CN112098536A - Method for measuring concentration of sunitinib in human plasma - Google Patents
Method for measuring concentration of sunitinib in human plasma Download PDFInfo
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- 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 discloses a method for determining sunitinib concentration in human plasma, which comprises the following steps: A. preparing standard working solution: accurately weighing 5mg of sunitinib standard substance, placing the sunitinib standard substance in a 5mL volumetric flask, dissolving the sunitinib standard substance in 50% methanol water solution, and determining the sunitinib standard substance in 5mL to obtain standard stock solution, diluting the standard stock solution with 50% methanol, preparing a plurality of standard working solutions in the concentration range of 130-14000ng/mL sunitinib respectively, and storing the standard working solutions at the temperature of-20 ℃; B. obtaining a standard curve equation of the standard working solution, wherein the obtained standard curve equation is y ═ a × x + b, and a and b are weight coefficients; C. treating a blood sample to be tested: collecting a blood sample, centrifuging the blood sample, putting the centrifuged supernatant into a corresponding sterile EP tube, marking, and storing at-20 ℃; D. preparing a sample; E. quantitative measurement of blood concentration: and (3) measuring the sample injection product by adopting a two-dimensional liquid chromatography to obtain the steady state blood concentration of sunitinib.
Description
Technical Field
The invention relates to the field of medical detection, in particular to a method for determining the concentration of sunitinib in human plasma.
Background
Sunitinib malate (sotan) is a small molecular multi-target Tyrosine Kinase Inhibitor (TKIs), mainly acts on VEGFR1-3, PDGFR alpha and beta, KIT, FLT-3, CSF-1R, RET and other targets, and has strong double effects of inhibiting tumor cell proliferation and resisting angiogenesis. The clinical application is mainly used for treating the advanced Renal Cell Carcinoma (RCC) which can not be operated, the adult patient with unresectable metastatic high-differentiation advanced pancreatic neuroendocrine tumor (pNET) and the gastrointestinal stromal tumor (GIST) which fails to treat or can not be endured by the imatinib mesylate. The curative effect and adverse reaction of sunitinib in different people have obvious difference: for example, fatigue and weakness occur 18.4% in Asian population and 58.5% in Europe; the incidence of mucositis or stomatitis is 22.9% in the us population and 42.1% in the european population; the occurrence of diarrhea is 17.6% in asian populations and 34.4% in the united states; meanwhile, according to data which is not counted, the clinical curative effect of sunitinib is also remarkably different, and the factors bring difficulty to reasonable use of sunitinib for patients of clinicians.
Blood drug concentration refers to the concentration of drug in the plasma. Meta analysis studies on the pharmacodynamics and pharmacokinetics of sunitinib show that the higher the exposure to sunitinib, the longer the disease progression, the higher the Overall Survival (OS), the larger the size of tumor shrinkage, but the greater the risk of adverse reactions therewith. A retrospective study of 521 patients with metastatic renal carcinoma who had taken sunitinib showed that on day 29 of dosing, the blood trough concentrations (sunitinib + active metabolite N-desethylsunitinib) in patients who had been reduced due to intolerance of the toxicity of sunitinib were similar to those in patients who had not been reduced. Furthermore, the decreased number of patients treated for longer periods of time and the PFS was longer, indicating that the total exposure of the drug in the patients was more responsive to the drug effect of sunitinib than the doses administered to the patients. According to the data reasoning of clinical pre-drug pharmacokinetics/pharmacodynamics, the effective blood concentration (sunitinib + SU12662) (total trough level, TTL) is 50-100ng/mL, so that platelet-derived growth factor receptor (PDGFR) and VEGFR2 can be effectively inhibited, and the concentration range is narrow. Consistent with the results of preclinical studies, the results of first-stage clinical trial studies on sunitinib show that the effect of renal cancer patients with TTL < 50ng/mL is significantly reduced compared with patients with TTL > 50 ng/mL. Thus, TTL must be greater than 50ng/mL per patient. Sunitinib exposure varies greatly due to inter-individual patient variability, combination during treatment, and other factors.
