LC-MS / MS-based method for determining baloxavir concentration in human plasma

Abstract

The present invention relates to the field of baloxavir concentration detection, and in particular to a method for detecting baloxavir concentration in human plasma based on LC-MS / MS. The method obtains the drug extraction efficiency and internal standard extraction efficiency of each theoretical concentration according to the drug detection concentration and internal standard detection concentration of a standard plasma solution at each theoretical concentration, obtains the reliability coefficient of the internal standard based on the difference between the drug extraction efficiency and the internal standard extraction efficiency between the same theoretical concentrations, obtains the extraction efficiency of the internal standard in the plasma solution to be tested based on the reference internal standard detection concentration of the plasma solution to be tested containing the internal standard at the reference theoretical concentration, and then predicts the extraction efficiency of baloxavir in the plasma solution to be tested, adjusts the baloxavir concentration of the plasma solution to be tested based on the reliability coefficient of the internal standard and the predicted extraction efficiency, and obtains the true baloxavir concentration of the plasma solution to be tested. The present invention can improve the detection accuracy of baloxavir concentration in plasma.

Description

Technical Field

[0001] The present invention relates to the field of baloxavir concentration detection, and in particular to a method for detecting baloxavir concentration in human plasma based on LC-MS / MS. Background Art

[0002] Baloxavir is a new small molecule neuraminidase inhibitor used to treat and prevent influenza virus infection. Accurate detection of baloxavir concentration in plasma can not only evaluate the human body's absorption efficiency of baloxavir and provide personalized treatment, but also prevent various adverse reactions. Detection of baloxavir concentration in plasma is of great significance in clinical practice and research.

[0003] In related technologies, protein precipitation or extraction methods are usually used to remove proteins from plasma samples in advance, thereby separating baloxavir and plasma proteins in the plasma samples. Then, liquid chromatography-tandem mass spectrometry (LC-MS / MS) is used to detect the baloxavir concentration in the plasma samples. However, since baloxavir has a strong binding rate with plasma proteins in plasma, it is difficult to extract baloxavir from the protein-bound state during the sample processing process, resulting in low extraction efficiency of baloxavir using existing methods, and thus poor detection accuracy of baloxavir concentration in plasma. Summary of the Invention

[0004] In order to solve the technical problem that the existing methods have low extraction efficiency of baloxavir, resulting in poor accuracy in detecting baloxavir concentration in plasma, the present invention aims to provide a method for detecting baloxavir concentration in human plasma based on LC-MS / MS. The technical solution adopted is as follows:

[0005] The present invention proposes a method for detecting baloxavir concentration in human plasma based on LC-MS / MS, which comprises:

[0006] Obtaining drug detection concentrations of a standard plasma solution containing baloxavir at different theoretical concentrations, and internal standard detection concentrations of a standard plasma solution containing an internal standard substance of baloxavir at different theoretical concentrations;

[0007] According to the drug detection concentration and the internal standard detection concentration at each theoretical concentration, the drug extraction efficiency and the internal standard extraction efficiency of the standard plasma solution at each theoretical concentration are obtained; according to the difference between the drug extraction efficiency and the internal standard extraction efficiency at the same theoretical concentration, and the change between the drug extraction efficiency and the internal standard extraction efficiency at the same theoretical concentration, the reliability coefficient of the internal standard is obtained;

[0008] Based on the difference between the drug extraction efficiency and the internal standard extraction efficiency at the same theoretical concentration, a reference theoretical concentration is selected from all theoretical concentrations, and a reference internal standard detection concentration of the test plasma solution containing the internal standard at the reference theoretical concentration is obtained; based on the reference internal standard detection concentration at the reference theoretical concentration, a reference extraction efficiency of the internal standard in the test plasma solution is obtained; based on the difference between the internal standard extraction efficiency and the reference extraction efficiency at the reference theoretical concentration, as well as the drug extraction efficiency and internal standard extraction efficiency at different theoretical concentrations, a predicted extraction efficiency of baloxavir in the test plasma solution is obtained;

[0009] Based on the reliability coefficient of the internal standard and the predicted extraction efficiency of baloxavir in the plasma solution to be tested, the detected baloxavir concentration of the plasma solution to be tested is adjusted to obtain the actual baloxavir concentration of the plasma solution to be tested.

[0010] Furthermore, obtaining the drug extraction efficiency and internal standard extraction efficiency of the standard plasma solution at each theoretical concentration includes:

[0011] The drug test concentration at each theoretical concentration is used as the numerator, each theoretical concentration is used as the denominator, and the ratio is used as the drug extraction efficiency of the standard plasma solution at each theoretical concentration;

[0012] The internal standard detection concentration at each theoretical concentration is used as the numerator, each theoretical concentration is used as the denominator, and the ratio is used as the internal standard extraction efficiency of the standard plasma solution at each theoretical concentration.

