A method for quantitatively analyzing drugs in plasma samples by using a high performance liquid chromatography-tandem mass spectrometry device

Abstract

The present invention provides a method for quantitatively analyzing SYHA1807 by using a high performance liquid chromatography-tandem mass spectrometry device, and the method comprises the following steps: 1) preparing a sample solution containing SYHA1807; 2) injecting the sample solution into the high performance liquid chromatography-tandem mass spectrometry device to obtain a mass chromatogram; 3) quantitatively analyzing SYHA1807 by using the internal standard method according to the mass chromatogram, wherein in the high performance liquid chromatography, gradient elution is carried out by using a mixed mobile phase composed of mobile phase A and mobile phase B, wherein the mobile phase A is selected from: acetonitrile, an acetonitrile solution containing 0.05% to 0.5% by mass of formic acid, and a water-acetonitrile mixed solution containing 0.05% to 0.5% by mass of formic acid and 1 mmol / L to 5 mmol / L of ammonium acetate, and the mobile phase B is selected from: an aqueous solution containing 0.05% to 0.5% by mass of formic acid, an aqueous solution containing 1 mmol / L to 5 mmol / L of ammonium acetate, and an acetonitrile-water mixed solution containing 0.05% to 0.5% by mass of formic acid and 1 mmol / L to 5 mmol / L of ammonium acetate.

Description

Technical Field

[0001] The present invention belongs to the fields of biochemistry and pharmaceutical analytical chemistry. Specifically, it relates to a method for quantitatively analyzing SYHA1807 using a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) device. Background Art

[0002] Liquid chromatography-mass spectrometry (LC-MS) analysis is mainly applied in the fields of drug metabolism, pharmacokinetics, clinical pharmacology, natural drug development, etc. It has the advantages of high sensitivity, high specificity, good reproducibility, accurate quantification, wide linear range, simple data processing, etc. High performance liquid chromatography can effectively separate the components of the analyte, while mass spectrometry can analyze each separated component one by one. LC-MS analysis utilizes the differences in the distribution and adsorption coefficients of the sample components between the mobile phase and the stationary phase in the chromatographic column. The sample is carried into the chromatographic column by the mobile phase for separation, and then through the interface device, different ion fragments have different movement behaviors in different electric and / or magnetic fields. The mass analyzer separates the ions according to the mass-to-charge ratio (m / z) to obtain a mass spectrum arranged in the order of mass. Through the analysis and processing of the mass spectrum, qualitative and quantitative analysis results of the sample can be obtained.

[0003] SYHA1807 is a novel small molecule kinase inhibitor of lysine-specific demethylase 1 (LSD1, also known as KDM1A). Currently, there is no similar product on the market globally. It can target the characteristics of small cell lung cancer with low differentiation and neuroendocrine properties, promote the differentiation of cancer stem cells through epigenetic means, down-regulate the expression of neuroendocrine-related genes, inhibit tumor growth, and at the same time enhance the sensitivity of tumors to chemotherapy. Preclinical studies have shown that this product has excellent anti-tumor activity in vitro and in vivo and good safety, and is very promising to show good therapeutic effects for small cell lung cancer in clinical studies.

[0004] Therefore, there is an urgent need to develop a rapid, specific, highly sensitive and highly stable quantitative analysis method to quantitatively determine SYHA1807. Summary of the Invention

[0005] The purpose of the present invention is to provide a high performance liquid chromatography-tandem mass spectrometry analysis method for quantitatively analyzing SYHA1807, enabling accurate quantification of SYHA1807 to meet the requirements of clinical pharmacokinetic studies.

[0006] To achieve the above-mentioned invention purpose, the present invention provides a method for quantitatively analyzing SYHA1807 using a high performance liquid chromatography-tandem mass spectrometry device, and the method includes the following steps:

[0007] 1) Prepare a sample solution containing SYHA1807;

[0008] 2) Inject the sample solution into a high performance liquid chromatography-tandem mass spectrometry device to obtain a mass chromatogram;

[0009] 3) Use the internal standard method to quantitatively analyze SYHA1807 according to the mass chromatogram,

[0010] wherein, in the high performance liquid chromatography, a mixed mobile phase composed of mobile phase A and mobile phase B is used for gradient elution,

[0011] wherein mobile phase A is selected from: acetonitrile, an acetonitrile solution containing 0.05% to 0.5% by mass of formic acid, and a water-acetonitrile mixture containing 0.05% to 0.5% by mass of formic acid and 1 mmol / L to 5 mmol / L of ammonium acetate, wherein the volume ratio of water to acetonitrile in the water-acetonitrile mixture is in the range of 1:99 to 10:90, and

[0012] mobile phase B is selected from: an aqueous solution containing 0.05% to 0.5% by mass of formic acid, an aqueous solution containing 1 mmol / L to 5 mmol / L of ammonium acetate, and an acetonitrile-water mixture containing 0.05% to 0.5% by mass of formic acid and 1 mmol / L to 5 mmol / L of ammonium acetate, wherein the volume ratio of acetonitrile to water in the acetonitrile-water mixture is in the range of 1:99 to 10:90.

[0013] In some embodiments, mobile phase A is a water-acetonitrile mixture with a volume ratio of 5:95 containing 0.1% by mass of formic acid and 2 mmol / L of ammonium acetate; and / or mobile phase B is an acetonitrile-water mixture with a volume ratio of 5:95 containing 0.1% by mass of formic acid and 2 mmol / L of ammonium acetate.

