Method for detecting atavalopag in human plasma by HPLC-MS / MS (High Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry)

Abstract

The invention provides a method for detecting atavalopag in human plasma by HPLC-MS / MS (High Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry). The method comprises the following steps: (1) pretreating a human plasma sample; (2) carrying out liquid chromatography-mass spectrometry detection; a mobile phase A and a mobile phase B are adopted as mixed mobile phases for gradient elution, and the mobile phase A is a methanol-acetonitrile mixed solution; a mobile phase B is a 2-20 mM ammonium acetate aqueous solution; and (3) determining the atavalopag in the human plasma. According to the method, ZORBAX SB-C18 is adopted as a chromatographic column, a specific mobile phase is screened out, the proportion and time of the mobile phase in the gradient elution process are optimized, and the method has the advantages of being good in reproducibility, high in sensitivity, high in analysis speed, small in matrix effect influence, high in recovery rate and the like.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical technology, specifically relating to a method for detecting avatrombopag in human plasma using HPLC-MS / MS coupling. Background Technology

[0002] Thrombocytopenia is a common hematological disorder in patients with chronic liver disease and a major risk factor for bleeding during surgical procedures and other medical treatments, accounting for 5%-10% of all hematology hospitalizations. Theoretically, both decreased platelet production and increased platelet consumption (destruction) can lead to thrombocytopenia. Decreased platelet count is the most common cause of hemorrhagic diseases.

[0003] Thrombocytopenia caused by chronic liver disease is associated with multiple factors, including abnormal platelet distribution, decreased thrombopoietin (TPO), and bone marrow suppression. Current treatments for thrombocytopenia in liver disease patients include etiological treatment, platelet transfusion, intravenous immunoglobulin, hormones and immunosuppressants, TPO and its analogues, splenic embolization and splenectomy, and transjugular intrahepatic portosystemic shunt. For patients with severe thrombocytopenia, platelet transfusion can increase platelet count and reduce bleeding risk in the short term. However, its clinical application is limited due to the scarcity and high cost of platelets, their short biological half-life, and the risk of infection with other diseases. To meet clinical needs, thrombopoietin receptor agonists have become a focus of research. Currently available thrombopoietin receptor agonists include romilastine, eltrombopag, and avatrombopag.

[0004] Avatrombopag maleate tablets are a new drug for the treatment of thrombocytopenia in patients with chronic liver disease. They are suitable for preventing excessive blood loss in these patients before undergoing medical surgery. It is the first drug approved by the FDA for this indication. It can safely increase the platelet count of patients, thereby reducing or even completely avoiding additional platelet transfusions and related resuscitation, reducing the risk of infection and other complications caused by platelet transfusions, and has good tolerability and safety.

[0005] Chinese patent CN 119510637 discloses a method and application for detecting avatrombopag in plasma using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). The analysis time for avatrombopag in this method is 6.5 minutes, which is a long time required for large-scale sample testing in clinical trials. Directly injecting the supernatant into the detection method carries risks of column clogging, contamination of the flow stream, blockage of ion transport channels, and contamination of the ion source and mass analyzer.

[0006] To meet the needs of large-scale clinical sample analysis for evaluating drug bioequivalence, it is necessary to develop simpler, more reliable, and higher-throughput sample pretreatment methods and methods for detecting avatrombopag concentrations in human plasma. Summary of the Invention

[0007] The purpose of this invention is to provide, based on existing technology, a method for detecting avatrombopag in human plasma using HPLC-MS / MS. This method has advantages such as requiring a small plasma sample volume (only 50 μL), fast analysis speed (only 3.5 min), a wide linear range (0.5 ng / mL-250 ng / mL), low limit of quantitation, high sensitivity, good reproducibility, minimal matrix effect, high extraction recovery rate, and clean injection solution. It is suitable for large-scale plasma sample detection and can be reliably used to evaluate the bioequivalence of avatrombopag in humans.

[0008] The technical solution of the present invention is as follows: A method for detecting avatrombopag in human plasma using HPLC-MS / MS, comprising the following steps: (1) human plasma sample pretreatment; (2) detection using HPLC-MS / MS; gradient elution using a mixed mobile phase A and mobile phase B, wherein mobile phase A is a methanol-acetonitrile mixed solution; and mobile phase B is a 2-20... (3) Determination of avatrombopag in human plasma; The gradient elution process is as follows: In the first 0-0.5 minutes, the volume ratio of mobile phase A to mobile phase B is 40:60; In the first 0.5-1.0 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes from 40:60 to 85:15; In the first 1.0-2.5 minutes, the volume ratio of mobile phase A to mobile phase B is 85:15; In the first 2.5-2.8 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes from 85:15 to 40:60; In the first 2.8-3.5 minutes, the volume ratio of mobile phase A to mobile phase B is 40:60. The specific gradient elution process is shown in Table 1.

