CN111122727A - Method for simultaneous determination of imatinib and imatinib metabolite concentrations in human plasma - Google Patents
Method for simultaneous determination of imatinib and imatinib metabolite concentrations in human plasma Download PDFInfo
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Abstract
本发明公开了一种人血浆中同时测定伊马替尼及伊马替尼代谢物浓度的方法，一、标准溶液的标定(a)标准工作液的配制：伊马替尼标准工作液的配制；去甲伊马替尼标准工作液的配制；(b)用标准溶液的标定，得到标准曲线方程：伊马替尼标准曲线方程；去甲伊马替尼标准曲线方程；二、(c)检测血液的离心处理；三、(d)待测样品的有机处理；四、(e)待测样品的检测。采用二维液相色谱法同时监测人血浆中伊马替尼及伊马替尼代谢物浓度，具有可操作性强、快速、灵敏、具有良好重现性、且监测成本较低的优点。
The invention discloses a method for simultaneous determination of the concentrations of imatinib and imatinib metabolites in human plasma. First, standard solution calibration (a) Preparation of standard working solution: preparation of imatinib standard working solution ; Preparation of nor-imatinib standard working solution; (b) calibration with standard solution to obtain standard curve equation: imatinib standard curve equation; nor-imatinib standard curve equation; 2. (c) Centrifugal treatment of the detection blood; 3. (d) organic treatment of the sample to be tested; 4. (e) detection of the sample to be tested. Using two-dimensional liquid chromatography to simultaneously monitor the concentrations of imatinib and imatinib metabolites in human plasma has the advantages of strong operability, rapidity, sensitivity, good reproducibility, and low monitoring costs.
Description
Technical Field
The invention relates to the technical field of medical treatment, in particular to a method for simultaneously determining the concentration of imatinib and imatinib metabolite in human plasma.
Background
Imatinib Mesylate (IM) is a small molecule protein Tyrosinase (TKIs) inhibitor with molecular formula C29H31N7O.CH4SO3The inhibitor can well inhibit the activity of BCR-ABL Tyrosine Kinase (TK), c-Kit and the like, and is applied to chronic granulocytic leukemia, unresectable/or metastatic gastrointestinal stromal tumor, acute lymphocytic leukemia and the like as a targeted drug. The medicine is orally administered, has bioavailability of 98%, and can slightly reduce its absorption due to high fat diet. Mainly metabolized by the liver and excreted in the feces. The research finds that IM as an oral small molecule targeted drug has huge intra-individual difference (about 75%) and inter-individual difference (60%), and the IM blood concentration of patients between different ethnic groups and regions also has difference in effective concentration threshold. Imatinib is N- { 4-methyl-3- [ (4-pyridin-3-pyrimidin-2-yl) amino]Phenyl } -4- (piperazine-1-methyl) -benzamide (norimatinib), which has similar potency to the original drug, has important implications in the accurate clinical individualized treatment of imatinib and its metabolites in blood samples.
At present, IM blood concentration monitoring is not widely carried out in domestic clinical practice, and methods for monitoring IM blood concentration only comprise a High Performance Liquid Chromatography (HPLC), a Mass Spectrometry (MS), a liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the like. The HPLC method has poor reproducibility and complex blood sample pretreatment process; mass spectrometers used for MS and LC-MS/MS are expensive, and the influence factor of the 'matrix effect' of liquid mass is large, so that the reproducibility is low and the requirement on operators is high. Compared with HPLC, the two-dimensional liquid chromatography has high precision and good reproducibility, and has no matrix effect compared with MS and LC-MS/MS. The method can sample in large volume, and improve sensitivity and accuracy; in addition, the method has the advantages that the processing steps of the sample to be detected are simple, the operation is simple and convenient, the supernatant fluid of the organic treating agent precipitated protein after centrifugation can be directly injected, and the operations of nitrogen blowing, redissolution, extraction and the like are not needed. And the method can simultaneously monitor IM and a metabolite (norimatinib) thereof in human plasma.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a method for simultaneously determining the concentration of imatinib and imatinib metabolite in human plasma.