[0014] In some embodiments, the gradient elution program is as follows:

[0015] From 0.00 minute to 1.00 minute, 8% to 12% by volume of mobile phase A;

[0016] From 1.01 minute to 1.99 minutes, 93% to 97% by volume of mobile phase A;

[0017] From 2.00 minutes to 3.00 minutes, 8% to 12% by volume of mobile phase A,

[0018] wherein in the mixed mobile phase, the total amount of mobile phase A and mobile phase B is 100% by volume.

[0019] Preferably, the gradient elution program is as follows:

[0020] From 0.00 minute to 1.00 minute, 10% by volume of mobile phase A;

[0021] From 1.01 minutes to 1.99 minutes, 95% by volume of mobile phase A;

[0022] From 2.00 minutes to 3.00 minutes, 10% by volume of mobile phase A,

[0023] wherein in the mixed mobile phase, the total amount of mobile phase A and mobile phase B is 100% by volume.

[0024] In some embodiments, the chromatographic column in the high performance liquid chromatography is a reverse phase chromatographic column, preferably a chromatographic column packed with octadecylsilyl-bonded silica gel. Preferably, the chromatographic column is an ACQUITY UPLC® BEH C18 chromatographic column (2.1×50 mm, 1.7 µm).

[0025] In some embodiments, the internal standard reference substance used is D5-SYHA1807 (deuterated SYHA1807).

[0026] In some embodiments, the preparation of the sample solution containing SYHA1807 includes: dissolving SYHA1807 in dimethyl sulfoxide to obtain a SYHA1807 stock solution, and then diluting the SYHA1807 stock solution with a diluent composed of water and acetonitrile. Preferably, the volume ratio of water to acetonitrile in the diluent ranges from 20:80 to 80:20, more preferably 50:50.

[0027] In some embodiments, SYHA1807 is from the plasma of a subject.

[0028] In some embodiments, the preparation of the sample solution containing SYHA1807 includes: adding an internal standard working solution to a plasma sample of a subject, shaking the resulting mixture, then centrifuging, taking the supernatant for drying to obtain a solid sample, and then dissolving and mixing the solid sample with a complex solution composed of 0.1% by mass aqueous formic acid solution and acetonitrile. Preferably, the volume ratio of acetonitrile to 0.1% by mass aqueous formic acid solution in the complex solution ranges from 5:95 to 20:80, more preferably 10:90.

[0029] Preferably, the internal standard working solution is prepared by: dissolving the internal standard reference substance D5-SYHA1807 in dimethyl sulfoxide to obtain an internal standard stock solution, and then diluting the internal standard stock solution with acetonitrile.

[0030] Preferably, the volume ratio of the plasma sample to the internal standard working solution ranges from 1:2 to 1:4, preferably 1:3.

[0031] Preferably, the volume ratio of the supernatant to the reconstitution solution ranges from 2:1 to 5:1, preferably from 3:1 to 4:1, and more preferably is 3.5:1.

[0032] In some embodiments, the injection volume of the sample solution is 5 to 20 μL, preferably 5 to 10 μL, and more preferably 10 μL.

[0033] In some embodiments, the flow rate of the mixed mobile phase is 0.2 to 0.5 mL / min, preferably 0.3 to 0.5 mL / min, and more preferably 0.4 mL / min.

[0034] In some embodiments, the column temperature in the high performance liquid chromatography is 35 to 45 °C, preferably 38 to 42 °C, and more preferably 40 °C.

[0035] In some embodiments, the detection method further includes preparing one or more of SYHA1807 stock solution, standard curve working solution, standard curve plasma sample solution, internal standard stock solution, internal standard working solution, quality control working solution, and quality control plasma sample solution.

[0036] In some embodiments, the preparation method of the SYHA1807 stock solution is: weighing the SYHA1807 standard product and dissolving it with an acetonitrile-water mixture (the volume ratio is preferably 1:1). In some embodiments, the concentration of the sample stock solution is 0.5 mg / mL.

[0037] In some embodiments, the preparation method of the standard curve working solution is: taking an appropriate amount of the SYHA1807 stock solution and further diluting it with a diluent composed of water and acetonitrile (where the volume ratio of water to acetonitrile ranges from 20:80 to 80:20, and more preferably is 50:50) to prepare standard curve working solutions with different concentrations. In a preferred embodiment, the concentrations of the standard curve working solutions are 1, 2.5, 5, 25, 50, 250, 500, 1000 ng / mL.

[0038] In some embodiments, the preparation method of the standard curve plasma sample solution is: respectively taking standard curve working solutions with different concentrations and diluting them with blank plasma to finally obtain a series of standard curve plasma sample solutions. In a preferred embodiment, the concentrations of the standard curve plasma sample solutions are 10, 25, 50, 250, 500, 25, 2500, 5000, and 10000 pg / mL.

[0039] In some embodiments, the method for preparing the quality control working solution is as follows: Take an appropriate amount of the SYHA1807 stock solution and further dilute it with a diluent composed of water and acetonitrile (where the volume ratio of water to acetonitrile ranges from 20:80 to 80:20, more preferably 50:50) to prepare quality control working solutions of different concentrations. In a preferred embodiment, the concentrations of the quality control working solutions are 1, 2, 80, 800 ng / mL.

[0040] In some embodiments, the method for preparing the quality control plasma sample solution is as follows: Take standard curve working solutions of different concentrations and dilute them with blank plasma to prepare quality control sample solutions. In a preferred embodiment, the concentrations of the quality control plasma sample solutions are 10, 20, 800, 8000, 40000 pg / mL.

[0041] In some embodiments, the method for preparing the internal standard stock solution is as follows: Weigh the internal standard standard and dissolve it using dimethyl sulfoxide. In a preferred embodiment, the concentration of the internal standard stock solution is 0.5 mg / mL.

