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Method for synchronously analyzing base, nucleotide, organic acid, fatty acid, amino acid and saccharide metabolic product with one-step derivation method

A technology of simultaneous analysis and carbohydrate metabolism, applied in the field of simultaneous analysis of bio-based samples, it can solve the problems of lack of extraction and simultaneous analysis methods, impact on research level, lack of screening, etc., to achieve easy retrieval and identification, good repeatability , complete effect of mass spectrum information

Active Publication Date: 2012-08-01
曹君
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

In the 20th century, our country can only carry out preliminary detection of metabolic diseases such as genetic metabolism and nutrition, relying on the detection technology of developed countries such as Europe, Japan and the United States, and the specimens need to be sent to foreign cooperation units for diagnosis; in the past ten years, my country has introduced gas phase Chromatography-mass spectrometry (GC-MS) urine organic acid detection method only detects genetic metabolic diseases of organic acids, but lacks screening methods for genetic metabolic diseases such as bases, nucleosides, amino acids, and sugars. The sample pretreatment and simultaneous analysis methods for investigation, monitoring and research have also seriously affected the overall research level in the medical field of various metabolic diseases in my country.
[0003] Both the chromatographic column ion exchange method and the organic acid extraction method can only analyze organic acids in urine and the amount is small, and there are few literature reports of more than 200 substances; the method is to enrich the organic acids in urine by chemical methods. In order to achieve the purpose of detecting organic acids in urine; the existing traditional analysis methods, whether it is to analyze the amount of acidic, alkaline and neutral organic acids in the sample, or to analyze the types of substances in the sample, and the objectively existing substances in the sample Compared with the number and types of actual small molecules, there are serious shortages, and there is even a lack of methods for the extraction and simultaneous analysis of bases, nucleosides, amino acids, sugars and other substances

Method used

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  • Method for synchronously analyzing base, nucleotide, organic acid, fatty acid, amino acid and saccharide metabolic product with one-step derivation method
  • Method for synchronously analyzing base, nucleotide, organic acid, fatty acid, amino acid and saccharide metabolic product with one-step derivation method
  • Method for synchronously analyzing base, nucleotide, organic acid, fatty acid, amino acid and saccharide metabolic product with one-step derivation method

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Experimental program
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Effect test

Embodiment 1

[0035] 1) Sampling: Collect 4 mL of amniotic fluid in a test tube; centrifuge to remove particles such as meconium and sebum;

[0036] 2) Quantify the sample to be tested: take 100 μL of the amniotic fluid supernatant after centrifugation; measure the creatinine content in the supernatant to determine the creatinine concentration; take 300 μL of the amniotic fluid supernatant equivalent to a creatinine concentration of 2.50 μmoL / μL and store it in the sample preparation bottle. As a sample to be tested, spare;

[0037] 3) Pretreatment: remove phosphate ions and sulfate ions from the sample to be tested (using barium: add 0.1mol / L Ba(Cl) to the sample to be tested 2 or Ba(OH) 2 300 μL of barium solution such as aqueous solution, shake for 30 minutes and then centrifuge to collect the centrifugate a), remove urea (add urease 5 to the centrifuge a) u / μL aqueous solution 120μL, put in a water bath at 35°C-40°C for 30-35 minutes), remove protein (add 1200μL of methanol solution ...

Embodiment 2

[0044] 1) Sampling: Collect the patient's urine with a test tube or filter paper (eg figure 2 : is the mass spectrogram of children with methylmalonic aciduria): use ordinary test tubes or domestic No. 3 filter paper, cut into 3.0cmX8.0cm size. The morning urine or any time of urine soaked, filtered and dried, put it in a special filter paper drying bag for later use.

[0045] 2) Quantification of the sample to be tested: dry the filter paper biomatrix sample, elute with 3.0mL deionized double-distilled water or 1‰ hydrochloric acid-methanol solution, and collect and wash the urine sample after ultrasonic, centrifugation and other physical methods. Dehydration; take 100 μL of centrifuged urine; measure the creatinine content in the urine sample to determine the creatinine concentration; take 120 μL of urine equivalent to a creatinine concentration of 2.50 μmoL / μL and store it in a sample preparation bottle as a sample to be tested and set aside;

[0046] 3) Pretreatment: rem...

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Abstract

The invention discloses a method for synchronously analyzing a base, a nucleotide, an organic acid, a fatty acid, an amino acid and a saccharide metabolic product with a one-step derivation method. The method comprises the following steps of: performing a series of physicochemical method technical treatment such as ultrasonic treatment, centrifugation, urea removing, phosphorus removing, sulfur removing, protein removing, nitrogen blowing, vacuum concentration and drying, and trimethyl silylation derivation on biological substrate samples (such as urea, blood, cerebrospinal fluid (CSF), and tissue fluid); and detecting the biological substrate samples by adopting a gas chromatograph-mass spectrum combination technology. Due to the adoption of method, programmed treatment can be performed on the biological substrate samples simultaneously, and over 200 kinds of metabolic intermediate and final products of over five kinds of substances can be detected at one time.

Description

technical field [0001] The invention belongs to the field of synchronous analysis methods for bio-based samples, and in particular relates to a method for synchronously analyzing bases, nucleosides, organic acids, fatty acids, amino acids and sugar metabolites in bio-based samples using test tubes or filter paper sheets as specimen carriers. Background technique [0002] Human urine contains terminal or intermediate metabolites of many endogenous compounds such as bases, nucleosides, organic acids, fatty acids, amino acids, and sugars. These organic compounds have relatively high water solubility and renal clearance rate. The concentration is usually higher than that of serum and other tissue fluids, such as blood, CSF (cerebrospinal fluid) and tissue fluid, etc. Among them, urine specimens are the easiest to collect and are non-invasive techniques recognized by researchers at home and abroad. In the 20th century, our country can only carry out preliminary detection of genet...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
Inventor 王敏王益超曹君刘守
Owner 曹君
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