CN113567590B - Method for determining imiquimod component content in SD rat plasma by HPLC-MS-MS method - Google Patents
Method for determining imiquimod component content in SD rat plasma by HPLC-MS-MS method Download PDFInfo
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Abstract
The invention provides a method for determining imiquimod component content in SD rat plasma by HPLC-MS-MS method, which comprises the following steps: and respectively adding 10.0ng/mL imiquimod-D9 internal standard working solution into the correction marking sample, the quality control sample, the internal standard sample and the SD rat sample to be tested to dilute for 20 times, respectively centrifuging to obtain supernatant, and respectively diluting the supernatant with ultrapure water solution for 2 times to obtain the sample injection analysis. And respectively adding the blank matrix and the ultrapure water into the blank matrix and the solvent sample, respectively diluting the blank matrix and the ultrapure water by 20 times, respectively centrifuging to obtain supernatant, and respectively diluting the supernatant by 2 times by using an ultrapure water solution to obtain the sample injection analysis. And (3) injecting the treated samples into a liquid chromatograph-mass spectrometer respectively, and quantitatively detecting the imiquimod-D9 component. The invention has the advantages of 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 measuring imiquimod component content in SD rat plasma by an HPLC-MS-MS method.
Background
Biological sample analysis is increasingly used with the development of mass spectrometry technology. But due to the complex matrix in the biological sample and the low drug concentration. Thus, there is a need to obtain a more accurate and stable assay. In HPLC-MS technology, high performance liquid chromatography is a continuous and multiple exchange process of solutes between stationary and mobile phases, which separates different solutes by means of differences in partition coefficients, affinities, adsorption capacities, ion exchange or rejection due to differences in molecular size between the two phases. Therefore, it is needed to develop a liquid chromatography-mass spectrometry method for establishing and verifying imiquimod concentration determination in SD rat plasma by adopting HPLC-MS-MS technology, and aims to obtain a content determination method with higher accuracy and high stability, thereby facilitating scientific research and industrial application.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a method for determining the imiquimod component content in SD rat plasma by an HPLC-MS-MS method, which is simple and convenient for sample analysis, and has low detection limit, high sensitivity and good repeatability and recovery rate.
The aim of the invention is achieved by the following technical scheme:
the invention provides a method for determining imiquimod component content in SD rat plasma by HPLC-MS-MS method, which comprises the following steps:
step one, sample preparation:
preparing a correction mark sample: the method comprises the steps of preparing correction marking working solutions with different gradient concentrations of imiquimod by taking imiquimod as a solute and acetonitrile aqueous solution as a solvent; respectively taking correction marking working solutions of various concentrations, and diluting with a blank matrix to obtain correction marking samples;
preparing a quality control sample: taking imiquimod as a solute, and acetonitrile aqueous solution as a solvent to prepare quality control working solutions with different gradient concentrations of imiquimod; respectively taking quality control working solutions with various concentrations, and diluting with a blank matrix to obtain a quality control sample;
step two, sample treatment:
respectively adding imiquimod-D9 internal standard working solution into correction marking sample, quality control sample, internal standard sample and SD rat plasma to be tested for dilution, respectively centrifuging to obtain supernatant, and respectively diluting the supernatant with ultrapure water solution;
respectively adding acetonitrile into the blank matrix and the solvent sample for dilution, respectively centrifuging to obtain supernatant, and respectively diluting the supernatant with ultrapure water solution;
step three, sample detection:
and (3) respectively injecting the correction marking sample, the quality control sample, the internal standard sample, the SD rat plasma to be detected, the blank matrix and the water sample treated in the step (II) into a liquid chromatography mass spectrometer, quantitatively detecting imiquimod and imiquimod-D9 components, and obtaining the imiquimod component content 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 mark sample and the quality control sample is 50%; the dilution with blank matrix was 20-fold.
Preferably, in the first step, the gradient concentration of imiquimod in the calibration marking working solution is 20.0-10000 ng/mL; the gradient concentration of imiquimod in the quality control working solution is 20.0-8000 ng/mL.
