Stable freeze-dried preparation comprising ritonavir and matrix, preparation method, kit, and use thereof

Abstract

A stable freeze-dried preparation comprising ritonavir and a matrix, a preparation method, a kit, and use thereof. The freeze-dried preparation is obtained by formulating an aqueous solution containing ritonavir and a matrix and then freeze-drying the aqueous solution, wherein the matrix comprises BSA and VC. The kit for measuring ritonavir comprises the freeze-dried preparation. The freeze-dried preparation and / or the kit can be used in liquid chromatography-tandem mass spectrometry combined measurement for measuring the concentration of ritonavir in a sample. The freeze-dried preparation and / or the kit are used for formulating a calibration product and / or a quality control product. The freeze-dried preparation and the kit can be stored for a long time and are used for formulation a calibration product and a quality control product to be used in liquid chromatography-tandem mass spectrometry measurement, providing a prerequisite for large-scale production of the ritonavir measurement kit.

Description

A stable lyophilizing agent comprising ritonavir and a matrix, its preparation method, a kit, and its application. Technical Field

[0001] This invention belongs to the field of biological sample drug analysis technology, and relates to a stable lyophilizing agent containing ritonavir and a matrix, its preparation method, reagent kit and its application. Background Technology

[0002] N-[(2S,3S,5R)-3-hydroxy-5-[[(2S)-3-methyl-2-[[methyl-[(2-isopropyl-1,3-thiazolyl-4-yl)methyl]carbamoyl]amino]butyryl]amino]-1,6-diphenyl-hex-2-yl]carbamate-5-thiazolylmethyl ester (drug name: Ritonavir, Cas No.: 155213-67-5). Ritonavir is a potent inhibitor of the cytochrome P450 isoenzyme CYP3A and can be used to treat HIV-related diseases. Additionally, ritonavir can inhibit the CYP3A-mediated metabolism of nirmatavir; when used in combination with nirmatavir, it helps slow down the metabolic breakdown of nirmatavir, allowing high concentrations of nirmatavir to remain in the patient's body for a longer period, thus better combating diseases such as COVID-19.

[0003] Plasma concentration refers to the total concentration of a drug in the blood plasma after absorption, including drugs bound to plasma proteins or free in the plasma. It can also refer to the concentration of a drug in whole blood. Plasma concentration testing is a key method for evaluating therapeutic efficacy or determining dosing regimens, enabling individualized dosing and improving the effectiveness of drug therapy.

[0004] Currently, ritonavir blood concentration can be quantified by combined liquid chromatography-mass spectrometry (LC / MS) detection of blood samples from patients. This combined detection requires ritonavir calibrators and quality control samples. However, existing ritonavir calibrators and quality control samples do not contain ritonavir that is stable in the matrix under normal conditions for extended periods. Therefore, they cannot be supplied through commercial mass production and storage. Instead, calibrators and quality control samples must be prepared during the detection process, which severely impacts the operational efficiency of ritonavir blood concentration detection and hinders the individualization of ritonavir treatment regimens.

[0005] In view of the above-mentioned shortcomings, it is necessary to provide a stable lyophilizing agent containing ritonavir and a matrix, a preparation method, a kit and its application.

[0006] Summary of the Invention

[0007] The main objective of this invention is to provide a stable lyophilizing agent comprising ritonavir and a matrix, a preparation method, and a kit. This aims to address the technical problem that existing ritonavir calibrators and quality control products cannot maintain stable ritonavir in the matrix for extended periods in normal environments, thus hindering commercial mass production and storage. Instead, calibrators and quality control products must be prepared during testing, severely impacting the operational efficiency of ritonavir blood concentration detection and causing inconvenience for individualized ritonavir regimen development.

[0008] To achieve the above objectives, the present invention proposes a method for preparing a stabilizing lyophilizer comprising ritonavir and a matrix, the preparation method comprising the following steps:

[0009] Step S1: Prepare an aqueous solution containing ritonavir and a matrix, wherein the matrix includes bovine serum albumin (BSA) and ascorbic acid (VC);

[0010] Step S2: Freeze-dry the aqueous solution to obtain the stabilized freeze-drying agent containing ritonavir and the matrix.

[0011] Preferably, in the aqueous solution, the mass percentage concentration of bovine serum albumin (BSA) is 0.1% to 5%, and the mass percentage concentration of ascorbic acid (VC) is 0.5% to 20%.

