A method for detecting thrombopoietin receptor agonists in biological samples

Abstract

The present invention relates to a method for detecting thrombopoietin receptor agonists in biological samples, comprising the steps of: mixing a test sample with an internal standard precipitant, vortexing to obtain a supernatant of the test sample, and using ultra-high performance liquid chromatography coupled to mass spectrometry to quantitatively detect the thrombopoietin receptor agonist in the supernatant of the test sample; the internal standard precipitant contains an internal standard and a special solvent, the internal standard is an isotope internal standard of the thrombopoietin receptor agonist, the special solvent contains methanol, acetonitrile and ammonia water, and the content of the components in the special solvent is: methanol> ammonia water, acetonitrile> ammonia water. The advantages of the present invention are: the special solvent provided can stably and fully dissolve the thrombopoietin receptor agonist standard and the isotope internal standard, is friendly to instrument accessories, can precipitate the protein in the plasma, and reduce the sample pre-treatment time; the thrombopoietin receptor agonist detection method provided has accurate analysis, a wide detection linear range, and a short detection time.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection in biological samples, and in particular relates to a method for detecting a thrombopoietin receptor agonist in a biological sample. Background Art

[0002] Avatrombopag, eltrombopag, hetrombopag, and rutrombopag are non-peptide thrombopoietin receptor agonists marketed in China. The oral absorption of this type of thrombopoietin receptor agonists varies greatly and can be affected by food. They are mainly metabolized by liver CYP3A or CYP2C9 enzymes. When taken simultaneously with inhibitors of the above-mentioned metabolic enzymes, the in vivo exposure of the above-mentioned thrombopoietin receptor agonists can be greatly increased, resulting in dose-related adverse reactions in patients. In addition, poor patient compliance can also lead to abnormal in vivo exposure of the above-mentioned thrombopoietin receptor agonists. Therefore, in actual clinical samples, the concentration of thrombopoietin receptor agonists can show polarization of being too high or too low. It is necessary to conduct therapeutic drug monitoring to evaluate the actual drug exposure in patients, so as to provide individualized treatment for patients and reduce adverse reactions.

[0003] The solubility of thrombopoietin receptor agonists in water or organic solvents is extremely poor, and DMSO is generally used as its dissolving agent in existing reports. Although DMSO can be used as a universal solvent, it has great damage to the PEEK pipeline of liquid chromatography, the filler of the chromatographic column, and the mass spectrometry ion source. Therefore, it is necessary to invent a solvent system that can dissolve all thrombopoietin receptor agonists and the corresponding isotope internal standards at the same time.

[0004] In order to ensure the accuracy of the test results of the aforementioned samples with too high or too low concentrations, it is necessary to design reasonable calibration concentrations for different thrombopoietin receptor agonists to ensure that the detection linear range of the method can cover all types of samples.

[0005] In summary, it is necessary to invent a detection method that has good solubility of the analyte, suitable linear range, simple pretreatment method, and is particularly suitable for large-scale biological sample detection. Summary of the invention

[0006] The present invention provides a method for detecting a thrombopoietin receptor agonist in a biological sample to solve the problems in the background technology.

[0007] The technical solution adopted by the present invention is: a method for detecting a thrombopoietin receptor agonist in a biological sample, comprising the steps of: mixing a test sample with an internal standard precipitant, vortexing to obtain a supernatant of the test sample, and quantitatively detecting the thrombopoietin receptor agonist in the supernatant of the test sample by ultra-high performance liquid chromatography coupled to mass spectrometry;

[0008] The internal standard precipitant contains an internal standard substance and a suitable solvent. The internal standard substance is an isotope internal standard substance of a thrombopoietin receptor agonist. The suitable solvent contains methanol, acetonitrile and ammonia water. In the suitable solvent, the content of methanol is greater than that of ammonia water, and the content of acetonitrile is greater than that of ammonia water.

[0009] Furthermore, the volume ratio of methanol to acetonitrile in the particularly suitable solvent is 1:1, and the volume fraction of aqueous ammonia is 0.5% to 2%; preferably, the volume ratio of methanol, acetonitrile and aqueous ammonia in the particularly suitable solvent is 49.5:49.5:1.

[0010] Furthermore, the conditions of ultra-high performance liquid chromatography include:

[0011] Gradient elution;

[0012] Mobile phase A is a formic acid-acetonitrile solution with a volume fraction of formic acid of 0.01% to 0.5%;

[0013] Mobile phase B is an aqueous solution of formic acid, and the volume fraction of formic acid is 0.01%~0.5%.

[0014] Furthermore, the conditions of ultra-high performance liquid chromatography include:

[0015] The model of the chromatographic column in the ultra-high performance liquid chromatography was Waters ACQUITY BEH C18, with a column length of 50 mm and an inner diameter of 2.1 mm;

[0016] The column filler is octadecylsilane bonded silica gel, and its particle size is 1.7 μm;

[0017] The flow rate was 0.3~0.5mL / min, the column temperature was 35~40℃, and the injection volume was 1~10μL.