Preferably, in the second step, the concentration of the imiquimod-D9 internal standard working solution adopted is 10.0ng/mL; the correction marking sample, the quality control sample, the internal standard sample and the SD rat plasma to be tested are diluted by 20 times; the dilution with ultrapure water solution was 2-fold.
Preferably, acetonitrile is added to the blank matrix and solvent sample to dilute the blank matrix and solvent sample by a factor of 20; the dilution with ultrapure water solution was 2-fold.
Preferably, in the first step, the preparation sample using imiquimod as a solute adopts acetonitrile aqueous solution containing imiquimod; formic acid, acetonitrile and ultrapure water in a volume ratio of 0.1:50:50; the concentration of imiquimod in the acetonitrile aqueous solution of formic acid is 1.00mg/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 a filler particle diameter of 5 mu m, a diameter of 2.1mm and a length of 50 mm;
mobile phase: the mobile phase is a mixed system of A and B, A is formic acid aqueous solution, wherein 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 is:
0.01min, 90% by volume of A and 10% by volume of B;
2.00min, wherein the volume percentage of A is 0 percent, and the volume percentage of B is 100 percent;
2.80min, wherein the volume percentage of A is 0 percent, and the volume percentage of B is 100 percent;
2.81min, volume percentage of A is 90%, volume percentage of B is 10%;
3.00min, the volume percentage of A is 90%, and the volume percentage of B is 10%.
Preferably, in the third step, the liquid chromatography injector cleaning solution is: the weak washing adopts 30% methanol aqueous solution, and the strong washing adopts methanol, acetonitrile, isopropanol and water which are mixed 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: 0.40mL/min;
column temperature: 40 ℃;
autoinjector temperature: 4 ℃;
sample injection amount: 10 mu L.
Preferably, in the third step, the detection is performed according to the following mass spectrometry conditions:
ion source: an electrospray ion source;
ionization mode: a positive ion mode;
the resolution mode is Unit;
the collision gas, the gas curtain gas, the atomization gas, the auxiliary gas 1 and the auxiliary gas 2 are all high-purity nitrogen;
spray voltage 5500V.
Preferably, in the third step, the following regression and data processing methods are adopted:
regression model y=ax+b, linear regression, weight factor 1/x 2 Y is the peak area ratio of the analyte to the internal standard, x is the concentration of the analyte in the calibration label;
the calculation software is Analyst 1.6.3 and Microsoft Office 2016 (or other version), all concentration values remain 3 significant digits, and% Bias and% CV remain 1 after decimal point;
analyte: imiquimod;
a substrate: SD rat plasma (heparin sodium as anticoagulant);
correction curve range: 1.00 ng/mL-500 ng/mL;
lower limit of quantification: 1.00ng/mL;
linear range: 1.00 ng/mL-500 ng/mL.
The invention has the beneficial effects that:
the method for determining the imiquimod component content in the SD rat plasma by using the HPLC-MS-MS method is simple and convenient for sample analysis, and has the advantages of low detection limit, high sensitivity, good repeatability and recovery rate.
The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments, as illustrated in the accompanying drawings.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a spectrum of imiquimod (up)/imiquimod-D9 (down) as an analyte in a solvent sample in an embodiment of the invention;
FIG. 2 is a graph of the analyte imiquimod (top)/imiquimod-D9 (bottom) in a double blank sample according to an embodiment of the present invention;
FIG. 3 is a spectrum of imiquimod (top)/imiquimod-D9 (bottom) as the analyte in an internal standard sample in an embodiment of the invention;
FIG. 4 is a graph showing the residual effect of imiquimod (up)/imiquimod-D9 (down) as an analyte in an embodiment of the invention;
FIG. 5 is a graph of the lower limit of the quantification of the calibration label sample for imiquimod (up)/imiquimod-D9 (down) in an embodiment of the present invention;
FIG. 6 is a graph of the upper limit of the amount of the analyte in the calibration label sample in the example of the present invention, imiquimod (upper)/imiquimod-D9 (lower).