In order to achieve the purpose, the invention adopts the technical scheme that: a method for simultaneously determining the concentration of imatinib and imatinib metabolite in human plasma,
calibration of standard solution
(a) Preparing a standard working solution:
preparing an imatinib standard working solution: accurately weighing 5mg of imatinib standard substance, placing the imatinib standard substance in a 5mL volumetric flask, dissolving the imatinib standard substance in 50% methanol aqueous solution, and determining the imatinib standard substance in 5mL to obtain standard stock solution A, diluting the standard stock solution A with 50% methanol, respectively preparing six standard working solutions in the concentration range of imatinib containing 130-14000ng/mL, and storing the working solutions at-20 ℃;
preparation of imatinib metabolite (norimatinib) standard working solution: accurately weighing 5mg of a norimatinib standard substance, placing the norimatinib standard substance in a 5mL volumetric flask, dissolving the norimatinib standard substance in a 50% methanol aqueous solution, and determining the norimatinib standard substance in 5mL to obtain a standard stock solution B, diluting the standard stock solution B with 50% methanol, preparing six standard working solutions in a concentration range containing 130 plus 14000ng/mL norimatinib respectively, and storing the working solutions at the temperature of-20 ℃;
(b) and (3) calibrating by using a standard solution to obtain a standard curve equation:
imatinib standard curve equation: respectively transferring 100 mu L of the prepared imatinib standard working fluids with different concentrations by using a liquid transfer gun, adding 900 mu L of blank plasma, and mixing to prepare a plurality of standard curve plasma samples with different concentrations; respectively putting 1000 mu L of organic treating agent into a plurality of 2mL sterile Ep tubes, respectively adding 400 mu L of the standard curve plasma samples with different concentrations, after a vortex instrument vortexes for 60s, centrifuging at high speed (145000rpm, 8min), respectively taking supernate into a sample feeding bottle by using a liquid-transferring gun, detecting the supernate by using a two-dimensional liquid chromatograph to obtain a plurality of imatinib standard solution chromatograms with different concentrations, performing linear regression by taking the concentrations of the plurality of imatinib standard solutions as abscissa X and the peak areas of the plurality of imatinib standard solutions with different concentrations as ordinate Y, and fitting to obtain a corresponding standard curve equation Y which is a + X + b and obtain weight coefficients a and b;
standard curve equation for norimatinib: respectively transferring 100 mu L of the prepared standard working solutions with different concentrations by using a liquid transfer gun, adding 900 mu L of blank plasma, and mixing to prepare a plurality of norimatinib standard curve plasma samples with different concentrations; respectively adding 1000 mu L of organic treating agent into a plurality of 2mL sterile Ep tubes, respectively adding 400 mu L of the standard curve plasma samples with different concentrations, after a vortex instrument vortexes for 60s, centrifuging at high speed (145000rpm for 8min), respectively taking supernate into a sample feeding bottle by using a liquid-transferring gun, detecting the supernate by using a two-dimensional liquid chromatograph to obtain a plurality of norimatinib standard solution chromatograms with different concentrations, performing linear regression by using the concentrations of the norimatinib standard solutions as horizontal coordinates X and the peak areas of the norimatinib standard solutions with different concentrations as vertical coordinates Y, fitting to obtain a corresponding standard curve equation Y ═ c X + d, and obtaining weight coefficients c and d;
II, separation treatment of the blood sample to be detected:
(c) taking at least 5mL of blood sample to be detected, centrifuging (5000rpm for 6-10min), taking supernatant, and storing the supernatant at-20 ℃; wherein the centrifugation time of the sample to be detected for treating the seeds is more preferably 7-9min, and the optimal time is 8 min;
thirdly, organic treatment of the blood sample to be detected:
(d) adding 1000 mu L of organic treating agent into a 2mL sterile Ep tube, adding 400 mu L of the blood supernatant obtained in the step (II), performing high-speed centrifugation (145000rpm, 8min) after a vortex instrument performs vortex for 60s, taking the supernatant into a sample injection bottle by using a pipette, and analyzing the sample injection;
fourthly, quantitative detection of the blood sample to be detected:
(e) and detecting the sample to be detected in the step by adopting an established two-dimensional liquid chromatography to obtain a chromatogram of the sample to be detected imatinib and metabolites thereof, and obtaining peak areas and concentrations of imatinib and norimatinib from the chromatogram.
Preferably, six imatinib standard working fluids are used in step (b). The concentrations of the six imatinib standard working fluids are respectively as follows: 13.44ng/mL, 44.80ng/mL, 112.00ng/mL, 280.00ng/mL, 700.00ng/mL, and 1400.00 ng/mL.
Preferably, the standard working solutions of norimatinib in step (b) are six. The concentrations of the six norimatinib standard working solutions are respectively as follows: 13.44ng/mL, 44.80ng/mL, 112.00ng/mL, 280.00ng/mL, 700.00ng/mL, and 1400.00 ng/mL.
