CN115015406A - Human plasma anti-liver cancer tyrosine kinase inhibitor tandem mass spectrometry detection kit - Google Patents

Human plasma anti-liver cancer tyrosine kinase inhibitor tandem mass spectrometry detection kit Download PDF

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CN115015406A
CN115015406A CN202210549169.7A CN202210549169A CN115015406A CN 115015406 A CN115015406 A CN 115015406A CN 202210549169 A CN202210549169 A CN 202210549169A CN 115015406 A CN115015406 A CN 115015406A
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solution
tandem mass
amu
tyrosine kinase
internal standard
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李挥
姜文
甄晓兰
哈婧
邢娣娣
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Hebei Institute for Drug and Medical Device Control
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Hebei Institute for Drug and Medical Device Control
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Priority to PCT/CN2022/138251 priority patent/WO2023221472A1/en
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    • 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
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • 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
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
    • 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
    • G01N2030/042—Standards
    • G01N2030/045—Standards internal

Abstract

The invention relates to the technical field of inhibitor detection, and provides a human plasma anti-liver cancer tyrosine kinase inhibitor tandem mass spectrometry detection kit. The invention comprises a calibrator, a quality control product, an internal standard solution, an extracting agent, a QuEChERS purifying material, a salting-out agent, a mobile phase A liquid, a mobile phase B liquid, a complex solution, a waterproof packaging bag and a packaging box for separating and intensively packaging the reagent bottles or tubes. The kit has high sensitivity, high specificity and high accuracy of detection results, can simultaneously detect various TKIs with the liver cancer resistance, and has a direct TKIs detection method and a kit which are simple and convenient to operate and easy to popularize so as to reduce the influence of matrix effect and compound instability on experimental results and improve the clinical detection quality.

Description

Human plasma anti-liver cancer tyrosine kinase inhibitor tandem mass spectrometry detection kit
Technical Field
The invention belongs to the technical field of inhibitor detection, and particularly relates to a human plasma anti-liver cancer tyrosine kinase inhibitor tandem mass spectrometry detection kit.
Background
Tyrosine Kinase Inhibitors (TKIs) are small molecule targeted drugs with receptor tyrosine kinases as targets, and the action mechanism of the TKIs is to competitively bind with Adenosine Triphosphate (ATP) binding sites of kinase domains to prevent or reduce phosphorylation of tyrosine kinases, thereby finally playing an anti-tumor role. Compared with the traditional cytotoxic anticancer drugs, the TKIs have the characteristics of high selectivity, few adverse reactions and the like, and are widely applied to cancers such as small cell lung cancer, non-small cell lung cancer, gastrointestinal stromal tumors, hepatocellular carcinoma, kidney cancer and the like. Liver cancer is a common malignant tumor, has the clinical characteristics of latent disease, rapid development, early relapse, poor prognosis and the like, and has high morbidity and mortality. Sorafenib is the first front-line oral small molecule tyrosine kinase inhibitor approved by the U.S. Food and Drug Administration (FDA) for HCC, opening a new era of molecular targeting of liver cancer. Researches on other targeted drugs are carried out successively after sorafenib, and breakthroughs for the treatment of the liver cancer molecular targeted drugs are searched. Rivatinib, regorafenib, cabozantinib, antrotinib, apatinib, gefitinib, tipavatinib and gallunestrib are all targeted drugs which are proved to have the effect of resisting liver cancer. However, the failure of the liver cancer patients in the drug therapy due to drug resistance and drug toxicity is often reported, so that the therapeutic drugs need to be monitored so as to adjust the dosage in time and improve the curative effect and safety of the drugs.
The determination of TKIs in the plasma can quantify the concentration of the therapeutic drug in the liver cancer patient, so as to adjust the administration dosage according to the actual condition of the patient. Sorafenib and ranvatinib are first-line targeted drugs for liver cancer, and regorafenib and cabozantinib are second-line targeted drugs for liver cancer. In addition, erlotinib, apatinib, gefitinib, tipavatinib and gallunestrtib have also been shown to be effective in treating middle and late stage liver cancer. The method for simultaneously detecting 9 TLIs in the plasma of the liver cancer patient is established, comprehensively covers the current TKIs with the liver cancer resistant effect, and has important significance for monitoring clinical treatment medicines.
