Method for determining concentration of letemovir in plasma by using liquid chromatography-tandem mass spectrometry

Abstract

The invention discloses a method for determining the concentration of letemovir in plasma by using liquid chromatography-tandem mass spectrometry, which comprises the following steps: S1, preparing a calibrator working solution and a quality control working solution of letemovir, respectively adding the calibrator working solution and the quality control working solution into a plasma matrix to obtain a plasma calibrator and a plasma quality control, preparing an internal standard working solution taking the lenvatinib-d5 as an internal standard substance; s2, respectively adding a plasma calibration material and a plasma quality control material into the internal standard working solution obtained in the step S1, carrying out protein precipitation treatment, collecting supernate, carrying out liquid chromatography-tandem mass spectrometry analysis on the supernate, and drawing a liquid chromatography-tandem mass spectrometry standard curve; and S3, obtaining the concentration of letemovir in the plasma of a sample to be detected according to the liquid chromatography-tandem mass spectrometry in the step S2. According to the method, the sample pretreatment step is simplified, the operation difficulty and time consumption are reduced, a rapid, accurate and high-sensitivity analysis result is ensured, and the method is suitable for the requirements of rapid clinical and research fields.

Description

Technical Field

[0001] The present invention belongs to the technical field of therapeutic drug monitoring and pharmacokinetic analysis, and particularly relates to a method for determining the concentration of letermovir in plasma using liquid chromatography tandem mass spectrometry. Background Art

[0002] Letermovir is an antiviral drug widely used for the treatment of viral infections. Its mechanism is to interfere with the key steps of viral replication, thereby inhibiting the growth and spread of the virus. Letermovir is usually administered orally or by intravenous injection, so precise monitoring of its drug concentration in plasma is crucial for the success of treatment. In addition, letermovir is used as a long-term preventive therapy after hematopoietic stem cell transplantation to prevent viral infections during the period of immune deficiency, which not only increases the complexity of treatment but also significantly increases the medical cost.

[0003] However, the pharmacokinetic properties of letermovir require precise control of the dose in clinical applications to avoid potential side effects and optimize the therapeutic effect. Especially when dealing with patients with complex viral loads or resistance to letermovir, it is particularly important to adjust the dose by monitoring the plasma drug concentration.

[0004] Currently, the methods used to monitor letermovir include traditional biochemical analysis methods and immunoassay methods, but these methods usually have problems such as insufficient sensitivity or quantitative limitations. The demand for more efficient and precise detection technologies is increasing, especially in clinical situations where the viral load changes rapidly. In addition, the operation of traditional methods is complex and time-consuming, which is not conducive to real-time adjustment of the treatment plan.

[0005] Therefore, there is a need to develop a method for detecting letermovir that is both rapid and accurate to optimize the drug treatment plan and reduce the risk of side effects for patients. This invention provides a liquid chromatography tandem mass spectrometry (LC-MS / MS) method that can accurately determine the concentration of letermovir in plasma within a short time, providing important pharmacokinetic data for clinical use. Summary of the Invention

[0006] In view of this, the present invention aims to propose a method for determining the concentration of letermovir in plasma using liquid chromatography tandem mass spectrometry to solve at least one technical problem in the background art.

[0007] To achieve the above object, the technical solution of the present invention is realized as follows: A method for determining the concentration of letermovir in plasma using liquid chromatography tandem mass spectrometry, comprising the following steps: S1: Prepare the working calibration solution and working quality control solution of letermovir. Add the working calibration solution and working quality control solution into plasma matrix respectively to obtain plasma calibration samples and plasma quality control samples; prepare the internal standard working solution with lenvatinib-d5 as the internal standard substance. S2: Add the plasma calibration samples and plasma quality control samples in step S1 into the internal standard working solution obtained in step S1 respectively for protein precipitation treatment, collect the supernatant, and perform liquid chromatography-tandem mass spectrometry analysis on the supernatant to draw the liquid chromatography-tandem mass spectrometry standard curve. S3: Add the test sample into the internal standard working solution obtained in step S1 for protein precipitation treatment, collect the supernatant, perform liquid chromatography-tandem mass spectrometry analysis on the supernatant, and obtain the concentration of letermovir in plasma according to the liquid chromatography-tandem mass spectrometry in step S2.

