A method for determining the concentration of conazepam in plasma

Abstract

The present application relates to a kind of determination methods of the concentration of conazepam in blood plasma, belong to the technical field of drug concentration analysis.The determination method uses the way of adding internal standard working solution to the blood plasma sample to be measured to process clinical sample, the concentration of conazepam in blood plasma is determined using ultra-high performance liquid chromatography-tandem mass spectrometry, the clinical specific population including AIDS patients, chronic hepatitis patients and liver transplant patients.The determination method of the concentration of conazepam in blood plasma provided by the present application combines the diagnosis and treatment needs of clinical therapeutic drug monitoring, systematically completes the method linear range, sample stability verification, and special plasma matrix effect of chronic hepatitis, AIDS, liver transplant patients is investigated, significantly improves the detection accuracy and clinical adaptability, can accurately support the individualized dosing of conazepam and therapeutic drug monitoring, and the clinical popularization value is excellent.

Description

Technical Field

[0001] This invention belongs to the field of drug concentration analysis technology, specifically relating to a method for determining the concentration of contazolamide in plasma. Background Technology

[0002] Contezolid is a Class I new drug independently developed in my country, belonging to the oxazolidinone class of antibacterial drugs. It was approved in 2021 for the treatment of complicated skin and soft tissue infections. Compared with the traditional oxazolidinone drug linezolid, its advantage lies in its structural optimization, which reduces the inhibition of mitochondrial function and monoamine oxidase activity in the human body, thereby reducing the occurrence of bone marrow suppression and neurological adverse reactions. Clinically, it is often used as an alternative therapy for patients who cannot tolerate linezolid. However, due to the short time since its market launch, its safety data is only based on Phase II and Phase III clinical trials. Currently, the target concentration range for the efficacy and safety of contezolid, validated by large-sample real-world experimental data, is still unclear. Therefore, developing a method for measuring plasma contezolid concentration suitable for clinical therapeutic drug monitoring is crucial for further developing therapeutic drug monitoring (TDM) and pharmacokinetic (PK)-guided precision medicine.

[0003] The convenience, cost-effectiveness, and applicability of methodologies throughout the entire process from clinical sample collection to laboratory testing are crucial for ensuring the accuracy of results and the timeliness of test reports, and are essential for developing precise treatment plans for continazolamide under the guidance of Therapeutic Drug Monitoring (TDM). However, a review of domestic and international literature and patents reveals that none of the current methods for determining continazolamide plasma drug concentration have been validated for clinical applicability based on clinical samples, nor have their stability and matrix effects in specific populations been validated based on TDM requirements. Furthermore, isotope internal standards are difficult to obtain and are not cost-effective in conventional methods for determining continazolamide concentration, thus making conventional methods unsuitable for widespread clinical application. Summary of the Invention

[0004] To address the shortcomings of existing methods for detecting continazolamide in blood samples—namely, the lack of validation with real clinical samples, poor applicability to specific pathological matrices, and difficulty in meeting the needs of personalized treatment monitoring—this invention provides a method for determining the concentration of continazolamide in plasma. The method employs a protein precipitation pretreatment: an internal standard solution is added to the plasma sample, which is then vortexed, centrifuged, and the supernatant is used to prepare the test solution. The concentration of continazolamide is quantitatively detected using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). Furthermore, considering the needs of clinical therapeutic drug monitoring, the method's linear range and sample stability have been systematically validated. Specific plasma matrix effects in patients with chronic hepatitis, AIDS, and liver transplantation have been investigated, significantly improving detection accuracy and clinical suitability. This method can accurately support personalized continazolamide dosing and therapeutic drug monitoring, demonstrating excellent clinical application value. This method is economical, simple, rapid, and clinically applicable, while also possessing the advantages of high selectivity and specificity.

[0005] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: A method for determining the concentration of contazolamide in plasma involves treating clinical samples by adding an internal standard working solution to the plasma sample for protein precipitation, and then determining the concentration of contazolamide in the plasma using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). The method includes the following steps: Step 1: Prepare contezolam standard stock solution and quality control stock solution; dilute the standard stock solution to prepare standard curve working solutions of different concentrations; dilute the quality control stock solution to prepare quality control working solutions of different concentrations; Step 2: Dilute the standard curve working solutions of different concentrations into blank plasma (i.e., plasma matrix) to obtain standard curve samples of different concentrations; dilute the quality control working solutions of different concentrations into blank plasma to obtain quality control samples of different concentrations. Step 3: Mix the standard curve samples or quality control samples of different concentrations with the internal standard working solution, vortex and centrifuge, collect the supernatant to obtain standard curve solutions or quality control solutions of different concentrations; take a certain volume of standard curve solutions or quality control solutions of different concentrations and mix them with diluent, analyze them with UPLC-MS / MS system, plot the standard curve with the concentration of continazolamide as the X-axis and the ratio of the peak area of ​​continazolamide to the peak area of ​​the internal standard as the Y-axis; Step 4: Mix the clinical test sample with the internal standard working solution, vortex and centrifuge, collect the supernatant to obtain the clinical test sample solution; take a certain volume of the clinical test sample solution and mix it with the diluent, analyze it with the UPLC-MS / MS system, and calculate the concentration of contezolid in the clinical test sample according to the standard curve. In steps 3 and 4, the internal standard working solution is a linezolid acetonitrile solution with a concentration of 200-400 ng / mL.

