A method for determining the unconjugated concentration of lenvatinib

Abstract

The application discloses a method for determining unbound concentration of lenvatinib, which comprises the following steps: sending standard curve working solution and quality control working solution into a liquid chromatography tandem mass spectrometry detection system to obtain a standard curve; adding a plasma sample into an ultrafiltration tube, centrifuging under the condition of a centrifugal force of 3000g for 15 min, and collecting an ultrafiltrate of the plasma sample; adding the ultrafiltrate of the plasma sample into blank plasma ultrafiltrate, mixing uniformly, taking supernatant into a collection plate, sending the collection plate into the liquid chromatography tandem mass spectrometry detection system to obtain a ratio of a peak area of lenvatinib to a peak area of an internal standard, and then bringing the area ratio into the standard curve to obtain the unbound concentration of lenvatinib in the plasma sample. The method for determining the unbound concentration of lenvatinib can accurately determine the unbound concentration of lenvatinib in plasma and overcome the prejudice of the prior art.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug detection, and particularly relates to a method for detecting unbound concentration of lenvatinib. BACKGROUND

[0002] Lenvatinib is an oral multi-target tyrosine kinase inhibitor, which can inhibit vascular endothelial growth factor receptors 1-3, fibroblast growth factor receptors 1-4, platelet-derived growth factor receptor alpha, and proto-oncogene RET and KIT. It shows good antitumor effect in various solid tumors such as thyroid cancer, liver cancer and kidney cancer. The most common treatment-related adverse reactions of lenvatinib include hypertension, diarrhea, proteinuria, decreased appetite and fatigue. These adverse reactions lead to 38.7% and 52.1% of patients to reduce the dose or interrupt the treatment, respectively. Studies have found that the incidence of liver dysfunction and thrombocytopenia in patients with thyroid cancer increases when the plasma trough concentration of lenvatinib is higher than 88 ng / mL. In patients with liver cancer, the plasma trough concentration of lenvatinib ≥71.4 ng / mL may be a limiting factor for interrupting treatment. Another analysis of a phase 2 study of lenvatinib in patients with liver cancer shows that the plasma trough concentration and area under the curve of lenvatinib are closely related to the occurrence of adverse reactions. Therefore, the optimal dose adjustment of lenvatinib is crucial to avoid serious adverse reactions and maximize its therapeutic effect. At present, in the therapeutic drug monitoring of lenvatinib, the total concentration is measured for dose adjustment. However, it is generally believed that the pharmacology and toxicology effects of a drug are related to the unbound concentration of the drug to which the patient is exposed at the site of action. Especially for high protein binding molecules (>85%), the proportion of unbound part not only affects the pharmacodynamic characteristics of the drug, but also affects the absorption, distribution, metabolism and excretion characteristics of the drug. Therefore, in therapeutic drug monitoring, it is necessary to determine the unbound concentration of lenvatinib.

[0003] Non-specific binding is common in membrane separation processes, and the binding of drugs to membranes will cause differences between the measured results and the actual existence. There is a study in the prior art that uses equilibrium dialysis method to analyze the unbound concentration of lenvatinib, but due to the non-specific binding of dialysis membrane to lenvatinib, the recovery rate of lenvatinib is only 51.3-55.9%, which leads to low accuracy of the determination results and at least 18 hours of sample processing time, which is not suitable for routine clinical practice and clinical research of therapeutic drug monitoring. Ultrafiltration centrifugation is also a typical membrane separation technology. Based on the existing views and research results, the people in the field believe that the use of ultrafiltration centrifugation to separate lenvatinib in plasma will also cause the binding of lenvatinib to the ultrafiltration membrane, resulting in the technical problem of differences between the measured results and the actual existence. This technical bias leads to the fact that there is no study in the prior art that uses ultrafiltration centrifugation to analyze the unbound concentration of lenvatinib in plasma. SUMMARY

[0004] Therefore, the present application aims to provide a method for determining the unbound concentration of lenvatinib, so as to solve the technical problem of determining the unbound concentration of lenvatinib in plasma in therapeutic drug monitoring, and solve the technical prejudice problem that the non-specific combination of the ultrafiltration membrane and lenvatinib affects the accuracy of the determination result in the prior art.