[0013] Furthermore, the reliability coefficient of obtaining the internal standard includes:

[0014] According to the difference between the drug extraction efficiency and the internal standard extraction efficiency at the same theoretical concentration, the extraction efficiency proximity between the internal standard and baloxavir in the standard plasma solution is obtained;

[0015] The absolute value of the difference between each theoretical concentration and the next adjacent theoretical concentration in the drug extraction efficiency is used as the drug extraction efficiency change of each theoretical concentration, and the absolute value of the difference between each theoretical concentration and the next adjacent theoretical concentration in the internal standard extraction efficiency is used as the internal standard extraction efficiency change of each theoretical concentration;

[0016] According to the difference between the change in the drug extraction efficiency and the change in the internal standard extraction efficiency at the same theoretical concentration, the similarity of the change in the extraction efficiency between the internal standard and baloxavir in the standard plasma solution is obtained;

[0017] The extraction efficiency proximity and the extraction efficiency variation similarity are integrated and normalized to obtain a reliability coefficient of the internal standard.

[0018] Furthermore, obtaining the closeness of the extraction efficiency between the internal standard and baloxavir in the standard plasma solution comprises:

[0019] The absolute value of the difference between the drug extraction efficiency and the internal standard extraction efficiency at the same theoretical concentration is used as the extraction efficiency difference value of the standard plasma solution at each theoretical concentration;

[0020] The average values ​​of the extraction efficiency differences at all theoretical concentrations were negatively correlated to obtain the extraction efficiency proximity between the internal standard and baloxavir in the standard plasma solution.

[0021] Furthermore, obtaining the similarity of the changes in extraction efficiency between the internal standard and baloxavir in the standard plasma solution includes:

[0022] The absolute value of the difference between the change in drug extraction efficiency and the change in internal standard extraction efficiency at the same theoretical concentration is taken as the difference in extraction efficiency of the standard plasma solution at each theoretical concentration;

[0023] Negative correlation mapping was performed on the average values ​​of the extraction efficiency change differences at all theoretical concentrations to obtain the similarity of the extraction efficiency changes between the internal standard and baloxavir in the standard plasma solution.

[0024] Furthermore, selecting a reference theoretical concentration from all theoretical concentrations includes:

[0025] The theoretical concentration corresponding to the minimum value of the extraction efficiency difference is used as the reference theoretical concentration.

[0026] Furthermore, obtaining a reference extraction efficiency of the internal standard in the plasma solution to be tested includes:

[0027] The reference internal standard detection concentration under the reference theoretical concentration is used as the numerator, the reference theoretical concentration is used as the denominator, and the ratio is used as the reference extraction efficiency of the internal standard in the plasma solution to be tested.

[0028] Furthermore, obtaining the predicted extraction efficiency of baloxavir in the test plasma solution includes:

[0029] Normalizing the absolute value of the difference between the internal standard extraction efficiency of the standard plasma solution at the reference theoretical concentration and the reference extraction efficiency of the internal standard in the plasma solution to be tested to obtain the extraction deviation of the internal standard in the plasma solution to be tested;

[0030] Inputting the drug extraction efficiency at different theoretical concentrations and the internal standard extraction efficiency into a linear regression model, outputting a regression function from the linear regression model, and substituting the reference extraction efficiency of the internal standard in the test plasma solution into the regression function to obtain the initial extraction efficiency of baloxavir in the test plasma solution;

[0031] According to the extraction deviation of the internal standard in the plasma solution to be tested, the initial extraction efficiency of baloxavir in the plasma solution to be tested is adjusted to obtain the predicted extraction efficiency of baloxavir in the plasma solution to be tested.

[0032] Furthermore, the initial extraction efficiency of baloxavir in the plasma solution to be tested is adjusted according to the extraction deviation of the internal standard in the plasma solution to be tested to obtain the predicted extraction efficiency of baloxavir in the plasma solution to be tested, comprising:

[0033] The product of the extraction deviation of the internal standard in the plasma solution to be tested and the initial extraction efficiency of baloxavir in the plasma solution to be tested is used as the adjustment amount of the extraction efficiency of baloxavir in the plasma solution to be tested;

[0034] The difference between the initial extraction efficiency of baloxavir in the test plasma solution and the adjusted amount of extraction efficiency is used as the predicted extraction efficiency of baloxavir in the test plasma solution.

[0035] Furthermore, obtaining the actual baloxavir concentration of the plasma solution to be tested comprises:

[0036] The detected baloxavir concentration of the plasma solution to be tested is used as the numerator, the product of the reliability coefficient of the internal standard and the predicted extraction efficiency of baloxavir in the plasma solution to be tested is used as the denominator, and the ratio is used as the actual baloxavir concentration of the plasma solution to be tested.

[0037] The present invention has the following beneficial effects:

[0038] The present invention takes into account the low extraction efficiency of baloxavir in existing methods, resulting in poor accuracy in detecting baloxavir concentration in plasma. Therefore, the present invention introduces a stable isotope internal standard with a highly similar structure to baloxavir and uses the indirectly predicted actual extraction efficiency of baloxavir in the test plasma solution to correct the detection result of baloxavir concentration in the test plasma solution. This can effectively compensate for the extraction loss caused by incomplete protein binding and release during sample processing. Compared with the extraction model established by relying solely on blank plasma, this overcomes the systematic deviation caused by the difference in protein composition and binding properties between blank plasma and real plasma. The ultimately obtained baloxavir concentration data can more truly reflect the drug exposure level in vivo, thereby achieving accurate detection of baloxavir concentration in plasma based on LC-MS / MS technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions and advantages of the embodiments of the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art descriptions. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0040] Figure 1 This is a flow chart of a method for detecting baloxavir concentration in human plasma based on LC-MS / MS provided in one embodiment of the present invention. DETAILED DESCRIPTION

