Liquid chromatography-tandem mass spectrometry analysis method for detecting XY028-140 in blood

Abstract

The invention relates to the field of medicine analysis, and particularly discloses a liquid chromatography-tandem mass spectrometry analysis method for detecting XY028-140 in blood. The method comprises the steps of plasma sample solution preparation, standard curve sample solution preparation and LC-MS / MS detection. According to the method, accurate quantitative detection of the XY028-140 in the plasma can be achieved, the detection result is high in accuracy and good in precision, operation is easy and convenient, and the method can be effectively applied to biological analysis in the drug development process of the PROTAC drug XY028-140.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical analysis. More specifically, it relates to a liquid chromatography-tandem mass spectrometry analysis method for detecting XY028-140 in blood. Background Art

[0002] Targeted protein degradation chimeras, also known as PROTACs, are an emerging chemical biology method that utilizes the intracellular ubiquitin-proteasome system to recruit E3 ligases to target proteins and induce ubiquitination through the proteasome to achieve targeted protein degradation. PROTACs are heterobifunctional molecules that contain two different ligands in their structure. One is a ligand that binds to the target protein, and the other is a ligand that binds to the E3 ubiquitin ligase. The two ligands are connected by a linker to form a ternary complex with a specific three-dimensional structure. Different from traditional small molecule inhibitors, PROTACs do not need to bind to the active site of the target with high affinity, so they can selectively degrade targets that are traditionally considered "undruggable". In recent years, PROTAC technology has shown broad application prospects in multiple fields such as cancer treatment, neurological diseases, and immune inflammation. With the continuous maturity of the technology and the accumulation of more clinical data, PROTAC is expected to become one of the key technologies for future blockbuster drugs and overcome a series of disease targets that are currently considered difficult to treat.

[0003] XY028-140 (MS140) is a novel CDK4 / CDK6-selective small molecule PROTAC degrader with a CAS number of 2229974-83-6 and a structural formula of XY028-140 can efficiently and selectively target the CDK4 / CDK6 kinases in CDK4 / CDK6 inhibitor-sensitive tumor cells to the proteasome system composed of CRL4-Cereblon-E3 ubiquitin ligase, thereby degrading the CDK4 / CDK6 kinases and achieving the inhibition of cancer cell proliferation to achieve an anti-cancer effect, providing new ideas and methods for the treatment of various cancers and having high potential application value in the field of cancer treatment.

[0004] Currently, XY028-140 has specifically inhibited the RB-E2F signaling pathway in in vitro studies, reduced the protein expression levels of CDK4 and CDK6, showed a dose- and time-dependent relationship, and demonstrated good CDK4 / CDK6 degradation effects. However, there is no reported bioanalytical method for detecting XY028-140 in plasma yet. To better ensure the effectiveness and safety of the drug, evaluate the treatment effect, obtain the best treatment plan, and avoid potential drug conflicts or adverse reactions, it is very necessary to develop a rapid, sensitive, and efficient bioanalytical method to detect XY028-140 in plasma. Summary of the Invention

[0005] Plasma in this application is reasonably understood by those skilled in the art as experimental animal plasma, human plasma, matrix for in vitro biotransformation experiments, cell culture matrix, or microbial culture medium, etc. This is because in the prior art, for example, serum medium is usually contained in in vitro biotransformation experiments or in vitro cell test matrices. This application takes plasma samples as an example for illustration. The treatment of biotransformation or cell culture matrices can be completely obtained by using the well-known technical means of those skilled in the art. That is, LC-MS / MS is the key step in the method for detecting XY028-140 in plasma described in this application. Based on this, the LC-MS / MS method for detecting XY028-140 in blood in this application can be understood as a method for detecting XY028-140 in biological samples. Those skilled in the art can use this method for pharmacokinetic experiments, cytotoxicity experiments, in vivo animal safety experiments, bioequivalence experiments, microbial simulation of human drug-metabolizing enzyme metabolism experiments, etc. without creative labor.

[0006] To solve the above technical problems, this application provides a liquid chromatography tandem mass spectrometry analysis method for detecting XY028-140 in blood, which can achieve accurate quantitative detection of XY028-140 in plasma or samples containing serum matrix, with rapid detection and high accuracy and precision of the results.