[0005] The method for determining the unbound concentration of lenvatinib comprises the following steps:

[0006] 1) A lenvatinib standard sample is weighed and dissolved in methanol to prepare a standard curve stock solution with a mass concentration of 1 mg / mL and a quality control stock solution with a mass concentration of 1 mg / mL; the standard curve stock solution is diluted with acetonitrile to prepare standard curve working solutions with concentrations of 1 ng / mL, 2 ng / mL, 5 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL and 200 ng / mL; the quality control stock solution is diluted with acetonitrile to prepare quality control working solutions with concentrations of 3 ng / mL, 30 ng / mL and 150 ng / mL;

[0007] 2) A lenvatinib-d4 standard sample is weighed and dissolved in methanol to prepare an internal standard stock solution with a mass concentration of 1 mg / mL, and acetonitrile is used to dilute the internal standard stock solution to an internal standard solution with a concentration of 20 ng / mL;

[0008] 3) 10 μL of the standard curve working solution with a concentration of 1 ng / mL is added to 90 μL of blank plasma ultrafiltrate, mixed well, then 100 μL of the internal standard solution is added, vortexed at 1400 rpm for 5 min, centrifuged at 4°C and 14000 rpm for 10 min, and 100 uL of the supernatant is collected in a collection plate; and the same treatment is performed on the standard curve working solutions with concentrations of 2 ng / mL, 5 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL and 200 ng / mL;

[0009] 10 μL of the quality control working solution with a concentration of 3 ng / mL is added to 90 μL of blank plasma ultrafiltrate, mixed well, then 100 μL of the internal standard solution is added, vortexed at 1400 rpm for 5 min, centrifuged at 4°C and 14000 rpm for 10 min, and 100 uL of the supernatant is collected in a collection plate; and the same treatment is performed on the quality control working solutions with concentrations of 30 ng / mL and 150 ng / mL;

[0010] 4) the collection plate described in step 3) is sent to a liquid chromatography tandem mass spectrometry detection system for detection, the standard curve working solution on the collection plate is detected to obtain a standard curve with the ratio of the peak area of lenvatinib to the peak area of the internal standard as the vertical coordinate and the concentration of lenvatinib as the horizontal coordinate; the quality control working solution on the collection plate is detected to obtain the ratio of the peak area of lenvatinib to the peak area of the internal standard of the quality control working solution, the ratio of the peak area of lenvatinib to the peak area of the internal standard of the quality control working solution and the concentration of lenvatinib of the quality control working solution are used to calibrate the aforementioned standard curve, if the relative standard deviation requirement is met, the standard curve is qualified; if the relative standard deviation requirement is not met, steps 1)-4) are repeated until a standard curve meeting the relative standard deviation requirement is obtained;

[0011] 5) 200uL of the plasma sample is added to a 0.5mL ultrafiltration tube, the ultrafiltration tube is equilibrated in a 37℃ water bath for 30min, then transferred to a centrifuge, centrifuged at 37℃ and a centrifugal force of 3000g for 15min, and the plasma sample ultrafiltrate is collected;

[0012] 6) 10uL of the plasma sample ultrafiltrate is added to 90uL of blank plasma ultrafiltrate, mixed, then 100uL of the internal standard solution is added, vortexed at 1400rpm for 5min, centrifuged at 4℃ and 14000rpm for 10min, and 100uL of the supernatant is taken to the collection plate;

[0013] 7) the collection plate described in step 6) is sent to a liquid chromatography tandem mass spectrometry detection system for detection, the ratio of the peak area of lenvatinib to the peak area of the internal standard is obtained; then the area ratio is brought into the standard curve obtained in step 4) to obtain the unbound concentration of lenvatinib in the plasma sample.