[0006] In this invention, given that both continazolamide and linezolid are oxazolidinone antibiotics with similar mechanisms of action and antibacterial spectra, and that the combined use of linezolid and continazolamide is rare in clinical practice, and considering that linezolid is inexpensive, readily available, and structurally similar to continazolamide, the method for determining the concentration of continazolamide in plasma provided by this invention uses a linezolid acetonitrile solution as the internal standard working solution. Since the internal standard working solution is an organic solvent containing a certain concentration of internal standard, the organic solvent can act as a protein precipitant. The principle is to lower the dielectric constant of the solution to enhance intermolecular electrostatic attraction and to compete for water molecules, thereby disrupting the protein hydration layer, leading to a sharp drop in solubility and aggregation and sedimentation. Therefore, centrifugation ensures more complete protein precipitation in plasma, guaranteeing the uniform presence of the internal standard throughout the solution system; compared to conventional methods for detecting the concentration of continazolamide in plasma, this significantly reduces the detection cost. This invention uses quality control samples to verify the accuracy and precision of the method. The detection method has good linearity, is simple, economical, fast, and clinically applicable. It is especially suitable for conducting TDM-guided safety and efficacy studies of continazolamide in specific populations (such as those with chronic hepatitis, AIDS, and post-liver transplantation) as well as large-sample PK studies. It provides technical support for determining the target concentration range for the safety and efficacy of continazolamide and for model-guided precision medication, improving drug efficacy while reducing adverse reactions and increasing patient satisfaction. It has significant economic and social benefits and is suitable for clinical promotion.

[0007] Optionally, in step 1, the concentrations of the standard stock solution and the quality control stock solution of contezolidinone are 1.00 mg / mL, respectively.

[0008] Optionally, in step 1, the solvent used to prepare the standard stock solution and the quality control stock solution is methanol.

[0009] Optionally, in step 1, the concentrations of the standard curve working solutions of different concentrations are: 1000 ng / mL, 2500 ng / mL, 5000 ng / mL, 10000 ng / mL, 25000 ng / mL, 50000 ng / mL, 75000 ng / mL, 150000 ng / mL, and 300000 ng / mL; and the concentrations of the quality control working solutions of different concentrations are: 3000 ng / mL, 20000 ng / mL, 60000 ng / mL, 120000 ng / mL, and 240000 ng / mL.

[0010] Optionally, in step 2, the concentrations of the standard curve samples of different concentrations are 100 ng / mL, 250 ng / mL, 500 ng / mL, 1000 ng / mL, 2500 ng / mL, 5000 ng / mL, 7500 ng / mL, 15000 ng / mL, and 30000 ng / mL, respectively; and the concentrations of the quality control samples of different concentrations are 300 ng / mL, 2000 ng / mL, 6000 ng / mL, 12000 ng / mL, and 24000 ng / mL, respectively.

[0011] Optionally, in step 2, the blank plasma is the supernatant portion of venous whole blood collected by an EDTA-K2 anticoagulant tube after mixing and centrifugation.

[0012] The blank plasma used in this invention were all samples that were to be discarded after testing, collected using EDTA-K2 anticoagulant tubes at the Clinical Laboratory Center of Beijing You'an Hospital, affiliated with Capital Medical University. After tracing the source, none of the test subjects had taken continazole.

[0013] Preferably, in step 2, the preparation of blank plasma includes: collecting venous whole blood through a blood collection tube containing the anticoagulant EDTA-K2, centrifuging at 2000-3000 rpm (for example, 10 minutes), and then taking the supernatant to obtain blank plasma.

[0014] Preferably, in step 2, the standard curve working solutions of different concentrations are diluted 10 times with blank plasma to obtain standard curve samples of different concentrations; and the quality control working solutions of different concentrations are diluted 10 times with blank plasma to obtain quality control samples of different concentrations.

[0015] Preferably, in steps 3 and 4, the standard curve samples, quality control samples, or clinical test samples of different concentrations are mixed with the internal standard working solution at a volume ratio of 1:5, vortexed, and then centrifuged to collect the supernatant.

[0016] Preferably, in steps 3 and 4, the standard curve solution or quality control solution or clinical test sample of different concentrations is mixed with the diluent at a volume ratio of 1:5, centrifuged after vortexing, and then analyzed using a UPLC-MS / MS system.

[0017] Optionally, in step 3, the processing method for the standard curve samples and quality control samples of different concentrations is as follows: Take 50 μL of each of the standard curve sample and quality control sample into a 1.5 mL centrifuge tube, add 250 μL of internal standard working solution, vortex, and centrifuge at 4℃~30℃ and 10000 rpm~15000 rpm. Take 100 μL of the supernatant, add 500 μL of diluent, mix well, and then perform the determination using a UPLC-MS / MS system.

[0018] Optionally, in step 4, the clinical sample to be tested is obtained by collecting venous whole blood from the patient at different times before and after taking the medication using EDTA-K2 anticoagulant tubes, centrifuging the sample, and then storing it at -80℃ until the sample is tested.

[0019] For example, the clinical test samples of this invention are samples from patients enrolled in "an open-label study evaluating the pharmacokinetic characteristics of contazolamide tablets in HIV-infected patients with Gram-positive bacterial infections".