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The instruments and reagents in the following examples are as follows:
instrument:
high performance liquid chromatography Shimadzu LC-30AD
Mass spectrometer AB SCIEX-5000Q TRAP-LC/MS/MS system
Data acquisition and management analysis 1.6.3,Applied Biosystem
Daily computing/reporting Office 2016 or other version,Microsoft
Reagent:
acetonitrile HPLC grade
Methanol HPLC grade
Isopropanol HPLC grade
AR grade formic acid
The present example provides a method for determining imiquimod component content in SD rat plasma by HPLC-MS method, comprising the steps of:
1. sample and solution preparation:
1. preparing a correction mark sample:
an amount of imiquimod was weighed and dissolved in acetonitrile water (0.1/50, v/v/v) formate to give 1.00mg/mL imiquimod stock.
1.00mg/mL imiquimod stock solution was diluted with 50% acetonitrile aqueous solution to obtain imiquimod series concentrations of 20, 40, 100, 400, 2000, 9000 and 10000ng/mL of calibration marking working solution.
And respectively mixing 10 mu L of correction marking working solution with 190 mu L of blank matrix to obtain the correction marking samples with the imiquimod series concentration of 1, 2, 5, 20, 100, 450 and 500ng/mL.
2. Preparing a quality control sample:
an amount of imiquimod was weighed and dissolved in acetonitrile water (0.1/50, v/v/v) formate to give 1.00mg/mL imiquimod stock. Taking 1.00mg/mL imiquimod stock solution, and diluting the imiquimod stock solution with 50% acetonitrile aqueous solution to obtain quality control working solution with imiquimod series concentration of 20, 60, 600 and 8000ng/mL.
And respectively taking 10 mu L of quality control working solution with each concentration and mixing with 190 mu L of blank matrix to respectively obtain quality control samples with imiquimod series concentration of 1, 3, 30 and 400 ng/mL.
3. Double blank samples were prepared: the blank matrices, in this example, were all SD rat plasma collected from the company of the biological medicine of the collar (su zhou), with heparin sodium as the anticoagulant.
4. Preparing a solvent sample: solvent-like. The water used in this example was ultrapure water.
5. Preparing a sample to be tested: i.e. SD rat plasma samples to be tested.
6. Preparing a pure liquid sample: acetonitrile.
7. Preparing a pure solution with recovery rate and matrix effect, namely a heat solution:
imiquimod-D9 solution was mixed with 50% acetonitrile in water to give a final imiquimod-D9 concentration of 47.5ng/mL.
The final imiquimod concentration was 0.75 ng/mL by mixing imiquimod solution with 50% acetonitrile in water.
2. Sample treatment:
the internal standard working solution used in the following treatment steps was 10.0ng/mL imiquimod-D9 internal standard working solution. Acetonitrile represents 100% acetonitrile.
Mixing 10 μl of calibration sample and 190 μl of internal standard working solution, centrifuging to obtain supernatant 100 μl, adding 100 μl of aqueous solution, mixing at low speed, and performing sample injection analysis.
Mixing 10 μl of quality control sample and 190 μl of internal standard working solution, centrifuging to obtain supernatant 100 μl, adding 100 μl of aqueous solution, mixing at low speed, and performing sample injection analysis.
Mixing 10 μl of internal standard sample, namely blank matrix, 190 μl of internal standard working solution, centrifuging to obtain 100 μl of supernatant, adding 100 μl of aqueous solution, mixing at low speed, and performing sample injection analysis.
Mixing 10 μl of sample to be tested (SD rat plasma to be tested) and 190 μl of internal standard working solution uniformly, centrifuging to obtain 100 μl of supernatant, adding 100 μl of aqueous solution, mixing at low speed, and performing sample injection analysis.
Mixing 10 μl solvent sample (water, 190 μl acetonitrile) uniformly, centrifuging to obtain 100 μl supernatant, adding 100 μl aqueous solution, mixing at low speed, and performing sample injection analysis.