[0042] In some embodiments, the method for preparing the internal standard working solution is as follows: Take an appropriate amount of the internal standard stock solution and dilute it with acetonitrile. In some embodiments, the concentration of the internal standard working solution is 0.5 ng / mL.

[0043] For the sake of providing a more concise description, the term "about" is not used for the quantitative data in this article. It should be understood that whether the term "about" is explicitly used or not, each value given here not only includes the actually given value (the given value), but also means including the approximation of such a given value reasonably inferred by those of ordinary skill in the art, including the equivalent values and approximations of such a given value due to experimental and / or measurement conditions. The approximation is preferably ±20%, ±15%, ±10%, ±8%, ±6%, ±5%, ±4%, ±3%, 2%, ±1% based on the given value.

[0044] In some embodiments, the numerical ranges and numerical parameters that set forth the broad scope of some embodiments of the present invention are approximations, and the numerical parameters should be interpreted in accordance with the number of significant digits reported and by applying ordinary rounding techniques. Although the numerical values presented in the specific examples are reported as precisely as possible, the numerical values presented in some embodiments of the present invention may contain certain errors, which are inevitably caused by the standard deviation in test measurements.

[0045] The present invention has achieved the following beneficial technical effects:

[0046] (1)The present invention first established a rapid, stable, and sensitive UPLC–MS / MS method for the determination of compound SYHA1807 in human plasma. The chemical structure of compound SYHA1807 contains nitrogen atoms, and the positive ionization mode of the ESI source used in the present invention has better ionization performance and stable response. In addition, the present invention optimized the source / gas and compound parameters (including capillary voltage, cone voltage, etc.) to obtain the most suitable ionization conditions.

[0047] (2)Due to the presence of a benzene ring in the chemical structure of compound SYHA1807, reversed-phase chromatography is preferred. The inventors explored the separation effects of various chromatographic columns. Compared with other chromatographic columns, ACQUITY UPLC BEH C18 (2.1×50 mm, 1.7 µm) has sufficient retention and a stable chromatographic peak shape, and the inventors selected this chromatographic column for separation. The present invention also screened the mobile phase and found that using acetonitrile as the organic phase has better elution effect and very low background noise. Adding appropriate amounts of formic acid, ammonium acetate, and acetonitrile to the aqueous phase can increase the response. The present invention can complete gradient elution within 3 minutes, with short time consumption, and obtain good chromatographic peak shapes and high sensitivity.

[0048] (3)When preparing plasma samples, the inventors first used the liquid-liquid extraction method, but this method can cause chemical contamination and low sensitivity. The inventors also tried the solid-phase extraction method, but due to the complex operation, this method consumes a lot of time and cost. For the protein precipitation method, although generally this method may have matrix effects that affect the accuracy of determination; but finally, the methodological verification showed that under the detection conditions described in the present invention, when using the protein precipitation method to prepare plasma samples, the matrix effect can be ignored, clear chromatographic peaks are obtained, and it has good reproducibility. Description of the Drawings

[0049] Figure 1A Shows the representative mass chromatogram of a representative double blank plasma sample (Double Blank, DB) measured under the conditions of the present invention.

[0050] Figure 1B Shows the representative mass chromatogram of the lower limit of quantification (LLOQ) sample measured under the conditions of the present invention.

[0051] Figure 2 Shows the typical standard curve of SYHA1807 under the conditions of the present invention.

[0052] Figure 3 Shows the representative plasma concentration-time curve of a small cell lung cancer patient after oral administration of 6 mg SYHA1807. Detailed Description of the Invention

[0053] 1. Chemicals and Reagents

[0054] Reference standards of compound SYHA1807 (purity 99.6%) and compound D5 - SYHA1807 (deuterated internal standard, purity 95.7%) were provided by CSPC Pharmaceutical Group (Shijiazhuang, China).

[0055] HPLC - grade acetonitrile was purchased from Honeywell Burdick & Jackson (ML, USA).

[0056] Analytical - grade formic acid was purchased from Sigma - Aldrich chemicals (MO, USA).

[0057] Analytical - grade dimethyl sulfoxide was purchased from Beijing Chemical Reagent Company (Beijing, China).

[0058] Analytical - grade ammonium acetate was purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China).

[0059] Blank plasma was provided by Peking Union Medical College Hospital (Beijing, China).

[0060] Deionized water was purified by a Milli - Q system (Millipore, Bedford, MA, USA).

[0061] 2. Chromatographic and Mass Spectrometric Conditions

[0062] 2.1 Chromatographic Conditions

[0063] Chromatographic separation was performed using an ACQUITY UPLC system (Waters, MA, USA).

[0064] Chromatographic column: ACQUITY UPLC BEH® C18 column (2.1×50 mm, 1.7 µm).

[0065] Mobile phase A: A solution of water - acetonitrile (volume ratio 5:95) containing 0.1 mass% formic acid and 2 mmol / L ammonium acetate;

[0066] Mobile phase B: A solution of acetonitrile - water (volume ratio 5:95) containing 0.1 mass% formic acid and 2 mmol / L ammonium acetate

[0067] Table 1: Gradient Elution Program

[0068]

[0069] Column temperature: 40 °C

[0070] Autosampler temperature: 10 °C

[0071] Injection volume: 10 μL

[0072] Flow rate: 0.4 mL / min

[0073] To reduce residue, the syringe and injection valve of the autosampler were cleaned with acetonitrile - aqueous solution with a volume ratio of 90:10 (strong wash) and acetonitrile - aqueous solution with a volume ratio of 10:90 (weak wash).

