CN113567590A - Method for determining content of imiquimod component in SD rat plasma by HPLC-MS-MS method - Google Patents
Method for determining content of imiquimod component in SD rat plasma by HPLC-MS-MS method Download PDFInfo
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- 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
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Abstract
The invention provides a method for determining the content of an imiquimod component in SD rat plasma by an HPLC-MS-MS method, which comprises the following steps: respectively adding 10.0ng/mL imiquimod-D9 internal standard working solution into the calibration marked sample, the quality control sample, the internal standard sample and the SD rat sample to be detected to dilute by 20 times, respectively centrifuging to obtain supernate, and then respectively diluting the supernate by 2 times with ultrapure water solution to carry out sample injection analysis. Respectively adding the blank substrate and ultrapure water into the blank substrate and the solvent sample, respectively diluting by 20 times with acetonitrile, respectively centrifuging to obtain supernate, and then respectively diluting the supernate by 2 times with ultrapure water solution to obtain sample injection analysis. And respectively injecting the processed samples into a liquid chromatography-mass spectrometer, and quantitatively detecting the imiquimod and the imiquimod-D9. The invention has simple and convenient sample analysis, low detection limit, high sensitivity, good repeatability and recovery rate.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a method for determining the content of an imiquimod component in SD rat plasma by an HPLC-MS-MS method.
Background
With the development of mass spectrometry technology, biological sample analysis is increasingly widely applied. But due to the complex matrix and low drug concentration in the biological sample. Therefore, there is a need to obtain more accurate and stable assay methods. In the HPLC-MS-MS technique, high performance liquid chromatography is a continuous multiple exchange process of solute between stationary phase and mobile phase, which separates different solutes by means of their partition coefficient, affinity, adsorption capacity, ion exchange or exclusion differences caused by different molecular sizes between the two phases. Therefore, a liquid chromatography-mass spectrometry method for determining the concentration of imiquimod in SD rat plasma by HPLC-MS-MS technology is urgently needed to be developed, and the purpose is to obtain a more accurate and high-stability content determination method, so that scientific research and industrial application are facilitated.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide the method for determining the content of the imiquimod component in the SD rat plasma by the HPLC-MS-MS method, which is simple and convenient to analyze a sample, low in detection limit, high in sensitivity, good in repeatability and recovery rate.
The purpose of the invention is realized by the following technical scheme:
the invention provides a method for determining the content of an imiquimod component in SD rat plasma by an HPLC-MS-MS method, which comprises the following steps:
step one, sample preparation:
preparing a calibration marker sample: preparing correction marking working solution of imiquimod with different gradient concentrations by taking imiquimod as a solute and acetonitrile aqueous solution as a solvent; respectively taking the concentration correction marking working solution, and diluting the concentration correction marking working solution by using a blank matrix to obtain a correction marking sample;
preparing a quality control sample: preparing quality control working solutions with different gradient concentrations of imiquimod by taking imiquimod as a solute and acetonitrile aqueous solution as a solvent; respectively taking the quality control working solution with each concentration, and diluting the working solution with a blank matrix to obtain a quality control sample;
step two, sample treatment:
respectively adding imiquimod-D9 internal standard working solution into the calibration marked sample, the quality control sample, the internal standard sample and the SD rat plasma to be detected for dilution, respectively centrifuging to obtain supernate, and then respectively diluting the supernate with ultrapure water solution;
respectively adding acetonitrile into the blank matrix and the solvent sample for dilution, respectively centrifuging to obtain supernate, and then respectively diluting the supernate with ultrapure water solution;
step three, sample detection:
and (3) respectively injecting the corrected marked sample, the quality control sample, the internal standard sample, the SD rat plasma to be detected, the blank matrix and the water sample which are processed in the second step into a liquid chromatography-mass spectrometer, carrying out quantitative detection on the imiquimod and the imiquimod-D9 component, and obtaining the content of the imiquimod component in the SD rat plasma through regression and data processing.
Preferably, in the first step, the volume concentration of the acetonitrile aqueous solution used for preparing the calibration marker sample and the quality control sample is 50%; the dilution times with the blank matrix were all 20-fold.
