A method for detecting pyridotriazole compounds based on high performance liquid chromatography-tandem mass spectrometry and its application

Abstract

This invention provides a method for detecting pyridotriazole compounds based on high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), comprising the following steps: A) reconstitute the sample and internal standard with the mobile phase after processing, centrifuge, and collect the supernatant to obtain the test solution, wherein the sample contains pyridotriazole compounds; B) perform HPLC-MS / MS detection on the test solution. The method for detecting the drug concentration of pyridotriazole compounds in plasma based on HPLC-MS / MS established in this invention has good specificity, high repeatability, and good stability, and can meet the needs of pharmacokinetic studies.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method and its application for detecting pyridotriazole compounds based on high performance liquid chromatography-tandem mass spectrometry. Background Technology

[0002] SMO inhibitors are among the effective drugs for treating breast cancer. Pyridotriazole compounds are SMO inhibitors, and in vitro activity evaluations have shown varying degrees of inhibitory effects on cancers such as colon cancer, breast cancer, and non-small cell lung cancer, suggesting that pyridotriazole compounds possess anti-breast cancer activity both in vivo and in vitro, with low toxicity, thus showing great development potential. Pharmacokinetics mainly studies the absorption, distribution, metabolism, and excretion of drugs in vivo, and is one of the important research contents in drug development. Therefore, pharmacokinetic studies of pyridotriazole compounds are necessary. One important aspect of pharmacokinetic studies is establishing methods for detecting blood drug concentrations. However, since pyridotriazole compounds are novel synthetic compounds used to treat breast cancer, and in vivo and in vitro studies have shown that these pyridotriazole compounds have good safety and efficacy, blood drug concentration detection is an important part of preclinical pharmacokinetics and phase I clinical trials. Currently, however, no relevant methods for detecting blood drug concentrations have been reported. Summary of the Invention

[0003] In view of this, the technical problem to be solved by the present invention is to provide a method for detecting pyridotriazole compounds based on high performance liquid chromatography-tandem mass spectrometry. The method for detecting the drug concentration of pyridotriazole compounds in plasma based on high performance liquid chromatography-tandem mass spectrometry established by the present invention has good specificity, high repeatability, and good stability, and can meet the needs of pharmacokinetic studies.

[0004] This invention provides a method for detecting pyridotriazole compounds based on high performance liquid chromatography-tandem mass spectrometry, comprising the following steps:

[0005] A) After processing the test sample and internal standard, redissolve them with the mobile phase, centrifuge, and take the supernatant to obtain the test solution. The test sample includes pyridotriazole compounds.

[0006] B) The solution to be tested was detected by high performance liquid chromatography-tandem mass spectrometry.

[0007] Preferably, the pyridotriazole compound has the general formula shown in Formula I:

[0008]

[0009] Wherein, R is selected from phenyl, substituted phenyl, pyridyl, substituted pyridyl, thiazolyl, and oxazolyl.

[0010] Preferably, the pyridotriazole compound is selected from compounds having the following structure:

[0011]

[0012]

[0013]

[0014] Preferably, the chromatographic conditions for the high performance liquid chromatography are as follows: mobile phase A is a 0.1% (v / v) formic acid aqueous solution, mobile phase B is acetonitrile, and the flow rate is 0.3 mL / min;

[0015] The gradient elution procedure is as follows:

[0016] The mobile phase A was 95% from 0 to 5 min.

[0017] The mobile phase A changes from 95% to 20% over 0.5–1 min.

[0018] Mobile phase A is 20% for 1–2.5 min;

[0019] 2.5–2.6 min, mobile phase A changes from 20% to 0%;

[0020] 2.6–3 min, mobile phase A is 0%;

[0021] 3–3.1 min mobile phase A changes from 0% to 20%;

[0022] 3.1–3.2 min; Mobile phase A is 20%;

[0023] 3.2–3.3 min, mobile phase A changes from 20% to 95%;

[0024] 3.3–3.5 min, mobile phase A is 95%.

[0025] Preferably, the high-performance liquid chromatography column is an Agilent ZORBAX StableBond C18 (50 mm × 2.1 mm ID, 3.5 μm), and the column temperature is 25 °C.