[0018] Furthermore, the conditions for gradient elution are:

[0019] 0.0min, mobile phase A, 16%; mobile phase B, 84%;

[0020] 0.8min: mobile phase A, 70%; mobile phase B, 30%;

[0021] 1.0min: mobile phase A, 93%; mobile phase B, 7%;

[0022] 2.0min: mobile phase A, 99%; mobile phase B, 1%;

[0023] 3.5min: mobile phase A, 99%; mobile phase B, 1%;

[0024] 3.51min: mobile phase A, 16%; mobile phase B, 84%;

[0025] 4~5min: mobile phase A, 16%; mobile phase B, 84%.

[0026] Furthermore, the mass spectrometry conditions are:

[0027] Positive ion electrospray ion source, multiple reaction monitoring MRM, capillary voltage 0.5~2.0kV, desolvation gas temperature 400~500℃, desolvation gas flow rate 600~800L / Hr, cone gas flow rate 40~60L / Hr.

[0028] Furthermore, the four thrombopoietin receptor agonists that can be detected by the method include: avatrombopag, eltrombopag, hetrombopag, and lutrombopag; when the method is used to detect the four thrombopoietin receptor agonists, the internal standards include avatrombopag-D8, eltrombopag-4C13 and / or lutrombopag-D13.

[0029] Furthermore, the step of quantitatively detecting the supernatant of the test sample includes calibrating the standard sample, and the operation is as follows:

[0030] A standard sample is prepared, wherein the standard sample contains a thrombopoietin receptor agonist and blank plasma; the standard sample is mixed with an internal standard precipitant, and vortexed to obtain a standard sample supernatant; four thrombopoietin receptor agonists in the standard sample supernatant are separated and quantitatively detected by ultra-high performance liquid chromatography-mass spectrometry, and the detection conditions of the standard sample supernatant and the test sample supernatant are the same;

[0031] The mass concentration of each thrombopoietin receptor agonist is taken as the horizontal axis. X , the chromatographic peak area ratio of each thrombopoietin receptor agonist to the internal standard is the ordinate - Y , weighted least squares method is used for linear regression, and the weighting factor is 1 / X 2 , and the standard curve equations of the four thrombopoietin receptor agonists were obtained respectively.

[0032] Furthermore, the quantitative lower limits of the method for detecting four thrombopoietin receptor agonists are:

[0033] Avatrombopag: 2 ng / mL;

[0034] Eltrombopag: 20 ng / mL;

[0035] Hetromopa: 2 ng / mL;

[0036] Lutrombopag: 2ng / mL.

[0037] The advantages and positive effects of the present invention include at least:

[0038] (1) A special solvent system is provided that does not contain dimethyl sulfoxide and can stably and fully dissolve thrombopoietin receptor agonist standards and isotope internal standards. The properties and solubility of this solvent system change little at low temperature and room temperature, which can ensure the stability of the test agent and is friendly to instrument accessories such as chromatographic columns, pipelines and ion sources;

[0039] (2) The above-mentioned specially adapted solvent system can also precipitate proteins in plasma. The internal standard precipitant prepared with the above-mentioned specially adapted solvent system can complete the two steps of internal standard addition and protein precipitation at one time, which greatly reduces the sample pre-treatment time and is suitable for clinical therapeutic drug monitoring of large batches of samples.

[0040] (3) A method for rapid detection of thrombopoietin receptor agonist content in biological samples using ultra-high performance liquid chromatography-mass spectrometry technology was provided, covering all types of thrombopoietin receptor agonists that have been marketed. A reasonable detection linear range was designed to cover the actual concentration range of the four thrombopoietin receptor agonists in clinical samples, and methodological verification was performed. This method has accurate analysis, a wide detection linear range, simple sample pretreatment, and a short detection time (the sample loading and analysis time is only 4.5 minutes), which is suitable for large-scale clinical sample analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The chromatogram of the method validation sample 1 in Example 3 of the present invention;

[0042] Figure 2 It is the chromatogram of the blank sample in Example 3 of the present invention. DETAILED DESCRIPTION

[0043] The present invention will be further described below in conjunction with embodiments and drawings:

[0044] Unless otherwise defined, all professional terms used hereinafter have the same meaning as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and comparative examples, and are not intended to limit the scope of protection of the present invention. It should be particularly noted that there may be multiple names for the same organic structure, as long as the structure is within the scope of this patent, it belongs to the protection object of this patent.

[0045] Unless otherwise defined, the raw materials, reagents, etc. in the following examples can be purchased from the market or prepared according to reported methods.

[0046]

Drugs and reagents

[0047] Avatrombopag (purity: 99%, batch number: 570406-98-3), Avatrombopag-D8 (purity: >98%, batch number: 20201215), and Eltrombopag (purity: 99%, batch number: 20200907) were purchased from Shanghai Hanxiang Biotechnology Co., Ltd.

[0048] Lutrombopag (purity: 98.32%, batch number: 22971) and Lutrombopag-D13 (purity: 98.32%, batch number: 532932) were purchased from MedChemExpress, USA.

[0049] Eltrombopag-4C13 (purity: 99%, batch number: 1901-031B4) was purchased from TLC Pharmaceutical Standards Ltd., Canada.

