CN112098536A - Method for measuring concentration of sunitinib in human plasma - Google Patents
Method for measuring concentration of sunitinib in human plasma Download PDFInfo
- Publication number
- CN112098536A CN112098536A CN202010836907.7A CN202010836907A CN112098536A CN 112098536 A CN112098536 A CN 112098536A CN 202010836907 A CN202010836907 A CN 202010836907A CN 112098536 A CN112098536 A CN 112098536A
- Authority
- CN
- China
- Prior art keywords
- sunitinib
- standard
- concentration
- sample
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000002147 L01XE04 - Sunitinib Substances 0.000 title claims abstract description 91
- 229960001796 sunitinib Drugs 0.000 title claims abstract description 91
- WINHZLLDWRZWRT-ATVHPVEESA-N sunitinib Chemical compound CCN(CC)CCNC(=O)C1=C(C)NC(\C=C/2C3=CC(F)=CC=C3NC\2=O)=C1C WINHZLLDWRZWRT-ATVHPVEESA-N 0.000 title claims abstract description 91
- 238000000034 method Methods 0.000 title claims abstract description 24
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 51
- 210000004369 blood Anatomy 0.000 claims abstract description 32
- 239000008280 blood Substances 0.000 claims abstract description 32
- 239000012224 working solution Substances 0.000 claims abstract description 23
- 239000000126 substance Substances 0.000 claims abstract description 17
- 238000002347 injection Methods 0.000 claims abstract description 16
- 239000007924 injection Substances 0.000 claims abstract description 16
- 238000004780 2D liquid chromatography Methods 0.000 claims abstract description 14
- 239000006228 supernatant Substances 0.000 claims abstract description 13
- 239000011550 stock solution Substances 0.000 claims abstract description 9
- 239000000243 solution Substances 0.000 claims abstract description 8
- 238000007865 diluting Methods 0.000 claims abstract description 4
- 238000005259 measurement Methods 0.000 claims abstract description 4
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000000047 product Substances 0.000 claims abstract description 4
- 238000005303 weighing Methods 0.000 claims abstract description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 36
- 239000003814 drug Substances 0.000 claims description 19
- 239000007864 aqueous solution Substances 0.000 claims description 16
- 239000012086 standard solution Substances 0.000 claims description 15
- 239000004254 Ammonium phosphate Substances 0.000 claims description 12
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 12
- 229910000148 ammonium phosphate Inorganic materials 0.000 claims description 12
- 235000019289 ammonium phosphates Nutrition 0.000 claims description 12
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 12
- 239000003795 chemical substances by application Substances 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- 238000000703 high-speed centrifugation Methods 0.000 claims description 6
- 238000012417 linear regression Methods 0.000 claims description 4
- 238000012545 processing Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 238000010241 blood sampling Methods 0.000 claims description 3
- 238000004141 dimensional analysis Methods 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 238000012546 transfer Methods 0.000 claims description 3
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 claims description 3
- 210000002381 plasma Anatomy 0.000 description 27
- 230000000875 corresponding effect Effects 0.000 description 8
- 238000010586 diagram Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000012544 monitoring process Methods 0.000 description 5
- 229940079593 drug Drugs 0.000 description 4
- 206010067484 Adverse reaction Diseases 0.000 description 3
- 108091008606 PDGF receptors Proteins 0.000 description 3
- 102000011653 Platelet-Derived Growth Factor Receptors Human genes 0.000 description 3
- 230000006838 adverse reaction Effects 0.000 description 3
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 208000033383 Neuroendocrine tumor of pancreas Diseases 0.000 description 2
- 206010067517 Pancreatic neuroendocrine tumour Diseases 0.000 description 2
- 208000006265 Renal cell carcinoma Diseases 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 208000021010 pancreatic neuroendocrine tumor Diseases 0.000 description 2
- 230000003285 pharmacodynamic effect Effects 0.000 description 2
- 229940121358 tyrosine kinase inhibitor Drugs 0.000 description 2
- 239000005483 tyrosine kinase inhibitor Substances 0.000 description 2
- 150000004917 tyrosine kinase inhibitor derivatives Chemical class 0.000 description 2