[0026] Preferably, the mass spectrometry conditions are: ion source: electrospray ionization source with positive ions (ESI). + ); Scanning method: Multiple response monitoring (MRM); Spray voltage: 5.5kV; Atomizing gas temperature: 450℃; Atomizer pressure: 40psi; Assist gas pressure: 40psi; Collision gas pressure: 4psi; Air curtain pressure: 10psi.

[0027] Preferably, in step A), the processing method for the sample to be tested and the internal standard is as follows:

[0028] The sample to be tested and the internal standard were mixed with methanol aqueous solution, ethyl acetate was added and mixed, and then centrifuged to obtain the supernatant.

[0029] The supernatant was dried to obtain the processed test sample and internal standard.

[0030] Preferably, the internal standard is selected from tinidazole.

[0031] The present invention also provides the application of the above-mentioned method for detecting pyridotriazole compounds based on high performance liquid chromatography-tandem mass spectrometry in the pharmacokinetic evaluation of pyridotriazole compounds and their metabolites in mice.

[0032] Compared with existing technologies, this invention provides a method for detecting pyridotriazole compounds based on high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), comprising the following steps: A) reconstitute the sample and internal standard with the mobile phase after processing, centrifuge, and collect the supernatant to obtain the test solution, wherein the sample contains pyridotriazole compounds; B) perform HPLC-MS / MS detection on the test solution. The method for detecting the drug concentration of pyridotriazole compounds in plasma based on HPLC-MS / MS established in this invention has good specificity, high repeatability, and good stability, and can meet the needs of pharmacokinetic studies. Attached Figure Description

[0033] Figure 1 The chemical structural formula is A15;

[0034] Figure 2 The mass spectrum of A15;

[0035] Figure 3 The mass spectrum of the internal standard tinidazole;

[0036] Figure 4 The chromatogram is shown when the mobile phase is methanol-formic acid-water.

[0037] Figure 5 The chromatogram is shown when the mobile phase is acetonitrile-formic acid aqueous solution.

[0038] Figure 6 The chromatogram is shown when the mobile phase is acetonitrile-formic acid aqueous solution.

[0039] Figure 7 This is a chromatogram of the sample after processing using the protein precipitation method;

[0040] Figure 8 The chromatogram of A15 in blank plasma is shown.

[0041] Figure 9 The chromatogram of A15 in the LLOQ sample;

[0042] Figure 10 The chromatogram shows the internal standard tinidazole in blank plasma.

[0043] Figure 11 The chromatogram of tinidazole, the internal standard in LLOQ;

[0044] Figure 12 For standard curves;

[0045] Figure 13 This is a drug-time curve of A15 in rats. Detailed Implementation

[0046] This invention provides a method for detecting pyridotriazole compounds based on high-performance liquid chromatography-tandem mass spectrometry, characterized by comprising the following steps:

[0047] A) After processing the test sample and internal standard, redissolve them with the mobile phase, centrifuge, and take the supernatant to obtain the test solution. The test sample includes pyridotriazole compounds.

[0048] B) The solution to be tested was detected by high performance liquid chromatography-tandem mass spectrometry.

[0049] In this invention, the pyridotriazole compounds have the general formula shown in Formula I:

[0050]

[0051] Wherein, R is selected from phenyl, substituted phenyl, pyridyl, substituted pyridyl, thiazolyl, and oxazolyl.

[0052] In some specific embodiments of the present invention, the pyridotriazole compounds are selected from compounds having the following structures:

[0053]

[0054]

[0055]

[0056] More preferably, the pyridotriazole compound is selected from A14 or A15.

[0057] A15 Naming:

[0058] 2,6-dichloro-N-(4-chloro-3-(8-chloro-[1,2,4]triazolo[4,3-a]pyridin-3-yl)phenyl)nicotinamide;

[0059] A14 Naming:

[0060] N-(4-chloro-3-(8-chloro-[1,2,4]triazolo[4,3-a]pyridin-3-yl)phenyl)-2-fluoro-4-(trifluoromethyl)benzamide.

[0061] The present invention first processes the sample to be tested and the internal standard. The internal standard is selected from tinidazole.

[0062] The processing methods for the test samples and internal standards are as follows:

[0063] The sample to be tested and the internal standard were mixed with methanol aqueous solution, ethyl acetate was added and mixed, and then centrifuged to obtain the supernatant.

[0064] The supernatant was dried to obtain the processed test sample and internal standard.

