Method for detecting content of quinoline compound as shown in formula (I)

Abstract

The invention relates to the technical field of quinoline compound detection, in particular to a method for detecting the content of a quinoline compound shown in a formula (I), and the content of the quinoline compound shown in the formula (I) in a to-be-detected object is detected by adopting a liquid chromatography and mass spectrometry combined method. The conditions of the liquid chromatography are as follows: a 0.1 vol% formic acid aqueous solution is used as a mobile phase A, an acetonitrile solution is used as a mobile phase B, and a chromatographic column is ACE 5C18. According to the method, the content of the quinoline compound shown in the formula (I) can be accurately measured by adopting a liquid chromatography-mass spectrometry method, the sensitivity is high, and the lower quantification limit is 0.05 ng / mL.

Description

Technical Field

[0001] The present invention relates to the technical field of quinoline compound detection, and particularly relates to a method for detecting the content of a quinoline compound represented by formula (I). Background Art

[0002] The quinoline compound represented by formula (I) is a Class 1.1 innovative drug with a brand-new molecule.

[0003] Quinoline compounds are often used in the treatment of malignant tumors. For example, Chinese Patent Document CN102977014A records in the specification a compound represented by formula (I) and its salts, and it is recorded in the specification that they can be used to inhibit various tumor cells such as lung cancer cells, colon cancer cells, gastric cancer cells, liver cancer cells, breast cancer cells, and malignant glioblastoma cells.

[0004] The preparation of the quinoline compound represented by formula (I) can be administered orally and is intended for the treatment of advanced malignant solid tumors. In order to support the clinical safety and efficacy evaluation of this drug, a bioanalysis method is needed to monitor the blood drug concentration level of the quinoline compound represented by formula (I) in the human body. Summary of the Invention

[0005] Therefore, in order to solve the above problems, a method for detecting the content of a quinoline compound represented by formula (I) is provided.

[0006] For this purpose, the present invention provides a method for detecting the content of a quinoline compound represented by formula (I), including the following steps: using liquid chromatography - mass spectrometry to detect the content of the quinoline compound represented by formula (I) in the analyte. The conditions of the liquid chromatography are as follows: using an aqueous solution of 0.1 vol% formic acid as mobile phase A, using an acetonitrile solution as mobile phase B, and using an ACE 5C18 chromatographic column.

[0007]

[0008] In some embodiments, mobile phase A further includes 5 - 10 mM ammonium formate; preferably, the concentration of ammonium formate in mobile phase A is 10 mM.

[0009] In some embodiments, mobile phase B further includes 0.1 vol% formic acid.

[0010] In some of these embodiments, the conditions for gradient elution in the liquid chromatography are as follows: at 0.01 min, the volume ratio of mobile phase A to mobile phase B is 70:30; at 1.2 min, the volume ratio of mobile phase A to mobile phase B is 40:60; at 1.3 min, the volume ratio of mobile phase A to mobile phase B is 5:95; at 2 min, the volume ratio of mobile phase A to mobile phase B is 5:95; at 2.1 min, the volume ratio of mobile phase A to mobile phase B is 70:30; at 3.3 min, the volume ratio of mobile phase A to mobile phase B is 40:60; at 3.4 min, the volume ratio of mobile phase A to mobile phase B is 5:95; at 4.1 min, the volume ratio of mobile phase A to mobile phase B is 5:95; at 4.2 min, the volume ratio of mobile phase A to mobile phase B is 70:30.

[0011] In some of these embodiments, the conditions for gradient elution in the liquid chromatography are as follows: at 0.01 min, the volume ratio of mobile phase A to mobile phase B is 65:35; at 1.2 min, the volume ratio of mobile phase A to mobile phase B is 45:55; at 1.3 min, the volume ratio of mobile phase A to mobile phase B is 5:95; at 2 min, the volume ratio of mobile phase A to mobile phase B is 5:95; at 2.1 min, the volume ratio of mobile phase A to mobile phase B is 65:35.

[0012] In some of these embodiments, during the process of coupling liquid chromatography and mass spectrometry, the sample processed by the liquid chromatography is subjected to mass spectrometry analysis at 1.1 min.

[0013] In some of these embodiments, during the gradient elution process in the liquid chromatography, it further includes a step of washing the needle. The needle washing solution includes formic acid, methanol, acetonitrile, water, and isopropanol. The concentration of formic acid in the needle washing solution is 1 - 3 vol%, and the volume ratio of methanol, acetonitrile, water, and isopropanol is 1 - 3:1 - 3:1 - 3:1 - 3.

[0014] In some of these embodiments, before detecting the analyte by using the method of coupling liquid chromatography and mass spectrometry, it further includes a step of pre - treating the analyte. The pre - treatment step includes mixing the analyte and a precipitant to obtain a mixed solution, performing solid - liquid separation to obtain a supernatant, and adding a re - solution to the supernatant.

[0015] In some of these embodiments, the precipitant includes acetonitrile.

[0016] In some of these embodiments, the volume ratio of the analyte to the precipitant is 1:3 - 5.

