CN118090994B - Stable freeze-drying agent containing ritonavir and matrix, preparation method, kit and application of stable freeze-drying agent - Google Patents

Abstract

The invention discloses a stable freeze-drying agent containing ritonavir and a matrix, a preparation method, a kit and application thereof, wherein the freeze-drying agent is obtained by preparing an aqueous solution containing ritonavir and the matrix and freeze-drying the aqueous solution, and the matrix comprises BSA and VC; the detection method for detecting the ritonavir blood concentration comprises the freeze-drying agent; the lyophilizate and/or the kit can be used for liquid chromatography-tandem mass spectrometry combined detection to detect the concentration of ritonavir in a sample, and the lyophilizate and/or the kit is used for preparing a calibrator and/or a quality control. The prepared freeze-dried agent and the kit can be stored for a long time, are used for preparing a calibrator and a quality control product which are required to be used in liquid chromatography-tandem mass spectrometry detection, and provide preconditions for the mass production of ritonavir detection kits.

Description

Stable freeze-drying agent containing ritonavir and matrix, preparation method, kit and application of stable freeze-drying agent

Technical Field

The invention belongs to the technical field of biological sample drug analysis, and relates to a stable freeze-drying agent containing ritonavir and a matrix, a preparation method, a kit and application thereof.

Background

5-Thiazolylmethyl N- [ (2S, 3S, 5R) -3-hydroxy-5- [ [ (2S) -3-methyl-2- [ [ methyl- [ (2-isopropyl-1, 3-thiazol-4-yl) methyl ] carbamoyl ] amino ] butyryl ] amino ] -1, 6-diphenyl-hex-2-yl ] carbamate (pharmaceutical name: ritonavir (Ritonavir), cas No.: 155213-67-5), a potent inhibitor of cytochrome P450 isozymes CYP3A, can be used to treat diseases associated with HIV. In addition, ritonavir can better combat diseases such as COVID-19 by inhibiting CYP3A mediated metabolism rate of Nematavir (NIRMATRELVIR), which in combination with nematavir helps to slow the metabolic breakdown of nematavir, allowing a high drug concentration of nematavir to remain in the patient for a longer period of time.

Blood concentration (Plasma Concentration) refers to the total concentration of the drug in the plasma after absorption, including the drug bound to plasma proteins or free in the plasma, and sometimes also to the concentration of the drug in whole blood. Blood concentration detection is a key way to evaluate efficacy or determine dosing regimen, individualize dosing regimen, and increase drug treatment levels.

At present, the blood concentration of ritonavir can be quantified by performing liquid chromatography-mass spectrometry (LC/MS) combined detection on a blood sample of a user. In the combined detection, a calibrator and a quality control product of ritonavir are needed, however, for the existing ritonavir Wei Jiaozhun product and quality control product, ritonavir in a matrix cannot exist stably for a long time in a general environment, so that the ritonavir cannot be supplied through commercial mass production and storage, and the calibrator and quality control product can only be prepared during detection, so that the operation efficiency of ritonavir blood concentration detection is seriously affected, and the personalized formulation of ritonavir schemes is inconvenient.

In view of the above drawbacks, there is a need to provide a stable freeze-dried formulation comprising ritonavir and a matrix, a method of preparation, a kit and uses thereof.

Disclosure of Invention

The invention mainly aims to provide a stable freeze-drying agent containing ritonavir and a matrix, a preparation method and a kit, and aims to solve the technical problems that the ritonavir in the matrix cannot exist stably for a long time in a general environment in the existing ritonavir Wei Jiaozhun product and quality control product, so that the ritonavir cannot be supplied through commercial mass production and storage, and the calibration product and the quality control product can only be prepared during detection, so that the operation efficiency of ritonavir blood concentration detection is seriously influenced, and inconvenience is brought to the personalized formulation of ritonavir schemes.