[0007] More specifically, a liquid chromatography tandem mass spectrometry analysis method for detecting XY028-140 in blood provided by the present application includes the preparation of a plasma sample solution, the preparation of a standard curve sample solution, and LC-MS / MS detection. The conditions of LC in the LC-MS / MS detection include using an Xbridge C18 chromatographic column, with a column temperature of 30°C - 50°C, a flow rate of 0.5 mL / min - 0.7 mL / min, mobile phase A being an aqueous formic acid solution with a concentration of 0.1 wt% - 0.5 wt%, and mobile phase B being a formic acid acetonitrile solution with a formic acid concentration of 0.1 wt% - 0.5 wt%, for gradient elution; the scanning mode used in the MS / MS detection analysis is the multiple reaction ion detection scanning mode. In the multiple reaction ion detection scanning mode, the DP of XY028-140 is 85 V - 95 V, the CE is 50 V - 60 V, the EP is 10 V, the CXP is 5 V - 15 V, the DP of the internal standard is 135 V - 145 V, the CE is 35 V - 45 V, the EP is 10 V, and the CXP is 5 V - 15 V; the CAD in the MS / MS detection analysis is 8 - 10, the GS1 is 40 psi - 60 psi, the GS2 is 40 psi - 60 psi, and the curtain gas is 25 psi - 40 psi.

[0008] Through the above technical solution, high-sensitivity and high-accuracy LC-MS / MS detection of XY028-140 in plasma is achieved. Specifically, an Xbridge C18 chromatographic column is used, with a column temperature of 30°C - 50°C, a flow rate of 0.5 mL / min - 0.7 mL / min, mobile phase A being an aqueous formic acid solution with a concentration of 0.1 wt% - 0.5 wt%, and mobile phase B being a formic acid acetonitrile solution with a formic acid concentration of 0.1 wt% - 0.5 wt% for gradient elution, ensuring the effective separation of the target compound and the appearance of a good peak shape, improving the resolution and reproducibility of the detection; the selection of each parameter in the multiple reaction ion detection scanning mode can optimize the ionization efficiency and signal intensity of the target compound, reduce background interference, and improve the sensitivity and selectivity of the detection; the selection of each parameter in the MS / MS detection analysis further optimizes the ion transmission and detection environment, ensuring stable and reliable detection results.

[0009] Optionally, the conditions for the gradient elution of LC are as follows: at 0 - 0.3 minutes, mobile phase A is 90% and mobile phase B is 10%; at 0.3 - 1.4 minutes, mobile phase A changes from 90% to 5% and mobile phase B changes from 10% to 95%; at 1.4 - 1.8 minutes, mobile phase A is 5% and mobile phase B is 95%; at 1.8 - 1.9 minutes, mobile phase A changes from 5% to 90% and mobile phase B changes from 95% to 10%; at 1.9 - 2.5 minutes, mobile phase A is 90% and mobile phase B is 10%.

[0010] Through the above technical solution, the method can achieve high-sensitivity and high-accuracy detection of XY028-140 in plasma. Specifically, by optimizing the gradient elution conditions of LC, the separation effect of XY028-140 on the chromatographic column is ensured, thereby improving the detection resolution and reproducibility. At 0 - 0.3 minutes, mobile phase A is 90% and mobile phase B is 10%, ensuring a stable baseline in the initial stage; at 0.3 - 1.4 minutes, mobile phase A gradually decreases to 5% and mobile phase B gradually increases to 95%, enabling the rapid elution of the target compound; at 1.4 - 1.8 minutes, mobile phase A is maintained at 5% and mobile phase B is maintained at 95%, ensuring the complete elution of the target compound; at 1.8 - 1.9 minutes, it quickly returns to the initial conditions (mobile phase A is 90% and mobile phase B is 10%) for the preparation of the next injection; at 1.9 - 2.5 minutes, the initial conditions are maintained to further ensure a stable baseline. The optimization of these gradient elution conditions significantly improves the detection sensitivity and accuracy.

[0011] Optionally, in the detection of LC, 5 mM - 10 mM ammonium formate or ammonium acetate is further added to mobile phase A; 5 mM - 10 mM ammonium formate or ammonium acetate is further added to mobile phase B.

[0012] Through the above technical solution, adding 5 mM - 10 mM ammonium formate or ammonium acetate to mobile phase A and mobile phase B respectively can effectively improve the sensitivity and stability of LC-MS / MS detection, reduce matrix effects, and ensure the accuracy and reliability of the detection results.

[0013] Optionally, in the multiple reaction ion detection scan mode, the DP of XY028-140 is 90 V, the CE is 55 V, the EP is 10 V, the CXP is 10 V, the DP of the internal standard is 140 V, the CE is 40 V, the EP is 10 V, and the CXP is 10 V.