[0009] Table 1. Liquid Chromatography Gradients of Avatrombopag

[0010] In determining avatrombopag in human plasma, this invention employs the aforementioned gradient elution method, with an analysis time of only 3.5 min. This short analysis time significantly reduces the analysis time and achieves a quantitation limit of 0.5 ng / mL, making the analysis of large batches of biological samples more convenient and feasible, and effectively improving sensitivity and extraction recovery rate.

[0011] When using HPLC-MS / MS to detect avatrombopag in human plasma, the strong wash solution is a mixed solution of methanol, acetonitrile, isopropanol, and formic acid. Preferably, the volume ratio of methanol, acetonitrile, isopropanol, and formic acid in the strong wash solution is 1000:1000:1000:3. The weak wash solution is a mixed solution of methanol, water, and formic acid. Preferably, the volume ratio of methanol, water, and formic acid in the weak wash solution is 1000:1000:2.

[0012] In this invention, when using HPLC-MS / MS to detect avatrombopag in human plasma, in a preferred embodiment, mobile phase A is a methanol-acetonitrile mixed solution, with a methanol-acetonitrile volume ratio of 45-60:60-40; more preferably, the methanol-acetonitrile volume ratio is 45-55:55-45; and even more preferably, the methanol-acetonitrile volume ratio is 50:50. In this invention, to improve chromatographic separation selectivity, the polarity of the mobile phase is adjusted by adding ammonium acetate and formic acid. Mobile phase B is a 2-20 mM ammonium acetate aqueous solution, preferably an 8-12 mM ammonium acetate aqueous solution, and even more preferably, a 10 mM ammonium acetate aqueous solution.

[0013] In a preferred embodiment, the mobile phase B is a 10 mM ammonium acetate aqueous solution, containing 0.05-0.20% formic acid by volume, based on a total volume of 100% of the ammonium acetate aqueous solution.

[0014] In a more preferred embodiment, mobile phase B is a 10 mM ammonium acetate aqueous solution, containing 0.1% formic acid by volume, based on a total volume of 100% of the ammonium acetate aqueous solution. For example, mobile phase B is a 0.1% formic acid-10 mM ammonium acetate aqueous solution.

[0015] In chromatography, the selection of the chromatographic column is crucial, requiring high column efficiency, good selectivity, and fast analysis speed. This invention employs HPLC-MS / MS to detect avatrombopag in human plasma, using an Agilent ZORBAX SB-C18 column. Specifically, this column has a length of 50 mm, a diameter of 2.1 mm, and a packing particle size of 3.5 µm; that is, the column is an Agilent ZORBAX SB-C18 (2.1 × 50 mm, 3.5 μm). This invention uses the aforementioned mobile phases A and B as a mixed mobile phase for gradient elution. With the Agilent ZORBAX SB-C18 column, under other favorable conditions, endogenous substances do not interfere with the sample determination, and the analysis exhibits good reproducibility, high sensitivity, short analysis time, and minimal matrix effect. Using other similar chromatographic columns, such as Venusil C18 Plus (2.1*100 mm, 3µm), the separation effect is poor, with lower chromatographic peak response, peak broadening and tailing.

[0016] When using the internal standard method, the selection of the internal standard is crucial. An ideal internal standard should be able to be added to the sample in an accurate and known amount, and should have essentially the same or as closely as possible the same physicochemical properties, chromatographic behavior, and response characteristics as the analyte. Under chromatographic conditions, the internal standard must be able to completely separate from the components in the sample. In this invention, when using HPLC-MS / MS to detect avatrombopag in human plasma, avatrombopag-d8 is used as the internal standard, with deuterated analyte as the internal standard. The deuterated internal standard and the analyte have the same retention time, chemical properties, and matrix effect, resulting in good reproducibility and accuracy in determining the concentration of avatrombopag in plasma.

[0017] In step (1), the present invention pretreatments human plasma samples using a protein precipitation method, with methanol as the precipitant. Using protein precipitation for pretreatment of human plasma samples avoids the tedious and time-consuming liquid-liquid extraction process while achieving a surprisingly high recovery rate. In this invention, the protein precipitation method is used for pretreatment of human plasma samples, with an Agilent ZORBAX SB-C18 column. Under other conditions, the overall extraction recovery rate of avatrombopag was 108.94%.

[0018] In a preferred embodiment, step (1) of the pretreatment of human plasma samples includes: adding an internal standard working solution and a precipitant to the human plasma sample, vortexing and centrifuging, taking the supernatant, and mixing it with a diluent to obtain the sample to be tested; wherein, the internal standard in the internal standard working solution is avatrombopag-d8; and the precipitant is methanol. The diluent is a mixed solution composed of 10 mM ammonium acetate solution, methanol, and acetonitrile, wherein the volume ratio of 10 mM ammonium acetate solution, methanol, and acetonitrile in the mixed solution is 1:1-3:1-3, and based on the total volume of the mixed solution being 100%, it contains 0.05-0.20% formic acid by volume; preferably, the volume ratio of 10 mM ammonium acetate solution, methanol, and acetonitrile in the mixed solution is 1:2:2, and based on the total volume of the mixed solution being 100%, it contains 0.1% formic acid by volume.

