A method for detecting a compound in biological samples

Abstract

This invention provides a method for detecting compound I or its pharmaceutically acceptable salt in biological samples, particularly a method for detecting the concentration in plasma samples. This method uses LC-MS / MS with gradient elution of acetonitrile and formic acid aqueous solution as the mobile phase. It is rapid, stable, sensitive, accurate, and reproducible, and can be used for pharmacokinetic studies of compound I or its pharmaceutically acceptable salt.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biology, and specifically relates to a method for detecting a compound in biological samples. Background Technology

[0002] Cancer is a major disease threatening human health, and its occurrence and development are related to many factors. The receptor tyrosine kinase family (RTK) plays a key role in the occurrence and malignant progression of various types of tumors.

[0003] Fibroblast growth factor receptors (FGFRs) are receptor tyrosine kinases. Family members include FGFR1, FGFR2, FGFR3, and FGFR4, which play roles in cell proliferation, survival, migration, and angiogenesis. FGFR activation is closely related to the development and resistance of various tumors. FGFRs mainly participate in tumorigenesis through three mechanisms: chromosomal translocation, gene mutation, and gene amplification or overexpression. Chromosomal translocations of the FGFR1 gene or its fusion gene are mainly found in multiple myeloma; mutations in FGFR2 and FGFR3 are expressed in squamous cell carcinoma of the lung, and the transmembrane FGFR4Y367C mutation leads to persistent activation of breast cancer cells. According to literature reports, FGFR amplification occurs in various cancers. FGFR1 amplification is found in patients with rectal cancer, lung cancer, and kidney cancer; FGFR2 amplification is also observed in patients with gastric and rectal cancer; and FGFR3 amplification is most common in bladder cancer patients. Therefore, research on FGFR kinase inhibitors is of great significance in the treatment of malignant tumors. In recent years, the development of small molecule tyrosine kinase inhibitors (TKIs) targeting FGFRs has become a hot topic in anti-tumor drug research. Some FGFR inhibitors have entered the clinical trial stage.

[0004] Compound I is a novel small-molecule, multi-target FGFR inhibitor for the clinical treatment of advanced solid tumors. Its structure is shown in formula (I):

[0005] (I)

[0006] Accurate determination of drug concentration is crucial for studying the pharmacokinetic properties of drugs and assessing exposure-effect relationships. However, existing technologies do not disclose analytical methods for compound I and its pharmaceutically acceptable salts in biological samples. Summary of the Invention

[0007] This invention provides a biological sample detection method for compound I or its pharmaceutically acceptable salts, particularly a biological sample detection method for compound I dihydrochloride (compound A), which has been fully validated. Using this method to detect compound A in plasma samples, it exhibits good accuracy, sensitivity, specificity, and reproducibility. This method has been successfully applied to pharmacokinetic studies in cancer patients.

[0008] Specifically, the present invention provides the following technical solutions:

[0009] A method for detecting compound I or a pharmaceutically acceptable salt thereof in biological samples, characterized by: using LC-MC / MC to detect the concentration of compound I or a pharmaceutically acceptable salt thereof in the sample.

[0010] The structure of compound I is shown in formula (I):

[0011]

[0012] Using D3-compound I or a pharmaceutically acceptable salt thereof as an internal standard (IS), the structure of D3-compound I is shown below:

[0013]

[0014] Preferred,

[0015] The pharmaceutically acceptable salt of compound I is the dihydrochloride salt (compound A), with the structure shown below:

[0016]

[0017] Using compound A (D3-) as an internal standard (IS), its structure is shown below:

[0018]

[0019] In some implementations, the detection method uses LC-MC / MC to detect the concentration of compound A in the sample, with D3-compound A as an internal standard (IS).

[0020] In some implementations, the detection method includes chromatographic conditions comprising:

[0021] Chromatographic column: C18 column, preferably Phenomenex Gemini 5u C18 110A column (2.0×50mm, 5μm);

[0022] Mobile phase A: Acetonitrile;

[0023] Mobile phase B: 0.1%-0.5% aqueous formic acid, preferably 0.2%-0.5% aqueous formic acid, more preferably 0.2% aqueous formic acid;

[0024] Gradient procedure:

[0025] Time (min) Mobile phase A% Mobile phase B% 0.00 18-22 78-82 1.00 18-22 78-82 1.01 78-82 18-22 2.00 78-82 18-22 2.01 18-22 78-82 3.00 18-22 78-82

[0026] Preferred,

[0027] Time (min) Mobile phase A% Mobile phase B% 0.00 20 80 1.00 20 80 1.01 80 20 2.00 80 20 2.01 20 80 3.00 20 80

[0028] Injection volume: 3-10 μL, preferably 5-10 μL, more preferably 5 μL;

[0029] Flow rate: 0.2-0.4 ml / min, preferably 0.3-0.4 ml / min, preferably 0.3 ml / min;

[0030] Column temperature: 35-45℃, preferably 38-42℃, more preferably 40℃.