Preferably, in step (a), standard stock solution a and standard stock solution B are diluted with an aqueous solution containing 50% methanol.
Preferably, in the method for simultaneously determining the concentration of imatinib and imatinib metabolite in human plasma in step (d), the organic treating agent is methanol-acetonitrile, wherein the volume ratio of methanol to acetonitrile is preferably (10-30)%: (90-70)%.
Preferably, the two-dimensional liquid chromatograph of step (e) uses a one-dimensional extraction Column (LC1 Column) of aston sx1(3.5 × 25mm,5 μm); the middle column was Aston SCB (4.6 x 10mm,3.5 μm); the two-dimensional analytical Column (LC2 Column) was Aston SCB (4.6 × 125mm,5 μm).
Preferably, in the step (e), the column temperature of the one-dimensional extraction column, the column temperature of the intermediate column and the column temperature of the two-dimensional analysis column of the two-dimensional liquid chromatograph are all set to 40 ℃.
Preferably, the one-dimensional chromatographic column mobile phase used in the two-dimensional liquid chromatography in step (e) is a solution containing methanol, acetonitrile and water, wherein the ratio of methanol: acetonitrile: water (5-20) and (15-25) and (55-80), then adjusting the pH value to 3.0-3.5 by using phosphoric acid water solution, and adding 1% ammonium citrate; the mobile phase of the middle column is pure water; the two-dimensional chromatographic column mobile phase is a solution containing methanol, acetonitrile and water, wherein the methanol is acetonitrile, and the water is (30-45) to (35-65) to (5-20), then the pH value is adjusted to 3.0-3.5 by phosphoric acid water solution, and 1% ammonium citrate is added; wherein the flow rate of the mobile phase is 1mL/min, and the sample injection amount is 500 mu L.
The mobile phase of the two-dimensional liquid chromatograph is adjusted to pH, preferably 2.5 to 4, more preferably 3 to 3.5, with an aqueous phosphoric acid solution.
Preferably, the above ratio is a ratio of volume ratio.
Preferably, the ultraviolet detector wavelength in step (e) is set to 264 nm.
The invention has the beneficial effects that:
the method for simultaneously monitoring the concentration of imatinib and imatinib metabolites in human plasma by adopting the two-dimensional liquid chromatography has the advantages of strong operability, rapidness, sensitivity, good reproducibility and lower monitoring cost.
Drawings
FIG. 1 is a chromatogram of empty horse plasma;
FIG. 2 is a chromatogram of blank human plasma;
FIG. 3 is a chromatogram of a blank reagent;
FIG. 4 is a chromatogram of a standard solution of imatinib;
FIG. 5 is a chromatogram of a standard solution of norimatinib;
FIG. 6 is a chromatogram of blank horse plasma with the addition of norimatinib + imatinib;
FIG. 7 is a chromatogram of blank human plasma with the addition of norimatinib + imatinib;
FIG. 8 is a chromatogram of a serum sample from a patient administered imatinib mesylate;
FIG. 9 is a standard graph of imatinib;
FIG. 10 is a graph of a standard for norimatinib;
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
Example 1:
a method for simultaneously determining the concentration of imatinib and imatinib metabolite in human plasma,
calibration of standard solution
(a) Preparing a standard working solution:
preparing an imatinib standard working solution: accurately weighing 5mg of imatinib standard substance, placing the imatinib standard substance in a 5mL volumetric flask, dissolving the imatinib standard substance in 50% methanol aqueous solution, and determining the imatinib standard substance in 5mL to obtain standard stock solution A, diluting the standard stock solution A with 50% methanol, respectively preparing six standard working solutions in the concentration range of imatinib containing 130-14000ng/mL, and storing the working solutions at-20 ℃;
preparation of imatinib metabolite (norimatinib) standard working solution: accurately weighing 5mg of a norimatinib standard substance, placing the norimatinib standard substance in a 5mL volumetric flask, dissolving the norimatinib standard substance in a 50% methanol aqueous solution, and determining the norimatinib standard substance in 5mL to obtain a standard stock solution B, diluting the standard stock solution B with 50% methanol, preparing six standard working solutions in a concentration range containing 130 plus 14000ng/mL norimatinib respectively, and storing the working solutions at the temperature of-20 ℃;
(b) and (3) calibrating by using a standard solution to obtain a standard curve equation:
