 mu m microporous filter membrane and the internal standard triptorelin solution into an EP tube, diluting the injection by using a diluent, and performing vortex and uniform mixing to prepare a test solution containing the internal standard triptorelin;
or taking a plasma sample containing the effective components of the Shuxuetong injection, adding the triptorelin internal standard solution and the diluent, uniformly mixing by vortex, adding methanol, vortex, centrifuging, taking the supernatant, placing the supernatant in an EP tube, carrying out vacuum centrifugal concentration at 40 ℃, adding an appropriate amount of the diluent for redissolution, vortex, centrifuging, and taking the supernatant as a test sample solution containing the internal standard triptorelin;
taking the prepared polypeptide series concentration mixed standard solution, taking the concentration (X) of a substance to be detected as a horizontal coordinate, and taking the peak area ratio (Y) of the target analyte and the internal standard, which is measured by a liquid chromatography-mass spectrometry method, as a vertical coordinate, so as to obtain a standard curve of the polypeptide; determining the test sample solution by adopting a liquid chromatography-mass spectrometry method to obtain the peak area ratio of the target analyte to the internal standard, and calculating according to the standard curve to obtain the content of the polypeptide to be detected;
in the fourth step, the liquid phase part chromatographic conditions are as follows: adopting a C18 chromatographic column as a separation medium, and carrying out gradient elution after sample injection, wherein the ratio of a mobile phase A: acetonitrile or methanol, mobile phase B: an organic solvent or water at a mass concentration of 10% or less or water (v/v) containing 0.1% formic acid; gradient elution conditions: 0-0.3min 5% A, 95% B; 0.6-1.2min 20% A, 80% B; 2.5-3.5min 50% A, 50% B; 5% of A and 95% of B at 3.51-4.5 min; the flow rate is 0.1-0.4 ml/min; column temperature: 20-40 ℃;
mass spectrum detection conditions: an electrospray ESI ion source positive ion detection mode; capillary voltage: 1.0-3.0 KV; taper hole voltage: 2-70V; nitrogen pressure: 5-10 psi; desolventizing gas temperature: 300 ℃ and 450 ℃; desolventizing air flow rate: 600-900 L.h-1(ii) a Taper hole air flow rate: 150 L.h-1(ii) a Collision airflow rate: 0.1-0.25 mL/min-1；
The scanning mode is multi-reaction ion monitoring MRM, and the parameters are as follows:
2. the method for determining the content of the polypeptide in the Shuxuetong injection according to claim 1, which is characterized in that: in the first step, Tp-ww-1504, Tp-ww-1505, Tp-ww-1506 and Tp-ww-1511 are precisely weighed to be 0.44mg, 0.56mg, 0.64mg and 0.61mg respectively; adding water containing 0.1% formic acid, and vortex for 2min to dissolve completely to obtain 500 μ g/ml polypeptide stock solution; another 0.59mg triptorelin was precisely weighed, added with water (v/v) containing 0.1% formic acid, vortexed for 2min to completely dissolve it, and made into 500. mu.g/ml triptorelin internal standard solution.
3. The method for determining the content of the polypeptide in the Shuxuetong injection according to claim 2, which is characterized in that: in the second step, precisely transferring the stock solutions of the polypeptides Tp-ww-1504 and Tp-ww-1506, and diluting the stock solutions into polypeptide standard solutions with the serial concentrations of 3 mu g/ml, 2.5 mu g/ml, 2.0 mu g/ml, 1.5 mu g/ml and 1.0 mu g/ml by using a diluent respectively; precisely transferring a proper amount of a stock solution of the polypeptide Tp-ww-1505, and gradually diluting the stock solution into polypeptide standard solutions with the serial concentrations of 500ng/ml, 200ng/ml, 150ng/ml, 100ng/ml and 50ng/ml by using a diluent; precisely transferring a proper amount of a stock solution of the polypeptide Tp-ww-1511, and gradually diluting the stock solution into polypeptide standard solutions with serial concentrations of 1 mu g/ml, 800ng/ml, 500ng/ml, 400ng/ml and 200ng/ml by using a diluent;
taking the polypeptide standard solutions, sequentially mixing according to concentration gradient, adding 100 mul of triptorelin internal standard solution with the concentration of 1 mug/ml, uniformly mixing by vortex, preparing 100ng/ml triptorelin containing internal standard by using a diluent, wherein the polypeptide Tp-ww-1504 and Tp-ww-1506 are 300ng/ml, 250ng/ml, 200ng/ml, 150ng/ml and 100 ng/ml; polypeptide Tp-ww-1505 is contained in the polypeptide, and the polypeptide is 50ng/ml, 20ng/ml, 15ng/ml, 10ng/ml and 5 ng/ml; the mixed standard solution containing the polypeptide Tp-ww-1511 is 100ng/ml, 80ng/ml, 50ng/ml, 40ng/ml and 20 ng/ml.