[0020] Optionally, in step 4, the clinical sample to be tested is processed as follows: Take 50 μL of the clinical sample to be tested into a 1.5 mL centrifuge tube, add 250 μL of internal standard working solution, vortex, and centrifuge at 4℃~30℃ (e.g., 5℃, 10℃, 15℃, 20℃ or 25℃) and 10000 rpm~15000 rpm (e.g., 11000 rpm, 12000 rpm, 13000 rpm or 14000 rpm). Take 100 μL of the supernatant, add 500 μL of diluent, mix well, and then perform the analysis using a UPLC-MS / MS system.

[0021] Optionally, the detection conditions for ultra-high performance liquid chromatography (UPLC) include: injector temperature of 10°C, column temperature of 40°C; mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is 0.1% formic acid acetonitrile solution; gradient elution is used, and the gradient elution program is as follows: First elution program: elution time is 0.00 ~ 0.20 min, and the ratio of mobile phase A to mobile phase B is 85% : 15%; Second elution procedure: elution time is 0.20 ~ 2.00 min, and the ratio of mobile phase A to mobile phase B is 85% ~ 40% : 15% ~ 60%; Third elution procedure: elution time is 2.00 ~ 2.50 min, and the ratio of mobile phase A to mobile phase B is 40% ~ 2% : 60% ~ 98%; Fourth elution program: Elution time is 2.50 ~ 3.00 min, and the ratio of mobile phase A to mobile phase B is 2%:98%; Fifth elution program: Elution time is 3.00 ~ 3.10 min, and the ratio of mobile phase A to mobile phase B is 2%~85%: 98~15%; The sixth elution procedure: elution time is 3.10 ~ 4.00 min, and the ratio of mobile phase A to mobile phase B is 85%:15%.

[0022] This invention employs gradient elution in ultra-high performance liquid chromatography, which allows for chromatographic separation by adjusting the elution intensity through changes in the mobile phase composition. This can shorten the analysis cycle, improve separation capability, improve analyte peak shape, reduce tailing, and increase detection sensitivity.

[0023] Optionally, the ultra-high performance liquid chromatography (UPLC) detection uses a WATERS ACQUITY UPLC® BEH C18 column (2.1 mm × 50 mm, 1.7 μm).

[0024] Optionally, the detection conditions for ultra-high performance liquid chromatography (UPLC) also include a gradient elution rate of 0.4 mL / min.

[0025] Optionally, the detection conditions for tandem mass spectrometry include: an electrospray ion source, a positive ion mode detection mode, a multiple ion reaction monitoring (MRM) scanning mode, a capillary voltage of 3.0 kV, a cone voltage of 30 V, a desolvation temperature of 400°C, a desolvation gas flow rate of 1000 L / h, and a cone gas flow rate of 20 L / h.

[0026] Optionally, the clinically specific populations used in the method for determining the concentration of contazolamide in plasma include patients with HIV / AIDS, patients with chronic hepatitis, and patients who have undergone liver transplantation.

[0027] The above-described technical solution of the present invention has the following advantages over the prior art: (1) The method for determining the concentration of continazolamide in plasma provided by the present invention uses the method of direct protein precipitation with internal standard working solution to process clinical samples. The sample pretreatment is simple and does not require expensive and hard-to-obtain continazolamide isotope internal standard. It has the advantages of simple operation, fast speed, small sample volume and low cost. (2) Regarding the clinical applicability of the method for determining the concentration of continazolamide in plasma provided by the present invention, since the time of continazolamide’s market launch is short and the target concentration range of its safety and efficacy is still unclear, the present invention establishes a method for determining the concentration of continazolamide in plasma that is suitable for therapeutic drug monitoring and large-scale PK studies based on drug concentration data in Phase II and Phase III clinical trials. Clinical samples can be pre-processed without dilution, which improves detection efficiency and ensures the timeliness of detection reports. (3) The method for determining the concentration of continazolamide in plasma provided by this invention has been used to investigate the plasma matrix effect in patients with AIDS and chronic hepatitis who are receiving antiviral drugs and in patients after liver transplantation who are receiving immunosuppressant therapy. The stability of the samples has also been investigated in conjunction with the clinical sampling, preservation and transportation environment. This method has improved the accuracy of the determination of the concentration of continazolamide in plasma of patients with AIDS, chronic hepatitis and after liver transplantation, and provides technical support for TDM and PK studies of continazolamide in the above populations. (4) The method for determining the concentration of contazolamide in plasma provided by the present invention has the advantages of being economical, rapid, accurate, highly sensitive, clinically applicable, and easy to promote in clinical practice. Attached Figure Description

[0028] Figure 1 These are chromatograms of blank plasma, plasma containing internal standard, plasma containing analyte, and plasma containing both internal standard and analyte in this invention. Specifically, A is a chromatogram of blank plasma, B is a chromatogram of blank plasma with internal standard added, C is a chromatogram of blank plasma with contazolamide added, and D is a chromatogram of blank plasma with contazolamide added after precipitation treatment with internal standard working solution.

[0029] Figure 2 This is a distribution diagram of the measurement results of clinical samples in this invention.