To achieve the above object, the present invention provides a method for preparing a stable freeze-dried formulation comprising ritonavir and a matrix, the method comprising the steps of:

step S1: preparing an aqueous solution containing ritonavir and a matrix including Bovine Serum Albumin (BSA) and ascorbic acid (VC);

step S2: lyophilizing the aqueous solution to obtain the stable lyophilized formulation comprising ritonavir and a matrix.

Preferably, in the aqueous solution, the concentration of Bovine Serum Albumin (BSA) is 0.1 to 5% by mass and the concentration of ascorbic acid (VC) is 0.5 to 20% by mass.

Preferably, ethylenediamine tetraacetic acid (EDTA) is also included in the matrix.

Preferably, in the aqueous solution, the Ethylene Diamine Tetraacetic Acid (EDTA) is present in a concentration of 0.1% to 10% by mass.

Preferably, the step S1 further includes:

and adding a lyoprotectant to the formulated aqueous solution containing ritonavir and a matrix.

Preferably, the lyoprotectant is selected from one or more of sucrose, mannitol and trehalose.

The present invention also provides a stable lyophilizate comprising ritonavir and a matrix, said stable lyophilizate being prepared by any of the preparation methods described above.

The invention also provides a kit for detecting ritonavir, which comprises the stable freeze-drying agent containing ritonavir and a matrix.

Preferably, the kit comprises a plurality of parts of the stable freeze-drying agent which are stored separately, wherein the plurality of parts of the stable freeze-drying agent are used for preparing a plurality of calibration products with the concentration of each point on a calibration curve in liquid chromatography-tandem mass spectrometry combined detection, and preparing quality control products with the concentration of each point.

Preferably, the corresponding aqueous solutions are prepared according to the concentrations of ritonavir in the calibrator and the quality control product.

Preferably, the invention also provides the use of a stable lyophilizate comprising ritonavir and a matrix or a kit for detecting ritonavir in a liquid chromatography-tandem mass spectrometry combined detection, wherein the lyophilizate or the kit is used for preparing a plurality of calibrators of various point concentrations on a calibration curve and/or preparing a plurality of quality control substances of various concentrations for detecting the ritonavir concentration in a sample.

Compared with the prior art, the invention has the following beneficial effects:

1. the freeze-drying agent prepared by the preparation method of the stable freeze-drying agent containing ritonavir and the matrix, which is adopted by the application, avoids the degradation phenomenon of ritonavir Wei Shiji prepared in the prior art caused by long-term storage, and simultaneously avoids the error caused by detection by using different batches of the reagents caused by the degradation of ritonavir Wei Shiji, thereby bringing convenience to the individuation formulation of ritonavir schemes;

compared with the existing ritonavir Wei Shiji, the stable freeze-drying agent containing ritonavir and a matrix prepared by the method can be stored for a long time, so that the freeze-drying agent can be supplied to a ritonavir Wei Yexiang chromatographic-tandem mass spectrometry detection scene through commercial mass production and storage, and the efficacy and detection precision of ritonavir Wei Yexiang chromatographic-tandem mass spectrometry detection are improved in blood concentration detection.

2. The preparation method of the stable freeze-drying agent containing ritonavir and a matrix comprises the steps of preparing an aqueous solution containing ritonavir and the matrix, wherein the matrix comprises Bovine Serum Albumin (BSA) and ascorbic Acid (AC), and the addition of the Bovine Serum Albumin (BSA) and the ascorbic Acid (AC) can be cooperated to ensure that the ritonavir exists stably in the freeze-drying agent; furthermore, the preferred mass percent concentration of the Bovine Serum Albumin (BSA) and the ascorbic Acid (AC) is set, and the setting can further amplify the synergistic effect, so that the stability of ritonavir in the freeze-drying agent is improved, and a precondition is provided for the mass production of the ritonavir detection kit.

Drawings

FIG. 1 is a schematic illustration of the process steps of the preparation of a stable lyophilizate comprising ritonavir and a matrix according to the present application.

