CN114563514B - Method for detecting gilsteritinib in blood plasma, kit and application thereof - Google Patents
Method for detecting gilsteritinib in blood plasma, kit and application thereof Download PDFInfo
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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
- 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
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Abstract
The invention relates to a method for detecting gilsteritinib in blood plasma, a kit and application thereof, wherein the method comprises the following steps: (1) pretreatment of plasma samples: adding a sample treatment solution and an internal standard solution into plasma to be measured, oscillating and mixing uniformly, centrifuging and taking supernatant for later use; (2) preparing a calibrator; (3) HPLC-MS/MS detection: liquid chromatography conditions: mobile phase: mobile phase A is 2mmol/L ammonium acetate-0.1% formic acid aqueous solution; mobile phase B is 0.1% methanolic formate solution; the mass spectrum detection conditions are as follows: electrospray ion source, ion ejection voltage: 5500V, ion source temperature: 500 ℃, GS1:50psi, GS2:55psi, curtangas: the monitoring mode uses a multiplex reaction detection mode MRM at 20 psi. The method has the advantages of simple pretreatment, small required plasma sample amount, short analysis time and good sensitivity and specificity.
Description
Technical Field
The invention belongs to the field of detection, and particularly relates to a method for detecting gilsterinib in blood plasma based on an HPLC-MS/MS method, a kit and application thereof.
Background
Gilsterinib (gefitinib) is a novel tyrosine kinase inhibitor approved for the treatment of acute myeloid leukemia with recurrent or refractory FLT3 mutations. Compared with the first-generation FLT3 inhibitor, the Gilterrinib single drug has good drug resistance and high selectivity to FLT 3. For patients with acute myeloid leukemia with recurrent or refractory FLT3 mutations, the survival and remission rates of gilsterinib treatment are superior to rescue treatment, and thus research into gilsterinib is necessary.
Gilsterinib is metabolized primarily by cytochrome P450 (CYP) 3A4 and use in combination with CYP3A4 inhibitors or inducers can significantly affect the exposure of gilsterinib and the incidence of adverse reactions, resulting in large differences between individuals with gilsterinib concentrations. Therefore, the research on the pharmacokinetics of gilitetinib has great significance in the development process of gilitetinib, and the research on the pharmacokinetics of gilitetinib requires a sensitive, specific and accurate method for detecting gilitetinib in blood plasma.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a method for detecting gilsterinib in blood plasma, a kit and application thereof, wherein the pretreatment is simple, the required blood plasma sample amount is small, the analysis time is short, and the sensitivity and the specificity are good.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the technical scheme is as follows:
a method of detecting gilsterinib in plasma comprising:
(1) Pretreatment of plasma samples: adding a sample treatment solution and an internal standard solution into plasma to be measured, oscillating and mixing uniformly, centrifuging and taking supernatant for later use;
(2) Preparing a calibrator;
(3) HPLC-MS/MS detection:
liquid chromatography conditions: the chromatographic column is Ultimate XB-C18, with specification of 4.6mm×50mm, and particle diameter of 5 μm; column temperature: 60 ℃; mobile phase: mobile phase A is 2mmol/L ammonium acetate-0.1% formic acid aqueous solution by volume concentration; mobile phase B is 0.1% methanol formate solution by volume; the chromatographic separation is gradient elution, and the gradient elution conditions are as follows: 0.01-0.8 min, B phase concentration of 40-95%, 1.6min, 2.4-2.6 min, B phase concentration of 95-40%, and 1.9min; flow rate: 0.7mL/min; sample injection volume: 5 μl;
the mass spectrum detection conditions are as follows: electrospray ion source, ion ejection voltage: 5500V, ion source temperature: 500 ℃, GS1:50psi, GS2:55psi,Curtain gas: the monitoring mode uses a multiplex reaction detection mode MRM at 20 psi.
Further, the mass spectrometry conditions of the MRM are shown in table 1;
table 1: MRM Mass Spectrometry Condition
| Parent ion | Ion | Dwelltime | DP(v) | CE(v) | |
| Gilteritinib | 553.6 | 436.3 | 100 | 85 | 40 |
| Gilteritinib-d 8 | 561.6 | 436.4 | 100 | 70 | 43 |
Furthermore, the calibrator adopts Gilterrinib standard solutions with different concentration gradients;
the preparation method of the Giltenitiib standard solution with different concentration gradients comprises the following steps: stock solutions of the standard products were prepared with a 1:1 mixture of methanol and acetonitrile, and then diluted with methanol to 8 concentration gradients of 0ng/mL, 10ng/mL, 25ng/mL, 100ng/mL, 250ng/mL, 500ng/mL, 1000ng/mL, 2500ng/mL, respectively.