[0017] In some of these embodiments, the volume ratio of the supernatant to the re - solution is 2:1 - 3. The re - solution includes acetonitrile, and the concentration of acetonitrile in the re - solution is 10 - 50 vol%.

[0018] In some of these embodiments, the concentration of acetonitrile in the complex solution is 15-30 vol%.

[0019] In some of these embodiments, the solid-liquid separation includes a centrifugation step, the centrifugation time is 10-20 min, and the centrifugation speed is 1500-2000 g.

[0020] The application of the method for detecting the content of the quinoline compound represented by formula (I) provided by the present invention in determining the content of the quinoline compound represented by formula (I) in plasma.

[0021] The technical solution of the present invention has the following advantages:

[0022] For the method for detecting the content of the quinoline compound represented by formula (I) provided by the present invention, liquid chromatography and mass spectrometry are used to detect the content of the quinoline compound represented by formula (I) in the analyte. The conditions of the liquid chromatography are as follows: 0.1 vol% formic acid aqueous solution is used as mobile phase A, acetonitrile solution is used as mobile phase B, and the chromatographic column is ACE 5C18;

[0023]

[0024] The present invention can accurately determine the content of the quinoline compound represented by formula (I) by liquid chromatography-mass spectrometry, has high sensitivity, and the lower limit of quantification is 0.05 ng / mL.

[0025] For the method for detecting the content of the quinoline compound represented by formula (I) provided by the present invention, acetonitrile is used as a precipitant to pretreat the analyte, which can not only shorten the detection time, but also improve the recovery rate of the quinoline compound represented by formula (I).

[0026] For the method for detecting the content of the quinoline compound represented by formula (I) provided by the present invention, the separation effect is good, there is no ion suppression interference, and high-fat state matrix and hemolysis state matrix do not affect the quantitative analysis of the sample. Description of the Drawings

[0027] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0028] Figure 1 It is the parent ion scan diagram of the analyte sample in Example 1 of the present invention;

[0029] Figure 2It is the daughter ion scan graph of the sample to be measured in Example 1 of the present invention;

[0030] Figure 3 It is the parent ion scan graph of the internal standard in Example 1 of the present invention;

[0031] Figure 4 It is the daughter ion scan graph of the internal standard in Example 1 of the present invention;

[0032] Figure 5 It is the chromatogram of the sample at the lower limit of quantification in Example 1 of the present invention;

[0033] Figure 6 It is the chromatogram of the sample at the upper limit of quantification in Example 1 of the present invention;

[0034] Figure 7 It is the HPLC-MS / MS chromatogram of the quinoline compound shown in formula (I) in the zero-concentration sample with internal standard added in Example 1 of the present invention;

[0035] Figure 8 It is the HPLC-MS / MS chromatogram of the internal standard in the zero-concentration sample with internal standard added in Example 1 of the present invention;

[0036] Figure 9 It is the HPLC-MS / MS chromatogram of the quinoline compound shown in formula (I) in the sample at the upper limit of quantification without internal standard added in Example 1 of the present invention;

[0037] Figure 10 It is the HPLC-MS / MS chromatogram of the internal standard in the sample at the upper limit of quantification without internal standard added in Example 1 of the present invention;

[0038] Figure 11 It is the characteristic standard curve for the quantitative analysis of the analyte in Example 1 of the present invention;

[0039] Figure 12 It is the pharmacokinetic curve of the quinoline compound shown in formula (I) in human plasma in Example 1 of the present invention;

[0040] Figure 13 It is the HPLC-MS / MS chromatogram of the sample of Group 1 in Example 2 of the present invention;

[0041] Figure 14 It is the HPLC-MS / MS chromatogram of the sample of Group 2 in Example 2 of the present invention;

[0042] Figure 15 It is the HPLC-MS / MS chromatogram of the sample of Group 3 in Example 2 of the present invention;

[0043] Figure 16 It is the HPLC-MS / MS chromatogram of the sample of Group 4 in Example 2 of the present invention;

[0044] Figure 17 It is the HPLC-MS / MS chromatogram of sample of Group 5 in Example 2 of the present invention;

[0045] Figure 18 It is the HPLC-MS / MS chromatogram of the sample treated with an acetonitrile aqueous solution of 50 vol% as the reconstitution solution in Example 2 of the present invention;

[0046] Figure 19 It is the HPLC-MS / MS chromatogram of the sample treated with an acetonitrile aqueous solution of 10 vol% as the reconstitution solution in Example 2 of the present invention;

[0047] Figure 20 It is the HPLC-MS / MS chromatogram of the sample treated with an acetonitrile aqueous solution of 20 vol% as the reconstitution solution in Example 2 of the present invention;

[0048] Figure 21 It is the HPLC-MS / MS chromatogram of the sample treated under single gradient elution conditions in Example 2 of the present invention;

[0049] Figure 22 It is the HPLC-MS / MS chromatogram of the sample treated under double gradient elution conditions in Example 2 of the present invention;

[0050] Figure 23 It is the HPLC-MS / MS chromatogram of the sample treated under the elution conditions with an added switching valve in Example 2 of the present invention;

