CN113960193A

CN113960193A - Method for measuring plasma MRTX849 concentration by high performance liquid chromatography tandem mass spectrometry

Info

Publication number: CN113960193A
Application number: CN202111178179.6A
Authority: CN
Inventors: 杜萍; 安卓玲; 王国永; 钟久昌
Original assignee: Beijing Chaoyang Hospital
Current assignee: Beijing Chaoyang Hospital
Priority date: 2021-10-09
Filing date: 2021-10-09
Publication date: 2022-01-21

Abstract

The invention discloses a method for measuring the concentration of plasma MRTX849 by high performance liquid chromatography-tandem mass spectrometry, which comprises the following steps: dissolving MRTX849 by using methanol to prepare standard curve working solutions with a plurality of concentrations, and preparing an internal standard substance working solution by using methanol; the linear range of the standard curve is 0.05-200 ng/mL; precisely sucking 50 mu L of plasma to be detected, adding internal standard substance working solution, vortexing, centrifuging, taking supernate to perform HPLC-MS/MS quantitative analysis, and reading the concentration of MRTX849 on a standard curve. The invention lays a technical foundation for monitoring follow-up clinical routine treatment medicines.

Description

Method for measuring plasma MRTX849 concentration by high performance liquid chromatography tandem mass spectrometry

Technical Field

The present invention relates to a method for detecting the concentration of a drug. More specifically, the invention relates to a method for determining plasma MRTX849 concentration by high performance liquid chromatography-tandem mass spectrometry.

Background

In recent years, with the wide clinical application of anti-tumor molecule targeted drugs, Tyrosine Kinase Inhibitors (TKIs) represented by Epidermal Growth Factor Receptor (EGFR), Anaplastic Lymphoma Kinase (ALK) rearrangement, Vascular Endothelial Growth Factor Receptor (VEGFR), and the like significantly improve the benefit risk ratio of patients with advanced cancer. The KRAS gene is located on chromosome 12, is a protooncogene of RAS family, and is an important "switch" in intracellular signaling pathways. Once it is opened, it will activate a variety of division, proliferation factors, including c-Raf and PI 3K. Normally, KRAS binds to GTP, cleaving the last phosphate group of GTP, allowing it to become GDP. KRAS is deactivated upon conversion of GTP to GDP. However, after the KRAS gene is mutated, the KRAS protein is continuously kept in an activated state, does not depend on the stimulation of a superior signal, and is in a state of being continuously combined with GTP, so that a downstream signal path is abnormally active, and the growth and proliferation of cells are promoted. Resulting in sustained cell proliferation. KRAS mutation sites, one of the most common oncogenes, are present in about 25% of all cancers. Wherein KRAS (G12C) accounts for more than 40% of all KRAS mutations and is highly dominant (about 13%) in lung cancer.

MRTX849 is an inhibitor of clinical trials in advanced cancer patients with KRAS (G12C) gene mutations. The results of early clinical studies showed that in the clinical studies of stage ib (14) and stage ii (51) of non-small cell lung cancer, the overall remission rates of MRTX849 treatment were 43% and 45%, respectively, and the disease control rates reached 100% and 96%. Currently, MRTX849, single or combined with tumor immunosuppressive agents, is undergoing clinical phase ii-iii studies, and no more effective data are disclosed.

It is well known that plasma drug concentration and exposure information are critical to evaluating the effectiveness, safety and tolerability of drugs. The drug concentration is the basis of exerting the drug effect and is also one of the important factors causing poor curative effect and increased toxicity of the drug. The blood concentration of a patient is regularly monitored, so that the curative effect and possible adverse reactions can be predicted, the benefit risk ratio of the patient is obviously improved, and the individual accurate medication is realized. However, no report is made on the high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) quantitative detection method of MRTX 849. Therefore, it is necessary and desirable to establish an accurate, rapid, high throughput method for determining the concentration of MRTX849 drug in blood. In order to meet the requirements of preclinical and clinical high-throughput detection and blood concentration monitoring, an HPLC-MS/MS method which has no special equipment requirement, has high throughput and less sample requirement, is convenient and rapid to determine the concentration of MRTX849 is urgently needed in the field.

