CN115980246A - Quantitative detection method for content of potential treatment drug for new coronary pneumonia - Google Patents

Abstract

The invention discloses a quantitative detection method for the content of a potential treatment drug for new coronary pneumonia, which comprises the following steps: (1) Taking a proper amount of standard substance, precisely weighing, dissolving to prepare a mixed solution containing the standard substance, and diluting the mixed standard solution with bovine serum albumin solution to prepare standard solutions with series concentrations; (2) preparing a solution to be detected and a standard solution; (3) carrying out two-dimensional liquid phase extraction and separation; (4) entering mass spectrometry and analyzing using MRM mode; (5) Obtaining various data by utilizing the two-dimensional liquid chromatography tandem mass spectrometry of the step 4; (6) solving a linear regression equation; (7) And (5) obtaining the concentration of the substance to be detected in the serum sample solution by using the curve. The invention establishes a two-dimensional liquid chromatography tandem mass spectrometry method for simultaneously and quantitatively detecting the concentration of a substance to be detected in serum, simplifies the pretreatment process while reducing the matrix effect, and has high flux, accuracy and precision.

Description

Quantitative detection method for content of potential treatment drug for new coronary pneumonia

Technical Field

The invention relates to the technical field of drug detection, in particular to a quantitative detection method for the content of a potential treatment drug for new coronary pneumonia.

Background

The reuse of antiviral active drugs in vitro has been of great interest and several substances are considered as potential drug candidates.

Reidesciclovir, a broad-spectrum antiviral drug, was originally developed for the treatment of hepatitis C and has received special approval for the treatment of severe disease caused by new coronavirus. Other potential drug candidates are the antiviral drug Favipiravir for the treatment of influenza, and the fixed dose combination of the antiviral drug lopinavir and its drug enhancer ritonavir for the prevention and treatment of HIV. Azithromycin is a broad spectrum antibiotic with antiviral and immunomodulatory properties and is also a candidate for the study of new coronary drug therapies. Chloroquine and hydroxychloroquine for the treatment of autoimmune diseases such as malaria and systemic lupus erythematosus have been suspended for failure to show any survival benefit in large scale trials of treatment with the new coronavirus, however, the world health organization recovered part of the hydroxychloroquine experiments due to unreliable data used in some studies.

Typically, the dose of the new coronary pneumonia treatment drug comes from the half maximal effective concentration (EC 50) value of the virus generated in vitro, as well as pharmacological-based pharmacokinetic models previously developed in other disease and clinical conditions. However, due to changes in the pathological state, the corresponding dosage regimen is not necessarily applicable to patients with severe new coronary disease. Improper dosage of the drug may result in ineffective treatment or cause drug toxicity without clear clinical benefit. Therefore, there is an urgent need to generate high quality pharmacokinetic data for new crown potential drug reuse. In addition, combination administration may result in unpredictable drug levels due to related drug-drug interactions. Therefore, the detection of the blood concentration of these drugs and the adoption of an individualized optimal administration strategy can further contribute to the establishment of the curative effect and safety of the drugs.

At present, methods for detecting blood concentration include an immunoassay method, a chemiluminescence method, a high performance liquid chromatography and the like, but have the defects of low sensitivity, poor selectivity, complex pretreatment process, low flux and the like.

Disclosure of Invention

The invention aims to solve the technical problem of providing a quantitative detection method for the content of potential treatment drugs for the new coronary pneumonia, establishing a two-dimensional liquid chromatography tandem mass spectrometry method for simultaneously and quantitatively detecting the concentration of substances to be detected in serum, simplifying the pretreatment process while reducing the matrix effect, and having high flux, accuracy and precision.

In order to solve the technical problems, the invention provides a quantitative detection method for the content of a potential treatment drug for new coronary pneumonia, which comprises the following steps:

(1) Taking a proper amount of standard substance, precisely weighing, dissolving to prepare a mixed solution containing the standard substance, and diluting the mixed standard solution with bovine serum albumin solution to prepare standard solutions with series concentrations;

(2) Taking 50ul of standard solutions with different concentrations and serum sample solutions, putting the standard solutions and the serum sample solutions into a 1.5ml centrifuge tube, adding 200ul of methanol solution containing an internal standard, carrying out vortex oscillation for 2 minutes, carrying out high-speed centrifugation at 13000rpm for 5 minutes, sucking 50ul of supernate, diluting the supernate with 950ul of 50% methanol water, and then loading the supernate on a machine for detection and analysis;