Mixing 10 μl of double blank sample and 190 μl of acetonitrile uniformly, centrifuging to obtain 100 μl of supernatant, adding 100 μl of aqueous solution, mixing at low speed, and performing sample injection analysis. Double blank samples refer to blank matrices without added analyte and internal standard for residual investigation.
Uniformly mixing 10 mu L of matrix effect sample and 190 mu L of acetonitrile; and centrifuging to obtain 100 mu L of supernatant, mixing with 20 mu Lneat solution and 80 mu L of aqueous solution at a low speed by vortex, and carrying out sample injection analysis. The matrix effect samples were at least 6 batches of blank matrix.
Mixing 10 μl of recovery rate sample, namely blank matrix, 190 μl acetonitrile, centrifuging to obtain supernatant 100 μl, mixing with 20 μl of heat solution, and 80 μl of water solution at low speed by vortex, and sample injection analysis.
Mixing 10 μl of pure liquid sample, namely water, 190 μl of acetonitrile, centrifuging to obtain 100 μl of supernatant, mixing with 20 μl of Lneat solution and 80 μl of aqueous solution at low speed by vortex, and sample injection analysis.
3. Sample detection:
and (3) respectively injecting the correction marking 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 treated in the step (II) into a liquid chromatograph mass spectrometer for quantitatively detecting imiquimod and imiquimod-D9 components, wherein the obtained corresponding spectrograms are shown in figures 1-6.
FIG. 1 is a mass spectrum of a sample-injected LC-MS/MS of a solvent sample after pretreatment of the sample (upper: 0ng/mL imiquimod/lower: 0ng/mL imiquimod-D9); FIG. 2 is a mass spectrum of a sample-injected LC-MS/MS (upper: 0ng/mL imiquimod/lower: 0ng/mL imiquimod-D9) of a blank matrix sample after pretreatment with the sample; FIG. 3 is a mass spectrum of an in-situ sample LC-MS/MS after pretreatment of the sample (upper: 0ng/mL imiquimod/lower: 10ng/mL imiquimod-D9); FIG. 4 is a mass spectrum of a sample LC-MS/MS (upper: 0ng/mL imiquimod/lower: 0ng/mL imiquimod-D9) of a blank matrix sample (sample blank after ULOQ sample investigation to investigate residues) after sample pretreatment; FIG. 5 is a mass spectrum of the sample injection LC-MS/MS after pretreatment of the lower limit sample (upper: 1ng/mL imiquimod/lower: 10ng/mL imiquimod-D9); FIG. 6 is a mass spectrum of the sample injection LC-MS/MS after pretreatment of the upper limit sample (upper: 500ng/mL imiquimod/lower: 10ng/mL imiquimod-D9).
Wherein, in the detection step, detection is carried out according to the following liquid chromatography conditions:
stationary phase: an ACE 5C18-PFP chromatographic column with a filler particle diameter of 5 mu m, a diameter of 2.1mm and a length of 50 mm;
mobile phase: a mixed system with mobile phase A and B, A is formic acid aqueous solution, wherein the volume ratio of formic acid to water is 0.02:100, and A is prepared by adding 1000mLH into 1L glass bottle 2 O and 0.2mL of formic acid are mixed uniformly.
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 to add 1000mL of acetonitrile and 0.2mL of formic acid into a 1L glass bottle and uniformly mix.
The elution gradient is:
0.01min, 90% by volume of A and 10% by volume of B;
2.00min, wherein the volume percentage of A is 0 percent, and the volume percentage of B is 100 percent;
2.80min, wherein the volume percentage of A is 0 percent, and the volume percentage of B is 100 percent;
2.81min, volume percentage of A is 90%, volume percentage of B is 10%;
3.00min, the volume percentage of A is 90%, and the volume percentage of B is 10%.