[0041] To further illustrate the technical means and efficacy employed by the present invention to achieve its intended objectives, the following, in conjunction with the accompanying drawings and preferred embodiments, details the specific implementation, structure, features, and efficacy of a method for detecting baloxavir concentration in human plasma based on LC-MS / MS according to the present invention. In the following description, references to different "one embodiment" or "another embodiment" do not necessarily refer to the same embodiment. Furthermore, specific features, structures, or characteristics of one or more embodiments may be combined in any suitable manner.

[0042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0043] The specific scheme of the method for detecting baloxavir concentration in human plasma based on LC-MS / MS provided by the present invention is described in detail below with reference to the accompanying drawings.

[0044] See also Figure 1 , which shows a flow chart of a method for detecting baloxavir concentration in human plasma based on LC-MS / MS provided by one embodiment of the present invention, the method comprising:

[0045] Step S1: obtaining drug detection concentrations of a standard plasma solution containing baloxavir at different theoretical concentrations, and internal standard detection concentrations of a standard plasma solution containing an internal standard substance of baloxavir at different theoretical concentrations.

[0046] The embodiment of the present invention firstly Different amounts of baloxavir are added to blank plasma solutions to prepare multiple standard plasma solutions containing baloxavir at different theoretical concentrations. The blank plasma solution refers to a plasma solution that does not contain baloxavir and its internal standard. In one embodiment of the present invention, the theoretical concentrations are 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, and 100 ng / mL, respectively. For example, if the theoretical concentration of a standard plasma solution containing baloxavir is 0.05 ng / mL, it means that the baloxavir concentration of the standard plasma solution is 0.05 ng / mL. The specific values ​​of the theoretical concentrations and the number of types can also be set by the implementer according to the specific implementation scenario and are not limited here.

[0047] Similarly, in more than 100 Different amounts of baloxavir internal standard substances were added to blank plasma solutions, thereby preparing multiple standard plasma solutions containing internal standard substances with different theoretical concentrations. The theoretical concentrations of the standard plasma solutions containing the internal standard substances corresponded one to one with the theoretical concentrations of the standard plasma solutions containing baloxavir. That is, the concentrations of the internal standard substances in the standard plasma solutions containing the internal standard substances were also 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, and 100 ng / mL, respectively. In one embodiment of the present invention, baloxavir acid was selected as the internal standard substance of baloxavir. The internal standard substance is not limited here.

[0048] Then, for any standard plasma solution containing baloxavir, a protein precipitant such as acetonitrile or methanol is added to the standard plasma solution, and the solution is centrifuged to separate the protein in the standard plasma solution. The standard plasma solution after protein separation is processed using liquid chromatography-tandem mass spectrometry (LC-MS / MS) to detect the concentration of baloxavir in the standard plasma solution, i.e., the drug detection concentration. The same treatment is then performed on each standard plasma solution containing baloxavir to obtain the drug detection concentration of the standard plasma solution containing baloxavir at different theoretical concentrations. The same method is then used to treat each standard plasma solution containing an internal standard to obtain the internal standard detection concentration of the standard plasma solution containing the internal standard at different theoretical concentrations.

[0049] Step S2: Based on the drug detection concentration and internal standard detection concentration at each theoretical concentration, the drug extraction efficiency and internal standard extraction efficiency of the standard plasma solution at each theoretical concentration are obtained; based on the difference in drug extraction efficiency and internal standard extraction efficiency between the same theoretical concentrations, and the change in drug extraction efficiency and internal standard extraction efficiency between the same theoretical concentrations, the reliability coefficient of the internal standard is obtained.

[0050] During the detection of baloxavir concentration in plasma, baloxavir has a strong binding ability with plasma proteins, and most of it exists in a bound state, making it difficult to be completely released during sample processing. Although protein separation is performed, some baloxavir will still be separated out along with the protein, thereby affecting its recovery rate during the extraction process, resulting in a low final measured concentration and an error. Therefore, in the embodiment of the present invention, standard plasma solutions containing baloxavir and its internal standard substance at different concentrations are respectively provided. Then, based on the drug detection concentration and the internal standard detection concentration at each theoretical concentration, the drug extraction efficiency and the internal standard extraction efficiency of the standard plasma solution at each theoretical concentration are obtained. The drug extraction efficiency refers to the extraction rate of baloxavir in the standard plasma solution, and the internal standard extraction efficiency refers to the extraction rate of the baloxavir internal standard substance in the standard plasma solution. Subsequently, by analyzing the difference between the drug extraction efficiency and the internal standard extraction efficiency at the same theoretical concentration, the reliability of the internal standard substance used to correct the baloxavir concentration can be accurately calculated and analyzed, thereby improving the accuracy of subsequent analysis of the baloxavir concentration in plasma.