A method of detecting gilsterinib in plasma, further comprising drawing a standard curve:
preparing a standard curve: and (3) respectively adding the Gilterminib standard solutions with different concentration gradients into the sample treatment solution and the internal standard solution, then carrying out sample injection detection, and drawing a calibration curve by taking the concentration of the Gilterminib calibration solution as an abscissa and the ratio of the measured peak area of the Gilterminib standard solution to the peak area of the internal standard solution as an ordinate.
A method of detecting gilsterinib in plasma, further comprising a quality control for a validation test of the detection method, the validation test of the detection method comprising a precision test, a recovery test and a stability test.
Further, the quality control product comprises a single level of low, medium and high concentration gilsterinib quality control solution;
the low-concentration level quality control solution adopts 100ng/mL Gilterminib standard solution;
the medium concentration level quality control solution adopts a Gilterrinib standard solution with the concentration of 750 ng/mL;
the high-concentration level quality control solution adopts 2000ng/mL Gilterrinib standard solution.
Further, in the pretreatment process of the plasma sample, the volume ratio of the plasma, the sample treatment liquid and the internal standard liquid is as follows: 1:1:3;
further, the sample treatment fluid is: 2mmol/L ammonium acetate-0.1% strength by volume aqueous formic acid solution;
the internal standard solution is as follows: 400ng/mL Gilterminib-d 8 methanol standard solution.
The second technical scheme is as follows:
a kit for performing gilsterinib detection in plasma using the method, comprising: calibration material, quality control material, sample treatment fluid, internal standard fluid and mobile phase.
The technical scheme is as follows:
use of the kit in the detection of giltidinib in plasma.
Compared with the prior art, the invention has the beneficial effects that:
the detection result of the invention is not interfered by common medicines and combined medicines, and an isotope internal standard is added in the sample treatment process, so that the accuracy of Gilterrinib quantitative detection is improved; the high performance liquid chromatography tandem mass spectrometry method has the advantages of simple pretreatment, small required plasma sample amount, short analysis time, good sensitivity and specificity, and can be used for Gilterrinib pharmacokinetics analysis and research.
Description of the drawings:
FIG. 1 is a graph of the secondary scan fragment ions of Giltertinib in the present invention;
FIG. 2 is a graph of the secondary scan fragment ions of Gilterminib-d 8 in the present invention;
FIG. 3 is a graph of Gilterminib according to the present invention;
Detailed Description
The technical solutions of the present invention will be clearly and fully described below with reference to specific embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1:
a kit for detecting gilsterinib in plasma comprising the following components: (1) a calibrator; (2) quality control product; (3) a sample processing liquid; (4) an internal standard solution; (5) a mobile phase;
wherein the calibrator is a giltidinib standard solution at 8 different concentration levels;
concentration level 7:2500ng/mL of Gilterminib standard solution;
concentration level 6:1000ng/mL of Gilterminib standard solution;
concentration level 5:500ng/mL of Gilterminib standard solution;
concentration level 4:250ng/mL of Gilterminib standard solution;
concentration level 3:100ng/mL Gilterminib standard solution;
concentration level 2:25ng/mL of Gilterminib standard solution;
concentration level 1:10ng/mL of Gilterminib standard solution;
concentration level 0: methanol.
The quality control product is a Gilteritinib quality control solution with low, medium and high concentration levels;
low concentration level gilsteritinib quality control solution: 100ng/mL Gilterminib standard solution;
medium concentration level gilsteritinib quality control solution: 750ng/mL of Gilterminib standard solution;
high concentration level gilsteritinib quality control solution: 2000ng/mL Gilterminib standard solution.
The sample treatment solution is 2mmol/L ammonium acetate-0.1% formic acid aqueous solution.
The internal standard solution is 400ng/mL Gilterminib-d 8 methanol solution.
The mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is 2mmol/L of ammonium acetate-0.1% formic acid aqueous solution and the mobile phase B is 0.1% formic acid methanol solution.
Example 2
The preparation method of the kit in the embodiment 1 comprises the following steps:
(1) Preparation of a standard:
taking 12mg of Giltering standard substance, dissolving with a mixed solution of methanol and acetonitrile in a ratio of 1:1, fixing the volume to a 10mL volumetric flask, carrying out ultrasonic mixing uniformly to obtain 1.2mg/mL Giltering mother liquor, and sub-packaging for later use.