Disclosure of Invention

An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.

The invention also aims to provide a method for measuring the concentration of the MRTX849 in the blood plasma by the high performance liquid chromatography-tandem mass spectrometry, which lays a technical foundation for the follow-up clinical routine therapeutic drug monitoring.

To achieve these objects and other advantages in accordance with the present invention, there is provided a method for determining concentration of MRTX849 in plasma by high performance liquid chromatography tandem mass spectrometry, comprising:

dissolving MRTX849 by using methanol to prepare standard curve working solution with a plurality of concentrations, and dissolving a carbamazepine internal standard substance by using methanol to prepare internal standard substance working solution;

respectively adding 0-5 mu L of standard curve working solution with multiple concentrations into blank plasma to complement to 50 mu L, carrying out vortex to prepare standard curve plasma samples with multiple concentrations, adding 5-10 mu L of internal standard substance working solution and 120-20 ℃ methanol vortex, carrying out centrifugation at 0-4 ℃, taking supernatant to carry out HPLC-MS/MS quantitative analysis, and drawing a standard curve with a linear range of 0.05-200ng/mL by taking the concentration of MRTX849 in the plasma samples as a horizontal coordinate and the ratio of the peak area of the MRTX849 to the peak area of the carbamazepine internal standard substance as a vertical coordinate;

precisely absorbing 50 mu L of plasma to be detected, adding equivalent internal standard substance working solution and 120-20 ℃ methanol vortex when a standard curve is drawn, centrifuging at 0-4 ℃, taking supernate to perform HPLC-MS/MS quantitative analysis, and reading the concentration of MRTX849 corresponding to the ratio of the measured MRTX849 peak area to the internal standard substance peak area on the standard curve;

the liquid phase conditions were: the chromatographic column is Waters XBrige C₁₈The inner diameter is 2.1mm, the column length is 50mm, the particle size is 3.5mm, the flow rate is 0.4mL/min, the sample injection volume is 4 muL, the analysis time is 3min, the column temperature is 40 ℃, the needle washing solution is a mixed solution of isopropanol, acetonitrile, methanol and water in a volume ratio of 1:1:1:1, the organic phase of the mobile phase is acetonitrile containing 0.1% by mass of formic acid, and the water phase is acetonitrileWater containing 0.1% formic acid and 5mM ammonium acetate, and gradient elution is set to be 90%, 10%, 0%, 90% of water phase at volume ratio of 0.01min, 1.2min, 2.2min, 2.3-3.0 min.

The mass spectrum conditions are as follows: the multi-reaction monitoring of the electrospray ion source is adopted, the positive ion scanning mode is adopted, the mass charge ratio of a parent ion pair and a child ion pair of MRTX849 is m/z 605.2 → 98.2, the mass charge ratio of a parent ion pair and a child ion pair of an internal standard substance is m/z 237.0 → 194.1, the scanning time of each channel is 200ms, the ionization temperature of the mass spectrum is 550 ℃, the gas curtain gas is 20psi, the atomizing gas and the auxiliary gas are both 55psi, the ionization voltage is 5500V, the inlet voltage is 10eV, and the outlet voltage of a collision chamber is 13 eV.

Preferably, the standard curve working solutions with concentrations of 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 100ng/mL, 500ng/mL, 1. mu.g/mL and 2. mu.g/mL are prepared by dissolving MRTX849 in methanol, and the internal standard working solutions with concentrations of 10ng/mL are prepared by dissolving the carbamazepine internal standard in methanol.

Preferably, 5. mu.L of standard curve working solution is pipetted precisely, 45. mu.L of blank plasma is added, and 10. mu.L of internal standard working solution is added.