[0051] Figure 24 It is the chromatogram of the lower limit of quantification sample in Comparative Example 1 of the present invention;

[0052] Figure 25 It is the chromatogram of the upper limit of quantification sample in Comparative Example 1 of the present invention;

[0053] Figure 26 It is the chromatogram of the lower limit of quantification sample in Comparative Example 2 of the present invention;

[0054] Figure 27 It is the chromatogram of the upper limit of quantification sample in Comparative Example 2 of the present invention;

[0055] Figure 28 It is the chromatogram of the lower limit of quantification sample in Comparative Example 3 of the present invention;

[0056] Figure 29 It is the chromatogram of the upper limit of quantification sample in Comparative Example 3 of the present invention. Detailed implementation manners

[0057] The following embodiments are provided to better further understand the present invention. It is not limited to the described best mode, and does not limit the content and protection scope of the present invention. Any product that is the same as or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior art features falls within the protection scope of the present invention.

[0058] For those embodiments where specific experimental steps or conditions are not indicated, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For reagents or instruments whose manufacturers are not indicated, they are all conventional reagent products that can be obtained through commercial purchase.

[0059] It should be noted that in the data of the embodiments of the present invention, E or e represents an exponent. For example, the peak area 2.79E-02 represents 2.79×10 -2 .

[0060] Example 1

[0061] This embodiment provides a method for detecting the content of quinoline compounds represented by formula (Ⅰ). The specific steps and methods are as follows:

[0062] (1) Reference substances and reagents:

[0063] Quinoline compounds represented by formula (Ⅰ) (content 99.6%);

[0064] Quinoline compounds-d6 of formula (Ⅰ) (internal standard, content 98.32%);

[0065] Methanol (HPLC grade), acetonitrile (HPLC grade), formic acid (HPLC grade), isopropanol (HPLC grade), purchased from Thermo Fisher; ammonium formate (analytical grade), N, N-dimethylformamide (analytical grade), purchased from Sinopharm Chemical Reagent Co., Ltd.; water purchased from Watson's; fat emulsion purchased from Fresenius Kabi AG.

[0066] (2) Detection method:

[0067] 2.1. Solution preparation:

[0068] Standard series working solutions: Weigh an appropriate amount of quinoline compounds represented by formula (Ⅰ), dissolve and make up the volume with N, N-dimethylformamide to prepare a stock solution with a concentration of about 1.00 mg / mL. Precision pipette an appropriate amount of each stock solution, and gradually dilute it with methanol: water (5:5, v / v) to obtain standard series working solutions. The concentration range of quinoline compounds represented by formula (Ⅰ) is 1.25 - 25000 ng / mL;

[0069] Standard series samples: Precisely pipette each standard series working solution, and serially dilute it with blank plasma to obtain a mixed standard series sample. The concentration range of the quinoline compounds of formula (I) is 0.050 - 50.0 ng / mL respectively;

[0070] Quality control working solution: Prepared in a similar method to the standard series working solution, to obtain quality control working solutions of quinoline compounds of formula (I) with concentration levels of 1.25, 3.75, 37.5, 375, 937.5, 5000 ng / mL.

[0071] Quality control samples: Precisely pipette each quality control working solution, and serially dilute it with blank plasma to obtain 4 concentration levels of quality control samples of quinoline compounds of formula (I). The lower limit of quantification concentration is 0.050 ng / mL, the low quality control (LQC) concentration is 0.150 ng / mL, the medium quality control (MQC) concentration is 1.500 ng / mL, the medium quality control 2 (MQC2) concentration is 15.0 ng / mL, and the high quality control (HQC) concentration is 37.5 ng / mL;

[0072] Internal standard working solution: Precisely weigh the reference substance of quinoline compounds - d6 of formula (I), dissolve and make up the volume with N, N - dimethylformamide to prepare an internal standard stock solution with a concentration of about 1.00 mg / mL. Precisely pipette an appropriate amount of the above internal standard stock solution and dilute it with methanol: water (5:5, v / v) to obtain an internal standard working solution with a concentration of 5.00 ng / mL.

[0073] 2.2. Treatment of plasma samples:

[0074] Use a pipette to add 100 μL of plasma sample into a 96 - well plate. The plasma sample contains a certain concentration of quinoline compounds of formula (I). Add 25 μL of internal standard working solution, vortex and mix for 3 min, then add 400 μL of acetonitrile, vortex and mix for 10 min, centrifuge for 15 min (4 °C, 1700 g), transfer 200 μL of the supernatant obtained after centrifugation to another solution containing 100 μL of 20 vol% acetonitrile aqueous solution, seal the membrane, and vortex and mix for 5 min to obtain the plasma sample to be treated.

[0075] 2.3. Liquid chromatography treatment:

[0076] Use ACE 5C18 (2.1×50.0 mm, 5.0 μm), column temperature 30 °C, inject 8 μL of sample, flow rate 0.8 mL / min, mobile phase A is an aqueous solution containing 0.1 vol% formic acid and 10 mM ammonium formate, mobile phase B is an acetonitrile solution containing 0.1 vol% formic acid, and elute the plasma sample to be treated according to the elution conditions in Table 1.