The sunitinib has narrow therapeutic index, the systemic exposure, curative effect and adverse reaction among different groups of individuals have large difference, and the blood concentration/exposure is positively correlated with clinical effect, and all the factors indicate that the patient needs to provide reference for scientific, reasonable, safe, effective and individual precise treatment through blood concentration monitoring in the process of taking sunitinib. However, the monitoring of the blood concentration of sunitinib is not widely carried out in the domestic clinical practice at present, and the analysis reason is probably related to the fact that the types of monitoring methods are few, the requirements are high, the treatment window of sunitinib of Chinese population is not clear, the monitoring consciousness is lacked and the like. Recent research shows that the method for monitoring sunitinib at home and abroad is only liquid chromatography-tandem mass spectrometry (LC-MS/MS), and a mass spectrometer used by the LC-MS/MS is expensive, and has large influence factor of 'matrix effect' of liquid, low reproducibility and high requirement on operators.
Disclosure of Invention
Aiming at the problems, the invention provides a method for measuring the concentration of sunitinib in human plasma, which has the advantages of novelty, rapidness, sensitivity, strong operability, good reproducibility and low cost.
The technical scheme of the invention is as follows:
a method for determining the concentration of sunitinib in human plasma, comprising the steps of:
A. preparing standard working solution: accurately weighing 5mg of sunitinib standard substance, placing the sunitinib standard substance in a 5mL volumetric flask, dissolving the sunitinib standard substance in 50% methanol water solution, and determining the sunitinib standard substance in 5mL to obtain standard stock solution, diluting the standard stock solution with 50% methanol, preparing a plurality of standard working solutions in the concentration range of 130-14000ng/mL sunitinib respectively, and storing the standard working solutions at the temperature of-20 ℃;
B. obtaining a standard curve equation of the standard working solution, wherein the obtained standard curve equation is y ═ a × x + b, and a and b are weight coefficients;
C. treating a blood sample to be tested: collecting a blood sample, centrifuging the blood sample, putting the centrifuged supernatant into a corresponding sterile EP tube, marking, and storing at-20 ℃;
D. preparing a sample: taking 1000ul of organic treating agent into a sterile EP tube, adding 400ul of supernatant into the sterile EP tube, performing vortex for 60s by a vortex instrument, performing high-speed centrifugation (145000rpm, 8min), taking the supernatant into a sample injection bottle by a pipette, and analyzing the sample injection;
E. quantitative measurement of blood concentration: and (3) measuring the sample injection product by adopting a two-dimensional liquid chromatography to obtain the steady state blood concentration of sunitinib.
In a further technical scheme, the step B of obtaining a standard curve equation of the standard working solution comprises the following steps:
b1, respectively transferring 100uL of each standard working solution into a corresponding sterile EP tube through a liquid transfer gun, and adding 900uL of blank plasma into each standard solution to mix to prepare different standard curve plasma samples;
b2, taking 1000uL of organic treating agent to a sterile EP tube corresponding to each standard curve plasma sample, adding 400uL of standard curve plasma sample, carrying out vortex for 60s by a vortex instrument, carrying out high-speed centrifugation (145000rpm, 8min), and taking supernate to a sample injection bottle by using a pipette respectively to obtain a sample to be detected;
and B3, detecting the sample to be detected by using a two-dimensional liquid chromatograph to obtain a sunitinib standard solution chromatogram, setting the concentration of each sunitinib standard solution as a horizontal coordinate X and the peak area of each sunitinib standard solution as a vertical coordinate Y, performing linear regression fitting to obtain a corresponding standard curve equation Y which is a X + B, and obtaining weight coefficients a and B.
In a further technical scheme, the step C of processing the blood sample to be detected comprises the following steps:
c1, collecting blood samples after taking the sunitinib medicament, wherein the blood sampling time is that the patient does not take the sunitinib medicament after the oral sunitinib medicament reaches a steady state, the time is 24 +/-2-3 hours from the time of the previous medicament taking, and the patient does not take the sunitinib medicament on the same day;
c2, presetting centrifuge parameters, and symmetrically placing the balanced centrifuge tubes in a centrifuge for centrifugation; transferring the supernatant to a 2ml sterile EP tube by a pipette and marking; after centrifugation, the serum was stored in a refrigerator at-20 ℃.