Preferably, the mixture is vortexed and vortexed for 1min, and centrifuged at 13500rpm for 10min at 4 ℃.

Preferably, the method comprises the following steps:

dissolving MRTX849 by using methanol to prepare quality control working solutions with a plurality of concentrations, and dissolving a carbamazepine internal standard substance by using methanol to prepare an internal standard substance working solution;

adding 0-5 muL of quality control working solution with a plurality of concentrations into blank plasma respectively to complement to 50 muL, carrying out vortex to prepare quality control plasma samples with a plurality of concentrations, adding 5-10 muL of internal standard substance working solution and 120-200 muL of methanol at-20 ℃, carrying out vortex at 0-4 ℃, centrifuging, taking supernatant, carrying out HPLC-MS/MS quantitative analysis, and correcting the standard curve to ensure that the accuracy of at least 2/3 of the quality control samples is within +/-15% of the marked value, and at least 50% of each concentration level of the samples meets the standard.

Preferably, the quality control working solution with the concentration of 1ng/mL, 20ng/mL and 1.6 mu g/mL is prepared by dissolving MRTX849 in methanol.

Preferably, 5. mu.L of quality control working solution is precisely aspirated, 45. mu.L of blank plasma is added, 10. mu.L of internal standard working solution and 140. mu.L of methanol at-20 ℃ are added.

The invention at least comprises the following beneficial effects:

firstly, the HPLC-MS/MS method established by the invention is used for measuring the concentration of MRTX849 in blood, has high sensitivity, strong specificity, rapidness and good reproducibility, and is suitable for clinically detecting and monitoring the concentration of a medicament at the same time with high flux, thereby realizing individualized accurate treatment.

Second, in the method of the invention, a smaller blood sample volume is used for the first time, the plasma sample volume is only 50 μ L, significantly reducing the traumatic burden on patients with advanced cancer. The result of the quantitative analysis shows that the volume used in the experiment can meet the requirement of quantitative detection, and based on the advantage, the method is particularly suitable for quantitative determination when the body state of a late-stage tumor patient is poor and the volume of a clinically collected blood sample is small, and is favorable for the application of monitoring of clinical treatment drugs.

Thirdly, the method for measuring the concentration of MRTX849 medicament, which is established for the first time, meets the analysis requirements of 2020 edition of Chinese pharmacopoeia, four parts 9012 of biological sample quantitative analysis method verification guiding principles on biological samples on the aspects of accuracy, precision, specificity, stability, extraction recovery rate, matrix effect and the like, and is successfully applied to rat pharmacokinetics research.

Fourth, liquid phase conditions preferably employ 0.1% formic acid and 5mM ammonium acetate in water (aqueous phase) -0.1% formic acid acetonitrile (organic phase); the addition of 0.1% formic acid can promote the ionization of the analyte, improve the detection sensitivity, increase the ionization effect of the object to be detected and is beneficial to eliminating the tailing phenomenon of the chromatographic peak; the addition of 5mM ammonium acetate prevents tailing of chromatographic peaks, and symmetric chromatographic peaks are easily obtained. In addition, highly efficient Waters XBrige C is employed in the method of the invention₁₈The chromatographic column (50mm multiplied by 2.1mm, the grain diameter is 3.5 mu m) improves the separation efficiency, realizes that the analysis time of each sample is only 3min, obviously increases the analysis flux, improves the analysis speed, and is beneficial to the rapid analysis of large-scale preclinical and clinical samples. Method of the inventionA high-sensitivity ultra-fast scanning speed triple quadrupole rod linear ion trap mass spectrometer (QTRAP 5500) mass spectrum system is adopted, and the quantitative detection limit is 0.05 ng/mL.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

Figure 1 is a mass spectrometry scan of MRTX849 and the internal standard carbamazepine.

FIG. 2 is a schematic diagram of a calibration curve.

FIG. 3 is a typical chromatogram of the results of the specificity test.