[0031] In some embodiments, the detection method, the mass spectrometry conditions include: mass spectrometry analysis using a Xevo TQS triple tandem quadrupole mass spectrometer (Waters, MA, USA), selecting the positive ion mode of the electrospray ionization (ESI) source; multiple reaction monitoring (MRM) scanning detection, the quantitative ion pair of compound A is: m / z 510.3→217.2; the quantitative ion of compound A-IS is: m / z 513.3→217.2.

[0032] In some implementations, the biological sample is a plasma sample.

[0033] In some embodiments, the chromatographic conditions are performed using a UPLC system, preferably an ACQUITY UPLC system (Waters, MA, USA).

[0034] In some implementations, the chromatographic conditions use an injection method selected from Full Loop or Partial Loop, with Partial Loop being preferred.

[0035] In some embodiments, the mass spectrometry conditions further include the following parameters: desolvation gas flow rate 1000 L / h, ion source temperature 500 °C; capillary voltage 3.30 kV, collision gas flow rate 0.25 ml·min. -1 The cone voltage is 35V; the collision voltages of compounds A and IS are 26V and 25V, respectively.

[0036] In some embodiments, the detection method optionally includes a plasma sample pretreatment step.

[0037] In some embodiments, the plasma sample pretreatment step includes: adding a methanol solution containing an internal standard to the plasma sample to precipitate proteins, shaking, and centrifuging; taking the supernatant, adding methanol solution, and mixing. In some embodiments, the volume ratio (v / v) of the plasma sample to the methanol solution containing the internal standard is 1:6-10, preferably 1:7-9, and more preferably 1:8. In some embodiments, the volume ratio (v / v) of the supernatant to the methanol solution is 0.5-2:1, preferably 0.8-1.5:1, and more preferably 1:1.

[0038] In some embodiments, the plasma sample pretreatment step is as follows: 25.0 μL of plasma sample is added to an EP tube, 200 μL of internal standard solution (5 ng / mL, methanol) is added to precipitate proteins, the mixture is shaken for 1 minute, and then centrifuged at 13300 rpm for 10 minutes. 100 μL of the supernatant is placed in a new EP tube, 100 μL of methanol solution is added, the mixture is stirred, and then injected.

[0039] In some embodiments, the detection method further includes preparing one or more of the following: a test substance stock solution, a standard curve working solution, a standard curve plasma sample solution, an IS stock solution, an IS working solution, a quality control working solution, and a quality control plasma sample solution. The test substance is selected from compound I or a pharmaceutically acceptable salt thereof, preferably a dihydrochloride, such as compound A. The IS is selected from D3-compound I or a pharmaceutically acceptable salt thereof, preferably a dihydrochloride, such as D3-compound A. The structures of compound I, D3-compound I, compound A, and D3-compound A are as previously shown.

[0040] In some implementations, the detection method further includes preparing a test substance stock solution, a standard curve working solution, a standard curve plasma sample solution, an IS stock solution, and an IS working solution.

[0041] In some embodiments, the test substance stock solution is prepared by accurately weighing the test substance standard and dissolving it in methanol-water (1:1, v:v). In some embodiments, the concentration of the test substance stock solution is 0.5 mg / mL.

[0042] In some embodiments, the standard curve working solution is prepared by taking an appropriate amount of the test substance stock solution and further diluting it to prepare standard curve working solutions of different concentrations. In some embodiments, the concentration of the standard curve working solution is 20, 50, 250, 500, 1000, 2500, 5000, or 10000 ng / ml.

[0043] In some embodiments, the preparation method of the standard curve plasma sample solution is as follows: a certain amount of standard curve working solution of different concentrations is taken and diluted with blank plasma to obtain a series of standard curve plasma sample solutions. In some embodiments, the concentration of the standard curve plasma sample solution is 0.2, 0.5, 2.5, 5.0, 10, 25, 50 and 100 ng / ml.

[0044] In some embodiments, the quality control working solution is prepared by taking an appropriate amount of the test substance stock solution and further diluting it to prepare quality control working solutions of different concentrations. In some embodiments, the concentration of the quality control working solution is 20, 40, 80, 8000, or 16000 ng / ml.

[0045] In some embodiments, the preparation method of the quality control plasma sample solution is as follows: a certain amount of standard curve working solution of different concentrations is taken and diluted with blank plasma to prepare the quality control plasma sample solution. In some embodiments, the concentration of the quality control plasma sample solution is 0.2, 0.4, 8.0, 80, or 160 ng / ml.

[0046] In some embodiments, the IS stock solution is prepared by accurately weighing the IS standard and dissolving it in methanol. In some embodiments, the concentration of the IS stock solution is 0.5 mg / ml.

[0047] In some embodiments, the IS working solution is prepared by diluting an appropriate amount of IS stock solution with methanol. In some embodiments, the concentration of the IS working solution is 5 ng / ml.

[0048] On the other hand, the present invention also provides D3-compound I or a pharmaceutically acceptable salt thereof, the structure of D3-compound I being as follows:

[0049]

[0050] Preferably, the pharmaceutically acceptable salt of D3-compound I is a dihydrochloride (D3-compound A), with the following structure:

[0051]

[0052] This invention also provides the use of D3-compound I or a pharmaceutically acceptable salt thereof as an internal standard compound for the biological sample detection of compound I or a pharmaceutically acceptable salt thereof. Furthermore, this invention also provides the use of D3-compound A as an internal standard compound for the biological sample detection of compound A. The structures of compound I, D3-compound I, compound A, and D3-compound A are as previously shown.