[0051] Preferably, in one embodiment of the present invention, the method for obtaining the drug extraction efficiency and internal standard extraction efficiency of the standard plasma solution at each theoretical concentration specifically includes:

[0052] The drug detection concentration at each theoretical concentration is used as the numerator, each theoretical concentration is used as the denominator, and the ratio is used as the drug extraction efficiency of the standard plasma solution at each theoretical concentration. The internal standard detection concentration at each theoretical concentration is used as the numerator, each theoretical concentration is used as the denominator, and the ratio is used as the internal standard extraction efficiency of the standard plasma solution at each theoretical concentration.

[0053] As an example, in one embodiment of the present invention, the expression for the drug extraction efficiency and internal standard extraction efficiency of the standard plasma solution at each theoretical concentration can be specifically, for example, as follows:

[0054] ;

[0055] ;

[0056] in, Indicates that the standard plasma solution is Drug extraction efficiency at each theoretical concentration; Indicates the standard plasma solution containing baloxavir Drug detection concentration under theoretical concentration; Indicates the Theoretical concentration; Indicates that the standard plasma solution is The extraction efficiency of the internal standard at each theoretical concentration; The standard plasma solution containing the internal standard of baloxavir is The internal standard detection concentration is the same as the theoretical concentration.

[0057] In the process of detecting the concentration of baloxavir in human plasma using LC-MS / MS technology, since baloxavir exists mainly in the form of binding to plasma proteins in the body, it is difficult to completely release it from the bound state during the extraction process, resulting in a low measured concentration. Therefore, in the embodiment of the present invention, a stable isotope internal standard substance with a highly similar structure to baloxavir is added to the blank plasma solution, and the same treatment process such as protein precipitation is performed as with the standard plasma solution containing baloxavir. Since the internal standard substance and baloxavir are basically consistent in physicochemical properties, protein binding ability, extraction behavior and mass spectrometry response characteristics, the internal standard substance is substantially consistent in the standard plasma solution. The behavior in the plasma solution and the subsequent plasma solution to be tested can be regarded as a reflection of baloxavir. At the same time, considering that even if the overall structure of the internal standard and baloxavir is similar, there may still be slight deviations in the extraction efficiency between the two due to isotope labeling positions or minor structural differences. Therefore, the embodiment of the present invention analyzes the difference between the drug extraction efficiency and the internal standard extraction efficiency at the same theoretical concentration, as well as the change in the drug extraction efficiency and the internal standard extraction efficiency at the same theoretical concentration. The obtained reliability coefficient reflects the reliability of the internal standard in correcting the subsequent baloxavir detection concentration, thereby improving the accuracy of baloxavir concentration detection analysis.

[0058] Preferably, in one embodiment of the present invention, the method for obtaining the reliability coefficient of the internal standard specifically includes:

[0059] First, based on the difference between the drug extraction efficiency and the internal standard extraction efficiency at the same theoretical concentration, the similarity of the extraction efficiency between the internal standard and baloxavir in the standard plasma solution was obtained. The greater the similarity of the extraction efficiency, the more consistent the extraction efficiency between the internal standard and baloxavir in the standard plasma solution, and thus the higher the reliability of the internal standard in correcting the subsequent baloxavir detection concentration.

[0060] Preferably, in one embodiment of the present invention, the method for obtaining the closeness of the extraction efficiency between the internal standard and baloxavir in the standard plasma solution specifically comprises:

[0061] The absolute value of the difference between the drug extraction efficiency and the internal standard extraction efficiency at the same theoretical concentration was used as the extraction efficiency difference value of the standard plasma solution at each theoretical concentration. The average value of the extraction efficiency difference value of all theoretical concentrations was then negatively correlated to obtain the extraction efficiency proximity between the internal standard and baloxavir in the standard plasma solution.

[0062] As an example, in one embodiment of the present invention, the expression for the closeness of the extraction efficiency between the internal standard and baloxavir in the standard plasma solution can be specifically, for example, as follows:

[0063] ;

[0064] in, It indicates the closeness of the extraction efficiency between the internal standard and baloxavir in the standard plasma solution; Indicates that the standard plasma solution is Drug extraction efficiency at each theoretical concentration; Indicates that the standard plasma solution is The extraction efficiency of the internal standard at each theoretical concentration; Indicates that the standard plasma solution is The difference in extraction efficiency between theoretical concentrations; represents the number of all theoretical concentrations; Expressed as a natural constant An exponential function with base , used for negative correlation mapping.

[0065] It should be noted that in other embodiments of the present invention, negative correlation mapping may be achieved through other basic mathematical operations, which will not be described in detail here.

[0066] Then, the absolute value of the difference in drug extraction efficiency between each theoretical concentration and the next adjacent theoretical concentration was taken as the change in drug extraction efficiency for each theoretical concentration, and the absolute value of the difference in internal standard extraction efficiency between each theoretical concentration and the next adjacent theoretical concentration was taken as the change in internal standard extraction efficiency for each theoretical concentration. Based on the difference in the change in drug extraction efficiency and the change in internal standard extraction efficiency between the same theoretical concentrations, the similarity in the change in extraction efficiency between the internal standard and baloxavir in the standard plasma solution was obtained. The greater the similarity in the change in extraction efficiency, the more consistent the change trend in the extraction efficiency between the internal standard and baloxavir in the standard plasma solution, and thus the higher the reliability of the internal standard in correcting the subsequent baloxavir detection concentration.