FIG. 2 is a chromatogram of Nemactetvir and ritonavir Wei Biaozhun (2.94 min: nemactetvir; 3.22min: ritonavir).

FIG. 3 is a chromatogram of a serum sample labeled (5. Mu.g/mL).

Fig. 4 is a chromatogram of a blank serum sample.

Detailed Description

Various aspects of the invention are described in further detail below.

Unless defined or otherwise indicated, 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. In addition, any method and material similar or equivalent to those described may be used in the methods of the present invention.

The term "or" as used herein includes the relationship of "and" unless specifically stated and defined otherwise. The sum corresponds to the boolean logic operator AND, the OR corresponds to the boolean logic operator OR, AND the AND is a subset of OR.

It will be understood that, although the terms "first," "second," etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another element. Thus, a first element could be termed a second element without departing from the teachings of the present disclosure.

In the present invention, the terms "consisting essentially of" and "consisting of" are included in the terms "containing," comprising, "or" including.

The terms "connected," "connected," and "connected" in this application are to be construed broadly, as they are, for example, fixedly connected or via an intermediary, in connection with one another, or in connection with one another, as they are in communication with one another, or in an interaction relationship between two elements, unless otherwise specifically indicated and defined. The specific meaning of the above terms in the present application can be understood by a person of ordinary skill in the art according to the specific circumstances.

For example, if an element (or component) is referred to as being "on", "coupled" or "connected" to another element, it can be directly on, coupled or connected to the other element or one or more intervening elements may be present therebetween. Conversely, if the expressions "directly on," "directly with," coupled "and" directly with, "connected" are used herein, then no intervening elements are indicated. Other words used to describe the relationship between elements should be interpreted similarly, such as "between" and "directly between", "attached" and "directly attached", "adjacent" and "directly adjacent", and the like.

It should be further noted that the words "front", "rear", "left", "right", "upper" and "lower" used in the following description refer to directions in the drawings. The words "inner" and "outer" are used to refer to directions toward or away from, respectively, the geometric center of a particular component. It will be understood that these terms are used herein to describe one element, layer or region's relationship to another element, layer or region as illustrated in the figures. These terms should also encompass other orientations of the device in addition to the orientation depicted in the figures.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

In order to more clearly illustrate the embodiments of the present disclosure or the technical solutions in the prior art, specific embodiments of the present invention will be described below with reference to the accompanying drawings. It is evident that the drawings in the following description are only examples of the invention, from which other drawings and other embodiments can be obtained for a person skilled in the art without inventive effort.

It should also be noted that the illustrations provided in the following embodiments merely illustrate the basic concepts of the disclosure by way of illustration, and only the components related to the present application are shown in the drawings rather than being drawn according to the number, shape and size of the components in actual implementation, and the form, number and proportion of the components in actual implementation may be arbitrarily changed, and the layout of the components may be more complicated. For example, the thickness of elements in the drawings may be exaggerated for clarity.

For the existing ritonavir Wei Jiaozhun products and quality control products, ritonavir in a matrix cannot exist stably for a long time in a general environment, so that the ritonavir cannot be supplied through commercial mass production and storage, and only the calibrator and the quality control products can be prepared during detection, the operation efficiency of ritonavir blood concentration detection is seriously affected, and further the technical problem of inconvenience is brought to individualized formulation of ritonavir schemes.

To achieve the above object, as shown in fig. 1, the present invention provides a method for preparing a stable freeze-dried formulation comprising ritonavir and a matrix, the method comprising the steps of:

step S1: preparing an aqueous solution containing ritonavir and a matrix including Bovine Serum Albumin (BSA) and ascorbic acid (VC);

step S2: lyophilizing the aqueous solution to obtain the stable lyophilized formulation comprising ritonavir and a matrix.