[0053] For the sake of brevity, the term "about" is not used for quantitative data herein. It should be understood that, whether the term "about" is explicitly used or not, every numerical value given herein includes not only the actual given value (the given value), but also approximations of such a given value based on reasonable inference by one of ordinary skill in the art, including equivalent and approximate values ​​resulting from experimental and / or measurement conditions. These approximations are preferably ±20%, ±15%, ±10%, ±8%, ±6%, ±5%, ±4%, ±3%, 2%, or ±1% of the given value.

[0054] In some embodiments, the numerical ranges and parameters described in the exposition of a broad range of embodiments of the invention are approximate values ​​and should be interpreted based on the number of significant digits reported and by applying common rounding techniques. Although the values ​​presented in specific embodiments are reported as accurately as possible, the values ​​presented in some embodiments of the invention may contain some errors, which are necessarily due to the standard deviation in test measurements.

[0055] The present invention achieves the following beneficial technical effects:

[0056] (1) This invention establishes for the first time a rapid, stable, and sensitive UPLC-MS / MS method for determining compound A in human plasma. Compound A contains nitrogen atoms in its chemical structure, and the positive ionization mode of the ESI source used in this invention has better ionization performance and a stable response. In addition, this invention optimizes the source / gas and compound parameters (including capillary voltage, cone voltage, etc.) to obtain the most suitable ionization conditions.

[0057] (2) Due to the presence of a benzene ring in the chemical structure of compound A, reversed-phase chromatography is the preferred method. The inventors explored the separation effects of various chromatographic columns. Compared with other columns, the Phenomenex Gemini 5u C18 110A column (2.0 × 50 mm, 5 μm) has sufficient retention and stable peak shape, and the inventors selected this column for separation. The mobile phase was also screened, and it was found that acetonitrile as the organic phase has good elution effect and very low background noise. Adding 0.2% formic acid to the aqueous phase can increase the response. This invention achieves gradient elution within 3.0 minutes, which is time-efficient and yields good peak shape and high sensitivity.

[0058] (3) When preparing plasma samples, the inventors initially used liquid-liquid extraction, but this method leads to chemical contamination and has low sensitivity. The inventors also tried solid-phase extraction, but due to its complexity, this method is time-consuming and costly. For protein precipitation, although this method may generally suffer from matrix effects that affect the accuracy of the determination, the final methodological validation showed that under the detection conditions described in this invention, the matrix effect is negligible when using protein precipitation to prepare plasma samples, resulting in clear chromatographic peaks and excellent reproducibility. Attached Figure Description

[0059] Figure 1 Representative chromatograms of the ion spectra of compound A and compound A-IS. Where: A - compound A; B - compound A-IS.

[0060] Figure 2 Typical MRM chromatograms of compounds A and IS. Wherein: A - blank plasma; B - blank plasma with IS added; C - LLOQ sample; D - clinically unknown plasma sample from the subject.

[0061] Figure 3 : Mean concentration-time curve of compound A in plasma after a single oral dose of compound A in the subjects. Where: A-1mg (n=3); B-2mg (n=4); C-4mg (n=4); D-6mg (n=4).

[0062] Figure 4 Separation performance of different types of chromatographic columns: A-X Bridgepheyl C18 (2.0×50mm, 3.5μm); B-Phenomenex Gemini 5u C18 110A (2.0×50mm, 5μm); C-ACQUITY UPLC BEH C18 column (2.1×50mm, 1.7μm).

[0063] Figure 5 Separation effect of different mobile phases A (organic phase), where: A - acetonitrile as mobile phase A; B - methanol as mobile phase A.

[0064] Figure 6 Separation effects of different mobile phases B (aqueous phase), where: A - using 2mM ammonium acetate solution as mobile phase B; B - using 5mM ammonium acetate solution as mobile phase B; C - using 0.1% formic acid aqueous solution as mobile phase B; D - using 0.2% formic acid aqueous solution as mobile phase B; E - using 0.5% formic acid aqueous solution as mobile phase B.

[0065] Figure 7 Effect of using acetonitrile as a precipitant on plasma sample treatment.

[0066] Figure 8 Separation effect at different flow rates, where A - flow rate 0.4 ml / min; B - flow rate 0.3 ml / min.

[0067] Figure 9 Separation effect at different column temperatures, where A-35℃; B-45℃. Detailed Implementation

[0068] To more clearly illustrate the present invention, the following detailed description of specific embodiments is provided. However, it should be understood that the specific embodiments described below are merely illustrative and not intended to limit the nature of the invention in any way. The materials, reagents, instruments, and operating conditions used are only representative and are not limited to the listed cases. Those skilled in the art can make modifications and improvements to the present invention without departing from the scope of protection defined by the claims, and such modifications and improvements are also within the scope of protection claimed by the present invention.