[0067] It should be noted that the theoretical concentrations are arranged in order from small to large or from large to small. At the same time, there is no adjacent next theoretical concentration for the last theoretical concentration. Therefore, the average value of the change in drug extraction efficiency of all theoretical concentrations before the last theoretical concentration can be used as the change in drug extraction efficiency of the last theoretical concentration. Similarly, the average value of the change in internal standard extraction efficiency of all theoretical concentrations before the last theoretical concentration can be used as the change in internal standard extraction efficiency of the last theoretical concentration for subsequent calculation and analysis.

[0068] Preferably, in one embodiment of the present invention, the method for obtaining the similarity of the changes in the extraction efficiencies of the internal standard and baloxavir in the standard plasma solution specifically comprises:

[0069] The absolute value of the difference between the change in drug extraction efficiency and the change in internal standard extraction efficiency at the same theoretical concentration was used as the difference in extraction efficiency of the standard plasma solution at each theoretical concentration. The average values ​​of the difference in extraction efficiency of all theoretical concentrations were negatively correlated to obtain the similarity in extraction efficiency changes between the internal standard and baloxavir in the standard plasma solution.

[0070] As an example, in one embodiment of the present invention, the expression for the similarity of the extraction efficiency changes between the internal standard and baloxavir in the standard plasma solution can be specifically, for example, as follows:

[0071] ;

[0072] in, It indicates the similarity of the changes in extraction efficiency between the internal standard and baloxavir in standard plasma solution; Indicates that the standard plasma solution is The change in drug extraction efficiency at each theoretical concentration; Indicates that the standard plasma solution is The change in internal standard extraction efficiency at each theoretical concentration; Indicates that the standard plasma solution is The difference in extraction efficiency between theoretical concentrations; represents the number of all theoretical concentrations; Expressed as a natural constant An exponential function with base , used for negative correlation mapping.

[0073] Finally, the extraction efficiency proximity and extraction efficiency change similarity are integrated and normalized, and the calculation results are limited to , thereby obtaining the reliability coefficient of the internal standard.

[0074] In the embodiment of the present invention, the integration of the extraction efficiency proximity and the extraction efficiency change similarity can be achieved by calculating the sum or product of the two, which is not limited here.

[0075] In an embodiment of the present invention, an activation function or a hyperbolic tangent function may be used to implement normalization processing, and the normalization in subsequent steps may be processed using the same method, which will not be described in detail.

[0076] As an example, in one embodiment of the present invention, the expression for the reliability coefficient of the internal standard can be specifically, for example, as follows:

[0077] ;

[0078] in, Represents the reliability coefficient of the internal standard; It indicates the closeness of the extraction efficiency between the internal standard and baloxavir in the standard plasma solution; It indicates the similarity of the changes in extraction efficiency between the internal standard and baloxavir in standard plasma solution; Represents the hyperbolic tangent function, which is used for normalization.

[0079] At this point, the analysis of the reliability of the internal standard for subsequent calibration of baloxavir detection concentration was completed, and the reliability coefficient of the internal standard was obtained.

[0080] Step S3: Based on the difference between the drug extraction efficiency and the internal standard extraction efficiency at the same theoretical concentration, a reference theoretical concentration is selected from all theoretical concentrations, and a reference internal standard detection concentration of the test plasma solution containing the internal standard at the reference theoretical concentration is obtained. Based on the reference internal standard detection concentration at the reference theoretical concentration, a reference extraction efficiency of the internal standard in the test plasma solution is obtained; based on the difference between the internal standard extraction efficiency and the reference extraction efficiency at the reference theoretical concentration, as well as the drug detection concentration and the internal standard detection concentration at different theoretical concentrations, a predicted extraction efficiency of baloxavir in the test plasma solution is obtained.

[0081] Then, the embodiment of the present invention needs to analyze the extraction rate of the internal standard in the test plasma solution, and then analyze the extraction rate of baloxavir in the test plasma solution. In order to make the extraction rate of the internal standard in the test plasma solution closer to the extraction rate of baloxavir in the test plasma solution and improve the accuracy of the subsequent analysis of the extraction rate of baloxavir in the test plasma solution, the embodiment of the present invention first selects a reference theoretical concentration from all theoretical concentrations based on the difference between the drug extraction efficiency and the internal standard extraction efficiency between the same theoretical concentrations.

[0082] Preferably, in one embodiment of the present invention, the theoretical concentration corresponding to the minimum value of the extraction efficiency difference value can be used as the reference theoretical concentration. In this case, the drug extraction efficiency of the standard plasma solution at the reference theoretical concentration is closest to the internal standard extraction efficiency, thereby improving the accuracy of the subsequent analysis of the extraction rate of baloxavir in the plasma solution to be tested, and further improving the accuracy of the adjustment of the baloxavir detection concentration in the plasma solution to be tested.