FIG. 4 is a plot of mean drug concentration versus time in rats and reanalysis of test samples after intragastric administration of MRTX849(15 mg/kg).

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.

1 preparation of solution

The volume and weight used to formulate the solutions can be adjusted proportionally, and all solutions are kept at room temperature except as specifically stated.

1.1 preparation of the Mobile phase solution

Organic phase (a): acetonitrile containing 0.1% by mass of formic acid.

Aqueous phase (B): water containing 0.1% by mass of formic acid, 5mM ammonium acetate.

Selecting a mobile phase: in order to meet the requirement of low quantitative detection range in the experiment, we examined organic phase (such as methanol, acetonitrile, etc.) and aqueous phase in various common mobile phases, and tried to add solution enhancers (such as 0.01% formic acid, 0.05% formic acid, 0.1% formic acid, 0.01% acetic acid, 0.1% trifluoroacetic acid, 5mM ammonium formate, 2mM ammonium acetate, etc.) in different proportions. As a result, it was found that the peak profile was improved when formic acid was added to the organic phase and the aqueous phase, the tailing of the peak was improved when 5mM ammonium acetate was added, and the most mobile phases were acetonitrile containing 0.1% formic acid-0.1% formic acid in 5mM ammonium formate water, based on the composition, retention time, chromatographic peak profile, gradient time, and the like of the mobile phase.

1.2 preparation of working fluid

Respectively and precisely weighing more than 10.00mg of MRTX849 and an internal standard substance carbamazepine in a 10mL beaker, adding a small amount of methanol to dissolve the materials, transferring the materials into a 10mL measuring flask, and carrying out constant volume to obtain a stock solution with the concentration of 1 mg/mL. Diluting step by adopting 1:1 methanol water to obtain a working solution with a standard curve: 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 100ng/mL, 500ng/mL, 1. mu.g/mL and 2. mu.g/mL, stored in a freezer at-80 ℃ until use. The internal standard working solution is diluted by methanol to the concentration of 10 ng/mL. The quality control working solution comprises: 1ng/mL, 20ng/mL, 1.6. mu.g/mL.

1.3 instruments and reagents

High performance liquid chromatograph (LC-20ADXR, Shimadzu corporation) tandem mass spectrometry detector (QTRAP5500, SCIEX corporation); milli Q-Direct 8 ultrapure water system (Millipore corporation, USA); fixed angle centrifuge (Thermo Scientific, usa).

MRTX849 (purity > 99%, batch number: DC2229803, purchased from DC Chemical company), carbamazepine (purity: 100%, batch number: 100142-; HPLC grade methanol, acetonitrile, isopropanol, etc. are all available from Fisher Scientific, USA. Other conventional reagents are commercially available on a daily basis.

In the experiment, the inventor firstly adopts a conventional HPLC-MS/MS system to preliminarily examine the feasibility of quantitative analysis, but the result shows that the peak area of the substance to be detected cannot meet the requirement of quantitative concentration. Therefore, the MRTX849 serving as a novel targeting KRAS (G12C) small molecular compound is difficult to analyze by a conventional LC-MS/MS system, and in order to ensure that an experiment is smoothly carried out, the inventor adopts a high-performance liquid chromatograph with higher resolution and detection capability and a triple quadrupole linear ion trap mass spectrometer connected in series as a final detection instrument. The molecular ion mass spectrum of MRTX849 is shown in figure 1.

1.4 Standard Curve and preparation and pretreatment of quality control plasma sample

Respectively sucking 5 mu L of series concentration standard curve and quality control working solution into an Eppendorf tube, then respectively adding 45 mu L of blank plasma, 10 mu L of internal standard working solution and 140 mu L of cold methanol (-20 ℃), whirling for 30s, and uniformly mixing to prepare series standard plasma samples with the concentrations of 0.05, 0.1, 0.5, 1, 2, 10, 50, 100 and 200 ng/mL. Blood control samples (0.1, 2, 160ng/mL) were prepared in a similar manner as described above.