[0069] 1. Chemicals and reagents

[0070] Standards for compound A (99.8% purity) and compound A-IS (internal standard, IS, 99.0% purity) were provided by the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (Shanghai, China). HPLC-grade methanol and acetonitrile were purchased from Honeywell Burdick & Jackson (ML, USA). Formic acid (analytical grade) was purchased from Sigma-Aldrich chemicals (MO, USA). Blank plasma was provided by Peking Union Medical College Hospital, Chinese Academy of Medical Sciences (Beijing, China). Deionized water was purified using a Milli-Q system (Millipore, Bedford, MA, USA).

[0071] 2. Chromatographic and mass spectrometric conditions

[0072] 2.1 Chromatographic conditions

[0073] Chromatographic separation was performed using an ACQUITY UPLC system (Waters, MA, USA).

[0074] Chromatographic column: Phenomenex Gemini 5u C18 110A column (2.0×50mm, 5μm).

[0075] Mobile phase A: Acetonitrile.

[0076] Mobile phase B: 0.2% formic acid aqueous solution.

[0077] Gradient procedure:

[0078]

[0079]

[0080] Injection volume: 5 μL.

[0081] Flow rate: 0.3 ml / min.

[0082] The column temperature was maintained at 40°C, and the autosampler temperature was set at 10°C.

[0083] To reduce residue, clean the autosampler syringe and injection valve with an acetonitrile-water solution (90:10; v / v).

[0084] 2.2 Mass Spectrometry Conditions

[0085] Mass spectrometry analysis was performed using a Xevo TQS triple tandem quadrupole mass spectrometer (Waters, MA, USA), with positive ion mode selected for electrospray ionization (ESI). Multiple reaction monitoring (MRM) scans were used for detection. The quantitative ion pair for compound A was m / z 510.3→217.2; the quantitative ion pair for compound A-IS was m / z 513.3→217.2. Other optimized parameters were as follows: desolvation gas flow rate 1000 L / h, ion source temperature 500℃, capillary voltage 3.30 kV, and collision gas flow rate 0.25 ml·min. -1 The cone voltage is 35V; the collision voltages of compounds A and IS are 26V and 25V, respectively.

[0086] 3. Preparation methods for stock solutions, calibration standards, and quality control samples

[0087] (1) Stock solution of compound A (0.5 mg / mL): Accurately weigh the standard and dissolve it in methanol-water (1:1, v:v). Used to prepare standard curve working solutions and quality control (QC) working solutions.

[0088] (2) Standard curve working solution: Take an appropriate amount of compound A stock solution (0.5 mg / mL) and further dilute it to prepare standard curve working solutions of different concentrations (concentrations of 20, 50, 250, 500, 1000, 2500, 5000, 10000 ng / mL).

[0089] (3) Standard curve plasma sample solutions: Take a certain amount of standard curve working solution of different concentrations and dilute it with blank plasma to obtain a series of standard curve sample solutions (concentrations of 0.2, 0.5, 2.5, 5.0, 10, 25, 50 and 100 ng / ml).

[0090] (4) Quality control (QCs) working solution: Take an appropriate amount of compound A stock solution (0.5 mg / mL) and further dilute it to prepare quality control (QCs) working solutions of different concentrations (concentrations of 20, 40, 80, 8000, and 16000 ng / mL).

[0091] (5) Quality control plasma sample solutions: Take a certain amount of standard curve working solution of different concentrations, dilute with blank plasma, and prepare QC sample solutions (concentrations of 0.2, 0.4, 8.0, 80, and 160 ng / ml, respectively denoted as LLOQ, LQC, MQC, HQC, and DQC).

[0092] (6) IS stock solution (0.5 mg / ml): Accurately weigh the IS standard and dissolve it in methanol-water (1:1, v:v) to obtain the solution.

[0093] (7) IS working solution: Take an appropriate amount of IS stock solution and dilute it with methanol to obtain an IS working solution with a concentration of 5 ng / ml.

[0094] The above solutions and plasma samples were frozen and stored at -80°C.

[0095] 4. Plasma sample pretreatment methods

[0096] Add 25.0 μL of plasma sample to an EP tube, add 200 μL of IS working solution (5 ng / mL, methanol) to precipitate proteins, vortex for 1 minute, and then centrifuge at 13300 rpm for 10 minutes. Take 100 μL of the supernatant and place it in a new EP tube, add 100 μL of methanol solution, mix well, and then inject the sample.

[0097] Example 1 Selective

[0098] 1. Method

[0099] Six blank plasma samples from different individuals were collected as blank matrix. For each blank matrix, a double-blank (DB) sample and an LLOQ sample were prepared, for a total of 12 samples. The detection results of the DB samples and the LLOQ samples were compared to evaluate the selectivity of the method.

[0100] Acceptance criteria: If interference exists in DB samples at the corresponding retention times of the analyte, the area of ​​the interfering peak should not exceed 20% of that of the LLOQ samples, and the peak area of ​​the internal standard at the corresponding retention time should not exceed 5% of the average peak area of ​​the internal standard in the standard curve and quality control samples. At least five DB samples must meet the above criteria.

[0101] 2. Results

[0102] Figure 2Typical chromatograms for DB samples, DB samples containing IS, LLOQ samples, and unknown clinical samples are shown. The retention time of compound A is 0.98 min. The endogenous interference of compound A at the corresponding retention time in one DB sample is greater than 20% of the average LLOQ response value, but for other samples it does not exceed 12.0%. This meets the acceptance criteria.

