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A method for detection and analysis of hydroxyl-containing cholesterol and its metabolites

An analysis method, cholesterol technology, applied in the direction of analyzing materials, measuring devices, material separation, etc., can solve the problems of high price, achieve the effect of improving quantitative accuracy, improving sensitivity, and reducing matrix effect

Active Publication Date: 2020-12-01
UNITED POWER PHARMA TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, isotope standards are not commercially available for a large number of biological metabolites and often they are very expensive

Method used

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  • A method for detection and analysis of hydroxyl-containing cholesterol and its metabolites
  • A method for detection and analysis of hydroxyl-containing cholesterol and its metabolites
  • A method for detection and analysis of hydroxyl-containing cholesterol and its metabolites

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Chromatographic separation and mass spectrometry qualitative and quantitative analysis of hydroxyl-containing cholesterol and its metabolites:

[0039] In the range of 2-3000 pg / mL, prepare 7 different concentrations d 0 - MCR6G-labeled hydroxyl-containing cholesterol and its metabolites standard solution (10 pg / mL, 100 pg / mL, 500 pg / mL, 1000 pg / mL, 1500 pg / mL, 2000 pg / mL, 2500 pg / mL), Among them d 3 -MCR6G-labeled standard (800 pg / mL) was used as fixed internal standard. The specific derivatization process is as follows: 1 part of the above-mentioned mixed standard solution with different concentration levels, 25 μL CMPI (7.5 wt%) and 25 μL DMAP (13 wt%) acetonitrile solution, 100 μL d 0 -MCR6G or d 3 - MCR6G acetonitrile solution, add to a 1.5 mL centrifuge tube one by one, vortex for 10 seconds. Seal and react in a microwave reactor (750 W) at 50 °C for 7.5 min. 7 levels of concentration d 0 -MCR6G mixed standard derivative and d 3 -MCR6G-labeled immobil...

Embodiment 2

[0044] The extraction of hydroxyl-containing cholesterol and metabolites in plasma includes the following steps:

[0045]Aspirate 500 μL of plasma, add 200 μL of 10% trichloroacetic acid to precipitate protein, shake and centrifuge, take supernatant, blow dry with nitrogen at room temperature, and fully dissolve the residue in 200 μL of acetonitrile for stable isotope labeling derivatization. Take 50 μL plasma acetonitrile solution or standard mixed solution, 25 μL CMPI (5.5 wt%) and 25 μL DMAP (12 wt%) acetonitrile solution into a 1.5 mL centrifuge tube. After vortexing for 10 seconds, add 100 μL d 0 -MCR6G or d 3 - MCR6G in acetonitrile. Shake well, seal and react in a microwave reactor (800W) at 55°C for 7.5 minutes. d 0 -MCR6G-labeled cerebrospinal fluid samples and d 3 -MCR6G labeled standard 1:1 ( V / V ) to mix and shake well. 5.5 mg Fe 3 o 4 / GO was dispersed into the above mixed solution, the pH of the solution was adjusted to 7, and shaken for 4 minutes to ...

Embodiment 3

[0047] The extraction of hydroxyl-containing cholesterol and metabolites in cerebrospinal fluid includes the following steps:

[0048] Take 200 μL of cerebrospinal fluid into a centrifuge tube, blow dry with nitrogen gas at room temperature, and fully dissolve the residue with 200 μL of acetonitrile. Take 50 μL of cerebrospinal fluid acetonitrile reconstitution solution or standard mixed solution, 25 μL of CMPI (6 wt%) and 25 μL of DMAP (14 wt%) acetonitrile solution into a 1.5 mL centrifuge tube. After vortexing for 10 seconds, add 100 μL d 0 -MCR6G or d 3 - MCR6G in acetonitrile. Shake well, seal and react in a microwave reactor (750W) at 50 °C for 7.5 minutes. d 0 -MCR6G-labeled cerebrospinal fluid samples and d 3 -MCR6G labeled standard 1:1 ( V / V ) to mix and shake well. 5 mg Fe 3 o 4 / GO was dispersed into the above mixed solution, the pH of the solution was adjusted to 6, and shaken for 4 minutes to reach adsorption equilibrium. Magnetic separation, decantin...

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Abstract

The invention relates to the field of analytical chemistry, and particularly relates to a detection and analysis method for hydroxy-containing cholesterol and metabolites thereof. The method includessubjecting hydroxy-containing cholesterol and metabolites thereof to stable isotope labeling derivatization with a derivatization agent that is d<0>- / d<3>-3-N-methyl-2'-carboxyrhodamine 6G; and aftera derivatization product obtained by magnetic dispersive solid-phase extraction is filtered by a filter membrane, detecting with an ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry analysis system. The stable isotope labeling derivatization agent that is the d<0>- / d<3>-MCR6G with permanent positive charges is utilized to label the hydroxy-containing cholesterol and metabolites thereof in a biological sample, and derivatization is mild in condition, rapid and high in efficiency. A magnetic dispersive solid-phase extraction technique is utilized, thus increasing the extraction efficiency, reducing sample pretreatment time, significantly increasing the degree of chromatographic separation and the mass spectrum ionization efficiency of analytes and reducing matrix interferences.

Description

technical field [0001] The invention relates to the field of analytical chemistry, in particular to a detection and analysis method for hydroxyl-containing cholesterol and its metabolites, especially to a method for utilizing d 0 - / d 3 -3- N -Methyl-2'-carboxyrhodamine 6G ( d 0 - / d 3 -MCR6G) as a derivatization reagent, microwave-assisted-stable isotope labeling derivatization-magnetic dispersion solid-phase extraction combined with ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry detection method. Background technique [0002] Hydroxyl-containing cholesterol and its metabolites are a class of bioorganic molecules that contain free hydroxyl groups and are present in low concentrations in biological samples, such as plasma, cerebrospinal fluid, liver tissue, urine, and feces. These compounds play important roles in cell membrane signaling, regulation and metabolism in a large number of organisms. Recent studies have shown that...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/062G01N2030/067
Inventor 朱树芸汪鑫赵先恩徐燕秋孙鲁平尤进茂
Owner UNITED POWER PHARMA TECH CO LTD
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