The sample injector cleaning liquid is as follows: weak wash is sample injector cleaning solution 1, sample injector cleaning solution 1 is 30% methanol aqueous solution, and the preparation method of 30% methanol aqueous solution comprises adding 300mL methanol and 700mL H into 1L glass bottle 2 And O, uniformly mixing.
The strong washing is a sample injector washing liquid 2, and the sample injector washing liquid 2 is formed by mixing methanol, acetonitrile, isopropanol and water according to a volume ratio of 1:1:1:1. The sample injector cleaning liquid 2 was prepared by adding 250mL of methanol, 250mL of acetonitrile, 250mL of isopropanol, 250mLH to a 1L glass bottle 2 And O, uniformly mixing.
The diluent 1 is an aqueous solution of formic acid acetonitrile, and the preparation method of the aqueous solution of formic acid acetonitrile is that 500mL of acetonitrile, 500mL of water and 1.0mL of formic acid are added into a 1L glass bottle and mixed uniformly.
Flow rate: 0.40mL/min;
column temperature: 40 ℃;
autoinjector temperature: 4 ℃;
sample injection amount: 10 mu L.
The retention time of imiquimod at the time of injection was about 1.37min and the retention time of imiquimod-D9 was about 1.36min.
In the detection step, detection is carried out according to the following mass spectrum conditions:
ion source: an electrospray ion source;
ionization mode: a positive ion mode;
the resolution mode is Unit;
the collision gas, the gas curtain gas, the atomization gas, the auxiliary gas 1 and the auxiliary gas 2 are all high-purity nitrogen;
spray voltage 5500V.
4. Regression and data processing
Regression model y=ax+b, linear regression, weight factor 1/x 2 Y is the peak area ratio of analyte to internal standard and x is the concentration of analyte in the calibration label.
The calculation software was analytical 1.6.3 and Microsoft Office 2016 (or other version), with all concentration values retaining the 3 significant digits and the% Bias and% CV to the 1-bit after the decimal point.
Analyte: imiquimod;
a substrate: SD rat plasma (heparin sodium as anticoagulant);
correction curve range: 1.00 ng/mL-500 ng/mL;
lower limit of quantification: 1.00ng/mL;
linear range: 1.00 ng/mL-500 ng/mL.
The principles and embodiments of the present invention have been described in detail with reference to specific examples, which are provided to facilitate understanding of the method and core ideas of the present invention; meanwhile, as those skilled in the art will have variations in the specific embodiments and application scope in accordance with the ideas of the present invention, the present description should not be construed as limiting the present invention in view of the above.
Claims (7)
1. A method for determining imiquimod component content in SD rat plasma by HPLC-MS method, comprising the steps of:
step one, sample preparation:
preparing a correction mark sample: weighing a certain amount of imiquimod, and dissolving the imiquimod into a solution of formic acid, acetonitrile and water in a volume ratio of 0.1:50:50 to obtain 1.00mg/mL imiquimod stock solution; taking 1.00mg/mL imiquimod stock solution, and preparing a correction marking working solution with gradient concentration of 20.0-10000 ng/mL of imiquimod by taking a 50% acetonitrile aqueous solution as a solvent; respectively taking correction marking working solutions of various concentrations, and diluting with a blank matrix to obtain correction marking samples;
preparing a quality control sample: weighing a certain amount of imiquimod, and dissolving the imiquimod into a solution of formic acid, acetonitrile and water in a volume ratio of 0.1:50:50 to obtain 1.00mg/mL imiquimod stock solution; taking 1.00mg/mL imiquimod stock solution, and preparing a quality control working solution with gradient concentration of 20.0-8000 ng/mL imiquimod by taking a 50% acetonitrile aqueous solution as a solvent; respectively taking quality control working solutions with various concentrations, and diluting with a blank matrix to obtain a quality control sample;
step two, sample treatment:
respectively adding imiquimod-D9 internal standard working solution into a correction marking sample, a quality control sample, an internal standard sample and SD rat plasma to be tested for dilution, respectively centrifuging the internal standard working solution by taking acetonitrile as a solvent to obtain supernatant, and respectively diluting the supernatant with ultrapure water solution;