[0103] Example 2 Linear Relationship

[0104] 1. Method

[0105] Standard curves were constructed using DB samples, blank samples with added IS, and eight standard curve samples at concentration levels ranging from LLOQ to ULOQ. One standard curve was prepared and analyzed at the beginning and end of each analytical batch. Linear regression was performed on the peak area ratio (y) of compound A relative to IS against the analyte concentration (x), with a weighting factor set to 1 / x. 2 .

[0106] Acceptance criteria: When the coefficient of determination R of all curves is... 2 When all values ​​are greater than 0.98, good linearity is considered. The LLOQ sample concentration calculated by reverse calculation should be between 80% and 120% of the LLOQ sample calibration value, and the reverse calculated concentration of other samples should be between 85% and 115% of the calibration concentration. At least 75% of the standard samples should meet the above standards.

[0107] 2. Results

[0108] The standard curves for all batches of samples showed good linearity in the range of 0.2–100 ng / mL. The regression coefficient (R²) 2 All values ​​were greater than 0.99. Table 1 shows the results of the inverse calculation of the concentration of the standard curve samples. The RSD was less than 10% and the RE was less than 2%, which met the acceptance criteria.

[0109] Table 1. Standard curve of compound A, calculated concentration from plasma samples.

[0110]

[0111] Example 3 Precision and Accuracy

[0112] 1. Method

[0113] Six control samples were taken at each of the LLOQ, Low Quality Control (LQC), Medium Quality Control (MQC), and High Quality Control (HQC) concentration levels to assess intra-batch and inter-batch precision and accuracy. Intra-batch precision and accuracy were assessed within a single batch, and inter-batch precision and accuracy were assessed between three consecutive batches. Precision and accuracy were assessed using relative standard deviation (RSD%) and relative error (RE%), respectively. Both RSD% and RE% should be within ±15% of the standard concentration (±20% for LLOQ).

[0114] 2. Results

[0115] Table 2 summarizes the intra-batch and inter-batch precision and accuracy. The accuracy of the quality control samples was less than or equal to 11.0%, and the inter-batch / intra-batch precision at each concentration level was less than or equal to 8.4%. The results indicate that the accuracy and precision values ​​meet the specified acceptance criteria, demonstrating that the method is reliable and reproducible for the determination of compound A.

[0116] Table 2. Intra-batch and inter-batch accuracy and precision of plasma QC samples containing compound A.

[0117]

[0118] Example 4 Extraction Recovery Rate

[0119] 1. Method

[0120] Low, medium, and high concentration quality control samples (LQC, MQC, and HQC) were prepared and extracted. The extraction recovery of compound A was evaluated by comparing the peak area of ​​the extracted QC samples with the peak area of ​​the extracted blank samples containing an equal amount of analyte.

[0121] For IS, take the IS working solution (5 ng / ml) and use the same method as for compound A to determine the extraction recovery.

[0122] The absolute value of the extraction recovery should not exceed 115%, the RSD% of the peak area at each concentration level should be less than 15%, and the RSD% of the extraction recovery at the three concentration levels should not exceed 20%.

[0123] 2. Results

[0124] Table 3 shows that the average extraction recoveries of compound A obtained from plasma samples at LQC, MQC, and HQC concentration levels were 104.0%, 100.7%, and 109.3%, respectively, with RSD% less than or equal to 4.6%. The average extraction recovery of IS was 103.6%, with an RSD% of 2.7%, which meets the requirements.

[0125] Table 3 Extraction recovery rate of compound A in plasma

[0126]

[0127] Example 5 Matrix Effect

[0128] 1. Method

[0129] The effect of different matrix sources on the assay was assessed using blank plasma from six different sources at LQC, MQC, and HQC concentration levels. The matrix factor (MF) for each compound A and internal standard was calculated by comparing the peak area in the presence of matrix (obtained by adding compound A and internal standard after blank matrix extraction) with the corresponding peak area in matrix-free samples (pure solutions of compound A and internal standard). The internal standard-normalized matrix factor (Absolute MF%) was further calculated by dividing the matrix factor of compound A (Analyte MF%) by the matrix factor of internal standard (ISMF%). The matrix effect of compound A and internal standard showed inter-individual variability of less than 15%, meeting the requirements.

[0130] Given that hemolysis and hyperlipidemia can also occur clinically, further evaluate the matrix effects of hemolyzed plasma (normal plasma with 2% fully ruptured blood cells added) and hyperlipidemic plasma (300 mg / dL). RSD% and RE% should be maintained at ≤15%.

[0131] 2. Results

[0132] The matrix effect of compound A under internal standard normalization ranged from 151.3% to 223.3%, with an RSD% of less than 2.6%. These results (Table 4-1) indicate that the matrix effect meets the requirements.

[0133] Furthermore, the matrix effect assessment results for hemolyzed plasma (normal plasma containing 2% ruptured blood cells) and hyperlipidemic plasma (300 mg / dL) are shown in Table 4-2. The RE% range for hemolyzed plasma samples was -2.3% to 1.9%, with an RSD% less than 5.2%, while the RE% range for hyperlipidemic samples was -1.5% to 6.5%, with an RSD% less than 6.1%. This suggests that the effect of the specific matrix on the determination of compound A is negligible.