[0050] Hetrombopag (purity: 99%, batch number: 6135231001) was donated by Jiangsu Hengrui Medicine Co., Ltd.

[0051] Acetonitrile (mass spectrometry grade, batch number: 217207), methanol (mass spectrometry grade, batch number: 34860-4L-R), formic acid (mass spectrometry grade, batch number: A117-50), and ammonia (mass spectrometry grade, batch number: 7115080) were purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0052] The water used in the experiment was prepared by an ultrapure water system.

[0053] Blank plasma can be obtained from a variety of channels, including waste plasma from blood banks and reagent companies.

[0054]

Equipment and consumables

[0055] The liquid spectrometer (model: Xevo TQD IVD) was purchased from Waters, USA.

[0056] Low-temperature high-speed centrifuge (model: SCIENTIFIC Legend Micro 21R), low-speed centrifuge (model: SL8), and vortex mixer (model: LP Vortex Mixer) were purchased from Thermo Fisher, USA.

[0057] The 1 / 100,000 balance (model: MS105) was purchased from Mettler, USA.

[0058] The ultrapure water system was purchased from Millipore Corporation, USA.

[0059] Pipettes (specifications: 10, 20, 100, 200, 1000 μL) were purchased from Eppendorf, Germany.

[0060] Volumetric flasks (specifications: 10 mL, 50 mL) were purchased from Beijing Xinweier Glass Instrument Co., Ltd.

[0061] Centrifuge tubes (specifications: 0.2 mL, 1.5 mL, 10 mL) were purchased from Aisheng Biotechnology (Hangzhou) Co., Ltd.

[0062] An embodiment of the present invention provides a method for detecting a thrombopoietin receptor agonist in a biological sample. The method can detect four non-peptide thrombopoietin receptor agonists, namely avatrombopag, eltrombopag, hetrombopag and rutrombopag.

[0063] The separation and detection technology used in the method is ultra-high performance liquid chromatography coupled to mass spectrometry, and the quantitative method used can be an external standard method or an isotope internal standard-standard curve method. In order to briefly describe the technical solution of the present invention, the isotope internal standard-standard curve method is specifically introduced below.

[0064] The method provided in the embodiment of the present invention comprises the following steps:

[0065] (1) Testing blank samples

[0066] The blank sample is blank plasma. The blank sample is mixed with the solvent in the internal standard precipitant, and vortexed to obtain the blank sample supernatant. The blank sample supernatant is detected using ultra-high performance liquid chromatography-mass spectrometry.

[0067] (2) Calibration standard samples

[0068] The standard sample and the internal standard precipitant were mixed and vortexed to obtain the standard sample supernatant, and the four thrombopoietin receptor agonists in the standard sample supernatant were separated and quantitatively detected by ultra-high performance liquid chromatography-mass spectrometry;

[0069] The mass concentration of each thrombopoietin receptor agonist is taken as the horizontal axis ( X ), the chromatographic peak area ratio of each thrombopoietin receptor agonist and the corresponding internal standard is the ordinate ( Y ), weighted least squares method was used for linear regression, and the weighting factor was 1 / X 2 , and the standard curve equations of the four thrombopoietin receptor agonists were obtained respectively.

[0070] (3) Testing quality control samples

[0071] The quality control sample and the internal standard precipitant were mixed and vortexed to obtain the quality control sample supernatant. Ultra performance liquid chromatography-mass spectrometry was used to separate and quantitatively detect the four thrombopoietin receptor agonists in the quality control sample supernatant.

[0072] (4) Testing of test samples

[0073] The test sample and the internal standard precipitant are mixed, vortexed to obtain the test sample supernatant, and the thrombopoietin receptor agonist in the test sample supernatant is quantitatively detected by ultra-high performance liquid chromatography-mass spectrometry.

[0074] In this method, the separation and detection conditions for the blank sample supernatant, standard sample supernatant, quality control sample supernatant and test sample supernatant are the same, which are:

[0075] ①Ultra-high performance liquid chromatography conditions:

[0076] Chromatographic column: Model: Waters ACQUITY BEH C18, column length: 50 mm, column inner diameter: 2.1 mm, filler: octadecylsilane bonded silica gel, filler particle size: 1.7~5 μm, preferably 1.7 μm, with higher column efficiency and faster analysis speed;

[0077] Mobile phase A: formic acid in acetonitrile solution, the volume fraction of formic acid is 0.01%~0.5%, preferably 0.1%;

[0078] Mobile phase B: aqueous solution of formic acid, the volume fraction of formic acid is 0.01%~0.5%, preferably 0.1%.

[0079] The acidity produced by the above formic acid concentration can avoid compound decomposition and produce better peak shape and separation effect on the chromatography.

[0080] Flow rate: 0.3~0.5mL / min, preferably 0.4mL / min;

[0081] Column temperature: 35~40℃, preferably 40℃;

[0082] Injection volume: 1~10μL, preferably 2μL.

[0083] The thrombopoietin receptor agonists detected by this method have similar long-chain mother nuclei, but have certain differences in polarity. Avatrombopag has the highest polarity, followed by rutrombopag, and hetrombopag and eltrombopag have lower polarity. To ensure the separation efficiency, the gradient elution conditions need to reduce the proportion of the aqueous phase to less than 10% within 1 min, preferably 7%, and then slowly decrease within 1 min, and the proportion is preferably 1%.