- FSPQCTGGIANIJZ-UHFFFAOYSA-N 2-[[(3,4-dimethoxyphenyl)-oxomethyl]amino]-4,5,6,7-tetrahydro-1-benzothiophene-3-carboxamide Chemical compound C1=C(OC)C(OC)=CC=C1C(=O)NC1=C(C(N)=O)C(CCCC2)=C2S1 FSPQCTGGIANIJZ-UHFFFAOYSA-N 0.000 description 1
- WSGYTJNNHPZFKR-UHFFFAOYSA-N 3-hydroxypropanenitrile Chemical group OCCC#N WSGYTJNNHPZFKR-UHFFFAOYSA-N 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 101000579425 Homo sapiens Proto-oncogene tyrosine-protein kinase receptor Ret Proteins 0.000 description 1
- 101000851007 Homo sapiens Vascular endothelial growth factor receptor 2 Proteins 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 0.000 description 1
- 102100028198 Macrophage colony-stimulating factor 1 receptor Human genes 0.000 description 1
- 101710150918 Macrophage colony-stimulating factor 1 receptor Proteins 0.000 description 1
- 206010028116 Mucosal inflammation Diseases 0.000 description 1
- 201000010927 Mucositis Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102100028286 Proto-oncogene tyrosine-protein kinase receptor Ret Human genes 0.000 description 1
- 102100020718 Receptor-type tyrosine-protein kinase FLT3 Human genes 0.000 description 1
- 101710151245 Receptor-type tyrosine-protein kinase FLT3 Proteins 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 206010050018 Renal cancer metastatic Diseases 0.000 description 1
- 102100033177 Vascular endothelial growth factor receptor 2 Human genes 0.000 description 1
- -1 acts on VEGFR1-3 Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000007012 clinical effect Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 201000011243 gastrointestinal stromal tumor Diseases 0.000 description 1
- 229960003685 imatinib mesylate Drugs 0.000 description 1
- YLMAHDNUQAMNNX-UHFFFAOYSA-N imatinib methanesulfonate Chemical compound CS(O)(=O)=O.C1CN(C)CCN1CC1=CC=C(C(=O)NC=2C=C(NC=3N=C(C=CN=3)C=3C=NC=CC=3)C(C)=CC=2)C=C1 YLMAHDNUQAMNNX-UHFFFAOYSA-N 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 201000010982 kidney cancer Diseases 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000010197 meta-analysis Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- LBWFXVZLPYTWQI-IPOVEDGCSA-N n-[2-(diethylamino)ethyl]-5-[(z)-(5-fluoro-2-oxo-1h-indol-3-ylidene)methyl]-2,4-dimethyl-1h-pyrrole-3-carboxamide;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.CCN(CC)CCNC(=O)C1=C(C)NC(\C=C/2C3=CC(F)=CC=C3NC\2=O)=C1C LBWFXVZLPYTWQI-IPOVEDGCSA-N 0.000 description 1
- LIZNIAKSBJKPQC-GDNBJRDFSA-N n-[2-(ethylamino)ethyl]-5-[(z)-(5-fluoro-2-oxo-1h-indol-3-ylidene)methyl]-2,4-dimethyl-1h-pyrrole-3-carboxamide Chemical compound CCNCCNC(=O)C1=C(C)NC(\C=C/2C3=CC(F)=CC=C3NC\2=O)=C1C LIZNIAKSBJKPQC-GDNBJRDFSA-N 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 208000003265 stomatitis Diseases 0.000 description 1
- 229960002812 sunitinib malate Drugs 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 231100001274 therapeutic index Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/38—Flow patterns
- G01N30/46—Flow patterns using more than one column
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
- G01N30/6034—Construction of the column joining multiple columns
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8822—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving blood
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
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.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010836907.7A CN112098536A (en) | 2020-08-19 | 2020-08-19 | Method for measuring concentration of sunitinib in human plasma |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010836907.7A CN112098536A (en) | 2020-08-19 | 2020-08-19 | Method for measuring concentration of sunitinib in human plasma |
Publications (1)
Publication Number | Publication Date |
---|---|
CN112098536A true CN112098536A (en) | 2020-12-18 |
Family
ID=73754125
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010836907.7A Pending CN112098536A (en) | 2020-08-19 | 2020-08-19 | Method for measuring concentration of sunitinib in human plasma |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112098536A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115060819A (en) * | 2022-06-08 | 2022-09-16 | 重庆大学附属肿瘤医院 | Method for simultaneously determining SUN and SU12662 in human plasma based on HPLC-MS/MS single peak method |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011095802A1 (en) * | 2010-02-02 | 2011-08-11 | Generics [Uk] Limited | Hplc method for analyzing sunitinib |
US20120009201A1 (en) * | 2009-03-20 | 2012-01-12 | Josip Blonder | Renal cell carcinoma biomarkers |
WO2019072888A1 (en) * | 2017-10-11 | 2019-04-18 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for predicting hepatocellular carcinoma treatment response |