[0065] Specifically, the sample to be tested, the internal standard, and a methanol-water solution are mixed to obtain a mixed solution. The sample to be tested is animal plasma, which may or may not contain pyridotriazole compounds.

[0066] The methanol-water solution is a 50% (v / v) methanol-water solution, and the IS concentration in the internal standard is 1000 ng / mL.

[0067] Then ethyl acetate was added to the mixture. The volume ratio of the test sample, methanol aqueous solution, internal standard, and ethyl acetate was 100:10:10:500.

[0068] Next, the solution is thoroughly mixed and then centrifuged to obtain the supernatant.

[0069] After drying the supernatant, it is reconstituted with a mobile phase, wherein the mobile phase includes mobile phase A and mobile phase B, mobile phase A is a 0.1% (v / v) aqueous solution of formic acid, and mobile phase B is acetonitrile.

[0070] The mobile phase used for redissolution is a mixed solution of mobile phase A and mobile phase B with a volume ratio of 3:7.

[0071] The reconstituted sample and internal standard are centrifuged, and the supernatant obtained is the test solution.

[0072] Then, the solution to be tested was detected by high performance liquid chromatography-tandem mass spectrometry.

[0073] The chromatographic conditions for the high performance liquid chromatography are as follows: mobile phase A is a 0.1% (v / v) formic acid aqueous solution, mobile phase B is acetonitrile, and the flow rate is 0.3 mL / min.

[0074] The gradient elution procedure is as follows:

[0075] The mobile phase A was 95% from 0 to 5 min.

[0076] The mobile phase A changes from 95% to 20% over 0.5–1 min.

[0077] Mobile phase A is 20% for 1–2.5 min;

[0078] 2.5–2.6 min, mobile phase A changes from 20% to 0%;

[0079] 2.6–3 min, mobile phase A is 0%;

[0080] 3–3.1 min mobile phase A changes from 0% to 20%;

[0081] 3.1–3.2 min; Mobile phase A is 20%;

[0082] 3.2–3.3 min, mobile phase A changes from 20% to 95%;

[0083] 3.3–3.5 min, mobile phase A is 95%.

[0084] The high-performance liquid chromatography column was an Agilent ZORBAX StableBond C18 (50 mm × 2.1 mm I.D., 3.5 μm), with a column temperature of 25 °C.

[0085] The mass spectrometry conditions are as follows: Ion source: Electrospray ionization source with positive ions (ESI). + ); Scanning method: Multiple response monitoring (MRM); Spray voltage: 5.5kV; Atomizing gas temperature: 450℃; Atomizer pressure: 40psi; Assist gas pressure: 40psi; Collision gas pressure: 4psi; Air curtain pressure: 10psi.

[0086] The present invention also provides the application of the above-mentioned method for detecting pyridotriazole compounds based on high performance liquid chromatography-tandem mass spectrometry in the pharmacokinetic evaluation of pyridotriazole compounds and their metabolites in mice.

[0087] The method for detecting the drug concentration of pyridotriazole compounds in plasma based on high performance liquid chromatography-tandem mass spectrometry established in this invention has good specificity, high repeatability, and good stability, and can meet the needs of pharmacokinetic studies.

[0088] To further understand the present invention, the method and application of detecting pyridotriazole compounds based on high performance liquid chromatography-tandem mass spectrometry provided by the present invention are described below with reference to the embodiments. The scope of protection of the present invention is not limited to the following embodiments.

[0089] Example 1

[0090] 1. Materials and Methods

[0091] 1.1 Instruments and Reagents

[0092] The high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) system consisted of a Shimadzu UFLC series LC-20AD binary pump, a SIL-20A multi-purpose autosampler, and an AB SCIEX API3000 mass spectrometer detector (ESI ion source). The chromatographic column was an Agilent ZORBAX StableBond C18 column (50 mm × 2.1 mm ID, 3.5 mm particle size). A15 standard (purity 99.5%) was provided by Southern Medical University; tinidazole (purity 99.8%) was purchased from the China National Institutes for Food and Drug Control; rat plasma was provided by Guangzhou Huateng Biomedical Technology Co., Ltd.; acetonitrile and methanol were chromatographically pure and purchased from Merck; heparin sodium, ethyl acetate, dimethyl sulfoxide (DMSO), and formic acid (FA) were purchased from Sigma-Aldrich.

