Method for detecting contents of epicatechin and mercapto epicatechin in plasma according to liquid-mass chromatography

A technology of epicatechin sulfhydryls and epicatechin, which is applied in the field of medical detection, can solve problems such as difficult detection, and achieve the effect of high sensitivity

Inactive Publication Date: 2016-07-06
HARMONIA TESTING TECH TIANJIN LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The active ingredient of condensed tannins is condensed tannins, which are not easy to be detected in pla...

Method used

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  • Method for detecting contents of epicatechin and mercapto epicatechin in plasma according to liquid-mass chromatography
  • Method for detecting contents of epicatechin and mercapto epicatechin in plasma according to liquid-mass chromatography
  • Method for detecting contents of epicatechin and mercapto epicatechin in plasma according to liquid-mass chromatography

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The determination of embodiment 1 detection method

[0044] 1.1 Experimental equipment and reagents

[0045] instrument:

[0046] Liquid chromatograph is WatersACQUITYUPLC (Waters company), is equipped with BinarySolventManager, Column, PDA detector; Chromatographic column: WatersBEHShieldRP18 (2.1 * 100mm, 1.7mm);

[0047] The mass spectrometer is QuattroPremierXE (Waters), equipped with electrospray ionization ion source (ESI) and atmospheric pressure chemical ionization source (APCI), MasslynxV4.1 workstation, UltraSonicCleanerUSKType ultrasonic cleaner;

[0048] FA604A electronic balance (Shanghai Ketian Electronic Instrument Co., Ltd.);

[0049] Vortex Mixer XW-80A (Haimen Qilinbeier Instrument Manufacturing Co., Ltd.)

[0050] TGL-16G high-speed desktop centrifuge (Shanghai Anting Scientific Instrument Factory)

[0051] Pipette gun (BIOHTPROLINE) 20-200μl; 10-1000μl

[0052] Reagent:

[0053] Epicatechin, purchased from Shanghai Yuanye Biotechnology Co., Ltd...

Embodiment 2

[0075] Embodiment 2 methodological verification experiment

[0076] Method specificity, linearity and correlation coefficients

[0077] Specificity of the method Take 50 μL of blank plasma from 6 Beagle dogs respectively, operate according to the item of “Plasma Sample Analysis Method”, inject 5 μL, and obtain the specificity chromatogram figure 1 Add a certain concentration of epicatechin standard solution, epicatechin thiol standard solution and internal standard propranolol solution into the blank plasma, and operate in the same way to obtain a limit of quantitation 10ng / mL chromatogram, see figure 2 , image 3 (for the standard curve 200ng / mL chromatogram); get the plasma sample collected after administration of the test dog, and operate in the same way to obtain the corresponding chromatogram Figure 4 (See Figure 4 ). The results showed that the endogenous substances in blank plasma did not interfere with the determination of epicatechin, epicatechin thiols and int...

Embodiment 3

[0099] The mensuration of embodiment 3 actual samples

[0100] Collection of plasma samples

[0101] Select 8 healthy Beagle dogs, weighing (9±1.0) kg, fast for more than 12 hours one day before the experiment, without water, and eat uniformly after 4 hours of administration. Two-cycle cross-administration, washout period of two weeks. Oral administration of Weimening capsules (200mg / capsules). The blank blood was drawn before the experiment, and the forelimb veins were taken at 0, 10, 20, 30min, 1, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72 and 84h after administration, respectively at 16 time points. Put 1mL of blood in a centrifuge tube containing heparin, place it in an ice bath, centrifuge at 3000rpm for 10min within 0.5hr at 4°C to collect the upper plasma, and store it in a freezer at -80°C until testing.

[0102] Determination of real plasma samples

[0103] The plasma sample obtained above was processed according to "Preparation of Plasma Samples Containing Drugs", and liqu...

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Abstract

The invention discloses a method for detecting contents of epicatechin and mercapto epicatechin in plasma according to the liquid-mass chromatography. The method comprises the following steps: (1) preparing a medicated plasma sample; (2) preparing a working curve sample; (3) setting LC determination conditions; (4) setting MS/MS conditions; (5) quantifying according to an internal standard method, taking the concentrations of series of epicatechin and mercapto epicatechin in blank plasma as horizontal coordinates, and the peak area ratios of the epicatechin and the mercapto epicatechin to an internal standard substance propranolol as vertical coordinates, and carrying out regressing calculation according to a weighted least square method, so as to obtain a linear regression equation, namely a working curve, wherein the weighting coefficient is 1/x2; calculating the concentrations of the epicatechin and the mercapto epicatechin in the medicated plasma sample according to the working curve. The method provided by the invention is fast, dedicated, precise and highly sensitive.

Description

technical field [0001] The invention belongs to the technical field of medical detection, and relates to a method for detecting the content of epicatechin and epicatechin thiols in blood plasma. Background technique [0002] Condensed tannins have various pharmacological activities such as anti-oxidation, anti-pathogenic microorganisms, anti-inflammation, lowering blood pressure, regulating blood lipids, and lowering blood sugar. In the past 10 years, research has found that condensed tannins have anti-tumor, anti-HIV, and anti-rheumatic effects. It has a good application prospect. Weimaining made from condensed tannin of golden buckwheat has been used as an anti-tumor drug in clinical practice. The main material basis of anti-inflammatory and anti-rheumatic activities. The active ingredient of condensed tannins is condensed tannins, which are not easy to be detected in blood plasma. At present, there is no accurate and precise method to measure the content of condensed tan...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/027G01N2030/042
Inventor 付淑军王鹏戴玮肖苏萍常新全左臣强张守庆赖东海
Owner HARMONIA TESTING TECH TIANJIN LTD
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