Detection and analysis method of protease inhibitor

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By mixing plasma samples with acetonitrile and vortex shock and centrifugation, combined with high-performance liquid chromatography mass spectrometry and internal standard curve method, efficient quantitative detection of protease inhibitors and their metabolites is achieved, and the existing solutions are solved The problem of complex sample preprocessing and low detection sensitivity in the method improves the precision and accuracy of the detection.

CN119438401APending Publication Date: 2025-02-14SYNTHESIS MED CHEM (SUZHOU) CO LTD

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Patent Information

Application Number: CN202310934373.5
Authority / Receiving Office: CN · China
Patent Type: Applications(China)
Current Assignee / Owner
Filing Date: 2023-07-28
Publication Date: 2025-02-14

AI Technical Summary

Technical Problem

The existing methods for determining drug concentration in plasma of protease inhibitors have problems such as complex and time-consuming sample preprocessing, low sensitivity, long detection time, narrow linear range, and large amount of plasma or organic reagents, resulting in large workload and detection. The cost is high and cannot meet clinical needs.

Method used

A protease inhibitor detection and analysis method is adopted, including mixing plasma samples with acetonitrile and vortexing, collecting supernatant as sample solution after centrifugation, and then determining the sample by high-performance liquid chromatography mass spectrometry and determining the sample based on retention time. Protease inhibitors and their metabolites in the solution were quantified by the internal standard curve method.

Benefits of technology

The detection of protease inhibitors with high throughput, high sensitivity, good precision and accuracy and good stability is achieved, reducing the interference effect generated by the matrix in the mass spectrometry and improving detection efficiency and accuracy.

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Abstract

The invention relates to a detection and analysis method of a protease inhibitor. The detection and analysis method sequentially comprises the following steps: S1, mixing a plasma sample with acetonitrile, carrying out vortex oscillation, centrifuging, and collecting supernate as a sample solution; step S2, determining the sample solution in the step S1 through a high performance liquid chromatography-mass spectrometry method; s3, determining the protease inhibitor and metabolites thereof in the sample solution according to the retention time; and S4, quantifying by adopting an internal standard curve method, and determining the concentration of the protease inhibitor and the metabolite thereof in the sample solution. The method has the advantages of high flux, high sensitivity, good precision and accuracy, good stability and high extraction recovery rate; through reasonable selection of a chromatographic column and a mobile phase and optimization of an elution gradient and a pretreatment method, the interference effect of a matrix in a mass spectrum can be greatly reduced.

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Description

Technical Field

[0001] The present invention relates to the technical field related to biomedical detection, and in particular to a detection and analysis method for protease inhibitors. Background Art

[0002] Most patients with hematological tumors are special populations such as children and the elderly. Compared with ordinary patients, their physiological and biochemical pathological mechanisms are quite different, and they have different pharmacokinetic and pharmacodynamic characteristics. In addition, many drugs lack clinical data for special populations or are used beyond the instructions. The situation of drug safety is very serious. Therefore, it is necessary to guide the rational use of drugs in the treatment process, and the most powerful means is therapeutic drug monitoring TDM. TDM refers to the adjustment of the dosage by measuring the drug concentration in the patient's body fluids (including blood, urine, saliva, etc.) to achieve the expected target level. At present, the methods used in TDM mainly include immunoassay, high performance liquid chromatography, liquid chromatography tandem mass spectrometry, etc. Among them, the immunoassay is simple to operate and has a short detection cycle, but its application range is limited and it cannot simultaneously determine multiple drugs; the HPLC method has strong specificity and good stability, but low sensitivity and long analysis time. LC-MS / MS technology can make up for the shortcomings of the above two methods, and has obvious advantages in specificity, sensitivity and multi-component analysis, playing a pivotal role in clinical diagnosis.