[0012] Preferably, the matrix further includes ethylenediaminetetraacetic acid (EDTA).

[0013] Preferably, the mass percentage concentration of ethylenediaminetetraacetic acid (EDTA) in the aqueous solution is from 0.1% to 10%.

[0014] Preferably, step S1 further includes:

[0015] A lyophilization protectant is added to the prepared aqueous solution containing ritonavir and the matrix.

[0016] Preferably, the freeze-drying protectant is selected from one or more of sucrose, mannitol, and trehalose.

[0017] The present invention also provides a stabilizing lyophilizer comprising ritonavir and a matrix, wherein the stabilizing lyophilizer is prepared by any of the foregoing preparation methods.

[0018] The present invention also provides a kit for detecting ritonavir, the kit comprising a stabilizing lyophilizer containing ritonavir and a matrix.

[0019] Preferably, the kit includes multiple portions of the stabilizer lyophilized agent stored separately, the multiple portions of the stabilizer lyophilized agent being used to prepare multiple calibrators at various concentrations on the calibration curve in liquid chromatography-tandem mass spectrometry combined detection, and to prepare multiple concentrations of quality control samples.

[0020] Preferably, corresponding aqueous solutions are prepared according to the concentration of ritonavir in the calibrator and the quality control sample, respectively.

[0021] Preferably, the present invention also provides the application of a stable lyophilized agent comprising ritonavir and a matrix or a kit for detecting ritonavir in liquid chromatography-tandem mass spectrometry, wherein the lyophilized agent or the kit is used to prepare multiple calibrators at various concentrations on a calibration curve and / or to prepare multiple quality control samples at various concentrations for detecting the concentration of ritonavir in a sample.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The lyophilizer prepared by the method of stabilizing lyophilizer containing ritonavir and matrix adopted in this application greatly avoids the degradation of ritonavir reagent prepared in the prior art due to long-term storage, and avoids the error caused by using different batches of the above reagent for testing due to the degradation of ritonavir reagent, thus bringing convenience to the individualized formulation of ritonavir regimen.

[0024] Furthermore, compared to existing ritonavir reagents, the stable lyophilized agent containing ritonavir and matrix prepared in this application can be stored for a long time. Therefore, it can be commercially mass-produced and stored to supply lyophilized agents for ritonavir liquid chromatography-tandem mass spectrometry detection, thereby improving the efficiency and detection precision of ritonavir liquid chromatography-tandem mass spectrometry detection in blood drug concentration detection.

[0025] 2. The method for preparing a stabilizing lyophilizer containing ritonavir and a matrix used in this application includes the step of preparing an aqueous solution containing ritonavir and a matrix, wherein the matrix includes bovine serum albumin (BSA) and ascorbic acid (AC). The addition of bovine serum albumin (BSA) and ascorbic acid (AC) synergistically ensures the stable presence of ritonavir in the lyophilizer. Furthermore, this application preferably sets the mass percentage concentration of the aforementioned bovine serum albumin (BSA) and ascorbic acid (AC) to be added. This setting can further amplify the aforementioned synergistic effect, increase the stability of ritonavir in the lyophilizer, and provide a prerequisite for the large-scale production of ritonavir test kits. Attached Figure Description

[0026] Figure 1 is a schematic diagram of the preparation steps of the stabilizing lyophilizer containing ritonavir and matrix in this application.

[0027] Figure 2 shows the chromatograms of nematriberi and ritonavir standards (2.94 min: nematriberi; 3.22 min: ritonavir).

[0028] Figure 3 shows the chromatogram of a serum sample spiked (5 μg / mL).

[0029] Figure 4 shows the chromatogram of a blank serum sample. Detailed Implementation

[0030] The various aspects of the present invention will be further described in detail below.

[0031] Unless otherwise defined or stated, all technical and scientific terms used herein have the same meaning as are familiar to a user skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0032] Unless otherwise expressly specified and limited, the term "or" as used in this invention includes the relationship of "and". "And" is equivalent to the Boolean logic operator "AND", and "or" is equivalent to the Boolean logic operator "OR", with "AND" being a subset of "OR".

[0033] It is understood that although the terms "first," "second," etc., may be used herein to describe different elements, these elements should not be limited by these terms. These terms are merely used to distinguish one element from another. Thus, a first element may be referred to as a second element without departing from the teachings of this disclosure.

[0034] In this invention, the terms "mainly composed of" and "composed of" are included in the terms "containing", "comprising" or "including".