2 liquid chromatogram tandem mass spectrum method condition

Liquid chromatography conditions: by using efficient Waters XBrige C₁₈Chromatography column (50 mm. times.2.1 mm, particle size 3.5 μm) at 40 ℃. Mobile phase a (acetonitrile, containing 0.1% formic acid): phase B (water, containing 0.1% formic acid, 5mM ammonium acetate). The flow rate was 0.4mL/min and the amount of sample was 4. mu.L. The needle washing solution is a mixed solution (1:1:1:1, v/v/v/v) containing isopropanol, acetonitrile, methanol and water, and gradient elution is adopted, wherein the gradient elution procedure is as follows: 0.01min (90% B), 1.2min (10% B), 2.2min (0% B), 2.3-3.0min (90% B). The analysis time is 3min, and the retention time of the two is 1.70min and 1.58min respectively.

Mass spectrum conditions: adopting Electrospray ionization (ESI) to perform measurement in positive ionization mode by mass spectrometry scanning mode of Multiple Reaction Monitoring (MRM), wherein the scanning time of each channel is 200ms, and [ M + H ] is mainly generated]⁺Excimer ion peak. The temperature of the mass spectrometer is 550 ℃, the gas curtain gas is 20psi, the atomizing gas and the auxiliary gas are both 55psi, the ionization voltage is 5500V, the inlet voltage is 10eV, and the outlet voltage of the collision chamber is 13 eV. The mass-to-charge ratio of the parent ion pair and the daughter ion pair of the MRTX849 is m/z 605.2 → 98.2, and the mass-to-charge ratio of the parent ion pair and the daughter ion pair of the internal standard is m/z 237.0 → 194.1.

And (3) an elution mode: the inventors also compared isocratic elution and gradient elution and showed that both chromatographic peak shape and response were higher than isocratic mode at gradient elution, so finally set the gradient within 3min of analysis time, with an initial gradient of 90% of the aqueous phase, thus ensuring a chromatographic peak at the highest organic phase ratio. In addition, we compared different flow rates (0.4mL/min and 0.5mL/min), different column temperature conditions (room temperature, 40 ℃, 50 ℃), different analysis times (3min, 4min and 5min), different sample volumes (4. mu.L, 2. mu.L and 1. mu.L). After many times of groping and optimization, the optimal conditions for confirmation and analysis are that the mobile phase is acetonitrile (A) and water (B) (both of which contain 0.1% formic acid), a gradient elution mode is adopted, the flow rate is 0.4mL/min, the column temperature is 40 ℃, the analysis time is 3min, the sample injection volume is 4 mu L, and the retention time of the two is 1.70min and 1.58min respectively.

Mass spectrometry ionization mode: in the experiment, the inventor respectively inspects the positive ion ionization mode and the negative ion ionization mode of the mass spectrum according to the chemical structures and the physicochemical properties of the two objects to be measured and the internal standard, and the result shows that the response of the objects to be measured and the internal standard under the positive ion mode is improved compared with the response of the negative ion mode, so that the positive ion mode adopting the ESI of the mass spectrum is determined. Selecting the parent and child ion pairs to be analyzed, combining different ionization energies and possible generated molecular fragments, and adopting M + H]⁺And (4) performing pattern analysis, and confirming the optimal parameter by adjusting different mass spectrum parameters. In ESI ionization mode, the two generate [ M + H ] mainly]⁺Excimer ion peak.

3 pretreatment of biological samples

Plasma samples were pre-treated using a rapid high throughput protein precipitation method. Taking 1.5mL of a centrifuge tube, adding 45 mu L of blank plasma, adding 5 mu L of MRTX849 working solution, adding 10 mu L of internal standard working solution and 140 mu L of cold methanol (-20 ℃), performing vortex oscillation for about 1min, centrifuging at 13500rpm at 4 ℃ for 10min, sucking a supernatant sample bottle, taking 4 mu L of supernatant for sample injection to perform HPLC-MS/MS analysis, recording a mass chromatogram and the peak area of a compound, and calculating the concentration of MRTX849 by adopting an internal standard method.

