A detection kit and detection method for triazole antifungal drugs
The detection process for triazole antifungal drugs is simplified by using in-situ ionization mass spectrometry and isotope dilution mass spectrometry, which solves the problems of complexity, time consumption and insufficient accuracy of existing methods, and realizes rapid and accurate drug concentration detection, supporting the development of personalized dosing regimens.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- PURSPEC TECH (CHINA) LTD
- Filing Date
- 2023-12-07
- Publication Date
- 2026-06-30
Smart Images
Figure SMS_1 
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Abstract
Description
Technical Field
[0001] This application relates to the technical field of clinical therapeutic drug monitoring, specifically to a detection kit and detection method for triazole antifungal drugs. Background Technology
[0002] Drugs can prevent, treat, and diagnose diseases, and are closely related to the occurrence and development of diseases. Drugs are absorbed into the bloodstream, redistributed to tissues and organs, and ultimately excreted from the body through liver and kidney metabolism. Drugs monitored clinically include psychotropic drugs, immunosuppressants, cardiovascular drugs, antitumor drugs, antifungal drugs, and antibiotics.
[0003] The concentration of antifungal drugs in the body is closely related to clinical efficacy and adverse reactions. Many drugs require real-time monitoring due to their narrow therapeutic range and significant side effects. By measuring the blood concentration or other body fluid concentration of the therapeutic drug in patients, individualized dosing regimens can be formulated based on pharmacokinetic principles and calculation methods to improve efficacy and reduce adverse reactions, achieving the goal of effective and safe treatment.
[0004] Currently, the mainstream detection method for triazole antifungal drugs is liquid chromatography-mass spectrometry. This method requires professional operators. It involves centrifuging to obtain plasma, adding organic reagents and internal standard reagents, centrifuging the precipitated protein again to obtain the supernatant, and establishing a standard curve. When testing actual samples, the established curve needs to be calibrated. Moreover, the pretreatment process is relatively complex and time-consuming, making it unsuitable for on-site timely testing. Other methods, such as immunoassay and luminescence assay, are prone to interference from analogues, resulting in false positive results and poor accuracy. Summary of the Invention
[0005] To quickly and conveniently detect the concentration of triazole antifungal drugs and improve the accuracy of the detection method, this application provides a detection kit and detection method for triazole antifungal drugs.
[0006] In one aspect, this application provides a detection kit for triazole antifungal drugs, including an in situ ionization kit and an isotope internal standard working solution; wherein the isotope internal standard working solution is diluted with acetonitrile, isopropanol and ammonium acetate in a volume ratio of 100:(5-10):(1-5).
[0007] The kit provided in this application is based on the principle of isotope dilution mass spectrometry (IDMS) for quantitative detection. That is, a concentrated isotope with the same molecular structure as the analyte is used as a diluent. After being thoroughly mixed with the sample, mass spectrometry is used for detection. The concentration of the analyte in the sample is obtained by combining the ratio of isotope abundance and the concentration of the known isotope. This method is sensitive and accurate, and can reduce matrix effects, reduce pretreatment operation errors and system errors.
[0008] This application uses acetonitrile, isopropanol, and ammonium acetate in the above-mentioned volume ratio as diluents to prepare an isotope internal standard working solution for the detection of triazole antifungal drugs, which can effectively improve the accuracy and precision of the detection method.
[0009] Preferably, the diluent is acetonitrile, isopropanol, and ammonium acetate in a volume ratio of 100:(7-10):(1-3).
[0010] In one specific implementation, the volume ratio of acetonitrile, isopropanol, and ammonium acetate can be 100:5:1, 100:7:1, 100:10:1, 100:5:3, 100:7:3, 100:10:3, 100:5:5, 100:7:5, or 100:10:5.
[0011] In some specific implementations, the volume ratio of acetonitrile, isopropanol, and ammonium acetate can also be 100:5:(1-3), 100:7:(1-3), 100:10:(1-3), 100:(7-10):3, 100:(5-10):3, 100:10:(1-5), 100:(7-10):5, 100:(5-10):5, or 100:10:(1-5).