[0030] Figure 3 Concentration-time curves of contezolid in the plasma of nine HIV patients. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, 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 pertains. In the event of any conflict, this specification shall prevail.

[0033] The reagents, materials, instruments, and equipment information in the following examples are shown below.

[0034] 1. Reagents and consumables: Acetonitrile, methanol, and formic acid were all chromatographic grade (Fisher); water was Watson distilled water; linezolid (purity: 99.90%, batch number: A20D11L135202, purchased from Shanghai Yuanye Biotechnology Co., Ltd.); continazolamide (purity: 99.92%, batch number: 5735-20-001, provided by Shanghai Mengke Pharmaceutical Co., Ltd.); chromatographic column ACQUITY UPLC®BEH C18 (2.1 mm × 50 mm, 1.7 μm; purchased from Waters Corporation, USA).

[0035] 2. Instruments and equipment: ACQUITY® 2695 / ACQUITY® TQD ultra-high performance liquid chromatography-tandem mass spectrometry system (Waters Corporation, USA); DV215CD analytical balance (OHAUS); data processing software: Mass Lynx V4.1 (Waters Corporation, USA).

[0036] Blank plasma (i.e., plasma matrix): Blank plasma is obtained from EDTA-K2 anticoagulated whole blood of patients in the outpatient department of the Clinical Laboratory Center of Beijing You'an Hospital, Capital Medical University, used for routine blood tests. After centrifugation, the supernatant is collected to obtain blank plasma. The patient sources of blank plasma include the following: 1) Blank plasma from patients with liver disease: plasma from patients diagnosed with chronic hepatitis B or hepatitis C, used in this invention for matrix effect assessment; 2) Plasma from patients with AIDS: plasma from patients diagnosed with AIDS or HIV infection, used in this invention for matrix effect assessment; 3) Plasma from patients after liver transplantation: plasma from patients diagnosed after liver transplantation, used in this invention for matrix effect assessment; 4) Plasma from healthy individuals: plasma from hospital staff undergoing physical examinations (age <50 years), used for matrix effect assessment; 5) Other sources: plasma from patients without diagnoses of "viral hepatitis," "AIDS," "HIV infection," or "liver transplantation," mainly used for the preparation of calibration curve samples and quality control samples in precision and accuracy verification.

[0037] Clinical test samples: Clinical test samples were obtained from patients diagnosed with "AIDS" or "HIV infection", with a contezolam treatment regimen of "800 mg q12 h". Samples were collected before medication, and at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 12 h after medication, and 30 min before the next dose 48 h after medication (steady-state trough concentration, C0). min ) and 2.0–2.5 (steady-state peak concentration, C) after the next dose 48 hours after administration. max Whole blood from the vein is transferred to an EDTA-K2 anticoagulant tube, mixed, and centrifuged at 1000 rpm to 3000 rpm. The supernatant plasma is collected to obtain the clinical sample to be tested. The sample is stored at -80°C until it is tested.

[0038] 3. Liquid Chromatography Conditions Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: 0.1% formic acid acetonitrile solution; Chromatographic column: ACQUITY UPLC® BEH C18 (2.1 mm × 50 mm, 1.7 μm); Sample acquisition time: 4.00 min; Autosampler temperature set to 10℃; Column temperature: 40℃; Injection volume: 10.0 μL. Gradient elution was used, and the elution program is shown in Table 1. Table 1:

[0039] 4. Mass spectrometry conditions The ion source was an electrospray ionization source, with positive ion mode and multiple ion reaction monitoring (MRM) mode for scanning; the nebulizing gas was nitrogen with a flow rate of 1000 L / h; the collision gas was argon with a flow rate of 20 L / h. The ion pairs and mass spectrometry parameters of the analyte (contezolid) and internal standard (linezolid) are shown in Table 2. Table 2:

[0040] Example 1 A method for determining the concentration of contazolamide in plasma, comprising the following steps.

[0041] Step S1, solution preparation and sample processing, are detailed below.

[0042] Preparation of contaminated standard stock solution: Accurately weigh a certain amount of contaminated standard, prepare a standard stock solution with methanol at a concentration of 1.00 mg / mL, and store it in a -80℃ refrigerator for later use.

[0043] Preparation of contaminated oxadoxime stock solution: Accurately weigh a certain amount of contaminated oxadoxime standard, prepare a stock solution with a concentration of 1.00 mg / mL using methanol, and store it in a -80℃ refrigerator for later use.

[0044] Preparation of internal standard working solution: Accurately weigh a certain amount of linezolid standard, prepare an internal standard stock solution with acetonitrile with a concentration of 1.00 mg / mL, and store it in a -80℃ refrigerator for later use; take an appropriate amount of internal standard stock solution and prepare an internal standard working solution with acetonitrile with a concentration of 200 ng / mL~400 ng / mL.

[0045] Preparation of working solutions for standard curves and quality control samples at different concentrations: Accurately pipette 300 μL of the continazolamide standard stock solution and dilute with 700 μL of water to prepare the highest concentration working solution. Then, dilute with water sequentially from high to low concentration to obtain nine different concentrations of standard curve working solutions: 300,000 ng / mL, 150,000 ng / mL, 75,000 ng / mL, 50,000 ng / mL, 25,000 ng / mL, 10,000 ng / mL, 5,000 ng / mL, 2,500 ng / mL, and 1,000 ng / mL. Accurately pipette 240 μL of the continazolamide quality control stock solution and dilute with 760 μL of water to prepare the highest concentration working solution. Then, dilute with water sequentially from high to low concentration to obtain five different concentrations of quality control working solutions: 240,000 ng / mL, 120,000 ng / mL, 60,000 ng / mL, 20,000 ng / mL, and 20,000 ng / mL. ng / mL, 3000 ng / mL.