[0093] 1.2. Laboratory Animals

[0094] Five male Sprague-Dawley rats, weighing 200±20g, were provided by Zhuhai Baitong Biotechnology Co., Ltd.

[0095] 1.3. Preparation of stock solution and working solution

[0096] Accurately weigh A15 standard and prepare a 2 mg / mL stock solution with DMSO. Dilute the stock solution with methanol:water (1:1, v / v) to prepare working standard solutions of 100, 200, 500, 1000, 2000, 5000, 10000, and 20000 ng / mL. Separately, accurately weigh A15 standard and prepare a 2 mg / mL stock solution with DMSO. Dilute the stock solution with methanol:water (1:1, v / v) to prepare quality control (QC) sample working solutions of 100, 300, 1500, and 15000 ng / mL. Accurately weigh tinidazole and prepare a 2 mg / mL internal standard (IS) stock solution with methanol. Dilute the tinidazole stock solution with methanol to prepare a 1000 ng / mL internal standard working solution. Store all stock solutions and working solutions at -20°C.

[0097] 1.4 Preparation of Standard Samples and Quality Control Samples

[0098] Add 10 μL of standard working solutions of 100, 200, 500, 1000, 2000, 5000, 10000, and 20000 ng / mL to 100 μL of blank rat plasma, vortex for 30 s, and prepare standard plasma samples of 10, 20, 50, 100, 200, 500, 1000, and 2000 ng / mL. Add 10 μL of QC sample working solutions of 100, 300, 1500, and 15000 ng / mL to 100 μL of blank rat plasma, vortex for 30 s, and prepare quality control samples with concentrations of 10 (LLOQ), 30 (LQC), 150 (MQC), and 1500 (HQC) ng / mL.

[0099] 1.5 Sample Pretreatment

[0100] Add 10 μL of internal standard (concentration 1000 ng / mL) to “1.4 Standard Samples and Quality Control Samples”, vortex for 10 s, then add 500 μL of ethyl acetate, vortex for 30 s, centrifuge at 14800 rpm for 5 minutes, and transfer 400 μL of the supernatant to a new EP tube for vacuum drying; take 100 μL of the rat plasma sample to be tested, add 10 μL of methanol:water (1:1, v / v) and 10 μL of internal standard (IS concentration 1000 ng / mL) to the sample. Vortex for 10 seconds, then add 500 μL of ethyl acetate, vortex for 30 seconds, centrifuge at 14800 rpm for 5 minutes, and transfer 400 μL of the supernatant to a new EP tube. Dry under vacuum. Add 100 μL of reconstitution solution (30% formic acid solution + 70% acetonitrile) to the dried EP tube, vortex for 30 seconds, centrifuge at 14800 rpm and 4°C for 10 minutes, and transfer 80 μL of the supernatant to a sample vial for LC-MS / MS analysis.

[0101] 1.6. Instrumental Analysis Conditions

[0102] 1.6.1 Chromatographic conditions

[0103] The chromatographic column was an Agilent ZORBAX StableBond C18 (50 mm × 2.1 mm ID, 3.5 μm), the column temperature was 25 °C, the mobile phase A was 0.1% formic acid water, and the mobile phase B was acetonitrile. Gradient elution was performed according to the conditions in Table 1.

[0104] Table 1A15 and Internal Standard Liquid Phase Conditions

[0105]

[0106] The liquid phase conditions for A14 are the same as those for A15.

[0107] 1.6.2 Mass Spectrometry Conditions

[0108] Ion source: Electrospray ionization source with positive ions (ESI) +); Scanning method: multiple reaction monitoring (MRM); Spray voltage: 5.5 kV; Nebulizer gas temperature: 450 °C; Nebulizer pressure: 40 psi; Assist gas pressure: 40 psi; Collision gas pressure: 4 psi; Curtain gas pressure: 10 psi; Mass spectrometry parameters of A15 and tinidazole are shown in Table 2.

[0109] Table 2A15 and Internal Standard Mass Spectrometry Parameters

[0110]

[0111] 1.7 Methodological Validation

[0112] Based on the validation guidelines for quantitative analysis methods of biological samples in the 2020 edition of the Chinese Pharmacopoeia, the selectivity, limit of quantitation, accuracy and precision, matrix effect, extraction recovery, dilution reliability, residues, and stability of the method were investigated.

