CN119198978A - A method for detecting thrombopoietin receptor agonists in biological samples - Google Patents
A method for detecting thrombopoietin receptor agonists in biological samples Download PDFInfo
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Abstract
The invention relates to a method for detecting thrombopoietin receptor agonist in biological samples, which comprises the steps of uniformly mixing a sample to be tested with an internal standard precipitant, vibrating and vortex to obtain supernatant of the sample to be tested, quantitatively detecting the thrombopoietin receptor agonist in the supernatant of the sample to be tested by utilizing an ultra-high performance liquid chromatography combined mass spectrometry technology, wherein the internal standard precipitant contains an internal standard substance and a special solvent, the internal standard substance is an isotope internal standard substance of the thrombopoietin receptor agonist, and the special solvent contains methanol, acetonitrile and ammonia water, and the content of the components in the special solvent is methanol > ammonia water and acetonitrile > ammonia water. The invention has the advantages that the provided special solvent can stably and fully dissolve the standard substance of the thrombopoietin receptor agonist and the isotope internal standard, is friendly to instrument accessories, can precipitate protein in blood plasma, reduces the pretreatment time of a sample, and has the advantages of accurate analysis, wide detection linear range and short detection duration.
Description
Technical Field
The invention belongs to the technical field of drug detection in biological samples, and particularly relates to a method for detecting thrombopoietin receptor agonists in biological samples.
Background
The atorvastatin, ai Qubo Pa, the Heteropap and the Luteropap are non-peptide thrombopoietin receptor agonists marketed in China, and the oral absorption of the thrombopoietin receptor agonists is greatly different and can be influenced by food, and are mainly metabolized by CYP3A or CYP2C9 enzymes of the liver. When taken simultaneously with an inhibitor of the metabolic enzyme, the in vivo exposure of the thrombopoietin receptor agonist may be greatly increased, resulting in dose-related adverse effects in the patient. In addition, poor patient compliance and the like can also lead to abnormal in vivo exposure of the above thrombopoietin receptor agonist. Therefore, in clinical practical samples, the concentration of the thrombopoietin receptor agonist can show the phenomenon of excessively high or excessively low bipolar differentiation, and the real drug exposure in a patient needs to be estimated by carrying out therapeutic drug monitoring, so that the patient is subjected to personalized treatment, and adverse reactions are reduced.
Thrombopoietin receptor agonists are poorly soluble in water or organic solvents and DMSO has been reported as the basic solvent. Although DMSO can be used as a universal solvent, it is a major detriment to both the PEEK tubing of liquid chromatography, the packing of the column and the mass spectrometry ion source, so a solvent system that can dissolve all thrombopoietin receptor agonists and corresponding isotopic internal standards simultaneously needs to be invented.
In order to ensure the accuracy of the detection result of the sample with the too high or the too low concentration, reasonable calibration concentration is designed aiming at different thrombopoietin receptor agonists, so that the detection linear range of the method can be covered on various types of samples.
In summary, it is necessary to invent a detection method which has good solubility of the object to be detected, proper linear range, simple pretreatment method and is especially suitable for large-scale biological samples.
Disclosure of Invention
The invention provides a method for detecting thrombopoietin receptor agonist in biological samples, which aims to solve the problems in the background technology.
Mixing a sample to be tested with an internal standard precipitator uniformly, vibrating and vortex to obtain a sample supernatant, and quantitatively detecting the thrombopoietin receptor agonist in the sample supernatant by utilizing an ultra-high performance liquid chromatography combined mass spectrometry technology;
the internal standard precipitant contains an internal standard substance and a special solvent, wherein the internal standard substance is an isotope internal standard substance of the thrombopoietin receptor agonist, the special solvent contains methanol, acetonitrile and ammonia water, and the content of the methanol is greater than the content of the ammonia water and the content of the acetonitrile is greater than the content of the ammonia water in the special solvent.
Further, the volume ratio of the methanol to the acetonitrile in the special solvent is 1:1, the volume fraction of the ammonia water is 0.5% -2%, and preferably, the volume ratio of the methanol to the acetonitrile to the ammonia water in the special solvent is 49.5:49.5:1.
Further, the conditions of the ultra performance liquid chromatography include:
Gradient elution;
The mobile phase A is acetonitrile solution of formic acid, and the volume fraction of the formic acid is 0.01% -0.5%;
the mobile phase B is an aqueous solution of formic acid, and the volume fraction of the formic acid is 0.01% -0.5%.
Further, the conditions of the ultra performance liquid chromatography include:
The model of the chromatographic column in the ultra-high performance liquid chromatography is Waters ACQUITY BEH C mm, the column length is 50mm, and the inner diameter is 2.1mm;
the chromatographic column filler is octadecylsilane chemically bonded silica gel, and the particle size of the filler is 1.7 mu m;
the flow rate is 0.3-0.5 mL/min, the column temperature is 35-40 ℃, and the sample injection amount is 1-10 mu L.