[0019] The detection method of the present invention further includes the preparation of an internal standard working solution, which is prepared as follows: Avatrombopag-d8 reference standard is weighed, dissolved in DMSO to obtain an internal standard stock solution with a concentration of 1.00 mg / mL, and then diluted with a methanol-water mixture to obtain an internal standard working solution with a concentration of 500 ng / mL. Preferably, the volume ratio of methanol to water in the mixed solution is 75-85:25-15; more preferably, the volume ratio of methanol to water in the mixed solution is 80:20.

[0020] In a more preferred embodiment, in step (1), the pretreatment of the human plasma sample includes: taking 50.0 μL of human plasma sample, adding 50.0 μL of internal standard working solution and 350 µL of methanol, vortexing and centrifuging, taking 100 µL of supernatant, and then mixing it with 400 μL of diluent to obtain the sample to be tested; wherein, the diluent is a mixed solution composed of 10 mM ammonium acetate solution, methanol and acetonitrile, and the volume ratio of 10 mM ammonium acetate solution, methanol and acetonitrile in the mixed solution is 1:2:2, and based on the total volume of the mixed solution being 100%, it contains 0.1% formic acid by volume.

[0021] The present invention uses protein precipitation to pretreat human plasma samples. The conditions for vortexing and centrifugation are as follows: vortexing at 1000 rpm / min for 5 min and centrifuging at 4000 rpm / min for 5 min at 4℃.

[0022] In this invention, the sample to be tested is placed in an autosampler for LC-MS / MS analysis during chromatographic detection. The injection volume is 10 μL and the autosampler temperature is 4 °C.

[0023] In the detection method of the present invention, step (2) uses liquid chromatography-mass spectrometry for detection. The detailed chromatographic conditions are as follows: an Agilent ZORBAX SB-C18 (2.1×50 mm, 3.5 μm) column is used as the chromatographic column, and gradient elution is performed according to the elution process mentioned above. The column temperature is 30-45℃, preferably 40℃; the flow rate is 0.5-0.9 mL / min, preferably 0.7 mL / min.

[0024] The mass spectrometry conditions of this invention include: an electrospray ionization source, positive ion multiple reaction monitoring (MRM) scanning, a spray voltage of 5500 V, and an ion source temperature of 650 °C; avatrombopag, [M+H]+, m / z 649.4→267.3, DP value 115 V, CE value 45 V; avatrombopag-d8, [M+H]+, m / z 657.4→267.4, DP value 115 V, CE value 45 V.

[0025] The detection method of the present invention, step (3) determination of avatrombopag concentration in human plasma: the plasma to be tested is prepared according to the sample pretreatment method in step (1), and detected by liquid chromatography-mass spectrometry according to step (2), the peak area of ​​avatrombopag is recorded, and the peak area ratio of avatrombopag and its internal standard is weighted by a coefficient w=1 / x 2 Linear regression was performed, with the equation y=ax+b, to calculate the concentration of avatrombopag in the plasma to be tested.

[0026] The detection method of this invention can be used for monitoring clinical pharmacokinetic plasma samples. The steps for calculating clinical drug metabolism kinetic parameters include: calculating pharmacokinetic parameters using WinNonlin 8.0, including: C max , T max t 1 / 2 , AUC 0-t At the same time, the mean and standard deviation of each parameter are calculated.

[0027] The advantages of using the technical solution of this invention are as follows: (1) The detection method of the present invention uses deuterium as an internal standard. The deuterated internal standard and the analyte have the same retention time, chemical properties and matrix effect. The reproducibility and accuracy of the determination of avatrombopag concentration in plasma are good.

[0028] (2) In the detection method of the present invention, the plasma sample volume is small, only 50 μL, the analysis speed is fast, and the analysis time is only 3.5 min, which is suitable for the detection of large batches of plasma samples.

[0029] (3) In the detection method of the present invention, the linear range of avatrombopag is 0.5 ng / mL-250 ng / mL, which is relatively wide and has a low limit of quantitation. It is suitable for analyzing plasma samples after administration of different specifications and dosage forms, and has a wide range of applications.

[0030] (4) The detection method of the present invention has a high extraction recovery rate of avatrombopag, which is close to 108.94%.

[0031] (5) In the detection method of the present invention, the retention time of avatrombopag is appropriate and the peak shape is good.