[0014] Advantages of the present application:

[0015] Non-specific binding is very common in membrane separation processes, and the binding of drugs to ultrafiltration membranes will cause differences between the measured results and the actual existence. Equilibrium dialysis and ultrafiltration centrifugation are typical membrane separation techniques, and the existing technology uses equilibrium dialysis to analyze the unbound concentration of lenvatinib, and the recovery rate of lenvatinib is only 51.3-55.9%, which proves that the dialysis membrane has non-specific binding effect on lenvatinib, which leads people to abandon the use of membrane separation technology to study the unbound concentration of lenvatinib in plasma.

[0016] The unbound concentration determination method of lenvatinib of the present application collects the plasma ultrafiltrate containing lenvatinib by using ultrafiltration centrifugation, and it is verified by experiments that the ultrafiltration membrane in the ultrafiltration centrifugation device does not have non-specific binding effect on lenvatinib, and the experiments also verify that the method described in the present application meets the analysis requirements in terms of determination accuracy and precision, so the method described in the present application solves the technical problem of determining the unbound concentration of lenvatinib in plasma and overcomes the prejudice of the prior art. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 For the selective investigation of results graph.

[0018] Figure 2 For the residual effect of investigation results graph. DETAILED DESCRIPTION

[0019] The present application will be further described below in conjunction with the accompanying drawings and examples.

[0020] The unbound concentration determination method of the present application includes the following steps:

[0021] 1) The standard curve stock solution and the quality control stock solution were prepared by weighing the standard sample of lenvatinib and dissolving it in methanol to a mass concentration of 1 mg / mL. The standard curve working solution was prepared by diluting the standard curve stock solution with acetonitrile to a concentration of 1 ng / mL, 2 ng / mL, 5 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL and 200 ng / mL. The quality control working solution was prepared by diluting the quality control stock solution with acetonitrile to a concentration of 3 ng / mL, 30 ng / mL and 150 ng / mL.

[0022] 2) The internal standard stock solution was prepared by weighing the standard sample of lenvatinib-d4 and dissolving it in methanol to a mass concentration of 1 mg / mL, and then diluting it with acetonitrile to a concentration of 20 ng / mL.

[0023] 3) 10 μL of the standard curve working solution with a concentration of 1 ng / mL was added to 90 μL of blank plasma ultrafiltrate, mixed well, then 100 μL of internal standard solution was added, vortexed at 1400 rpm for 5 min, centrifuged at 4°C and 14000 rpm for 10 min, and 100 uL of supernatant was collected in the collection plate. The same treatment was performed on the standard curve working solution with a concentration of 2 ng / mL, 5 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL and 200 ng / mL.

[0024] 10 μL of the quality control working solution with a concentration of 3 ng / mL was added to 90 μL of blank plasma ultrafiltrate, mixed well, then 100 μL of internal standard solution was added, vortexed at 1400 rpm for 5 min, centrifuged at 4°C and 14000 rpm for 10 min, and 100 uL of supernatant was collected in the collection plate. The same treatment was performed on the quality control working solution with a concentration of 30 ng / mL and 150 ng / mL.

[0025] 4) The collection plate described in step 3) is sent to a liquid chromatography tandem mass spectrometry detection system for detection. The standard curve working solution on the collection plate is detected to obtain a standard curve with the ratio of the lenvatinib peak area to the internal standard peak area as the vertical coordinate and the lenvatinib concentration as the horizontal coordinate. The quality control working solution on the collection plate is detected to obtain the ratio of the lenvatinib peak area to the internal standard peak area of the quality control working solution. The ratio of the lenvatinib peak area to the internal standard peak area of the quality control working solution and the lenvatinib concentration of the quality control working solution are used to calibrate the aforementioned standard curve. If the relative standard deviation requirement is met, the standard curve is qualified. If the relative standard deviation requirement is not met, steps 1) to 4) are repeated until a standard curve that meets the relative standard deviation requirement is obtained.

[0026] 5) 200 uL of the plasma sample is added to a 0.5 mL ultrafiltration tube. After the ultrafiltration tube is equilibrated in a 37°C water bath for 30 min, it is transferred to a centrifuge. The plasma sample ultrafiltrate is collected under the conditions of 37°C and centrifugal force of 3000 g for 15 min.