Further, the conditions of gradient elution are:
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%;
4-5 min, mobile phase A,16%, mobile phase B,84%.
Further, the mass spectrometry conditions were:
the positive ion electrospray ion source is used for monitoring MRM in multiple reactions, the capillary voltage is 0.5-2.0 kV, the desolvation gas temperature is 400-500 ℃, the desolvation gas flow rate is 600-800L/Hr, and the taper hole gas flow rate is 40-60L/Hr.
Further, the 4 thrombopoietin receptor agonists detectable by the method include atorvastatin, ai Qubo Pa, heteropap, and Luteropap, and when the 4 thrombopoietin receptor agonists are detected by the method, the internal standard comprises atorvastatin-D8, ai Qubo Pa-4C 13, and/or Luteropap-D13.
Further, the step of quantitatively detecting the supernatant of the test sample includes calibrating a standard sample, which is operated as follows:
Mixing the standard sample with an internal standard precipitator, vibrating and vortex to obtain a standard sample supernatant, and separating and quantitatively detecting 4 kinds of thrombopoietin receptor agonists in the standard sample supernatant by using an ultra-high performance liquid chromatography-mass spectrometry technology, wherein the detection conditions of the standard sample supernatant and the supernatant of a sample to be tested are the same;
And (3) carrying out linear regression by adopting a weighted least square method with the mass concentration of each thrombopoietin receptor agonist as an abscissa-X and the chromatographic peak area ratio of each thrombopoietin receptor agonist to an internal standard as an ordinate-Y, and obtaining standard curve equations of the 4 thrombopoietin receptor agonists respectively with the weighting factor of 1/X 2.
Further, the method has the following quantitative lower limits for detecting 4 thrombopoietin receptor agonists:
2ng/mL of atorvastatin;
ai Qubo Pa, 20ng/mL;
Sea treopa 2ng/mL;
and the volume of the rupoppa is 2ng/mL.
The invention has the advantages and positive effects that at least comprises:
(1) The solvent system has the advantages that the solvent system does not contain dimethyl sulfoxide, can stably and fully dissolve the thrombopoietin receptor agonist standard substance and the isotope internal standard, has small property and dissolution performance change at low temperature and normal temperature, can ensure the stability of a tested agent, and is friendly to chromatographic columns, pipelines, ion sources and other instrument accessories;
(2) The special solvent system can also precipitate protein in blood plasma, and the internal standard precipitant prepared by the special solvent system can finish two procedures of adding an internal standard substance and precipitating protein at one time, so that the pretreatment time of samples is greatly reduced, and the special solvent system is suitable for clinically developing therapeutic drug monitoring of a large number of samples;
(3) The method for rapidly detecting the content of the thrombopoietin receptor agonist in the biological sample by utilizing the ultra-high performance liquid chromatography combined mass spectrometry technology comprises the steps of covering all the types of the thrombopoietin receptor agonist on the market, designing a reasonable detection linear range to cover the actual concentration range of 4 types of the thrombopoietin receptor agonist in clinical samples, carrying out methodological verification, and being accurate in analysis, wide in detection linear range, simple in sample pretreatment, short in detection time (the loading analysis time is only 4.5 min) and suitable for mass clinical sample analysis.
Drawings
FIG. 1 is a chromatogram of a methodological validation sample I in example 3 of the present invention;
FIG. 2 is a chromatogram of a hollow white sample in example 3 of the present invention.
Detailed Description
The invention is further illustrated by the following examples and figures:
Unless defined otherwise, all technical and scientific terms used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments and comparative examples only and is not intended to be limiting of the scope of the present invention. It should be specifically noted that there may be various names for the same organic structure, so long as the structure belongs to the protection object of the present patent within the scope of the present patent.
Unless otherwise defined, the starting materials, reagents, and the like in the following examples may be obtained commercially or prepared according to reported methods.
[ Medicine and reagent ]
The above reagents were purchased from Shanghai Han Xiang Biotechnology Co., ltd.A. of Avermectin (purity: 99%, lot number: 570406-98-3), avermectin-D8 (purity: 98%, lot number: 20201215), ai Qubo Pa (purity: 99%, lot number: 20200907).
The above reagents were purchased from America MedChemExpress, inc. and used in combination with the other reagents, porpa (purity: 98.32%, lot number: 22971) and Porpa-D13 (purity: 98.32%, lot number: 532932).
Ai Qubo Pa-4C 13 (purity: 99%, lot number: 1901-031B 4) was purchased from TLC Pharmaceutical Standards Ltd.
Heteropap ethanolamine (purity: 99%, lot number: 6135231001) was given by Jiangsu Hengrui medical Co., ltd.
Acetonitrile (mass spectrum purity, lot number: 217207), methanol (mass spectrum purity, lot number: 34860-4L-R), formic acid (mass spectrum purity, lot number: A117-50), ammonia water (mass spectrum purity, lot number: 7115080), the above reagents were purchased from Siemens technology (China).
