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Analysis method of important metabolites related to glucose energy metabolism in serum samples
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A technology of energy metabolism and analysis method, applied in the direction of analysis materials, measuring devices, material separation, etc., can solve the problems that have not been seen, such as high solvent toxicity, complex extraction, etc., and achieve the effect of low solvent toxicity, high sensitivity, and simple pretreatment
Active Publication Date: 2019-07-19
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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The determination of glucose mainly includes enzymatic method, electrochemical method, photochemical method, etc.; the quantitative analysis of the intermediate products of the tricarboxylic acid cycle mostly uses the method of gas chromatography-mass spectrometry, such as Calderón-Santiago, etc., using chloroform-methanol-water dual-phase extraction, selected ion scanning mode detection , but there are problems such as complicated extraction and high solventtoxicity
Gas chromatography-triple quadrupole mass spectrometry, as a powerful tool for targeted analysis, through gas chromatography separation combined with selected ion scanning of two-stage mass spectrometry, can eliminate matrix interference to the greatest extent, and has the advantages of high sensitivity and wide linear range ; and there is no report on the analysis of important metabolites related to glucose energy metabolism based on gas chromatography-triple quadrupolemass spectrometry coupled with multiple reaction monitoring mode
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[0018] Referring to the HMDB database (http: / / www.hmdb.ca / ), for example, the range of blood sugar levels in people of different ages is 1100-16440 μM, that is, 198.2-2961.8 μg / mL. Mixed standard solutions were prepared according to the reference concentration range of metabolites in the database. ), malic acid (800.0μg / mL), 2-hydroxyglutarate (800.0μg / mL), 2-ketoglutarate (800.0μg / mL), aconitic acid (800.0μg / mL), citric acid ( 800.0μg / mL), isocitric acid (800.0μg / mL), glucose (4000μg / mL), the mixed standard solution was diluted 1.33 times, 1.6 times, 2.67 times, 4 times, 8 times, 40 times, 80 times, 400 times, 800 times, 4000 times, 8000 times, 20000 times, 40000 times, 100000 times, 200000 times, 500000 times, 1000000 times, 2500000 times to get mixed standard solutions with different...
Embodiment 2
[0025] Detection of important metabolites related to glucose energy metabolism in normal human serum samples
[0026] Thaw normal human serum samples stored at -80°C, vortex for 10 seconds, pipette 50 μL accurately, add 200 μL of methanol solution containing internal standard, and vortex for 1 min. The concentrations of the internal standards in the methanol solution are: lactic acid-3,3,3-d 3 (100μg / mL), succinate-2,2,3,3-d 4 (0.2 μg / mL), fumarate-2,3-d 2 (0.1μg / mL), citrate-2,2,4,4-d 4 (5μg / mL), glucose- 13 C 6 ,1,2,3,4,5,6,6-d 7 (150 μg / mL). Centrifuge at 14,000 rpm for 15 min at 4°C, and take 200 μL of the supernatant solution to freeze-dry. Add 50 μL of methoxyamine solution (20 mg.ml, pyridine) to the freeze-dried sample, vortex for 30 s, sonicate for 1 min, and react in a 37°C water bath for 1.5 h; silylyl) trifluoroacetamide, vortexed for 10s, and reacted in a 37°C water bath for 1h; after the reaction was completed, centrifuged at 14000rpm for 10min at 4°C, an...
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Abstract
The invention discloses an analysis method for important metabolites related to glucose energy metabolism in a serum sample. The method uses methanol to extract, and after the sample is freeze-dried, performs two-step derivation for gas chromatography-triple quadrupole mass spectrometry analysis. The multiple reaction monitoring mode is used for detection. For low-content metabolites, the ion-pair with the best abundance is optimized as the quantitative ion-pair to improve sensitivity; for high-content metabolites, the ion-pair with low abundance is selected after optimization as the quantitative ion-pair. Quantify transitions to broaden their linear range for simultaneous analysis of high and low abundance metabolites. The analysis method of the invention has the advantages of simple pretreatment, good repeatability, high sensitivity, wide linear range and the like.
Description
technical field [0001] The invention relates to the fields of analytical chemistry and biochemistry, and is a method for analyzing important metabolites related to glucose energy metabolism in serum samples based on gas chromatography-triple quadrupole mass spectrometry combined with multiple reaction monitoring mode. Background technique [0002] Glucose is the transport form of sugar in the blood, occupies a major position in the body's glucose metabolism, and is an important substance for maintaining life activities. Glucose is transformed into pyruvate through glycolysis and produces energy, and pyruvate is completely oxidized to form CO through the tricarboxylic acid cycle and oxidative phosphorylation under aerobic conditions. 2 and H 2 O, producing a large amount of free energy; and converted into lactic acid under anaerobic conditions. Glycolysis to complete oxidation of glucose not only provides a large amount of free energy for the body, but also provides precurs...
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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/88
Inventor 许国旺周洋路鑫张俊杰
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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