[0077] Table 1 Gradient elution

[0078]

[0079] In this example, at 1.1 min, the sample after chromatographic treatment was delivered to the mass spectrometer for detection. During the injection process, the injection needle was rinsed. The rinsing mode was Before and after aspiration, the rinsing volume was 500 μL, the rinsing method was Rinse Pump Then Port, and the rinsing solvent was a methanol - acetonitrile - water - isopropanol solution containing 2 vol% formic acid (the volume ratio of methanol, acetonitrile, water, and isopropanol was 25:25:25:25).

[0080] 2.4. Mass spectrometry detection:

[0081] An electrospray ionization source (ESI) was used. The detection mode was positive ion, the source temperature was 600 °C, the monitoring mode was multiple reaction monitoring (MRM), the curtain gas pressure (CUR) was 30 psi, the collision cell gas type (CAD Gas Type) was 9, the ion spray voltage was 5500 V, Gas1 was 60 psi, Gas2 was 60 psi, and the dwell time was 150 ms; for the quantitative analysis ion pair of the quinoline compound of formula (I), m / z was 622.1 / 140.2, the declustering voltage (DP) was 50 V, the collision energy (CE) was 30 V, the entrance voltage (EP) was 10 V, and the collision cell exit voltage (CXP) was 10 V; for the quantitative analysis ion pair of the internal standard analyte, m / z was 628.1 / 146.2, the declustering voltage (DP) was 80 V, the collision energy (CE) was 33 V, the entrance voltage (EP) was 10 V, and the collision cell exit voltage (CXP) was 10 V.

[0082] In this example, at 1.1 min of elution, the sample after chromatographic treatment was subjected to mass spectrometry detection.

[0083] The results are shown in Figures 1 - 4 , Figures 1 - 4 The concentration of the quinoline compound of formula (I) in the sample used in Figure 5 and Figure 6, it can be seen that the quinoline compounds shown in formula (I) have good chromatographic peak responses, excellent peak shapes, no obvious tailing, and no obvious differences in peak shapes and elution times among different batches on an ACE C18 (2.1×50mm, 5μm) ultra-high performance chromatographic column (Thermo Scientific).

[0084] (3) Method validation:

[0085] 3.1 Selectivity validation: Prepare zero-concentration samples with blank matrix (processed matrix samples containing internal standard but no analyte, prepared according to the preparation method of 2.1 quality control samples, where blank plasma is replaced by blank matrix, and the quinoline compounds shown in formula (I) are replaced by internal standard, and the concentration of the internal standard is the upper limit of quantification, 50 ng / mL), and perform detection according to steps 2.3 and 2.4. Prepare upper limit of quantification samples without internal standard added with blank matrix (containing the quinoline compounds shown in formula (I), without internal standard, specifically prepared according to the preparation method of 2.1 quality control samples, where blank plasma is replaced by blank matrix, and the concentration of the quinoline compounds shown in formula (I) is the upper limit of quantification, 50 ng / mL), and perform detection according to steps 2.3 and 2.4. The results are shown in Figures 7 - 10 , it can be seen that the retention times of the quinoline compounds shown in formula (I) and the internal standard are approximately 0.820 min and 0.812 min respectively, and there are no co-eluting interfering peaks at the retention times, indicating that the selectivity of the detection method provided in this example is good.

[0086] 3.2 According to the guiding principles of the Chinese Pharmacopoeia 9012, methodological verification was carried out on this method. According to the method of 2.2, prepare plasma samples with the concentrations of the quinoline compounds shown in formula (I) being 0.2 ng / mL, 10 ng / mL, 25 ng / mL, 45 ng / mL, and 50 ng / mL as the plasma samples to be processed, and perform separate processing on the plasma samples to be processed according to the methods of 2.3 and 2.4. The results are shown in Figure 11 . According to Figure 11 it can be seen that the linear ranges of the quinoline compounds shown in formula (I) in the clinical research plasma samples for determining the quinoline compounds shown in formula (I) are 0.005 - 50.0 ng / mL respectively, and the typical linear regression equations of the analyte standard curves are: y = 0.800x + 0.00392, R 2 = 0.9979.

[0087] 3.3 Use the plasma sample processing method in 2.2 to process the quality control samples at each concentration level prepared in 2.1, and perform accuracy and precision detection. The results are shown in Table 2.

[0088] Table 2 Precision and accuracy of the method

[0089]

[0090] As can be seen from Table 2, the method provided in this example has good precision and accuracy. The concentration of the quinoline compound shown in formula (I) in the lower limit of quantification sample is 0.005 ng / mL, the signal-to-noise ratio is greater than 5, and the sensitivity is good.