[0046] Step S2: Preparation of standard curve samples and quality control samples of different concentrations: Dilute the standard curve working solutions and quality control working solutions of different concentrations with plasma matrix (i.e., blank plasma) by a dilution factor of 10 times.

[0047] Specifically, 10.0 μL of standard curve working solutions and quality control working solutions of different concentrations were added to 90.0 μL of plasma matrix (containing EDTA-K2) to obtain standard curve samples and quality control samples of different concentrations. The concentrations of the standard curve samples were 30000 ng / mL, 15000 ng / mL, 7500 ng / mL, 5000 ng / mL, 2500 ng / mL, 1000 ng / mL, 500 ng / mL, 250 ng / mL, and 100 ng / mL, respectively; the concentrations of the quality control samples were 24000 ng / mL, 12000 ng / mL, 6000 ng / mL, 2000 ng / mL, and 300 ng / mL, respectively.

[0048] Step S3: Mix standard curve samples or quality control samples of different concentrations with internal standard working solution, vortex, centrifuge, and take the supernatant to obtain standard curve solutions or quality control solutions of different concentrations; wherein, the internal standard is linezolid, and the organic solvent for preparing the internal standard working solution is acetonitrile solution. Plasma sample processing and standard curve plotting for chromatographic analysis: Plasma samples included standard curve samples and quality control samples of different concentrations, as well as clinical test samples. The same processing method was used for all plasma samples: 50.0 μL of plasma sample was placed in a 1.5 mL centrifuge tube (EP tube), and 250 μL of internal standard working solution was added. The mixture was vortexed for 1 min, centrifuged at 12000 rpm for 5 min at 4℃, and 100 μL of the supernatant was placed in a 1.5 mL centrifuge tube (EP tube). 500 μL of ultrapure water (i.e., diluent) was added for dilution. After mixing, the mixture was analyzed using a UPLC-MS / MS system with an injection volume of 10.0 μL. A standard curve was plotted with drug concentration as the X-axis and the ratio of drug peak area to internal standard peak area as the Y-axis.

[0049] Step S4: Mix the clinical test sample with the internal standard working solution, vortex to completely precipitate the protein, then centrifuge to completely separate the precipitate and the supernatant, and take the supernatant to obtain the clinical test sample solution. After the clinical test sample solution is mixed with the diluent, it is analyzed using a UPLC-MS / MS system, and the drug concentration in the clinical test sample is calculated according to the standard curve.

[0050] Methodological validation of the assay method This invention was validated according to the methodology outlined in Part IV of the Chinese Pharmacopoeia (2020 Edition) – 9012 Validation Guidelines for Quantitative Analysis Methods for Biological Samples.

[0051] (1) Exclusivity: Take 50.0 μL of blank plasma into a 1.5 mL centrifuge tube (EP tube), add 250.0 μL of acetonitrile, vortex for 1 min, centrifuge at 12000 rpm and 4℃ for 5 min, take 100.0 μL of the supernatant into a 1.5 mL centrifuge tube (EP tube), dilute with 500.0 μL of ultrapure water, mix well, and analyze using a UPLC-MS / MS system to obtain the chromatogram of the blank plasma, as shown below. Figure 1 As shown in Figure A.

[0052] Take 50.0 μL of blank plasma, and following the procedure in step 3, "Paleolone Sample Processing," add 250 μL of internal standard working solution. Vortex for 1 min, centrifuge at 12000 rpm for 5 min at 4℃, and collect 100.0 μL of the supernatant in a 1.5 mL centrifuge tube. Dilute with 500.0 μL of ultrapure water, mix well, and analyze using a UPLC-MS / MS system. Obtain the chromatogram of the blank plasma with added internal standard, as shown below. Figure 1 As shown in B.

[0053] Take 10.0 μL of the working solution of the standard curve of continazolamide with a concentration of 1000 ng / mL, add 90 μL of blank plasma, vortex to mix, add 500.0 μL of acetonitrile, vortex for 1 min, centrifuge at 12000 rpm and 4℃ for 5 min, take 100 μL of the supernatant into a 1.5 mL centrifuge tube, add 500.0 μL of ultrapure water to dilute, mix well, and analyze using a UPLC-MS / MS system to obtain the chromatogram of blank plasma containing continazolamide, as shown below. Figure 1 As shown in C.

[0054] Take 10.0 μL of the working solution for the standard curve of continazolamide (1000 ng / mL), add 90 μL of blank plasma, vortex to mix, then add 500 μL of internal standard working solution, vortex for 1 min, centrifuge at 12000 rpm for 5 min at 4℃, take 100 μL of the supernatant and mix with 500 μL of ultrapure water, then analyze using a UPLC-MS / MS system. The chromatogram of blank plasma containing continazolamide after precipitation with internal standard working solution is shown below. Figure 1 As shown in D.