[0035] Unless otherwise expressly specified and limited, the terms "connected," "linked," and "connected" in this invention should be interpreted broadly. For example, they can refer to a fixed connection, a connection through an intermediate medium, or a connection within two elements or an interaction between two elements. Users of ordinary skill in the art can understand the specific meaning of these terms in this application based on the specific circumstances.

[0036] For example, if an element (or component) is referred to as being on, coupled to, or connected to another element, then the element may be directly formed on, coupled to, or connected to the other element, or there may be one or more intermediate elements between them. Conversely, if the expressions "directly on," "directly coupled to," and "directly connected to" are used herein, then it indicates that there is no intermediate element. Other terms used to describe relationships between elements should be interpreted similarly, such as "between" and "directly between," "attached" and "directly attached," "adjacent" and "directly adjacent," etc.

[0037] Additionally, it should be noted that the terms "front," "rear," "left," "right," "up," and "down" used in the following description refer to directions in the accompanying drawings. The terms "inner" and "outer" refer to directions toward or away from the geometric center of a particular component, respectively. It is understood that these terms are used here to describe the relationship of one element, layer, or region relative to another element, layer, or region, as shown in the accompanying drawings. These terms should also encompass other orientations of the device in addition to those described in the accompanying drawings.

[0038] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

[0039] To more clearly illustrate the technical solutions in the embodiments of this disclosure or the prior art, specific implementation methods of the present invention will be described below with reference to the accompanying drawings. Obviously, the drawings described below are merely some embodiments of the present invention. For users skilled in the art, other drawings and other implementation methods can be obtained based on these drawings without any creative effort.

[0040] It should also be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of this disclosure. The drawings only show components relevant to this application and are not drawn according to the actual number, shape, and size of the components in the actual implementation. In the actual implementation, the form, quantity, and proportion of each component can be arbitrarily changed, and the layout of the components may also be more complex. For example, the thickness of the elements in the drawings may be exaggerated for clarity.

[0041] For existing ritonavir calibrators and quality control products, the ritonavir in their matrix cannot exist stably in a normal environment for a long time. Therefore, they cannot be supplied through commercial mass production and storage. The calibrators and quality control products can only be prepared during testing. This seriously affects the operational efficiency of ritonavir blood concentration testing and brings inconvenience to the individualization of ritonavir regimens.

[0042] To achieve the above objectives, as shown in Figure 1, the present invention proposes a method for preparing a stabilizing lyophilizer comprising ritonavir and a matrix, the preparation method comprising the following steps:

[0043] Step S1: Prepare an aqueous solution containing ritonavir and a matrix, wherein the matrix includes bovine serum albumin (BSA) and ascorbic acid (VC);

[0044] Step S2: Freeze-dry the aqueous solution to obtain the stabilized freeze-drying agent containing ritonavir and the matrix.

[0045] It should be noted that bovine serum albumin (BSA) is one of the main protein components of bovine serum. It can bind to various substances and is a crucial transport carrier in plasma. Furthermore, BSA possesses hydrophilic-hydrophobic interactions and resists heat denaturation. Natural vitamin C (VC) is a polyhydroxy compound with strong reducing properties, readily oxidized to dehydrovitamin C. In preparing a lyophilized agent containing ritonavir and a matrix, the applicant discovered that setting the matrix to include BSA and VC, and then preparing an aqueous solution with ritonavir, resulted in a lyophilized agent with excellent stability after lyophilization. This allows ritonavir to remain stable for extended periods within the lyophilized agent, significantly avoiding the degradation and other adverse effects that occur with existing ritonavir reagents during long-term storage.

[0046] 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 at -80°C for 0.5 to 3 hours. After the freeze dryer is lowered to an initial temperature of -40 to -50°C, the pre-frozen product is placed in the freeze dryer and freeze-dried according to the automatic program set in Table 1.

[0047] Table 1.

[0048] In a preferred embodiment, the mass percentage concentration of bovine serum albumin (BSA) in the aqueous solution is 0.1% to 5%; preferably, the mass percentage concentration of bovine serum albumin (BSA) is 0.2% to 4.5%; preferably, the mass percentage concentration of bovine serum albumin (BSA) is 0.3% to 4%; preferably, the mass percentage concentration of bovine serum albumin (BSA) is 0.4% to 3.5%; preferably, the mass percentage concentration of bovine serum albumin (BSA) is 0.5% to 3%; preferably, the mass percentage concentration of bovine serum albumin (BSA) is 0.6% to 2.5%; preferably, the mass percentage concentration of bovine serum albumin (BSA) is 0.7% to 2%; preferably, the mass percentage concentration of bovine serum albumin (BSA) is 0.8% to 1.5%; preferably, the mass percentage concentration of bovine serum albumin (BSA) is 0.9% to 1.3%; preferably, the mass percentage concentration of bovine serum albumin (BSA) is 1%.