[0012] Experimental analysis shows that by using acetonitrile, isopropanol, and ammonium acetate in the above weight ratio as diluents to prepare the isotope internal standard working solution, the accuracy and precision of the detection method can be effectively improved.
[0013] Preferably, the in-situ ionization reagent kit includes a shell, a paper core, a capillary tube, and a steel ball; the steel ball is embedded in a groove in the upper shell; the capillary tube is inserted in the middle of the paper core, which serves as a sample carrier; the paper core is clamped and fixed by the upper and lower shells, which have a snap-fit design.
[0014] Preferably, the capillary is inserted to a depth of 0.8-1.2 mm in the middle of the paper core.
[0015] The in-situ ionization reagent kit consists of a shell, paper core, capillary, and steel ball, integrating chromatographic separation and sample preparation functions. This significantly reduces sample analysis time and is more convenient and energy-efficient. The steel ball is embedded in a groove in the upper shell, providing voltage conduction. The capillary is inserted into the middle of a long strip of paper core to a depth of approximately 1 mm, serving as a sample carrier. The paper core with the capillary is clamped and fixed by the upper and lower shells, which have a snap-fit design, providing support and protection. When liquid is dropped onto the paper core, the liquid solvent enters the capillary, forming an electrospray under high voltage, which then enters the detector for analysis.
[0016] Preferably, the triazole antifungal drug is selected from any one or more of voriconazole, fluconazole, isaconazole, and posaconazole.
[0017] Preferably, the triazole antifungal drug is voriconazole, and the isotope internal standard working solution is voriconazole-D3 at a concentration of 0.05-50 μg / mL.
[0018] Furthermore, the triazole antifungal drug is voriconazole, and the isotope internal standard working solution is voriconazole-D3 at a concentration of 0.1-10 μg / mL.
[0019] Voriconazole-D3 (CAS#:1217661-14-7) was used as the isotopic internal standard working solution. After being thoroughly mixed with a fixed volume of the test sample, it was added to the detection kit and inserted into a portable mass spectrometry system to achieve electrospray ionization of the sample. The sample was then analyzed by the mass spectrometry system to obtain the ratio of the signal peak areas of voriconazole and voriconazole-D3. Combined with the actual concentration of voriconazole-D3, the concentration of voriconazole in the sample was calculated from the data.
[0020] Secondly, this application provides a method for detecting triazole antifungal drugs, using the above-mentioned detection kit, comprising the following steps:
[0021] The sample to be tested is placed in the isotope internal standard working solution and vortexed to mix thoroughly. The mixed liquid is then dropped onto the in-situ ionization reagent kit. After standing for 5-30 seconds, the sample is inserted into a CELL portable mass spectrometer to achieve electrospray ionization. The sample is then analyzed by the mass spectrometry system to obtain the ratio of the signal peak area of the triazole antifungal drug to that of the isotope, and the content of the triazole antifungal drug in the sample is calculated.
[0022] Preferably, the settling time is 5-15 seconds.
[0023] Experimental analysis shows that, during the detection process, adding the mixed liquid dropwise onto the in-situ ionization reagent kit and allowing it to stand for a certain period of time can effectively improve the accuracy and precision of the detection method.
[0024] Preferably, when the triazole antifungal drug is voriconazole, the mass spectrometer analysis conditions are as follows: voriconazole detection mode: positive mode, precursor ion: m / z: 350.1, quantitative ion m / z: one or more of 127.1, 224.0, and 281.1, ion selection energy: 0.5~5 V, collision-induced dissociation energy: 1-4 V;
[0025] Voriconazole-D3: Detection mode: positive mode, precursor ion m / z: 353.1, quantitative ion m / z: one or more of 127.1, 224.0, and 284.1, ion selection energy: 0.5-5V, collision-induced dissociation energy: 1-4V.