[0014] Through the above technical solution, the DP is the declustering voltage, and its main function is to disperse the molecules and ions aggregated in clusters in the sample liquid flow entering the mass spectrometer, which is more conducive to ionization into ions, reduces the influence of molecular clusters on ionization, and can improve the detection sensitivity. For XY028-140, its DP range is 85 V - 95 V, and the sensitivity is better at 90 V. The CE is the collision energy, and the fragmentation of the analyte is achieved by collision with an inert gas to generate characteristic product ions. The abundance or response of characteristic ions is greatly affected under different collision energies. Optimizing this parameter can improve the detection sensitivity. Therefore, after optimization, for XY028-140, its CE is 50 V - 60 V, and the response is better at 55 V.

[0015] Optionally, in the MS / MS detection and analysis, CAD is 9, GS1 is 50 psi, GS2 is 50 psi, and the curtain gas is 35 psi.

[0016] Through the above technical solutions, a more stable electrospray ionization process can be achieved, improving the detection sensitivity and reproducibility. Specifically, setting CAD to 9 can effectively reduce background interference and increase the response value of the target compound; setting both GS1 and GS2 to 50 psi can optimize the desolvation process and ensure the ion transmission efficiency; setting the curtain gas to 35 psi can effectively prevent contamination and maintain the cleanliness and stable operation of the mass spectrometry system. The precise setting of these parameters makes the entire LC-MS / MS detection method more reliable and accurate.

[0017] This application takes plasma treatment as an example to illustrate the preparation of biological matrix sample solutions. Those skilled in the art have reason to consider it as a conventional technical means, that is, non-creative labor substitutions can be made on this basis. The detailed content is as follows: The preparation of the plasma sample solution is specifically as follows: Under the condition of 2°C - 8°C, the plasma sample containing anticoagulant is mixed with the precipitant containing internal standard, and after centrifugation, it is diluted with the first diluent to obtain the sample solution to be measured.

[0018] Optionally, the anticoagulant is K 2 EDTA; Optionally, the internal standard is verapamil or tolbutamide; Optionally, the precipitant is one of acetonitrile, acetonitrile solution with 0.1 wt% formic acid, methanol, and methanol solution with 0.1 wt% formic acid.

[0019] The precipitant containing internal standard is a solution with an internal standard concentration of 0.1 ng / mL prepared by adding the above internal standard to the above precipitant.

[0020] The centrifugation treatment is specifically as follows: Centrifuge at about 3000 g for about 10 - 15 minutes, take the supernatant and dilute it with the first diluent to obtain the sample solution to be measured.

[0021] The preparation of the standard curve sample solution includes: Mixing the XY028 - 140 standard working solution with a concentration of 2 ng / mL - 2000 ng / mL with the blank matrix containing anticoagulant to obtain a standard curve sample with a concentration of 0.1 ng / mL - 100 ng / mL. Under the condition of 2°C - 8°C, mix the standard curve sample with the precipitant containing internal standard, and after centrifugation, dilute it with the first diluent to obtain the standard curve sample solution; The XY028-140 standard working solution is prepared through the following steps: precisely weigh the XY028-140 standard product and dissolve it in dimethyl sulfoxide to obtain an XY028-140 stock solution with a concentration of 1 mg / mL of XY028-140, and then dilute it with a second diluent to prepare an XY028-140 standard working solution with a concentration gradient. In a specific embodiment of the present application, the concentration gradient of the XY028-140 standard working solution is set to 2 ng / mL, 4 ng / mL, 10 ng / mL, 40 ng / mL, 200 ng / mL, 800 ng / mL, 1600 ng / mL, and 2000 ng / mL.

[0022] Modifications made by those skilled in the art according to the guiding principles of relevant drug inspection regulations without creative efforts, for example, among the 8 concentration points selected in practice according to the standard curve, the second concentration point from low to high is twice the lowest concentration point, and the concentration of the seventh concentration point from low to high is 80% of the eighth concentration point, and appropriate adjustments to the remaining intermediate concentration points are also within the scope of the method of the present application. That is, the concentration gradient of the standard working solution in the examples of the present application being set to 2 ng / mL, 4 ng / mL, 10 ng / mL, 40 ng / mL, 200 ng / mL, 800 ng / mL, 1600 ng / mL, and 2000 ng / mL is only one example, rather than all design schemes.