[0049] The mass percentage concentration of ascorbic acid (VC) is 0.5% to 20%; preferably, the mass percentage concentration of ascorbic acid (VC) is 0.7% to 18%; the mass percentage concentration of ascorbic acid (VC) is 0.9% to 16%; the mass percentage concentration of ascorbic acid (VC) is 1.1% to 14%; the mass percentage concentration of ascorbic acid (VC) is 1.3% to 12%; the mass percentage concentration of ascorbic acid (VC) is 1.5% to 10%; the mass percentage concentration of ascorbic acid (VC) is 1.7% to 8%; the mass percentage concentration of ascorbic acid (VC) is 1.8% to 6%; the mass percentage concentration of ascorbic acid (VC) is 1.9% to 4%; the mass percentage concentration of ascorbic acid (VC) is 2%.

[0050] In a preferred embodiment, the matrix further includes ethylenediaminetetraacetic acid (EDTA), and the aqueous solution contains EDTA at a mass percentage concentration of 0.1% to 10%.

[0051] It should be noted that the applicant found that no researchers have yet studied the stability of antiviral drugs (such as ritonavir). To investigate the stability of ritonavir in the lyophilized formulation prepared in this application, accelerated stability studies were conducted on lyophilized formulations or solutions containing one or more combinations of bovine serum albumin (BSA), ascorbic acid (VC), and ethylenediaminetetraacetic acid (EDTA). The specific research steps were as follows:

[0052] 1. Prepare aqueous solutions of ritonavir containing different matrices. The concentration of ritonavir is 0.4 ug / ml. The content of the matrices is shown in Table 3. The content of each matrice is expressed as a mass percentage concentration.

[0053] 2. Prepare a lyophilizing agent by lyophilizing a portion of the prepared aqueous solution through the lyophilization operation in step S2 above. The specific lyophilization parameters are shown in the table below.

[0054] Table 2.

[0055] 3. The prepared lyophilizing agent and aqueous solution were placed in an oven at 37°C. After 7 days, the concentration changes of ritonavir before and after the drying were examined and compared to test its stability. The results are shown in Table 3.

[0056] Table 3.

[0057] As shown in the table above, ritonavir cannot exist stably for a long time in aqueous solutions containing the above matrix components, and its mass percentage concentration decreases significantly. In contrast, its stability is greatly improved after being prepared into a lyophilized agent by lyophilization. Therefore, preparing a lyophilized agent by lyophilization helps to improve the stability of ritonavir in the matrix.

[0058] Furthermore, in the lyophilization group results, the stability of ritonavir was improved in the group with added vitamin C compared to the group without added vitamin C; the stability of ritonavir was improved in the group with added BSA compared to the group with added BSA-EDTA; the stability of ritonavir was further increased in the lyophilization group containing both BSA and vitamin C; meanwhile, the concentration of ritonavir in the 1% BSA-1% EDTA-2% vitamin C lyophilization group was close to 95%, and it also had relatively good long-term stability; the highest concentration of ritonavir was found in the lyophilization group containing only 1% BSA-2% vitamin C, reaching 98.27%.

[0059] The above experiments and analyses show that the lyophilized formulations containing 1% BSA and 2% VC, as well as the lyophilized formulations containing 1% BSA, 1% EDTA, and 2% VC, exhibit superior stability of ritonavir.

[0060] As a preferred embodiment, a freeze-drying protectant may be added as needed. The freeze-drying protectant is added together with the aqueous solution. The freeze-drying protectant includes, but is not limited to, one or more of sucrose, mannitol, and trehalose.

[0061] On the other hand, the present invention also provides a kit for detecting ritonavir, the kit comprising a stabilizing lyophilizer containing ritonavir and a matrix.

[0062] In a preferred embodiment, the kit includes multiple portions of the lyophilized reagent stored separately, which are used to prepare multiple calibrators at various concentrations on the calibration curve for liquid chromatography-tandem mass spectrometry combined detection, as well as to prepare multiple quality control samples at various concentrations.