At present, liquid chromatography and mass spectrometry (LC-MS/MS) are mostly adopted for analyzing small-molecule tyrosinase inhibitors, but a sample is required to have a good matrix effect during detection, so that a proper pretreatment technology needs to be selected to improve the purification efficiency and reduce the influence of impurities. For detection of TKIs, commonly used pretreatment technologies include Protein Precipitation (PP), liquid-liquid extraction (LLE), salting-out assisted liquid-liquid extraction (SALLE), solid-phase extraction (SPE), etc., whereas QuEChERS, a new pretreatment technology derived from dispersed solid-phase extraction (dSPE), was originally used in the field of pesticide residues, and in recent years, with improvement and development of the technology, has been gradually applied to analysis of metabolites and other compounds in biological matrices such as plasma and urine.
Therefore, clinical detection needs a direct detection method and kit for TKIs, which have high sensitivity, high specificity and high accuracy detection results, can detect multiple TKIs with liver cancer resistance simultaneously, is simple and convenient to operate and easy to popularize, and reduces the influence of matrix effect and compound instability on experimental results, thereby improving the clinical detection quality.
Disclosure of Invention
The invention aims to solve the problems and provides a human plasma anti-liver cancer tyrosine kinase inhibitor tandem mass spectrometry detection kit which is characterized by comprising a calibrator, a quality control product, an internal standard solution, an extracting agent, a QuEChERS purifying material, a salting-out agent, a mobile phase A solution, a mobile phase B solution, a complex solution, a waterproof packaging bag and a packaging box for separating and intensively packaging the reagent bottles or tubes.
Preferably, the calibrator: using methanol diluent, and simultaneously adding standard substances of the ticarcillin, the galluninertib, the sorafenib, the ranvatinib, the regorafenib, the cabozantinib, the apratinib, the gefitinib to prepare a calibrator A-F with 6 concentration dose points, wherein the concentrations of the ticanilinib and the galluninertib are respectively 1ng/mL, 2ng/mL, 5ng/mL, 20ng/mL, 80ng/mL, 100ng/mL, the sorafenib, the ranvatinib, the regorafenib, the cabozantinib, the apratinib, the gefitinib are respectively 0.1ng/mL, 0.2ng/mL, 0.5ng/mL, 2ng/mL, 8ng/mL, 10 ng/mL;
quality control product: using methanol diluent, simultaneously adding standard substances of the tipavancib, the galluninrtib, the sorafenib, the ranvatinib, the regorafenib, the cabozantinib, the apratinib, the gefitinib, and preparing 3 concentrations of quality control substances C1-C3, wherein the concentrations of the tipavancib and the galluninrtib are respectively 2ng/mL, 50ng/mL and 100ng/mL, and the concentrations of the sorafenib, the ranvatinib, the regorafenib, the cabozantinib, the apratinib, the gefitinib and the gefitinib are respectively 0.2ng/mL, 5ng/mL and 10 ng/mL;
internal standard solution: using methanol as a diluent, diluting propranolol to a final concentration of 5 ng/mL;
an extracting agent: 1mL to 2.5mL of acetonitrile;
QuEChERS purification material: 20-50mg PSA, and packaging with a waterproof packaging bag;
salting-out agent: 200 and 500mg of anhydrous magnesium sulfate are packaged by a water-proof packaging bag;
mobile phase A: is pure acetonitrile;
mobile phase B: is prepared by formic acid and purified water, the content of the formic acid is 0.05 percent to 0.2 percent;
compounding the solution: is prepared from methanol and purified water, wherein the content of the methanol is 50-100%.
Preferably, the dosage of acetonitrile in the extractant is 1.5 mL;
the dosage of the PSA is 40 mg;
the dosage of anhydrous magnesium sulfate in the salting-out agent is 350 mg;
the content of formic acid in the mobile phase B is 0.1%;
the content of methanol in the complex solution is 50%.