[0023] In summary, the present application has the following beneficial technical effects: The present application first prepares a plasma sample solution and a standard curve sample solution containing XY028-140 by selecting appropriate internal standards and precipitants, and then uses liquid chromatography-tandem triple quadrupole mass spectrometry for detection, which can achieve accurate quantitative detection of XY028-140 in plasma. The detection results have high accuracy, good precision, and simple operation, and can be effectively applied to the bioanalysis in the drug development process of the PROTAC drug XY028-140, including pharmacokinetic studies and safety evaluation studies, which can better ensure the effectiveness and safety of the drug, accurately evaluate the therapeutic effect, obtain the best treatment plan, and avoid potential drug conflicts or adverse reactions. Description of the Drawings

[0024] Figure 1 is a typical LC-MS / MS spectrum of XY028-140 in a blank matrix sample (XY028-140 channel); Figure 2 is a typical LC-MS / MS spectrum of XY028-140 in a blank matrix sample (internal standard channel); Figure 3It is the typical LC-MS / MS spectrum of XY028-140 in the zero-concentration sample (XY028-140 channel); Figure 4 It is the typical LC-MS / MS spectrum of XY028-140 in the zero-concentration sample (internal standard channel); Figure 5 It is the typical LC-MS / MS spectrum of XY028-140 in the lower limit of quantitation (LLOQ) sample (XY028-140 channel); Figure 6 It is the typical LC-MS / MS spectrum of XY028-140 in the lower limit of quantitation (LLOQ) sample (internal standard channel); Figure 7 It is the typical LC-MS / MS spectrum of XY028-140 in the upper limit of quantitation (ULOQ) sample (XY028-140 channel); Figure 8 It is the typical LC-MS / MS spectrum of XY028-140 in the upper limit of quantitation (ULOQ) sample (internal standard channel); Figure 9 It is a typical standard curve graph of XY028-140. Detailed implementation manners

[0025] The present application will be further described in detail below in conjunction with the attached drawings and verification experiments.

[0026] In the detailed implementation manners of the present application, an LC-MS / MS system composed of an LC-20AD XR liquid chromatograph of Shimadzu Corporation and a TripleQuad 5500+ mass spectrometer of SCIEX Corporation is adopted; an XBridge C18 2.1×50 mm 3.5 μm chromatographic column of Waters Corporation; an Eppendorf low-temperature high-speed centrifuge; the Analyst 1.7.3 software of SCIEX Corporation is used to output original data, spectra, concentrations, accuracies, etc. Verapamil or tolbutamide has no interference in the detection process as an internal standard. In the detailed implementation manners of the present application, verapamil is taken as an example for illustration. Acetonitrile or methanol or acetonitrile containing formic acid or methanol containing formic acid has good precipitation effects as a precipitant. In the detailed implementation manners of the present application, acetonitrile is taken as an example for illustration. The XY028-140 standard product is purchased from MedChem Corporation, the internal standard verapamil is purchased from Sigma Corporation, and other reagents are all commercially available HPLC-grade or GR-grade reagents.

[0027] Taking formic acid as an example, the preparation method of the first diluent is as follows: 1 mL - 3 mL of formic acid is added to 1000 mL of ultrapure water, and after mixing evenly, an aqueous formic acid solution with a formic acid concentration of 0.1 wt% - 0.3 wt% is obtained. In the specific implementation manner of this application, an aqueous formic acid solution with a formic acid concentration of 0.1 wt% is used as the first diluent for illustration.

[0028] Taking acetonitrile as an example, the preparation method of the second diluent is as follows: 50 mL of acetonitrile is added to 0 - 200 mL of ultrapure water, and after vortex mixing evenly, an acetonitrile solution with an acetonitrile concentration of 20 wt% - 100 wt% is obtained. In the specific implementation manner of this application, an acetonitrile solution with an acetonitrile concentration of 50 wt% is used as the second diluent for illustration.

[0029] Taking verapamil as an example, the specific preparation method of the precipitant containing the internal standard is as follows: 2 mg of verapamil is dissolved in dimethyl sulfoxide to obtain an internal standard stock solution with an internal standard concentration of 1 mg / mL, and then the internal standard stock solution is diluted with acetonitrile to an internal standard concentration of 0.1 ng / mL.