imatinib standard curve equation: respectively transferring 100 mu L of the prepared imatinib standard working fluids with different concentrations by using a liquid transfer gun, adding 900 mu L of blank plasma, and mixing to prepare a plurality of standard curve plasma samples with different concentrations; respectively putting 1000 mu L of organic treating agent into a plurality of 2mL sterile Ep tubes, respectively adding 400 mu L of the standard curve plasma samples with different concentrations, after a vortex instrument vortexes for 60s, centrifuging at high speed (145000rpm, 8min), respectively taking supernate into a sample feeding bottle by using a liquid-transferring gun, detecting the supernate by using a two-dimensional liquid chromatograph to obtain a plurality of imatinib standard solution chromatograms with different concentrations, performing linear regression by taking the concentrations of the plurality of imatinib standard solutions as abscissa X and the peak areas of the plurality of imatinib standard solutions with different concentrations as ordinate Y, and fitting to obtain a corresponding standard curve equation Y which is a + X + b and obtain weight coefficients a and b;
standard curve equation for norimatinib: respectively transferring 100 mu L of the prepared standard working solutions with different concentrations by using a liquid transfer gun, adding 900 mu L of blank plasma, and mixing to prepare a plurality of norimatinib standard curve plasma samples with different concentrations; respectively adding 1000 mu L of organic treating agent into a plurality of 2mL sterile Ep tubes, respectively adding 400 mu L of the standard curve plasma samples with different concentrations, after a vortex instrument vortexes for 60s, centrifuging at high speed (145000rpm for 8min), respectively taking supernate into a sample feeding bottle by using a liquid-transferring gun, detecting the supernate by using a two-dimensional liquid chromatograph to obtain a plurality of norimatinib standard solution chromatograms with different concentrations, performing linear regression by using the concentrations of the norimatinib standard solutions as horizontal coordinates X and the peak areas of the norimatinib standard solutions with different concentrations as vertical coordinates Y, fitting to obtain a corresponding standard curve equation Y ═ c X + d, and obtaining weight coefficients c and d;
II, separation treatment of the blood sample to be detected:
(c) taking at least 5mL of blood sample to be detected, centrifuging (5000rpm for 6-10min), taking supernatant, and storing the supernatant at-20 ℃; wherein the centrifugation time of the sample to be detected for treating the seeds is more preferably 7-9min, and the optimal time is 8 min;
thirdly, organic treatment of the blood sample to be detected:
(d) adding 1000 mu L of organic treating agent into a 2mL sterile Ep tube, adding 400 mu L of the blood supernatant obtained in the step (II), performing high-speed centrifugation (145000rpm, 8min) after a vortex instrument performs vortex for 60s, taking the supernatant into a sample injection bottle by using a pipette, and analyzing the sample injection;
fourthly, quantitative detection of the blood sample to be detected:
(e) and detecting the sample to be detected in the step by adopting an established two-dimensional liquid chromatography to obtain a chromatogram of the sample to be detected imatinib and metabolites thereof, and obtaining peak areas and concentrations of imatinib and norimatinib from the chromatogram.
Example 2:
in this embodiment, based on example 1, six imatinib standard working fluids are used in step (a). The concentrations of the six imatinib standard working fluids are respectively as follows: 13.44ng/mL, 44.80ng/mL, 112.00ng/mL, 280.00ng/mL, 700.00ng/mL, and 1400.00 ng/mL.
Example 3:
this example is based on example 1, and the standard working solutions of norimatinib in step (a) are six. The concentrations of the six norimatinib standard working solutions are respectively as follows: 13.44ng/mL, 44.80ng/mL, 112.00ng/mL, 280.00ng/mL, 700.00ng/mL, and 1400.00 ng/mL.
Example 4:
this example is based on example 1, and in step (a), standard stock solution A and standard stock solution B were diluted with 50% methanol in water.
Example 5:
this example is based on example 1, and the method for simultaneously determining imatinib and imatinib metabolite concentrations in human plasma in step (d) comprises the following steps, wherein the organic treating agent is methanol-acetonitrile, and the volume ratio of methanol to acetonitrile is preferably (10-30)%: (90-70)%, 10%: 90% and 20%: 80% or 30%: 70%, most preferably 20%: 80 percent.
Example 6:
in this example, the one-dimensional extraction Column (LC1 Column) used in the two-dimensional liquid chromatography in step (e) was Aston SX1(3.5 × 25mm,5 μm) based on example 1; the middle column was Aston SCB (4.6 x 10mm,3.5 μm); the two-dimensional analytical Column (LC2 Column) was Aston SCB (4.6 × 125mm,5 μm).