Bovine serum albumin (Bovine Serum Albumin, BSA) is one of the major protein components in bovine serum, which is capable of binding to a variety of substances, and is a very important substance transport carrier in plasma; meanwhile, bovine serum albumin also has the property of hydrophilic and hydrophobic acting force and resisting thermal denaturation. Ascorbic acid (Natural Vitamin C, VC) is a polyhydroxy compound which is strongly reducing and readily oxidized to dehydrovitamin C. The applicant finds that when preparing a freeze-dried agent containing ritonavir and a matrix, the matrix is set to comprise Bovine Serum Albumin (BSA) and ascorbic acid (VC), and an aqueous solution is prepared together with ritonavir, and the freeze-dried agent prepared after freeze-drying the aqueous solution has good stability, so that the ritonavir Wei Zaisuo can stably exist in the freeze-dried agent for a long time, and the defect that the existing ritonavir Wei Shiji is easy to degrade and the like during long-term storage is avoided to a great extent.

In a preferred embodiment, in step S1, a mixed aqueous solution containing ritonavir, BSA and VC is prepared, then in step S2, the prepared aqueous solution is pre-frozen for 0.5-3 hours at-80 ℃, the freeze dryer is cooled to an initial temperature of-40 to-50 ℃, and then the pre-frozen product is placed into the freeze dryer for freeze drying operation according to an automatic program set in Table 1.

Table 1.

As a preferred embodiment, the concentration of Bovine Serum Albumin (BSA) in the aqueous solution is 0.1 to 5% by mass; preferably, the Bovine Serum Albumin (BSA) is present at a concentration of 0.2% to 4.5% by mass; preferably, the Bovine Serum Albumin (BSA) is present at a concentration of 0.3% to 4% by mass; preferably, the Bovine Serum Albumin (BSA) is present at a concentration of 0.4% to 3.5% by mass; preferably, the Bovine Serum Albumin (BSA) is present at a concentration of 0.5% to 3% by mass; preferably, the Bovine Serum Albumin (BSA) is present at a concentration of 0.6% to 2.5% by mass; preferably, the Bovine Serum Albumin (BSA) is present at a concentration of 0.7% to 2% by mass; preferably, the Bovine Serum Albumin (BSA) is present at a concentration of 0.8% to 1.5% by mass; preferably, the Bovine Serum Albumin (BSA) is present in a concentration of 0.9% to 1.3% by mass; preferably, the Bovine Serum Albumin (BSA) is present at a concentration of 1% by mass;

The mass percentage concentration of the ascorbic acid (VC) is 0.5 to 20 percent; preferably, the concentration of ascorbic acid (VC) is 0.7% to 18% by mass; the mass percentage concentration of the ascorbic acid (VC) is 0.9 to 16 percent; the mass percentage concentration of the ascorbic acid (VC) is 1.1 to 14 percent; the mass percentage concentration of the ascorbic acid (VC) is 1.3 to 12 percent; the mass percentage concentration of the ascorbic acid (VC) is 1.5 to 10 percent; the mass percentage concentration of the ascorbic acid (VC) is 1.7-8%; the mass percentage concentration of the ascorbic acid (VC) is 1.8 to 6 percent; the mass percentage concentration of the ascorbic acid (VC) is 1.9 to 4 percent; the concentration of ascorbic acid (VC) was 2% by mass.

As a preferred embodiment, the matrix further comprises ethylenediamine tetraacetic acid (EDTA), and the concentration of the ethylenediamine tetraacetic acid (EDTA) in the aqueous solution is 0.1% to 10% by mass.

It should be noted that, the applicant found that no research on the stability of antiviral drugs (e.g., ritonavir) has been performed, and in order to examine the stability of ritonavir in the lyophilized preparation prepared in the present application, accelerated stability research has been performed on a lyophilized preparation product or solution to which one or more of Bovine Serum Albumin (BSA), ascorbic acid (VC) and ethylenediamine tetraacetic acid (EDTA) are added. The specific research steps are as follows:

1. aqueous solutions of ritonavir were prepared separately containing different matrices, each having a concentration of 0.4ug/ml, the contents of the matrices being shown in table 3, the mass percent concentrations of each matrix being shown in table 3.