CN111122727A (en) * | 2019-12-23 | 2020-05-08 | 四川省肿瘤医院 | Method for simultaneously determining concentration of imatinib and imatinib metabolite in human plasma |
-
2020
- 2020-08-19 CN CN202010836907.7A patent/CN112098536A/en active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20120009201A1 (en) * | 2009-03-20 | 2012-01-12 | Josip Blonder | Renal cell carcinoma biomarkers |
WO2011095802A1 (en) * | 2010-02-02 | 2011-08-11 | Generics [Uk] Limited | Hplc method for analyzing sunitinib |
WO2019072888A1 (en) * | 2017-10-11 | 2019-04-18 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for predicting hepatocellular carcinoma treatment response |
CN111122727A (en) * | 2019-12-23 | 2020-05-08 | 四川省肿瘤医院 | Method for simultaneously determining concentration of imatinib and imatinib metabolite in human plasma |
Non-Patent Citations (4)
Title |
---|
JOURDIL JF 等: "Simultaneous quantitation of azole antifungals, antibiotics, imatinib, and raltegravir in human plasma by two-dimensional high-performance liquid chromatography-tandem mass spectrometry", 《JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES》 * |
任静 等: "肾癌舒尼替尼耐药细胞株ACSUR的建立及其生物学特性研究", 《天津医科大学学报》 * |
申琳 等: "苹果酸舒尼替尼制备及对照品标化", 《海峡药学》 * |
陈爱瑛 等: "HPLC测定人血浆中舒尼替尼浓度及其血浆蛋白结合率", 《中国现代应用药学》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115060819A (en) * | 2022-06-08 | 2022-09-16 | 重庆大学附属肿瘤医院 | Method for simultaneously determining SUN and SU12662 in human plasma based on HPLC-MS/MS single peak method |
CN115060819B (en) * | 2022-06-08 | 2023-09-01 | 重庆大学附属肿瘤医院 | Method for simultaneously measuring SUN and SU12662 in human plasma based on HPLC-MS/MS single-peak method |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108828077B (en) | Kit for simultaneously detecting capecitabine and metabolite thereof in blood plasma as well as detection method and application thereof | |
CN109633063B (en) | Method for detecting concentration of ticagrelor and active metabolite thereof in human plasma | |
CN111562322B (en) | Enrichment detection method and application of five anti-tumor drugs in blood sample | |
CN101618119A (en) | Quality standard of Sinopanax formosanus pills | |
CN114935620A (en) | Kit for simultaneously and quantitatively detecting 78 neuropsychiatric drugs | |
CN115166080B (en) | Method for detecting impurity A and impurity B in ifosfamide bulk drug | |
CN110780007B (en) | Method for evaluating 6 component contents of mango cough relieving tablet by HPLC (high performance liquid chromatography) method | |
CN109406690A (en) | A kind of method in relation to substance in detection chloraldurate | |
CN115356420A (en) | Pudilan anti-inflammatory tablet quality evaluation method based on one-test-multiple evaluation | |
CN115479995B (en) | Preferred method of rising and sinking soup extraction process | |
CN108061767A (en) | The method of HPLC method separation determination Rivaroxaban intermediates and its related impurities | |
CN112630339B (en) | Method for simultaneously and quantitatively measuring 4 blood-entering components in agilawood alcohol extract | |
CN102253129A (en) | Method for simultaneously testing plasma concentration of multiple anti-human-immunodeficiency-virus (HIV) medicaments | |
CN112098536A (en) | Method for measuring concentration of sunitinib in human plasma | |
CN109142593A (en) | HPLC-DAD method measures Quetiapine drug concentration in human serum | |
CN111122727A (en) | Method for simultaneously determining concentration of imatinib and imatinib metabolite in human plasma | |
CN103926350A (en) | Inspection method of rehabilitation liquid formulation fingerprint and standard fingerprint | |
CN103543222A (en) | Reduning injection saccharide content detection method | |
CN114563496B (en) | Quantitative fingerprint analysis method for components in ginger, ginger and pinellia tuber percolate | |
AU2021106279A4 (en) | Method for establishing hplc-elsd fingerprints of shenlingbaizhu pills and standard fingerprints thereof | |
CN111579684B (en) | Method for measuring content of total capsaicin in capsule wall material of capsule | |
CN107045031A (en) | The LC MS/MS high-flux detection methods of BMS-477118 and 5 hydroxyl BMS-477118s in human plasma | |
CN107884496B (en) | Method for determining content of succinic acid in trelagliptin succinate | |
CN109856262A (en) | A kind of method that can be used for two fourth preparation main component quantification and qualifications simultaneously | |
CN107632075A (en) | Golden three kinds of component contents of bavin KANGGAN JIAONANG while assay method and its HPLC fingerprint map construction methods |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20201218 |