Preferably, in the first step, the gradient concentration of the imiquimod in the calibration marking working solution is 20.0-10000 ng/mL; the gradient concentration of the imiquimod in the quality control working solution is 20.0-8000 ng/mL.
Preferably, in the second step, the concentration of the adopted imiquimod-D9 internal standard working solution is 10.0 ng/mL; the dilution times of the calibration marker sample, the quality control sample, the internal standard sample and the SD rat plasma to be detected by using the test kit are 20 times; the dilution factor with ultrapure water solution was 2 times.
Preferably, acetonitrile is added into the blank matrix and the solvent sample for dilution by a factor of 20; the dilution factor with ultrapure water solution was 2 times.
Preferably, in the first step, an acetonitrile formic acid solution containing imiquimod is adopted for preparing the sample by taking the imiquimod as a solute; the volume ratio of formic acid to acetonitrile to ultrapure water is 0.1:50: 50; the concentration of the imiquimod in the acetonitrile aqueous solution of formic acid is 1.00 mg/mL.
Preferably, in the third step, the detection is performed according to the following liquid chromatography conditions:
stationary phase: an ACE 5C18-PFP chromatographic column with the filler particle size of 5 μm, the diameter of 2.1mm and the length of 50 mm;
mobile phase: the mobile phase is a mixed system of A and B, wherein A is a formic acid aqueous solution, and the volume ratio of formic acid to water is 0.02: 100; b is formic acid acetonitrile solution, wherein the volume ratio of formic acid to acetonitrile is 0.02: 100;
the elution gradient was:
0.01min, wherein the volume percent of A is 90 percent, and the volume percent of B is 10 percent;
2.00min, the volume percent of A is 0 percent, and the volume percent of B is 100 percent;
2.80min, the volume percent of A is 0 percent, and the volume percent of B is 100 percent;
2.81min, the volume percent of A is 90 percent, and the volume percent of B is 10 percent;
3.00min, the volume percent of A is 90 percent, and the volume percent of B is 10 percent.
Preferably, in the third step, the liquid chromatography sample injector cleaning solution is: the weak washing is carried out by mixing 30% methanol aqueous solution and the strong washing is carried out by mixing methanol, acetonitrile, isopropanol and water according to the volume ratio of 1:1:1: 1.
Preferably, in the third step, the detection is performed according to the following liquid chromatography conditions:
flow rate: 0.40 mL/min;
column temperature: 40 ℃;
autosampler temperature: 4 ℃;
sample introduction amount: 10 μ L.
Preferably, in the third step, the detection is performed according to the following mass spectrometry conditions:
an ion source: an electrospray ion source;
ionization mode: a positive ion mode;
the resolution mode is Unit;
the collision gas, the air curtain gas, the atomization gas, the auxiliary gas 1 and the auxiliary gas 2 are all high-purity nitrogen;
the spraying voltage is 5500V.
Preferably, in the third step, the regression and data processing method is as follows:
the regression model is y ═ ax + b, linear regression, weight factor 1/x2Y is the peak area ratio of the analyte to the internal standard, and x is the calibrationPositive marker concentration of analyte in the sample;
the calculation software was Analyst 1.6.3 and Microsoft Office 2016(or other version), all concentration values retained 3 significant digits, with% Bias and% CV retained to 1 digit after the decimal point;
an analyte: imiquimod;
matrix: SD rat plasma (heparin sodium is an anticoagulant);
calibration curve range: 1.00 ng/mL-500 ng/mL;
lower limit of quantitation: 1.00 ng/mL;
linear range: 1.00 ng/mL-500 ng/mL.
The invention has the beneficial effects that:
the method for determining the content of the imiquimod component in the SD rat plasma by the HPLC-MS-MS method has the advantages of simple and convenient sample analysis, low detection limit, high sensitivity, good repeatability and good recovery rate.