[0008] Further, the solvent of the calibration working solution in step S1 is 45 - 55% methanol. The solvent of the quality control working solution in step S1 is 45 - 55% methanol. The solvent of the internal standard working solution in step S1 is 45 - 55% methanol.

[0009] Further, the concentration of the calibration working solution of letermovir in step S1 is a stepwise concentration. Preferably, the stepwise concentration is 40, 80, 200, 400, 1000, 2000 ng / mL.

[0010] Further, the concentration of the quality control working solution of letermovir in step S1 is a stepwise concentration. Preferably, the stepwise concentration is 120, 500, 1500 ng / mL.

[0011] Further, the concentration of the internal standard working solution in step S1 is 50 ng / mL.

[0012] Further, the liquid chromatography-tandem mass spectrometry analysis in step S2 is performed using a liquid chromatography-tandem mass spectrometer. Preferably, the model of the liquid chromatography-tandem mass spectrometer is YS EXACT 9900MD liquid chromatography-tandem mass spectrometer.

[0013] Further, the chromatographic column of the liquid chromatography-tandem mass spectrometer is ChromCore C18 chromatographic column.

[0014] Further, the mobile phase A of the liquid chromatography-tandem mass spectrometer is 2 mM ammonium acetate - 0.1% formic acid aqueous solution; and the mobile phase B is 0.1% formic acid methanol solution. Gradient elution program settings for the liquid chromatography tandem mass spectrometer: From 0 to 1.5 minutes, the proportion of mobile phase B increases from 3% to 95%, maintain this proportion until 2.0 minutes, then rapidly reduce the proportion of mobile phase B to 3% within 2.0 to 2.1 minutes, and maintain this proportion within 2.1 to 2.5 minutes; The conditions of the mass spectrometry of the liquid chromatography tandem mass spectrometer are to use an ESI electrospray ionization source and perform selected reaction monitoring scans in the positive ion mode.

[0015] Furthermore, the flow rate of the gradient elution of the liquid chromatography tandem mass spectrometer is set between 0.3 mL / min and 1 mL / min, and the column temperature is controlled between 35°C and 40°C; The injection volume of the liquid chromatography tandem mass spectrometer is set between 1 μL and 5 μL; The spray voltage of the liquid chromatography tandem mass spectrometer is set at 2900 - 3100 V, the sheath gas pressure is 40 - 50 Arb, the auxiliary gas pressure is 12.5 - 17.5 Arb, the backflush gas pressure is 2.5 - 7.5 Arb, and the temperature of the ion transfer tube is controlled at 340 - 360°C, and the atomization temperature is set at 440 - 460°C.

[0016] Furthermore, in the liquid chromatography tandem mass spectrometry standard curve in step S2, the concentration of the plasma matrix calibrator is used as the abscissa, and the ratio of the peak area of letermovir to the internal standard is used as the ordinate, set the weight as 1 / x², exclude the origin, and perform linear fitting.

[0017] Compared with the prior art, the method for determining the concentration of letermovir in plasma using liquid chromatography tandem mass spectrometry according to the present invention has the following advantages: Compared with the prior art, the method provided by the present invention has the advantages of simple operation, short reaction time, small sample volume required, etc., can significantly improve the accuracy and efficiency of detection, and thus provide highly specific and highly sensitive analysis results in a short time, which helps to assist clinicians in quickly and accurately adjusting the treatment plan. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments and descriptions thereof of the present invention are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings: Figure 1 It is the chromatogram interface of the blank sample (plasma matrix) described in the embodiment of the present invention (showing two levels, the upper part is the ion current diagram of the letermovir internal standard, and the lower part is the ion current diagram of letermovir); Figure 2Chromatogram interface of letermovir at low concentration level in the matrix samples described in the embodiments of the present invention (showing two levels: the upper part is the ion current diagram of the letermovir internal standard, and the lower part is the ion current diagram of letermovir); Figure 3 Chromatogram interface of letermovir at medium concentration level in the matrix samples described in the embodiments of the present invention (showing two levels: the upper part is the ion current diagram of the letermovir internal standard, and the lower part is the ion current diagram of letermovir); Figure 4 Chromatogram interface of letermovir at high concentration level in the matrix samples described in the embodiments of the present invention (showing two levels: the upper part shows the ion current diagram of the letermovir internal standard, and the lower part is the ion current diagram of letermovir); Figure 5 Example of chromatogram interface of actual patient samples described in the embodiments of the present invention (showing two levels: the upper part is the ion current diagram of the letermovir internal standard, and the lower part is the ion current diagram of letermovir). Detailed implementation manners