[0084] The conditions for gradient elution are preferably: 0.0 min, mobile phase A, 16%; mobile phase B, 84%; 0.8 min: mobile phase A, 70%; mobile phase B, 30%; 1.0 min: mobile phase A, 93%; mobile phase B, 7%; 2.0 min: mobile phase A, 99%; mobile phase B, 1%; 3.5 min: mobile phase A, 99%; mobile phase B, 1%; 3.51 min: mobile phase A, 16%; mobile phase B, 84%; 4~5 min: mobile phase A, 16%; mobile phase B, 84%.

[0085] ②Mass spectrometry conditions:

[0086] Positive ion electrospray ion source;

[0087] The capillary voltage is 0.5-2.0 kV, preferably 2.0 kV;

[0088] The desolvation temperature is 400-500°C, preferably 400°C;

[0089] The desolvation gas flow rate is 600-800 L / Hr, preferably 800 L / Hr;

[0090] The cone hole gas flow rate is 40~60L / Hr, preferably 60L / Hr;

[0091] The multiple reaction monitoring (MRM) mode was used, and the ion information, collision energy, and cone voltage are shown in Table 1.

[0092] Table 1 Ion information, collision energy and cone voltage

[0093]

[0094] In this method, the quantitative detection method for the supernatant of the test sample is as follows: the chromatographic peak area ratio of the thrombopoietin receptor agonist in the supernatant of the test sample to the internal standard is substituted into the standard curve equation to calculate the concentration of the thrombopoietin receptor agonist in the test sample; the chromatographic peak area ratio of the thrombopoietin receptor agonist in the supernatant of the quality control sample to the internal standard is substituted into the standard curve equation to calculate the measured concentration of the thrombopoietin receptor agonist in the quality control sample, and the degree of deviation from the theoretical concentration of the thrombopoietin receptor agonist in the quality control sample is compared to determine the quality of this quantitative detection.

[0095] This method can analyze and detect the above four thrombopoietin receptor agonists at the same time, and can also analyze and detect one of the above four thrombopoietin receptor agonists or a mixture of two or more according to actual detection needs.

[0096] The internal standard used in this method is an isotope internal standard, and one or more of avatrombopag-D8, eltrombopag-4C13 and lutrombopag-D13 can be selected according to the actual conditions and the above-mentioned different detection drugs. During the detection, each thrombopoietin receptor agonist can be respectively selected by single internal standard method or double internal standard method. The preferred single internal standard scheme is: avatrombopag uses avatrombopag-D8 as the internal standard, eltrombopag uses eltrombopag-4C13 as the internal standard, and hetrombopag and lutrombopag use lutrombopag-D13 as the internal standard respectively. The preferred double internal standard scheme is: avatrombopag, hetrombopag and lutrombopag all use avatrombopag-D8 and lutrombopag-D13 as the internal standards, and eltrombopag uses eltrombopag-4C13 and lutrombopag-D13 as the internal standards.

[0097] The preparation method of standard samples and quality control samples in this method includes:

[0098] ① Prepare 4 stock solutions of standard substances: Dissolve the standard substances of avatrombopag, eltrombopag, hetrombopag and rutrombopag in appropriate solvents and make up to volume respectively;

[0099] ②Prepare standard working solution: according to the actual testing requirements, several of the above four standard stock solutions are mixed in proportion, diluted with a specially suitable solvent and fixed to volume to obtain the standard working solution with the highest concentration respectively; then use a specially suitable solvent to use the multiple dilution method to dilute the standard working solution with the highest concentration downward, and finally obtain standard working solutions with at least 5 concentration points (including the standard working solution with the highest concentration);

[0100] ③Prepare quality control working solution: According to actual testing needs, mix several of the above 4 standard stock solutions in proportion, dilute and fix the volume with a special solvent to obtain the quality control working solution with the highest concentration; then use a special solvent to use the multiple dilution method to dilute the quality control working solution with the highest concentration downward, and finally obtain quality control working solutions with at least 2 concentration points (including the quality control working solution with the highest concentration).

[0101] The above-mentioned specially suitable solvent contains methanol, acetonitrile and ammonia water, wherein the content of methanol is greater than the content of ammonia water, and the content of acetonitrile is greater than the content of ammonia water.

[0102] Since the four thrombopoietin receptor agonists detected by this method have a similar long-chain parent nucleus, and a strongly polar benzoic acid structure at one end and a weakly polar heterocyclic conjugated system at the other end, their lipid solubility and water solubility are poor. Using the above-mentioned special solvent system, methanol and acetonitrile can ensure the solubility of the heterocyclic conjugated system, acetonitrile can also adapt to the mobile phase of ultra-high performance liquid chromatography, and a small amount of ammonia water can increase the solubility of the benzoic acid region, thereby dissolving the thrombopoietin receptor agonist well.

[0103] Preferably, the volume ratio of methanol, acetonitrile and ammonia water in the particularly suitable solvent is 49.5:49.5:1. The standard stock solution prepared using this solvent system is a high-concentration standard stock solution that is liquid homogeneous without solid precipitation at -80°C and can be used normally when transferred to room temperature.