[0113] 1.7.1 Specificity, standard curve, and lower limit of quantitation

[0114] Specificity was determined by extracting and analyzing six batches of rat plasma. Sample preparation included: double-blank (blank rat plasma) with an internal standard and LLOQ added. Interference peaks at the A15 and internal standard elution positions were examined. The response value of the A15 retention time position in the blank plasma was ≤20% of the LLOQ sample response value; the response value of the IS elution position was ≤5% of the IS response value.

[0115] Based on the correlation between the A15 standard concentration and the ratio of the peak areas of A15 and the internal standard, a standard curve was fitted using a 1 / x*x weighted least squares linear regression. The LLOQ is the lowest point of the standard concentration, and the signal-to-noise ratio (SNR) is obtained by comparing the response value of A15 at retention time (RT) with the blank baseline; its SNR should be greater than 10.

[0116] 1.7.2 Accuracy and Precision

[0117] The accuracy and precision of the method for detecting A15 in rat plasma were evaluated using quality control samples at four concentrations: 10 (LLOQ), 30 (LQC), 150 (MQC), and 1500 (HQC), with six replicates for each concentration. Accuracy was assessed by calculating the deviation (%) between the standard concentration and the actual detected concentration; the deviation at each concentration level should be 100 ± 15% (100 ± 20% at the LLOQ level). Inter-batch and intra-batch precision were assessed by calculating the coefficient of variation of the average analytical concentration; the precision at each concentration level should be ≤15% (LLOQ precision ≤20%). Intra-batch accuracy and precision were determined three times, and the results from the three batches were used for inter-batch determination.

[0118] 1.7.3 Matrix effect and extraction recovery

[0119] A15 was added to a reconstituted solution of blank rat plasma obtained through liquid-liquid extraction to prepare matrix-containing samples of 30 ng / mL and 1500 ng / mL. Matrix-free samples of 30 ng / mL and 1500 ng / mL were also prepared using the reconstituted solution for analysis. The matrix factors of A15 and the internal standard were obtained by dividing the peak area of ​​the matrix-containing sample by the matrix-free sample. The matrix factor normalized to the internal standard was calculated by dividing the matrix factor of the analyte by the matrix factor of the internal standard. The coefficient of variation of the matrix factor normalized to the internal standard calculated from 6 batches of matrix samples should not exceed 15%.

[0120] Standard samples and matrix extracts at concentrations of 30 ng / mL and 1500 ng / mL were prepared and analyzed. The peak area ratio of the analyte in the extracted standard samples to that in the matrix-containing samples was compared to evaluate the extraction recovery rate.

[0121] 1.7.4 Residue

[0122] After testing the highest concentration standard sample, inject a blank sample. The response value of A15 in the blank sample should not exceed 20% of the LLOQ, and the response value of the internal standard should not exceed 5% of the response value of the high internal standard.

[0123] 1.7.5 Dilution Reliability

[0124] Prepare a standard plasma sample with a concentration of 15000 ng / mL, dilute it 10 times with blank rat plasma, extract and analyze it. The precision and accuracy should be within ±15% to prove the reliability of the dilution.

[0125] 1.7.6 Stability

[0126] The stability of the analyte in rat plasma matrix was assessed using two quality control samples (n=6) at concentrations of 30 ng / mL and 1500 ng / mL. The stability assessment conditions were as follows: 24 hours at room temperature, two months at -20°C, three freeze-thaw cycles, and simultaneous evaluation of the stability of the extract at 4°C for 24 hours in an autosampler. The test QC samples were compared with freshly prepared samples; if the deviation was ≤15%, the sample was considered stable under the storage conditions.

[0127] 1.8 Application of Analytical Methods

[0128] Experimental rats were fasted for 12 hours before drug administration but had free access to water. A15 concentration of 5 mg / mL was prepared using 0.5% HPMC and 0.3% Tween-80 solution and administered to rats via gavage at a volume of 10 mL / kg, resulting in a dose of 50 mg / kg. At 0, 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 hours post-administration, approximately 300 μL of blood was collected via the orbital venous plexus into heparin-treated centrifuge tubes. The tubes were centrifuged at 4000 rpm for 10 minutes. Plasma samples were then transferred to new EP tubes and stored at -20°C until analysis. The obtained blood drug concentration data were processed using DAS 2.1.1 software, and pharmacokinetic parameters for oral administration in rats were calculated using a statistical moment model.