[0032] (6) The detection method of the present invention selects a specific mobile phase and optimizes the elution time and the ratio of the mobile phase during the gradient elution process. It has the advantages of good reproducibility, high sensitivity, fast analysis speed, small matrix effect, high recovery rate and clean injection solution. It has also carried out full method verification including specificity, accuracy, precision, matrix effect, extraction recovery rate and stability. It can be reliably used to evaluate the bioequivalence of avatrombopag in humans. Attached Figure Description

[0033] Figure 1 This is an Avatropoparis ion scan; Figure 2 This is a scan of the avatrombopag-d8 daughter ion; Figure 3This is a specific chromatogram of avatrombopag in the first batch of blank plasma from different individuals determined by LC-MS / MS. The chromatogram on the left is avatrombopag, and the chromatogram on the right is avatrombopag-d8. Figure 4 This is a specific chromatogram for the determination of avatrombopag in blank plasma from a second batch of different individuals by LC-MS / MS. The chromatogram on the left is avatrombopag, and the chromatogram on the right is avatrombopag-d8. Figure 5 This is a specific chromatogram for the determination of avatrombopag in blank plasma from different individuals in the third batch by LC-MS / MS method. The chromatogram on the left is avatrombopag, and the chromatogram on the right is avatrombopag-d8. Figure 6 This is a specific chromatogram for the determination of avatrombopag in blank plasma from different individuals in the fourth batch by LC-MS / MS method. The chromatogram on the left is avatrombopag, and the chromatogram on the right is avatrombopag-d8. Figure 7 This is a specific chromatogram for the determination of avatrombopag in blank plasma from different individuals in the fifth batch by LC-MS / MS method. The chromatogram on the left is avatrombopag, and the chromatogram on the right is avatrombopag-d8. Figure 8 This is a specific chromatogram for the determination of avatrombopag in blank plasma from different individuals in the sixth batch by LC-MS / MS method. The chromatogram on the left is avatrombopag, and the chromatogram on the right is avatrombopag-d8. Figure 9 The image shows a chromatogram of mixed blank plasma, where the left chromatogram represents avatrombopag and the right chromatogram represents avatrombopag-d8. Figure 10 The chromatograms are for samples with the lower limit of quantification. The chromatogram on the left is for avatrombopag, and the chromatogram on the right is for avatrombopag-d8. Detailed Implementation

[0034] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0035] Example 1: A method for detecting avatrombopag in human plasma using HPLC-MS / MS coupling 1. Instruments and reagents

[0036] High-performance liquid chromatography (Shimadzu LC-20AD series); mass spectrometry (API 4000 / API 4000Q, Applied Biosystems / Sciex); pure water system (Milli-Q, Merck Millipore); microbalance (XP6, METTLERTOLEDO); centrifuge (Heraeus Multifuge X1R, Thermo Fisher).

[0037] Methanol (Merck, HPLC grade), acetonitrile (Merck, HPLC grade), ammonium acetate (Aladdin, HPLC grade), water (ultrapure water, laboratory-prepared), formic acid (Aladdin, HPLC grade), dimethyl sulfoxide (Aladdin, HPLC grade), isopropanol (Sinopharm Chemical Reagent Co., Ltd., analytical grade). Blank plasma was obtained from healthy subjects. Avatrombopag (TLC, batch number: 4235-091A13), avatrombopag hydrochloride-d8 (TLC, batch number: 4265-060A6).

[0038] 2. Liquid chromatography-mass spectrometry (LC-MS) conditions

[0039] Liquid chromatography conditions: Agilent ZORBAX SB-C18 column (2.1 × 50 mm, 3.5 μm; column length 50 mm, diameter 2.1 mm, packing particle size 3.5 µm); column temperature 40℃; injector temperature 4℃; flow rate 0.7 mL / min; mobile phase A was a methanol-acetonitrile mixture (methanol to acetonitrile volume ratio 50:50); mobile phase B was 0.1% formic acid-10 The gradient elution process is as follows: Within 0-0.5 minutes, the volume ratio of mobile phase A to mobile phase B is 40:60; within 0.5-1.0 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes from 40:60 to 85:15; within 1.0-2.5 minutes, the volume ratio of mobile phase A to mobile phase B is 85:15; within 2.5-2.8 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes from 85:15 to 40:60; within 2.8-3.5 minutes, the volume ratio of mobile phase A to mobile phase B is 40:60.

[0040] The strong washing solution is a mixed solution of methanol-acetonitrile-isopropanol-formic acid (volume ratio of methanol-acetonitrile-isopropanol-formic acid is 1000:1000:1000:3, v / v / v / v), and the weak washing solution is a mixed solution of methanol-water-formic acid (volume ratio of methanol-water-formic acid is 1000:1000:2, v / v / v).