[0104] When preparing the standard working solution and the quality control working solution, the dilution ratio of the standard working solution is preferably 2-5-2-5-2-5 times in a step-by-step manner, and the dilution ratio of the quality control working solution is preferably 10 times.

[0105] ④Prepare standard samples and quality control samples:

[0106] The standard working solution and blank plasma were mixed in a certain ratio to obtain a standard sample;

[0107] The quality control working solution and blank plasma were mixed in a certain ratio to obtain the quality control sample.

[0108] When preparing standard samples and quality control samples, the volume ratio of blank plasma to standard working solution and the volume ratio of blank plasma to quality control working solution are (5-10):1, preferably 9:1.

[0109] The preparation method of the internal standard precipitant in this method includes:

[0110] ①Prepare internal standard stock solution: dissolve the internal standard substance in a suitable solvent and make up the volume to obtain the internal standard stock solution.

[0111] ②Prepare the internal standard precipitant: dilute the internal standard stock solution with a suitable solvent and adjust the volume to obtain the internal standard precipitant.

[0112] In this method, the concentrations of each internal standard in the internal standard precipitant are preferably: avatrombopag-D8: 100 ng / mL, rutrombopag-D13: 100 ng / mL, and eltrombopag-4C13: 500 ng / mL.

[0113] When preparing the standard sample supernatant and the quality control sample supernatant, the volume ratio of the standard sample to the internal standard precipitant and the volume ratio of the quality control sample to the internal standard precipitant are 1:(1.5-4), respectively, preferably 1:2.

[0114] The specially suitable solvent can not only stably and fully dissolve the standard and isotope internal standard, but also precipitate the protein in the plasma. The internal standard precipitant prepared in this way, after being mixed with the sample, can complete the two processes of internal standard addition and protein precipitation at one time, which greatly reduces the sample pre-processing time and is suitable for clinical therapeutic drug monitoring of large quantities of samples.

[0115] When using this method for detection, it is necessary to replace the ordinary needle washing solution with the above-mentioned special solvent to avoid the generation of high injection residues and affect the detection accuracy.

[0116] The quantitative lower limits of the four thrombopoietin receptor agonists detected by the above method were avatrombopag: 2 ng / mL; eltrombopag: 20 ng / mL; hetrombopag: 2 ng / mL; and rutrombopag: 2 ng / mL.

[0117] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, without departing from the concept of the present invention, several variations and improvements can also be made, which all belong to the protection scope of the present invention.

[0118] Example 1: Rapid detection of thrombopoietin receptor agonist content in biological samples

[0119] S1. Prepare solution

[0120] ①Prepare a suitable solvent

[0121] Methanol, acetonitrile and aqueous ammonia were mixed in a volume ratio of 49.5%:49.5%:1% to obtain a particularly suitable solvent.

[0122] ②Prepare 4 standard stock solutions and 3 internal standard stock solutions

[0123] Accurately weigh the solid standard and internal standard of thrombopoietin receptor agonist, dissolve them in suitable solvents, transfer them to 10 mL volumetric flasks, make up to volume with suitable solvents, shake well to obtain 4 standard stock solutions with a concentration of 1,000,000 ng / mL and 3 internal standard stock solutions with a concentration of 100,000 ng / mL.

[0124] The weighed masses were: 10 mg avatrombopag, 10 mg eltrombopag, 10 mg lutrombopag, 12.7 mg hetrombopag ethanolamine, 1 mg avatrombopag-D8, 1 mg eltrombopag-4C13, and 1 mg lutrombopag-D13.

[0125] All the above stock solutions can be stored at -80℃.

[0126] ③Prepare standard working solutions at 7 concentration points

[0127] Accurately measure 20 μL each of the avatrombopag standard stock solution, the heltrombopag standard stock solution, the rutrombopag standard stock solution, and 200 μL of the eltrombopag standard stock solution, mix them, add 740 μL of a special solvent to make up the volume and shake well to obtain the standard working solution with the highest concentration S7 concentration point.

[0128] The standard working solution at the concentration point S7 was diluted stepwise by 2-5-2-5-2-5 times using a specially suitable solvent to obtain standard working solutions at 6 concentration points from S6 to S1.

[0129] ④Prepare quality control working solution at 3 concentration points

[0130] Accurately measure 18 μL each of avatrombopag standard stock solution, heltrombopag standard stock solution and rutrombopag standard stock solution, and 180 μL of eltrombopag standard stock solution, mix them, add 766 μL of a special solvent to make up the volume and shake well to obtain the quality control working solution of the highest concentration H concentration point.

[0131] The quality control working solution at the H concentration point was diluted 10 times in stages using a specially suitable solvent to obtain quality control working solutions at two concentration points, M and L.

[0132] Both the standard working solution and the quality control working solution can be stored at -20°C. The specific preparation method is shown in Table 2, and the specific concentration is shown in Table 3.