Example 7:
this example set the column temperature of the one-dimensional extraction column, the column temperature of the intermediate column, and the column temperature of the two-dimensional analysis column of the two-dimensional liquid chromatography in step (e) to 40 ℃ on the basis of example 6.
Example 8:
this example is based on example 6, wherein the one-dimensional column mobile phase used in the two-dimensional liquid chromatography in step (e) is a solution containing methanol, acetonitrile and water, wherein the ratio of methanol: acetonitrile: water (5-20) to (15-25) to (55-80), wherein the specific ratio of methanol, acetonitrile and water is as follows: 5%: 15%: 80% and 12.5%: 20%: 67.5% or 20%: 25%: 55%, most preferably 12.5%: 20%: 67.5%, then adjusting the pH to 3.0-3.5 with phosphoric acid aqueous solution, and adding 1% ammonium citrate; the mobile phase of the middle column is pure water; the two-dimensional chromatographic column mobile phase is a solution containing methanol, acetonitrile and water, wherein the methanol is acetonitrile and water (30-45) to (35-65) to (5-20), and the specific ratio of the methanol to the acetonitrile to the water is as follows: 5%: 15%: 80% and 12.5%: 20%: 67.5% or 20%: 25%: 55%, most preferably 12.5%: 20%: 67.5%, then adjusting the pH to 3.0-3.5 with phosphoric acid aqueous solution, and adding 1% ammonium citrate; wherein the flow rate of the mobile phase is 1mL/min, and the sample injection amount is 500 mu L.
Example 9:
in this example, the ratio of methanol, acetonitrile and water is the volume ratio based on example 8.
Example 10:
this example is based on example 1, and the wavelength of the ultraviolet detector used in the two-dimensional liquid chromatography in step (e) was 264 nm.
The specific detection method comprises the following steps:
a method for simultaneously determining the concentration of imatinib and imatinib metabolite in human plasma,
calibration of standard solution
(a) Preparing a standard working solution:
preparing an imatinib standard working solution: accurately weighing 5mg of an imatinib standard substance, placing the imatinib standard substance in a 5mL volumetric flask, dissolving the imatinib standard substance in 50% methanol aqueous solution, and determining the imatinib standard substance in 5mL to obtain a standard stock solution A, diluting the standard stock solution A with 50% methanol, preparing each standard working solution in the concentration range of 130-14000ng/mL imatinib respectively, and storing the standard working solutions at the temperature of-20 ℃;
preparation of a standard working solution of norimatinib: accurately weighing 5mg of a norimatinib standard substance, placing the norimatinib standard substance in a 5mL volumetric flask, dissolving the norimatinib standard substance in a 50% methanol aqueous solution, and determining the norimatinib standard substance in 5mL to obtain a standard stock solution B, diluting the standard stock solution B with 50% methanol, preparing each standard working solution in the concentration range of 130 plus 14000ng/mL norimatinib, and storing the working solutions at the temperature of-20 ℃;
(b) and (3) calibrating by using a standard solution to obtain a standard curve equation:
imatinib standard curve equation: respectively transferring 100 mu L of the prepared six standard working solutions with different concentrations by using a liquid transfer gun, adding 900 mu L of blank plasma, and mixing to prepare six standard curve plasma samples with different concentrations; respectively taking 1000 mu L of organic treating agent to correspond to six 2mL sterile Ep tubes, respectively adding 400 mu L of the six standard curve plasma samples with different concentrations, vortexing by a vortexer for 60s, centrifuging at high speed (145000rpm for 8min), respectively taking supernate to a sample feeding bottle by using a pipetting gun, detecting the supernate by using a two-dimensional liquid chromatograph and a two-dimensional liquid chromatograph to obtain six imatinib standard solution chromatograms with different concentrations, performing linear regression by taking the six imatinib standard solution concentrations as horizontal coordinates X and the six imatinib standard solution peak areas with different concentrations as vertical coordinates Y, fitting to obtain a corresponding standard curve equation Y ═ a X + b, and obtaining weight coefficients a and b;