[0055] (2) Standard curve and lower limit of quantitation: Standard curve samples of different concentrations were taken and, according to the "processing of plasma samples", the ratio of the peak area of ​​contazolamide to the peak area of ​​the internal standard (y) was used as the ordinate and the concentration of contazolamide (x) was used as the abscissa. The least squares method was used for fitting, with a weighting coefficient of 1 / χ². The sample with the lowest concentration of the standard curve was set as the lower limit of quantitation (LLOQ), i.e., LLOQ was 100 ng / mL.

[0056] Standard curve evaluation: Within 2 days, following step S3, three standard curves were freshly prepared and plotted for evaluation. The evaluation of the three standard curves showed that contezoline exhibited good linearity (r² > 0.99) in the range of 100.0 ng / mL to 30000.0 ng / mL.

[0057] (3) Precision (RSD) and accuracy (RE): Precision and accuracy were assessed using five quality control samples at five different concentrations and the lower limit of quantitation (LLOQ) of 100 ng / mL. Three batches were completed over at least three days, with six samples per batch for each concentration. The results are shown in Table 3.

[0058] Table 3:

[0059] As shown in Table 3, the relative error of accuracy within a batch (i.e., within a batch) is 1.22%~3.55%, and the relative standard deviation of precision is 2.88%~5.28%; the relative error of accuracy between batches (between batches) is -2.49%~1.29%, and the relative standard deviation of precision is 2.89%~6.33%.

[0060] (4) Matrix effect: Subjects of investigation: This invention investigated healthy individuals, chronic hepatitis patients receiving antiviral therapy, HIV patients receiving antiviral therapy, and patients receiving immunosuppressant therapy after liver transplantation.

[0061] Obtaining blank plasma from different populations: For each population, EDTA-K2 anticoagulated whole blood was collected from 6 different individuals, and after centrifugation, 6 blank plasma samples were obtained for the corresponding population. Sample preparation for examination: For the first group (Set A), take 10 μL of each of the three different concentrations (3000 ng / mL, 60000 ng / mL and 240000 ng / mL), add 90 μL of blank plasma, vortex for 1 min, process the samples according to step S3, and inject for detection. The peak area ratio of contezolid to internal standard is recorded as SA. For the second group (Set B), take 10 μL of each of the three working solutions of quality control samples with different concentrations (3000 ng / mL, 60000 ng / mL and 240000 ng / mL), add 90 μL of acetonitrile solution, vortex for 1 min, process the samples according to step S3, and perform sample injection and detection. The peak area ratio of contezolid to internal standard is recorded as SB. Internal standard normalized matrix effect factor (MF) i Calculation of MF: i =SA / SB.

[0062] The results of the investigation of matrix effects are shown in Table 4.

[0063] Table 4:

[0064] (5) Stability: In terms of stability study design, this invention considers the influencing factors throughout the entire process from clinical sample collection to laboratory testing, including storage, transportation, and reporting. It investigates the stability of samples under different matrices and temperatures to improve the clinical applicability of this methodology and ensure the accuracy of test results.

[0065] In terms of short-term stability studies, considering the possible influencing factors at each stage of the clinical sample collection process to report issuance, the stability of samples in whole blood (EDTA-K2) and plasma (pre-treated samples) at 4°C and room temperature (RT, 22°C~26°C) for different storage times was investigated. In addition, the stability of pre-treated plasma samples stored at 4°C and autosampler (10°C) for different storage times was investigated.

[0066] Specifically, for the preparation of whole blood samples, take 250 μL each of the low-value quality control working solution (3000 ng / mL) and the high-value quality control working solution (24000 ng / mL), add 2250 μL of EDTA-K2 anticoagulated whole blood, vortex for 1 min, and then aliquot the whole blood samples into 50 μL portions. Store them according to the stability study conditions. Perform three replicates for each concentration. When the samples are ready for testing, process them according to step S3 and then perform the analysis.

[0067] Specifically, for plasma (samples before pretreatment), take 500 μL each of the low-value quality control working solution (3000 ng / mL) and the high-value quality control working solution (24000 ng / mL), add 4500 μL of blank plasma, vortex for 1 min, and then aliquot the plasma samples into 50 μL portions. Store the samples according to the stability study conditions, and perform three replicates for each concentration. When the samples are ready for testing, process them according to step S3 and then perform the analysis.

[0068] Specifically, for plasma (pretreated samples), 100 μL each of the low-value quality control working solution (3000 ng / mL) and the high-value quality control working solution (24000 ng / mL) were added, along with 900 μL of blank plasma. After vortexing for 30 s, 2500 μL of internal standard working solution was added, and the mixture was vortexed for 1 min. The supernatant was then collected. For stability testing at 4℃, the supernatant was aliquoted into 100 μL portions, with three replicates per concentration, according to the stability testing time. These were stored at 4℃. When samples were ready for testing, 500 μL of ultrapure water was added, mixed, centrifuged, and then the samples were analyzed. For stability testing of the autosampler (10℃), 100 μL of the supernatant was added, mixed, centrifuged, and then placed in the autosampler for analysis according to the stability testing time.

[0069] Specifically, the stability test samples were all measured using the method described in Example 1. The results are shown in Table 5.