[0026] Meanwhile, when using the above-mentioned detection kit and detection method, in the single isotope internal standard quantitative method, the concentration of the isotope internal standard is known. Based on the fact that the analyte and the isotope-labeled analyte have the same properties, including ionization efficiency, fragmentation efficiency, matrix effect, etc., quantification is then performed using the ratio of the peak area of the fragments of the analyte and the isotope internal standard, without the need to establish a calibration curve. In the multi-isotope internal standard quantitative method, two or more isotope internal standards are used, and the concentration of the isotope internal standards covers the entire linear range. During analysis and detection, two or more isotopes are used to establish two-point or multi-point calibration curves, and the concentration of the analyte is calculated based on the linear equation. This allows the establishment of the calibration curve and the quantitative analysis of the analyte to be completed simultaneously, greatly reducing system bias and eliminating the need for periodic calibration of the calibration curve.
[0027] In summary, the technical solution of this application has the following effects:
[0028] The detection kit provided in this application is based on in-situ ionization mass spectrometry. The in-situ ionization kit, combined with a portable mass spectrometry analysis system, ensures accuracy and sensitivity while avoiding false positives caused by structural similarities between metabolites and drugs. Furthermore, it is simple to operate, can be performed anytime, and provides results within 5 minutes, which is beneficial for rapid development of individualized dosing regimens and metabolic kinetic studies in clinical practice.
[0029] The detection kit and detection procedure developed in this application do not require complex pretreatment or professional mass spectrometry background knowledge, enabling instant analysis and exhibiting good accuracy and precision. Attached Figure Description
[0030] Figure 1 This is the in-situ ionization reagent kit in Example 1 of this application.
[0031] Figure 2 This is the operational procedure for the voriconazole detection method in Example 1 of this application. Detailed Implementation
[0032] The present application will be further described in detail below with reference to embodiments, comparative examples and performance test results. These embodiments should not be construed as limiting the scope of protection claimed in this application. Example
[0033] Examples 1-7
[0034] Examples 1-7 provide a method for detecting voriconazole.
[0035] The difference between the above embodiments is that the volume ratios of acetonitrile, isopropanol, and ammonium acetate in the diluent are different, as shown in Table 1.
[0036] The in-situ ionization reagent kit used in the examples is as follows: Figure 1 As shown, the device includes a shell, paper core, capillary tube, and steel ball, integrating chromatographic separation and pretreatment functions. This greatly reduces sample analysis time and is more convenient and energy-efficient. The steel ball is embedded in a groove in the upper shell, providing voltage conduction. The capillary tube is inserted into the middle of the long strip paper core to a depth of about 1 mm, and the paper core serves as a sample carrier. The paper core with the capillary tube inserted is clamped and fixed by the upper and lower shells with a snap-fit design, and the shells provide support and protection. When liquid is dropped onto the paper core, the liquid solvent enters the capillary tube, and under the drive of high voltage, it forms an electrospray, which then enters the detector for analysis.
[0037] The CELL portable mass spectrometer used in this embodiment is from Beijing Qingpu Technology Co., Ltd. (length × width × height = 35cm × 25cm × 15cm, weight 8.5kg, power ≤100W, mainly composed of a discontinuous atmospheric pressure interface, a small vacuum pump, a linear ion trap mass analyzer, and an ion detector, etc.)
[0038] The steps of the voriconazole detection method in the above embodiments are as follows: Figure 2 As shown, specifically:
[0039] S1: According to Table 1, prepare the corresponding diluent. Weigh 0.5012 mg of voriconazole-D3 reference standard, dissolve it in 100 ml of diluent, and take 10 ml to dilute it tenfold to prepare a voriconazole-D3 isotope internal standard working solution with a concentration of 0.5 μg / mL.
[0040] S2: Take 100 μL of spiked sample and place it in an ampoule containing 400 μL of isotope internal standard working solution. Attach the dropper to the top of the ampoule and vortex for 15 seconds to mix thoroughly.
[0041] S3: Add 5 drops of the mixed liquid to the in situ ionization kit, let stand for 15 seconds, and then insert the in situ ionization kit into the sample inlet of the CELL portable mass spectrometer.
[0042] S4: Under the vacuum system of a small portable mass spectrometer, the mixture is drawn into the instrument. At the same time, under negative pressure, the molecules sublimate and transfer charge to the sample molecules, achieving electrospray ionization of the sample. The sample is then analyzed by the mass spectrometry system to obtain the ratio of the signal peak area of voriconazole to voriconazole-D3 isotope in the sample to be tested, and the content of voriconazole in the sample to be tested is calculated.