In order to make the aforementioned and other objects, features and advantages of the invention comprehensible, preferred embodiments accompanied with figures are described in detail below.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a spectrum of the analyte in a solvent sample of the present invention as imiquimod (top)/imiquimod-D9 (bottom);
FIG. 2 is a spectrum of the analyte in the double blank sample of the present invention as imiquimod (up)/imiquimod-D9 (down);
FIG. 3 is a spectrum of an internal standard sample with an analyte of imiquimod (top)/imiquimod-D9 (bottom) according to an example of the present invention;
FIG. 4 is a graph of the residual effect of an example of the present invention in which the analyte is imiquimod (top)/imiquimod-D9 (bottom);
FIG. 5 is a plot of the analyte in the lower limit of quantitation for the calibration markers in the example of the invention as imiquimod (top)/imiquimod-D9 (bottom);
FIG. 6 is a plot of the upper limit of quantitation for the analyte in the calibration standards of the present invention as imiquimod (top)/imiquimod-D9 (bottom).
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The apparatus and reagents in the following examples are as follows:
the instrument comprises the following steps:
high performance liquid chromatography Shimadzu LC-30AD
Mass spectrometer AB SCIEX-5000Q TRAP-LC/MS/MS system
Data acquisition and management of analysis 1.6.3, Applied Biosystem
Daily computing/reporting handles Office 2016 or other version, Microsoft
Reagent testing:
acetonitrile HPLC grade
Methanol HPLC grade
Isopropanol HPLC grade
Grade AR formic acid
The embodiment provides a method for determining the content of an imiquimod component in SD rat plasma by an HPLC-MS-MS method, which comprises the following steps:
firstly, sample and solution preparation:
1. preparing a calibration marker sample:
an amount of imiquimod was weighed and dissolved in acetonitrile formate in water (0.1/50/50, v/v/v) to give a 1.00mg/mL stock solution of imiquimod.
1.00mg/mL of imiquimod stock solution is diluted by 50% acetonitrile aqueous solution to obtain the working solution of the imiquimod series with the concentration of 20, 40, 100, 400, 2000, 9000 and 10000ng/mL of calibration marks.
10 mu L of calibration marker working solution with each concentration is respectively mixed with 190 mu L of blank matrix to respectively obtain calibration marker samples with the imiquimod series concentrations of 1, 2, 5, 20, 100, 450 and 500 ng/mL.
2. Preparing a quality control sample:
an amount of imiquimod was weighed and dissolved in acetonitrile formate in water (0.1/50/50, v/v/v) to give a 1.00mg/mL stock solution of imiquimod. Taking 1.00mg/mL imiquimod stock solution, diluting with 50% acetonitrile water solution to obtain quality control working solution with imiquimod series concentration of 20, 60, 600 and 8000 ng/mL.
10 mu L of quality control working solution with each concentration is respectively mixed with 190 mu L of blank matrix to respectively obtain quality control samples with imiquimod series concentrations of 1, 3, 30 and 400 ng/mL.
3. A double blank sample was prepared: blank matrices, in this example, the hollow white matrices were SD rat plasma collected from the department of biological medicine, suzhou, ltd, with heparin sodium as an anticoagulant.
4. Preparation of solvent samples: and (4) sampling a solvent. The water used in this example was ultrapure water.
5. Preparing a sample to be tested: namely SD rat plasma samples to be tested.
6. Preparation of pure liquid samples: and (3) acetonitrile.
7. Preparation of recovery and matrix effect pure solution, i.e. neat solution:
the solution of imiquimod-D9 was mixed with 50% acetonitrile to give a final concentration of 47.5ng/mL of imiquimod-D9.
The imiquimod solution and 50% acetonitrile in water were mixed to give a final concentration of 0.75, 100ng/mL imiquimod.
Secondly, sample treatment:
the internal standard working solution used in the following processing steps was 10.0ng/mL imiquimod-D9 internal standard working solution. Acetonitrile represents 100% acetonitrile.
And uniformly mixing 10 mu L of the calibration marker sample and 190 mu L of the internal standard working solution, centrifuging to obtain 100 mu L of supernatant, adding 100 mu L of aqueous solution, uniformly mixing by low-speed vortex, and carrying out sample injection analysis.