[0030] In the specific implementation manner of plasma samples, the plasma samples to be tested are collected and stored at -80 °C. During analysis, the plasma samples are taken and placed in an ice box filled with crushed ice, and placed in the dark until completely thawed. After vortex mixing evenly, they are used for sampling. 20 μL of plasma sample is taken as the blank plasma sample.

[0031] In the specific implementation manner of plasma sample solutions, 20 μL of plasma sample is taken, and K 2 EDTA is added thereto in a manner well known to those skilled in the art to obtain a plasma sample containing an anticoagulant. Then, the plasma sample containing the anticoagulant is mixed with 100 μL of the precipitant containing the internal standard, and centrifuged at a temperature of 2 °C - 8 °C and a centrifugation condition of 3000 g for 10 minutes. 50 μL of the supernatant is taken, and 150 μL of the first diluent is added and diluted and vortex mixed evenly to obtain a plasma sample solution.

[0032] In the specific implementation of the standard curve samples, 2 mg of the XY028-140 standard was weighed and dissolved in dimethyl sulfoxide to obtain an XY028-140 stock solution with a concentration of 1 mg / mL. The second diluent was used to prepare XY028-140 standard working solutions with concentrations ranging from 2 ng / mL to 2000 ng / mL. In a specific implementation of this application, the XY028-140 standard working solutions with concentrations of 2 ng / mL, 4 ng / mL, 10 ng / mL, 40 ng / mL, 200 ng / mL, 800 ng / mL, 1600 ng / mL, and 2000 ng / mL were used as examples for illustration. Then, the XY028-140 standard working solutions were mixed with blank plasma samples containing anticoagulants to obtain standard curve samples with concentrations ranging from 0.1 ng / mL to 100 ng / mL. In a specific implementation of this application, the concentrations of 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 2 ng / mL, 10 ng / mL, 40 ng / mL, 80 ng / mL, and 100 ng / mL were used as examples.

[0033] In the specific implementation of the standard curve sample solution, 20 μL of the standard curve sample was mixed with 100 μL of the precipitant containing the internal standard. At a temperature of 2°C - 8°C and a centrifugation condition of 3000 g, centrifugation was carried out for 10 minutes. 50 μL of the supernatant was taken and diluted with 150 μL of the first diluent, followed by vortex mixing to obtain the standard curve sample solution.

[0034] In the specific implementation of the blank sample solution, 20 μL of the standard curve sample was mixed with 100 μL of the precipitant. At a temperature of 2°C - 8°C and a centrifugation condition of 3000 g, centrifugation was carried out for 10 minutes. 50 μL of the supernatant was taken and diluted with 150 μL of the first diluent, followed by vortex mixing to obtain the blank sample solution.

[0035] In the specific implementation of the XY028-140 lower limit of quantification sample solution (LLOQ), the XY028-140 stock solution was taken and diluted with the second diluent to a concentration of 2 ng / mL. Then, it was mixed with the blank plasma sample to obtain an XY028-140 lower limit of quantification sample with a concentration of 0.1 ng / mL. 20 μL of the XY028-140 lower limit of quantification sample was mixed with 100 μL of the precipitant containing the internal standard. At a temperature of 2°C - 8°C and a centrifugation condition of 3000 g, centrifugation was carried out for 10 minutes. 50 μL of the supernatant was taken and diluted with 150 μL of the first diluent, followed by vortex mixing to obtain the XY028-140 lower limit of quantification sample solution.

[0036] In the specific implementation of the XY028-140 quality control sample solution (QC), take the XY028-140 stock solution and dilute it with the second diluent to concentrations of 6 ng / mL, 600 ng / mL, and 1500 ng / mL respectively. Then mix it with the blank matrix to obtain XY028-140 quality control samples with concentrations of 0.3 ng / mL, 30 ng / mL, and 75 ng / mL. Mix 20 μL of the XY028-140 quality control sample with 100 μL of the precipitating agent containing the internal standard, centrifuge at a temperature of 2°C - 8°C and a centrifugation condition of 3000 g for 10 minutes, take 50 μL of the supernatant, add 150 μL of the first diluent, dilute and vortex to mix evenly to obtain the XY028-140 quality control sample solution.