[0063] As a preferred embodiment, corresponding aqueous solutions are prepared according to the concentration of ritonavir in the calibrator and the quality control sample, respectively.

[0064] It should be noted that the kit includes multiple portions of the stabilizer lyophilized agent stored separately, enabling the kit to be stored for a long time. This facilitates the preparation of calibrators and quality control products used in liquid chromatography-tandem mass spectrometry detection, providing a prerequisite for the large-scale production and supply of ritonavir detection kits.

[0065] On the other hand, the present invention also provides the application of a stable lyophilized agent comprising ritonavir and a matrix or a kit for detecting ritonavir in liquid chromatography-tandem mass spectrometry, wherein the lyophilized agent or the kit is used to prepare the calibrator and / or the quality control for detecting the concentration of ritonavir in a sample.

[0066] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0067] Example 1: Preparation of lyophilizing agent

[0068] Prepare a matrix aqueous solution. The specific composition of the matrix in the matrix aqueous solution is shown in Table 6. It should be noted that each sample of the lyophilizing agent group in Table 6 also has a corresponding matrix aqueous solution before lyophilization.

[0069] The above-mentioned matrix aqueous solution was used to prepare calibrators (C1-C6) and quality control samples (LQC, MQC, and HQC) at various curve points for ritonavir and nematovir calibration curves. The curve point concentrations are shown in Table 4 below.

[0070] Table 4.

[0071] The prepared calibrators and quality control samples were freeze-dried according to the following process:

[0072] Pre-freeze the product in a -80℃ freezer for 1 hour. When the temperature of the freeze dryer plates drops to -40℃, evenly place the product on the freeze dryer plates and perform vacuum freeze drying according to the freeze drying program set in Table 5 below to obtain the freeze-dried product (see Table 5):

[0073] Table 5.

[0074] The lyophilized product was placed in a 37℃ oven for accelerated stability testing. After 7 days, it was removed and its stability was tested. Taking the accelerated stability test results of HQC (8μg / mL) lyophilized agent as an example, the results are shown in Table 6:

[0075] Table 6.

[0076] As shown in the table above, among the lyophilized formulations containing only 1% BSA and 2% VC, ritonavir had the highest concentration, reaching 99.98%.

[0077] Example 2: Blood drug concentration detection of nematvir and ritonavir

[0078] 1. Experimental Instruments and Materials

[0079] (1) Instruments: RZ-500 high performance liquid chromatography-tandem mass spectrometry (Ruikang Biotechnology); Milli-Q plus ultrapure water system (Milipore, USA); 5804-R high-speed refrigerated centrifuge (Eppendorf); XPE105 electronic balance (METTLER, Switzerland); MB100-4A microplate constant temperature shaker (Hangzhou Aosheng Instrument Co., Ltd.); VTS temperature-controlled heat sealer (TIL); 96-well plates (1.0mL, 0.36mL); heat-sealing aluminum film (Waters); 12-channel adjustable pipette (Eppendorf).

[0080] (2) Reagents: MS grade methanol (CNW), MS grade acetonitrile (CNW), MS grade ammonium acetate (CNW), MS grade formic acid (CNW), bovine serum albumin (Sigma), PBS buffer (Solarbio), Proclin 950 (Sigma), VC (CNW), EDTA (CNW), citric acid (CNW).

[0081] (3) Chromatographic column: CNW Shell C18, 2.1*100mm, 2.6μm.

[0082] (4) Standard products are shown in Table 7.

[0083] Table 7.

[0084] 2. Instrument conditions

[0085] (1) Liquid phase conditions

[0086] Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: 0.1% formic acid in methanol;

[0087] Column type: CNWShell C18, 2.1*100mm, 2.6μm; liquid phase conditions: binary mixed gradient elution, initial ratio of mobile phase A to mobile phase B: 90:10, specific elution parameters are shown in Table 8, flow rate: 0.4mL / min, column temperature: 40℃, injection volume: 5μL.

[0088] Table 8.

[0089] (2) Mass spectrometry conditions

[0090] Mass spectrometry parameters are shown in Table 9:

[0091] Table 9.

[0092] 3. Preparation of working solution

[0093] (1) Reconstitution of calibrators and quality control samples:

[0094] Remove the lyophilized calibrators and quality control samples from the 4°C freezer and allow them to reach room temperature. Redissolve each sample in 400 μL of ultrapure water and vortex to mix.