Preferably, the using method comprises the following steps:
(1) preparing a working solution: respectively diluting a calibrator, a quality control product and an internal standard solution in the kit by using methanol to respectively prepare a calibrator working solution, a quality control product working solution and an internal standard working solution;
(2) sample pretreatment: mixing 200 mu L of plasma sample with 10 mu L of internal standard working solution, sequentially adding an extracting agent, a QuEChERS purifying material and a salting-out agent, uniformly mixing, centrifugally separating supernatant, completely drying by nitrogen at room temperature, and adding 200 mu L of complex solution until complete dissolution;
(3) uniformly mixing: fully mixing the solution obtained in the step (2) for 1min by using a vortex mixer;
(4) setting chromatographic and mass spectrum conditions: respectively setting working parameters and conditions of the ultra-high performance liquid chromatography and the tandem mass spectrometer according to the model of the actual instrument;
(5) sample detection: injecting 5 mu L of each of the treated working solution of the calibrator, the working solution of the quality control material and the plasma sample into a high performance liquid chromatography-tandem mass spectrometer for detection and analysis, and recording peak areas and internal standard peak areas of sorafenib, rivatinib, regorafenib, cabozantinib, aritinib, gefitinib, tembotinib and gallunessrtib in a chromatogram and the detection sample;
(6) and (4) analyzing results: and drawing a standard curve and calculating a regression equation of the curve, and respectively calculating the concentrations of sorafenib, rivatinib, regorafenib, cabozantinib, aritinib, apatinib, gefitinib, tematinib and gallunertib in the sample.
Preferably, in the step (1), the internal standard solution is prepared according to the ratio of internal standard solution to methanol being 1: 99.
Preferably, the chromatographic conditions in step (5) are:
a chromatographic column: hypersil GOLD VANQUISH C18 (2.1X 100mm,1.9 μm),
flow rate: 0.3mL/min of the water-soluble polymer,
column temperature: at a temperature of 40 c,
sample introduction amount: the volume of the solution is 5 mu L,
gradient elution conditions:
Figure BDA0003653849080000041
the mass spectrum conditions are as follows:
the ESI positive ion SRM scan mode,
spraying voltage: the voltage of the liquid crystal is 3500V,
flow rate of sheath gas: 45Arb of the total weight of the steel,
flow rate of auxiliary gas: 10 of the total number of the N,
ion transfer tube temperature: at the temperature of 350 ℃,
atomization temperature: at a temperature of 400 c,
collision gas: the argon gas is introduced into the reaction chamber,
pressure: 1.5 mTor;
Q1/Q3 ion channels were selected as
Rivatinib 427.1 → 370.1, 312.0 amu;
465.1 → 252.1, 270.1 amu;
cabozantinib 502.2 → 323.1, 297.1 amu;
regorafenib 483.1 → 270.1, 288.1 amu;
apatinib: 398.2 → 212.1, 184.1 amu;
gefitinib 447.1 → 128.1, 100.1 amu;
angutinib 408.2 → 339.1, 304.1 amu;
tenavancib 370.1 → 253.1, 158.1 amu;
Galunisertib:370.1→336.1，325.1amu。
compared with the prior art, the invention has the following beneficial effects:
1. the kit already contains main matched reagents and consumables required by TKIs in human plasma for detecting tandem mass spectrometry, and simplifies experimental operation steps, so that clinical detection is simpler, more convenient and faster, and operation errors caused by independent purchase of reagent consumables and self-preparation of reagents are greatly reduced.
2. The detection method used by the kit is an ultra-high performance liquid chromatography tandem mass spectrometry, and the 9 TKIs in the plasma are directly detected according to the inherent physicochemical properties of the analyte, so that the matrix effect of clinical blood samples can be effectively removed, the purification efficiency is improved, and the condition of each analyzed substance in the body can be accurately quantified.
3. Compared with the traditional pretreatment method introduced in the background technology, the kit can simultaneously, rapidly and accurately detect sorafenib, rivatinib, regorafenib, cabozantinib, antrotinib, apatinib, gefitinib, tematinib and gallunestrib 9 substances in human plasma, thereby more clearly reflecting the concentration of the therapeutic drugs in a patient body.