[0043] The mass spectrometer analysis conditions are as follows:
[0044] Voriconazole: Detection mode: positive mode, precursor ion m / z: 350.1, quantitative ion m / z: 281.1, ion selection energy: 3.45V, collision-induced dissociation energy: 2.5V;
[0045] Voriconazole-D3: Detection mode: positive mode, precursor ion m / z: 353.1, quantitative ion m / z: 284.1, ion selection energy: 3.45V, collision-induced dissociation energy: 2.5V.
[0046] Table 1. Volume ratio of acetonitrile, isopropanol, and ammonium acetate in the diluent
[0047]
[0048] Examples 8-10
[0049] Examples 8-10 each provide a method for detecting voriconazole.
[0050] The difference between the above embodiment and embodiment 3 is that step S3 is different, specifically:
[0051] In Example 8: S3: Add 5 drops of the mixed liquid to the in situ ionization reagent kit, let it stand for 35 seconds, and then insert the in situ ionization reagent kit into the sample inlet of the CELL portable mass spectrometer.
[0052] In Example 9: S3: Add 5 drops of the mixed liquid to the in situ ionization reagent kit, let it stand for 30 seconds, and then insert the in situ ionization reagent kit into the sample inlet of the CELL portable mass spectrometer.
[0053] In Example 10: S3: Add 5 drops of the mixed liquid to the in situ ionization reagent kit, let it stand for 5 seconds, and then insert the in situ ionization reagent kit into the sample inlet of the CELL portable mass spectrometer.
[0054] The remaining steps of the above embodiments are the same as those of Embodiment 3.
[0055] Examples 11-12
[0056] Examples 11-12 provide a method for detecting voriconazole.
[0057] The difference between the above embodiment and embodiment 3 is that step S3 is different, specifically:
[0058] In Example 11, the concentration of the voriconazole-D3 isotope internal standard working solution was 0.01 μg / mL.
[0059] In Example 12, the concentration of the voriconazole-D3 isotope internal standard working solution was 50 μg / mL.
[0060] The remaining steps of the above embodiments are the same as those of Embodiment 3. Comparative Example
[0061] Comparative Examples 1-6
[0062] Comparative Examples 1-6 each provide a method for the detection of voriconazole.
[0063] The difference between the above comparative example and Example 3 is that the composition of the diluent is different, as shown in Table 2.
[0064] Table 2 Composition of the diluent
[0065]
[0066] The above comparative examples are identical to the remaining steps of Example 3.
[0067] Performance testing
[0068] (1) Accuracy testing
[0069] Using the detection methods of Examples 1-12 and Comparative Examples 1-6, reference samples of voriconazole at known concentrations (low, medium, and high concentrations of 0.498 μg / mL, 2.056 μg / mL, and 10.103 μg / mL) were used as test samples. The content of voriconazole was then detected, and the Mean ± SD was measured to examine the accuracy of the detection kit and detection method in this application.
[0070] Test results are shown in Table 3.
[0071] Table 3. Accuracy test results of Examples 1-12 and Comparative Examples 1-6
[0072]
[0073] By analyzing the test results in Table 3, the triazole antifungal drugs were detected using the test kit and test method provided in this application. The mean of the test values of the test samples was consistent with the actual sample values, and the standard deviation was small, indicating that the test kit and test method provided in this application have high accuracy.
[0074] (2) Precision testing:
[0075] Using the detection methods of Examples 1-12 and Comparative Examples 1-6, reference samples of voriconazole at known concentrations (low, medium, and high concentrations of 0.498 μg / mL, 2.056 μg / mL, and 10.103 μg / mL) were used as test samples. The content of voriconazole was then detected, and the intra-batch and inter-batch coefficients of variation were measured to examine the precision of the detection kit and detection method in this application.
[0076] Intra-batch coefficient of variation: Each sample was tested 5 times using the same batch of reagent kits, and the intra-batch coefficient of variation of the detection method in this application was calculated.