[0095] (2) Preparation of internal standard extraction solution:

[0096] Accurately weigh nelmatevir-D9 and ritonavir-C13,D3, dissolve them in methanol, and prepare internal standard stock solutions with a concentration of 1 mg / mL. Accurately transfer 40 μL of each nelmatevir-D9 and ritonavir-C13,D3 internal standard stock solution, and dilute to 50 mL with acetonitrile to prepare internal standard extraction solutions. The concentrations of the internal standard solutions are shown in Table 10.

[0097] Table 10 Internal Standard Solution Concentration

[0098] 4. Method Establishment

[0099] To establish a detection method capable of simultaneously determining the concentrations of two antiviral drugs, parameter optimization for each compound is necessary. First, a suitable ionization mode is selected, and a full scan (Q1Scan) is performed on the analyte to determine an appropriate Q1. Then, ion monitoring parameters and a suitable ion pair Q1>Q3 are optimized. Finally, source parameters for all compounds are optimized, such as backflushing gas, ion source temperature, and voltage. Finally, multiple reaction monitoring conditions for the two compounds and two isotopic internal standards are established.

[0100] By optimizing the mobile phase gradient of high performance liquid chromatography (HPLC), peak shape and elution time were optimized, and a gradient elution program for HPLC was established.

[0101] The detection results are shown in Figures 3 and 4, with Figure 2 showing the chromatograms of nematvir and ritonavir standards. The results indicate that the peak shapes of each compound are good, there is no interference between ion pairs, the response is good, and the separation of each compound is complete. This demonstrates that the lyophilized calibrators and quality control samples produced in this application maintained the stability of the two antiviral drugs.

[0102] 5. Sample processing

[0103] (1) Take 50 μL each of calibrator, quality control sample and sample into a 1.0 mL 96-well plate, add 200 μL of internal standard extraction solution containing internal standard, seal the 96-well plate with heat-sealing aluminum foil, and place it in a microplate shaker and shake for 5 min. Centrifuge at 4000 r / min for 5 min, transfer 40 μL of supernatant into a 0.36 mL 96-well plate with a pipette, add 240 μL of acetonitrile-water (1:1) dilution solution, seal with sealing film, shake for 1 min, and then perform analysis.

[0104] (2) Establish a calibration curve: Using the concentration ratio of the target analyte to the internal standard as the abscissa and the peak area ratio of the target analyte to the internal standard as the ordinate, the isotope internal standard method is used for quantification to establish a calibration curve and calculate the concentration of the analyte in the serum sample.

[0105] 6. Method Performance Verification

[0106] (1) Investigation of the absolute matrix effect

[0107] The target compound was added to a pure solvent to prepare a pure solution sample; then, a biological matrix sample of the same concentration was prepared using extracted blank human serum; the corresponding differences of compounds in the two matrices were compared, and the matrix effect results are shown in Table 11.

[0108] Table 11.

[0109] The results above show that the difference between the target analyte in pure solvent and biological matrix sample is less than 20%, indicating that the presence or absence of matrix effect does not affect the accurate quantification of the target analyte.

[0110] (2) Accuracy assessment

[0111] The accuracy of the method was evaluated by spiking human serum samples at known concentrations and determining the recovery rate. Three standard solutions with low, medium, and high spiking concentrations were prepared, and five samples of each concentration were measured three times. The theoretical value is the sum of the concentration of endogenous substances in the sample and the concentration of the added calibrator. The ratio of the measured value to the theoretical value can be used to evaluate accuracy. The detected value should be within ±15% of the target value. The spiking results are shown in Table 12.

[0112] Table 12.

[0113] (3) Precision test

[0114] Precision was assessed using samples at three different concentrations: low (L), medium (M), and high (H). Each concentration was measured 10 times (the baseline concentrations for nematvir and ritonavir were: low L (0.2 ug / ml), medium M (2.5 ug / ml), and high H (4.3 ug / ml)). Intra-assay precision was measured. Precision was required to be <15%. The results are shown in Table 13.

[0115] Table 13.

[0116] The results above show that the precision of quantitative determination of the concentrations of the two antiviral drugs using the isotope internal standard method is 3.83%-5.21%. The precision is good and meets the performance requirements.