[0070] Table 5:

[0071] Note: T0 indicates a newly collected sample.

[0072] As shown in Table 5, the stability study results indicate that contezolidinone is stable in whole blood and plasma for 7 days, and the treated samples are stable at 4°C and in an autosampler (10°C) for 72 hours, which can ensure the orderly implementation of the therapeutic drug monitoring program.

[0073] (6) Clinical application: This invention validated the clinical applicability of the established method for measuring continazolamide plasma concentration through clinical sample testing. Clinical samples were obtained from 9 patients diagnosed with AIDS or HIV infection, treated with continazolamide at a dose of 800 mg every 12 hours. Steady-state trough concentration (C0.05) was collected before and at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 12 h after medication, and within 30 minutes before the next dose 48 hours after medication. min ) and 2.0–2.5 (steady-state peak concentration, C) after the next dose 48 hours after administration. max Collect whole blood from the vein into an EDTA-K2 anticoagulant tube, mix well, centrifuge, collect the supernatant plasma, and store at -80°C until sample testing.

[0074] The concentration of contaminated zolpidem was determined in clinical samples from nine HIV patients who had taken contaminated zolpidem, using the method described in Example 1. Table 6 shows the plasma concentrations of contaminated zolpidem in the nine patients who had taken contaminated zolpidem. Figure 2 This is a distribution chart of the concentration of contazolamide in the plasma of 9 patients who took contazolamide (i.e., clinical samples). The data source is the sampling results of 108 clinical samples from the 9 patients. The drug concentration measurement results are plotted on the vertical axis, and the 108 data points are randomly ordered on the horizontal axis. Figure 3 The concentration-time curves of contezolid in the plasma of nine HIV patients are shown. Specifically, the graph is a line graph obtained by using the data in Table 6, with the concentration results of the same patient at different time points as the x-axis and the individual patient as the y-axis.

[0075] Table 6:

[0076] NA*: Concentration below the limit of quantitation.

[0077] As shown in Table 6, the sample concentration results analysis shows that 96.30% (78 / 81) of the sample concentrations are within the linear range of the method of this invention (100 ng / mL~30000 ng / mL).

[0078] Depend on Figure 3It can be seen that the concentration of contaminated benzodiazepine varies considerably among individuals, highlighting the necessity of TDM-based personalized medication guidance and further pharmacokinetic (PK) studies. Therefore, the method for determining plasma contaminated benzodiazepine concentration provided by this invention can provide strong technical support for further TDM-guided safety and efficacy studies of contaminated benzodiazepine and the implementation of PK-guided precision medication models.

[0079] In summary, this invention provides a method for determining the concentration of contaminated plasma (CPD) in patients with chronic hepatitis, HIV / AIDS, and post-liver transplantation, suitable for monitoring CPD concentrations during CPD treatment. The use of linezolid as an internal standard significantly reduces the detection cost of this methodology. This method exhibits good linearity, simplicity, economy, speed, and strong clinical applicability. It is particularly suitable for conducting TDM-guided safety and efficacy studies of CPD in specific populations (such as those with chronic hepatitis, HIV / AIDS, and post-liver transplantation), as well as large-scale pharmacokinetic (PK) studies. This provides technical support for determining the target concentration range for CPD safety and efficacy and for model-guided precision medication, improving drug efficacy while reducing adverse reactions and increasing patient satisfaction. It offers significant economic and social benefits and is suitable for clinical application.

[0080] The above descriptions are merely some preferred embodiments of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for determining the concentration of contazolamide in plasma, characterized in that, Clinical samples were treated by adding an internal standard working solution to the plasma sample for protein precipitation. The concentration of contazobium in the plasma was determined by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). The determination method includes the following steps: Step 1: Prepare contezolam standard stock solution and quality control stock solution; dilute the standard stock solution to prepare standard curve working solutions of different concentrations; dilute the quality control stock solution to prepare quality control working solutions of different concentrations; Step 2: Dilute the standard curve working solutions of different concentrations into blank plasma to obtain standard curve samples of different concentrations; dilute the quality control working solutions of different concentrations into blank plasma to obtain quality control samples of different concentrations. Step 3: Mix the standard curve samples or quality control samples of different concentrations with the internal standard working solution, vortex and centrifuge, collect the supernatant to obtain standard curve solutions or quality control solutions of different concentrations; take a certain volume of standard curve solutions or quality control solutions of different concentrations and mix them with diluent, analyze them with UPLC-MS / MS system, plot the standard curve with the concentration of continazolamide as the X-axis and the ratio of the peak area of ​​continazolamide to the peak area of ​​the internal standard as the Y-axis; Step 4: Mix the clinical test sample with the internal standard working solution, vortex and centrifuge, collect the supernatant to obtain the clinical test sample solution; take a certain volume of the clinical test sample solution and mix it with the diluent, analyze it with the UPLC-MS / MS system, and calculate the concentration of contezolid in the clinical test sample according to the standard curve. In steps 3 and 4, the internal standard working solution is a linezolid acetonitrile solution with a concentration of 200-400 ng / mL.