[0027] 6) 10 uL of the plasma sample ultrafiltrate is added to 90 uL of blank plasma ultrafiltrate. After mixing, 100 uL of the internal standard solution is added. The mixture is vortexed at 1400 rpm for 5 min, and then centrifuged at 4°C and 14000 rpm for 10 min. 100 uL of the supernatant is taken and placed in a collection plate.

[0028] 7) The collection plate described in step 6) is sent to a liquid chromatography tandem mass spectrometry detection system for detection to obtain the ratio of the lenvatinib peak area to the internal standard peak area. Then the area ratio is brought into the standard curve obtained in step 4) to obtain the unbound concentration of lenvatinib in the plasma sample.

[0029] The unbound concentration determination method of lenvatinib described in the present embodiment is experimentally verified as follows:

[0030] Experimental standards and reagents:

[0031] The lenvatinib standard (purity ≥ 98%) and the lenvatinib-d4 standard (purity ≥ 98%) are purchased from the Canadian TLC Pharmaceutical Standards Limited, with batch numbers 3796-099A1 and 3161-033A5, respectively. The reagents methanol and acetonitrile are HPLC grade, purchased from the United States Honeywell Company. The reagent formic acid is analytical pure, purchased from Chengdu Kelong Chemical Reagent Factory. The pure water used in the experiment is purchased from Guangzhou Watson Pure Water Co., Ltd.

[0032] Experimental instruments:

[0033] AB SCIEX Triple Quad TM4500MD liquid chromatography tandem mass spectrometry detection system, consisting of a triple quadrupole mass spectrometer (4500MD), a high performance liquid chromatograph (Jasper TM ) and software MD (version number: 1.6) (AB SCIEX Corporation); electronic balance (SQP type, Sartorius Corporation); vortex mixer (KB3 type, Nanjing Deli Jia Biological Technology Co., Ltd.); ultrasonic cleaner (SB-5200D type, Ningbo Xinzhi Biological Technology Co., Ltd.); medical centrifuge (L3-5K type, Hunan Kecheng Instrument and Equipment Co., Ltd.); medical centrifuge (H1-16KR type, Hunan Kecheng Instrument and Equipment Co., Ltd.); 1000 μL, 200 μL, 100 μL, 10 μL pipette (Eppendorf Corporation). The ultrafiltration tube is Ultra-0.5 mL, UFC501096; cut-off 10 kDa (Millipore Corporation).

[0034] Chromatographic conditions:

[0035] The chromatographic column selected is ACQUITY UPLC BEH C18, 1.7 μm, 2.1 x 50 mm (Waters, USA); the mobile phase A is 0.1% formic acid aqueous solution, and the B is acetonitrile solution, using gradient elution mode, the elution conditions are: 0-0.5 min, 10% B; 0.5-1.0 min, 10%-90% B; 1.0-2.5 min, 90% B; 2.5-3.0 min, 90%-10% B; 3.0-4.0 min, 10% B. The flow rate is 0.3 mL / min; the injector temperature is 10°C; the column oven temperature is 40°C; the injection volume is 10 μL.

[0036] Mass spectrometry conditions:

[0037] The ion source is an electrospray ion source, and mass monitoring is carried out in positive ion mode, and the acquisition mode is multiple reaction monitoring. The mass spectrometry condition parameters are: Ion spray voltage is 5500 V, Source temperature is 550°C, Ion source Gas1 and Gas2 are both 55 psi, Curtain Gas is 30 psi, and Collision Gas is 8 psi. The mass spectrometry parameters of lenvatinib and lenvatinib-d4 are shown in Table 1.

[0038] Table 1 Mass spectrometry parameters of lenvatinib and lenvatinib-d4

[0039]

[0040] Prepare standard curve stock solution, quality control stock solution, internal standard solution and plasma ultrafiltrate according to the method described in the examples, and carry out the following validation items.