[0019] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0020] The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0021] The present invention has developed a liquid chromatography tandem mass spectrometry technology for detecting letermovir in plasma, with the following characteristics: Preparation of working solutions: Weigh accurately the letermovir standard product, dissolve it with methanol and gradually dilute it with 50% methanol to prepare calibration solutions with multiple concentration gradients; similarly, weigh accurately the letermovir standard product, dissolve it with methanol and dilute it with 50% methanol according to a certain concentration gradient to prepare quality control solutions with different concentrations; select lenvatinib-d5 as the internal standard and prepare the internal standard working solution with methanol; Preparation of matrix samples: Add the calibration and quality control working solutions to the plasma matrix to obtain plasma calibration products and plasma quality control products Pretreatment of samples: Accurately pipette appropriate amounts of plasma calibration products, plasma quality control products and plasma samples to be tested, add the internal standard working solution and perform protein precipitation, and take the supernatant after oscillation and high-speed centrifugation; 4. Sample analysis: Place the sample supernatant into a liquid chromatography tandem mass spectrometer for detection; The set conditions of the liquid chromatography tandem mass spectrometer include: Liquid chromatography tandem mass spectrometer: YS EXACT 9900MD liquid chromatography tandem mass spectrometer; Chromatographic column: ChromCore C18 chromatographic column; Mobile phase composition: Mobile phase A is 2 mM ammonium acetate - 0.1% formic acid aqueous solution; while mobile phase B is 0.1% formic acid methanol solution; Gradient elution program settings: From 0 to 1.5 minutes, the proportion of mobile phase B is increased from 3% to 95%, maintained at this proportion until 2.0 minutes, then the proportion of mobile phase B is rapidly reduced to 3% within 2.0 to 2.1 minutes, and maintained at this proportion within 2.1 to 2.5 minutes; Mass spectrometry condition settings: Use ESI electrospray ionization source and perform selected reaction monitoring (SRM) scan in positive ion mode; 5. Data processing: Use the TraceFinder software of the mass spectrometer for data processing; Specific parameters of the mass spectrometry include: In liquid chromatography - tandem mass spectrometry analysis, the parameter settings of the ion source are as follows: The spray voltage is set to 3000 V, the sheath gas pressure is 45 Arb, the auxiliary gas pressure is 15 Arb, the back - purge gas pressure is 5 Arb, and the temperature of the ion transfer tube is controlled at 350 °C, and the nebulization temperature is set at 450 °C.

[0022] The selected clinical blood samples are taken from patients after oral administration of letermovir. The specimen collection time is 30 minutes before continuous dosing, and trough concentration samples are collected to ensure data consistency and comparability; The calibration curve takes the plasma matrix calibrator concentration as the abscissa and the ratio of the peak area of letermovir to the internal standard as the ordinate, sets the weight as 1 / x², excludes the origin, and performs linear fitting; The detailed formulations of mobile phases A and B are: Mobile phase A is 2 mM ammonium acetate - 0.1% formic acid aqueous solution; while mobile phase B is 0.1% formic acid methanol solution; The elution flow rate is set between 0.3 mL / min and 1 mL / min, and the column temperature is maintained between 35 °C and 40 °C. In a specific example, the flow rate is 0.4 mL / min and the column temperature is 40 °C; The injection volume is between 1 μL and 5 μL, and in a specific example, it is 1 μL; The concentration gradient of the plasma matrix calibrator is 40, 80, 200, 400, 1000, 2000 ng / mL; The concentration gradient of the plasma matrix quality control product is 120, 500, 1500 ng / mL; The concentration of the internal standard working solution is fixed at 50 ng / mL; Example 1: The implementation steps are as follows: 1. Use of calibrator 1.1 Preparation of calibrator Accurately weigh 5 mg of letermovir solid, transfer 5 mL of methanol solution, vortex until the solid is completely dissolved, prepare a stock solution with a concentration of 1 mg / mL, dispense it into 2 mL vials, 0.5 mL per vial, label it, and store it at -20°C. When in use, take out the letermovir standard solution (1 mg / mL) stored at -20°C, let it return to room temperature, and vortex for 30 seconds before use.