[0091] 3.4. According to the quality control sample preparation method in 2.1, use the following types of matrix samples to replace blank plasma respectively to prepare matrix samples containing the lower limit of quantification concentration, low quality control (LQC) concentration, and high quality control (HQC) concentration. Then process them according to the plasma sample treatment method in 2.2;

[0092] Among them, the matrix in matrix sample 1 is the first blank normal matrix, and the concentration of the quinoline compound with formula (I) is the low quality control concentration; the matrix in matrix sample 2 is the second blank normal matrix, and the concentration of the quinoline compound with formula (I) is the high quality control concentration; the matrix in matrix sample 3 is the third blank normal matrix, and the internal standard concentration is 5 ng / mL; the matrix in matrix sample 4 is the fourth blank normal matrix, and the concentration of the quinoline compound with formula (I) is the low quality control concentration; the fifth blank normal matrix in matrix sample 5, and the concentration of the quinoline compound with formula (I) is the high quality control concentration; the matrix in matrix sample 6 is the sixth blank normal matrix, and the internal standard concentration is 5 ng / mL; the matrix in matrix sample 7 is the blank high-fat matrix, and the concentration of the quinoline compound with formula (I) is the low quality control concentration; the matrix in matrix sample 8 is the blank high-fat matrix, and the concentration of the quinoline compound with formula (I) is the high quality control concentration; the matrix in matrix sample 9 is the blank high-fat matrix, and the internal standard concentration is 5 ng / mL; the matrix in matrix sample 10 is the blank hemolytic matrix, and the concentration of the quinoline compound with formula (I) is the low quality control concentration; the matrix in matrix sample 11 is the blank hemolytic matrix, and the concentration of the quinoline compound with formula (I) is the high quality control concentration; the matrix in matrix sample 12 is the blank hemolytic matrix, and the internal standard concentration is 5 ng / mL. Among them, the first to sixth blank normal matrices are blank normal matrices from different sources.

[0093] Detect the above matrix samples according to the methods in steps 2.3 and 2.4, calculate the matrix factors for the analyte and internal standard in different donor matrix samples respectively, and then calculate the matrix factor of the analyte in each donor sample normalized by the internal standard. The relative standard deviation of the matrix factors of the analytes calculated from all different donor matrix samples normalized by the internal standard should not exceed 15%. The results are shown in Table 3.

[0094] Table 3 Evaluation results of high-fat effect and hemolysis effect

[0095]

[0096] In the table, n represents 6 blank normal matrices.

[0097] It is known that the matrix effects of the blank normal matrix from 6 different sources, the blank high-fat matrix from 1 source, and the blank hemolytic matrix from 1 source on the analyte and internal standard are close, and do not affect the quantitative analysis of the analyte.

[0098] Furthermore, the matrix factors normalized by the internal standard of the quinoline compounds of formula (I) at low quality control and high quality control concentration levels are 0.995 and 0.986 respectively, and the RSD(%) are 2.6 and 2.0 respectively. The results show that the matrix effect does not interfere with the accuracy of the analysis of the analyte to be measured.

[0099] Mix the first blank matrix, blank high-fat matrix and blank hemolytic matrix according to the volume ratio of 1:1:1 to obtain a mixed matrix, and use the mixed matrix to prepare mixed matrix samples with low quality control concentration, medium quality control concentration and high quality control concentration of the quinoline compounds of formula (I) respectively.

[0100] After the mixed blank matrix is processed, pure solutions of the analyte and internal standard with the same concentrations as low quality control, medium quality control and high quality control are added respectively to prepare recovery evaluation samples (REC samples), and this type of sample is an unextracted sample.

[0101] The relative standard deviation (RSD) of 6 parallel samples for each concentration does not exceed 15%, and there are at least 5 valid values. The respective average recoveries of the quality control samples for each concentration should not show a trend related to the concentration, and the RSD of the recoveries between the quality control concentrations should not exceed 30%.

[0102] At low quality control, medium quality control and high quality control concentration levels, the extraction recoveries of the quinoline compounds of formula (I) are 99.1%, 100.3% and 97.3% respectively. The relative standard deviation (%RSD) of the extraction recovery of the analyte is 1.3, and the average values of the extraction recoveries of the internal standard are 103.9%, 101.9% and 104.9%. The relative standard deviation (%RSD) of the extraction recovery of the internal standard is 1.5.

[0103] 3.5. When investigating the stability of each analyte in plasma samples, the low quality control concentration plasma samples and high quality control concentration plasma samples are placed in different temperatures and environments, and after the placement, six-sample analysis precision, accuracy and stability are carried out. Among them, the placement conditions investigated are: placed at room temperature for 24 h, the prepared samples are placed at 4 °C for 74 h, experienced 5 freeze-thaw cycles (from -80 °C to -20 °C), placed at -80 °C for 187 days, and each sample is processed according to the steps of 2.2 and 2.3, and the results are shown in Table 4.

[0104] Table 4 Data for investigating matrix stability

[0105]

[0106]

[0107] It can be seen that the method for detecting the concentration of quinoline compounds of formula (I) provided by this example is stable under various investigated conditions.