In a further embodiment, the one-dimensional extraction Column (LC1Column) used in the two-dimensional liquid chromatography in step E is 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).
In a further technical scheme, 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 the step E are all set to be 40 ℃; the flow rate is 1.20 ml/min; sample introduction volume: 500 μ L.
In a further technical scheme, the mobile phase of the one-dimensional chromatographic column used in the two-dimensional liquid chromatography in the step E is methanol: acetonitrile: ammonium phosphate aqueous solution (5-25): 55-90); adjusting the pH value to 3.0-7.0 by using a phosphoric acid aqueous solution; the mobile phase of the middle column is pure water; the two-dimensional chromatographic column mobile phase is a solution containing methanol, acetonitrile and ammonium phosphate aqueous solution, wherein the mass ratio of methanol: acetonitrile, namely ammonium phosphate aqueous solution (20-45), namely (25-65), namely (5-40), V/V, adjusting the pH to 3.5-6.5 by using phosphoric acid aqueous solution, and adding 1% of ammonium citrate; wherein the flow rate of the mobile phase is 1.20mL/min, and the sample injection amount is 500 muL.
In a further technical scheme, the ratio of the methanol to the acetonitrile to the aqueous solution of ammonium phosphate is volume ratio.
The invention has the beneficial effects that:
1. measuring the concentration of sunitinib in human plasma by adopting a two-dimensional liquid chromatography;
2. the method is novel, rapid, sensitive, strong in operability, good in reproducibility and low in cost price;
drawings
FIG. 1 is a schematic diagram of a blank horse plasma structure of a method for determining sunitinib concentration in human plasma according to an embodiment of the invention;
FIG. 2 is a schematic structural diagram of a blank human plasma in a method for determining sunitinib concentration in human plasma according to an embodiment of the invention;
FIG. 3 is a schematic diagram of a reagent blank structure of a method for determining sunitinib concentration in human plasma according to an embodiment of the present invention;
FIG. 4 is a schematic structural diagram of a sunitinib standard solution in the method for determining the concentration of sunitinib in human plasma according to the embodiment of the invention;
FIG. 5 is a schematic diagram of a blank human plasma with sunitinib added thereto according to an embodiment of the present invention;
fig. 6 is a schematic diagram of a sunitinib standard curve for a method of determining sunitinib concentration in human plasma according to an embodiment of the invention.
Detailed Description
The embodiments of the present invention will be further described with reference to the accompanying drawings.
Example (b):
a method for determining the concentration of sunitinib in human plasma, comprising the steps of:
A. preparing standard working solution: accurately weighing 5mg of sunitinib standard substance, placing the sunitinib standard substance in a 5mL volumetric flask, dissolving the sunitinib standard substance in 50% methanol water solution, and determining the sunitinib standard substance in 5mL to obtain standard stock solution, diluting the standard stock solution with 50% methanol, preparing a plurality of standard working solutions in the concentration range of 130-14000ng/mL sunitinib respectively, and storing the standard working solutions at the temperature of-20 ℃;
the standard working solutions are six, and the concentrations of the six sunitinib standard working solutions are respectively as follows: 131.7ng/mL, 439.0ng/mL, 1097.6ng/mL, 2744.0ng/mL, 6860.0ng/mL, 13720.0 ng/mL.
The standard stock solution is a sunitinib calibrator diluted with 50% methanol in water
B. Obtaining a standard curve equation of the standard working solution, wherein the obtained standard curve equation is y ═ a × x + b, and a and b are weight coefficients;
C. treating a blood sample to be tested: collecting a blood sample, centrifuging the blood sample, putting the centrifuged supernatant into a corresponding sterile EP tube, marking, and storing at-20 ℃;
D. preparing a sample: taking 1000ul of organic treating agent into a sterile EP tube, adding 400ul of supernatant into the sterile EP tube, performing vortex for 60s by a vortex instrument, performing high-speed centrifugation (145000rpm, 8min), taking the supernatant into a sample injection bottle by a pipette, and analyzing the sample injection;
E. quantitative measurement of blood concentration: and (3) measuring the sample injection product by adopting a two-dimensional liquid chromatography to obtain the steady state blood concentration of sunitinib.