[0037] In the specific implementation of this application, the detection and analysis conditions of LC are as follows: an XBridge C18 chromatographic column from Waters company, with a specification of 2.1×50 mm, 3.5 μm, the column temperature is 30°C - 50°C, and in the specific implementation of this application, 40°C is taken as an example for illustration. The injection volume is 5 μL, and the flow rate is 0.5 mL / min - 0.7 mL / min, and in the specific implementation of this application, 0.6 mL / min is taken as an example for illustration. The temperature of the autosampler is 4°C. Mobile phase A uses a formic acid aqueous solution with a concentration of 0.1 wt% - 0.5 wt%, and mobile phase B uses a formic acid acetonitrile solution with a formic acid concentration of 0.1 wt% - 0.5 wt%. To further improve the effect, 5 mM - 10 mM of ammonium formate or ammonium acetate can be added to mobile phase A and mobile phase B respectively. In the specific implementation of this application, a formic acid aqueous solution with a concentration of 0.1 wt% is used as mobile phase A, and a formic acid acetonitrile solution with a formic acid concentration of 0.1 wt% is used as mobile phase B for illustration. The conditions of gradient elution are specifically shown in Table 1.

[0038] Table 1 Mobile phase gradient elution table Time (minutes) 0.3 1.4 1.8 1.9 2.5 Mobile phase A (%) 90 5 5 90 90 Mobile phase B (%) 10 95 95 10 10 In the specific embodiments of the present application, the conditions for MS / MS detection and analysis are as follows: using a TripleQuad 5500+ mass spectrometer from SCIEX company with an ESI ion source, in positive ion mode, multiple reaction monitoring (MRM) scan mode, spray voltage of 5500V, spray temperature of 550 °C, CAD of 8 - 10, GS1 of 40 psi - 60 psi, GS2 of 40 psi - 60 psi, curtain gas of 25 psi - 40 psi. In the specific embodiments of the present application, CAD is 9, GS1 is 50 psi, GS2 is 50 psi, and curtain gas is 35 psi. The relevant mass spectrometry parameters of XY028 - 140 are: DP is 85V - 95V, CE is 50V - 60V, EP is 10V, CXP is 5V - 15V. The relevant mass spectrometry parameters of verapamil are: DP is 135V - 145V, CE is 35V - 45V, EP is 10V, CXP is 5V - 15V. In the specific embodiments of the present application, the compound - related mass spectrometry parameters are shown in Table 2.

[0039] Table 2 Compound - related mass spectrometry analysis parameters Verification experiment Referring to the General Principles of Verification of Bio - analytical Methods in Part IV of the Chinese Pharmacopoeia (2020 Edition) and the requirements of ICH Guideline M10 "Bio - analytical Method Validation and Sample Analysis", the accuracy, precision, etc. of the LC - MS / MS method for detecting PROTAC drug XY028 - 140 in plasma of the present application were verified. The specific content is as follows.

[0040] (1) Selectivity investigation Six plasma samples from different individuals were selected respectively. After preparing the plasma sample solutions, LC - MS / MS detection and analysis were carried out. In addition, the upper limit of quantification plasma sample solution (ULOQ) with a concentration of 100 ng / mL without internal standard was analyzed to investigate the interference of the compound on the internal standard; the zero - concentration sample solution (plasma sample solution containing only the internal standard) was analyzed to investigate the interference of the internal standard on the compound. As Figures 1 - 8 shown, the results indicate that there is no interference at the retention time of XY028 - 140 and no interference at the retention time of the internal standard. The retention time of XY028 - 140 is approximately 1.45 minutes, and the retention time of the internal standard is approximately 1.40 minutes.

[0041] (2) Standard curve linearity investigation The standard curve includes a blank sample solution, a zero-concentration sample solution, and eight standard curve sample solutions with non-zero concentrations (0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 2 ng / mL, 10 ng / mL, 40 ng / mL, 80 ng / mL, 100 ng / mL). As Figure 9 shown, a typical standard curve of XY028-140 is y = 0.268x + 0.00234, with a correlation coefficient r = 0.9970. The results show that: the eight non-zero concentration standard curve points (including LLOQ and ULOQ) all meet the acceptance criteria of an accuracy not exceeding ±15% of the theoretical value (where the accuracy of LLOQ does not exceed ±20% of the theoretical value), and the correlation coefficient r of the standard curve meets the acceptance criteria of not less than 0.9900 (or the coefficient of determination R 2 not less than 0.9801).