[0108] 3.6. The verified method was used to analyze the concentration of quinoline compounds of formula (I) in plasma to evaluate the pharmacokinetic characteristics of quinoline compounds of formula (I). This analysis protocol was used to detect the concentration of quinoline compounds of formula (I) in clinical plasma to evaluate the pharmacokinetic characteristics of quinoline compounds of formula (I).

[0109] After the subjects took a single oral dose of 100 mg of quinoline compounds of formula (I) on an empty stomach, 2 mL of venous blood was collected into a vacuum blood collection tube containing EDTA-K2 anticoagulant at 0 h (before dosing), 1 h, 2 h, 4 h, 8 h, 12 h, 24 h (D2), 36 h, 48 h (D3), 72 h (D4), 96 h (D5), and 144 h (D7) after dosing, respectively. After centrifugation (4 °C, 1700 g) for 10 min, the plasma was separated for the determination of drug concentration. After the treatment in steps 2.2 and 2.3, the pharmacokinetic curve of quinoline compounds of formula (I) in the human body was as Figure 12 shown.

[0110] The experimental results show that the method provided by this example was successfully applied to the determination of the concentration of quinoline compounds of formula (I) in plasma.

[0111] Example 2

[0112] This example provides a method for detecting the content of quinoline compounds of formula (I), and different chromatographic conditions were investigated for it, including:

[0113] 1. Investigation of the mobile phase system:

[0114] According to the detection method of Example 1, an aqueous solution of 0.1 vol% formic acid containing 10 mM ammonium formate was used as mobile phase A, and an acetonitrile solution containing 0.1 vol% formic acid was used as mobile phase B (investigation group 1), an aqueous solution of 0.1 vol% formic acid was used as mobile phase A, and an acetonitrile solution was used as mobile phase B (investigation group 2), an aqueous solution of 10 mM ammonium formate was used as mobile phase A, and an acetonitrile solution was used as mobile phase B (investigation group 3), an aqueous solution of 0.1 vol% formic acid containing 5 mM ammonium formate was used as mobile phase A, and an acetonitrile solution was used as mobile phase B (investigation group 4), an aqueous solution of 0.1 vol% formic acid containing 10 mM ammonium formate was used as mobile phase A, and an acetonitrile solution was used as mobile phase B (investigation group 5) to detect blood samples. The results are shown in Figures 13 - 17It is known that the analyte can be eluted with formic acid as mobile phase A and acetonitrile as mobile phase B. Among them, an aqueous solution of 0.1 vol% formic acid containing 10 mM ammonium formate is used as mobile phase A, and an acetonitrile solution containing 0.1 vol% formic acid is used as mobile phase B for elution. The analyte has a high response and the compound residue effect is effectively avoided.

[0115] 2. Investigation of the precipitant:

[0116] Take 100 μL of quality control samples (the preparation method of the quality control samples is in accordance with the preparation method of the quality control samples in step 2.1 of Example 1, and the quality control samples are quality control samples with low, medium, and high quality control concentrations), and perform sample treatment according to the method in step 2.2 of Example 1. Specifically, add 25.0 μL of internal standard working solution and 400 μL of acetonitrile to each concentration of quality control sample, vortex and centrifuge, take 200 μL of the supernatant, add 100 μL of 20 vol% acetonitrile aqueous solution, and vortex to mix evenly.

[0117] And prepare the mixed matrix samples and recovery evaluation samples of each quality control concentration sample according to the steps in 3.4 of Example 1.

[0118] Detect the prepared samples according to the steps in steps 2.3 and 2.4 of Example 1, and measure the matrix effect and recovery rate. See Table 5.

[0119] Table 5 Matrix effect / recovery rate with acetonitrile as the precipitant

[0120]

[0121] According to Table 5, when acetonitrile is used as the protein precipitant for treatment, the recoveries of low, medium, and high concentrations are about 100%, and there is no matrix effect (in Table 5, taking the compound with low quality control concentration as an example, the calculation method of its recovery rate is the peak area of the analyte of the low quality control concentration compound / the peak area of the analyte of the low quality control concentration recovery evaluation sample × 100%, and the calculation method of the matrix effect is the peak area of the analyte of the low quality control concentration recovery evaluation sample / the peak area of the analyte of the low quality control concentration mixed matrix sample × 100%).

[0122] 3. Selection of the reconstitution solution in the pretreatment of plasma samples:

[0123] Take 100 μL of plasma (the quality control concentration in the plasma is 1.9 ng / mL, and the internal standard concentration is 0.159 ng / mL) and perform treatment according to the treatment method in step 2.2 of Example 1. The specific steps are to add 25.0 μL of internal standard working solution and 400 μL of acetonitrile to the plasma, vortex and centrifuge, take 200 μL of the supernatant, and add 100 μL of 50 vol% acetonitrile aqueous solution, 20 vol% acetonitrile aqueous solution, and 10 vol% acetonitrile aqueous solution respectively, and vortex to mix evenly.

[0124] The samples were processed according to the steps in 2.3 of Example 1, and the results are shown in Figures 18 - 20 .

[0125] After the above plasma samples were placed for one day, the samples were processed according to the steps in 2.3 and 2.4 of Example 1, and the results are shown in Table 6.