The water for the experiment is prepared from an ultrapure water system.
Blank plasma is available from a variety of sources, such as blood bank waste plasma, reagent companies, and the like.
[ Equipment and consumables ]
A liquid chromatography apparatus (model: xex TQD IVD) was purchased from Waters, inc. of the United states.
The low temperature high speed centrifuge (model: SCIENTIFIC LEGEND Micro 21R), the low speed centrifuge (model: SL 8), and the Vortex machine (model: LP Vortex Mixer) were all purchased from Thermo Fisher, inc. of America.
One ten million balance (model: MS 105) was purchased from Metrehler, america.
Ultra-pure water systems are purchased from mi blog corporation of the united states.
Pipettes (Specifications: 10, 20, 100, 200, 1000. Mu.L) were purchased from Ai Bende, germany.
Volumetric flasks (specification: 10mL, 50 mL) were purchased from Beijing Xinweil glass instruments Co., ltd.
Centrifuge tubes (specification: 0.2mL, 1.5mL, 10 mL) were purchased from Aishi Biotechnology (Hangzhou) Inc.
The embodiment of the invention provides a method for detecting thrombopoietin receptor agonists in biological samples, which can detect 4 non-peptide thrombopoietin receptor agonists, namely, atorvastatin, ai Qubo Pa, herotrpa and Rurotrpa.
The separation and detection technology adopted by the method is an ultra-high performance liquid chromatography combined mass spectrometry technology, and the adopted quantitative method can be an external standard method or an isotope internal standard-standard curve method. For conciseness and brevity, the technical scheme of the present invention is specifically described below with respect to an isotope internal standard-standard curve method.
The method provided by the embodiment of the invention comprises the following steps:
(1) Detecting blank samples
The blank sample is blank plasma, the blank sample is uniformly mixed with a solvent in an internal standard precipitator, the blank sample supernatant is oscillated and vortex, and the ultra-high performance liquid chromatography and mass spectrometry technology is used for detecting the blank sample supernatant.
(2) Calibration standard sample
Mixing a standard sample with an internal standard precipitant uniformly, vibrating and vortex to obtain a standard sample supernatant, and separating and quantitatively detecting 4 thrombopoietin receptor agonists in the standard sample supernatant by utilizing an ultra-high performance liquid chromatography-mass spectrometry technology;
And (3) carrying out linear regression by adopting a weighted least square method with the mass concentration of each thrombopoietin receptor agonist as an abscissa (X) and the chromatographic peak area ratio of each thrombopoietin receptor agonist to the corresponding internal standard as an ordinate (Y), and obtaining standard curve equations of the 4 thrombopoietin receptor agonists respectively with the weighting factor of 1/X 2.
(3) Detection quality control sample
Mixing the quality control sample and an internal standard precipitant uniformly, vibrating and vortex to obtain a quality control sample supernatant, and separating and quantitatively detecting 4 thrombopoietin receptor agonists in the quality control sample supernatant by utilizing an ultra-high performance liquid chromatography-mass spectrometry technology.
(4) Sample specimen for detection
Mixing the sample to be tested with an internal standard precipitant uniformly, vibrating and vortex to obtain a supernatant of the sample to be tested, and quantitatively detecting the thrombopoietin receptor agonist in the supernatant of the sample to be tested by utilizing an ultra-high performance liquid chromatography-mass spectrometry technology.
In the method, the separation and detection conditions of the supernatant of the blank sample, the supernatant of the standard sample, the supernatant of the quality control sample and the supernatant of the test sample are the same as each other, and the separation and detection conditions are as follows:
① Conditions of ultra-high performance liquid chromatography:
The chromatographic column is Waters ACQUITY BEH C mm in model number, 50mm in column length, 2.1mm in column inner diameter, octadecylsilane chemically bonded silica as filler, and 1.7-5 μm in particle size, preferably 1.7 μm in particle size, and has higher column efficiency and higher analysis speed;
The mobile phase A is acetonitrile solution of formic acid, and the volume fraction of the formic acid is 0.01% -0.5%, preferably 0.1%;
the mobile phase B is an aqueous solution of formic acid, and the volume fraction of the formic acid is 0.01% -0.5%, preferably 0.1%.
The acidity generated by the concentration of the formic acid can avoid compound decomposition, and has better peak shape and separation effect on the chromatograph.
The flow rate is 0.3-0.5 mL/min, preferably 0.4mL/min;
The column temperature is 35-40 ℃, preferably 40 ℃;
the sample amount is 1 to 10. Mu.L, preferably 2. Mu.L.
The thrombopoietin receptor agonist detected by the method has similar long-chain parent nucleus, but has certain difference in polarity, the polarity of the atorvastatin is the largest, the polarity of the halopara is lower, the gradient elution condition needs to reduce the proportion of the water phase to be less than 10 percent, preferably 7 percent, within 1min and then slowly reduce the proportion within 1min, preferably 1 percent, in order to ensure the separation efficiency.