[0041] Mass spectrometry conditions: Electrospray ionization source, positive ion multiple reaction monitoring (MRM); Ionization method: pneumatic-assisted electrospray ionization (ESI); Ion polarity: positive ion; Detection target: avatrombopag, [M+H] + Avatrombopag-d8, m / z 649.4→267.3, DP value 115 V, CE value 45 V; [M+H]+, m / z 657.4→267.4, DP value 115 V, CE value 45 V. Mass spectrometry parameters: spray voltage 5500 V; ion source temperature 650℃. Specific ion scans of avatrombopag and avatrombopag-d8 are shown below. Figure 1 and Figure 2 As shown. 3. Preparation of standard solutions

[0042] Preparation of analyte working solutions: Accurately weigh two portions of avatrombopag reference standard, correct for mass correction factor, and dissolve in DMSO to obtain two internal standard stock solutions with a concentration of 1.00 mg / mL. Store the stock solutions in a -20℃ freezer. After the stock solution passed inspection, accurately measure a portion of the avatrombopag stock solution and dilute it with a methanol-water mixture (methanol to water volume ratio of 80:20, v / v) to prepare a series of standard curve working solutions with avatrombopag concentrations of 10.0 ng / mL (LLOQ), 20.0 ng / mL, 100 ng / mL, 250 ng / mL, 500 ng / mL, 2500 ng / mL, 4000 ng / mL, and 5000 ng / mL (ULOQ). Accurately measure another portion of the avatrombopag stock solution and dilute it with a methanol-water mixture (methanol to water volume ratio of 80:20, v / v) to prepare avatrombopag concentrations of 10.0 ng / mL (LLOQ QC), 30.0 ng / mL (LQC), 225 ng / mL (GMQC), 2000 ng / mL (MQC), and 3750 ng / mL (ULOQ). Quality control working solution at ng / mL (HQC).

[0043] Preparation of the internal standard working solution: Accurately weigh avatrombopag-d8 reference standard, correct for mass, and dissolve in DMSO to obtain an internal standard stock solution with a concentration of 1.00 mg / mL. Store the stock solution at -20°C. Accurately measure a certain amount of the internal standard stock solution and dilute it with a methanol-water mixture (methanol and water volume ratio of 80:20, v / v) to prepare an internal standard working solution with a concentration of 500 ng / mL for avatrombopag-d8.

[0044] 4. Preparation of standard curve samples and quality control samples The preparation process for standard curve samples and quality control samples at each concentration level is illustrated below: Add 20.0 μL of the corresponding working solution to 380 μL of blank plasma, mix thoroughly, and adjust the volume as needed. Prepare standard curve samples containing avatrombopag at concentrations of 0.500 ng / mL (LLOQ), 1.00 ng / mL, 5.00 ng / mL, 12.5 ng / mL, 25.0 ng / mL, 125 ng / mL, 200 ng / mL, and 250 ng / mL (ULOQ) and quality control samples at concentrations of 0.500 ng / mL (LLOQ QC), 1.50 ng / mL (LQC), 11.25 ng / mL (GMQC), 100 ng / mL (MQC), and 187.5 ng / mL (HQC), respectively.

[0045] 5. Sample pretreatment Add 50.0 μL of sample (biological sample to be tested, standard curve sample, quality control sample) to a 96-well plate; for double blank samples and blank samples, add 50.0 µL of blank matrix. Add 50.0 μL of methanol-water mixture (methanol to water volume ratio of 80:20, v / v) to the double blank samples. Except for the double blank samples, add 50.0 µL of internal standard working solution (500 ng / mL) to all wells, then add 350 µL of precipitant methanol. Vortex the 96-well plate at 1000 rpm / min for 5 min, and then centrifuge the 96-well plate at 4000 rpm / min for 5 min at 4℃. Add 100 µL of supernatant to a clean 96-well plate, then add 400 µL of diluent (a mixed solution consisting of 10 mM ammonium acetate solution, methanol, and acetonitrile, with a volume ratio of 1:2:2 (v / v / v) and containing 0.1% formic acid based on a total volume of 100%). Mix well and place in the injection chamber or a refrigerator at the same temperature for analysis.

[0046] 6. Methodological Examination Content The detection method was validated according to the "Guidelines for Validation of Quantitative Analysis Methods for Biological Samples" in the 2020 edition of the Chinese Pharmacopoeia to ensure the accuracy, repeatability, and stability of the assay. Validation included the following: specificity, standard curve, precision and accuracy, matrix effect, extraction recovery, and stability.

[0047] II. Results and Discussion 1. Exclusivity

[0048] Under the chromatographic conditions used in this invention, the retention time of avatrombopag is approximately 1.71 min. Figure 10 The retention time of the internal standard avatrombopag-d8 is approximately 1.71 min. Figure 9 Take 50.0 μL of blank plasma from each of the six different sources. Except for the absence of internal standards, follow the sample pretreatment procedure to obtain the chromatograms of the blank plasma samples, as shown below. Figure 3-8 The chromatogram of the sample with the lower limit of quantitation is shown in [reference needed]. Figure 10 The results indicate that endogenous substances in plasma do not affect the detection of avatrombopag, and internal standards also do not affect the detection of avatrombopag. 2. Accuracy and precision tests