[0023] 1.2 Preparation of internal standard stock solution Take 1 mg of lenvatinib-d5 solid, dissolve it with methanol and make up the volume to 10 mL to prepare an internal standard stock solution with a concentration of 100 μg / mL. Dispense it into 2 mL vials, 1 mL per vial, label it, and store it at -20°C. When in use, take out the lenvatinib-d5 internal standard stock solution (100 μg / mL) stored at -20°C, let it return to room temperature, and vortex for 30 seconds before use.

[0024] 2. Preparation of internal standard working solution Take 50 μL of the lenvatinib-d5 internal standard stock solution with a concentration of 100 μg / mL respectively, add 99.95 mL of methanol, vortex and mix well to prepare a 50 ng / mL lenvatinib-d5 internal standard working solution.

[0025] 3. Preparation of standard curve working solution Take 40 μL of the letermovir stock solution with a concentration of 1 mg / mL, add 960 μL of 50% methanol solution, vortex and mix well for 3 minutes to prepare a letermovir working solution (C6) with a concentration of 40 μg / mL, and store it at 2 - 8°C. Before preparing the analyte working solution with C6, the solution should be restored to room temperature and vortexed for 3 minutes. The analyte working solutions are numbered from high to low as C6 - C5 - C4 - C3 - C2 - C1, and the specific preparation process is as follows: Preparation of C5: Take 200 μL of the C6 working solution, add 200 μL of 50% methanol, vortex and mix well to prepare C5; Preparation of C4: Take 100 μL of the C6 working solution, add 400 μL of 50% methanol, vortex and mix well to prepare C4; Preparation of C3: Take 100 μL of the C5 working solution, add 400 μL of 50% methanol, vortex and mix well to prepare C3; Preparation of C2: Take 100 μL of the C4 working solution, add 400 μL of 50% methanol, vortex and mix well to prepare C2; Preparation of C1: Take 100 μL of the C3 working solution, add 400 μL of 50% methanol, vortex and mix well to prepare C1.

[0026] 4. Preparation of quality control sample working solution Take 30 μL of the letermovir stock solution with a concentration of 1 mg / mL, add 970 μL of 50% methanol solution, vortex and mix for 3 minutes to prepare a letermovir mixed quality control working solution (QCH) with a concentration of 30 μg / mL, and store it in a refrigerator at -20 °C. The analyte QC working solution numbers are QCH - QCM - QCL from high to low concentration. The specific preparation process is as follows: Preparation of QCM: Take 150 μL of the QCH solution, add 300 μL of 50% methanol, vortex and mix to prepare QCM; Preparation of QCL: Take 120 μL of the QCM solution, add 380 μL of 50% methanol, vortex and mix to prepare QCL; Preparation of plasma standard curve and quality control samples 5.1 Preparation of plasma standard curve In a 1.5 mL centrifuge tube, use a pipette to add 95 μL of normal human blank plasma, then add 5 μL of a series of concentration working solutions respectively, vortex and mix (about 30 sec) to prepare the corresponding plasma matrix calibrators S1 - S2 - S3 - S4 - S5 - S6. The plasma matrix calibrators should be freshly prepared on the day of sample processing, as shown in Table 1.