Taking 100 mu L of 1.2mg/mL Gilterminib mother solution, adding 1100 mu L of methanol, and shaking and mixing uniformly to obtain 100 mu g/mL Gilterminib standard solution;
100 mu L of 100 mu g/mL of Gilterminib standard solution is taken, 900 mu L of methanol is added, and the mixture is mixed with shaking to obtain 10 mu g/mL of Gilterminib standard solution.
Concentration level 7: taking 200 mu L of 10 mu g/mL of Giltering standard solution, adding 600 mu L of methanol, and shaking and mixing uniformly to obtain 2500ng/mL of Giltering standard solution;
concentration level 6: taking 100 mu L of 10 mu g/mL of Giltering standard solution, adding 900 mu L of methanol, and shaking and mixing uniformly to obtain 1000ng/mL of Giltering standard solution;
concentration level 5: taking 500 mu L of solution with the concentration level of 6, adding 500 mu L of methanol, and shaking and uniformly mixing to obtain a Giltering standard solution with the concentration of 500ng/mL;
concentration level 4: taking 100 mu L of solution with the concentration level of 7, adding 900 mu L of methanol, and shaking and uniformly mixing to obtain a Giltering standard solution with the concentration of 250 ng/mL;
concentration level 3: taking 100 mu L of solution with the concentration level of 6, adding 900 mu L of methanol, and shaking and uniformly mixing to obtain 100ng/mL Gilterminib standard solution;
concentration level 2: taking 100 mu L of solution with the concentration level of 4, adding 900 mu L of methanol, and shaking and uniformly mixing to obtain 25ng/mL Gilterminib standard solution;
concentration level 1: taking 100 mu L of solution with the concentration level of 3, adding 900 mu L of methanol, and shaking and uniformly mixing to obtain 10ng/mL Gilterminib standard solution;
concentration level 0: methanol.
(2) And (3) preparation of a quality control product:
high concentration level gilsteritinib quality control solution: taking 200 mu L of 10 mu g/mL of Giltering standard solution, adding 800 mu L of methanol, and shaking and mixing uniformly to obtain 2000ng/mL of Giltering standard solution;
medium concentration level gilsteritinib quality control solution: taking 60 mu L of 10 mu g/mL of Giltering standard solution, adding 740 mu L of methanol, and shaking and mixing uniformly to obtain 750ng/mL of Giltering standard solution;
low concentration level gilsteritinib quality control solution: taking 100 mu L of 1000ng/mL of Giltering standard solution, adding 900 mu L of methanol, and shaking and mixing uniformly to obtain 100ng/mL of Giltering standard solution.
(3) Preparation of sample treatment solution (2 mmol/L ammonium acetate-0.1% strength by volume aqueous formic acid solution):
step 1: weighing 7.7g of ammonium acetate, dissolving the ammonium acetate in the Guangzhou yield distilled water, and adding the distilled water to a 100mL volumetric flask to fix the volume, and uniformly mixing the ammonium acetate with ultrasonic waves to obtain 100mmol/L ammonium acetate solution;
step 2: taking 1mL of formic acid solution, 2mL of 100mmol/L ammonium acetate solution, and using Guangzhou distilled water to fix the volume into a 1000mL volumetric flask, and carrying out ultrasonic mixing to obtain 2mmol/L ammonium acetate-0.1% formic acid water solution.
(4) Preparation of internal standard solution (400 ng/mL Gilterrinib-d 8 solution):
dissolving 10mg of Gilterminib-d 8 standard substance with methanol, fixing the volume with methanol to a 10mL volumetric flask to obtain 1mg/mL Gilterminib-d 8 solution, mixing with ultrasound, and packaging;
taking 100 mu L of 1mg/mL Giltering-d 8 solution, using methanol to fix the volume to a 250mL volumetric flask, and carrying out ultrasonic mixing to obtain 400ng/mL Giltering-d 8 solution.