[0074] 2.2 Mass spectrometry conditions

[0075] Mass spectrometry analysis was performed using an Xevo TQS triple - quadrupole mass spectrometer (Waters, MA, USA), and the positive ion mode of the electrospray ionization (ESI) source was selected.

[0076] For multiple reaction monitoring (MRM) scan detection, the quantitative ion pair of compound SYHA1807: m / z 353.2→228.2; the quantitative ion of compound D5 - SYHA1807 (internal standard) is: m / z 358.2→233.2.

[0077] Other optimized parameters are shown in Table 2 below:

[0078] Table 2: Tandem mass spectrometry parameters

[0079]

[0080] 3. Preparation methods of stock solutions, calibration standards and quality control samples

[0081] (1) Compound SYHA1807 stock solution (0.5 mg / mL): Accurately weigh 6.047 mg of SYHA1807 standard powder, transfer it to a 10 mL volumetric flask, add an appropriate amount of dimethyl sulfoxide (DMSO) to dissolve and make up to the mark to obtain a 0.5 mg / mL stock solution. It is used to prepare the standard curve working solution and quality control (QCs) working solution.

[0082] (2) Standard curve working solution: According to the following table, use a water:acetonitrile mixture (volume ratio 50:50) and the standard stock solution to prepare SYHA1807 standard curve working solutions (concentrations are 1, 2.5, 5, 25, 50, 250, 500 and 1000 ng / mL) respectively.

[0083] Table 3: Preparation of standard curve working solution

[0084]

[0085] WS9*: Only for preparing working solutions

[0086] (3) Standard curve plasma sample solution: According to the following table, prepare the standard curve (concentrations are 0.0100, 0.0250, 0.0500, 0.250, 0.500, 2.50, 5.00, and 10.0 ng / mL respectively) using blank plasma.

[0087] Table 4: Preparation of standard curve plasma sample solution

[0088]

[0089] (4) Quality control (QCs) working solution: According to the following table, prepare the SYHA1807 quality control working solution (concentrations are 1, 2, 80, 800, 4000 ng / mL respectively) using a water:acetonitrile mixture (volume ratio 50:50).

[0090] Table 5: Preparation of quality control working solution

[0091]

[0092] WQ4*: Only for preparing working solution

[0093] (5) Quality control plasma sample solution: According to the following table, prepare quality control samples using blank plasma, with concentrations of 0.0100, 0.0200, 0.800, 8.00, and 40.0 ng / mL respectively, denoted as the lower limit of quantification (LLOQ), low concentration quality control (LQC), medium concentration quality control (MQC), high concentration quality control (HQC), and dilution quality control (DQC).

[0094] Table 6: Preparation of quality control plasma sample solution

[0095]

[0096] (6) Internal standard stock solution (0.5 mg / mL): Weigh 6.290 mg of the internal standard SYHA1807-D5 standard, transfer it to a 10 mL volumetric flask, add an appropriate amount of DMSO to dissolve and then make up to the mark to obtain a 0.5 mg / mL mother liquor, which is dissolved using dimethyl sulfoxide (DMSO).

[0097] (7) Internal standard working solution: Take 10 μL of the internal standard stock solution and dilute it with acetonitrile to obtain an internal standard working solution with a concentration of 0.5 ng / mL.

[0098] The above solutions and plasma samples are stored frozen at a temperature of -80 °C.

[0099] 4. Plasma sample pretreatment method

[0100] Take 100 µL of plasma sample and add it to an EP tube. Add 300 µL of internal standard solution (0.5 ng / mL, with acetonitrile as the solvent) to precipitate proteins. Oscillate for 1 minute, and then centrifuge at 13300 rpm for 10 minutes. Take 350 µL of the supernatant and place it in a new EP tube. Dry it under nitrogen at room temperature, add 100 µL of reconstitution solution (acetonitrile: 0.1% formic acid aqueous solution = 1:9, v:v) for reconstitution, mix well and then inject the sample.

[0101] Example 1: Selectivity

[0102] 1. Method

[0103] Take blank matrices of human plasma samples from six different individuals. Prepare 1 double blank plasma sample (Double Blank, DB) and 1 lower limit of quantitation (LLOQ) sample for each blank matrix from each source, for a total of 12 samples. Compare the detection results of the double blank plasma samples (DB) obtained by the method of the present invention with the lower limit of quantitation (LLOQ) samples to evaluate the selectivity of the method.

[0104] Acceptance criteria: If there is a certain interference at the corresponding retention time of the analyte in the blank sample, this interference should not be greater than 20% of the average peak area of the analyte in the LLOQ samples; when quantifying by the internal standard method, the chromatographic peak area at the corresponding retention time of the internal standard should not be greater than 5% of the average peak area of the internal standard in the standard curve and quality control samples.

[0105] 2. Results

[0106] The method of the present invention meets the acceptance criteria, and the results are shown in Table 7 below.

[0107] Table 7: Selectivity investigation

[0108]

[0109] According to the selected LC-MS / MS conditions, the mass chromatograms of 6 double blank plasma samples (DB) from different sources were measured; a representative figure is shown in Figure 1A . As can be seen from the figure, there are no endogenous interference peaks of the internal standard in human plasma, so the plasma matrix has no interference on the separation and determination of the analyte and the internal standard under the experimental conditions. A representative LLOQ chromatogram is shown in Figure 1B .

