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A liquid chromatography-mass spectrometry screening method for multi-target antithrombotic active substances

A screening method and anti-thrombotic technology, applied in the field of liquid chromatography-mass spectrometry screening, can solve the problems of affecting the sensitivity of mass spectrometry, increasing operating steps, increasing reagent costs, etc., to save analysis time, strong anti-interference ability, and save reagent costs Effect

Active Publication Date: 2022-03-22
MARINE BIOMEDICAL RES INST OF QINGDAO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The enzyme reaction system needs to use non-volatile buffer salt (Tris-HCl) to maintain the activity of commercial enzymes. The non-volatile buffer salt will not only affect the sensitivity of mass spectrometry, but also remain in the instrument of liquid chromatography-mass spectrometry. damage instrument
Therefore, in this method, the reaction solution must be processed to remove buffer salts before sample analysis, which not only increases the operation steps, but also inevitably uses organic solvents and chemical fillers, which increases the cost of reagents and is not environmentally friendly.

Method used

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  • A liquid chromatography-mass spectrometry screening method for multi-target antithrombotic active substances
  • A liquid chromatography-mass spectrometry screening method for multi-target antithrombotic active substances
  • A liquid chromatography-mass spectrometry screening method for multi-target antithrombotic active substances

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The present invention obtains a liquid chromatography-mass spectrometry screening method for multi-target antithrombotic active substances based on dried blood spot samples through the following steps:

[0045] 1. Dried blood spot sample preparation and standard solution preparation :

[0046] 1.1 Preparation of dried blood spot samples: pipette 10-80 μL of whole blood onto a Whatman903 filter paper card, and air-dry at room temperature for 2 hours to obtain a dried blood spot sample. The prepared dried blood spot sample is placed in a sealed bag containing a desiccant and stored at -20°C for later use.

[0047] 1.2 Preparation of substrate standard solution: Accurately weigh an appropriate amount of substrate powder, and use deionized water to prepare substrates S2238, S2765, S2266, S2302 and cyclic adenosine monophosphate (cAMP) to 500mg L -1 Concentrated solutions are stored.

[0048] 1.3 Preparation of the compound solution to be screened: 78 compounds to be scr...

Embodiment 2

[0070] The method validation of the established product quantification method, and the investigation of the quantification limit, detection limit, precision, accuracy and matrix effect, should meet the requirements of the 2015 edition of the Chinese Pharmacopoeia IV "Guiding Principles for Validation of Analytical Methods".

[0071] (1) Quantitation limit and detection limit

[0072] Detection limit of p-nitroaniline: 5 μg L -1 (S / N=3.3±1.61; n=6); lower limit of quantitation: 10 μg L -1 (S / N=10.5±1.86; n=6); detection limit of 7-amino-4-methylcoumarin: 4 μg L -1 (S / N=1.4±2.71; n=6); lower limit of quantitation: 10 μg L -1 (S / N=12.3±1.71; n=6); detection limit of 5’-adenosine monophosphate: 5 μg L -1 (S / N=3.76±1.37; n=6); lower limit of quantitation: 10 μg L -1 (S / N=10.89±1.69; n=6); detection limit of 5’-guanosine monophosphate: 8 μg L -1 (S / N=6.62±2.39; n=6); lower limit of quantitation: 15 μg L -1 (S / N=13.66±1.52; n=6). From the results, it can be seen that the detec...

Embodiment 3

[0086] Using standard enzymes to verify the liquid chromatography-mass spectrometry screening method for multi-target antithrombotic active substances:

[0087] 1. Solution preparation: Accurately weigh appropriate amount of thrombin, coagulation factor Xa, coagulation factor XIa, coagulation factor XIIa, and phosphodiesterase 3 powders, and dilute them to 1000U L with deionized water -1 concentration and stored at -20°C for use; accurately weigh appropriate amounts of substrates S2238, S2765, S2266, S2302, and cAMP powder, and prepare 500mg L with deionized water -1 Concentration solutions were stored; 78 compounds to be screened were respectively taken, dissolved in a small amount of DMSO, and prepared as 100 μM stock solutions in water. The above stock solutions were stored at -20°C in the dark. Dissolve argatroban, rivaroxaban, benzamidine, 3-acetylcoumarin, and milrinone in 50 µL of DMSO, respectively, and then add water to a final concentration of 500 µM for storage. W...

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Abstract

The invention discloses a liquid chromatography-mass spectrometry screening method for multi-target antithrombotic substances based on dried blood spot samples, which belongs to the field of drug screening. The method comprises the following steps: rehydrating the dried blood spot sample, optionally several substrates of key target enzymes involved in the thrombus formation process, mixing several substrate standard solutions with the substances to be screened and then adding them to the rehydrated In the dried blood spot sample, the reaction solution is processed and analyzed by liquid chromatography-mass spectrometry to screen for substances with multi-target antithrombotic activity. The screening method of the present invention is simple and quick to operate, has high accuracy and strong specificity, and can continuously analyze a large number of samples. The target of substance action is clear, which lays a theoretical foundation for the subsequent drug development. The reagents used are easy to obtain, the degree of operation automation is high, clinical promotion and popularization are easy, and the commercial value is high.

Description

technical field [0001] The invention relates to the field of drug screening, in particular to a liquid chromatography-mass spectrometry screening method for multi-target antithrombotic active substances based on dried blood spot samples. Background technique [0002] According to reports, stroke, coronary heart disease and other thrombotic diseases have become the main killers threatening human health. Studies have shown that abnormal activation of platelets leads to platelet aggregation and a series of coagulation factors are activated successively to convert fibrinogen into fibrin, and then form a network and recruit red blood cells to rapidly increase the thrombus mass, and finally combine tightly with platelets to form a firm thrombus , which is the main cause of arterial thrombotic disease and venous thrombotic disease. From the perspective of thrombus formation mechanism, a variety of enzymes and receptors are involved in the process of thrombus formation. Theoretica...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/88G01N30/06
CPCG01N30/88G01N30/06G01N2030/8822G01N2030/8804
Inventor 许哲邹旋管华诗
Owner MARINE BIOMEDICAL RES INST OF QINGDAO CO LTD
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