[0133] Table 2 Stepwise dilution preparation method of standard working solution and quality control working solution

[0134]

[0135] Table 3 Concentrations of standard working solutions and quality control working solutions

[0136]

[0137] ⑤Prepare standard samples and quality control samples

[0138] Accurately add 180 μL of blank plasma and 20 μL of standard working solution into a 1.5 mL centrifuge tube to obtain a standard sample;

[0139] Accurately add 180 μL of blank plasma and 20 μL of quality control working solution to a 1.5 mL centrifuge tube to obtain a quality control sample.

[0140] ⑥Preparation of internal standard precipitant

[0141] Accurately measure 10 μL of avatrombopag-D8 internal standard stock solution, 10 μL of rutrombopag-D13 internal standard stock solution, and 50 μL of eltrombopag-4C13 internal standard stock solution, add them into a 10 mL volumetric flask, make up to volume with a suitable solvent, and shake well to obtain the internal standard precipitant.

[0142] S2. Sample processing and sample injection testing

[0143] The conditions for ultra-high performance liquid chromatography and mass spectrometry have been described in detail above and will not be repeated here.

[0144] Perform the following operations on the blank sample: accurately add 200 μL of blank sample (i.e., blank plasma) and 400 μL of a suitable solvent into a 1.5 mL centrifuge tube, vortex, centrifuge at 14,000 rpm / min for 5 min at 10°C, accurately measure the supernatant, and inject for detection.

[0145] Perform the following operations on the standard sample, quality control sample and test sample respectively: accurately add 200 μL sample and 400 μL internal standard precipitant into a 1.5 mL centrifuge tube, vortex, centrifuge at 14000 rpm / min for 5 min at 10°C, accurately measure the supernatant, and inject for detection.

[0146] S3. Obtain the standard curve equation

[0147] The mass concentration of each thrombopoietin receptor agonist in the standard sample is taken as the horizontal axis ( X ), the chromatographic peak area ratio of each thrombopoietin receptor agonist and the corresponding internal standard is the ordinate ( Y ), weighted least squares method was used for linear regression, and the weighting factor was 1 / X 2 , and obtained the standard curve equations of the four thrombopoietin receptor agonists respectively.

[0148] Among them, avatrombopag uses avatrombopag-D8 as the internal standard, eltrombopag uses eltrombopag-4C13 as the internal standard, and heltrombopag and lutrombopag use lutrombopag-D13 as the internal standard respectively.

[0149] S4. Quantitative calculation of thrombopoietin receptor agonists contained in the test sample

[0150] Substitute the chromatographic peak area ratio of the thrombopoietin receptor agonist in the supernatant of the test sample to the corresponding internal standard into the standard curve equation to calculate the concentration of the thrombopoietin receptor agonist in the test sample; substitute the chromatographic peak area ratio of the thrombopoietin receptor agonist in the supernatant of the quality control sample to the corresponding internal standard into the standard curve equation to calculate the measured concentration of the thrombopoietin receptor agonist in the quality control sample, compare the degree of deviation from the theoretical concentration of the thrombopoietin receptor agonist in the quality control sample, and determine the quality of this quantitative detection.

[0151] Examples 2-8 carried out methodological validation on the method of Example 1 in accordance with the requirements of the 2020 edition of the Chinese Pharmacopoeia.

[0152] Example 2: Linearity and Range

[0153] The detection method for the standard sample in "Sample Processing and Sample Injection Detection" in Example 1 was used for continuous operation and determination for 3 days. Eltrombopag had a good linear relationship in the concentration range of 20ng / mL to 20000ng / mL, and avatrombopag, hetrombopag and rutrombopag had a good linear relationship in the concentration range of 2ng / mL to 2000ng / mL, respectively. The determination coefficient ( r ) were all greater than 0.99. The lower limit of quantification of eltrombopag was 20 ng / mL, and the lower limits of quantification of avatrombopag, hetrombopag and rutrombopag were all 2 ng / mL.

[0154] Example 3: Specificity

[0155] A standard sample with the concentration value of the "lower limit of quantitation" in Example 2 was prepared as sample one for methodology verification.

[0156] According to the detection method of "Sample Processing and Sample Injection Detection" in Example 1, the method validation sample 1 and the blank sample were detected respectively, and the chromatograms of the method validation sample 1 were obtained respectively (see Appendix Figure 1 ) and blank samples (see Appendix Figure 2 ).

[0157] By the attached Figure 1 It can be seen that the retention times of avatrombopag, heltrombopag, rutrombopag and eltrombopag are 1.25min, 2.17min, 1.76min and 2.12min respectively, and the retention times of the internal standards avatrombopag-D8, rutrombopag-D13 and eltrombopag-4C13 are 1.25min, 1.75min and 2.12min respectively. The above 7 detection channels do not interfere with each other. Figure 2 It can be seen that the endogenous substances in the blank sample (i.e., blank plasma) do not interfere with the detection of the target substances (i.e., four thrombopoietin receptor agonists) and three internal standards.

[0158] Example 4: Accuracy and Precision

[0159] Five standard samples with the concentration value of the "lower limit of quantitation" in Example 2 were prepared in parallel as the second sample for method verification.

[0160] Five quality control samples of three concentration points (L, M and H) were prepared in parallel as the third sample for method validation.