And (3) uniformly mixing 10 mu L of quality control sample and 190 mu L of internal standard working solution, centrifuging to obtain 100 mu L of supernatant, adding 100 mu L of aqueous solution, uniformly mixing by low-speed vortex, and carrying out sample injection analysis.
And uniformly mixing 10 mu L of internal standard sample, namely blank matrix, 190 mu L of internal standard working solution, centrifuging, taking 100 mu L of supernatant, adding 100 mu L of aqueous solution, uniformly mixing by low-speed vortex, and carrying out sample injection analysis.
And (3) uniformly mixing 10 mu L of a sample to be detected (SD rat plasma to be detected) and 190 mu L of internal standard working solution, centrifuging to obtain 100 mu L of supernatant, adding 100 mu L of aqueous solution, uniformly mixing by low-speed vortex, and carrying out sample injection analysis.
Mixing 10 μ L solvent sample, i.e. water, 190 μ L acetonitrile, centrifuging, collecting supernatant 100 μ L, adding 100 μ L water solution, low speed vortex mixing, and analyzing by sample introduction.
And (3) uniformly mixing 10 mu L of double blank sample and 190 mu L of acetonitrile, centrifuging to obtain 100 mu L of supernatant, adding 100 mu L of aqueous solution, uniformly mixing by low-speed vortex, and carrying out sample injection analysis. Double blank samples refer to blank matrices without added analyte and internal standard for residue examination.
Uniformly mixing 10 mu L of matrix effect sample and 190 mu L of acetonitrile; centrifuging to obtain 100 μ L of supernatant, mixing with 20 μ L of Lneat solution and 80 μ L of aqueous solution, low speed vortex mixing, and analyzing by sample injection. The matrix effect sample is at least 6 batches of blank matrix.
Mixing 10 μ L recovery sample, namely blank matrix, 190 μ L acetonitrile, centrifuging to obtain supernatant 100 μ L, mixing with 20 μ L neat solution and 80 μ L aqueous solution, low speed vortex mixing, and sample injection analysis.
Mixing 10 μ L pure liquid sample, i.e. water, 190 μ L acetonitrile, centrifuging, collecting supernatant 100 μ L, mixing with 20 μ Lneat solution, 80 μ L aqueous solution, low speed vortex, and analyzing.
Thirdly, sample detection:
and (3) respectively injecting the corrected marked sample, the quality control sample, the internal standard sample, the sample to be detected, the double blank sample, the solvent sample, the pure liquid sample, the matrix effect sample and the recovery rate sample which are processed in the step two into a liquid chromatography-mass spectrometer, and quantitatively detecting the imiquimod and the imiquimod-D9, wherein the obtained corresponding spectrogram is shown in the attached drawings 1-6.
FIG. 1 is a sample pre-treatment sample LC-MS/MS mass spectrum (upper: 0ng/mL imiquimod/lower: 0ng/mL imiquimod-D9); FIG. 2 is a sample LC-MS/MS mass spectrum of a blank matrix sample after sample pretreatment (upper: 0ng/mL imiquimod/lower: 0ng/mL imiquimod-D9); FIG. 3 is an LC-MS/MS mass spectrum of an internal standard sample after sample pretreatment (upper: 0ng/mL imiquimod/lower: 10ng/mL imiquimod-D9); FIG. 4 is a sample LC-MS/MS mass spectrum (top: 0ng/mL imiquimod/bottom: 0ng/mL imiquimod-D9) of a blank matrix sample (blank matrix after examination of ULOQ samples to examine residues) after sample pretreatment; FIG. 5 is a mass spectrum of a sample LC-MS/MS (upper: 1ng/mL imiquimod/lower: 10ng/mL imiquimod-D9) after pretreatment of a quantitative lower limit sample; FIG. 6 shows the LC-MS/MS mass spectrum of the upper limit of the quantitation sample after sample pretreatment (upper: 500ng/mL imiquimod/lower: 10ng/mL imiquimod-D9).