The gradient elution conditions are preferably 0.0min, 16% of mobile phase A, 84% of mobile phase B, 0.8min, 70% of mobile phase A, 30% of mobile phase B, 1.0min, 93% of mobile phase A, 7% of mobile phase B, 2.0min, 99% of mobile phase A, 1% of mobile phase B, 3.5min, 99% of mobile phase A, 1% of mobile phase B, 3.51min, 16% of mobile phase A, 84% of mobile phase B, 4-5 min, 16% of mobile phase A, and 84% of mobile phase B.
② Mass spectrometry conditions:
A positive ion electrospray ion source;
the capillary voltage is 0.5-2.0 kV, preferably 2.0kV;
The temperature of the desolventizing gas is 400-500 ℃, preferably 400 ℃;
the flow rate of the desolventizing agent is 600-800L/Hr, preferably 800L/Hr;
the air flow rate of the taper hole is 40-60L/Hr, preferably 60L/Hr;
The ion information, collision energy and cone hole voltage are shown in Table 1 using the multiple reaction monitoring mode MRM.
TABLE 1 ion information, collision energy and cone aperture voltage
The quantitative detection method for the supernatant of the test sample comprises the steps of substituting the chromatographic peak area ratio of the thrombopoietin receptor agonist in the supernatant of the test sample and an internal standard substance into a standard curve equation, calculating to obtain the concentration of the thrombopoietin receptor agonist in the test sample, substituting the chromatographic peak area ratio of the thrombopoietin receptor agonist in the supernatant of the quality control sample and the internal standard substance into the standard curve equation, calculating to obtain the measured concentration of the thrombopoietin receptor agonist in the quality control sample, comparing the deviation degree of the theoretical concentration of the thrombopoietin receptor agonist in the quality control sample, and judging the quality of the quantitative detection.
The method can be used for simultaneously analyzing and detecting the 4 kinds of thrombopoietin receptor agonists, and can also be used for analyzing and detecting one or a mixture of two or more than two of the 4 kinds of thrombopoietin receptor agonists according to actual detection requirements.
The internal standard adopted by the method is an isotope internal standard, and one or more of the atorvastatin-D8, ai Qubo Pa-4C 13 and the rupopa-D13 can be selected according to actual conditions and different detection medicines. In the detection, each thrombopoietin receptor agonist can be respectively selected from a single internal standard method or a double internal standard method, wherein the preferred single internal standard scheme is that the atorvastatin uses the atorvastatin-D8 as an internal standard, the Ai Qubo Pa uses Ai Qubo Pa-4C 13 as an internal standard, the Hepatopa and the Rupatopa use the Rupatopa-D13 as internal standards, and the preferred double internal standard scheme is that the atorvastatin, the Hepatopa and the Rupatopa use the atorvastatin-D8 and the Rupatopa-D13 as internal standards, and the Ai Qubo Pa uses Ai Qubo Pa-4C 13 and the Rupatopa-D13 as internal standards.
The preparation method of the standard sample and the quality control sample in the method comprises the following steps:
① Preparing 4 standard stock solutions, namely taking standard products of the atorvastatin, ai Qubo Pa, the hypapa and the rupoppa, respectively dissolving the standard products by a special solvent, and then fixing the volume;
② Preparing standard working solution, namely mixing a plurality of the 4 standard substance stock solutions in proportion according to actual detection requirements, diluting with a special solvent and fixing the volume to obtain the standard working solution with the highest concentration respectively;
③ And preparing a quality control working solution, namely mixing a plurality of standard substance stock solutions in proportion according to actual detection requirements, diluting with a special solvent and fixing the volume to obtain the highest-concentration quality control working solution respectively, and diluting the highest-concentration quality control working solution downwards by using the special solvent by a multiple dilution method to finally obtain the quality control working solution (containing the highest-concentration quality control working solution) with at least 2 concentration points.
The special solvent contains methanol, acetonitrile and ammonia water, wherein the content of the methanol is greater than the content of the ammonia water, and the content of the acetonitrile is greater than the content of the ammonia water.
Because the 4 thrombopoietin receptor agonists detected by the method have similar long-chain mother cores, one end is of a strong-polarity benzoic acid structure, the other end is of a weak-polarity heterocyclic conjugated system, the lipid solubility and the water solubility of the 4 thrombopoietin receptor agonists are poor, the solubility of the heterocyclic conjugated system can be ensured by adopting the system of the special solvent, the acetonitrile can also be matched with the mobile phase of the ultra-high performance liquid chromatography, and a small amount of ammonia water can increase the solubility of a benzoic acid region, so that the thrombopoietin receptor agonists are well dissolved.
Preferably, the volume ratio of methanol, acetonitrile and ammonia water in the special solvent is 49.5:49.5:1, and the standard stock solution prepared by adopting the solvent system is in a liquid homogeneous phase at-80 ℃ and has no solid precipitation, and the standard stock solution can be transferred to room temperature for normal use.