(3) Extracting and separating the supernatant obtained in thestep 2, wherein four mobile phases A1, A2, B1 and B2 exist together, and firstly, pumping the supernatant into an online solid-phase extraction column for further extraction by using the mobile phase A1; the second step is that the supernatant fluid enters an analytical column by the back flush of a mobile phase A2, and the mobile phases A2 and B2 are used for gradient elution and separation; meanwhile, the flow of A1 and B1 is used for cleaning relative to the on-line solid phase extraction column;

(4) Entering mass spectrometry and analyzing using an MRM mode;

(5) Utilizing the two-dimensional liquid chromatography tandem mass spectrometry of the step 4 to obtain the peak area ratio of the standard substance and the internal standard in the standard solution and the peak area ratio of the object to be detected and the internal standard in the serum sample solution;

(6) Respectively taking the concentration of the standard substance in the standard solution as a horizontal coordinate, performing regression calculation by using a weighted least square method according to the peak area ratio of the standard substance to the internal standard in the standard solution, performing regression operation by using the least square method, and obtaining a linear regression equation as a standard curve;

(7) And (4) substituting the peak area ratio of the object to be measured in the serum sample solution obtained in the step (4) to the peak area ratio of the internal standard into the ordinate of the linear regression equation, wherein the abscissa is the concentration of the object to be measured in the serum sample solution.

Further, the standard substance is Redesivir, favipiravir, lopinavir, ritonavir, azithromycin, chloroquine and hydroxychloroquine.

Further, the chromatographic parameters of the two-dimensional liquid phase analysis in thestep 3 are as follows: a chromatographic column 1: oasis HLB Direct ConnectHP column, column 2: ACQUITY UPLC BEH C18 Column, A1: ultrapure water, B1:0.1% formic acid acetonitrile, A2:0.1% formic acid water (containing 2mM ammonium acetate), B2:0.1% formic acid acetonitrile (containing 2mM ammonium acetate).

Further, the mass spectrum parameters of the two-dimensional liquid phase analysis in thestep 3 are as follows: an ionization mode: ESI +; the scanning mode is as follows: MRM; ion source temperature: 150 degrees; desolventizing gas temperature: 450 degrees; desolventizing air flow rate: 900L/h; taper hole voltage: 3.0KV.

The invention has the beneficial effects that: the invention establishes a method for simultaneously and quantitatively detecting the Redexilvir, the Favipiravir, the lopinavir, the ritonavir, the azithromycin, the chloroquine and the hydroxychloroquine in serum by a two-dimensional liquid chromatography tandem mass spectrometry, wherein a liquid phase part of the method contains an extraction column and an analysis column, the pretreatment process is simplified while the matrix effect is reduced, and the detection method has high flux, accuracy and precision.

Drawings

FIG. 1 is a series of standard solution concentration tables according to the present invention.

Fig. 2 is a table of thepump 1 gradient elution program of the present invention.

Fig. 3 is a table of thepump 2 gradient elution procedure of the present invention.

Fig. 4 is a table of MRM scan parameters of the present invention.

Figure 5 is a table of linearity, accuracy and precision of the present invention.

FIG. 6 is a schematic diagram of a first step of two-dimensional liquid phase analysis according to the present invention.

FIG. 7 is a schematic diagram of a second step of two-dimensional liquid phase analysis of the present invention.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

In the description of the present invention, it is to be understood that the terms "central," "longitudinal," "transverse," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," "circumferential," and the like are used in the orientations and positional relationships indicated in the drawings for convenience in describing the invention and to simplify the description, but are not intended to indicate or imply that the device or element so referred to must have a particular orientation, be constructed and operated in a particular orientation, and are not to be construed as limiting the invention.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.

In the present invention, unless otherwise explicitly stated or limited, the terms "mounted," "connected," "fixed," and the like are to be construed broadly, e.g., as being permanently connected, detachably connected, or integral; can be mechanically or electrically connected; they may be directly connected or indirectly connected through intervening media, or they may be connected internally or in any other suitable relationship, unless expressly stated otherwise. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

In the present invention, unless otherwise expressly stated or limited, the first feature "on" or "under" the second feature may be directly contacting the first and second features or indirectly contacting the first and second features through an intermediate. Also, a first feature "on," "above," and "over" a second feature may be directly on or obliquely above the second feature, or simply mean that the first feature is at a higher level than the second feature. A first feature "under," "beneath," and "under" a second feature may be directly under or obliquely under the second feature, or may simply mean that the first feature is at a lesser elevation than the second feature.