[0027] Table 1 Concentration table of letermovir in plasma matrix calibrators 5.2 Preparation of plasma quality control samples In a 1.5 mL centrifuge tube, use a pipette to add 95 μL of normal human blank plasma, then add 5 μL of a series of concentration quality control working solutions respectively, vortex and mix (about 30 sec) to prepare the corresponding plasma matrix quality control samples, namely LQC, MQC, and HQC. The quality control samples are freshly prepared on the day of sample processing, as shown in Tables 2 - 6.

[0028] Table 2 Concentration table of letermovir in plasma spiked samples Plasma Spiked Sample Concentration LQC MQC HQC Letermovir (ng / mL) 120 500 1500 Table 3 Chromatography and mass spectrometry conditions Solution Name Solution Ratio Preparation Method Storage Condition Shelf Life Needle Wash Solution Methanol-Water (1:1) 500 mL Methanol + 500 mL Purified Water, Ultrasonic for 5 min Room Temperature 1 Week Mobile Phase A 2 mM Ammonium Acetate - 0.1% Formic Acid Aqueous Solution 499 mL Purified Water + 0.5 mL Formic Acid + 0.5 mL 2M Ammonium Acetate, Ultrasonic for 5 min Room Temperature Freshly Prepared Mobile Phase B 0.1% Formic Acid Methanol Solution 499.5 mL Methanol + 0.5 mL Formic Acid, Ultrasonic for 5 min Room Temperature Freshly Prepared Table 4 Ion source parameters Spray Voltage (V) Shealth Gas (Arb) Aux Gas (Arb) Sweep Gas (Arb) Ion Transfer Tube Temp (℃) Vaporizer Temp (℃) 3000 45 15 5 350 450 Table 5 Ion pair information MRM Channel Compound Adduct Mode Q1 Q3 Cycle time(s) CE (V) RF Lens (V) 1 Letermovir M+H 573.2 364.1 0.3 44 166 2 Letermovir M+H 573.2 424.1 0.3 33 166 3 Lenvatinib-d5 M+H 432.1 312.1 0.3 43 76 4 Lenvatinib-d5 M+H 432.1 370.1 0.3 27 76 Table 6 Liquid phase method conditions 6. Sample pretreatment Take 50 μL of clinical sample plasma, add 300 μL of internal standard working solution, vortex for 1 min, place the sample in a centrifuge at 4°C and centrifuge at 14000 rpm for 10 min, and transfer 150 μL of the supernatant to an injection vial. Place the processed sample in an autoinjector for determination.

[0029] 7. Extract chromatographic peaks and fit calibration curve Use TraceFinder software to perform the operation of extracting chromatographic peaks from blank samples, calibrators, quality control samples of different sample types, and actual samples to be analyzed.

[0030] As Figures 1 to 4 Shown, an example of the chromatographic peak interface extracted by TraceFinder software for the above various types of samples. Figure 1 Show the chromatogram of the blank sample, Figure 2 Show the chromatogram of the letermovir matrix sample at a low concentration (120 ng / mL), Figure 3 Show the chromatogram of the letermovir matrix sample at a medium concentration (500 ng / mL), Figure 4 Show the chromatogram of the letermovir matrix sample at a high concentration (1500 ng / mL), while Figure 5 Is the chromatogram of the sample to be analyzed. The upper part of the figure shows the chromatogram of the letermovir internal standard, and the lower part shows the corresponding chromatograms of letermovir respectively.

[0031] Record the peak areas of letermovir and its internal standard in each sample and the ratio of the two (letermovir peak area / letermovir internal standard peak area). Using the concentration of the plasma matrix calibrator as the x-axis and the peak area ratio of letermovir to the internal standard as the y-axis, perform linear calibration curve fitting in a way with a weight of 1 / x² and ignoring the origin.

[0032] 8. Calculate the concentration of the sample to be tested Calculating the plasma concentration of a drug is usually achieved by the area under the plasma concentration-time curve (AUC), and the commonly used integral method or trapezoidal method is used. Input the ratio of the letermovir peak area to the internal standard peak area of the sample to be tested into the fitted calibration curve, thereby calculating the corresponding plasma concentration.