[0042] (3) Investigation of accuracy and precision Accuracy was verified by examining the closeness of the measured concentrations to the theoretical concentrations of the XY028-140 lower limit of quantification sample solution at a concentration of 0.1 ng / mL and the XY028-140 quality control sample solutions at three concentration levels (0.3 ng / mL, 30 ng / mL, 75 ng / mL) in three different analytical batches, expressed as: measured concentration / theoretical concentration × 100%; Precision was verified by examining the closeness of the repeated measurements of the XY028-140 lower limit of quantification sample solution at a concentration of 0.1 ng / mL and the XY028-140 quality control sample solutions at three concentration levels (0.3 ng / mL, 30 ng / mL, 75 ng / mL) in three different analytical batches, i.e., the relative standard deviation (coefficient of variation, %CV) between the measured values. There were 6 samples for each concentration. The results are shown in Table 3.

[0043] Table 3 Results of accuracy and precision The results show that: the within-batch and between-batch average accuracies of the XY028-140 quality control sample solutions at each concentration are within the range of ±15% of the theoretical concentration, the within-batch and between-batch average accuracies of the XY028-140 lower limit of quantification sample solution do not exceed ±20% of the theoretical concentration, the within-batch and between-batch precisions (%CV) of the XY028-140 quality control sample solutions at each concentration do not exceed 15%, and the within-batch and between-batch precisions (%CV) of the XY028-140 lower limit of quantification sample solution do not exceed 20%, all meeting the requirements.

[0044] (4) Investigation of sensitivity The sensitivity was investigated by the accuracy and precision of the XY028-140 lower limit of quantitation (LLOQ) sample solution at a concentration of 0.1 ng / mL. According to the results in Table 3, it shows that the average intra-batch and inter-batch accuracies of the XY028-140 LLOQ sample solution are within ±20% of the theoretical concentration, and the precisions (%CV) of intra-batch and inter-batch are both not more than 20%, meeting the requirements. This indicates that the detection method of this application has high sensitivity, can accurately and precisely quantify the sample at 0.1 ng / mL, and can fully meet the bioanalysis requirements of XY028-140.

[0045] (5) Matrix effect, hemolysis effect and recovery rate investigation Matrix effect and hemolysis effect: Blank plasma from 6 different beagle dogs was used for the matrix effect. The matrix effect was investigated by separately preparing 3 low-concentration plasma quality control sample solutions and 3 high-concentration plasma quality control sample solutions (0.3 ng / mL, 75 ng / mL) using blank plasma from different sources; and, 3 low-concentration plasma quality control sample solutions and 3 high-concentration plasma quality control sample solutions were prepared for the investigation of the hemolysis effect. The results are shown in Table 4.

[0046] Table 4 Matrix effect verification result table The results show that the average accuracies of the low-concentration plasma quality control sample solutions and high-concentration plasma quality control sample solutions prepared from 6 different batches of blank plasma do not exceed 15% of the theoretical concentration, and the precisions are not greater than 15%. The average accuracies of each concentration in the hemolytic matrix do not exceed 15% of the theoretical concentration, and the precisions are not greater than 15%, all meeting the requirements. This indicates that the detection method of this application is not affected by the matrix effect and hemolysis effect.

[0047] Recovery rate: The recovery rate was determined by comparing the peak areas of adding XY028-140 and the internal standard solution to the same concentration as the quality control sample solution for the low-concentration plasma quality control sample solution, medium-concentration plasma quality control sample solution, high-concentration plasma quality control sample solution (0.3 ng / mL, 30 ng / mL, 75 ng / mL) and blank plasma samples respectively. The results are shown in Table 5.

[0048] Table 5 Recovery rate verification result table The results show that the precisions of the recovery rates of XY028-140 at low, medium, and high three quality control concentration levels and between different concentrations of samples do not exceed 15%. The precisions of the internal standard recovery rates of all samples also do not exceed 15%, and the recovery rates are consistent among different concentrations.

[0049] (6) Stability investigation The stability of low-concentration plasma quality control sample solution and high-concentration plasma quality control sample solution (0.3 ng / mL, 75 ng / mL) after being placed at room temperature for 6 hours and after being repeatedly frozen and thawed 3 times was detected. The results are shown in Table 6.

[0050] Table 6 Stability Results Table The results show that: after being placed at room temperature for 6 hours, the accuracy of each concentration of plasma quality control sample solution is within 15% of the theoretical concentration, the precision is within 15%, and the stability meets the requirements; after being repeatedly frozen and thawed 3 times, the accuracy of the low-concentration plasma quality control sample solution and the high-concentration plasma quality control sample solution is within 15% of the theoretical concentration, the precision is within 15%, and the stability meets the requirements.