It will be understood that when an element is referred to as being "secured to" or "disposed on" another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may also be present. The terms "vertical," "horizontal," "upper," "lower," "left," "right," and the like as used herein are for illustrative purposes only and do not denote a unique embodiment.

Referring to fig. 1 to 7, an embodiment of the method for quantitatively detecting the content of a potential therapeutic drug for new coronary pneumonia according to the present invention includes the following steps:

(1) Taking a proper amount of standard substance, precisely weighing, dissolving to prepare a mixed solution containing the standard substance, and diluting the mixed standard solution with bovine serum albumin solution to prepare standard solutions with series concentrations;

(2) Taking 50ul of standard solutions with different concentrations and serum sample solutions, putting the standard solutions and the serum sample solutions into a 1.5ml centrifuge tube, adding 200ul of methanol solution containing an internal standard, carrying out vortex oscillation for 2 minutes, carrying out high-speed centrifugation at 13000rpm for 5 minutes, sucking 50ul of supernate, diluting the supernate with 950ul of 50% methanol water, and then loading the supernate on a machine for detection and analysis;

(3) Extracting and separating the supernatant obtained in thestep 2, wherein four mobile phases A1, A2, B1 and B2 coexist, and firstly, pumping the supernatant into an online solid-phase extraction column for further extraction by using the mobile phase A1; the second step is that the supernatant fluid enters an analytical column by the back flush of a mobile phase A2, and the mobile phases A2 and B2 are used for gradient elution and separation; meanwhile, the flow of A1 and B1 is used for cleaning relative to the on-line solid phase extraction column;

(4) Entering mass spectrometry and analyzing using MRM mode;

(5) Utilizing the two-dimensional liquid chromatography tandem mass spectrometry of the step 4 to obtain the peak area ratio of the standard substance and the internal standard in the standard solution and the peak area ratio of the object to be detected and the internal standard in the serum sample solution;

(6) Respectively taking the concentration of the standard substance in the standard solution as a horizontal coordinate, performing regression calculation by using a weighted least square method according to the peak area ratio of the standard substance to the internal standard in the standard solution, performing regression operation by using the least square method, and obtaining a linear regression equation as a standard curve;

(7) And (4) substituting the peak area ratio of the object to be measured in the serum sample solution obtained in the step (4) to the peak area ratio of the internal standard into the ordinate of the linear regression equation, wherein the abscissa is the concentration of the object to be measured in the serum sample solution.

Instep 1, the standard substances are redexivir, favipiravir, lopinavir, ritonavir, azithromycin, chloroquine and hydroxychloroquine, and fig. 1 shows the concentrations of the standard substances in five different standard solutions, i.e., the experiment was performed by using the standard solutions with the concentrations of the five different standard solutions in the present example;

instep 2, the five standard solutions with different concentrations obtained instep 1 and the serum sample to be detected are respectively processed, each of which obtains 50ul of processed supernatant (diluted with 950ul of 50% methanol water), and then the next experiment is performed.

Instep 3, the chromatographic parameters of the two-dimensional liquid phase analysis are as follows: a chromatographic column 1: oasis HLB Direct ConnectHP column, column 2: ACQUITY UPLC BEH C18 Column, A1: ultrapure water, B1:0.1% formic acid acetonitrile, A2:0.1% formic acid water (containing 2mM ammonium acetate), B2:0.1% formic acid acetonitrile (containing 2mM ammonium acetate).

The mass spectrum parameters were as follows: an ionization mode: ESI +; the scanning mode comprises the following steps: MRM; ion source temperature: 150 degrees; desolvation gas temperature: 450 degrees; desolventizing air flow rate: 900L/h; taper hole voltage: 3.0KV.

The peak appearance time of each parameter is staggered, recording is convenient, and then the peak area ratio of the standard substance and the internal standard in the standard solution and the peak area ratio of the object to be measured and the internal standard in the serum sample solution are obtained.

And then respectively taking the concentration of the standard substance as a horizontal coordinate and the ratio of the peak area of the standard substance to the peak area of the internal standard substance as a vertical coordinate, performing regression calculation by using a weighted least square method, performing regression operation by using the least square method, and obtaining a linear regression equation which is a standard curve. The linear correlation coefficient of the standard curve of each standard product is shown in figure 5, and the linear correlation coefficient is more than 0.995, which shows that each detection substance has good linearity by the method.