[0134] Table 4-1 Matrix effect of compound A in blank plasma

[0135] Mean RSD% RE% LQC 223.3 1.8 NA MQC 176.7 2.5 NA HQC 151.3 2.6 NA

[0136] Table 4-2 Matrix effects of compound A in hemolytic or hyperlipidemic plasma

[0137]

[0138] Note: RE% represents the deviation between the detected concentration of hemolytic plasma sample or hyperlipidemic plasma sample and the theoretical concentration of blank plasma sample.

[0139] Example 6 Stability

[0140] 1. Method

[0141] The stability of analytes was assessed by analyzing LQC (n=6) and HQC (n=6) samples under various storage, processing, and analytical conditions. The stability of stock solutions was evaluated by comparing peak areas of freshly prepared solutions and stock solutions (8 hours at room temperature and 217 days at -30°C). The stability of analytes in plasma was tested under different conditions. To assess short-term and long-term stability, samples were placed at room temperature for 15 hours, stored at -20°C for 32 days, and stored at -80°C for 378 days, respectively. Extracted samples were placed in an autosampler (15°C) for 48 hours and then analyzed using a freshly prepared standard curve to assess the stability of the autosampler. Additionally, analyzed samples were stored in a freezer (4°C) for 48 hours and then re-injected to assess the stability of repeated injections. For freeze-thaw stability, samples were frozen at -80°C for more than 12 hours and then thawed at room temperature for at least 2 hours, for a total of 5 cycles.

[0142] For whole blood stability assessment, samples at LQC and 1 / 4 HQC concentrations were prepared using freshly collected whole blood and then divided into two groups (Group A and Group B). Group A plasma samples were obtained by immediate centrifugation of the QC samples, while Group B samples were centrifuged after being left at room temperature for 2 hours. The average ratio of the analyte peak area to the IS peak area for each concentration in Group A was used as the indicative value. The average value for Group B must be within ±15% of the average value for Group A, and the RSD% must not exceed 15%.

[0143] 2. Results

[0144] This experiment investigated the stability of compound A in solution and plasma under different storage and handling conditions during routine analysis. Table 5 presents the stability test results.

[0145] The results showed that storing the stock solution samples at room temperature for 8 hours and at -30°C for 217 days did not affect the stability of compounds A and IS.

[0146] Plasma samples maintained good stability after being stored at room temperature for 15 hours, at -20°C for 32 days, and at -80°C for 378 days, and remained stable even after five freeze-thaw cycles from -80°C to room temperature. Plasma samples stored at 4°C for 48 hours showed reproducible results upon repeated testing and remained stable at 15°C for 48 hours. Whole blood samples stored at room temperature for 2 hours before centrifugation exhibited good stability.

[0147] Table 5 Stability verification results

[0148]

[0149]

[0150] Example 7: Dilution Reliability

[0151] 1. Method

[0152] The reliability of dilution was assessed to determine whether samples with concentrations above the standard curve range could be diluted and accurately measured. Prior to analysis, the high-concentration quality control samples were diluted 10-fold with a blank matrix to obtain DQC samples (160 ng / ml, n=6). The RE% and RSD% of the six diluted quality control samples should be within ±15%.

[0153] 2. Results

[0154] After diluting plasma samples 10-fold to 160 ng / mL, the RE% and RSD% of the six diluted quality control samples were 5.3% and 3.5%, respectively. These results indicate that samples with concentrations higher than ULOQ can be reliably diluted 10-fold.

[0155] Table 6 Dilution reliability results

[0156]

[0157] Example 8 Residual Effect

[0158] 1. Method

[0159] After injecting the ULOQ sample, the DB sample is then analyzed to assess residual effects. The peak response of compound A in the DB sample must be less than 20% of that in the LLOQ sample, and the peak response of IS in the DB sample must be less than 5% of the average of all IS peak responses.

[0160] 2. Results

[0161] After testing the ULOQ samples, the peak areas of compounds A and IS in the DB plasma samples were no more than 17.6% and 0.1% of the peak area of ​​LLOQ, respectively, indicating that neither compound A nor IS had a significant residual effect.

[0162] Table 7 Residual effects during batch sample testing

[0163]

[0164] Example 9 Pharmacokinetic Application

[0165] The method of this invention was used in a phase I clinical study to investigate the plasma pharmacokinetics of compound A in Chinese patients with advanced solid tumors. Subjects were randomized to four groups and received single oral doses of 1, 2, 4, and 6 mg of compound A. The study is ongoing, and to date, 180 plasma samples have been successfully analyzed using the method of this invention. The mean concentration-time curves for single oral doses of 1, 2, 4, and 6 mg of compound A are shown below. Figure 3 As shown.