[0110] Example 2: Linear relationship

[0111] 1. Method

[0112] Process and measure 2 sets of standard curve samples containing 8 concentration points, which are placed at the beginning and end of the analysis batch respectively. Use the ratio of the peak area of SYHA1807 to the internal standard as Y and the concentration as X for fitting. Weighting coefficient 1 / X2 , the curve regression equation of the standard curve is Y = aX + b. Each analytical batch includes 1 double blank sample (DB) and 1 blank sample (BK) to ensure that the blank matrix and the added reagents do not interfere with the detection of the compound.

[0113] Acceptance criteria: Good linearity, r ≥ 0.990 or r 2 ≥ 0.980; the concentration recalculated from the calibration standard should be within 85% - 115% of the labeled value, and within 80% - 120% at the lower limit of quantification. The determination accuracy of at least 75% of the calibration standards (at least 6 valid concentrations) should meet the above requirements; for the calibration standards at the same concentration point, at least 50% should meet the above requirements.

[0114] 2. Results

[0115] The ratio of the concentration of all batches of samples within the range of 0.01 - 10.0 ng / mL to the peak area has a good linear relationship. Table 8 shows the results of the concentration recalculated from the plasma samples of the standard curve.

[0116] Table 8: Recalculated Concentration of the Standard Curve

[0117]

[0118] %CV: Precision (Coefficient of Variation)

[0119] %Bias: Accuracy Deviation

[0120] During the method validation, a total of 20 standard curves were prepared, and the R 2 values were all between 0.990 and 1.000, as shown in Table 9. A typical standard curve graph is shown in Figure 2 .

[0121] Table 9: Summary of Standard Curve Parameters

[0122]

[0123] Example 3: Precision and Accuracy

[0124] 1. Method

[0125] For each batch in precision and accuracy verification, independently prepared and freshly prepared standard curves and quality control (QC) samples should be used; precision and accuracy should not fail in two consecutive analytical batches, and should pass at least two consecutive analytical batches, and be carried out on at least two days. At the lower limit of quantitation (LLOQ), low concentration quality control (LQC), medium concentration quality control (MQC), and high concentration quality control (HQC) concentration levels, six quality control samples are taken each to evaluate within-batch and between-batch precision and accuracy. Evaluate within-batch precision and accuracy in a single batch, and evaluate between-batch precision and accuracy among three consecutive batches.

[0126] Within-batch / between-batch accuracy acceptance criteria: The mean of the measured values of quality control samples at three concentration levels of low, medium, and high should be within the range of 85% - 115% of their labeled values, and for 6 quality control samples at each concentration level, at least 67% of the measured values of the samples should be within the range of 85% - 115% of their labeled values. For LLOQ samples, the mean of the measured values should be within the range of 80% - 120% of their labeled values, and at least 67% of the measured values of the samples should be within the range of 80% - 120% of their labeled values.

[0127] Within-batch / between-batch precision acceptance criteria: The coefficient of variation (CV) within batch and between batches generally should not exceed 15%, and the coefficient of variation of the lower limit of quantitation should not exceed 20%.

[0128] 2. Results

[0129] Table 10 summarizes the within-batch and between-batch precision and accuracy. The coefficient of variation of the quality control samples is less than or equal to 8.6%, and the coefficient of variation of the lower limit of quantitation within batch and between batches at each concentration level is less than or equal to 17.7%. The results show that the accuracy and precision values meet the specified acceptance criteria, indicating that the method is reliable and reproducible for the determination of compound SYHA1807.

[0130] Table 10: Within-batch, between-batch accuracy and precision of SYHA1807 plasma QC samples

[0131]

[0132] Example 4: Extraction recovery

[0133] 1. Method

[0134] Add different amounts of standard products to a certain amount of blank plasma matrix to prepare validation samples at three concentrations of low, medium, and high (0.0200 ng / mL, 0.800 ng / mL, 8.00 ng / mL respectively), and carry out extraction; extract the blank plasma matrix, prepare a reference solution of the same concentration with the solution as the solvent, compare the measured responses of the two, and calculate the extraction recovery of the method.

[0135] Internal standard extraction recovery rate: A certain amount of internal standard reference substance (the added amount is the same as that used in the sample preparation process) was added to the blank plasma matrix to prepare a validation sample, and extraction was carried out. It was compared with the response value of the internal standard reference substance solution prepared with the solution after extraction of the blank plasma matrix as the solvent at the same concentration, and the extraction recovery rate of the internal standard was calculated.

[0136] Acceptance criteria: The absolute value of the extraction recovery rate of low, medium, and high concentration quality control samples is not greater than 115%, the %CV of the peak area at each concentration level should be less than 15% respectively, and the %CV of the extraction recovery rate at the three concentration levels should not be greater than 20%.

[0137] 2. Results

[0138] It meets the acceptance criteria, and the results are shown in Tables 11 and 12. The average extraction recovery rates of the analytes in the plasma at the three concentration levels of low, medium, and high are 92.5%, 92.3%, and 90.3% respectively. The %CV of the peak area at each concentration level is not greater than 4.5%, and the %CV of the extraction recovery rate at the three concentration levels is not greater than 1.3%. The results are shown in Table 11. The average extraction recovery rate of the internal standard is 103.3%, and the %CV of the peak area is not greater than 3.3%. The results are shown in Table 12.

[0139] Table 11: Extraction recovery rate of analytes

[0140]

[0141] Table 12: Recovery rate of internal standard

[0142]

[0143] Example 5: Matrix effect:

[0144] 1. Method

[0145] The effects of different matrix sources on the determination were evaluated at the LQC, MQC, and HQC concentration levels using six different sources of blank plasma. By calculating the ratio of the peak area in the presence of the matrix (measured by adding the analyte and internal standard after extraction from the blank matrix) to the corresponding peak area without the matrix (pure solutions of the analyte and internal standard), the matrix factor of each analyte and internal standard was calculated; further, by dividing the matrix factor of the analyte (Analyte MF%) by the matrix factor of the internal standard (IS MF%), the matrix factor normalized by the internal standard (Absolute MF%) was calculated. The variation of the matrix effect between individuals for the compound and the internal standard is less than 15%, meeting the requirements.