[0126] Using glass bottles and centrifuge tubes as containers for preparing plasma samples respectively, using acetonitrile aqueous solutions with 50 vol%, 20 vol%, and 10 vol% acetonitrile as reconstitution solutions respectively, and processing plasma samples with low quality control concentration, medium quality control concentration, and high quality control concentration respectively. The samples were processed according to the steps in 2.3 and 2.4 of Example 1, and the results are shown in Tables 7 - 8.

[0127] Table 6 Analyte response values after different ratios of reconstitution solutions were placed for 1 day

[0128]

[0129]

[0130] Table 7 Analyte response values when the ratio of acetonitrile:water in the reconstitution solution is 1:9

[0131]

[0132] Table 8 Analyte response values when the ratio of acetonitrile:water in the reconstitution solution is 2:8

[0133]

[0134]

[0135] It can be seen that when the reconstitution solution is acetonitrile:water = 1:1, the peak shape is front and wide, and there is a solvent effect. When the reconstitution solution is acetonitrile:water = 1:9, the analyte response is low and the response fluctuates greatly after standing. Therefore, acetonitrile:water = 2:8 is selected as the reconstitution solution, and both glass bottles and centrifuge tubes can be used as the preparation containers.

[0136] 4. Investigation of the elution gradient:

[0137] 4.1. Using an aqueous solution containing 0.1 vol% formic acid and 10 mM ammonium formate as mobile phase A, and an acetonitrile solution containing 0.1 vol% formic acid as mobile phase B, the lower limit of quantification sample (a plasma sample with a concentration of 0.05 ng / mL of the quinoline compound of formula (I), the treatment method is referred to in Example step 2.2), the upper limit of quantification sample (a plasma sample with a concentration of 50 ng / mL of the quinoline compound of formula (I), the treatment method is referred to in Example step 2.2), and the blank reagent (20 vol% acetonitrile aqueous solution) were detected successively according to the gradient elution conditions in Table 9, and the analyte peak areas obtained were 2.66×10 3 、2.81×10 6 、1.62×10 3 , and the residue rate was 61%, see Figure 21 .

[0138] Table 9 Single gradient elution conditions

[0139]

[0140] 4.2. Using an aqueous solution containing 0.1 vol% formic acid and 10 mM ammonium formate as mobile phase A, and an acetonitrile solution containing 0.1 vol% formic acid as mobile phase B, and a solution of methanol - acetonitrile - water - isopropanol containing 2 vol% formic acid (the volume ratio of methanol, acetonitrile, water and isopropanol is 25:25:25:25) as the needle washing liquid, the lower limit of quantification sample (a plasma sample with a concentration of 0.05 ng / mL of the quinoline compound of formula (I), the treatment method is referred to in Example step 2.2), the upper limit of quantification sample (a plasma sample with a concentration of 50 ng / mL of the quinoline compound of formula (I), the treatment method is referred to in Example step 2.2), and the blank reagent (20 vol% acetonitrile aqueous solution) were detected successively according to the gradient elution conditions in Table 10, and the analyte peak areas obtained were 4.49×10 3 、4.51×10 6 、5.16×10 2 , and the residue rate was 11%, see Figure 22 .

[0141] Table 10 Double gradient elution conditions

[0142]

[0143] 4.3. Using an aqueous solution containing 0.1 vol% formic acid and 10 mM ammonium formate as mobile phase A, an acetonitrile solution containing 0.1 vol% formic acid as mobile phase B, and a methanol-acetonitrile-water-isopropanol solution containing 2 vol% formic acid (volume ratio of methanol, acetonitrile, water to isopropanol is 25:25:25:25) as the needle wash solution, the lower limit of quantification sample (a plasma sample with a concentration of 0.05 ng / mL of the quinoline compound of formula (I), the treatment method refers to step 2.2 of the example), the upper limit of quantification sample (a plasma sample with a concentration of 50 ng / mL of the quinoline compound of formula (I), the treatment method refers to step 2.2 of the example) and the blank reagent (20 vol% acetonitrile aqueous solution) were detected according to the gradient elution conditions in Table 11, and the peak areas of the analytes were 2.13×10 3 、1.78×10 6 、0, and the residual rate was 0%, see Figure 23 .

[0144] Table 11 Increased switching valve elution conditions

[0145]

[0146] Comparative Example 1

[0147] This comparative example provides a method for detecting the content of the quinoline compound shown in formula (I). The specific steps and parameters are the same as those in Example 1, except that WeC18 was used as the chromatographic column to detect the lower limit of quantification (a plasma sample with a concentration of 0.05 ng / mL of the quinoline compound of formula (I), the treatment method refers to step 2.2 of the example), the upper limit of quantification sample (a plasma sample with a concentration of 50 ng / mL of the quinoline compound of formula (I), the treatment method refers to step 2.2 of the example). The specific information of the WeC18 chromatographic column is Ultimate XB-C18, 3um, 2.1×50mm.

[0148] The test results are shown in Figures 24 - 25 . It can be seen that the response of the WeC18 chromatographic column to the plasma sample at the lower limit of quantification is not ideal.