[0049] Quality control samples containing avatrombopag at concentrations of 0.500 ng / mL (LLOQ QC), 1.50 ng / mL (LQC), 11.25 ng / mL (GMQC), 100 ng / mL (MQC), and 187.5 ng / mL (HQC) were prepared, with six samples prepared for each concentration. Two standard curves were prepared (obtained by regression analysis of two sets of standard curve samples), and the peak area of ​​avatrombopag was calculated. As and corresponding internal standard peak area Ai The ratio ƒ, denoted as ƒ, is used to substitute ƒ into the standard curve for that day to obtain the measured concentration, the average measured concentration, and the accuracy. Intra-batch precision and accuracy are then calculated, and the results are shown in Table 2. The results show that, except for the limit of quantitation (LLOQ), the RSD of the intra-batch quality control samples for avatrombopag is less than 15%, the intra-batch accuracy (RE) is at least 67% not exceeding ±15%, and at least 50% of the quality control samples at each concentration level deviate from their theoretical values ​​by no more than ±15%. For the LLOQ avatrombopag samples, the RSD of the intra-batch quality control samples is less than 20%, the intra-batch accuracy (RE) is at least 67% not exceeding ±20%, and at least 50% of the quality control samples at each concentration level deviate from their theoretical values ​​by no more than ±20%. In summary, both precision and accuracy meet the requirements.

[0050] Table 2 Precision and accuracy of intra-batch and inter-batch sample testing

[0051]

[0052] 3. Investigation of matrix effect Matrix sample preparation: Six batches of blank plasma from different donors were used to prepare six replicate double blank samples from each batch of blank plasma. The blank matrix extract was obtained by following the sample pretreatment procedure. After extraction, a certain amount of analyte and internal standard were added to make the final concentration consistent with the injection concentration of the low and high concentration quality control samples (three replicates for each concentration level).

[0053] Sample preparation: Pure water was used instead of blank plasma for pretreatment. The working solution was then diluted to the appropriate concentration so that the concentration after adding 50.0 µL of blank matrix extract or 50.0 µL of pure water extract was consistent with the injection concentration of the low, medium, and high concentration quality control samples after pretreatment. Three replicate samples were prepared for each concentration.

[0054] The results showed that the total matrix effect factor (calculated as peak area ratio) of avatrombopag was 1.00, and the precision was 1.2%, indicating that the plasma matrix did not affect the accurate quantification of avatrombopag. The matrix effect data are shown in Table 3.

[0055] Table 3 Matrix Effect

[0056]

[0057] 4. Analysis of extraction recovery rate Matrix sample preparation: Plasma was prepared by mixing blank plasma from 6 batches of different donors to form 9 replicate double blank samples. The blank plasma extract was obtained by following the sample pretreatment procedure. After extraction, a certain amount of analyte and internal standard were added to make the final concentration consistent with the injection concentration of the low, medium and high concentration quality control samples (3 replicates for each concentration level).

[0058] Quality control sample preparation: Take quality control samples of low, medium and high concentrations and process them according to the sample processing method. Prepare 6 samples for each concentration level.

[0059] Recovery is evaluated by comparing the response values ​​of the analyte or internal standard in a single quality control sample with the mean response values ​​of a double blank sample after extraction with the addition of the analyte and internal standard.

[0060] The acceptance criteria for recovery were: the precision of recovery at each concentration level and across all concentration levels should be within 15.0%. The recovery rate of avatrombopag extraction (calculated as peak area ratio) was 108.94%, with recoveries of 106.74%, 110.06%, and 110.01% at low, medium, and high concentrations, respectively. The results are shown in Table 4.

[0061] Table 4 Extraction Recovery Rate 5. Stability test

[0062] Stability of processed samples: After the first injection analysis of the analytical batch to examine precision and accuracy, the sample was placed in the autosampler (4℃) for 217 h. Freshly prepared standard curve samples and previously analyzed samples were then injected and analyzed, and the chromatograms were recorded. The results are shown in Table 5. The injection solution of avatrombopag plasma sample after processing showed good stability after being placed in the autosampler for 217 h, which meets the requirements for biological sample analysis.

[0063] Room temperature stability: The prepared low and high concentration levels of quality control samples, containing avatrombopag at concentrations of 1.50 ng / mL and 187.5 ng / mL, were mixed thoroughly and left at room temperature for 28 h before LC-MS / MS analysis was performed and the chromatograms were recorded. The results are shown in Table 6. The plasma samples showed good stability at room temperature for 28 h.

[0064] Freeze-thaw stability: Freshly prepared samples containing 1.50 ng / mL and 187.5 ng / mL of avatrombopag were subjected to five freeze-thaw cycles at -80°C. Acceptance criteria were: the %RE (mean reactivity ratio) of the average measured value of the stable sample should not exceed ±15.0% of its theoretical value, and the %RSD (mean relative standard deviation) of the measured values ​​of the stable samples at each concentration level should be ≤15.0%. The results are shown in Table 7. The samples exhibited good stability after five freeze-thaw cycles at -80°C.

