Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Liquid chromatography-mass spectrometry screening method for multi-target antithrombotic active substance

A screening method and liquid chromatography technology, applied in the field of drug screening, can solve the problems of affecting the sensitivity of mass spectrometry, increase the operation steps, increase the cost of reagents, etc., and achieve the effects of saving reagent costs, strong anti-interference ability, and reducing economic costs.

Active Publication Date: 2019-12-13
MARINE BIOMEDICAL RES INST OF QINGDAO CO LTD
View PDF5 Cites 6 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Liquid chromatography-mass spectrometry screening method for multi-target antithrombotic active substance
  • Liquid chromatography-mass spectrometry screening method for multi-target antithrombotic active substance
  • Liquid chromatography-mass spectrometry screening method for multi-target antithrombotic active substance

Examples

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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a liquid chromatography-mass spectrometry screening method for a multi-target antithrombotic substance based on a dried blood spot sample, and belongs to the field of drug screening. The method comprises the following steps of preparing a rehydrated dry blood spot sample, optionally selecting a plurality of substrates of key target enzymes involved in thrombosis processes,mixing a plurality of substrate standard solutions with a substance to be screened, adding a mixture into the rehydrated dry blood spot sample, treating a reaction solution, and carrying out liquid chromatography-mass spectrometry analysis to screen to obtain a substance with multi-target antithrombotic activity. The screening method of the invention has the advantages of simplicity and rapidness,high accuracy, high specificity, a large number of samples can be continuously analyzed, the antithrombotic substance capable of inhibiting one or more target enzymes at the same time can be screenedout at the same time according to the screening method, action targets of the screened active substance are clear, and a theoretical basis is laid for subsequent drug development. Reagents are easy to obtain, the operation automation degree is high, clinical popularization and promotion 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com