[0041] Selectivity validation:

[0042] Take 6 different sources of human blank ultrafiltrate matrix to investigate the selectivity of the method. The response of the interfering peak should not be higher than 20% of the response of the target peak at the lower limit of quantification (LLOQ) and 5% of the response of the internal standard.

[0043] The experimental results are shown in Table 1: Figure 1 The retention time of lenvatinib and lenvatinib-d4 is 2.32 min, and there is no obvious endogenous substance in the 6 different sources of plasma ultrafiltrate, which does not interfere with the determination of the analyte.

[0044] Residual effect validation:

[0045] By injecting high concentration samples (upper limit of quantification, ULOQ) followed by injecting blank samples, the residual effect is evaluated. The residual of high concentration samples in blank samples should not be more than 20% of the lower limit of quantification and not more than 5% of the internal standard.

[0046] The experimental results are shown in Table 2: Figure 2 By injecting three consecutive blank samples after injecting the highest concentration sample, the residual effect is evaluated, and there is no obvious residual effect for lenvatinib and lenvatinib-d4.

[0047] Standard curve and lower limit of quantification validation:

[0048] The standard curve should include at least 6 calibration concentration levels, the concentration calculated by the calibration standard should generally be within ± 15% of the labeled value, and the lower limit of quantification should be within ± 20%. At least 75% of the calibration standards, containing at least 6 effective concentrations, should meet the above standards.

[0049] The concentrations of a series of standard solutions of lenvatinib are 0.1, 0.2, 0.5, 2.5, 5, 10, and 20 ng / mL. The standard curve is drawn with the concentration of the analyte as the abscissa and the peak area ratio of the analyte to the internal standard as the ordinate, and the Analyst software is used for weighted (ω = 1 / x 2 ) least squares regression calculation. The linear equation of lenvatinib is Y = 4.84 x 10^(-2) C + 3.30 x 10^(-3), r 2 = 0.9996, the lower limit of quantification is 0.1 ng / mL, and the accuracy of each calibration concentration level is within ± 15%. The experimental results are shown in Table 2.

[0050] Table 2 Standard curve

[0051]

[0052]

[0053] Accuracy and precision verification:

[0054] The accuracy of an analytical method is the closeness of agreement between the test result and the true value of the analyte, expressed as: (measured value / true value) x 100%. The precision of an analytical method is the closeness of agreement between independent test results obtained under the same conditions, defined as the relative standard deviation (RSD) of measurements.

[0055] Intra-batch accuracy and precision: The lower limit of quantification and low, medium, high concentration quality control samples should be taken from one analysis batch, at least 5 samples for each concentration. Inter-batch accuracy and precision: evaluated by at least 3 analysis batches, and at least two days, the lower limit of quantification and low, medium, high concentration quality control samples were used in each batch, at least 5 determination values for each concentration. The average accuracy should generally be within ± 15% of the labeled value of the quality control sample, and the accuracy of the lower limit of quantification should be within ± 20% of the labeled value. The coefficient of variation of precision should generally not exceed 15%, and the coefficient of variation of the lower limit of quantification should not exceed 20%.

[0056] The lower limit of quantification and low, medium, high concentration quality control samples were selected, with concentrations of 0.1, 0.3, 3, 15 ng / mL, at least 5 samples for each concentration, and at least 3 analysis batches were used for investigation. The experimental results are shown in Table 3. The intra-batch accuracy of lenvatinib was 99.73%-101.87%, and the inter-batch accuracy was 100%-102.22%; the intra-batch precision was 1.67%-6.64%, and the inter-batch precision was 0.00-3.20%. All met the requirements.

[0057] Table 3. Accuracy and precision results (mean ± SD, n = 5)

[0058]

[0059] Matrix effect and extraction recovery verification:

[0060] At least 6 batches of blank ultrafiltrate matrix of different sources should be used in low and high concentration quality control samples. For each batch of matrix, the matrix factor of each analyte and internal standard was calculated by calculating the peak area in the presence of the matrix (measured by adding the analyte and internal standard after extraction of the blank matrix) and the peak area without the matrix (pure solution of the analyte and internal standard). Further, the internal standard normalized matrix factor (IS-MF) was calculated by dividing the matrix factor of the analyte by the matrix factor of the internal standard. The coefficient of variation of the internal standard normalized matrix factor calculated from 6 batches of matrix should not be greater than 15%.