[0065] Long-term stability: Freshly prepared samples containing 0.300 ng / mL and 37.5 ng / mL of avatrombopag were stored at -80°C for 91 days before testing. Acceptance criteria were: the %RE (mean reactivity ratio) of the average measured value of the stable samples should not exceed ±15.0%, and the %RSD (mean relative standard deviation) of the measured values ​​of the stable samples at each concentration level should be ≤15.0%. The results are shown in Table 8. The samples showed good stability after 91 days of storage at -80°C.

[0066] Table 5 Stability of treated samples

[0067] Table 6. Stability during biological sample pretreatment (room temperature stability)

[0068] Table 7 Freeze-thaw stability

[0069] Table 8 Long-term stability

[0070] The HPLC-MS / MS method for the determination of avatrombopag in plasma provided by this invention has good specificity. Endogenous substances in plasma do not interfere with the determination of samples. The linear range of the avatrombopag standard curve is 0.500 ng / mL-250 ng / mL, with good linearity. The intra- and inter-assay precision of the test results for quality control samples at four concentration levels of avatrombopag (high concentration, 187.5 ng / mL, medium concentration, 100 ng / mL, sub-medium concentration, 11.25 ng / mL, and low concentration, 1.50 ng / mL) is less than 15.0%. The intra- and inter-assay precision of the test results for the quality control sample at the limit of quantitation (0.500 ng / mL) is less than 20.0%.

[0071] The total matrix effect factor of avatrombopag was 1.00, and the precision was 0.2%. The plasma matrix did not affect the accurate quantification of avatrombopag. The recovery rate of avatrombopag extraction was 108.94%. Avatrombopag plasma samples showed good stability after being placed at room temperature for 28 h; good stability after 5 freeze / thaw cycles; good stability after being placed in an autosampler at 4℃ for 217 h; and good stability after being placed at -80℃ for 91 days, meeting the requirements for biological sample analysis.

[0072] In summary, the HPLC-MS / MS method for determining the concentration of avatrombopag in human plasma established in this invention meets the relevant requirements of the 2020 edition of the Pharmacopoeia "Guiding Principles for Validation of Quantitative Analysis Methods for Biological Samples" and can be used for plasma sample analysis and detection in clinical trials. Comparative Example 1

[0073] The mass spectrometry conditions and plasma sample pretreatment methods were the same as in Example 1, except that the liquid chromatography conditions were different. The Agilent ZORBAX SB-C18 column (2.1 mm * 50 mm, 3.5 µm) was replaced with a Venusil C18 Plus column (2.1 * 100 mm, 3 µm).

[0074] The results showed that when the chromatographic column changed, the signal of avatrombopag in the sample decreased, the limit of quantification of avatrombopag increased from 0.5 ng / mL to 4 ng / mL, the sensitivity decreased significantly, and the peak broadened and showed tailing. Comparative Example 2

[0075] The mass spectrometry conditions and plasma sample pretreatment were the same as in Example 1, except for the liquid phase conditions. The gradient elution process was adjusted as follows: within 0.0-3.0 minutes, the volume ratio of mobile phase A to mobile phase B was gradually changed from 55:45 to 95:5 at a uniform rate; within 3.0-4.0 minutes, the volume ratio of mobile phase A to mobile phase B was 95:5; within 4.0-4.1 minutes, the volume ratio of mobile phase A to mobile phase B was gradually changed from 95:5 to 55:45 at a uniform rate; and within 4.1-6.5 minutes, the volume ratio of mobile phase A to mobile phase B was 55:45.

[0076] The results showed that when the gradient elution process changed, the avatrombopag signal in the sample decreased, the limit of quantification of avatrombopag increased from 0.5 ng / mL to 5 ng / mL, the sensitivity decreased significantly, the separation was poor, and there was a bulge before the peak position. Comparative Example 3

[0077] The mass spectrometry conditions and plasma sample pretreatment were the same as in Example 1, except for the liquid phase conditions. Mobile phase A was replaced with methanol instead of a 50:50 methanol-acetonitrile mixture, and mobile phase B (0.1% formic acid-10 mM ammonium acetate aqueous solution) was replaced with a 0.1% formic acid aqueous solution.

[0078] The results showed that when the mobile phase changed, the peak position of avatrombopag in the sample was delayed, the peak was wider, and the signal was reduced. Comparative Example 4

[0079] The mass spectrometry and liquid chromatography conditions were the same as in Example 1, except that during the pretreatment process, the diluent (a mixed solution consisting of 10 mM ammonium acetate solution, methanol and acetonitrile, with a volume ratio of 1:2:2, v / v / v) was replaced with methanol-water (volume ratio of 50:50, v / v).

[0080] The results showed that when the diluent was changed, the avatrombopag signal in the test sample decreased, and the accuracy and precision analysis batch failed, which was presumably due to the incomplete dissolution of avatrombopag in the final injection solution.