[0146] Considering that hemolytic and hyperlipidemic samples also occur clinically, the matrix effects of hemolyzed plasma (normal plasma with 2% completely lysed blood cells) and hyperlipidemic plasma (300 mg / dL) were further evaluated. %CV and %Bias should be maintained at ≤15%.

[0147] 2. Results

[0148] The matrix effect of compound SYHA1807 normalized to the internal standard ranged from 105.5% to 105.8%, and the %CV was less than 1.8%. These results (see Table 13) indicate that the matrix effect meets the requirements.

[0149] Table 13: Matrix effect of compound SYHA1807 in blank plasma

[0150]

[0151] The validation results of precision and accuracy for hyperlipidemic and hemolytic matrices: meet the acceptance criteria. The results are shown in Table 14 for details.

[0152] Table 14: Matrix effect of compound SYHA1807 in hyperlipidemic plasma or hemolytic plasma

[0153]

[0154] The %CV of 300 mg / dL (3.40 mM) high-lipid quality control samples was 1.4% to 4.9%, and the %Bias ranged from 2.2% to 4.6%; the %CV of quality control samples prepared with 2% hemolytic matrix at three concentration levels was 2.1% to 6.8%, and the Bias % ranged from -2.8% to -9.8%.

[0155] Example Six: Stability

[0156] 1. Method

[0157] The stability of the analyte was evaluated by analyzing LQC samples (n = 6) and HQC samples (n = 6) under various storage, processing, and analysis conditions. The stability of the stock solution was evaluated by comparing the peak areas of freshly prepared solutions and stored solutions (24 hours at room temperature and 230 days at -80 °C). The stability of the working solution was evaluated by comparing the peak areas of freshly prepared solutions and working solutions (24 hours at room temperature and 108 days at -80 °C). The stability of the analyte in plasma was tested under different conditions. To evaluate short-term and long-term stability, samples were placed at room temperature for 24 hours, stored at -30 °C for 112 days, and placed at -80 °C for 217 days. The extracted samples were placed in an autosampler (10 °C) for 96 hours and then analyzed using a freshly prepared standard curve to evaluate the stability of the autosampler. In addition, the prepared samples were stored in a refrigerator (10 °C) for 46 hours and then re-injected to evaluate the stability of repeated injections. For freeze-thaw stability, the 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.

[0158] For the evaluation of whole blood stability, 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). Plasma samples were obtained by immediately centrifuging the QC samples in Group A, and the samples in Group B were centrifuged after being placed at room temperature for 2 h. The average value of the ratio of the peak area of the analyte to the peak area of the IS in the samples at each concentration in Group A was regarded as the labeled value. The average value of Group B must be within ±15% of the average value of Group A, and the RSD% must not exceed 15%.

[0159] 2. Results

[0160] In this experiment, the stability of the analyte SYHA1807 in solution and plasma under different storage and operating conditions during routine analysis was investigated.

[0161] 1) Stability of the stock solution: The stability of the stock solutions of the analyte and internal standard stored at -80 °C for 230 days was good; the stability of the stock solutions of the analyte and internal standard placed at room temperature for 24 h was good.

[0162] 2) Stability of the working solution: The stability of the working solutions of the analyte and internal standard stored at -80 °C for 108 days was good; the stability of the working solutions of the analyte and internal standard stored at room temperature for 24 h was good.

[0163] 3) Stability during the pretreatment of biological samples (short-term stability): The analyte in plasma remained stable after being placed at room temperature for 24 hours.

[0164] 4) Freeze-thaw stability: The analyte in plasma could tolerate at least 5 repeated freeze-thaw cycles (-80 °C to room temperature).

[0165] 5) Sample stability after preparation: The analytes in the plasma after sample pretreatment were stored in an autosampler (10°C) for 96 hours and quantified using a freshly prepared standard curve with accuracy and precision meeting the requirements.

[0166] 6) Repeated injection reproducibility: After the analyte in the plasma after sample pretreatment was stored at 10°C for 46 hours, the accuracy and precision met the requirements.

[0167] 7) Long-term cryopreservation stability: The analytes in plasma remained stable after being stored at -80°C for 416 days and at -30°C for 112 days.

[0168] 8) Stability of the analyte in the matrix before centrifugation: The analyte in whole blood remains stable after being placed at room temperature for 1 hour.

[0169] Example 7: Dilution reliability

[0170] 1. Methods

[0171] Dilution reliability was assessed to determine that samples with concentrations above the standard curve range could be diluted and accurately measured. A high-concentration diluted quality control sample (concentration of 400 ng / mL) was prepared and diluted 10 times (n=6) with the same blank biomatrix dilution. The sample was quantitatively determined using the accompanying standard curve. The dilution factor was the dilution factor. The sample concentration after dilution should be within the marked concentration range. The concentration was measured and compared with the labeled value. The %CV of the 6 diluted quality control samples was less than 15%. The concentration result obtained by multiplying the measured value by the dilution factor should be within 85%-115% of the diluted quality control labeled value.

[0172] 2. Results

[0173] Meeting the acceptance criteria, the %CV and %Bias of the six diluted QC samples were 0.8% and 10.4%, respectively, after the plasma samples were diluted 10-fold to 40 ng / mL. These results indicate that samples with concentrations above the upper limit of quantitation (ULOQ) can be reliably diluted 10-fold. The results are detailed in Table 15.