[0051] The embodiments of this specific implementation manner are all preferred embodiments of this application, and do not limit the protection scope of this application accordingly. Therefore, all equivalent changes made according to the structure, shape, and principle of this application should be covered within the protection scope of this application.

Claims

1. A liquid chromatography tandem mass spectrometry analysis method for detecting XY028-140 in blood, comprising preparation of plasma sample solution, preparation of standard curve sample solution and LC-MS / MS detection, characterized in that: The LC conditions in the LC-MS / MS detection include using an Xbridge C18 chromatographic column, a column temperature of 30°C-50°C, a flow rate of 0.5mL / min-0.7mL / min, a mobile phase A of 0.1wt%-0.5wt% formic acid aqueous solution, and a mobile phase B of 0.1wt%-0.5wt% formic acid acetonitrile solution for concentration gradient elution; the scanning mode used in the MS / MS detection analysis is a multiple reaction ion detection scanning mode, and XY02 The DP of 8-140 is 85V-95V, CE is 50V-60V, EP is 10V, CXP is 5V-15V, the DP of internal standard is 135V-145V, CE is 35V-45V, EP is 10V, CXP is 5V-15V; the CAD in MS / MS detection analysis is 8-10, GS1 is 40psi-60psi, GS2 is 40psi-60psi, and the curtain gas is 25psi-40psi.

2. A liquid chromatography tandem mass spectrometry analysis method for detecting XY028-140 in blood according to claim 1, characterized in that: The conditions of the gradient elution of the LC are: at 0-0.3 minutes, the mobile phase A is 90%, and the mobile phase B is 10%; at 0.3-1.4 minutes, the mobile phase A changes from 90% to 5%, and the mobile phase B changes from 10% to 95%; at 1.4-1.8 minutes, the mobile phase A is 5%, and the mobile phase B is 95%; at 1.8-1.9 minutes, the mobile phase A changes from 5% to 90%, and the mobile phase B changes from 95% to 10%; at 1.9-2.5 minutes, the mobile phase A is 90%, and the mobile phase B is 10%.

3. A liquid chromatography tandem mass spectrometry analysis method for detecting XY028-140 in blood according to claim 1, characterized in that: In the LC detection, 5mM-10mM ammonium formate or ammonium acetate is added to mobile phase A; 5mM-10mM ammonium formate or ammonium acetate is added to mobile phase B.

4. The method for detecting XY028-140 in blood according to claim 1, characterized in that: In the multiple reaction ion detection scanning mode, the DP of XY028-140 is 90V, the CE is 55V, the EP is 10V, and the CXP is 10V, and the DP of the internal standard is 140V, the CE is 40V, the EP is 10V, and the CXP is 10V.

5. The liquid chromatography tandem mass spectrometry analysis method for detecting XY028-140 in blood according to claim 1, characterized in that: In the MS / MS detection analysis, CAD is 9, GS1 is 50 psi, GS2 is 50 psi, and the curtain gas is 35 psi.

6. A liquid chromatography tandem mass spectrometry analysis method for detecting XY028-140 in blood according to claim 1, characterized in that: The plasma sample solution is prepared specifically as follows: under the condition of 2° C.-8° C., a plasma sample containing an anticoagulant is mixed with a precipitant containing an internal standard, centrifuged, and then diluted with a first diluent to obtain a plasma sample solution.

7. A liquid chromatography tandem mass spectrometry analysis method for detecting XY028-140 in blood according to claim 6, characterized in that: The anticoagulant is K2EDTA.

8. The method for detecting XY028-140 in blood according to claim 6, characterized in that: The internal standard is verapamil or tolbutamide.

9. A liquid chromatography tandem mass spectrometry analysis method for detecting XY028-140 in blood according to claim 6, characterized in that: The precipitant is one of acetonitrile, a formic acid acetonitrile solution with a formic acid concentration of 0.1 wt %, methanol, and a formic acid methanol solution with a formic acid concentration of 0.1 wt %.

10. A liquid chromatography tandem mass spectrometry analysis method for detecting XY028-140 in blood according to claim 1, characterized in that: The preparation of the standard curve sample solution includes: mixing an XY028-140 standard working solution with a concentration of 2 ng / mL-2000 ng / mL with a blank matrix containing an anticoagulant to obtain a standard curve sample with a concentration of 0.1 ng / mL-100 ng / mL; mixing the standard curve sample with a precipitant containing an internal standard under the condition of 2° C.-8° C., centrifuging, and diluting with a first diluent to obtain a standard curve sample solution.

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