Wherein, in the detection step, the detection is carried out according to the following liquid chromatography conditions:
stationary phase: an ACE 5C18-PFP chromatographic column with the filler particle size of 5 μm, the diameter of 2.1mm and the length of 50 mm;
mobile phase: the mobile phase is a mixed system of A and B, the A is formic acid aqueous solution, wherein the volume ratio of formic acid to water is 0.02:100, and the preparation method of the A is that 1000mLH is added into a 1L glass bottle2O and 0.2mL formic acid, mix well.
B is formic acid acetonitrile solution, wherein the volume ratio of formic acid to acetonitrile is 0.02:100, and the preparation method of B is that 1000mL of acetonitrile and 0.2mL of formic acid are added into a 1L glass bottle and mixed evenly.
The elution gradient was:
0.01min, wherein the volume percent of A is 90 percent, and the volume percent of B is 10 percent;
2.00min, the volume percent of A is 0 percent, and the volume percent of B is 100 percent;
2.80min, the volume percent of A is 0 percent, and the volume percent of B is 100 percent;
2.81min, the volume percent of A is 90 percent, and the volume percent of B is 10 percent;
3.00min, the volume percent of A is 90 percent, and the volume percent of B is 10 percent.
The sample injector cleaning solution is: the weak washing is injector cleaning solution 1, the injector cleaning solution 1 is 30% methanol water solution, and the preparation method of the 30% methanol water solution is that 300mL of methanol and 700mL of H are added into a 1L glass bottle2And O, and mixing uniformly.
The strong washing is sample injector cleaning liquid 2, and the sample injector cleaning liquid 2 is formed by mixing methanol, acetonitrile, isopropanol and water according to the volume ratio of 1:1:1: 1. The injector cleaning solution 2 was prepared by adding 250mL of methanol, 250mL of acetonitrile, 250mL of isopropanol, and 250mLH to a 1L glass bottle2And O, and mixing uniformly.
The diluent 1 is a formic acid acetonitrile water solution, and the preparation method of the formic acid acetonitrile water solution comprises the steps of adding 500mL of acetonitrile, 500mL of water and 1.0mL of formic acid into a 1L glass bottle, and uniformly mixing.
Flow rate: 0.40 mL/min;
column temperature: 40 ℃;
autosampler temperature: 4 ℃;
sample introduction amount: 10 μ L.
The retention time of imiquimod was approximately 1.37min and the retention time of imiquimod-D9 was approximately 1.36min at the time of injection.
Wherein, in the detection step, the detection is carried out according to the following mass spectrum conditions:
an ion source: an electrospray ion source;
ionization mode: a positive ion mode;
the resolution mode is Unit;
the collision gas, the air curtain gas, the atomization gas, the auxiliary gas 1 and the auxiliary gas 2 are all high-purity nitrogen;
the spraying voltage is 5500V.
Four, regression and data processing
The regression model is y ═ ax + b, linear regression, weight factor 1/x2Y is the peak area ratio of the analyte to the internal standard, and x is the concentration of the analyte in the calibration marker.
The software was calculated as Analyst 1.6.3 and Microsoft Office 2016(or other version), with all concentration values retained 3 significant digits and% Bias and% CV retained to 1 digit after the decimal point.
An analyte: imiquimod;
matrix: SD rat plasma (heparin sodium is an anticoagulant);
calibration curve range: 1.00 ng/mL-500 ng/mL;
lower limit of quantitation: 1.00 ng/mL;
linear range: 1.00 ng/mL-500 ng/mL.
The principle and the implementation mode of the invention are explained by applying specific embodiments in the invention, and the description of the embodiments is only used for helping to understand the method and the core idea of the invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present invention.