In another embodiment, the step B of obtaining the standard curve equation of the standard working fluid comprises the following steps:
b1, respectively transferring 100uL of each standard working solution into a corresponding sterile EP tube through a liquid transfer gun, and adding 900uL of blank plasma into each standard solution to mix to prepare different standard curve plasma samples;
b2, taking 1000uL of organic treating agent to a sterile EP tube corresponding to each standard curve plasma sample, adding 400uL of standard curve plasma sample, carrying out vortex for 60s by a vortex instrument, carrying out high-speed centrifugation (145000rpm, 8min), and taking supernate to a sample injection bottle by using a pipette respectively to obtain a sample to be detected; wherein the organic treating agent is methanol-acetonitrile (10-40%: 90-60% solution); the proportion of the organic processor refers to the proportion of volume ratio;
and B3, detecting the sample to be detected by using a two-dimensional liquid chromatograph to obtain a sunitinib standard solution chromatogram, setting the concentration of each sunitinib standard solution as a horizontal coordinate X and the peak area of each sunitinib standard solution as a vertical coordinate Y, performing linear regression fitting to obtain a corresponding standard curve equation Y which is a X + B, and obtaining weight coefficients a and B.
In another embodiment, the step C of processing the blood sample to be tested comprises the following steps:
c1, collecting blood samples after taking the sunitinib medicament, wherein the blood sampling time is that the patient does not take the sunitinib medicament after the oral sunitinib medicament reaches a steady state, the time is 24 +/-2-3 hours from the time of the previous medicament taking, and the patient does not take the sunitinib medicament on the same day;
c2, presetting centrifuge parameters, and symmetrically placing the balanced centrifuge tubes in a centrifuge for centrifugation; transferring the supernatant to a 2ml sterile EP tube by a pipette and marking; after centrifugation, the serum was stored in a refrigerator at-20 ℃.
In another embodiment, the one-dimensional extraction Column (LC1Column) used for two-dimensional liquid chromatography in step E is 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).
In another embodiment, 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 all set to 40 ℃; the flow rate is 1.20 ml/min; sample introduction volume: 500 μ L.
In another embodiment, the one-dimensional chromatographic column mobile phase used for the two-dimensional liquid chromatography in step E is methanol: acetonitrile: ammonium phosphate aqueous solution (5-25): 55-90); adjusting the pH value to 3.0-7.0 by using a phosphoric acid aqueous solution; the mobile phase of the middle column is pure water; the two-dimensional chromatographic column mobile phase is a solution containing methanol, acetonitrile and ammonium phosphate aqueous solution, wherein the mass ratio of methanol: acetonitrile, namely ammonium phosphate aqueous solution (20-45), namely (25-65), namely (5-40), V/V, adjusting the pH to 3.5-6.5 by using phosphoric acid aqueous solution, and adding 1% of ammonium citrate; wherein the flow rate of the mobile phase is 1.20mL/min, and the sample injection amount is 500 muL.
In another embodiment, the ratio between methanol, acetonitrile and aqueous ammonium phosphate is a volume ratio.
Verification result
Specificity: FIGS. 1 to 5 are chromatograms of a blank horse plasma, a blank human plasma, a blank reagent, a sunitinib standard solution, and a blank human plasma added with a sunitinib standard solution, respectively. The retention time of sunitinib is 12.09min, and the separation and determination of the sample are not interfered by endogenous substances and other impurities in blood plasma.
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 sunitinib to obtain a regression equation:
Y＝1140.08X-12787.9，r＝0.9998867。
as shown in fig. 6. The linear range is 13.17-1372.00 ng/ml < -1 >, and the lower limit concentration of the quantification is 13.17 ng/ml < -1 >.