[0061] Ultrafiltration-centrifugation extraction recovery: The ratio of the response value of the analyte added to the blank ultrafiltrate to the response value of the analyte added to the supernatant after protein precipitation. At least six batches should be prepared and performed on low- and high-concentration quality control samples. The coefficient of variation of the calculated extraction recovery should not exceed 15%.

[0062] Six batches of human plasma ultrafiltrate from different sources were selected for matrix effect investigation. The normalized matrix factor of the internal standard for low-concentration and high-concentration lenvatinib quality control samples were 1.05±0.03 and 0.92±0.06, respectively, with coefficients of variation of 3.22% and 6.78%, respectively. The extraction recoveries for low-concentration and high-concentration samples were 106.46% and 95.46%, respectively, with coefficients of variation of 4.64% and 0.99%, respectively. The experimental results are shown in Table 4.

[0063] Table 4. Matrix effect and extraction recovery

[0064]

[0065] Nonspecific binding verification:

[0066] Low-concentration and high-concentration samples were prepared using blank ultrafiltrate, and after ultrafiltration, the peak area ratio of the analyte after ultrafiltration to that before ultrafiltration was calculated.

[0067] Experimental results: The peak area ratios before and after ultrafiltration for low and high concentration solutions were 102.15% and 97.96%, respectively, with coefficients of variation of 2.89% and 4.30%, respectively. The peak area ratio of lenvatinib before and after ultrafiltration was almost unchanged; therefore, it can be concluded that there is no non-specific binding of lenvatinib to the ultrafiltration membrane in the ultrafiltration device.

[0068] Stability verification:

[0069] Low and high concentration quality control samples were used and analyzed immediately after storage and pretreatment. A standard curve was obtained from freshly prepared calibration standards. The quality control samples were analyzed, and the mean value of each concentration should deviate from the labeled concentration within ±15%.

[0070] Low-concentration and high-concentration quality control samples were used to investigate stability. The investigation conditions included: 24 hours at room temperature (20℃), 24 hours in an autosampler (10℃), 3 days in a 4℃ refrigerator, 5 freeze-thaw cycles, and long-term stability investigation (1, 2, and 3 months at -20℃). The accuracy ranged from 89.56% to 112.5%. The experimental results are shown in Table 5.

[0071] Table 5. Stability Study

[0072]

[0073] Optimization of centrifugal force and centrifugation time:

[0074] The ultrafiltrate volume is calculated according to the following formula, wherein W 1+u and W1are the weights of the ultrafiltration chamber before and after ultrafiltration, respectively, and the density of the ultrafiltrate (C) is taken as 1 g / mL 3 .

[0075]

[0076] Centrifugal force optimization: 200 μL of the plasma sample was added to the ultrafiltration tube, and the sample (Nos. F 001, F 002, F-003, F-004, F-005, and F-006) was centrifuged at 1000 g, 2000 g, 3000 g, 4000 g, 5000 g, and 6000 g, respectively, at 37°C for 10 min, and the volume of the ultrafiltrate obtained was investigated. The experimental results are shown in Table 6.

[0077] Table 6. Centrifugal force optimization

[0078]

[0079]

[0080] Centrifugal time optimization: 200 μL of the plasma sample was added to the ultrafiltration tube, and the sample (Nos. T 001, T 002, T-003, T-004, and T-005) was centrifuged at 3000 g at 37°C for 5 min, 10 min, 15 min, 20 min, and 25 min, respectively, and the volume of the ultrafiltrate obtained was investigated. The experimental results are shown in Table 7.