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 of stepwise dilution, and the dilution ratio of the quality control working solution is preferably 10 times.
④ Preparing a standard sample and a quality control sample:
Mixing standard working solution and blank plasma uniformly according to a ratio to obtain a standard sample;
And uniformly mixing the quality control working solution and blank plasma according to the ratio to obtain a quality control sample.
When the standard sample and the quality control sample are prepared, the volume ratio of the blank plasma to the standard working solution and the volume ratio of the blank plasma to the quality control working solution are respectively (5-10): 1, and are respectively preferably 9:1.
The preparation method of the internal standard precipitant in the method comprises the following steps:
① And (3) preparing an internal standard stock solution, namely dissolving an internal standard substance with a special solvent, and then calibrating the volume to obtain the internal standard stock solution.
② And (3) preparing an internal standard precipitant, diluting the internal standard stock solution with a special solvent, and then fixing the volume to obtain the internal standard precipitant.
The concentration of each internal standard in the internal standard precipitant in the method is preferably 100ng/mL of atorvastatin-D8, 100ng/mL of ruqu-Pa-D13 and 500ng/mL of eltrombopag-4C 13 respectively.
When preparing the supernatant of the standard sample and the supernatant of the quality control sample, 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 respectively 1 (1.5-4), and are respectively preferably 1:2.
The special solvent can not only stably and fully dissolve the standard substance and the isotope internal standard, but also precipitate the protein in the blood plasma, thus the prepared internal standard precipitant can complete two procedures of adding the internal standard substance and precipitating the protein at one time after being mixed with a sample, greatly reduces the pretreatment time of the sample, and is suitable for clinically developing the therapeutic drug monitoring of a large number of samples.
When the method is used for detection, the common needle washing liquid needs to be changed into the special solvent so as to avoid higher sample injection residues and influence on detection accuracy.
The lower limit of quantification for detecting the 4 thrombopoietin receptor agonists by adopting the method is respectively 2ng/mL of atorvastatin, 20ng/mL of Ai Qubo Pa, 2ng/mL of halopara and 2ng/mL of halopara.
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by those skilled in the art without departing from the spirit of the invention, which falls within the scope of the invention.
Example 1 rapid detection of thrombopoietin receptor agonist levels in biological samples
S1, preparing a solution
① Preparing special solvent
Mixing methanol, acetonitrile and ammonia water in the volume ratio of 49.5% to 1% to obtain the special solvent.
② Preparing 4 standard stock solutions and 3 internal standard stock solutions
Precisely weighing solid standard substances and internal standard substances of the thrombopoietin receptor agonist, respectively dissolving the solid standard substances and the internal standard substances by using special solvents, respectively transferring the solid standard substances and the internal standard substances into 10mL volumetric flasks, and uniformly shaking the volumetric flasks by using the special solvents to obtain 4 standard substance stock solutions with the concentration of 1000000ng/mL and 3 internal standard stock solutions with the concentration of 100000 ng/mL.
The weight amounts are 10mg of Alvarroa, 10mg Ai Qubo Pa, 10mg of Uvalia, 12.7mg of Hedera lobata ethanol amine, 1mg of Alvarroa-D8, 1mg of Ai Qubo Pa-4C 13 and 1mg of Uvalia-D13 respectively.
All stock solutions mentioned above can be stored at-80 ℃.
③ Preparing 7 concentration points standard working solution
Precisely measuring 20 mu L of the standard substance stock solution of the atorvastatin, 200 mu L of the standard substance stock solution of the Heteropap and 200 mu L of the standard substance stock solution of the Rutrepa, mixing, adding 740 mu L of a special solvent to a certain volume, and shaking uniformly to obtain the standard working solution with the highest concentration of S7 concentration point.
And (3) carrying out 2-5-2-5-2-5 times stepwise dilution on the standard working solution of the S7 concentration point by adopting a special solvent to obtain the standard working solution of 6 concentration points in total of S6-S1.
④ Preparing quality control working solution with 3 concentration points
Accurately measuring 18 mu L of the standard substance stock solution of the atorvastatin, 180 mu L of the standard substance stock solution of the Heteropap and 180 mu L of the standard substance stock solution of the Rutrepa, mixing, adding 766 mu L of a special solvent to a certain volume, and shaking uniformly to obtain the quality control working solution with the highest concentration of H concentration point.
And (3) carrying out 10-time stepwise dilution on the quality control working solution of the H concentration points by adopting a special solvent to obtain the quality control working solution of M, L total 2 concentration points.
The standard working solution and the quality control working solution can be stored at-20 ℃, the specific preparation method is shown in table 2, and the specific concentration is shown in table 3.
Table 2 stepwise dilution preparation method of standard working solution and quality control working solution
TABLE 3 concentration of standard working fluid and quality control working fluid
⑤ Preparing standard sample and quality control sample
Accurately adding 180 mu L of blank plasma and 20 mu L of standard working solution into a 1.5mL centrifuge tube to obtain a standard sample;
180 mu L of blank plasma and 20 mu L of quality control working solution are precisely added into a 1.5mL centrifuge tube, so as to obtain a quality control sample.