Claims (10)
1. A method for determining the content of an imiquimod component in SD rat plasma by an HPLC-MS-MS method is characterized by comprising the following steps:
step one, sample preparation:
preparing a calibration marker sample: preparing correction marking working solution of imiquimod with different gradient concentrations by taking imiquimod as a solute and acetonitrile aqueous solution as a solvent; respectively taking the concentration correction marking working solution, and diluting the concentration correction marking working solution by using a blank matrix to obtain a correction marking sample;
preparing a quality control sample: preparing quality control working solutions with different gradient concentrations of imiquimod by taking imiquimod as a solute and acetonitrile aqueous solution as a solvent; respectively taking the quality control working solution with each concentration, and diluting the working solution with a blank matrix to obtain a quality control sample;
step two, sample treatment:
respectively adding imiquimod-D9 internal standard working solution into the calibration marked sample, the quality control sample, the internal standard sample and the SD rat plasma to be detected for dilution, respectively centrifuging to obtain supernate, and then respectively diluting the supernate with ultrapure water solution;
respectively adding acetonitrile into the blank matrix and the solvent sample for dilution, respectively centrifuging to obtain supernate, and then respectively diluting the supernate with ultrapure water solution;
step three, sample detection:
and (3) respectively injecting the corrected marked sample, the quality control sample, the internal standard sample, the SD rat plasma to be detected, the blank matrix and the solvent sample which are processed in the second step into a liquid chromatography-mass spectrometer, carrying out quantitative detection on the imiquimod and the imiquimod-D9 component, and obtaining the content of the imiquimod component in the SD rat plasma through regression and data processing.
2. The method for determining the content of imiquimod component in SD rat plasma by HPLC-MS-MS method according to claim 1, wherein in the first step, the volume concentration of acetonitrile aqueous solution used for preparing the calibration label sample and the quality control sample is 50%; the dilution times with the blank matrix were all 20-fold.
3. The method for determining the content of the imiquimod component in the plasma of SD rats by the HPLC-MS-MS method as claimed in claim 1, wherein in the first step, the gradient concentration of the imiquimod in the calibration label working solution is 20.0-10000 ng/mL; the gradient concentration of the imiquimod in the quality control working solution is 20.0-8000 ng/mL.
4. The method for determining the content of the imiquimod component in the plasma of SD rats by the HPLC-MS-MS method according to claim 1, wherein in the second step, the concentration of the working solution of the imiquimod-D9 internal standard is 10.0 ng/mL; the dilution times of the calibration marker sample, the quality control sample, the internal standard sample and the SD rat plasma to be detected by using the test kit are 20 times; the dilution times of the ultrapure water solution are 2 times;
adding acetonitrile into the blank matrix and the solvent sample for dilution by a factor of 20; the dilution factor with ultrapure water solution was 2 times.
5. The method for determining the content of imiquimod component in the plasma of SD rats by HPLC-MS-MS method according to claim 1, wherein in the first step, the imiquimod is used as solute to prepare a sample, and the sample is acetonitrile solution of formic acid containing imiquimod; the volume ratio of formic acid to acetonitrile to ultrapure water is 0.1:50: 50; the concentration of the imiquimod in the acetonitrile aqueous solution of formic acid is 1.00 mg/mL.
6. The method for determining the content of imiquimod component in SD rat plasma by HPLC-MS-MS method according to claim 1, wherein in the third step, the detection is performed according to the following liquid chromatography conditions:
stationary phase: an ACE 5C18-PFP chromatographic column with the filler particle size of 5 μm, the diameter of 2.1mm and the length of 50 mm;
mobile phase: the mobile phase is a mixed system of A and B, wherein A is a formic acid aqueous solution, and the volume ratio of formic acid to water is 0.02: 100; b is formic acid acetonitrile solution, wherein the volume ratio of formic acid to acetonitrile is 0.02: 100;
the elution gradient was:
0.01min, wherein the volume percent of A is 90 percent, and the volume percent of B is 10 percent;
2.00min, the volume percent of A is 0 percent, and the volume percent of B is 100 percent;
2.80min, the volume percent of A is 0 percent, and the volume percent of B is 100 percent;
2.81min, the volume percent of A is 90 percent, and the volume percent of B is 10 percent;
3.00min, the volume percent of A is 90 percent, and the volume percent of B is 10 percent.