[0033] Example 2: Method validation 1. Residual verification Take blank samples, low-concentration samples, and high-concentration samples, perform pretreatment, and inject and detect them in sequence. The detailed residual experiment evaluation is shown in Table 7.

[0034] Table 7 Results of the letermovir residual experiment evaluation 2. Precision and accuracy In the blank matrix sample, letermovir quality control working solution was added to prepare letermovir matrix standard solutions at three different concentration levels. The specific concentrations were set as low concentration 120 ng / mL, medium concentration 500 ng / mL, and high concentration 1500 ng / mL. Six parallel samples were prepared for each concentration level to form a batch; the experiment was carried out for three consecutive days, and one batch was processed and analyzed every day. The results of detailed precision and accuracy are shown in Table 8.

[0035] Table 8 Summary Table of Letermovir Precision and Accuracy Data 3. Matrix Effect To evaluate the influence of plasma matrix effect on the determination of letermovir, quality control (QC) working solutions at two concentration levels (QCL and QCH) were added to blank plasma and solvents from six different sources respectively. After correction using the internal standard substance, the matrix factor of letermovir normalized to the internal standard was calculated. As shown in Table 9, the matrix effect of letermovir in plasma was small and had little impact on the accuracy of the analysis results.

[0036] Table 9 Summary Table of Letermovir Matrix Effect Evaluation (n = 6) Internal Standard Normalized Matrix Factor LQC HQC Mean 0.91 0.96 Standard Deviation (SD) 0.04 0.09 Precision (%CV) 4.40 9.38 4. Extraction Recovery Plasma and plasma supernatant samples were prepared with QC working solutions at two concentration levels of LQC and HQC, pretreated, and the extraction recoveries of the analyte and its internal standard were determined. As shown in Table 10, the recovery rate of letermovir was between 97.44% and 101.68%.

[0037] Table 10 Summary Table of Letermovir Extraction Recovery (n = 6) 5. Stability QC working solutions at two concentration levels of LQC and HQC were added to blank plasma respectively to investigate their stability at room temperature for 24 hours and 96 hours; in addition, they were placed in a 4-degree refrigerator to detect their stability at 24 hours and 48 hours. And the stability of letermovir after being frozen and thawed three times repeatedly. The results of relevant stability tests are shown in Table 11.

[0038] Table 11 Summary Table of Analyte Stability Investigation in Letermovir Plasma Matrix Samples (n = 6) 6. Standard Curve and Lower Limit of Quantification Working solutions at six concentration levels of STD01, STD02, STD03, STD04, STD05 and STD06 were separately added to blank plasma for pretreatment. Using the chromatographic peak area ratio of the analyte to the internal standard as the ordinate, weighted (w = 1 / x 2 ), the least squares method was used to perform linear regression on the concentration (x) of the analyte in plasma and the peak area ratio (y). The resulting regression equation (y = a + bx) was the standard curve. The standard curve of letermovir was obtained by continuously measuring for 3 days by liquid chromatography-tandem mass spectrometry, with 1 standard curve per day, as follows. As shown in Table 12, letermovir had a good linear relationship in the range of 40 - 2000 ng / mL, and the lower limit of quantification was 40 ng / mL.

[0039] Table 12 Standard Curve of Letermovir The LC-MS / MS method of the present invention optimizes the detection process of letermovir in plasma, shortens the analysis time required to within 2.5 minutes, and effectively improves the accuracy and precision of the analysis process. In addition, this method simplifies the sample pretreatment steps, reduces the operation difficulty and time consumption, ensures fast, accurate and highly sensitive analysis results, and is applicable to the requirements of the rapid clinical and research fields.