[0083] After obtaining the reference theoretical concentration, the embodiment of the present invention needs to be 100 The baloxavir internal standard is added to the test plasma solution to prepare a test plasma solution containing the internal standard at a reference theoretical concentration. That is, the internal standard concentration in the test plasma solution is the reference theoretical concentration. The test plasma solution containing the internal standard is then subjected to the same treatment as step S1, including protein separation and treatment of the test plasma solution after protein separation using liquid chromatography-tandem mass spectrometry, to obtain a reference internal standard detection concentration of the test plasma solution containing the internal standard at the reference theoretical concentration. The reference internal standard detection concentration is the internal standard concentration of the test plasma solution containing the internal standard detected by liquid chromatography-tandem mass spectrometry. Based on the reference internal standard detection concentration at the reference theoretical concentration, a reference extraction efficiency of the internal standard in the test plasma solution is obtained. The reference extraction efficiency is used to reflect the extraction rate of the internal standard in the test plasma solution.

[0084] Preferably, in one embodiment of the present invention, the method for obtaining the reference extraction efficiency of the internal standard in the plasma solution to be tested specifically includes:

[0085] The reference internal standard detection concentration under the reference theoretical concentration is used as the numerator, the reference theoretical concentration is used as the denominator, and the ratio is used as the reference extraction efficiency of the internal standard in the plasma solution to be tested.

[0086] As an example, in one embodiment of the present invention, the expression for the reference extraction efficiency of the internal standard in the plasma solution to be tested can be specifically, for example, as follows:

[0087] ;

[0088] in, It represents the reference extraction efficiency of the internal standard in the plasma solution to be tested; It represents the reference internal standard detection concentration of the plasma solution to be tested containing the internal standard under the reference theoretical concentration; Indicates the reference theoretical concentration.

[0089] When a known concentration of baloxavir or an internal standard is added to a standard plasma solution and then processed and analyzed, the degree of binding of baloxavir or the internal standard to proteins in the standard plasma solution is lower than the degree of binding of baloxavir or the internal standard to proteins in real human plasma, thereby affecting the accuracy of the assessment of the baloxavir extraction efficiency. Therefore, the embodiment of the present invention predicts and adjusts the extraction efficiency of baloxavir in the test plasma solution based on the difference between the internal standard extraction efficiency at a reference theoretical concentration and the reference extraction efficiency, and in combination with the drug extraction efficiencies at different theoretical concentrations and the internal standard extraction efficiencies, thereby obtaining a predicted extraction efficiency of baloxavir in the test plasma solution. Subsequently, the baloxavir concentration in the detected test plasma solution can be adjusted based on the predicted extraction efficiency and the reliability coefficient of the internal standard obtained above, thereby improving the accuracy of the detection and analysis of baloxavir in plasma.

[0090] Preferably, in one embodiment of the present invention, the method for obtaining the predicted extraction efficiency of baloxavir in the test plasma solution specifically comprises:

[0091] First, the absolute value of the difference between the internal standard extraction efficiency of the standard plasma solution at the reference theoretical concentration and the reference extraction efficiency of the internal standard in the plasma solution to be tested was normalized, and the calculation result was limited to , thereby obtaining the extraction deviation of the internal standard in the plasma solution to be tested. The larger the extraction deviation, the greater the difference in the extraction rate of the internal standard in the standard plasma solution and the plasma solution to be tested, and the greater the subsequent adjustment of the extraction efficiency of baloxavir in the plasma solution to be tested, thereby improving the prediction accuracy of the extraction efficiency of baloxavir in the plasma solution to be tested.

[0092] Then, the drug extraction efficiency and internal standard extraction efficiency at different theoretical concentrations are input into the linear regression model, and the linear regression model outputs a regression function. The reference extraction efficiency of the internal standard in the test plasma solution is substituted into the regression function to obtain the initial extraction efficiency of baloxavir in the test plasma solution. The use of a linear regression model for data prediction is a technical means well known to those skilled in the art and will not be elaborated here.

[0093] Furthermore, the initial extraction efficiency of baloxavir in the test plasma solution was adjusted according to the extraction deviation of the internal standard in the test plasma solution to obtain the predicted extraction efficiency of baloxavir in the test plasma solution.

[0094] Preferably, in one embodiment of the present invention, the method for obtaining the predicted extraction efficiency of baloxavir in the test plasma solution further comprises:

[0095] From the above analysis, it can be seen that the degree of binding of baloxavir or the internal standard to proteins in the standard plasma solution is lower than the degree of binding of baloxavir or the internal standard to proteins in real human plasma. In other words, the extraction rate of baloxavir or the internal standard in the standard plasma solution is higher than the extraction rate in the test plasma solution. Therefore, the above-predicted initial extraction efficiency of baloxavir in the test plasma solution needs to be appropriately lowered. Therefore, the product of the extraction deviation of the internal standard in the test plasma solution and the initial extraction efficiency of baloxavir in the test plasma solution can be used as the extraction efficiency adjustment amount of baloxavir in the test plasma solution, and the difference between the initial extraction efficiency of baloxavir in the test plasma solution and the extraction efficiency adjustment amount can be used as the predicted extraction efficiency of baloxavir in the test plasma solution.