[0003] It has been reported that liquid chromatography-tandem mass spectrometry is the most widely used method for determining the drug concentration of protease inhibitors and their metabolites in plasma. However, these methods have more or less some shortcomings: such as complex or time-consuming sample pretreatment, low sensitivity, long detection time, narrow linear range, large amount of plasma or organic reagents, etc., which leads to large workload, high detection cost or cannot meet clinical needs.

[0004] In view of the above-mentioned defects, the designers have actively carried out research and innovation in order to create a detection and analysis method for protease inhibitors to make it more valuable for industrial use. Summary of the invention

[0005] In order to solve the above technical problems, the object of the present invention is to provide a method for detecting and analyzing protease inhibitors.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A method for detecting and analyzing protease inhibitors comprises the following steps in sequence:

[0008] Step S1, mixing the plasma sample with acetonitrile, vortexing, centrifuging, and collecting the supernatant as a sample solution;

[0009] Step S2, measuring the sample solution in the above step S1 by high performance liquid chromatography-mass spectrometry;

[0010] Step S3, determining the protease inhibitors and their metabolites in the sample solution according to the retention time;

[0011] Step S4, using the internal standard curve method for quantitative determination of the concentration of the protease inhibitor and its metabolites in the sample solution;

[0012] The above-mentioned liquid chromatography detection conditions are:

[0013] C18 column, 4.6×150 mm, 10 μm, column temperature 25-40 °C;

[0014] Flow rate: 0.5~2.0ml / min;

[0015] Injection volume: 5-20ul;

[0016] Detection wavelength: 150nm~250nm;

[0017] Mobile phase: Mobile phase A was 0.1 vol% acetic acid in water and the pH was adjusted to 3.0-3.5 with formic acid, and mobile phase B was 100% acetonitrile, with gradient elution for 5 min;

[0018] The mass spectrometry detection conditions mentioned above are:

[0019] Electrospray ion source, positive ion detection mode, spray voltage is 5000V, atomization temperature is 500℃, atomization gas is 60units; auxiliary heating gas is 60units; curtain gas is 40units; collision gas is 20units; inlet voltage is 10V; collision chamber outlet voltage is 12V.

[0020] As a further improvement of the present invention, the C18 chromatographic column is a GL Sciences InertsilTM ODS-4 chromatographic column, the column temperature is 25°C, and the automatic sampler temperature is 4°C.

[0021] As a further improvement of the present invention, the C18 chromatographic column is: Agilent ZORBAX SB-Aq chromatographic column, the column temperature is 40°C, and the automatic sampler temperature is 4°C.

[0022] As a further improvement of the present invention, in the above-mentioned liquid chromatography detection, 3-5:6-8 is split after the column, and 2-4 / 10 flows into mass spectrometry analysis.

[0023] As a further improvement of the present invention, the gradient elution conditions in the above-mentioned liquid chromatography are as follows:

[0024] 0~1.0min: flow rate: 1.0mL / min; mobile phase A: 80%, mobile phase B: 20%;

[0025] 1.0~1.5min: flow rate: 1.0mL / min; mobile phase A: 80%, mobile phase B: 20%;

[0026] 1.5~2.0min: flow rate: 1.0mL / min; mobile phase A: 75%, mobile phase B: 25%;

[0027] 2.0~3.0min: flow rate: 1.0mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0028] 3.0~4.5min: flow rate: 1.0mL / min; mobile phase A: 80%, mobile phase B: 20%;

[0029] 4.5~5.0min: flow rate: 1.0mL / min; mobile phase A: 80%, mobile phase B: 20%.

[0030] As a further improvement of the present invention, in step S1, after vortexing for 3-6 minutes, centrifugation is performed at 5-8°C and 5000-10000 g / min for 8-15 minutes.

[0031] As a further improvement of the present invention, in the above-mentioned mass spectrometry detection, the monitoring method is to perform primary / secondary mass spectrometry analysis in a multiple reaction monitoring mode.

[0032] By means of the above scheme, the present invention has at least the following advantages:

[0033] The invention has high throughput, high sensitivity, good precision and accuracy, good stability and high extraction recovery rate; the invention can greatly reduce the interference effect of the matrix in the mass spectrometry by rationally selecting the chromatographic column and the mobile phase and optimizing the elution gradient and the pretreatment method.