[0081] Table 7. Centrifugal time optimization

[0082]

[0083] According to the criteria that the ultrafiltration membrane is not damaged and the ratio of the volume of the ultrafiltrate to the total volume of the plasma before and after ultrafiltration is 0.2-0.35, the centrifugal force of 3000 g and the centrifugal time of 15 min were selected as the optimal centrifugal conditions.

[0084] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and not to limit the same. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced equivalently, and these substantially equivalent modifications or replacements should also be encompassed in the scope of the claims of the present application.

Claims

1. A method for determining the unconjugated concentration of lenvatinib, characterized by: Comprising the following steps: 1) Take the standard sample of lenvatinib, prepare a standard curve stock solution with a mass concentration of 1 mg / mL and a quality control stock solution with a mass concentration of 1 mg / mL with methanol; dilute the standard curve stock solution with acetonitrile to prepare standard curve working solutions with concentrations of 1 ng / mL, 2 ng / mL, 5 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL and 200 ng / mL; dilute the quality control stock solution with acetonitrile to prepare quality control working solutions with concentrations of 3 ng / mL, 30 ng / mL and 150 ng / mL; 2) Take the standard sample of lenvatinib-d4, prepare an internal standard stock solution with a mass concentration of 1 mg / mL with methanol, and dilute it with acetonitrile to prepare an internal standard solution with a concentration of 20 ng / mL; 3) Add 10 μL of the standard curve working solution with a concentration of 1 ng / mL to 90 μL of blank plasma ultrafiltrate, mix well, then add 100 μL of internal standard solution, vortex at 1400 rpm for 5 min, centrifuge at 4°C and 14000 rpm for 10 min, take 100 μL of supernatant to the collection plate; and the same treatment is performed on the standard curve working solutions with concentrations of 2 ng / mL, 5 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL and 200 ng / mL; Add 10 μL of the quality control working solution with a concentration of 3 ng / mL to 90 μL of blank plasma ultrafiltrate, mix well, then add 100 μL of internal standard solution, vortex at 1400 rpm for 5 min, centrifuge at 4°C and 14000 rpm for 10 min, take 100 μL of supernatant to the collection plate; and the same treatment is performed on the quality control working solutions with concentrations of 30 ng / mL and 150 ng / mL; 4) Send the collection plate in step 3) to the liquid chromatography tandem mass spectrometry detection system for detection, detect the standard curve working solution on the collection plate to obtain a standard curve with the ratio of the peak area of lenvatinib to the peak area of the internal standard as the vertical coordinate and the concentration of lenvatinib as the horizontal coordinate; detect the quality control working solution on the collection plate to obtain the ratio of the peak area of lenvatinib to the peak area of the internal standard, calibrate the aforementioned standard curve with the ratio of the peak area of lenvatinib to the peak area of the internal standard of the quality control working solution and the concentration of lenvatinib of the quality control working solution, if the relative standard deviation requirement is met, the standard curve is qualified; if the relative standard deviation requirement is not met, repeat steps 1) - 4) until a standard curve that meets the relative standard deviation requirement is obtained; 5) Add 200 μL of plasma sample to 0.5 mL ultrafiltration tube, equilibrate the ultrafiltration tube in 37 °C water bath for 30 min, then transfer it to the centrifuge, centrifuge at 37 ℃ and centrifugal force 3000 g for 15 min, collect the plasma sample ultrafiltrate; 6) Take 10 μL of the ultrafiltrate of the plasma sample and add it to 90 μL of the blank plasma ultrafiltrate, mix well, then add 100 μL of the internal standard solution, vortex at 1400 rpm for 5 min, centrifuge at 4°C and 14000 rpm for 10 min, take 100 μL of the supernatant and place it in the collection plate; 7) Take the collection plate described in step 6) and send it to the liquid chromatography tandem mass spectrometry detection system for detection to obtain the ratio of the area of the lonafarnib peak to the area of the internal standard peak; then take the area ratio to the standard curve obtained in step 4) to obtain the unbound concentration of lonafarnib in the plasma sample; The ultrafiltration tube is Amicon Ultra-0.5 mL, UFC 501096; cut-off 10 kDa.

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