Drawings
FIG. 1 is a flow chart of a sample processing operation of the present invention;
FIG. 2 is a chromatogram of Arotinib in an example;
FIG. 3 is a chromatogram of cabozantinib in an example;
FIG. 4 is a chromatogram of example Lonvatinib;
FIG. 5 is a chromatogram of Galunertib in an example;
fig. 6 is a chromatogram of regorafenib in an example;
FIG. 7 is a chromatogram of sorafenib in the example;
FIG. 8 is a chromatogram of apatinib from an example;
FIG. 9 is a chromatogram of Tenavancib in an example;
FIG. 10 is a chromatogram of gefitinib in the example;
FIG. 11 is a chromatogram of propranolol as an internal standard solution;
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention relates to a kit for detecting TKIs in human plasma by tandem mass spectrometry, and provides a matched mature kit and a corresponding operation method for clinical detection. In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, the present invention includes kit components and operation methods as follows:
kit Components
Figure BDA0003653849080000071
Kit matching operation method
1. Preparing reagent components:
(1) calibrators (A, B, C, D, E, F) and quality controls (C1, C2, C3) and internal standard stock solutions: under the condition of room temperature, 1000 mu L of methanol is accurately measured and added into each bottle of calibrator or quality control material, and the methanol is completely dissolved for standby.
(2) Working fluid: and (3) adding methanol to continuously dilute the calibrator, the quality control material and the internal standard stock solution in the step (1) to a required concentration, wherein the final concentration of the calibrator working solution is as follows: 1ng/mL, 2ng/mL, 5ng/mL, 20ng/mL, 80ng/mL, 100ng/mL (Tenavancib, Galunestrib); 0.1ng/mL, 0.2ng/mL, 0.5ng/mL, 2ng/mL, 8ng/mL, 10ng/mL (sorafenib, lenvatinib, regorafenib, cabozantinib, aritinib, apatinib and gefitinib). The final concentration of the quality control product working solution is as follows: 2ng/mL, 50ng/mL, 100ng/mL (Tenavancib and Galunecertib); 0.2ng/mL, 5ng/mL, 10ng/mL (sorafenib, lenvatinib, regorafenib, cabozantinib, ariotinib, apatinib and gefitinib). The final concentration of the internal standard working solution was 5 ng/mL.
2. Processing a detection sample: see fig. 1.
a) Internal standard mixing: fully mixing 200 mu L of plasma sample with 10 mu L of internal standard substance working solution;
b) pretreatment: sequentially and accurately adding an extracting agent, a purifying material and a salting-out agent, uniformly mixing, and centrifuging at 13000rpm for 10min to separate a supernatant;
c) drying by nitrogen: after centrifugation, 1mL of supernatant is respectively sucked into a centrifuge tube and is thoroughly dried by nitrogen at room temperature;
d) redissolving: then adding 200 mu L of sample complex solution respectively, and mixing uniformly;
d) transferring: after redissolution, 200. mu.L of the suspension was transferred to a liquid vial and ready for on-machine sample analysis.
3. Chromatographic conditions are as follows:
a) a chromatographic column: hypersil GOLD VANQUISH C18 (2.1X 100mm,1.9 μm) or equivalent;
b) mobile phase: mobile phase A liquid and mobile phase B liquid;
c) flow rate: 0.3 mL/min;
d) gradient elution conditions:
Figure BDA0003653849080000091
e) column temperature: 40 ℃;
f) sample introduction amount: 5 μ L.