[0034] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail as follows. DETAILED DESCRIPTION

[0035] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0036] In order to make those skilled in the art better understand the scheme of the present invention, the technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, rather than all the embodiments. The components of the embodiment of the present invention generally shown here can be arranged and designed in various configurations. Therefore, the following detailed description of the embodiment of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents the selected embodiment of the present invention. Based on the embodiment of the present invention, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present invention.

[0037] Embodiment 1:

[0038] A method for detecting and analyzing protease inhibitors comprises the following steps in sequence:

[0039] Step S1, after mixing the plasma sample with acetonitrile for 3 minutes, centrifuge at 5°C and 10000g / min for 8 minutes to collect the supernatant as the sample solution;

[0040] Step S2, measuring the sample solution in the above step S1 by high performance liquid chromatography-mass spectrometry;

[0041] Step S3, determining the protease inhibitors and their metabolites in the sample solution according to the retention time;

[0042] Step S4: quantify the concentration of the protease inhibitor and its metabolites in the sample solution by using the internal standard curve method.

[0043] The above-mentioned liquid chromatography detection conditions are:

[0044] GL Sciences InertsilTM ODS-4 column, 4.6×150 mm, 10 μm, column temperature 25°C, autosampler temperature 4°C;

[0045] 3-5% of the flow is split after the column, and 2 / 10 flows into mass spectrometry analysis;

[0046] Flow rate: 1.0ml / min;

[0047] Injection volume: 5ul;

[0048] Detection wavelength: 150nm;

[0049] Mobile phase: Mobile phase A was 0.1 vol% acetic acid in water and the pH was adjusted to 3.0 with formic acid, and mobile phase B was 100% acetonitrile, with gradient elution for 5 min;

[0050] The gradient elution conditions were as follows:

[0051] 0~1.0min: flow rate: 1.0mL / min; mobile phase A: 80%, mobile phase B: 20%;

[0052] 1.0~1.5min: flow rate: 1.0mL / min; mobile phase A: 80%, mobile phase B: 20%;

[0053] 1.5~2.0min: flow rate: 1.0mL / min; mobile phase A: 75%, mobile phase B: 25%;

[0054] 2.0~3.0min: flow rate: 1.0mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0055] 3.0~4.5min: flow rate: 1.0mL / min; mobile phase A: 80%, mobile phase B: 20%;

[0056] 4.5~5.0min: flow rate: 1.0mL / min; mobile phase A: 80%, mobile phase B: 20%.

[0057] The mass spectrometry detection conditions mentioned above are:

[0058] Electrospray ion source, positive ion detection mode, spray voltage was 5000V, nebulization temperature was 500℃, nebulization gas was 60units; auxiliary heating gas was 60units; curtain gas was 40units; collision gas was 20units; inlet voltage was 10V; collision chamber outlet voltage was 12V; monitoring method was primary / secondary mass spectrometry analysis in multiple reaction monitoring mode.

[0059] Embodiment 2:

[0060] A method for detecting and analyzing protease inhibitors comprises the following steps in sequence:

[0061] Step S1, after mixing the plasma sample with acetonitrile for 6 minutes, centrifuge at 8°C and 10000g / min for 15 minutes to collect the supernatant as the sample solution;

[0062] Step S2, measuring the sample solution in the above step S1 by high performance liquid chromatography-mass spectrometry;

[0063] Step S3, determining the protease inhibitors and their metabolites in the sample solution according to the retention time;

[0064] Step S4: quantify the concentration of the protease inhibitor and its metabolites in the sample solution by using the internal standard curve method.