2. The prepared partial aqueous solution is prepared into a freeze-drying agent through the freeze-drying operation of the step S2, and specific freeze-drying parameters are shown in the table below.

Table 2.

3. The prepared lyophilized preparation and aqueous solution were placed in an oven at 37℃and taken out after 7 days, and the stability was tested by examining the concentration change before and after ritonavir, and the results are shown in Table 3.

Table 3.

As can be seen from the above table, ritonavir cannot exist stably for a long period of time in an aqueous solution containing the above matrix components, and the concentration of ritonavir in mass percent is reduced more; in contrast, the stability of the ritonavir is greatly improved after the ritonavir is prepared into the freeze-drying agent through freeze-drying operation, so that the ritonavir is beneficial to improving the stability of the ritonavir in a matrix after the ritonavir is prepared into the freeze-drying agent through freeze-drying operation.

In addition, in the freeze-drying agent group result, the stability of the group added with VC is improved compared with that of the group ritonavir without VC; the stability of ritonavir in the BSA-added group was improved compared to the BSA-EDTA-added group; in the freeze-dried agent group containing BSA and VC, the stability of ritonavir is further improved; meanwhile, in the freeze-drying agent group of 1% BSA-1% EDTA-2% VC, the concentration of ritonavir is close to 95%, and the ritonavir has relatively good long-term stability; the concentration of ritonavir is highest, reaching 98.27%, in the freeze-dried agent group containing 1% BSA-2% VC.

From the above experiments and analysis, it was found that the freeze-dried formulation group containing 1% BSA-2% VC and the freeze-dried formulation group containing 1% BSA-1% EDTA-2% VC showed superior ritonavir stability.

As a preferred embodiment, lyoprotectants may also be added as needed, together with the lyoprotectant at the time of formulation of the aqueous solution, including but not limited to one or more of sucrose, mannitol, trehalose.

In another aspect, the invention also provides a kit for detecting ritonavir, comprising the stable lyophilizate comprising ritonavir and a matrix.

As a preferred embodiment, the kit comprises a plurality of parts of the lyophilized preparation stored separately, a plurality of parts of the lyophilized preparation being used for preparing a plurality of calibrators for each point concentration on a calibration curve in a liquid chromatography-tandem mass spectrometry combined detection, and a plurality of concentrations of quality control substances.

As a preferred embodiment, the corresponding aqueous solutions are prepared according to the concentrations of ritonavir in the calibrator and the quality control product, respectively.

The kit comprises a plurality of parts of the stable freeze-drying agents which are stored separately, so that the kit can be stored for a long time, and a calibrator and a quality control product which are required to be used in liquid chromatography-tandem mass spectrometry detection are conveniently prepared, thereby providing preconditions for the mass production and supply of ritonavir detection kits.

In another aspect, the invention also provides the use of a stable lyophilizate comprising ritonavir and a matrix or a kit for detecting ritonavir in a liquid chromatography-tandem mass spectrometry combination detection, wherein the lyophilizate or the kit is used for configuring the calibrator and/or the quality control for detecting the concentration of ritonavir in a sample.

The invention is further illustrated below in conjunction with specific examples. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

EXAMPLE 1 preparation of a Freeze-dried preparation

An aqueous matrix solution is prepared, wherein the specific composition of the matrix in the aqueous matrix solution is shown in table 6, and each sample of the freeze-drying agent group in table 6 also corresponds to the aqueous matrix solution before freeze-drying.

Calibrators (C1-C6) and quality control (LQC, MQC and HQC) were prepared from the above aqueous matrix solutions at the concentrations of each curve point of ritonavir and nemalte Wei Jiaozhun curves, as shown in Table 4 below:

Table 4.