standard curve equation for norimatinib: respectively transferring 100 mu L of the prepared six standard working solutions with different concentrations by using a liquid transfer gun, adding 900 mu L of blank plasma, and mixing to prepare six standard curve plasma samples with different concentrations; respectively adding 1000 mu L of organic treating agent into six 2mL sterile Ep tubes, respectively adding 400 mu L of the six standard curve plasma samples with different concentrations, vortexing by a vortexer for 60s, centrifuging at high speed (145000rpm for 8min), respectively taking supernate into a sample feeding bottle by using a liquid transfer gun, detecting the supernate by using a two-dimensional liquid chromatograph and a two-dimensional liquid chromatograph to obtain six norimatinib standard solution chromatograms with different concentrations, performing linear regression by taking the six norimatinib standard solution concentrations as horizontal coordinates X and the six norimatinib standard solution peak areas with different concentrations as vertical coordinates Y, fitting to obtain a corresponding standard curve equation Y ═ c X + d, and obtaining weight coefficients c and d;
second, centrifugal treatment of blood to be measured
Taking at least 5ml of blood to be detected, carrying out high-speed centrifugation (5000rpm for 8min), taking supernatant, and storing the supernatant at-20 ℃;
verification of detection results
Specificity of
FIGS. 1-8 are chromatograms of a blood sample from a patient administered imatinib mesylate, from blank horse plasma, blank human plasma, blank reagent, imatinib standard solution, nor imatinib standard solution, from blank horse plasma with imatinib and nor imatinib added, from blank human plasma with imatinib and nor imatinib added, respectively. The retention time of imatinib in the graphs of 4, 6, 7 and 8 is about 10.5min, the retention time of norimatinib in the graphs of 5, 6, 7 and 8 is about 7.5min, and endogenous substances and other impurities in blood plasma do not interfere with the separation and determination of a sample;
linear range and quantitative limit
Processing and determining the prepared standard curve sample according to the method, and performing linear regression on the concentration by using the peak area of imatinib to obtain a regression equation:
Y＝1011.48X-3526.20，r＝0.9999。
as shown in FIG. 9, the linear range was 13.44-1400.0ng/mL, and the lower limit of quantitation was 13.44 ng/mL.
Processing and determining the prepared standard curve sample according to the method, and performing linear regression on the concentration by using the peak area of the norimatinib to obtain a regression equation:
Y＝670.901X-914.774，r＝0.9999
as shown in fig. 10. The linear range was 13.17-1372.0ng/mL, and the lower limit of quantitation was 13.17 ng/mL.
Sensitivity of the probe
And taking the lowest concentration point of the prepared calibration solution, carrying out continuous sample introduction for 6 times, and calculating the precision of the calibration solution.
Table 1 lowest quantitative limit (n ═ 6)
From table 1, it can be known that the lower limit variation coefficient of imatinib quantification limit is less than 1.8%;
the lower limit coefficient of variation of the norimatinib quantification limit is less than 2.8 percent
Accuracy and precision
And (3) processing and measuring prepared quality control samples with low, medium and high imatinib horizontal concentrations, setting 5 groups of parallel concentration samples, substituting measured results into a linear regression equation to calculate the concentration, and comparing the concentration with the concentration theoretically added to obtain the addition recovery rate and the variation coefficient of the method, the inspection accuracy and the in-day precision. Preparing and measuring quality control samples continuously for 3 days, and investigating the day precision of the method. The results in tables 2 and 3 show that the recovery rate of the established imatinib analysis method is between 99.24% and 102.46%, and the variation coefficient in both day and day is less than 10.1%. The analysis method has high sensitivity and good stability, and meets the methodology requirements of bioequivalence research.
TABLE 2 recovery and in-day precision (n ═ 5)
Table 2 The recovery and intra-day accuracy(n＝5)
TABLE 3 method daytime precision (n ═ 5)
Table 3 The inter-day accuracy(n＝5)
The specific operation method comprises the following steps:
IM blood sample pretreatment
Blood sample collection: after the gastrointestinal stromal tumor patient takes the IM to reach a steady state, the patient does not take the medicine on the same day, the time (24 +/-2-3) h before taking the medicine is taken, and the time when the patient does not take the blood is the blood sampling time on the same day.
Transfer of blood samples: after the specimen is obtained, the specimen is quickly and stably transported to a laboratory without violent shaking, and the specimen pollution caused by blood cell rupture is prevented. If the sample can not be transferred in time, the sample can be stored in a heat preservation box at 4 ℃ (not more than 72 hours).
Treatment of blood samples: plasma separation: balancing a centrifugal tube: trimming two by two on a balance; setting the parameters of the centrifuge to 5000rpm for 8 minutes, and symmetrically placing the well-balanced centrifuge tube in a centrifuge for centrifugation; transferring the supernatant into a 2mL sterile EP tube by using a pipette gun, and marking; after centrifugation, the serum was stored in a refrigerator at-20 ℃.