[0065] The above-mentioned liquid chromatography detection conditions are:

[0066] GL Sciences InertsilTM ODS-4 column, 4.6×150 mm, 10 μm, column temperature 40°C, autosampler temperature 4°C;

[0067] 6-8% of the flow is split after the column, and 4 / 10 flows into mass spectrometry analysis;

[0068] Flow rate: 1.5ml / min;

[0069] Injection volume: 10ul;

[0070] Detection wavelength: 200nm;

[0071] Mobile phase: Mobile phase A was 0.1 vol% acetic acid in water and the pH was adjusted to 3.3 with formic acid, and mobile phase B was 100% acetonitrile, with gradient elution for 5 min;

[0072] The gradient elution conditions were as follows:

[0073] 0~1.0min: flow rate: 1.5mL / min; mobile phase A: 80%, mobile phase B: 20%;

[0074] 1.0~1.5min: flow rate: 1.5mL / min; mobile phase A: 80%, mobile phase B: 20%;

[0075] 1.5~2.0min: flow rate: 1.5mL / min; mobile phase A: 75%, mobile phase B: 25%;

[0076] 2.0~3.0min: flow rate: 1.5mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0077] 3.0~4.5min: flow rate: 1.5mL / min; mobile phase A: 80%, mobile phase B: 20%;

[0078] 4.5~5.0min: flow rate: 1.5mL / min; mobile phase A: 80%, mobile phase B: 20%.

[0079] The mass spectrometry detection conditions mentioned above are:

[0080] Electrospray ion source, positive ion detection mode, spray voltage was 5000V, nebulization temperature was 500℃, nebulization gas was 60units; auxiliary heating gas was 60units; curtain gas was 40units; collision gas was 20units; inlet voltage was 10V; collision chamber outlet voltage was 12V; monitoring method was primary / secondary mass spectrometry analysis in multiple reaction monitoring mode.

[0081] Embodiment three:

[0082] A method for detecting and analyzing protease inhibitors comprises the following steps in sequence:

[0083] Step S1, after mixing the plasma sample with acetonitrile for -6 minutes, centrifuge at 8°C and 10000g / min for 10 minutes, and collect the supernatant as the sample solution;

[0084] Step S2, measuring the sample solution in the above step S1 by high performance liquid chromatography-mass spectrometry;

[0085] Step S3, determining the protease inhibitors and their metabolites in the sample solution according to the retention time;

[0086] Step S4: quantify the concentration of the protease inhibitor and its metabolites in the sample solution by using the internal standard curve method.

[0087] The above-mentioned liquid chromatography detection conditions are:

[0088] Agilent ZORBAX SB-Aq column, 4.6 × 150 mm, 10 μm, column temperature 25 °C, autosampler temperature 4 °C;

[0089] 3-5% of the flow is split after the column, and 2 / 10 flows into mass spectrometry analysis;

[0090] Flow rate: 1.0ml / min;

[0091] Injection volume: 20ul;

[0092] Detection wavelength: 250nm;

[0093] Mobile phase: Mobile phase A was 0.1 vol% acetic acid in water and the pH was adjusted to 3.5 with formic acid, and mobile phase B was 100% acetonitrile, with gradient elution for 5 min;

[0094] The gradient elution conditions were as follows:

[0095] 0~1.0min: flow rate: 1.0mL / min; mobile phase A: 80%, mobile phase B: 20%;

[0096] 1.0~1.5min: flow rate: 1.0mL / min; mobile phase A: 80%, mobile phase B: 20%;

[0097] 1.5~2.0min: flow rate: 1.0mL / min; mobile phase A: 75%, mobile phase B: 25%;

[0098] 2.0~3.0min: flow rate: 1.0mL / min; mobile phase A: 65%, mobile phase B: 35%;

[0099] 3.0~4.5min: flow rate: 1.0mL / min; mobile phase A: 80%, mobile phase B: 20%;

[0100] 4.5~5.0min: flow rate: 1.0mL / min; mobile phase A: 80%, mobile phase B: 20%.