[0081] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications may still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions may be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting avatrombopag in human plasma using HPLC-MS / MS, characterized in that, It includes the following steps: (1) pretreatment of human plasma samples; (2) detection by liquid chromatography-mass spectrometry; gradient elution is performed using a mixed mobile phase A and mobile phase B, wherein mobile phase A is a methanol-acetonitrile mixed solution; and mobile phase B is a 2-20 (3) Determination of avatrombopag in human plasma; The gradient elution process is as follows: within 0-0.5 minutes, the volume ratio of mobile phase A to mobile phase B is 40:60; within 0.5-1.0 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes from 40:60 to 85:15; within 1.0-2.5 minutes, the volume ratio of mobile phase A to mobile phase B is 85:15; within 2.5-2.8 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes from 85:15 to 40:60; within 2.8-3.5 minutes, the volume ratio of mobile phase A to mobile phase B is 40:

60.

2. The method according to claim 1, characterized in that, The liquid chromatography conditions include: an Agilent ZORBAX SB-C18 column; preferably, the column length is 50 mm, the diameter is 2.1 mm, and the packing particle size is 3.5 µm.

3. The method according to claim 2, characterized in that, The mobile phase A is a methanol-acetonitrile mixed solution, wherein the volume ratio of methanol to acetonitrile in the mixed solution is 45-60:60-40; preferably, the volume ratio of methanol to acetonitrile in the mixed solution is 45-55:55-45; more preferably, the volume ratio of methanol to acetonitrile in the mixed solution is 50:

50.

4. The method according to claim 3, characterized in that, The mobile phase B is an 8-12 mM ammonium acetate aqueous solution, preferably a 10 mM ammonium acetate aqueous solution; more preferably, the mobile phase B contains 0.05-0.20% formic acid by volume, based on 100% of the total volume of the ammonium acetate aqueous solution; particularly preferably, the mobile phase B contains 0.1% formic acid by volume, based on 100% of the total volume of the ammonium acetate aqueous solution.

5. The method according to claim 1, characterized in that, In step (1), the pretreatment of human plasma samples includes: adding internal standard working solution and precipitant to human plasma samples, vortexing and centrifuging, taking the supernatant, and mixing it with diluent to obtain the sample to be tested; the internal standard working solution contains avatrombopag-d8 as the internal standard; the precipitant is methanol; the diluent is a mixed solution composed of 10mM ammonium acetate solution, methanol and acetonitrile, in which the volume ratio of 10mM ammonium acetate solution, methanol and acetonitrile in the mixed solution is 1:1-3:1-3, and based on the total volume of the mixed solution being 100%, it contains 0.05-0.20% formic acid by volume; preferably, in which the volume ratio of 10mM ammonium acetate solution, methanol and acetonitrile in the mixed solution is 1:2:2, and based on the total volume of the mixed solution being 100%, it contains 0.1% formic acid by volume.

6. The method according to claim 5, characterized in that, In step (1), the internal standard working solution is prepared as follows: weigh avatrombopag-d8 reference standard, dissolve it in DMSO to obtain an internal standard stock solution with a concentration of 1.00 mg / mL, and then dilute it with a methanol-water mixed solution to obtain an internal standard working solution with a concentration of 500 ng / mL avatrombopag-d8; preferably, the volume ratio of methanol to water in the mixed solution is 75-85:25-15, more preferably, the volume ratio of methanol to water in the mixed solution is 80:

20.

7. The method according to claim 6, characterized in that, In step (1), the pretreatment of human plasma samples includes: taking 50.0 μL of human plasma sample, adding 50.0 μL of internal standard working solution and 350 µL of methanol, vortexing and centrifuging, taking 100 µL of supernatant, and then mixing it with 400 μL of diluent to obtain the sample to be tested; the diluent is a mixed solution composed of 10 mM ammonium acetate solution, methanol and acetonitrile, in which the volume ratio of 10 mM ammonium acetate solution, methanol and acetonitrile in the mixed solution is 1:2:2, and based on the total volume of the mixed solution being 100%, it contains 0.1% formic acid by volume.

8. The method according to claim 7, characterized in that, In step (1), the pretreatment of human plasma samples includes: the conditions for vortexing and centrifugation are: vortexing at 1000 rpm / min for 5 min, centrifuging at 4000 rpm / min for 5 min at 4℃; placing the sample to be tested in an autosampler for LC-MS / MS analysis, with an injection volume of 10 μL and an autosampler temperature of 4℃.

9. The method according to claim 1, characterized in that, Mass spectrometry conditions included: electrospray ionization source, positive ion multiple reaction monitoring (MRM), spray voltage of 5500 V, and ion source temperature of 650 °C; avatrombopag, [M+H] + m / z 649.4→267.3, DP value 115 V, CE value 45 V; Avatrombopag -d8, [M+H] + m / z 657.4→267.4, DP value 115V, CE value 45V.

10. The method according to claim 1, characterized in that, The liquid chromatography conditions include: column temperature of 30-45℃, preferably 40℃; flow rate of 0.5-0.9 mL / min, preferably 0.7 mL / min.

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