⑥ Preparing internal standard precipitant
Precisely measuring 10 mu L of the internal standard stock solution of the atorvastatin-D8, 10 mu L of the internal standard stock solution of the ruxotropa-D13 and 50 mu L of the internal standard stock solution of Ai Qubo Pa-4C 13, adding into a 10mL volumetric flask, and shaking uniformly after the volumetric flask is fixed with a special solvent to obtain the internal standard precipitant.
S2, sample processing and sample injection detection
Conditions for ultra-high performance liquid chromatography and mass spectrometry have been described in detail above and are not described in detail herein.
The blank sample was subjected to the following operations of precisely adding 200. Mu.L of the blank sample (i.e., blank plasma) and 400. Mu.L of a specific solvent into a 1.5mL centrifuge tube, shaking and vortexing, centrifuging at 14000rpm/min for 5min at 10 ℃, precisely measuring the supernatant, and performing sample injection detection.
And respectively carrying out the following operations of precisely adding 200 mu L of sample and 400 mu L of internal standard precipitant into a 1.5mL centrifuge tube, vibrating and vortex, centrifuging at a speed of 14000rpm/min for 5min at 10 ℃, precisely measuring the supernatant, and carrying out sample injection detection.
S3, obtaining a standard curve equation
And (3) taking the mass concentration of each thrombopoietin receptor agonist in the standard sample as an abscissa (X), the chromatographic peak area ratio of each thrombopoietin receptor agonist to the corresponding internal standard substance as an ordinate (Y), adopting a weighted least square method to carry out linear regression, and taking a weighting factor as 1/X 2 to respectively obtain standard curve equations of the 4 types of the thrombopoietin receptor agonists.
Wherein, the atorvastatin uses the atorvastatin-D8 as an internal standard, the Ai Qubo Pa uses Ai Qubo Pa-4C 13 as an internal standard, and the Heteropap and the Luteropap respectively use the Luteropap-D13 as internal standards.
S4, quantitatively calculating the thrombopoietin receptor agonist contained in the test sample
Substituting the chromatographic peak area ratio of the thrombopoietin receptor agonist in the supernatant of the tested sample and the corresponding internal standard substance into a standard curve equation, calculating to obtain the concentration of the thrombopoietin receptor agonist in the tested sample, substituting the chromatographic peak area ratio of the thrombopoietin receptor agonist in the supernatant of the quality control sample and the corresponding internal standard substance into the standard curve equation, calculating to obtain the measured concentration of the thrombopoietin receptor agonist in the quality control sample, comparing the deviation degree of the theoretical concentration of the thrombopoietin receptor agonist in the quality control sample, and judging the quality of the quantitative detection.
Examples 2-8 methodological validation was performed on the method of example 1 according to the requirements of the chinese pharmacopoeia of the 2020 edition.
Example 2 linearity and Range
The standard sample was continuously processed and assayed for 3 days according to the method for detecting a standard sample in "sample processing and sample introduction detection" in example 1. Ai Qubo Pa has good linear relation in the concentration range of 20 ng/mL-20000 ng/mL, and the atorvastatin, the Hexaprop and the Lu-nepar have good linear relation in the concentration range of 2 ng/mL-2000 ng/mL respectively, and the determining coefficients (r) are all larger than 0.99. The lower limit of the method for the quantification of Ai Qubo Pa is 20ng/mL, and the lower limit of the method for the quantification of the atorvastatin, the maritima and the rotigotine is 2ng/mL.
EXAMPLE 3 specificity
A standard sample of the "lower limit on quantitation" concentration value in example 2 was formulated as a methodological validation sample one.
According to the detection method of "sample processing and sample introduction detection" in example 1, the first and blank samples were detected respectively to obtain the chromatogram of the first sample (see fig. 1) and the chromatogram of the blank sample (see fig. 2).
As can be seen from the accompanying figure 1, the retention times of the atorvastatin, the hypaparine and the Ai Qubo Pa are respectively 1.25min, 2.17min, 1.76min and 2.12min, the retention times of the internal standards of the atorvastatin-D8, the hypaparine-D13 and the Ai Qubo Pa-4C 13 are respectively 1.25min, 1.75min and 2.12min, the 7 detection channels do not interfere with each other, and as can be seen from the accompanying figure 2, the endogenous substances in a blank sample (namely blank blood plasma) do not interfere with the detection of the target substances (namely 4 platelet-forming hormone receptor agonists) and 3 internal standards.
Example 4 accuracy and precision
5 Standard samples of the "lower limit of quantitation" concentration value in example 2 were prepared in parallel and used as methodological validation samples two.
Quality control samples of 3 concentration points (L, M and H) were prepared in parallel 5 each as methodological validation sample three.