(5) Preparation of mobile phase:
preparation of mobile phase a: weighing 7.7g of ammonium acetate, dissolving with Guangzhou Chengsu distilled water, and adding the solution into a 100mL volumetric flask with the volume fixed by using the Guangzhou Chengsu distilled water, and uniformly mixing by ultrasonic to obtain 100mmol L -1 Ammonium acetate solution;
1mL of formic acid solution, 2mL of 100mmol. L -1 The ammonium acetate solution is prepared into a 1000mL volumetric flask by using distilled water of Chengshi, guangzhou, and is evenly mixed by ultrasonic, namely 2 mmol.L -1 Ammonium acetate-0.1% strength by volume aqueous formic acid solution;
preparation of mobile phase B: taking 1mL of formic acid solution, using methanol to fix the volume to a 1000mL volumetric flask, and uniformly mixing by ultrasonic to obtain the formic acid methanol solution with the volume concentration of 0.1%.
Example 3
A method of detecting gilsterinib in plasma comprising:
(1) 10 mu L of each solution in the calibrator and the quality control product in the kit of the example 1 is respectively added with 90 mu L of blank plasma, 100 mu L of sample treatment solution and 300 mu L of internal standard solution, and the mixture is vibrated and mixed uniformly, and supernatant is obtained by centrifugation for standby.
(2) Taking 100 mu L of plasma to be detected, adding 100 mu L of sample treatment liquid and 300 mu L of internal standard liquid, vibrating and mixing uniformly, centrifuging, and taking supernatant for later use.
(3) Sampling and detecting each supernatant, using a mass spectrum isotope internal calibration method, taking the concentration of Giltering standard vertebral products as an abscissa, taking the ratio of the measured peak area of the Giltering standard products to the peak area of the internal standard products as an ordinate, establishing a standard curve, and calculating the concentration of Giltering standard products in a sample to be detected according to the standard curve;
drawing a standard curve: taking the standard solution with the concentration of Gilterminib 7 as an abscissa (X), taking the ratio of the measured peak area of the standard solution with the concentration of Gilterminib 7 to the peak area of each internal standard as an ordinate (Y), carrying out linear fitting on X-line weighted square regression by Y, and drawing a standard curve to obtain a linear regression equation of Y=0.00185X+0.0122, wherein r=0.9990.
(4): the detection conditions of the high performance liquid chromatography tandem mass spectrometry are as follows:
liquid chromatography conditions: the chromatographic column is Ultimate XB-C18, with specification of 4.6mm×50mm, and particle diameter of 5 μm; column temperature: 60 ℃; mobile phase: mobile phase A is 2mmol/L ammonium acetate-0.1% formic acid aqueous solution by volume concentration; mobile phase B is 0.1% methanol formate solution by volume; chromatographic separation is gradient elution: 0.01-0.8 min, B phase concentration of 40-95%, 1.6min, 2.4-2.6 min, B phase concentration of 95-40%, and 1.9min; flow rate: 0.7mL/min; sample injection volume: 5 μl;
the mass spectrum detection conditions are as follows: electrospray ion source, ion ejection voltage: 5500V, ion source temperature: 500 ℃, GS1:50psi, GS2:55psi,Curtain gas: the monitoring mode uses a multiplex reaction detection mode MRM at 20 psi.
The MRM parameters are shown in Table 1.
Table 1 mass spectrometry conditions
| Parent ion | Ion | Dwelltime | DP(v) | CE(v) | |
| Gilteritinib | 553.6 | 436.3 | 100 | 85 | 40 |
| Gilteritinib-d 8 | 561.6 | 436.4 | 100 | 70 | 43 |
Example 4: and (3) verifying a detection method:
1. precision and recovery test
Taking 6 parts of Giltering quality control solutions with low, medium and high concentrations in the quality control product of the example 1, respectively adding 90 mu L of blank plasma, 100 mu L of sample treatment liquid and 300 mu L of internal standard liquid into each part of sample 10 mu L, vibrating and mixing uniformly, centrifuging, and taking supernatant for later use; and then carrying out sample injection analysis according to the detection conditions of the high performance liquid chromatography tandem mass spectrometry in the embodiment 3, and obtaining the measured concentration of the quality control sample according to the standard curve of the same day, and continuously measuring for 3 days. Based on the measurement results, the daily and daytime RSD were calculated, and the ratio of the measured concentration to the standard concentration was used as the relative recovery rate. The results are shown in Table 2.