respectively adding acetonitrile into the blank matrix and the solvent sample for dilution, respectively centrifuging to obtain supernatant, and respectively diluting the supernatant with ultrapure water solution;
step three, sample detection:
respectively injecting the correction marking sample, the quality control sample, the internal standard sample, the SD rat plasma to be detected, the blank matrix and the solvent sample treated in the step two into a liquid chromatography mass spectrometer for quantitatively detecting imiquimod and imiquimod-D9 components, and obtaining the content of the imiquimod components in the SD rat plasma through regression and data processing; wherein the detection is performed according to the following liquid chromatography conditions:
stationary phase: an ACE 5C18-PFP chromatographic column with a filler particle size of 5 mu m, a diameter of 2.1mm and a length of 50 mm;
mobile phase: the mobile phase is a mixed system of A and B, A is formic acid aqueous solution, wherein 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 is:
0.01min, 90% by volume of A and 10% by volume of B;
2.00min, wherein the volume percentage of A is 0 percent, and the volume percentage of B is 100 percent;
2.80min, wherein the volume percentage of A is 0 percent, and the volume percentage of B is 100 percent;
2.81min, volume percentage of A is 90%, volume percentage of B is 10%;
3.00min, the volume percentage of A is 90%, and the volume percentage of B is 10%.
2. The method for determining imiquimod component content in SD rat plasma by HPLC-MS method according to claim 1, wherein in the first step, the dilution factor of blank matrix used for preparing calibration labeling sample and quality control sample is 20 times.
3. The method for determining imiquimod component content in SD rat plasma by HPLC-MS method according to claim 1, wherein in the second step, the concentration of imiquimod-D9 internal standard working solution used is 10.0. 10.0ng/mL; the correction marking sample, the quality control sample, the internal standard sample and the SD rat plasma to be tested are diluted by 20 times; the dilution factor is 2 times by adopting ultrapure water solution;
adding acetonitrile to the blank matrix and the solvent sample for dilution by 20 times; the dilution with ultrapure water solution was 2-fold.
4. The method for determining imiquimod component content in SD rat plasma by HPLC-MS method according to claim 1, wherein in the third step, the injector washing liquid of the liquid chromatograph is: the weak washing adopts 30% methanol aqueous solution, and the strong washing adopts methanol, acetonitrile, isopropanol and water which are mixed according to the volume ratio of 1:1:1:1.
5. The method for determining imiquimod component content in SD rat plasma by HPLC-MS method according to claim 1, wherein in the third step, detection is performed according to the following liquid chromatography conditions:
flow rate: 0.40mL/min;
column temperature: 40 ℃;
autoinjector temperature: 4 ℃;
sample injection amount: 10 mu L.
6. The method for determining imiquimod component content in SD rat plasma by HPLC-MS method according to claim 1, wherein in the third step, detection is performed according to the following mass spectrum conditions:
ion source: an electrospray ion source;
ionization mode: a positive ion mode;
the resolution mode is Unit;
the collision gas, the gas curtain gas, the atomization gas, the auxiliary gas 1 and the auxiliary gas 2 are all high-purity nitrogen;
spray voltage 5500V.
7. The method for determining imiquimod component content in SD rat plasma by HPLC-MS method according to claim 1, wherein in the third step, the following regression and data processing method is adopted:
regression model y=ax+b, linear regression, weight factor 1/x 2 Y is the peak area ratio of the analyte to the internal standard, x is the concentration of the analyte in the calibration label;
the calculation software is analytical 1.6.3 and Microsoft Office 2016, all concentration values remain 3 significant digits, and% Bias and% CV remain 1 after the decimal point;
analyte: imiquimod;
a substrate: SD rat plasma with heparin sodium as anticoagulant;
correction curve range: 1.00 ng/mL-500 ng/mL;
lower limit of quantification: 1.00ng/mL;
linear range: 1.00 ng/mL-500 ng/mL.
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