According to the detection method of "sample processing and sample introduction detection" in example 1, the second and third detection methodologies were used to verify the sample respectively, and the concentration was calculated daily by using the newly measured standard curve equation for 3 consecutive days of investigation.
Accuracy and precision were assessed using the relative error (RE%) and the relative standard deviation (RSD%) and the results are shown in table 4 for both the daily and daily RE% and RSD% of the 4 thrombopoietin receptor agonists to be within 15% (LLOQ limit 20%).
TABLE 4 accuracy and precision
EXAMPLE 5 residual Effect
① 5 Standard samples of the "lower limit of quantitation" concentration value in example 2 were prepared in parallel and used as methodological validation samples four.
Sample four was validated according to the test method of "sample handling and sample introduction test" in example 1, 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, respectively.
② 5 Standard samples with concentration points S7 were prepared in parallel and used as methodological validation samples five.
According to the detection method of sample processing and sample injection detection in example 1, the sample five and blank samples are verified by the detection method respectively, the sample injection sequence of the sample five-blank sample is verified according to the method, and the sample injection sequence is repeated 5 times, and the average value of chromatographic peak areas of the target thrombopoietin receptor agonist and the corresponding internal standard of the blank sample is recorded as b 1 and b 2 respectively.
The residual ratio of the target thrombopoietin receptor agonist was calculated to be b 1/a1, less than 20%, the residual ratio of the internal standard to be b 2/a2, less than 5%.
EXAMPLE 6 dilution Effect
Precisely measuring 40 mu L of atorvastatin standard stock solution, 40 mu L of sea-buckthorn standard stock solution, 400 mu L of Ai Qubo Pa standard stock solution and 40 mu L of Lu-triptopa standard stock solution, placing the solution in a 1.5mL centrifuge tube, and adding 480 mu L of special solvent to fix the volume to obtain a dilution effect basic solution I.
Precisely measuring 100 mu L of dilution effect base solution I, adding 900 mu L of blank plasma, and preparing dilution effect base solution II.
Using blank plasma, the dilution effect base solution was diluted 10-fold (20 μl dilution effect base solution two+180 μl blank plasma), 25-fold (8 μl dilution effect base solution two+192 μl blank plasma) and 50-fold (4 μl dilution effect base solution two+196 μl blank plasma), respectively, and 5 parts of each concentration point was prepared in parallel to obtain a methodological verification sample six.
Sample six was verified according to the test method of "sample processing and sample introduction test" in example 1, using the standard curve equation newly measured on the same day to calculate the concentration, RE% being within 11%, and the results are shown in table 5.
TABLE 5 dilution effect
Example 7 extraction recovery and matrix Effect
① Quality control samples of 3 concentration points (L, M and H) were accurately formulated in parallel 6 each as methodological validation sample seven.
Sample seven was validated according to the test methods of "sample handling and sample introduction test" in example 1, the peak areas of the target thrombopoietin receptor agonist and the corresponding internal standard were recorded as a 1 and a 2, respectively.
② And (3) processing 6 blank plasma from different sources according to the method of sample processing and sample injection detection in the embodiment 1 to obtain blank sample supernatant, adding a corresponding volume of quality control working solution and an internal standard precipitating agent into the blank sample supernatant to obtain a methodology verification sample eight, wherein the sample injection concentration of the methodology verification sample eight is the same as the theoretical concentration of the thrombopoietin receptor agonist and the theoretical concentration of the corresponding internal standard in the methodology verification sample seven, the sample injection detection methodology verification sample eight records that the peak areas of the target thrombopoietin receptor agonist and the corresponding internal standard are B 1 and B 2 respectively.
③ The method for treating blank plasma in "sample treatment and sample introduction detection" in example 1 was followed to obtain ultrapure water supernatant, and a corresponding volume of a quality control working solution and an internal standard precipitant were added to the ultrapure water supernatant to obtain a recipe verification sample nine, the sample introduction concentration of which was the same as the theoretical concentration of the thrombopoietin receptor agonist and the theoretical concentration of the corresponding internal standard in the recipe verification sample seven, respectively, and the peak areas of the target thrombopoietin receptor agonist and the corresponding internal standard were recorded as C 1 and C 2, respectively.
The extraction recovery rate (%) = a/b×100%, the matrix effect (%) = B/c×100% and the internal standard normalized matrix factor=target thrombopoietin receptor agonist matrix effect/internal standard matrix effect were calculated, the extraction recovery rate was 73.96% -99.83%, the internal standard normalized matrix factor was 0.97% -1.19, and the results are shown in table 6.
TABLE 6 extraction recovery and matrix Effect
Example 8 stability
Quality control samples of 3 concentration points (L, M and H) are accurately prepared, the quality control samples are used as methodology verification samples ten, the inspection methodology is used for verifying the stability of the sample ten under the following 5 conditions that the sample ten is placed in an automatic sampler (15 ℃) for 24 hours after being treated, the sample is placed at room temperature (22 ℃) for 24 hours, a 4 ℃ refrigerator is refrigerated for 48 hours, a 20 ℃ refrigerator is repeatedly frozen and thawed for 3 times (once a day), the sample ten is frozen for 14 days by the 20 ℃ refrigerator, 6 samples are inspected in parallel at each concentration point of each condition, and the stability under various conditions is good, and the results are shown in Table 7.