[0040] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for determining the concentration of letermovir in plasma using liquid chromatography tandem mass spectrometry, characterized in that: The steps include: S1: preparing a calibrator working solution and a quality control working solution of letermovir, respectively adding the calibrator working solution and the quality control working solution into a plasma matrix to obtain a plasma calibrator and a plasma quality control; preparing an internal standard working solution using lenvatinib-d5 as an internal standard substance; S2: adding the plasma calibrator and plasma quality control product in step S1 to the internal standard working solution obtained in step S1 respectively for protein precipitation treatment, collecting the supernatant, performing liquid chromatography tandem mass spectrometry analysis on the supernatant, and drawing a liquid chromatography tandem mass spectrometry standard curve; S3: adding the sample to be tested to the internal standard working solution obtained in step S1 to perform protein precipitation treatment, collecting the supernatant, performing liquid chromatography tandem mass spectrometry analysis on the supernatant, and obtaining the concentration of letermovir in plasma according to the liquid chromatography tandem mass spectrometry of step S2.

2. The method for determining the concentration of Letermovir in plasma using liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The solvent of the calibrant working solution in step S1 is 45-55% methanol; The solvent of the quality control working solution in step S1 is 45-55% methanol; The solvent of the internal standard working solution in step S1 is 45-55% methanol.

3. The method for determining the concentration of Letermovir in plasma using liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The concentration of the calibrator working solution of letermovir in step S1 is a step concentration, preferably, the step concentration is 40, 80, 200, 400, 1000, 2000 ng / mL.

4. The method for determining the concentration of Letermovir in plasma using liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The concentration of the quality control solution of Letermovir in step S1 is a step concentration; Preferably, the step concentrations are 120, 500, and 1500 ng / mL.

5. The method for determining the concentration of Letermovir in plasma using liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The concentration of the internal standard working solution in step S1 is 50 ng / mL.

6. The method for determining the concentration of Letermovir in plasma using liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The liquid chromatography tandem mass spectrometry analysis in step S2 is performed using a liquid chromatography tandem mass spectrometer; Preferably, the model of the liquid chromatography tandem mass spectrometer is YS EXACT 9900MD liquid chromatography tandem mass spectrometer.

7. The method for determining the concentration of Letermovir in plasma using liquid chromatography-tandem mass spectrometry according to claim 6, characterized in that: The chromatographic column of the liquid chromatography tandem mass spectrometer is a ChromCore C18 column.

8. The method for determining the concentration of Letermovir in plasma using liquid chromatography-tandem mass spectrometry according to claim 6, characterized in that: The mobile phase A of the liquid chromatography-tandem mass spectrometer was 2 mM ammonium acetate-0.1% formic acid in water, while the mobile phase B was 0.1% formic acid in methanol; The gradient elution program of the liquid chromatography-tandem mass spectrometer was set as follows: from 0 to 1.5 min, the proportion of mobile phase B increased from 3% to 95%, maintained at this proportion until 2.0 min, then the proportion of mobile phase B was rapidly reduced to 3% from 2.0 to 2.1 min, and maintained at this proportion from 2.1 to 2.5 min; The mass spectrometry conditions of the liquid chromatography tandem mass spectrometer were as follows: an ESI electrospray ion source was used and a selected reaction monitoring scan was performed in the positive ion mode.

9. The method for determining the concentration of Letermovir in plasma using liquid chromatography tandem mass spectrometry according to claim 6, characterized in that: The flow rate of gradient elution of the liquid chromatography-tandem mass spectrometer was set between 0.3 mL / min and 1 mL / min, and the column temperature was controlled between 35°C and 40°C; The injection volume of the liquid chromatography-tandem mass spectrometer was set to 1 μL to 5 μL; The spray voltage of the liquid chromatography tandem mass spectrometer was set to 2900-3100 V, the sheath gas pressure was 40-50 Arb, the auxiliary gas pressure was 12.5-17.5 Arb, the backflush gas pressure was 2.5-7.5 Arb, and the temperature of the ion transfer tube was controlled at 340-360°C, and the nebulizer temperature was set to 440-460°C.

10. The method for determining the concentration of Letermovir in plasma using liquid chromatography-tandem mass spectrometry according to claim 6, characterized in that: The liquid chromatography tandem mass spectrometry standard curve in step S2 uses the plasma matrix calibrator concentration as the horizontal coordinate and the peak area ratio of letermovir to the internal standard as the vertical coordinate. The weight is set to 1 / x², the origin is excluded, and a linear fit is performed.

Images