[0077] Inter-batch coefficient of variation: Each sample was tested using 5 different batches of reagent kits, and the inter-batch coefficient of variation of the detection method in this application was calculated.
[0078] Test results are shown in Table 4.
[0079] Table 4. Precision test results of Examples 1-12 and Comparative Examples 1-6
[0080]
[0081]
[0082] Analysis of the test results in Table 4 shows that the intra-assay and inter-assay coefficients of variation for the test kit and test method in this application are both less than 8.30%. These test results indicate that the test kit and test method provided in this application have high precision in detecting triazole antifungal drugs, meaning that the test method in this application has good repeatability and stability.
[0083] Based on the detection results of Examples 1-7 and Comparative Examples 1-6 in Tables 3 and 4, it can be seen that the present application uses acetonitrile, isopropanol, and ammonium acetate in a volume ratio of 100:(5-10):(1-5) as a diluent to prepare the isotope internal standard working solution, which can effectively improve the accuracy and precision of the detection method. Furthermore, the present application controls the volume ratio of acetonitrile, isopropanol, and ammonium acetate to be 100:(7-10):(1-3).
[0084] Based on the detection results of Examples 4 and 8-10 in Tables 3 and 4, it can be seen that by adding the mixed liquid to the in-situ ionization reagent kit and allowing it to stand for 5-30 seconds during the detection process, the accuracy and precision of the detection method can be further improved.
[0085] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.
Claims
1. A test kit for triazole antifungal drugs, characterized by comprising: The kit includes a paper tube spray in situ ionization reagent kit and an isotope internal standard working solution; the isotope internal standard working solution is diluted with acetonitrile, isopropanol and ammonium acetate in a volume ratio of 100:(5-10):(1-5); The triazole antifungal drug is voriconazole, and the isotope internal standard working solution is voriconazole-D3 at a concentration of 0.05-50 μg / mL.
2. The triazole antifungal drug test kit according to claim 1, wherein The diluent is acetonitrile, isopropanol, and ammonium acetate in a volume ratio of 100:(7-10):(1-3).
3. The triazole antifungal drug test kit according to claim 1, wherein The paper-insertion spray in-situ ionization kit includes a shell, a paper core, a capillary tube, and a steel ball; the steel ball is embedded in a groove in the upper shell; the capillary tube is inserted in the middle of the paper core, which serves as a sample carrier; the paper core is clamped and fixed by the upper and lower shells, which have a snap-fit design.
4. The detection kit for triazole antifungal drugs according to claim 3, characterized in that, The capillary tube is inserted to a depth of 0.8-1.2 mm in the middle of the paper core.
5. A method for detecting triazole antifungal drugs, characterized in that, The detection is performed using the detection kit described in any one of claims 1-4.
6. The method for detecting triazole antifungal drugs according to claim 5, characterized in that, Includes the following steps: The sample to be tested is placed in the isotope internal standard working solution and vortexed to mix thoroughly. The mixed liquid is then dropped onto the paper tube spray in situ ionization kit. After standing for 5-30 seconds, the kit is inserted into a CELL small portable mass spectrometer to achieve electrospray ionization of the sample. The sample is then analyzed by the mass spectrometry system to obtain the ratio of the signal peak area of the triazole antifungal drug to that of the isotope, and the content of the triazole antifungal drug in the sample is calculated.
7. The method for detecting triazole antifungal drugs according to claim 6, characterized in that, The settling time is 5-15 seconds.
8. The method for detecting triazole antifungal drugs according to claim 6, characterized in that, When the triazole antifungal drug is voriconazole, the mass spectrometer analysis conditions are as follows: voriconazole detection mode: positive mode, precursor ion: m / z: 350.1, quantitative ion m / z: one or more of 127.1, 224.0, and 281.1, ion selection energy: 0.5~5 V, collision-induced dissociation energy: 1-4 V; Voriconazole-D3: Detection mode: positive mode, precursor ion m / z: 353.1, quantitative ion m / z: one or more of 127.1, 224.0, and 284.1, ion selection energy: 0.5-5V, collision-induced dissociation energy: 1-4V.