7. The method for determining the content of imiquimod component in SD rat plasma by HPLC-MS-MS method according to claim 1, wherein in the third step, the sample injector cleaning solution of liquid chromatography is: the weak washing is carried out by mixing 30% methanol aqueous solution and the strong washing is carried out by mixing methanol, acetonitrile, isopropanol and water according to the volume ratio of 1:1:1: 1.
8. The method for determining the content of imiquimod component in SD rat plasma by HPLC-MS-MS method according to claim 1, wherein in the third step, the detection is performed according to the following liquid chromatography conditions:
flow rate: 0.40 mL/min;
column temperature: 40 ℃;
autosampler temperature: 4 ℃;
sample introduction amount: 10 μ L.
9. The method for determining the content of imiquimod component in SD rat plasma by HPLC-MS-MS method according to claim 1, wherein in the third step, the detection is performed according to the following mass spectrum conditions:
an ion source: an electrospray ion source;
ionization mode: a positive ion mode;
the resolution mode is Unit;
the collision gas, the air curtain gas, the atomization gas, the auxiliary gas 1 and the auxiliary gas 2 are all high-purity nitrogen;
the spraying voltage is 5500V.
10. The method for determining the content of imiquimod component in SD rat plasma by HPLC-MS-MS method according to claim 1, wherein in the third step, the following regression and data processing methods are adopted:
the regression model is y ═ ax + b, linear regression, weight factor 1/x2Y is the peak area ratio of the analyte to the internal standard, and x is the concentration of the analyte in the calibration marker sample;
the calculation software was Analyst 1.6.3 and Microsoft Office 2016(or other version), all concentration values retained 3 significant digits, with% Bias and% CV retained to 1 digit after the decimal point;
an analyte: imiquimod;
matrix: SD rat plasma (heparin sodium is an anticoagulant);
calibration curve range: 1.00 ng/mL-500 ng/mL;
lower limit of quantitation: 1.00 ng/mL;
linear range: 1.00 ng/mL-500 ng/mL.
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| US20080213906A1 (en) * | 2007-01-26 | 2008-09-04 | Sigma Aldrich Company | Compositions and methods for combining protein precipitation and solid phase extraction |
| US20120003298A1 (en) * | 2010-04-30 | 2012-01-05 | Alcide Barberis | Methods for inducing an immune response |
| CN109633065A (en) * | 2018-12-28 | 2019-04-16 | 沈阳出入境检验检疫局检验检疫综合技术中心 | The detection method of medicament residue in a kind of animal body |
| CN110412158A (en) * | 2019-07-18 | 2019-11-05 | 浙江公正检验中心有限公司 | A kind of detection method for class medicament residue antiviral in animal derived product |
-
2021
- 2021-08-10 CN CN202110910998.9A patent/CN113567590B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080213906A1 (en) * | 2007-01-26 | 2008-09-04 | Sigma Aldrich Company | Compositions and methods for combining protein precipitation and solid phase extraction |
| US20120003298A1 (en) * | 2010-04-30 | 2012-01-05 | Alcide Barberis | Methods for inducing an immune response |
| CN109633065A (en) * | 2018-12-28 | 2019-04-16 | 沈阳出入境检验检疫局检验检疫综合技术中心 | The detection method of medicament residue in a kind of animal body |
| CN110412158A (en) * | 2019-07-18 | 2019-11-05 | 浙江公正检验中心有限公司 | A kind of detection method for class medicament residue antiviral in animal derived product |
Non-Patent Citations (3)
| Title |
|---|
| BALIREDDI V 等: "Development and Validation of Stability Indicating UPLC–PDA/MS for the Determination of Imiquimod and its Eight Related Substances: Application to Topical Cream", 《JOURNAL OF CHROMATOGRAPHIC SCIENCE》 * |
| HARRISON L I 等: "Pharmacokinetics and safety of imiquimod 5% cream in the treatment of actinic keratoses of the face, scalp, or hands and arms", 《ARCHIVES OF DERMATOLOGICAL RESEARCH》 * |
| 刘文静 等: "UPLC-MS/MS测定清热凉血方中5种成分的含量", 《中药材》 * |
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