4 methodological validation

The detection method is completely verified according to the verification guiding principle of biological sample quantitative analysis method of the general rule 9012 of the four parts of the Chinese pharmacopoeia of 2020 edition, so as to ensure the accuracy, repeatability and stability of detection. The verification contents comprise specificity, precision and accuracy, a standard curve, stability, matrix effect, recovery rate and the like.

Pharmacokinetics in 5 rats

Specific Pathogen Free (SPF) grade SD rat, male, body weight 180-: SCXK (Kyoto) 2016-.

To verify the versatility and feasibility of the established method, the inventors described the in vivo temporal kinetics of the drug according to different blood sampling time points after intragastric administration of the rat MRTX849 drug.

Rats were fasted without water deprivation for 12h before administration, and were administered by gastric gavage at 5mL/kg, at a dose of 15 mg/kg. The blood sampling time points are 0.5h, 2h, 6h, 8h, 12h, 24h, 48h, 72h and 96h before and after administration, and total 10 blood sampling points are about 0.2mL of whole blood per tube. Placing the whole blood sample in a heparinized centrifuge tube, centrifuging at 3500rpm for 10min, sucking supernatant plasma, and storing in a refrigerator at-80 ℃ for later use.

The experimental result shows that the HPLC-MS/MS method established by the inventor can accurately quantify the drug concentration in the blood plasma of the rat after the rat is perfused with the MRTX849 drug. As shown in fig. 4, the range of the standard curve of the method of the present invention completely covers the range of concentration detected in the animal. The method can determine the blood concentration of MRTX849 96h after administration, and the method is successfully applied and popularized.

6 results of the experiment

6.1 specificity

In the experiment, rat plasma from different sources is investigated and prepared into a specific sample for analysis. Double blank samples (treated matrix samples without analyte and internal standard), blank samples (treated matrix samples with internal standard), lower limit of quantitation (LLOQ) samples, upper limit of quantitation (ULOQ) samples, samples in unknown plasma were prepared. The specific examination result is shown in figure 3, (A): a double blank sample; (B) the method comprises the following steps Blank samples; (C) the method comprises the following steps Quantifying a lower limit sample; (D) the method comprises the following steps Quantifying an upper limit sample; (E) the method comprises the following steps Unknown plasma samples. In the experiment, the chromatographic peak signal-to-noise ratios of MRTX849 and carbamazepine in the LLOQ sample are respectively 30 and 50, and different ion scanning channels have no mutual interference.

6.2 precision and accuracy

6 quality control samples with lower limit, low concentration, medium concentration and high concentration of plasma quantitative determination are prepared in parallel, and the results of precision and accuracy in batches and among batches (table 1) show that the results of precision and accuracy of the method both meet the requirements of quantitative detection of biological samples.

Precision and accuracy of the methods of Table 1

6.3 matrix Effect and extraction recovery

In this experiment, rat blank plasma from different sources was examined to prepare low, medium and high concentration samples, and the matrix factor for the analyte and the internal standard was calculated by calculating the ratio of the peak area in the presence of matrix (B, measured by extracting blank matrix and adding analyte) to the peak area in the absence of matrix (C, pure solution of analyte) in the absence of matrix (water instead of plasma). And the internal standard normalized matrix factor was calculated from the ratio of the analyte to the internal standard matrix factor, and the results are shown in table 2. CV of matrix factors of the analyte and the internal standard in the low, medium and high concentration quality control samples and CV of the matrix factors normalized by the internal standard are all lower than 15%, and the fact that the determination of the analyte and the internal standard compound in the plasma is not influenced by the biological matrix is shown. The extraction recovery rates of low, medium and high 3 concentration quality control samples are respectively considered, and each concentration is parallel to 6 samples.