And then substituting the peak area ratio of the object to be measured in the serum sample solution to the peak area ratio of the internal standard into the ordinate of the linear regression equation, wherein the abscissa is the concentration of the object to be measured in the serum sample solution, namely the final purpose of the invention.

Precision interpretation of intra-batch accuracy for the test substances:

for batch precision, 7 analysis sequences were performed over a period of 56 days, on 7 different days, and all QC samples were analyzed repeatedly at high and low concentrations. Calibration was performed daily to ensure the correctness of the results. The accuracy was calculated as the measured average of the QC samples at each concentration level divided by the actual value. The within-batch accuracy was assessed by measuring the CV of QC samples at three different times on the same day, and the relevant data are shown in fig. 5. As can be seen from the table, the accuracy of all the substances at high concentration and low concentration in batch and in batch is within an acceptable range (CV is less than 15 percent, and the accuracy is between 85 percent and 115 percent), which indicates that the detection value of the method is accurate and precise.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Claims (4)

1. A quantitative detection method for the content of a potential treatment drug for new coronary pneumonia is characterized by comprising the following steps:

(1) Taking a proper amount of standard substance, precisely weighing, dissolving to prepare a mixed solution containing the standard substance, and diluting the mixed standard solution with bovine serum albumin solution to prepare standard solutions with series concentrations;

(2) Taking 50ul of standard solutions with different concentrations and serum sample solutions, putting the standard solutions and the serum sample solutions into a 1.5ml centrifuge tube, adding 200ul of methanol solution containing an internal standard, carrying out vortex oscillation for 2 minutes, carrying out high-speed centrifugation at 13000rpm for 5 minutes, sucking 50ul of supernate, diluting the supernate with 950ul of 50% methanol water, and then loading the supernate on a machine for detection and analysis;

(3) Extracting and separating the supernatant obtained in the step 2, wherein four mobile phases A1, A2, B1 and B2 exist together, and firstly, pumping the supernatant into an online solid-phase extraction column for further extraction by using the mobile phase A1; the second step is that the supernatant fluid enters an analytical column by the back flush of a mobile phase A2, and the mobile phases A2 and B2 are used for gradient elution and separation; meanwhile, the flow of A1 and B1 is used for cleaning relative to the on-line solid phase extraction column;

(4) Entering mass spectrometry and analyzing using an MRM mode;

(5) Utilizing the two-dimensional liquid chromatography tandem mass spectrometry of the step 4 to obtain the peak area ratio of the standard substance and the internal standard in the standard solution and the peak area ratio of the object to be detected and the internal standard in the serum sample solution;

(6) Respectively taking the concentration of the standard substance in the standard solution as a horizontal coordinate, performing regression calculation by using a weighted least square method according to the peak area ratio of the standard substance to the internal standard in the standard solution, performing regression operation by using the least square method, and obtaining a linear regression equation as a standard curve;

(7) And (4) substituting the peak area ratio of the object to be measured in the serum sample solution obtained in the step (4) to the peak area ratio of the internal standard into the ordinate of the linear regression equation, wherein the abscissa is the concentration of the object to be measured in the serum sample solution.

2. The method for quantitatively determining the content of a potential therapeutic drug for neocoronarism as claimed in claim 1, wherein said standard substance is Redesivir, favipiravir, lopinavir, ritonavir, azithromycin, chloroquine and hydroxychloroquine.

3. The method for quantitatively detecting the content of the drug for potential treatment of the new coronary pneumonia according to claim 1, wherein the chromatographic parameters of the two-dimensional liquid phase analysis in the step 3 are as follows: a chromatographic column 1: oasis HLB Direct ConnectHP column, column 2: ACQUITY UPLC BEH C18 Column, A1: ultrapure water, B1:0.1% formic acid acetonitrile, A2:0.1% formic acid water (containing 2mM ammonium acetate), B2:0.1% formic acid acetonitrile (containing 2mM ammonium acetate).

4. The method for quantitatively detecting the content of the drug for potential treatment of the new coronary pneumonia according to claim 1, wherein the mass spectrometric parameters of the two-dimensional liquid phase analysis in the step 3 are as follows: an ionization mode: ESI +; the scanning mode is as follows: MRM; ion source temperature: 150 degrees; desolvation gas temperature: 450 degrees; desolventizing air flow rate: 900L/h; taper hole voltage: 3.0KV.

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