Freeze-drying the prepared calibrator and quality control product according to the following process:

The product is placed in a refrigerator with the temperature of minus 80 ℃ for pre-freezing for 1 hour, when the temperature of the plate layer of the freeze dryer is reduced to minus 40 ℃, the product is evenly placed in the plate layer of the freeze dryer, and the freeze drying procedure is set according to the following table 5 for vacuum freeze drying, so as to obtain the freeze dryer product:

Table 5.

The lyophilized product was placed in an oven at 37 ℃ and the accelerated stability test was examined. After 7 days of standing, the sample was taken out and tested for stability, and the results of the accelerated stability test of HQC (8. Mu.g/mL) lyophilized preparation are shown in Table 6:

Table 6.

As can be seen from the above table, ritonavir concentration was up to 99.98% in the lyophilizate group containing 1% BSA-2% VC.

Example 2 blood concentration detection of Nemactevir and ritonavir

1. Experimental apparatus and materials

(1) Instrument: RZ-500 high performance liquid chromatograph-tandem mass spectrometer (Ruikang organism); milli-Q plus ultra-pure water instrument (Millipore Co., U.S.A.); 5804-R high-speed cryocentrifuge (Eppendorf); electronic balance XPE105 (METTLER company, switzerland); MB100-4A microplate thermostated oscillator (Hangzhou O Cheng Yiqi Co., ltd.); VTS temperature regulated heat sealer (TIL); 96-well plates (1.0 mL, 0.36 mL); heat-seal aluminum films (Waters); 12 channel adjustable pipettor (Eppendorf).

(2) Reagent: MS grade methanol (CNW), MS grade acetonitrile (CNW), MS grade ammonium acetate (CNW), MS grade formic acid (CNW), bovine serum albumin (Sigma), PBS buffer salt (Solarbio), proclin 950 (Sigma), VC (CNW), EDTA (CNW), citric acid (CNW).

(3) Chromatographic column: CNW SHELL C18,2.1 x 100mm,2.6 μm.

(4) The standard is shown in Table 7.

Table 7.

2. Instrument conditions

(1) Liquid phase conditions

Mobile phase a:0.1% formic acid in water; mobile phase B:0.1% methanol formate;

Chromatographic column model: CNWSHELL C18,2.1 x 100mm,2.6 μm; the liquid phase condition adopts binary mixed gradient elution, and the initial proportion of the mobile phase A to the mobile phase B is 90:10, specific elution parameters are shown in Table 8, the flow rate is 0.4mL/min, the column temperature is 40 ℃, and the sample injection volume is 5. Mu.L.

Table 8.

(2) Mass spectrometry conditions

The mass spectral parameters are shown in table 9:

table 9.

3. Preparation of working solution

(1) And (3) re-dissolving the calibrator and the quality control product:

Taking out the lyophilized calibrator and quality control product from the refrigerator at 4deg.C, and standing at room temperature. Adding 400 mu L of ultrapure water for re-dissolution respectively, and mixing by vortex.

(2) Preparing an internal standard extraction solution:

Nemactevir-D9 and ritonavir-C13, D3 were precisely weighed, dissolved in methanol and formulated as internal standard stock solutions at 1mg/mL each. Accurately transferring 40 mu L of each of the Nemactevir-D9 and ritonavir-C13 and D3 internal standard stock solution, and preparing an internal standard extraction solution by using acetonitrile to reach a volume of 50mL, wherein the concentration of the internal standard solution is shown in Table 10.

Table 10 internal standard solution concentration

4. Method of establishing

In order to establish a detection method capable of simultaneously measuring the concentrations of 2 antiviral drugs, parameter optimization is required for each compound. First, a proper ionization mode is selected, and the compound to be tested is subjected to full scanning (Q1 Scan), so that a proper Q1 is determined. The ion monitoring parameters and the appropriate ion pairs Q1> Q3 are again optimized. And finally, optimizing source parameters, such as back-blowing, ion source temperature, voltage and the like, of all the compounds. And finally, establishing multiple reaction monitoring conditions of 2 compounds and 2 isotope internal standards.