[0129] 2 Results and Discussion

[0130] 2.1 Optimization of Liquid Chromatography-Mass Spectrometry Conditions

[0131] This study optimized various parameters by directly injecting A15 standard solution (1 μg / mL) using a peristaltic pump. The molecular structure of A15 is as follows: Figure 1 As shown, the molecular weight is 453.2, and the hydrogen-generating precursor ion [M+H] was obtained in Q1 full scan mode. + The process employs a daughter ion scanning mode, which scans daughter ions by varying the collision energy. Figure 2 The image shows the daughter ion scan of A15 under a collision energy of 85 eV. The daughter ions include 92.0, 99.8, 127.2, and 148.3. Among them, the 99.8 daughter ion is relatively stable, so 99.8 was selected as the daughter ion for quantitative analysis. Figure 3 The mass spectrum of the internal standard tinidazole is shown. As can be seen from the figure, the precursor ion of tinidazole is 248.0, and its main daughter ion is 121.1. Therefore, this daughter ion was selected as the ion pair for internal standard quantification.

[0132] 2.1.1 Mobile phases A and B are 0.1% formic acid water and methanol, respectively.

[0133] The gradient elution procedure is shown in Table 3, and the results are shown in Table 4. Figure 4

[0134] Table 3

[0135]

[0136] 2.1.2 Mobile phases A and D are 0.1% formic acid water and acetonitrile, respectively.

[0137] The gradient elution procedure is shown in Table 4, and the results are shown in Table 5. Figure 5

[0138] Table 4

[0139]

[0140] 2.1.3 Mobile phases A and D are 0.1% formic acid water and acetonitrile, respectively.

[0141] The gradient elution procedure is shown in Table 5, and the results are shown in Table 6. Figure 6

[0142] Table 5

[0143]

[0144] First, methanol and acetonitrile were investigated as mobile phases. It was found that using methanol as the organic phase resulted in peak tailing and prolonged elution time. Considering the potentially weak elution ability of methanol, acetonitrile, with its stronger elution ability, was used as the organic phase. Adding a small amount of formic acid to the mobile phase improved the peak shape for better integration and quantification. Finally, the mobile phase consisted of 0.1% formic acid in water and pure acetonitrile. An Agilent ZORBAX Stable Bond C18 column (50 mm × 2.1 mm ID, 3.5 μm particle size) was used, and the analysis time was 3.5 min. This method employed electrospray ionization in positive ion mode. The precursor ion of A15 is 454.2, and the daughter ion fragments are 92.0, 100.0, and 127.2. When optimizing the mass spectrometry and chromatographic conditions, it was found that the response of 100.0 among the daughter ion fragments of A15 was the highest. Therefore, 454.2→100.0 was selected as the quantitative ion pair. The monitoring conditions of the mass spectrometry were then optimized to find the most suitable mass spectrometry parameters for the compound.

[0145] 2.2 Optimization of Sample Pretreatment Conditions

[0146] The literature reports that commonly used biological sample processing methods include protein precipitation, liquid-liquid extraction, and solid-liquid extraction. This experiment tested all three methods.

[0147] When using protein precipitation, it was found that peak splitting and low extraction efficiency occurred after extraction and injection (as shown in Table 6). This could not be avoided regardless of whether methanol or acetonitrile was used as the precipitant. Therefore, protein precipitation was not used. Solid-liquid extraction produced good chromatographic peak shapes and high extraction efficiency, but the processing was too cumbersome. Considering that a large number of bioanalytical samples would be generated in the experiment, a complicated sample processing process would consume a lot of time. Therefore, other simpler methods were given priority.

[0148] The specific method for processing samples using protein precipitation (taking acetonitrile as an example).

[0149] • System: 5 μL standard solution + 45 μL blank plasma + 150 μL ice-cold acetonitrile solution containing internal standard;

[0150] • Sample preparation: Take 50 μL of plasma into an EP tube, add 150 μL of ice-cold acetonitrile solution containing internal standard, vortex for 2 min, centrifuge at 14800 rpm for 10 min at 4℃, take 130 μL of supernatant into a new EP tube, centrifuge at 14800 rpm for 10 min at 4℃, and inject 5 μL of supernatant for analysis.