[0096] As an example, in one embodiment of the present invention, the expression for predicting the extraction efficiency of baloxavir in the test plasma solution can be specifically, for example, as follows:

[0097] ;

[0098] ;

[0099] in, represents the predicted extraction efficiency of baloxavir in the test plasma solution; It represents the initial extraction efficiency of baloxavir in the plasma solution to be tested; Indicates the extraction deviation of the internal standard in the plasma solution to be tested; It represents the internal standard extraction efficiency of the standard plasma solution at the reference theoretical concentration; It represents the reference extraction efficiency of the internal standard in the plasma solution to be tested; Represents the hyperbolic tangent function, which is used for normalization.

[0100] Thus, the predicted extraction efficiency of baloxavir in the test plasma solution was obtained.

[0101] Step S4: Based on the reliability coefficient of the internal standard and the predicted extraction efficiency of baloxavir in the plasma solution to be tested, the detected baloxavir concentration of the plasma solution to be tested is adjusted to obtain the actual baloxavir concentration of the plasma solution to be tested.

[0102] The larger the reliability coefficient of the internal standard, the more reliable the internal standard is in correcting the baloxavir detection concentration. Therefore, based on the reliability coefficient of the internal standard and the predicted extraction efficiency of baloxavir in the test plasma solution, the detected baloxavir concentration of the test plasma solution can be adjusted to obtain the true baloxavir concentration of the test plasma solution, thereby compensating for the concentration detection deviation caused by incomplete extraction of baloxavir in the test plasma solution and improving the accuracy of the baloxavir concentration detection in plasma.

[0103] Preferably, in one embodiment of the present invention, the method for obtaining the actual baloxavir concentration of the plasma solution to be tested specifically includes:

[0104] The detected baloxavir concentration of the test plasma solution is used as the numerator, the product of the reliability coefficient of the internal standard and the predicted extraction efficiency of baloxavir in the test plasma solution is used as the denominator, and the ratio is used as the actual baloxavir concentration of the test plasma solution.

[0105] As an example, in one embodiment of the present invention, the expression for the actual baloxavir concentration of the plasma solution to be tested can be specifically, for example, as follows:

[0106] ;

[0107] in, represents the actual baloxavir concentration of the plasma solution to be tested; represents the detected baloxavir concentration of the plasma solution to be tested, which can be processed in the same manner as step S1 and detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS) to determine the baloxavir concentration of the plasma solution to be tested; Represents the reliability coefficient of the internal standard; It represents the predicted extraction efficiency of baloxavir in the test plasma solution.

[0108] Among them, the reliability coefficient of the internal standard As the predicted extraction efficiency of baloxavir in the test plasma solution The weight used, reliability coefficient The larger the value, the more predicted the extraction efficiency of baloxavir in the plasma solution to be tested. The more real.

[0109] It should be noted that the order in which the embodiments of the present invention are described above is for illustrative purposes only and does not necessarily represent the superiority or inferiority of the embodiments. The processes depicted in the accompanying drawings do not necessarily require the specific order or sequential order shown to achieve the desired results. In certain embodiments, multitasking and parallel processing are also possible or may be advantageous.

[0110] The various embodiments in this specification are described in a progressive manner, and the same or similar parts between the various embodiments can be referred to each other. Each embodiment focuses on the differences from other embodiments.

Claims

1. A method for detecting baloxavir concentration in human plasma based on LC-MS / MS, characterized in that: The method comprises: Obtaining drug detection concentrations of a standard plasma solution containing baloxavir at different theoretical concentrations, and internal standard detection concentrations of a standard plasma solution containing an internal standard substance of baloxavir at different theoretical concentrations; According to the drug detection concentration and the internal standard detection concentration at each theoretical concentration, the drug extraction efficiency and the internal standard extraction efficiency of the standard plasma solution at each theoretical concentration are obtained; according to the difference between the drug extraction efficiency and the internal standard extraction efficiency at the same theoretical concentration, and the change between the drug extraction efficiency and the internal standard extraction efficiency at the same theoretical concentration, the reliability coefficient of the internal standard is obtained; Based on the difference between the drug extraction efficiency and the internal standard extraction efficiency at the same theoretical concentration, a reference theoretical concentration is selected from all theoretical concentrations, and a reference internal standard detection concentration of the test plasma solution containing the internal standard at the reference theoretical concentration is obtained; based on the reference internal standard detection concentration at the reference theoretical concentration, a reference extraction efficiency of the internal standard in the test plasma solution is obtained; based on the difference between the internal standard extraction efficiency and the reference extraction efficiency at the reference theoretical concentration, as well as the drug extraction efficiency and internal standard extraction efficiency at different theoretical concentrations, a predicted extraction efficiency of baloxavir in the test plasma solution is obtained; Adjusting the detected baloxavir concentration of the plasma solution to be tested based on the reliability coefficient of the internal standard and the predicted extraction efficiency of baloxavir in the plasma solution to be tested to obtain the actual baloxavir concentration of the plasma solution to be tested; The reliability coefficient of obtaining the internal standard comprises: According to the difference between the drug extraction efficiency and the internal standard extraction efficiency at the same theoretical concentration, the extraction efficiency proximity between the internal standard and baloxavir in the standard plasma solution is obtained; The absolute value of the difference between each theoretical concentration and the next adjacent theoretical concentration in the drug extraction efficiency is used as the drug extraction efficiency change of each theoretical concentration, and the absolute value of the difference between each theoretical concentration and the next adjacent theoretical concentration in the internal standard extraction efficiency is used as the internal standard extraction efficiency change of each theoretical concentration; According to the difference between the change in the drug extraction efficiency and the change in the internal standard extraction efficiency at the same theoretical concentration, the similarity of the change in the extraction efficiency between the internal standard and baloxavir in the standard plasma solution is obtained; The extraction efficiency proximity and the extraction efficiency change similarity are synthesized and normalized to obtain a reliability coefficient of the internal standard; The method of obtaining the predicted extraction efficiency of baloxavir in the plasma solution to be tested comprises: Normalizing the absolute value of the difference between the internal standard extraction efficiency of the standard plasma solution at the reference theoretical concentration and the reference extraction efficiency of the internal standard in the plasma solution to be tested to obtain the extraction deviation of the internal standard in the plasma solution to be tested; Inputting the drug extraction efficiency at different theoretical concentrations and the internal standard extraction efficiency into a linear regression model, outputting a regression function from the linear regression model, and substituting the reference extraction efficiency of the internal standard in the test plasma solution into the regression function to obtain the initial extraction efficiency of baloxavir in the test plasma solution; adjusting the initial extraction efficiency of baloxavir in the plasma solution to be tested according to the extraction deviation of the internal standard in the plasma solution to be tested, to obtain a predicted extraction efficiency of baloxavir in the plasma solution to be tested; The obtaining of the actual baloxavir concentration of the plasma solution to be tested comprises: The detected baloxavir concentration of the plasma solution to be tested is used as the numerator, the product of the reliability coefficient of the internal standard and the predicted extraction efficiency of baloxavir in the plasma solution to be tested is used as the denominator, and the ratio is used as the actual baloxavir concentration of the plasma solution to be tested.