4. Mass spectrum conditions:
a) an ion source: electrospray ionization (ESI);
b) scanning mode: for positive ion SRM scanning analysis, the ion channel Q1/Q3 is selected from the group consisting of Rivatinib, 427.1 → 370.1, 312.0 amu; 465.1 → 252.1, 270.1 amu; cabozantinib 502.2 → 323.1, 297.1 amu; regorafenib 483.1 → 270.1, 288.1 amu; apatinib: 398.2 → 212.1, 184.1 amu; gefitinib 447.1 → 128.1, 100.1 amu; ambotinib 408.2 → 339.1, 304.1 amu; tenavancib 370.1 → 253.1, 158.1 amu; galunertib: 370.1 → 336.1, 325.1 amu.
c) In a preferred example of embodiment of the present invention, the ion source parameters are as follows:
Figure BDA0003653849080000092
Figure BDA0003653849080000101
ion source parameters may vary from one instrument model to another.
a) In a preferred example of the embodiment of the present invention, the mass spectrum parameters are as follows:
Figure BDA0003653849080000102
a quantitative ion
The mass spectrum voltage parameter can be different according to different actual instrument models.
5. Sample detection:
and (3) selecting 5 mu L of each of the processed calibrator, the quality control material and the plasma sample, injecting the 5 mu L of each of the calibrator, the quality control material and the plasma sample into an ultra-high performance liquid chromatography-tandem mass spectrometer for detection and analysis, and recording peak areas and internal standard peak areas of sorafenib, rivatinib, regorafenib, cabozantinib, apatinib, gefitinib, tipertinib and gallisirtib in the chromatogram and the detection sample.
6. And (3) calculation of detection results:
(1) the standard curve drawing method comprises the following steps: standard curves were plotted with the plotted concentrations of 6 calibrators (e.g., 1ng/mL, 2ng/mL, 5ng/mL, 20ng/mL, 80ng/mL, 100 ng/mL; sorafenib, lenvatinib, regorafenib, cabozantinib, ariotinib, apatinib, gefitinib: 0.1ng/mL, 0.2ng/mL, 0.5ng/mL, 2ng/mL, 8ng/mL, 10ng/mL) as the abscissa (x) and the ratios of the actual peak areas of the 6 calibrators to the respective peak areas of the internal standards as the ordinate (y).
(2) Fitting of standard curve equation: the labeled concentrations (x) were linearly regressed with peak area ratios (y) of 6 calibrators. A regression equation can be obtained: and y is a + bx, wherein y is an ordinate, x is an abscissa, a is an intercept, b is a slope, and calculating a correlation coefficient (r), wherein r is required to be not less than 0.9900.
(3) Calculation of plasma sample results: substituting the ratio of the actual detection peak area of the TKIs in the plasma sample to the internal standard peak area into a standard curve equation, and calculating the concentration of the TKIs in the plasma sample.
Example (b): quantitative analysis of 9 tyrosine kinase inhibitors in healthy human plasma
1. Specialization inspection
The analysis was performed by adding 9 tyrosine kinase inhibitors to the plasma of healthy persons according to the scheme shown in fig. 1, wherein typical chromatograms of 9 tyrosine kinase inhibitors and an internal standard in SRM mode are shown in fig. 2-11. The experimental result shows that the separation effect of the 9 substances is good, and the peak pattern of each analyte is good, which indicates that the method has good selectivity and can be used for quantitative detection of the 9 tyrosine kinase inhibitors in the blood plasma.
2. Linear experiment
A blank plasma sample is taken, the operation is carried out according to the flow shown in figure 1, and the result analysis is carried out according to the part of 'calculation of detection result' to obtain a linear regression equation of each analyte and the internal standard. The linear correlation coefficient (R2) of all analytes is between 0.9966 and 0.9999.
Figure BDA0003653849080000121
3. Precision and accuracy testing
The day precision and accuracy of the method was obtained by repeated analysis of 3 different levels of quality control samples (n-6). Daytime accuracy and precision were evaluated over 3 consecutive days. Precision is expressed in Relative Standard Deviation (RSD) and accuracy in Relative Error (RE). The result shows that the precision of all tyrosine kinase inhibitors is less than 9.09%, and the accuracy is-7.34-6.64%. Therefore, the method is suitable for detecting 9 tyrosine kinase inhibitors at different concentration levels, and has good accuracy and precision.
Figure BDA0003653849080000122
Figure BDA0003653849080000131
Figure BDA0003653849080000141
4. Recovery rate experiment
To ensure efficient recovery, 3 analyte concentration levels were evaluated in 6 replicates. The standard recovery rate of the 9 analytes under different concentrations is 90.84-100.13%, and the RSD is 1.85-9.29%. The result shows that the extraction efficiency of the 9 TKIs is high.