[0166] Optimization of mass spectrometry conditions in Screening Example 1

[0167] The parameters of the Xevo TQS triple quadrupole mass spectrometer (Waters, MA, USA) were tuned and optimized, and the positive ion mode of the electrospray ionization (ESI) source was selected. The desolvation gas flow rate, ion source temperature, capillary voltage, collision gas flow rate, and cone voltage were adjusted. After collision voltage detection using multiple reaction monitoring (MRM), the following conditions were finally determined: the quantitative ion pair of compound A was m / z 510.3→217.2; the quantitative ion of compound A-IS was m / z 513.3→217.2. Other optimized parameters were as follows: desolvation gas flow rate 1000 L / h, ion source temperature 500℃, capillary voltage 3.30 kV, and collision gas flow rate 0.25 ml·min. -1 The cone voltage is 35V; the collision voltages of compounds A and IS are 26V and 25V, respectively.

[0168] Screening Example 2: Column Screening Test

[0169] Referring to "2. Chromatographic and Mass Spectrometry Conditions", different types of chromatographic columns were selected, and method parameters such as mobile phase type, gradient conditions, and flow rate were comprehensively adjusted based on the detection results. The detection performance of the test sample solution in different types of chromatographic columns was investigated. Specific information about the chromatographic columns is as follows:

[0170] Table 8 Chromatographic Column Information

[0171] model Specification XBridgepheyl C18 column 2.0×50mm, 3.5μm Phenomenex Gemini 5u C18 110A column 2.0×50mm, 5μm ACQUITY UPLC BEH C18 column 2.1×50mm, 1.7μm

[0172] The test results are shown in Figure 4 The results showed that the Phenomenex Gemini 5u C18 110A column (2.0 × 50 mm, 5 μm) performed well in detection.

[0173] Screening Example 3: Screening Tests of Different Mobile Phase Components

[0174] 1. Screening of mobile phase A (organic phase)

[0175] Referring to "2. Chromatographic and Mass Spectrometry Conditions", mobile phase B (aqueous phase) was selected as 0.2% formic acid aqueous solution, and mobile phase A (organic phase) was selected as acetonitrile and methanol for detection, respectively. Chromatograms were recorded.

[0176] See results Figure 5 .Depend on Figure 5 It can be seen that acetonitrile as mobile phase A (organic phase) has a better elution effect and very low background noise, while methanol as mobile phase A (organic phase) has a poorer retention effect.

[0177] 2. Screening of mobile phase B (aqueous phase)

[0178] Referring to "2. Chromatographic and Mass Spectrometry Conditions", acetonitrile was selected as the mobile phase A (organic phase), and 2 mM ammonium acetate solution, 5 mM ammonium acetate solution, 0.1% formic acid aqueous solution, 0.2% formic acid aqueous solution, and 0.5% formic acid aqueous solution were selected as the mobile phase B for detection, and the chromatograms were recorded.

[0179] See results Figure 6 It is evident that the ammonium acetate solution system exhibits significant tailing in mobile phase B, while the formic acid aqueous solution system shows better chromatographic peaks. The detection response using 0.2% formic acid aqueous solution as mobile phase B is higher than that of 0.1% formic acid aqueous solution, but not significantly different from that of 0.5% formic acid aqueous solution. Therefore, 0.2% formic acid aqueous solution is selected as mobile phase B.

[0180] Screening Example 4: Screening of Plasma Sample Processing Methods

[0181] Referring to "4. Plasma Sample Pretreatment Methods", acetonitrile was used as the protein precipitation reagent to prepare plasma samples, and "2. Chromatographic and Mass Spectrometry Conditions" were used for detection.

[0182] See results Figure 7 As can be seen, when acetonitrile is used as a precipitant, the chromatographic peaks show two peaks, indicating that acetonitrile is not suitable for processing plasma samples in this invention.

[0183] Screening Example 5: Flow Rate Screening Test

[0184] Referring to "2. Chromatographic and Mass Spectrometry Conditions", the effect of flow rate on the detection effect was investigated. Detection was performed at flow rates of 0.3 ml / min and 0.4 ml / min, with all other experimental conditions remaining the same.

[0185] See results Figure 8 The results showed that at a flow rate of 0.4 ml / min, the peak elution time was 0.5 min, and the column retention was poor; at a flow rate of 0.3 ml / min, the peak elution time was 1 min, and the retention was better. Therefore, the preferred flow rate was 0.3 ml / min.

[0186] Screening Example 6: Column Temperature Screening Test

[0187] Referring to "2. Chromatographic and Mass Spectrometry Conditions", the effect of column temperature on the detection effect was investigated. Detection was performed at column temperatures of 35℃ and 45℃, with all other experimental conditions remaining the same.

[0188] See results Figure 9 The results showed that the column temperature level had little effect on the peak shape and response, with only a slight difference in retention time.

[0189] Preparation Example 1: Preparation of Compound A and Internal Standard D3-Compound A

[0190] 1. Preparation of compound A

[0191] 1.1 Preparation of compound I

[0192] Compound I was prepared according to the method described in Example 10 of patent WO2017140269A1.

[0193] 1.2 Preparation of compound A (compound I dihydrochloride)

[0194] Compound I (0.52 g) and 25 mL of methanol were added to a reaction flask and stirred until the system was evenly dispersed. Then, 1.2 mL of 2 mol / L hydrochloric acid-methanol solution was added dropwise while stirring, and the mixture was stirred until homogeneous. Crystallization was allowed to occur for 5 h. The resulting solid was filtered, dried under vacuum at 30 °C for 12 h to constant weight, and then dried under vacuum at 45 °C until the solvent residue was within acceptable limits, yielding compound A (0.53 g).