Table 75 stability under conditions
The foregoing describes the embodiments of the present invention in detail, but the description is only a preferred embodiment of the present invention and should not be construed as limiting the scope of the invention. All equivalent changes and modifications within the scope of the present invention are intended to be covered by the present invention.
Claims (9)
1. The method for detecting the thrombopoietin receptor agonist in the biological sample is characterized by comprising the steps of uniformly mixing a sample to be tested with an internal standard precipitant, vibrating and vortex to obtain a supernatant of the sample to be tested, and quantitatively detecting the thrombopoietin receptor agonist in the supernatant of the sample to be tested by utilizing an ultra-high performance liquid chromatography combined mass spectrometry technology;
the internal standard precipitant contains an internal standard substance and a special solvent, wherein the internal standard substance is an isotope internal standard substance of the thrombopoietin receptor agonist, the special solvent contains methanol, acetonitrile and ammonia water, and the content of the methanol is greater than the content of the ammonia water and the content of the acetonitrile is greater than the content of the ammonia water in the special solvent.
2. The method for detecting thrombopoietin receptor agonist in biological samples according to claim 1, wherein the volume ratio of methanol to acetonitrile in the specific solvent is 1:1, and the volume fraction of ammonia water is 0.5% -2%.
3. The method for detecting thrombopoietin receptor agonist in a biological sample according to claim 1 or 2, characterized in that the conditions of ultra performance liquid chromatography comprise:
Gradient elution;
The mobile phase A is acetonitrile solution of formic acid, and the volume fraction of the formic acid is 0.01% -0.5%;
the mobile phase B is an aqueous solution of formic acid, and the volume fraction of the formic acid is 0.01% -0.5%.
4. The method for detecting thrombopoietin receptor agonist in a biological sample according to claim 3, wherein the conditions of ultra performance liquid chromatography comprise:
The model of the chromatographic column in the ultra-high performance liquid chromatography is Waters ACQUITY BEH C mm, the column length is 50mm, and the inner diameter is 2.1mm;
the chromatographic column filler is octadecylsilane chemically bonded silica gel, and the particle size of the filler is 1.7 mu m;
the flow rate is 0.3-0.5 mL/min, the column temperature is 35-40 ℃, and the sample injection amount is 1-10 mu L.
5. The method for detecting thrombopoietin receptor agonist in a biological sample according to claim 4, the method is characterized in that the gradient elution conditions are as follows:
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%;
4-5 min, mobile phase A,16%, mobile phase B,84%.
6. The method for detecting thrombopoietin receptor agonist in a biological sample according to any one of claims 1-2 and 4-5, wherein mass spectrometry conditions are:
the positive ion electrospray ion source is used for monitoring MRM in multiple reactions, the capillary voltage is 0.5-2.0 kV, the desolvation gas temperature is 400-500 ℃, the desolvation gas flow rate is 600-800L/Hr, and the taper hole gas flow rate is 40-60L/Hr.
7. The method of claim 6, wherein the 4 thrombopoietin receptor agonists detectable by the method comprise atorvastatin, ai Qubo Pa, heteropap, and wherein the internal standard comprises atorvastatin-D8, ai Qubo Pa-4C 13, and/or Luteropap-D13 when the 4 thrombopoietin receptor agonists are detected by the method.
8. The method of claim 7, wherein the step of quantitatively detecting thrombopoietin receptor agonist in the supernatant of the test sample comprises calibrating a standard sample, which is operable to:
Mixing the standard sample with an internal standard precipitant, vibrating and vortex to obtain a standard sample supernatant, and separating and quantitatively detecting 4 kinds of thrombopoietin receptor agonists in the standard sample supernatant by using an ultra-high performance liquid chromatography-mass spectrometry technology, wherein the detection conditions of the standard sample supernatant and the supernatant of the sample to be tested are the same;
And (3) carrying out linear regression by adopting a weighted least square method with the mass concentration of each thrombopoietin receptor agonist as an abscissa-X and the chromatographic peak area ratio of each thrombopoietin receptor agonist to an internal standard as an ordinate-Y, and obtaining standard curve equations of the 4 thrombopoietin receptor agonists respectively with the weighting factor of 1/X 2.
9. The method for detecting thrombopoietin receptor agonist in a biological sample according to any one of claims 7-8, wherein the method comprises the following quantitative lower limits for detecting 4 thrombopoietin receptor agonists:
2ng/mL of atorvastatin;
ai Qubo Pa, 20ng/mL;
Sea treopa 2ng/mL;
and the volume of the rupoppa is 2ng/mL.
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