4. The method for determining the content of the polypeptide in the Shuxuetong injection according to claim 3, which is characterized in that: in the third step, the injection for dredging blood circulation is taken and filtered by a microporous filter membrane of 0.22 mu m; precisely transferring 500 mu l of Shuxuetong and 100 mu l of 1 mu g/ml internal standard triptorelin into a 1.5ml EP tube, diluting 2 times by using a diluent, and performing vortex and uniform mixing; preparing a test solution containing 100ng/ml of triptorelin as an internal standard.
5. The method for determining the content of the polypeptide in the Shuxuetong injection according to claim 3, which is characterized in that: in the third step, 0.2ml of plasma sample containing Shuxuetong injection is taken, 50 mul of triptorelin internal standard solution and 50 mul of diluent are precisely added, vortex mixing is carried out, 0.8ml of methanol is added, vortex is carried out for 5min, centrifugation is carried out at 14000rpm for 10min, 0.9ml of supernatant is taken and placed in an EP tube, vacuum concentration is carried out at 40 ℃, 0.1ml of diluent is added for redissolution, vortex is carried out for 5min, centrifugation is carried out at 14000rpm for 5min, and the supernatant is taken for sampling and determination.
6. The method for determining the content of the polypeptide in the Shuxuetong injection according to the claim 1, 2, 3, 4 or 5, which is characterized in that: the diluent in the second step and the third step is selected from 60% methanol water (v/v) containing 0.01% -0.5% formic acid or 60% methanol water (v/v) containing 0.01% -0.1% trifluoroacetic acid.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1982892A (en) * | 2006-01-24 | 2007-06-20 | 李振国 | Method and use for setting up Shuxuetong injection or its intermediate fingerprint atlas |
| CN101748127A (en) * | 2008-12-11 | 2010-06-23 | 上海人类基因组研究中心 | Schistosoma japonica actin gene and a coding protein and application thereof |
| CN104897840A (en) * | 2015-06-18 | 2015-09-09 | 牡丹江友搏药业股份有限公司 | Novel method for quality control on polypeptide (oligopeptide) components in Shuxuetong injection |
| CN109374768A (en) * | 2018-11-12 | 2019-02-22 | 牡丹江友搏药业有限责任公司 | A kind of method of hypoxanthine content in measurement SHUXUETONG ZHUSHEYE |
| CN110133124A (en) * | 2019-04-29 | 2019-08-16 | 天津中医药大学 | The content assaying method of 18 kinds of amino acid in SHUXUETONG ZHUSHEYE |
-
2020
- 2020-11-17 CN CN202011285739.3A patent/CN112557527A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1982892A (en) * | 2006-01-24 | 2007-06-20 | 李振国 | Method and use for setting up Shuxuetong injection or its intermediate fingerprint atlas |
| CN101748127A (en) * | 2008-12-11 | 2010-06-23 | 上海人类基因组研究中心 | Schistosoma japonica actin gene and a coding protein and application thereof |
| CN104897840A (en) * | 2015-06-18 | 2015-09-09 | 牡丹江友搏药业股份有限公司 | Novel method for quality control on polypeptide (oligopeptide) components in Shuxuetong injection |
| CN109374768A (en) * | 2018-11-12 | 2019-02-22 | 牡丹江友搏药业有限责任公司 | A kind of method of hypoxanthine content in measurement SHUXUETONG ZHUSHEYE |
| CN110133124A (en) * | 2019-04-29 | 2019-08-16 | 天津中医药大学 | The content assaying method of 18 kinds of amino acid in SHUXUETONG ZHUSHEYE |
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