2. The method for detecting baloxavir concentration in human plasma based on LC-MS / MS according to claim 1, wherein: The method of obtaining the drug extraction efficiency and internal standard extraction efficiency of the standard plasma solution at each theoretical concentration includes: The drug test concentration at each theoretical concentration is used as the numerator, each theoretical concentration is used as the denominator, and the ratio is used as the drug extraction efficiency of the standard plasma solution at each theoretical concentration; The internal standard detection concentration at each theoretical concentration was used as the numerator, each theoretical concentration was used as the denominator, and the ratio was used as the internal standard extraction efficiency of the standard plasma solution at each theoretical concentration.

3. The method for detecting the concentration of baloxavir in human plasma based on LC-MS / MS according to claim 1, wherein: The obtaining of the closeness of the extraction efficiency between the internal standard and baloxavir in the standard plasma solution comprises: The absolute value of the difference between the drug extraction efficiency and the internal standard extraction efficiency at the same theoretical concentration is used as the extraction efficiency difference value of the standard plasma solution at each theoretical concentration; The average values ​​of the extraction efficiency differences at all theoretical concentrations were negatively correlated to obtain the extraction efficiency proximity between the internal standard and baloxavir in the standard plasma solution.

4. The method for detecting baloxavir concentration in human plasma based on LC-MS / MS according to claim 1, wherein: The similarity of the extraction efficiency changes between the internal standard and baloxavir in the standard plasma solution is obtained as follows: The absolute value of the difference between the change in drug extraction efficiency and the change in internal standard extraction efficiency at the same theoretical concentration is taken as the difference in extraction efficiency of the standard plasma solution at each theoretical concentration; Negative correlation mapping was performed on the average values ​​of the extraction efficiency change differences at all theoretical concentrations to obtain the similarity of the extraction efficiency changes between the internal standard and baloxavir in the standard plasma solution.

5. The method for detecting baloxavir concentration in human plasma based on LC-MS / MS according to claim 3, characterized in that: The step of selecting a reference theoretical concentration from all theoretical concentrations includes: The theoretical concentration corresponding to the minimum value of the extraction efficiency difference is used as the reference theoretical concentration.

6. The method for detecting baloxavir concentration in human plasma based on LC-MS / MS according to claim 1, characterized in that: The method of obtaining a reference extraction efficiency of the internal standard in the plasma solution to be tested comprises: The reference internal standard detection concentration under the reference theoretical concentration is used as the numerator, the reference theoretical concentration is used as the denominator, and the ratio is used as the reference extraction efficiency of the internal standard in the plasma solution to be tested.

7. The method for detecting baloxavir concentration in human plasma based on LC-MS / MS according to claim 1, characterized in that: The step of adjusting the initial extraction efficiency of baloxavir in the plasma solution to be tested based on the extraction deviation of the internal standard in the plasma solution to obtain the predicted extraction efficiency of baloxavir in the plasma solution to be tested comprises: The product of the extraction deviation of the internal standard in the plasma solution to be tested and the initial extraction efficiency of baloxavir in the plasma solution to be tested is used as the adjustment amount of the extraction efficiency of baloxavir in the plasma solution to be tested; The difference between the initial extraction efficiency of baloxavir in the test plasma solution and the adjusted amount of extraction efficiency is used as the predicted extraction efficiency of baloxavir in the test plasma solution.

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