TABLE 2 matrix Effect and extraction recovery

6.4 stability

In the experiment, the MRTX849 is placed at room temperature for 24 hours, is placed at room temperature for 18 hours after preparation, is repeatedly frozen and thawed for 3 times, and is stored at-80 ℃ for 10 days, and the result is shown in table 3, and the MRTX849 is stable under the common stability condition.

TABLE 3 stability

6.5 Re-analysis of test specimens (ISR)

In order to verify the tolerance and reproducibility of the established HPLC-MS/MS method, plasma samples of an absorption phase and an elimination phase in a rat time curve are selected, two time point samples are selected for each rat, 12 samples are selected in total, quantitative analysis is carried out by the established method, a new analysis batch is newly analyzed on test samples after different days, and the accuracy of actual sample determination is evaluated. Near peak concentrations and elimination phase samples were determined. Acceptance criteria: at least 67% of the replicates were tested, with the difference between the concentration measured in the original analysis and the concentration measured in the re-analysis being within ± 20% of the mean of the two. The experimental results are shown in FIG. 4, and the deviation between the initial measurement value and the retest value is less than or equal to +/-11%. The results show that the established HPLC-MS/MS method is reliable and has good reproducibility.

The number of apparatuses and the scale of the process described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the present invention will be apparent to those skilled in the art.

While embodiments of the invention have been disclosed above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein, but is not limited to the details shown and described without departing from the generic concept as defined by the claims and their equivalents.

Claims

1. The method for measuring the concentration of plasma MRTX849 by high performance liquid chromatography-tandem mass spectrometry is characterized by comprising the following steps:

the liquid phase conditions were: the chromatographic column is Waters XBrige C₁₈The inner diameter is 2.1mM, the column length is 50mM, the particle size is 3.5mM, the flow rate is 0.4mL/min, the sample injection volume is 4 muL, the analysis time is 3min, the column temperature is 40 ℃, the needle washing solution is a mixed solution of isopropanol, acetonitrile, methanol and water in a volume ratio of 1:1:1:1, the organic phase of the mobile phase is acetonitrile containing 0.1% by mass of formic acid, the water phase is water containing 0.1% by mass of formic acid and 5mM ammonium acetate, and the gradient elution is set to be 90%, 10%, 0% and 90% in volume ratio of the water phase of 0.01min, 1.2min, 2.2min and 2.3-3.0 min;

2. The method for hplc tandem mass spectrometry of claim 1, wherein the standard curve working solutions with concentrations of 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 100ng/mL, 500ng/mL, 1 μ g/mL and 2 μ g/mL are prepared using methanol to dissolve the MRTX849, and the internal standard working solution with a concentration of 10ng/mL is prepared using methanol to dissolve the carbamazepine internal standard.

3. The method for determining the concentration of MRTX849 in plasma according to claim 1, wherein 5 μ L of standard curve working solution is precisely aspirated, 45 μ L of blank plasma is added, and 10 μ L of internal standard substance working solution is added.

4. The method for determining the concentration of MRTX849 in plasma according to claim 1, wherein the plasma is vortexed and shaken for 1min and centrifuged at 13500rpm at 4 ℃ for 10 min.

5. The method for determining the concentration of MRTX849 in plasma according to any one of claims 1-4, comprising:

6. The method for determining the concentration of MRTX849 in plasma according to claim 5, wherein the MRTX849 is dissolved in methanol to prepare a quality control working solution with the concentration of 1ng/mL, 20ng/mL or 1.6 μ g/mL.

7. The method for determining the concentration of MRTX849 in plasma according to claim 6, wherein 5 μ L of quality control working solution is precisely aspirated, 45 μ L of blank plasma is added, 10 μ L of internal standard working solution and 140 μ L of methanol at-20 ℃ are added.