[0151] See results Figure 7 Since chromatographic peaks were found to be split, protein precipitation was not used; instead, liquid-liquid extraction was selected.

[0152] When using liquid-liquid extraction, various extractants were compared: chloroform, n-butanol, ethyl acetate, ethylene oxalate, and petroleum ether. It was found that ethyl acetate had the highest extraction efficiency (as shown in Table 7), the extract had a good peak shape, and there were no phenomena such as tailing or peak splitting. In addition, liquid-liquid extraction has a shorter processing time and a simpler process. Therefore, liquid-liquid extraction was adopted as the pretreatment method for A15 plasma samples.

[0153] The experiment compared the protein precipitation with methanol and acetonitrile, but the recovery rate was low and the chromatographic peak shape was poor. Liquid-liquid extraction with tert-butyl ether, diethyl ether, and ethyl acetate was used. The results showed that ethyl acetate had a high extraction recovery rate and no obvious interfering substances were found. Therefore, ethyl acetate was selected as the extraction solvent.

[0154] To reduce residual effects, the choice of cleaning solution was optimized. The cleaning effects of acetonitrile, 70% acetonitrile, 50% acetonitrile, methanol, 70% methanol and 50% methanol were compared. It was found that when methanol was used as the cleaning agent, the sample residue was the least (as shown in Table 8). Therefore, methanol was selected as the cleaning agent for A15 and the injection needle was cleaned after each injection.

[0155] The optimized method described above has been validated and can be used for the quantitative analysis of A15 in rat plasma.

[0156] Table 6. Protein precipitation extraction recovery rate

[0157]

[0158] Table 7 Effect of different extraction solvents on extraction recovery rate

[0159]

[0160]

[0161] Table 8. Effects of different injection washing solutions on residues

[0162]

[0163] 2.3 Specificity, standard curve, and lower limit of quantitation

[0164] Figure 8 The image shows the chromatogram of A15 in blank plasma. Figure 9 The chromatogram of A15 in the LLOQ sample shows that there are no endogenous interfering substances in the blank plasma. Figure 10 This is the chromatogram of tinidazole as an internal standard in blank plasma. Figure 11 The chromatogram of tinidazole, the internal standard in LLOQ, shows that no endogenous interfering substances were found, indicating that the analytical method has good specificity.

[0165] Figure 12 The A15 standard curve fitted by 1 / x*x weighted least squares linear regression showed good linearity in the quantification range of 10-2000 ng / mL, r 2 The value ranges from 0.9929 to 0.9997.

[0166] The signal-to-noise ratio calculated from the response values ​​of the A15 chromatographic peak and the blank in the LLOQ sample is greater than 10, and the precision and accuracy of LLOQ meet the requirements. Therefore, the limit of quantitation is 10 ng / mL.

[0167] 2.4 Accuracy and Precision

[0168] Table 9 shows the intra-batch and inter-batch precision and accuracy of A14 and A15 LLOQ, LQC, MQC and HQC. The intra-batch accuracy is 4.5-10.5% and the precision is 93.8-105.5%; the inter-batch accuracy is 3.2-10.8% and the precision is 96.0-106.3%, all of which meet the requirements, and the intra-batch and inter-batch precision and accuracy are good.

[0169] Table 9. Intra-batch and inter-batch precision and accuracy

[0170]

[0171] 2.5 Matrix effect and extraction recovery

[0172] As shown in Table 10, the internal standard normalized matrix factors of A14 and A15 in LQC, MQC and HQC samples ranged from 0.91 to 1.07, and the coefficient of variation of matrix factors was less than or equal to 5.4%; the extraction recovery rate was high, all greater than 90%.

[0173] Table 10 Extraction recoveries and matrix effects of A14 and A15

[0174]

[0175] 2.6 Residue

[0176] After detecting the ULOQ sample of A15 in rat plasma, the detection system injected a blank sample. No obvious response was observed at the retention times of A15 and the internal standard. Therefore, no residual effect of A15 was observed under the conditions of this analytical method.