[0161] According to the detection method of "Sample Processing and Sample Injection Detection" in Example 1, the method validation sample 2 and the method validation sample 3 were detected respectively for 3 consecutive days, and the concentration was calculated every day using the newly determined standard curve equation.

[0162] Relative error (RE%) and relative standard deviation (RSD%) were used to evaluate accuracy and precision. The inter-day and intra-day RE% and RSD% of the four thrombopoietin receptor agonists were all within 15% (LLOQ limit was 20%). The results are shown in Table 4.

[0163] Table 4 Accuracy and precision

[0164]

[0165] Example 5: Residual Effect

[0166] ① Prepare 5 standard samples with the concentration value of the "lower limit of quantitation" in Example 2 in parallel as the fourth sample for method verification.

[0167] According to the detection method of "Sample Processing and Sample Injection Detection" in Example 1, the methodological verification sample 4 was detected respectively, and the average chromatographic peak areas of the target thrombopoietin receptor agonist and the corresponding internal standard were recorded as a 1 and a 2 .

[0168] ② Prepare 5 standard samples with a concentration point of S7 in parallel as the method verification sample 5.

[0169] According to the detection method of "Sample Processing and Injection Detection" in Example 1, the method validation sample 5 and the blank sample were detected respectively, and the injection sequence of "method validation sample 5-blank sample" was repeated 5 times, and the average chromatographic peak areas of the target thrombopoietin receptor agonist and the corresponding internal standard in the blank sample were recorded as b 1 and b 2 .

[0170] Calculate the residual ratio of the target thrombopoietin receptor agonist as b 1 / a 1 , the residual ratio is less than 20%, and the residual ratio of the internal standard is b 2 / a 2 , the residual ratio is less than 5%.

[0171] Example 6: Dilution Effect

[0172] Accurately measure 40 μL of avatrombopag standard stock solution, 40 μL of heltrombopag standard stock solution, 400 μL of eltrombopag standard stock solution, and 40 μL of rutrombopag standard stock solution, place them in a 1.5 mL centrifuge tube, add 480 μL of a specifically suitable solvent to make up the volume, and obtain the dilution effect base solution one.

[0173] Accurately measure 100 μL of dilution effect basic solution 1 and add 900 μL of blank plasma to prepare dilution effect basic solution 2.

[0174] Using blank plasma, the dilution effect base solution 2 was diluted 10 times (20 μL dilution effect base solution 2 + 180 μL blank plasma), 25 times (8 μL dilution effect base solution 2 + 192 μL blank plasma), and 50 times (4 μL dilution effect base solution 2 + 196 μL blank plasma), and 5 replicates were prepared in parallel for each concentration point to obtain method validation sample 6.

[0175] According to the detection method of “Sample Processing and Sample Injection Detection” in Example 1, the methodological verification sample 6 was detected respectively, and the concentration was calculated using the standard curve equation newly measured on the same day. The RE% was within 11%. The results are shown in Table 5.

[0176] Table 5 Dilution effect

[0177]

[0178] Example 7: Extraction recovery and matrix effect

[0179] ①Prepare 6 copies of quality control samples at 3 concentration points (L, M and H) in parallel as sample 7 for method validation.

[0180] According to the detection method of "Sample Processing and Sample Injection Detection" in Example 1, the method validation sample 7 was detected respectively, and the peak areas of the target thrombopoietin receptor agonist and the corresponding internal standard were recorded as A 1 and A 2 .

[0181] ② Six portions of blank plasma from different sources were treated according to the method of “Sample Processing and Injection Testing” in Example 1 to obtain blank sample supernatants, and corresponding volumes of quality control working solution and internal standard precipitant were added to the blank sample supernatants to obtain methodology validation sample eight. The injection concentration of methodology validation sample eight was the same as the theoretical concentration of the thrombopoietin receptor agonist and the theoretical concentration of the corresponding internal standard in methodology validation sample seven, respectively. The methodology validation sample eight was injected and tested, and the peak areas of the target thrombopoietin receptor agonist and the corresponding internal standard were recorded as B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, 1 and B 2 .

[0182] ③ Treat the ultrapure water according to the method for treating blank plasma in "Sample Treatment and Injection Detection" in Example 1 to obtain an ultrapure water supernatant, add a corresponding volume of quality control working solution and internal standard precipitant to the ultrapure water supernatant, and obtain method validation sample 9. The injection concentration of the method validation sample 9 is the same as the theoretical concentration of the thrombopoietin receptor agonist and the theoretical concentration of the corresponding internal standard in the method validation sample 7, respectively. Inject and detect the method validation sample 9, and record the peak areas of the target thrombopoietin receptor agonist and the corresponding internal standard, respectively. 1 and C 2 .

[0183] According to the calculation of extraction recovery rate (%) = A / B × 100%, matrix effect (%) = B / C × 100%, and internal standard normalized matrix factor = target thrombopoietin receptor agonist matrix effect / internal standard matrix effect, the extraction recovery rate was between 73.96% and 99.83%, and the internal standard normalized matrix factor was between 0.97 and 1.19. The results are shown in Table 6.