[0174] Table 15: Dilution reliability

[0175]

[0176] Example 8: Residual Effect

[0177] 1. Methods

[0178] At least one DB should be injected after the first standard curve ULOQ of each analytical batch to evaluate whether there is carryover of high-concentration samples in the blank plasma sample.

[0179] Acceptance criteria: The peak area of the analyte in the DB after ULOQ should not exceed 20% of the mean peak area of the analyte at LLOQ, and the peak area of the internal standard in the DB should not exceed 5% of the mean peak area of the internal standard at LLOQ.

[0180] 2. Results

[0181] It meets the acceptance criteria. In the blank samples after the high-concentration sample (S8), the residue of the analyte retention time is less than 20.0% of the mean peak area of batch S1, and the results are shown in Table 16.

[0182] Table 16: Residue in the detection process of batch samples

[0183]

[0184] Example 9: Pharmacokinetic application

[0185] The method of the present invention was used to detect the plasma pharmacokinetics of compound SYHA1807 in Chinese extensive-stage small cell lung cancer patients in a Phase I clinical study. A total of 12 subjects were enrolled in this study. In the single-dose escalating study, for the 0.5 mg, 1.5 mg, 3 mg, and 6 mg groups (except subject 01006), plasma was collected before dosing and at 1 h, 4 h, 8 h, 12 h, 24 h, 30 h, 36 h, 48 h, 72 h, 96 h, 120 h, 144 h on the 1st day after dosing, 0 h on the 8th day, 0 h on the 15th day, 0 h, 1 h, 4 h, 8 h, 12 h, 24 h, 30 h, 36 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h on the 22nd day after dosing. For the 6 mg group of subject 01006, plasma was collected before dosing and at 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h on the 1st day after dosing, 0 h on the 8th day, 0 h on the 15th day, 0 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h on the 22nd day after dosing. The plasma pretreatment method and the high-performance liquid chromatography-tandem mass spectrometry quantification method provided by the present invention were used to analyze the pharmacokinetic characteristics of SYHA1807 in human plasma (N = 299) to study the clinical pharmacology of this innovative drug. The representative plasma concentration-time curve of a small cell lung cancer patient after oral administration of 6 mg SYHA1807 is shown in Figure 3 .

[0186] Screening Example 1: Optimization of mass spectrometry conditions

[0187] The parameters were tuned and optimized using an Xevo TQS triple quadrupole mass spectrometer (Waters, MA, USA). 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, cone voltage, and collision voltage were adjusted, and then detected by multiple reaction monitoring (MRM) scanning. Finally, the following conditions were determined. The quantitative ion pair of SYHA1807: m / z 353.2→228.2; the quantitative ion of D5-SYHA1807 (internal standard): m / z 358.2→233.2. The other optimized parameters were as follows: desolvation gas flow rate 1000 L / h, ion source temperature 500 °C; capillary voltage 3.50 kV, collision gas flow rate 0.25 mL / min; the cone voltages of SYHA1807 and the internal standard were 30 V and 35 V respectively; the collision voltages of SYHA1807 and the internal standard were 25 V.

[0188] Screening Example 2: Chromatographic column screening test

[0189] Referring to the above "2. Chromatographic and mass spectrometric conditions" section, different types of chromatographic columns were selected, and the method parameters such as the type of mobile phase, gradient conditions, and flow rate were comprehensively adjusted according to the detection effect. The detection of the analyte solution in different types of chromatographic columns was investigated. The specific information of the chromatographic columns is as follows:

[0190] Table 17: Chromatographic column information

[0191]

[0192] The results showed that the ACQUITY UPLC BEH C18 (2.1×50 mm, 1.7 µm) chromatographic column had a good peak shape, appropriate retention time, and good detection effect.

[0193] Screening Example 3: Screening test of different mobile phase components

[0194] 1. Screening of mobile phase A (organic phase)

[0195] Referring to "2. Chromatographic and mass spectrometric conditions", when an aqueous solution containing 0.1% formic acid was used as mobile phase B (aqueous phase), acetonitrile and methanol were respectively selected as mobile phase A (organic phase) for detection, and the chromatograms were recorded.

[0196] As a result, using acetonitrile as mobile phase A (organic phase) had better elution effect and very low background noise, while using methanol as mobile phase A (organic phase) had poor retention effect. Therefore, the acetonitrile system was selected as mobile phase A. When mobile phase B was mixed into mobile phase A, it was found that when the amount of mobile phase B in the mixed mobile phase was below 7% by volume, the buffer salts contained in mobile phase B would not precipitate; while when the amount of mobile phase B exceeded 7% by volume, the buffer salts contained in mobile phase B would precipitate.

[0197] 2. Screening of Mobile Phase B (Aqueous Phase)

[0198] Referring to "2. Chromatography and Mass Spectrometry Conditions", when acetonitrile is selected as Mobile Phase A (organic phase), 2 mM ammonium acetate solution, 5 mM ammonium acetate solution, 0.1 mass% formic acid aqueous solution, 0.2 mass% formic acid aqueous solution, 0.5 mass% formic acid aqueous solution, and an aqueous solution containing 0.1 mass% formic acid and 2 mmol / L ammonium acetate are respectively selected as Mobile Phase B (aqueous phase) for detection, and the chromatogram is recorded.