Figure BDA0003653849080000142
Figure BDA0003653849080000151
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (6)

1. A human plasma anti-liver cancer tyrosine kinase inhibitor tandem mass spectrometry detection kit is characterized by comprising a calibrator, a quality control product, an internal standard solution, an extracting agent, a QuEChERS purifying material, a salting-out agent, a mobile phase A solution, a mobile phase B solution, a complex solution, a waterproof packaging bag and a packaging box for separating and intensively packaging the reagent bottles or tubes;
QuEChERS purification material: 20-50mg PSA, and packaging with a waterproof packaging bag;
salting-out agent: 200 and 500mg of anhydrous magnesium sulfate, and packaging the magnesium sulfate by using a water-proof packaging bag.
2. The human plasma anti-liver cancer tyrosine kinase inhibitor tandem mass spectrometry detection kit of claim 1,
calibration products: using methanol diluent, and simultaneously adding standard substances of the ticarcillin, the galluninertib, the sorafenib, the ranvatinib, the regorafenib, the cabozantinib, the apratinib, the gefitinib to prepare a calibrator A-F with 6 concentration dose points, wherein the concentrations of the ticanilinib and the galluninertib are respectively 1ng/mL, 2ng/mL, 5ng/mL, 20ng/mL, 80ng/mL, 100ng/mL, the sorafenib, the ranvatinib, the regorafenib, the cabozantinib, the apratinib, the gefitinib are respectively 0.1ng/mL, 0.2ng/mL, 0.5ng/mL, 2ng/mL, 8ng/mL, 10 ng/mL;
quality control product: using methanol diluent, simultaneously adding standard substances of the tipavancide, the Galunisortib, the sorafenib, the rivatinib, the regorafenib, the cabozantinib, the apratinib, the apartinib and the gefitinib, and preparing 3 concentrations of quality control substances C1-C3, wherein the concentrations of the tipavancib and the Galunisortib are respectively 2ng/mL, 50ng/mL and 100ng/mL, and the concentrations of the sorafenib, the rivatinib, the regorafenib, the cabozantinib, the apratinib, the apartinib and the gefitinib are respectively 0.2ng/mL, 5ng/mL and 10 ng/mL;
internal standard solution: using methanol as a diluent, diluting propranolol to a final concentration of 5 ng/mL;
an extracting agent: 1mL to 2.5mL of acetonitrile;
mobile phase A: is pure acetonitrile;
mobile phase B: is prepared by formic acid and purified water, the content of the formic acid is 0.05 percent to 0.2 percent;
compounding the solution: is prepared from methanol and purified water, wherein the content of the methanol is 50-100%.
3. The human plasma anti-hepatoma tyrosine kinase inhibitor tandem mass spectrometry kit according to any one of claims 1 or 2,
the dosage of acetonitrile in the extractant is 1.5 mL;
the dosage of the PSA is 40 mg;
the dosage of anhydrous magnesium sulfate in the salting-out agent is 350 mg;
the content of formic acid in the mobile phase B is 0.1%;
the content of methanol in the complex solution is 50%.
4. The human plasma anti-liver cancer tyrosine kinase inhibitor tandem mass spectrometry detection kit of claim 1, wherein the using method comprises the following steps:
(1) preparing a working solution: respectively diluting a calibrator, a quality control product and an internal standard solution in the kit by using methanol to respectively prepare a calibrator working solution, a quality control product working solution and an internal standard working solution;
(2) sample pretreatment: mixing 200 mu L of plasma sample with 10 mu L of internal standard working solution, sequentially adding an extracting agent, a QuEChERS purifying material and a salting-out agent, uniformly mixing, centrifugally separating supernatant, thoroughly drying by nitrogen at room temperature, and adding 200 mu L of complex solution until complete dissolution;
(3) uniformly mixing: fully mixing the solution obtained in the step (2) for 1min by using a vortex mixer;
(4) setting chromatographic and mass spectrum conditions: respectively setting working parameters and conditions of the ultra-high performance liquid chromatography and the tandem mass spectrometer according to the model of the actual instrument;
(5) sample detection: injecting 5 mu L of each of the treated working solution of the calibrator, the working solution of the quality control material and the plasma sample into a high performance liquid chromatography-tandem mass spectrometer for detection and analysis, and recording peak areas and internal standard peak areas of sorafenib, rivatinib, regorafenib, cabozantinib, aritinib, gefitinib, tembotinib and gallunessrtib in a chromatogram and the detection sample;
(6) and (4) analyzing results: and drawing a standard curve and calculating a regression equation of the curve, and respectively calculating the concentrations of sorafenib, rivatinib, regorafenib, cabozantinib, aritinib, apatinib, gefitinib, tematinib and gallunertib in the sample.