Table 2 gilsterinib precision and recovery assay (n=6,)
2. stability test
Taking 6 parts of Giltering quality control solution with low, medium and high 3 concentrations in the quality control product of example 1, respectively adding 90 mu L of blank plasma, 100 mu L of sample treatment liquid and 300 mu L of internal standard liquid into each part of sample 10 mu L, respectively refrigerating and standing at 4 ℃ for 12 hours and at room temperature for 12 hours, performing sample injection analysis according to the detection conditions of high performance liquid chromatography-tandem mass spectrometry in example 3, respectively measuring the concentration of each sample, investigating the stability, comparing the measured concentration with the standard concentration, and obtaining the result shown in Table 3
Table 3 gilsterinib stability test (n=6,)
in conclusion, the test shows that the kit has the advantages of simple operation, accurate result, good precision, relative recovery rate up to 85% -115%, good accuracy, RSD lower than 10%, precision and stability meeting the requirements of clinical batch sample monitoring.
The above described embodiments are only preferred examples of the invention and are not exhaustive of the possible implementations of the invention. Any obvious modifications thereof, which would be apparent to those skilled in the art without departing from the principles and spirit of the present invention, should be considered to be included within the scope of the appended claims.
Claims (1)
1. A method for detecting gilsterinib in plasma based on HPLC-MS/MS, comprising:
(1) Pretreatment of plasma samples: adding a sample treatment solution and an internal standard solution into plasma to be measured, oscillating and mixing uniformly, centrifuging and taking supernatant for later use;
in the pretreatment process of the plasma sample, the volume ratio of the plasma, the sample treatment liquid and the internal standard liquid is as follows: 1:1:3;
the sample treatment fluid is as follows: 2mmol/L ammonium acetate-0.1% strength by volume aqueous formic acid solution;
the internal standard solution is as follows: 400ng/mL Gilterminib-d 8 methanol standard solution;
(2) Preparing a calibrator;
the calibrator adopts Gilterminib standard solutions with different concentration gradients;
the preparation method of the Giltenitiib standard solution with different concentration gradients comprises the following steps: preparing a standard stock solution by using a mixed solution of methanol and acetonitrile at a ratio of 1:1, and then diluting the stock solution into 8 concentration gradients by using methanol, wherein the 8 concentration gradients are respectively 0ng/mL, 10ng/mL, 25ng/mL, 100ng/mL, 250ng/mL, 500ng/mL, 1000ng/mL and 2500ng/mL;
the method further comprises the steps of drawing a standard curve:
preparing a standard curve: respectively adding the Gilterminib standard solutions with different concentration gradients into a sample treatment solution and an internal standard solution, then carrying out sample injection detection, taking the concentration of the Gilterminib calibration solution as an abscissa, taking the ratio of the measured peak area of the Gilterminib standard solution to the peak area of the internal standard solution as an ordinate, and drawing a calibration curve;
(3) HPLC-MS/MS detection:
liquid chromatography conditions: the chromatographic column is Ultimate XB-C18, with specification of 4.6mm×50mm, and particle diameter of 5 μm; column temperature: 60 ℃; mobile phase: mobile phase A is 2mmol/L ammonium acetate-0.1% formic acid aqueous solution by volume concentration; mobile phase B is 0.1% methanol formate solution by volume; the chromatographic separation is gradient elution, and the gradient elution conditions are as follows: 0.01-0.8 min, B phase concentration of 40-95%, 1.6min, 2.4-2.6 min, B phase concentration of 95-40%, and 1.9min; flow rate: 0.7mL/min; sample injection volume: 5 μl;
the mass spectrum detection conditions are as follows: electrospray ion source, ion ejection voltage: 5500V, ion source temperature: 500 ℃, GS1:50psi, GS2:55psi,Curtain gas:20psi, the monitoring mode adopts a multiple reaction detection mode MRM;
the mass spectrometry conditions of the MRM are shown in the following table;
table: MRM Mass Spectrometry Condition
The quality control product is used for a verification test of a detection method, wherein the verification test of the detection method comprises a precision test, a recovery rate test and a stability test;
the quality control product comprises a single level of low, medium and high concentration Gilterrinib quality control solution;
the quality control solution with low concentration level adopts 100ng/mL Gilterminib standard solution;
the medium concentration level quality control solution adopts a Gilterrinib standard solution with the concentration of 750 ng/mL;
the high concentration level quality control solution adopts 2000ng/mL Gilterrinib standard solution.
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Non-Patent Citations (2)
| Title |
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| An LC-MS/MS Bioanalytical Assay for the Determination of Gilteritinib in Rat Plasma and Application to a Drug–Drug Interaction Study;Qiong Wang et al.;《Drug Design, Development and Therapy》;第14卷;第2063页 * |
| Využití LC-MS/MS v in vitro transportních experimentech;Šárka Salvová;《Katedra farmaceutické chemie a farmaceutické analýzy》;第9页 * |
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