[0199] The results show that when the formic acid aqueous solution system is selected as Mobile Phase B, there is an obvious tailing phenomenon. When the aqueous solution system containing both ammonium acetate and formic acid is selected, the chromatographic peaks are better; the difference in the influence of the formic acid and ammonium acetate concentrations on the response is not significant. Therefore, a system of 0.1 mass% formic acid and 2 mmol / L ammonium acetate is selected as Mobile Phase B. When Mobile Phase A is mixed into Mobile Phase B, it is found that when the amount of Mobile Phase A in the mixed mobile phase is 12% by volume or less, the buffer salt contained in Mobile Phase B will not precipitate; while when the amount of Mobile Phase A exceeds 12% by volume, the buffer salt contained in Mobile Phase B will precipitate.

[0200] Screening Example 4: Screening of Plasma Sample Treatment Methods

[0201] Referring to "4. Plasma Sample Pretreatment Method", methanol and acetonitrile are respectively used as protein precipitation reagents to prepare plasma samples, and "2. Chromatography and Mass Spectrometry Conditions" are used for detection.

[0202] The results show that the chromatographic peaks are good when acetonitrile is used as the precipitant, so acetonitrile is selected as the precipitant.

[0203] Screening Example 5: Flow Rate Screening Test

[0204] Referring to "2. Chromatography and Mass Spectrometry Conditions", the influence of the flow rate on the detection effect is investigated. The flow rates of 0.3 mL / min and 0.4 mL / min are respectively selected for detection, and other test conditions are the same.

[0205] The results show that the peak emergence time is relatively late at a flow rate of 0.3 mL / min, and the retention effect of the chromatographic column is stronger; the peak emergence time is 1 minute at a flow rate of 0.4 mL / min, and the retention effect is better. Therefore, a flow rate of 0.4 mL / min is preferred.

[0206] Screening Example 6: Column Temperature Screening Test

[0207] Referring to "2. Chromatography and Mass Spectrometry Conditions", the influence of the column temperature on the detection effect is investigated. The column temperatures of 35 °C, 40 °C, and 45 °C are respectively selected for detection, and other test conditions are the same.

[0208] The results showed that the three column temperature levels in the experiment had little effect on the chromatographic peak shape and response, and only the retention time had slight differences.

[0209] As described above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed in the present disclosure can easily think of changes or substitutions, which should all be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A method for quantitatively analyzing SYHA1807 by using a high performance liquid chromatography-tandem mass spectrometry device, characterized in that, The method includes the following steps: 1) Prepare a sample solution containing SYHA1807; 2) Inject the sample solution into a high performance liquid chromatography-tandem mass spectrometry device to obtain a mass chromatogram; 3) Use the internal standard method to quantitatively analyze SYHA1807 according to the mass chromatogram, wherein the chromatographic column in the high performance liquid chromatography is a chromatographic column filled with octadecylsilane-bonded silica gel, in the high performance liquid chromatography, a mixed mobile phase composed of mobile phase A and mobile phase B is used for gradient elution, and the gradient elution program is as follows: From 0.00 minute to 1.00 minute, 8 vol% to 12 vol% of mobile phase A; From 1.01 minute to 1.99 minute, 93 vol% to 97 vol% of mobile phase A; From 2.00 minutes to 3.00 minutes, 8 vol% to 12 vol% of mobile phase A, wherein in the mixed mobile phase, the total amount of mobile phase A and mobile phase B is 100 vol%, wherein mobile phase A is selected from: acetonitrile, an acetonitrile solution containing 0.05 mass% to 0.5 mass% formic acid, and a water-acetonitrile mixture containing 0.05 mass% to 0.5 mass% formic acid and 1 mmol / L to 5 mmol / L ammonium acetate, wherein the volume ratio of water to acetonitrile in the water-acetonitrile mixture is in the range of 1:99 to 10:90, and mobile phase B is an acetonitrile-water mixture containing 0.05 mass% to 0.5 mass% formic acid and 1 mmol / L to 5 mmol / L ammonium acetate, wherein the volume ratio of acetonitrile to water in the acetonitrile-water mixture is in the range of 1:99 to 10:90, wherein in the mass spectrometry, multiple reaction monitoring scan detection is used, the quantitative ion pair of compound SYHA1807 is: m / z 353.2→228.2; the quantitative ion pair of internal standard compound D5-SYHA1807 is: m / z 358.2→233.2, and wherein SYHA1807 is from the plasma of a subject, and the preparation of the sample solution containing SYHA1807 includes: adding an internal standard working solution to the plasma sample of the subject, oscillating the resulting mixture, then centrifuging, taking the supernatant for drying to obtain a solid sample, and then dissolving and mixing the solid sample with a reconstitution solution composed of water and acetonitrile.

2. The method according to claim 1, wherein mobile phase A is a water-acetonitrile mixture with a volume ratio of 5:95 containing 0.1 mass% formic acid and 2 mmol / L ammonium acetate; and / or mobile phase B is an acetonitrile-water mixture with a volume ratio of 5:95 containing 0.1 mass% formic acid and 2 mmol / L ammonium acetate.

3. The method according to claim 1, wherein the gradient elution program is as follows: From 0.00 minute to 1.00 minute, 10 vol% of mobile phase A; From 1.01 minute to 1.99 minute, 95 vol% of mobile phase A; From 2.00 minutes to 3.00 minutes, 10 vol% of mobile phase A, wherein in the mixed mobile phase, the total amount of mobile phase A and mobile phase B is 100 vol%.

4. The method according to claim 1, wherein the internal standard working solution is prepared by: dissolving the internal standard standard D5-SYHA1807 with dimethyl sulfoxide to obtain an internal standard stock solution, and then diluting the internal standard stock solution with acetonitrile.

5. The method according to claim 1, wherein the volume ratio of the supernatant to the complex solution is in the range of 2:1 to 5:1.

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