5. The human plasma anti-liver cancer tyrosine kinase inhibitor tandem mass spectrometry kit of claim 4, wherein in the step (1), the internal standard working solution is prepared according to the ratio of internal standard solution to methanol of 1: 99.
6. The human plasma anti-hepatoma tyrosine kinase inhibitor tandem mass spectrometry detection kit as claimed in claim 4, wherein the chromatographic conditions in step (5) are as follows:
and (3) chromatographic column: hypersil GOLD VANQUISH C18 (2.1X 100mm,1.9 μm),
flow rate: 0.3mL/min of the water-soluble polymer,
column temperature: at a temperature of 40 c,
sample introduction amount: the volume of the solution is 5 mu L,
gradient elution conditions:
Figure FDA0003653849070000031
the mass spectrum conditions are as follows:
the ESI positive ion SRM scan mode,
spraying voltage: the voltage of the liquid crystal is 3500V,
flow rate of sheath gas: 45Arb of the total weight of the steel,
flow rate of auxiliary gas: 10 of the total number of the N,
ion transfer tube temperature: at the temperature of 350 ℃,
atomization temperature: at a temperature of 400 c,
collision gas: the argon gas is introduced into the reaction chamber,
pressure: 1.5 mTor;
Q1/Q3 ion channels were selected as
Rivatinib 427.1 → 370.1, 312.0 amu;
465.1 → 252.1, 270.1 amu;
cabozantinib 502.2 → 323.1, 297.1 amu;
regorafenib 483.1 → 270.1, 288.1 amu;
apatinib: 398.2 → 212.1, 184.1 amu;
gefitinib 447.1 → 128.1, 100.1 amu;
angutinib 408.2 → 339.1, 304.1 amu;
tenavancib 370.1 → 253.1, 158.1 amu;
Galunisertib:370.1→336.1，325.1amu。
CN202210549169.7A 2022-05-20 2022-05-20 Human plasma anti-liver cancer tyrosine kinase inhibitor tandem mass spectrometry detection kit Pending CN115015406A (en)

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WO2023221472A1 (en) * 2022-05-20 2023-11-23 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) Tandem mass spectrometry detection kit for human plasma anti-hepatoma tyrosine kinase inhibitor
WO2024055447A1 (en) * 2022-09-14 2024-03-21 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) Human plasma calcium ion antagonist-tandem mass spectrometry detection kit

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CN112710771A (en) * 2020-12-25 2021-04-27 北京陆道培生物技术有限公司 Kit and method for detecting ABL tyrosine kinase inhibitor based on HPLC-MS/MS method
CN112684057A (en) * 2020-12-31 2021-04-20 南京品生医疗科技有限公司 Kit for detecting concentration of 11 anti-tumor drugs in serum and application thereof
CN115015406A (en) * 2022-05-20 2022-09-06 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) Human plasma anti-liver cancer tyrosine kinase inhibitor tandem mass spectrometry detection kit
CN115112785A (en) * 2022-05-20 2022-09-27 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) Human urine anti-liver cancer tyrosine kinase inhibitor tandem mass spectrometry detection kit

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WO2024055447A1 (en) * 2022-09-14 2024-03-21 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) Human plasma calcium ion antagonist-tandem mass spectrometry detection kit

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