[0101] The mass spectrometry detection conditions mentioned above are:

[0102] Electrospray ion source, positive ion detection mode, spray voltage was 5000V, nebulization temperature was 500℃, nebulization gas was 60units; auxiliary heating gas was 60units; curtain gas was 40units; collision gas was 20units; inlet voltage was 10V; collision chamber outlet voltage was 12V; monitoring method was primary / secondary mass spectrometry analysis in multiple reaction monitoring mode.

[0103] The invention has high throughput, high sensitivity, good precision and accuracy, good stability and high extraction recovery rate; the invention can greatly reduce the interference effect of the matrix in the mass spectrometry by rationally selecting the chromatographic column and the mobile phase and optimizing the elution gradient and the pretreatment method.

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the technical principles of the present invention, and these improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for detecting and analyzing protease inhibitors, characterized in that: The following steps are included in sequence: Step S1, mixing the plasma sample with acetonitrile, vortexing, centrifuging, and collecting the supernatant as a sample solution; Step S2, measuring the sample solution in the above step S1 by high performance liquid chromatography-mass spectrometry; Step S3, determining the protease inhibitors and their metabolites in the sample solution according to the retention time; Step S4, using the internal standard curve method for quantitative determination of the concentration of the protease inhibitor and its metabolites in the sample solution; The above-mentioned liquid chromatography detection conditions are: C18 column, 4.6×150 mm, 10 μm, column temperature 25-40 °C; Flow rate: 0.5~2.0ml / min; Injection volume: 5-20ul; Detection wavelength: 150nm~250nm; Mobile phase: Mobile phase A was 0.1 vol% acetic acid in water and the pH was adjusted to 3.0-3.5 with formic acid, and mobile phase B was 100% acetonitrile, with gradient elution for 5 min; The mass spectrometry detection conditions mentioned above are: Electrospray ion source, positive ion detection mode, spray voltage is 5000V, atomization temperature is 500℃, atomization gas is 60units; auxiliary heating gas is 60units; curtain gas is 40units; collision gas is 20units; inlet voltage is 10V; collision chamber outlet voltage is 12V.

2. A method for detecting and analyzing a protease inhibitor according to claim 1, characterized in that: The C18 chromatographic column is a GL Sciences InertsilTM ODS-4 chromatographic column, the column temperature is 25°C, and the automatic sampler temperature is 4°C.

3. A method for detecting and analyzing a protease inhibitor according to claim 1, characterized in that: The C18 chromatographic column is: Agilent ZORBAX SB-Aq chromatographic column, the column temperature is 40°C, and the automatic sampler temperature is 4°C.

4. A method for detecting and analyzing a protease inhibitor according to claim 1, characterized in that: In the above-mentioned liquid chromatography detection, the flow after the column is 3-5:6-8 split, and 2-4 / 10 flows into mass spectrometry analysis.

5. The method for detecting and analyzing a protease inhibitor according to claim 1, characterized in that: The gradient elution conditions in the above-mentioned liquid chromatography are as follows: 0~1.0min: flow rate: 1.0mL / min; mobile phase A: 80%, mobile phase B: 20%; 1.0~1.5min: flow rate: 1.0mL / min; mobile phase A: 80%, mobile phase B: 20%; 1.5~2.0min: flow rate: 1.0mL / min; mobile phase A: 75%, mobile phase B: 25%; 2.0~3.0min: flow rate: 1.0mL / min; mobile phase A: 65%, mobile phase B: 35%; 3.0~4.5min: flow rate: 1.0mL / min; mobile phase A: 80%, mobile phase B: 20%; 4.5~5.0min: flow rate: 1.0mL / min; mobile phase A: 80%, mobile phase B: 20%.

6. A method for detecting and analyzing a protease inhibitor according to claim 1, characterized in that: In the step S1, after vortexing for 3-6 minutes, centrifugation is performed at 5-8° C. and 5000-10000 g / min for 8-15 minutes.

7. A method for detecting and analyzing a protease inhibitor according to claim 1, characterized in that: In the above mass spectrometry detection, the monitoring method is to perform primary / secondary mass spectrometry analysis in multiple reaction monitoring mode.