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 (7)
1. A method for determining sunitinib concentration in human plasma, which is characterized by comprising the following steps:
A. preparing standard working solution: accurately weighing 5mg of sunitinib standard substance, placing the sunitinib standard substance in a 5mL volumetric flask, dissolving the sunitinib standard substance in 50% methanol water solution, and determining the sunitinib standard substance in 5mL to obtain standard stock solution, diluting the standard stock solution with 50% methanol, preparing a plurality of standard working solutions in the concentration range of 130-14000ng/mL sunitinib respectively, and storing the standard working solutions at the temperature of-20 ℃;
B. obtaining a standard curve equation of the standard working solution, wherein the obtained standard curve equation is y ═ a × x + b, and a and b are weight coefficients;
C. treating a blood sample to be tested: collecting a blood sample, centrifuging the blood sample, putting the centrifuged supernatant into a corresponding sterile EP tube, marking, and storing at-20 ℃;
D. preparing a sample: taking 1000ul of organic treating agent into a sterile EP tube, adding 400ul of supernatant into the sterile EP tube, performing vortex for 60s by a vortex instrument, performing high-speed centrifugation (145000rpm, 8min), taking the supernatant into a sample injection bottle by a pipette, and analyzing the sample injection;
E. quantitative measurement of blood concentration: and (3) measuring the sample injection product by adopting a two-dimensional liquid chromatography to obtain the steady state blood concentration of sunitinib.
2. The method for determining sunitinib concentration in human plasma according to claim 1, wherein the step B of obtaining a standard curve equation of the standard working solution comprises the steps of:
b1, respectively transferring 100uL of each standard working solution into a corresponding sterile EP tube through a liquid transfer gun, and adding 900uL of blank plasma into each standard solution to mix to prepare different standard curve plasma samples;
b2, taking 1000uL of organic treating agent to a sterile EP tube corresponding to each standard curve plasma sample, adding 400uL of standard curve plasma sample, carrying out vortex for 60s by a vortex instrument, carrying out high-speed centrifugation (145000rpm, 8min), and taking supernate to a sample injection bottle by using a pipette respectively to obtain a sample to be detected;
and B3, detecting the sample to be detected by using a two-dimensional liquid chromatograph to obtain a sunitinib standard solution chromatogram, setting the concentration of each sunitinib standard solution as a horizontal coordinate X and the peak area of each sunitinib standard solution as a vertical coordinate Y, performing linear regression fitting to obtain a corresponding standard curve equation Y which is a X + B, and obtaining weight coefficients a and B.
3. The method for determining sunitinib concentration in human plasma according to claim 1, wherein the step C of processing the blood sample to be tested comprises the steps of:
c1, collecting blood samples after taking the sunitinib medicament, wherein the blood sampling time is that the patient does not take the sunitinib medicament after the oral sunitinib medicament reaches a steady state, the time is 24 +/-2-3 hours from the time of the previous medicament taking, and the patient does not take the sunitinib medicament on the same day;
c2, presetting centrifuge parameters, and symmetrically placing the balanced centrifuge tubes in a centrifuge for centrifugation; transferring the supernatant to a 2ml sterile EP tube by a pipette and marking; after centrifugation, the serum was stored in a refrigerator at-20 ℃.
4. The method for determining sunitinib concentration in human plasma according to claim 1, wherein the two-dimensional liquid chromatography in step E uses a one-dimensional extraction Column (LC1Column) of Aston SX1(3.5 x 25mm,5 μ ι η); 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).
5. The method for determining sunitinib concentration in human plasma according to claim 4, 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 all set to 40 ℃; the flow rate is 1.20 ml/min; sample introduction volume: 500 μ L.
6. The method for determining sunitinib concentration in human plasma according to claim 5, wherein the mobile phase of the one-dimensional chromatographic column used in the two-dimensional liquid chromatography in the step E is methanol: acetonitrile: ammonium phosphate aqueous solution (5-25): 55-90); adjusting the pH value to 3.0-7.0 by using a phosphoric acid aqueous solution; the mobile phase of the middle column is pure water; the two-dimensional chromatographic column mobile phase is a solution containing methanol, acetonitrile and ammonium phosphate aqueous solution, wherein the mass ratio of methanol: acetonitrile, namely ammonium phosphate aqueous solution (20-45), namely (25-65), namely (5-40), V/V, adjusting the pH to 3.5-6.5 by using phosphoric acid aqueous solution, and adding 1% of ammonium citrate; wherein the flow rate of the mobile phase is 1.20mL/min, and the sample injection amount is 500 muL.
7. The method for determining sunitinib concentration in human plasma according to claim 6, wherein the ratio between methanol, acetonitrile and aqueous ammonium phosphate is a volume ratio.
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