[0177] 2.7 Dilution Reliability

[0178] A standard sample with a concentration of 15000 ng / mL was diluted to 1500 ng / mL with blank rat plasma. Six replicates were performed, and the average concentration was 1495 ng / mL. The precision was 0.3%, and the accuracy was 103.8%. A 10-fold dilution did not affect the precision and accuracy of the detection.

[0179] 2.8 Stability

[0180] The stability of A14 and A15 in rat plasma was investigated after being placed at room temperature for 24 hours, repeatedly frozen and thawed at -20℃, stored at -20℃ for 60 days, and after liquid-liquid extraction and reconstitution was placed at 4℃ for 24 hours. The results are shown in Table 11. Under these conditions, A14 and A15 were relatively stable.

[0181] Table 11 Stability of A14 and A15

[0182]

[0183] 2.9 Analytical methods applied to pharmacokinetic studies in rats

[0184] The established analytical method was applied to the pharmacokinetic study of A15 in rats. After gavage administration of 50 mg / kg A15, the drug-time curve in rats is shown below. Figure 13 As shown in Table 12, the pharmacokinetic parameters were fitted using DAS2.1.1 software. The Cmax was 1831.8 ± 653.8 ng / mL. According to the guidelines, the lower limit of quantitation is 1 / 20 of Cmax, i.e., 91.6 ng / mL. The linear range of this method is 10-2000 ng / mL, and all blood drug concentrations fall within this range, indicating that this analytical method is suitable for pharmacokinetic studies of A15.

[0185] Table 12A15 Pharmacokinetic parameters in rats

[0186]

[0187]

[0188] 3. Conclusion

[0189] For the first time, LC-MS / MS analytical methods for compounds A14 and A15 were established and validated. The results showed that the established method had good specificity, the linear range met the requirements of pharmacokinetic studies, was relatively stable under the storage conditions indicated, had high extraction recovery, no residual effect, and good precision and accuracy.

[0190] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting pyridotriazole compounds based on high performance liquid chromatography-tandem mass spectrometry, characterized in that, Includes the following steps: A) After processing the test sample and internal standard, redissolve them with the mobile phase, centrifuge, and collect the supernatant to obtain the test solution. The test sample includes pyridotriazole compounds, which are selected from compounds having the following structures: The processing method for the test sample and internal standard is as follows: The test sample and internal standard were mixed with methanol aqueous solution, ethyl acetate was added and mixed, and then centrifuged to obtain the supernatant. The test sample was selected from the plasma of the animal to be tested. The supernatant was dried to obtain the processed test sample and internal standard. B) The solution to be tested is detected by high performance liquid chromatography-tandem mass spectrometry. The chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column of the high performance liquid chromatography is an Agilent ZORBAX StableBond C18. Mobile phase A is a 0.1% (v / v) aqueous solution of formic acid, and mobile phase B is acetonitrile; The gradient elution procedure is as follows: The mobile phase A is 95% during the 0~0.5 min period; The mobile phase A changes from 95% to 20% in 0.5~1 min. The mobile phase A is 20% for 1~2.5 min; 2.5~2.6 min, mobile phase A changes from 20% to 0%; 2.6~3 min, mobile phase A is 0%; 3~3.1 min mobile phase A changes from 0% to 20%; 3.1~3.2 min, mobile phase A is 20%; 3.2~3.3 min, mobile phase A changes from 20% to 95%; 3.3~3.5 min, mobile phase A is 95%.

2. The method according to claim 1, characterized in that, The flow rate of the high-performance liquid chromatography was 0.3 mL / min.

3. The method according to claim 1, characterized in that, The column temperature of the high-performance liquid chromatography column is 25°C.

4. The method according to claim 1, characterized in that, The mass spectrometry conditions are as follows: Ion source: Electrospray ionization source with positive ions (ESI). + Scanning method: Multiple reaction monitoring (MRM); Spray voltage: 5.5kV; Atomizing gas temperature: 450℃; Atomizer pressure: 40psi; Auxiliary gas pressure: 40 psi; Impact air pressure: 4 psi; Air curtain pressure: 10 psi.

5. The method according to claim 1, characterized in that, The internal standard was selected from tinidazole.

6. The use of the method for detecting pyridotriazole compounds based on high performance liquid chromatography-tandem mass spectrometry as described in any one of claims 1 to 5 in the pharmacokinetic evaluation of pyridotriazole compounds and their metabolites in mice.

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