Preparation of samples: and (3) adding 1000 mu L of organic treating agent into a 2mL sterile Ep tube, adding 400 mu L of blood supernatant, vortexing by a vortexer for 60s, centrifuging at high speed (145000rpm for 8min), taking the supernatant into a sample injection bottle by using a pipette, and analyzing the sample injection.
Quantitative measurement of blood concentration: the two-dimensional liquid chromatography which is established and considered and is suitable for clinically detecting the IM tinib blood concentration of a patient is adopted to determine the IM steady-state blood-valley concentration of the gastrointestinal stromal tumor patient, and the experimental process and the result are recorded.
IM two-dimensional liquid phase monitoring condition
The chromatographic conditions were as follows:
detection wavelength: 264nm
Mobile phase: the mobile phase of the one-dimensional chromatographic column is acetonitrile-sodium dihydrogen phosphate buffer solution (60-40)% (40-60)%, the PH value is adjusted to 3.0-3.5 by phosphoric acid), the mobile phase of the middle column is pure water, and the mobile phase of the two-dimensional chromatographic column is acetonitrile-water (25-45)% (75-55)%.
A chromatographic column: one-dimensional extraction Column (LC1 Column) was Aston SX1(3.5 × 25mm,5 μm); the middle column was aston scb (4.6 x 10mm,3.5 μm); the two-dimensional analytical Column (LC2 Column) was Aston SCB (4.6 × 125mm,5 μm).
Column temperature: 40 deg.C
Flow rate: 1mL/min
Sample introduction volume: 500 μ L.
The above-mentioned embodiments only express the specific embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Claims (10)
1. A method for simultaneously determining the concentration of imatinib and imatinib metabolites in human plasma is characterized in that:
calibration of standard solution
(a) Preparing a standard working solution:
preparing an imatinib standard working solution: accurately weighing 5mg of imatinib standard substance, placing the imatinib standard substance in a 5mL volumetric flask, dissolving the imatinib standard substance in 50% methanol aqueous solution, and determining the imatinib standard substance in 5mL to obtain standard stock solution A, diluting the standard stock solution A with 50% methanol, respectively preparing six standard working solutions in the concentration range of imatinib containing 130-14000ng/mL, and storing the working solutions at-20 ℃;
preparation of imatinib metabolite (norimatinib) standard working solution: accurately weighing 5mg of a norimatinib standard substance, placing the norimatinib standard substance in a 5mL volumetric flask, dissolving the norimatinib standard substance in a 50% methanol aqueous solution, and determining the norimatinib standard substance in 5mL to obtain a standard stock solution B, diluting the standard stock solution B with 50% methanol, preparing six standard working solutions in a concentration range containing 130 plus 14000ng/mL norimatinib respectively, and storing the working solutions at the temperature of-20 ℃;
(b) and (3) calibrating by using a standard solution to obtain a standard curve equation:
imatinib standard curve equation: respectively transferring 100 mu L of the prepared imatinib standard working fluids with different concentrations by using a liquid transfer gun, adding 900 mu L of blank plasma, and mixing to prepare a plurality of standard curve plasma samples with different concentrations; respectively putting 1000 mu L of organic treating agent into a plurality of 2mL sterile Ep tubes, respectively adding 400 mu L of the standard curve plasma samples with different concentrations, after a vortex instrument vortexes for 60s, centrifuging at high speed (145000rpm, 8min), respectively taking supernate into a sample feeding bottle by using a liquid-transferring gun, detecting the supernate by using a two-dimensional liquid chromatograph to obtain a plurality of imatinib standard solution chromatograms with different concentrations, performing linear regression by taking the concentrations of the plurality of imatinib standard solutions as abscissa X and the peak areas of the plurality of imatinib standard solutions with different concentrations as ordinate Y, and fitting to obtain a corresponding standard curve equation Y which is a + X + b and obtain weight coefficients a and b;
standard curve equation for norimatinib: respectively transferring 100 mu L of the prepared standard working solutions with different concentrations by using a liquid transfer gun, adding 900 mu L of blank plasma, and mixing to prepare a plurality of norimatinib standard curve plasma samples with different concentrations; respectively adding 1000 mu L of organic treating agent into a plurality of 2mL sterile Ep tubes, respectively adding 400 mu L of the standard curve plasma samples with different concentrations, after a vortex instrument vortexes for 60s, centrifuging at high speed (145000rpm for 8min), respectively taking supernate into a sample feeding bottle by using a liquid-transferring gun, detecting the supernate by using a two-dimensional liquid chromatograph to obtain a plurality of norimatinib standard solution chromatograms with different concentrations, performing linear regression by using the concentrations of the norimatinib standard solutions as horizontal coordinates X and the peak areas of the norimatinib standard solutions with different concentrations as vertical coordinates Y, fitting to obtain a corresponding standard curve equation Y ═ c X + d, and obtaining weight coefficients c and d;
II, separation treatment of the blood sample to be detected:
(c) taking at least 5mL of blood sample to be detected, centrifuging (5000rpm for 6-10min), taking supernatant, and storing the supernatant at-20 ℃;
thirdly, organic treatment of the blood sample to be detected:
(d) adding 1000 mu L of organic treating agent into a 2mL sterile Ep tube, adding 400 mu L of the blood supernatant obtained in the step (II), performing high-speed centrifugation (145000rpm, 6-10min) after a vortex instrument performs vortex for 60s, taking the supernatant into a sample injection bottle by using a pipette, and analyzing the sample injection;
fourthly, quantitative detection of the blood sample to be detected:
(e) and detecting the sample to be detected in the step by adopting an established two-dimensional liquid chromatography to obtain a chromatogram of the sample to be detected imatinib and metabolites thereof, and obtaining peak areas and concentrations of imatinib and norimatinib from the chromatogram.