[0149] Comparative Example 2

[0150] This comparative example provides a method for detecting the content of the quinoline compound shown in formula (I). The specific steps and parameters are the same as those in Example 1, except that AGC18 was used as the chromatographic column to detect the upper limit and lower limit of quantification. The specific information of the AGC18 chromatographic column is Venusil C18 Plus, 5um, 2.1×50mm.

[0151] The results are shown in Figures 26 - 27, it can be seen that the response of the plasma samples at the lower limit of quantification using the AGC18 chromatographic column is not ideal.

[0152] Comparative Example 3

[0153] This comparative example provides a method for detecting the content of the quinoline compound shown in formula (I). The specific steps and parameters are the same as those in Example 1, except that WAC18 is used as the chromatographic column to detect the upper limit and lower limit of quantification. The specific information of the WAC18 chromatographic column is Ultimate AQ-C18, 5um, 2.1×50mm.

[0154] The results are shown in Figures 28 - 29 , it can be seen that the response of the plasma samples at the lower limit of quantification using the WAC18 chromatographic column is not ideal.

[0155] Obviously, the above-mentioned examples are only for clearly illustrating the examples and are not intended to limit the implementation manners. For those of ordinary skill in the art, other different forms of changes or variations can be made on the basis of the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for detecting the content of a quinoline compound represented by formula (I), characterized in that, It includes the following steps, using liquid chromatography-mass spectrometry to detect the content of quinoline compounds shown in formula (I) in the analyte, the conditions of the liquid chromatography are as follows: using 0.1 vol% formic acid aqueous solution as mobile phase A, using acetonitrile solution as mobile phase B, and the chromatographic column is ACE 5C18; 2. The detection method according to claim 1, wherein mobile phase A further includes 5-10 mM ammonium formate; mobile phase B further includes 0.1 vol% formic acid.

3. The detection method according to claim 2, characterized in that, The concentration of ammonium formate in mobile phase A is 10 mM.

4. The detection method according to any one of claims 1-3, characterized in that the conditions of gradient elution in the liquid chromatography are, At 0.01 min, the volume ratio of mobile phase A to mobile phase B is 70:30, At 1.2 min, the volume ratio of mobile phase A to mobile phase B is 40:60, At 1.3 min, the volume ratio of mobile phase A to mobile phase B is 5:95, At 2 min, the volume ratio of mobile phase A to mobile phase B is 5:95, At 2.1 min, the volume ratio of mobile phase A to mobile phase B is 70:30, At 3.3 min, the volume ratio of mobile phase A to mobile phase B is 40:60, At 3.4 min, the volume ratio of mobile phase A to mobile phase B is 5:95, At 4.1 min, the volume ratio of mobile phase A to mobile phase B is 5:95, At 4.2 min, the volume ratio of mobile phase A to mobile phase B is 70:

30.

5. The detection method according to any one of claims 1-3, characterized in that, The conditions of gradient elution in the liquid chromatography are, At 0.01 min, the volume ratio of mobile phase A to mobile phase B is 65:35, At 1.2 min, the volume ratio of mobile phase A to mobile phase B is 45:55, At 1.3 min, the volume ratio of mobile phase A to mobile phase B is 5:95, At 2 min, the volume ratio of mobile phase A to mobile phase B is 5:95, At 2.1 min, the volume ratio of mobile phase A to mobile phase B is 65:

35.

6. The detection method according to claim 5, wherein During the process of liquid chromatography-mass spectrometry, the sample processed by liquid chromatography is subjected to mass spectrometry analysis at 1.1 min; and / or, During the gradient elution process in the liquid chromatography, it further includes the step of needle washing, the needle washing solution includes formic acid, methanol, acetonitrile, water and isopropanol, the concentration of formic acid in the needle washing solution is 1-3 vol%, the volume ratio of methanol, acetonitrile, water and isopropanol is 1-3:1-3:1-3:1-3.

7. The detection method according to claim 1, characterized in that, Before detecting the analyte by using liquid chromatography-mass spectrometry, it further includes the step of pre-treating the analyte, and the pre-treatment step includes mixing the analyte with a precipitant to obtain a mixed solution, performing solid-liquid separation to obtain a supernatant, and adding a reconstitution solution to the supernatant.

8. The detection method according to claim 7, characterized in that The precipitant includes acetonitrile; and / or, the volume ratio of the analyte to the precipitant is 1:3-5; and / or, the volume ratio of the supernatant to the reconstitution solution is 2:1-3, the reconstitution solution includes acetonitrile, and the concentration of acetonitrile in the reconstitution solution is 10-50 vol%.

9. The detection method according to claim 8, wherein The concentration of acetonitrile in the reconstitution solution is 15-30 vol%; and / or, the solid-liquid separation includes a centrifugation step, the centrifugation time is 10-20 min, and the centrifugation speed is 1500-2000 g.

10. Use of the detection method according to any one of claims 1-9 in determining the content of the quinoline compound represented by formula (I) in plasma.

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