And optimizing the peak shape and the peak outlet time by optimizing the gradient of the high performance liquid phase mobile phase, and establishing a gradient elution program of the high performance liquid chromatography.

The detection results are shown in fig. 3 and 4, and fig. 2 shows chromatograms of the Nemactivir and ritona Wei Biaozhun products. From the results, it can be seen that: the compounds have good peak shapes, no interference between ion pairs, good response and complete separation degree. The freeze-dried calibrator and quality control product of the application can maintain the stability of 2 antiviral drugs.

5. Sample processing

(1) Respectively taking 50 mu L of calibrator, quality control product and sample into 1.0mL of 96-well plate, adding 200 mu L of internal standard extract containing internal standard, sealing the 96-well plate with heat-sealed aluminum foil, and placing into a microplate vibrator for shaking for 5min. Centrifuging at 4000r/min for 5min, transferring 40 μl of supernatant into 0.36ml 96 well plate with a pipettor, adding 240 μl of diluted solution of acetonitrile-water (1:1), sealing with sealing plate membrane, oscillating for 1min, and detecting on machine.

(2) Establishing a calibration curve: and taking the concentration ratio of the target object to the internal standard as an abscissa, taking the peak area ratio of the target object to the internal standard as an ordinate, quantifying by adopting an isotope internal standard method, establishing a calibration curve, and calculating the concentration of the to-be-detected object in the serum sample.

6. Method performance verification

(1) Absolute matrix effect investigation

Adding a target object into a pure solvent to prepare a pure solution sample; preparing a biological matrix sample with the same concentration by using the extracted blank human serum; the corresponding differences of the compounds in the 2 matrices are compared and the matrix effect results are shown in Table 11.

Table 11.

From the above results, the difference between the target substance in the pure solvent and the biological matrix sample is less than 20%, indicating whether the matrix effect exists or not, and the accurate quantification of the target analyte is not affected.

(2) Accuracy investigation

The accuracy performance of the method was assessed by adding a label to a human serum sample of known concentration and determining the recovery of the label. Standard solutions with low, medium and high concentration are respectively prepared into 5 standard samples with three concentrations, and each standard solution is measured 3 times. The theoretical value is the sum of the concentration of the endogenous substance in the sample and the concentration of the added calibrator, the ratio of the measured value to the theoretical value can be used for evaluating the accuracy, the detection value is within +/-15% of the target value, and the standard adding result is shown in Table 12.

Table 12.

(3) Precision investigation

Selecting samples with low (L), medium (M) and high (H) concentrations for precision investigation, wherein each concentration is measured 10 times (wherein, the concentration standard of the Nemactetavir and ritonavir is that the concentration standard is low L (0.2 ug/ml), medium M (2.5 ug/ml) and high H (4.3 ug/ml)), and measuring the precision in the batch; the precision required is < 15% and the results are shown in Table 13.

Table 13.

From the above results, the concentrations of 2 antiviral drugs were quantitatively measured by the isotope internal standard method with a precision of 3.83% -5.21%. The precision is good, and the performance requirements are met.

(4) Linear investigation

Adding a calibrator with a certain concentration into blank human serum to prepare a low-concentration curve point S1 and a high-concentration curve point S7, and mixing the S1 and the S7 according to different proportions to prepare S2-S6. And repeatedly measuring each concentration point for 3 times to respectively obtain the average value of each concentration measurement result, and adopting a polynomial regression equation, wherein R2 is more than 0.9900. The linear results are shown in Table 14.

TABLE 14 Linear regression equation and correlation coefficient for antiviral drugs

From the above results, the linear properties of the Nemactevir and ritonavir in the respective ranges are good, and the correlation coefficients meet the requirements.