[0184] Table 6 Extraction recovery and matrix effect

[0185]

[0186] Example 8: Stability

[0187] Quality control samples with three concentration points (L, M and H) were accurately prepared as method validation sample ten. The stability of method validation sample ten was investigated under the following five conditions: placed in an automatic sampler (15°C) for 24 hours after treatment, placed at room temperature (22°C) for 24 hours, refrigerated in a 4°C refrigerator for 48 hours, repeatedly frozen and thawed in a -20°C refrigerator for 3 times (once a day), and frozen in a -20°C refrigerator for 14 days. Six samples were investigated in parallel at each concentration point under each condition. The stability was good under various conditions. The results are shown in Table 7.

[0188] Table 7 Stability under 5 conditions

[0189]

[0190]

[0191] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A method for detecting a thrombopoietin receptor agonist in a biological sample, characterized in that: The method comprises the following steps: mixing the test sample and the internal standard precipitant, vortexing to obtain the test sample supernatant, and using ultra-high performance liquid chromatography coupled with mass spectrometry to quantitatively detect the thrombopoietin receptor agonist in the test sample supernatant; The internal standard precipitant contains an internal standard substance and a suitable solvent, the internal standard substance is an isotope internal standard substance of a thrombopoietin receptor agonist, the suitable solvent contains methanol, acetonitrile and ammonia water, and in the suitable solvent, the content of methanol is greater than the content of ammonia water, and the content of acetonitrile is greater than the content of ammonia water; The volume ratio of methanol to acetonitrile in the suitable solvent is 1:1, and the volume fraction of ammonia water is 0.5%~2%; The four thrombopoietin receptor agonists that can be detected by the method include: avatrombopag, eltrombopag, hetrombopag, and rutrombopag.

2. The method for detecting thrombopoietin receptor agonists in biological samples according to claim 1, characterized in that: The conditions for UPLC include: Gradient elution; Mobile phase A is a formic acid-acetonitrile solution with a volume fraction of formic acid ranging from 0.01% to 0.5%; Mobile phase B is an aqueous solution of formic acid, and the volume fraction of formic acid is 0.01%~0.5%.

3. The method for detecting thrombopoietin receptor agonists in biological samples according to claim 2, characterized in that: The conditions for UPLC include: The model of the chromatographic column in the ultra-high performance liquid chromatography was Waters ACQUITY BEH C18, with a column length of 50 mm and an inner diameter of 2.1 mm; The column filler is octadecylsilane bonded silica gel, and its particle size is 1.7 μm; The flow rate was 0.3~0.5mL / min, the column temperature was 35~40℃, and the injection volume was 1~10μL.

4. The method for detecting thrombopoietin receptor agonists in biological samples according to claim 3, characterized in that: The conditions for gradient elution were: 0.0min, mobile phase A, 16%; mobile phase B, 84%; 0.8min: mobile phase A, 70%; mobile phase B, 30%; 1.0min: mobile phase A, 93%; mobile phase B, 7%; 2.0min: mobile phase A, 99%; mobile phase B, 1%; 3.5min: mobile phase A, 99%; mobile phase B, 1%; 3.51min: mobile phase A, 16%; mobile phase B, 84%; 4~5min: mobile phase A, 16%; mobile phase B, 84%.

5. The method for detecting thrombopoietin receptor agonist in a biological sample according to any one of claims 1, 3-4, characterized in that: The mass spectrometry conditions were: Positive ion electrospray ion source, multiple reaction monitoring MRM, capillary voltage 0.5~2.0kV, desolvation gas temperature 400~500℃, desolvation gas flow rate 600~800L / Hr, cone gas flow rate 40~60L / Hr.

6. The method for detecting thrombopoietin receptor agonists in biological samples according to claim 5, characterized in that: When the method is used to detect the four thrombopoietin receptor agonists, the internal standards include avatrombopag-D8, eltrombopag-4C13 and / or rutrombopag-D13.

7. The method for detecting thrombopoietin receptor agonists in biological samples according to claim 6, characterized in that: The steps of quantitatively detecting the supernatant of the test sample include calibrating the standard sample, and the operation is as follows: A standard sample is prepared, wherein the standard sample contains a thrombopoietin receptor agonist standard and blank plasma; the standard sample is mixed with an internal standard precipitant, and vortexed to obtain a standard sample supernatant; and four thrombopoietin receptor agonists in the standard sample supernatant are separated and quantitatively detected by ultra-high performance liquid chromatography-mass spectrometry, and the detection conditions of the standard sample supernatant and the test sample supernatant are the same; The mass concentration of each thrombopoietin receptor agonist is taken as the horizontal axis. X , the chromatographic peak area ratio of each thrombopoietin receptor agonist to the internal standard is the ordinate - Y , weighted least squares method is used for linear regression, and the weighting factor is 1 / X 2 , and the standard curve equations of the four thrombopoietin receptor agonists were obtained respectively.

8. The method for detecting a thrombopoietin receptor agonist in a biological sample according to any one of claims 6 to 7, characterized in that: The quantitative lower limits of the method for detecting four thrombopoietin receptor agonists are: Avatrombopag: 2 ng / mL; Eltrombopag: 20 ng / mL; Hetromopa: 2 ng / mL; Lutrombopag: 2ng / mL.

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