[0195] Ion chromatography results showed that the chloride ion content in the sample of compound A was 11.7%, while the theoretical chloride ion content of compound I dihydrochloride was 12.2%, indicating that the obtained compound A was compound I dihydrochloride.

[0196] 2. Preparation of internal standard D3-compound A

[0197] Using conventional deuteration processes in the art, and referring to the preparation method of compound A, D3-compound A was obtained.

[0198] In summary, this invention presents, for the first time, a rapid, reliable, and highly sensitive LC-MS / MS method for the quantitative detection of compound I or its pharmaceutically acceptable salt (e.g., dihydrochloride, compound A) in human plasma. This method ensures an economical and rapid sample preparation process, exhibits a broad linear range, and a low LLOQ. Method validation results demonstrate good stability and high extraction recovery, making it a reliable method for determining the concentration of compound A in plasma samples. The LC-MS / MS detection method described in this invention can be used to support further clinical trials of compound A.

Claims

1. A method for detecting compound I or its pharmaceutically acceptable salt in biological samples, characterized in that: The concentration of compound I or its pharmaceutically acceptable salt in the sample was determined using LC-MC / MC method. The structure of compound I is shown in formula (I): （I） Using compound D3-I or its pharmaceutically acceptable salt as the internal standard (IS), the structure of compound D3-I is shown below: (D3-Compound I); The chromatographic conditions are as follows: Chromatographic column: C18 column, 2.0 × 50 mm, 5 µm; Mobile phase A: Acetonitrile; Mobile phase B: 0.1%-0.5% formic acid aqueous solution; Gradient procedure: Injection volume: 3-10 μL; Flow rate: 0.2-0.4 ml / min; Column temperature: 35-45℃; The biological sample is a plasma sample.

2. The detection method according to claim 1, characterized in that, The pharmaceutically acceptable salt of compound I is a dihydrochloride, with the structure shown in compound A below: (Compound A) Using compound A (D3-) as an internal standard (IS), its structure is shown below: (D3-Compound A).

3. The detection method according to claim 1, characterized in that, The gradient procedure is as follows: 。 4. The detection method according to claim 1, characterized in that, Mobile phase B is a 0.2%-0.5% formic acid aqueous solution.

5. The detection method according to claim 1, characterized in that, Mobile phase B is a 0.2% formic acid aqueous solution.

6. The detection method according to claim 1, characterized in that, The chromatographic column was a Phenomenex Gemini 5u C18110A column.

7. The detection method according to claim 1, characterized in that, Injection volume: 5-10 μL.

8. The detection method according to claim 1, characterized in that, Injection volume: 5 μL.

9. The detection method according to claim 1, characterized in that, Flow rate 0.3-0.4 ml / min.

10. The detection method according to claim 1, characterized in that, Flow rate: 0.3 ml / min.

11. The detection method according to claim 1, characterized in that, Column temperature 38-42℃.

12. The detection method according to claim 1, characterized in that, Column temperature 40℃.

13. The detection method according to any one of claims 1-12, characterized in that, The mass spectrometry conditions are as follows: Mass spectrometry analysis was performed using a Xevo TQS triple quadrupole mass spectrometer with positive ion mode of the electrospray ionization (ESI) source. Multiple reaction monitoring (MRM) scan was used for detection. The quantitative ion pair for compound A was m / z 510.3→217.2, and the quantitative ion pair for compound A-IS was m / z 513.3→217.

2.

14. The detection method according to any one of claims 1-12, characterized in that, The chromatographic conditions were achieved using a UPLC system for chromatographic separation.

15. The detection method according to any one of claims 1-12, characterized in that, The chromatographic conditions were performed using an ACQUITY UPLC system for chromatographic separation.

16. The detection method according to any one of claims 1-12, characterized in that, The chromatographic conditions used an injection method selected from either Full Loop or Partial Loop.

17. The detection method according to claim 13, characterized in that, The mass spectrometry conditions also include the following parameters: desolvation gas flow rate 1000 L / h, ion source temperature 500 °C; capillary voltage 3.30 kV, collision gas flow rate 0.25 ml·min. -1 The cone voltage is 35V; the collision voltages of compounds A and IS are 26V and 25V, respectively.

18. The detection method according to any one of claims 1-12, characterized in that, This includes plasma sample pretreatment steps.

19. The detection method according to any one of claims 1-12, characterized in that, This includes preparing one or more of the following: a stock solution of compound A, a working solution for a standard curve, a plasma sample solution for a standard curve, an IS stock solution, an IS working solution, a quality control working solution, and a quality control plasma sample solution.

20. Use of D3-compound I or a pharmaceutically acceptable salt thereof as an internal standard compound in the detection method according to any one of claims 1-19, wherein the structure of D3-compound I is as follows: 。 21. The use according to claim 20, wherein the pharmaceutically acceptable salt of the D3-compound I is a dihydrochloride salt with the following structure: 。

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