2. The method for the simultaneous determination of imatinib and imatinib metabolite concentration in human plasma according to claim 1, wherein: the imatinib standard working solution in the step (a) is six. The concentrations of the six imatinib standard working fluids are respectively as follows: 13.44ng/mL, 44.80ng/mL, 112.00ng/mL, 280.00ng/mL, 700.00ng/mL, and 1400.00 ng/mL.
3. The method for the simultaneous determination of imatinib and imatinib metabolite concentration in human plasma according to claim 1, wherein: the standard working solutions of the norimatinib in the step (a) are six. The concentrations of the six norimatinib standard working solutions are respectively as follows: 13.44ng/mL, 44.80ng/mL, 112.00ng/mL, 280.00ng/mL, 700.00ng/mL, and 1400.00 ng/mL.
4. The two-dimensional liquid chromatography for measuring blood concentration of imatinib and its metabolite in blood according to claim 1, wherein in step (a), standard stock solution a and standard stock solution B are diluted with 50% methanol aqueous solution.
5. The two-dimensional liquid chromatography for measuring blood concentration of imatinib and its metabolites in blood according to claim 1, wherein the method for simultaneously measuring concentration of imatinib and its metabolites in human blood plasma of step (d) comprises: the organic treating agent is methanol-acetonitrile.
6. The method for simultaneous determination of imatinib and imatinib metabolite concentration in human plasma of claim 1, wherein the two-dimensional liquid chromatography in step (e) uses a one-dimensional extraction Column (LC1 Column) of Aston SX1(3.5 x 25mm,5 μm); the middle column was Aston SCB (4.6 x 10mm,3.5 μm); the two-dimensional analytical Column (LC2 Column) was Aston SCB (4.6 × 125mm,5 μm).
7. The method for simultaneously determining imatinib and imatinib metabolite concentration in human plasma of claim 6, wherein the column temperature of the one-dimensional extraction column, the column temperature of the intermediate column, and the column temperature of the two-dimensional analysis column of the two-dimensional liquid chromatography in step (e) are set to 40 ℃.
8. The method for simultaneously determining imatinib and imatinib metabolite concentration in human plasma of claim 6, wherein the one-dimensional chromatographic column mobile phase used in the two-dimensional liquid chromatography of step (e) is a solution comprising methanol, acetonitrile and water, wherein methanol: acetonitrile: water (5-20) and (15-25) and (55-80), then adjusting the pH value to 3.0-3.5 by using phosphoric acid water solution, and adding 1% ammonium citrate; the mobile phase of the middle column is pure water; the two-dimensional chromatographic column mobile phase is a solution containing methanol, acetonitrile and water, wherein the methanol is acetonitrile, and the water is (30-45) to (35-65) to (5-20), then the pH value is adjusted to 3.0-3.5 by phosphoric acid water solution, and 1% ammonium citrate is added; wherein the flow rate of the mobile phase is 1mL/min, and the sample injection amount is 500 mu L.
9. The method for the simultaneous determination of imatinib and imatinib metabolite concentration in human plasma of claim 8, wherein said ratio between methanol, acetonitrile and water is a volume ratio.
10. The method for simultaneously determining the concentration of imatinib and imatinib metabolite in human plasma according to claim 1, wherein the wavelength of the UV detector used in the two-dimensional liquid chromatography in step (e) is 264 nm.
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