The freeze-drying agent prepared by the preparation method of the stable freeze-drying agent containing ritonavir and the matrix, which is adopted by the invention, avoids the degradation phenomenon of ritonavir Wei Shiji prepared in the prior art caused by long-term storage to a great extent, and simultaneously avoids the error caused by detection by using different batches of the reagents caused by the degradation of ritonavir Wei Shiji;

Compared with the existing ritonavir Wei Shiji, the stable freeze-drying agent containing ritonavir and a matrix prepared by the method can be stored for a long time, so that the freeze-drying agent can be supplied to a ritonavir Wei Yexiang chromatographic-tandem mass spectrometry detection scene through commercial mass production and storage, the efficacy and the detection precision of ritonavir Wei Yexiang chromatographic-tandem mass spectrometry detection are improved in blood concentration detection, and a precondition is provided for the mass production of ritonavir detection kits.

Based on the present disclosure, one skilled in the art should appreciate that one aspect described herein may be implemented independently of any other aspect, and that two or more of these aspects may be combined in various ways. For example, apparatus may be implemented and/or methods practiced using any number and aspects set forth herein. In addition, such apparatus may be implemented and/or such methods practiced using other structure and/or functionality in addition to one or more of the aspects set forth herein.

It should be noted that the above embodiments can be freely combined as needed. The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be understood that various changes and modifications may be made by those skilled in the art after reading the foregoing description of the application, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims (9)

1. A method of preparing a stable freeze-dried formulation comprising ritonavir and a matrix, said method comprising the steps of:

step S1: preparing an aqueous solution containing ritonavir and a matrix including Bovine Serum Albumin (BSA) and ascorbic acid (VC);

step S2: lyophilizing the aqueous solution to obtain the stable lyophilized formulation comprising ritonavir and a matrix;

Wherein, in the aqueous solution, the concentration of the Bovine Serum Albumin (BSA) is 0.1 to 5 percent by mass, and the concentration of the ascorbic acid (VC) is 0.5 to 20 percent by mass.

2. The method of preparing a stable freeze-dried formulation comprising ritonavir and a matrix of claim 1, wherein the matrix further comprises ethylenediamine tetraacetic acid (EDTA).

3. The method of preparing a stable lyophilisate comprising ritonavir and a matrix according to claim 2, characterized in that the mass percentage concentration of ethylenediamine tetraacetic acid (EDTA) in the aqueous solution is 0.1% to 10%.

4. A method of preparing a stable lyophilisate comprising ritonavir and a matrix according to any of claims 1 to 3, wherein step S1 further comprises:

and adding a lyoprotectant to the formulated aqueous solution containing ritonavir and a matrix.

5. The method of preparing a stable lyophile comprising ritonavir and a matrix according to claim 4 wherein said lyoprotectant is selected from one or more of sucrose, mannitol, trehalose.

6. A stable lyophilisate comprising ritonavir and a matrix, characterized in that it is prepared by the preparation method of any one of claims 1 to 5.

7. A kit for the detection of ritonavir, comprising a stable lyophilizate comprising ritonavir and a matrix according to claim 6.

8. The kit for detecting ritonavir according to claim 7, wherein said kit comprises separately stored multiple portions of said stable freeze-dried agent for use in formulating multiple calibrators of each point concentration on a calibration curve in a liquid chromatography-tandem mass spectrometry combined assay, and formulating quality control of multiple concentrations;

and respectively preparing corresponding aqueous solutions according to the concentrations of ritonavir in the calibrator and the quality control product.

9. Use of a stable lyophilizate comprising ritonavir and a matrix according to claim 6 or a kit for detecting ritonavir according to any of claims 7 to 8 in a liquid chromatography-tandem mass spectrometry combination assay, wherein the lyophilizate or the kit is used for formulating a plurality of calibrators at various points on a calibration curve and/or for formulating a plurality of concentrations of quality control for detecting ritonavir concentration in a sample.