[0117] (4) Linear Examination

[0118] A certain concentration of calibrator was added to blank human serum to prepare low-concentration curve point S1 and high-concentration curve point S7. S1 and S7 were mixed in different proportions to prepare S2-S6. Each concentration point was measured three times, and the mean value of each concentration measurement result was obtained. A polynomial regression equation was used, and R2 > 0.9900. The linearity results are shown in Table 14.

[0119] Table 14. Linear regression equations and correlation coefficients for antiviral drugs

[0120] Based on the above results, nematovir and ritonavir showed good linearity within their respective ranges, and their correlation coefficients met the requirements.

[0121] The lyophilizer prepared by the method of the present invention, which includes ritonavir and matrix, greatly avoids the degradation of ritonavir reagent prepared in the prior art due to long-term storage, and also avoids the error caused by using different batches of the above reagent for testing due to the degradation of ritonavir reagent.

[0122] Furthermore, compared to existing ritonavir reagents, the stable lyophilized agent containing ritonavir and matrix prepared in this application can be stored for a long time. Therefore, it can be commercially mass-produced and stored to supply lyophilized agents for ritonavir liquid chromatography-tandem mass spectrometry detection scenarios, improving the efficiency and detection precision of ritonavir liquid chromatography-tandem mass spectrometry detection in blood drug concentration detection, and providing a prerequisite for the large-scale production of ritonavir detection kits.

[0123] Based on this application, those skilled in the art should understand that one aspect described herein can be implemented independently of any other aspect, and two or more of these aspects can be combined in various ways. For example, any number and aspects set forth herein can be used to implement the device and / or practice the method. Furthermore, this device and / or practice the method can be implemented using other structures and / or functionalities besides one or more of the aspects set forth herein.

[0124] It should be noted that the above embodiments can be freely combined as needed. The above description is only a preferred embodiment of the present invention. It should be pointed out that for users of ordinary skills in this field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0125] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing description of this invention, users skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for preparing a stabilizing lyophilizer comprising ritonavir and a matrix, characterized in that, The preparation method includes the following steps: Step S1: Prepare an aqueous solution containing ritonavir and a matrix, wherein the matrix includes bovine serum albumin (BSA) and ascorbic acid (VC); Step S2: Freeze-dry the aqueous solution to obtain the stabilized freeze-drying agent containing ritonavir and the matrix; In the aqueous solution, the mass percentage concentration of bovine serum albumin (BSA) is 0.1% to 5%, and the mass percentage concentration of ascorbic acid (VC) is 0.5% to 20%.

2. The method for preparing the stabilizing lyophilizer comprising ritonavir and a matrix according to claim 1, characterized in that, The matrix also includes ethylenediaminetetraacetic acid (EDTA).

3. The method for preparing the stabilizing lyophilizer comprising ritonavir and a matrix according to claim 2, characterized in that, In the aqueous solution, the mass percentage concentration of ethylenediaminetetraacetic acid (EDTA) is from 0.1% to 10%.

4. The method for preparing a stabilizing lyophilizer comprising ritonavir and a matrix according to any one of claims 1 to 3, characterized in that, Step S1 further includes: A lyophilization protectant is added to the prepared aqueous solution containing ritonavir and the matrix.

5. The method for preparing the stabilizing lyophilizer comprising ritonavir and a matrix according to claim 4, characterized in that, The freeze-drying protectant is selected from one or more of sucrose, mannitol, and trehalose.

6. A stabilizing lyophilizer comprising ritonavir and a matrix, characterized in that, The stabilizing lyophilizing agent is prepared by the preparation method according to any one of claims 1 to 5.

7. A kit for detecting ritonavir, characterized in that, The kit includes the stabilizing lyophilizer comprising ritonavir and a matrix as described in claim 6.

8. The kit for detecting ritonavir according to claim 7, characterized in that, The kit includes multiple portions of the stabilized lyophilized agent stored separately, which are used to prepare multiple calibrators at various concentrations on the calibration curve in liquid chromatography-tandem mass spectrometry combined detection, as well as to prepare multiple concentrations of quality control samples. Prepare corresponding aqueous solutions based on the concentration of ritonavir in the calibrators and quality control samples.

6. The application of a stable lyophilizing agent comprising ritonavir and a matrix as described in claim 6, or a kit for detecting ritonavir as described in any one of claims 7 to 8, in combined liquid chromatography-tandem mass spectrometry detection, characterized in that, The lyophilizing agent or the kit is used to prepare multiple calibrators at various concentrations on the calibration curve and / or multiple quality control samples at various concentrations for detecting the concentration of ritonavir in the sample.

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