2. The method for determining the concentration of contazolamide in plasma according to claim 1, characterized in that, In step 1, the concentrations of the standard stock solution and the quality control stock solution of contazolamide are 1.00 mg / mL, and the solvent used to prepare the standard stock solution and the quality control stock solution is methanol. In step 1, the concentrations of the standard curve working solutions of different concentrations are: 1000 ng / mL, 2500 ng / mL, 5000 ng / mL, 10000 ng / mL, 25000 ng / mL, 50000 ng / mL, 75000 ng / mL, 150000 ng / mL, and 300000 ng / mL; the concentrations of the quality control working solutions of different concentrations are: 3000 ng / mL, 20000 ng / mL, 60000 ng / mL, 120000 ng / mL, and 240000 ng / mL. In step 2, the concentrations of the standard curve samples at different concentrations are: 100 ng / mL, 250 ng / mL, 500 ng / mL, 1000 ng / mL, 2500 ng / mL, 5000 ng / mL, 7500 ng / mL, 15000 ng / mL, and 30000 ng / mL; the concentrations of the quality control samples at different concentrations are: 300 ng / mL, 2000 ng / mL, 6000 ng / mL, 12000 ng / mL, and 24000 ng / mL. In step 4, the diluent includes ultrapure water.

3. The method for determining the concentration of contazolamide in plasma according to claim 1, characterized in that, In step 2, the blank plasma is the supernatant portion of venous whole blood collected by EDTA-K2 anticoagulant tube after mixing and centrifugation; In step 4, the clinical test sample is obtained by collecting venous whole blood from the patient at different times before and after taking the medication using EDTA-K2 anticoagulant tubes, centrifuging the sample, and then storing it at -80℃ until the sample is tested.

4. The method for determining the concentration of contazolamide in plasma according to claim 2, characterized in that, In step 2, the preparation of blank plasma includes: collecting venous whole blood through a blood collection tube containing the anticoagulant EDTA-K2, centrifuging at 2000 rpm to 3000 rpm, and then taking the supernatant to obtain blank plasma.

5. The method for determining the concentration of contazolamide in plasma according to any one of claims 1-4, characterized in that, In step 2, the standard curve working solutions of different concentrations are diluted 10 times with blank plasma to obtain standard curve samples of different concentrations; the quality control working solutions of different concentrations are diluted 10 times with blank plasma to obtain quality control samples of different concentrations. In steps 3 and 4, the standard curve samples, quality control samples, or clinical test samples of different concentrations are mixed with the internal standard working solution at a volume ratio of 1:5, vortexed, and then centrifuged to collect the supernatant. In steps 3 and 4, the standard curve solutions or quality control solutions or clinical test samples of different concentrations are mixed with the diluent at a volume ratio of 1:5, centrifuged after vortexing, and then analyzed using a UPLC-MS / MS system.

6. The method for determining the concentration of contazolamide in plasma according to claim 5, characterized in that, In step 3, the processing method of the standard curve samples and quality control samples of different concentrations is as follows: Take 50 μL of each standard curve sample and quality control sample into a 1.5 mL centrifuge tube, add 250 μL of internal standard working solution, vortex, and centrifuge at 4℃~30℃ and 10000 rpm~15000 rpm. Take 100 μL of supernatant, add 500 μL of diluent, mix well, and then perform the determination using a UPLC-MS / MS system.

7. The method for determining the concentration of contazolamide in plasma according to claim 1, characterized in that, The detection conditions for the ultra-high performance liquid chromatography (UHPLC) include: injector temperature of 10℃, column temperature of 40℃; mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is 0.1% formic acid acetonitrile solution; gradient elution is used, and the gradient elution program is as follows: First elution program: elution time is 0.00 ~ 0.20 min, and the ratio of mobile phase A to mobile phase B is 85% : 15%; Second elution procedure: elution time is 0.20 ~ 2.00 min, and the ratio of mobile phase A to mobile phase B is 85%~40% : 15%~60%; Third elution procedure: elution time is 2.00 ~ 2.50 min, and the ratio of mobile phase A to mobile phase B is 40% ~ 2% : 60% ~ 98%; Fourth elution program: Elution time is 2.50 ~ 3.00 min, and the ratio of mobile phase A to mobile phase B is 2%: 98%; Fifth elution program: Elution time is 3.00 ~ 3.10 min, and the ratio of mobile phase A to mobile phase B is 2%~85%: 98~15%; The sixth elution program: elution time is 3.10 ~ 4.00 min, and the ratio of mobile phase A to mobile phase B is 85%: 15%.

8. The method for determining the concentration of contazolamide in plasma according to claim 2, characterized in that, The detection conditions for the ultra-high performance liquid chromatography also include: a WATERS ACQUITY UPLC® BEH C18 column; and a gradient elution rate of 0.4 mL / min.

9. The method for determining the concentration of contazolamide in plasma according to claim 1, characterized in that, The detection conditions for the tandem mass spectrometry include: an electrospray ion source, a positive ion mode, a multi-ion reaction monitoring mode, a capillary voltage of 3.0 kV, a cone voltage of 30 V, a desolvation temperature of 400°C, a desolvation gas flow rate of 1000 L / h, and a cone gas flow rate of 20 L / h.

10. The method for determining the concentration of contazolamide in plasma according to claim 1, characterized in that, The method for determining the concentration of contazolamide in plasma is applicable to specific clinical populations including patients with HIV / AIDS, patients with chronic hepatitis, and patients who have undergone liver transplantation.

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