A detection method for metabolites in urine and a kit

A detection method and technology for metabolites, applied in the field of biochemical detection, can solve the problems of requiring standard products, taking a long time, and detecting a single type of metabolites, and achieving the effect of appropriate polarity and good thermal stability.

Active Publication Date: 2017-11-10
GUANGZHOU RIBOBIO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Based on this, the present invention provides an accurate, convenient, high-efficiency, high-throughput urine metabolite detection method for the shortcomings o

Method used

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  • A detection method for metabolites in urine and a kit
  • A detection method for metabolites in urine and a kit
  • A detection method for metabolites in urine and a kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] A method for detecting metabolites in urine of the present embodiment, comprising the following steps:

[0077] (1) Sampling: Take 200 μL of fresh urine as the urine sample to be tested.

[0078] (2) Urea removal: Add 20 μL of urease aqueous solution containing 100 U urease to the urine sample to be tested, and incubate at 37° C. for 10 minutes to obtain urine A.

[0079] (3) Sulfur and phosphorus removal: Add 0.1 mg of active adsorbent to urine A, and ultrasonically for 2 minutes to obtain urine B; the active adsorbent is activated alumina.

[0080] (4) Precipitate protein: add 1.7mL protein precipitation reagent (frozen methanol) to urine B, mix well and centrifuge to precipitate protein, and take the supernatant, namely urine C.

[0081] (5) Step-by-step drying: add 5 mg of anhydrous sodium sulfate to urine C, mix well and centrifuge, then take the supernatant and dry it with a nitrogen blower to obtain urine residue D.

[0082] (6) Microwave-assisted oximation: Ad...

Embodiment 2-4

[0088] A kind of detection method of metabolite in the urine of embodiment 2-4, used urine sample is identical with embodiment 1,

[0089] Except the difference shown in the table below, all the other steps are the same as in Example 1. For chromatogram see Figure 2-Figure 4 , see Table 1 for quantitative results.

[0090] Example

Example 2

Example 3

Example 4

active adsorbent

2mg activated alumina

5mg activated alumina

100 μL BaCl 2 Aqueous solution 0.1mol / L

[0091] Table 1 embodiment 1-4 analysis result contrast

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099] As can be seen from Table 1, compared with Example 4, the loss of target metabolites caused by the use of activated alumina to remove sulfur and phosphorus in Examples 1-3 is small, especially for low-content metabolites such as the target of the reference interval<5 The results of metabolite determination are more accurat...

Embodiment 5-8

[0101] A kind of detection method of metabolite in the urine of embodiment 5-8, the urine sample used is identical with embodiment 1, except that the amount of activated alumina added in step (3) is 1mg and the difference shown in the table below, the rest Step is with embodiment 1. For chromatogram see Figure 5-Figure 8 , see Table 2 for quantitative results.

[0102] Example

Example 5

Example 6

Example 7

Example 8

protein precipitation reagent

frozen ethanol

Chilled propanol

Frozen butanol

Refrigerated Methanol: Acetonitrile (V / V, 4 / 1)

[0103] Table 2 embodiment 5-8 analysis results (only show 40 data at random as illustration)

[0104]

[0105]

[0106] It can be seen from Table 2 that ethanol, propanol, butanol, methanol / acetonitrile (V / V, 4 / 1) etc. are selected as protein precipitation reagents, and the effect is not much different. The inventor has found through a large number of experiments that the preci...

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Abstract

The invention relates to a detection method for metabolites in urine. The method includes (1) a step of removing impurities from a urine sample, namely a step of removing urea, sulfur and phosphorus and precipitating protein to obtain urine C; (2) a drying step, namely a step of drying the urine C to obtain urine residues D; (3) a step of oximation, namely a step of subjecting beta-carbonyl in acid or alcohol metabolites in the urine residues D to oximation by utilizing an oximation agent to obtain urine E; (4) a step of derivatization, namely a step of subjecting the urine E to derivatization with an derivatization agent to obtain a urine sample F to be detected, with the derivatization agent being an agent mixture comprising a silanization agent, a catalyst and a catalyst stabilizer, and with the catalyst stabilizer being selected from dithioerythritol, dithiothreitol, 1-propanethiol, ethanethiol and 2-mercaptoethanol; and (5) a step of detection, namely a step of detecting the urine sample F to be detected by adopting a gas chromatography-mass spectrography process. The method has advantages of a low sample using amount, simple pretreatment, short time, capability of detecting a plurality of varieties of compounds, rapid and accurate result analysis, and the like.

Description

technical field [0001] The invention relates to the technical field of biochemical detection, in particular to a method and kit for detecting metabolites in urine by gas chromatography-mass spectrometry. Background technique [0002] Urine contains a large number of metabolic compounds related to human life activities. By detecting the content of these metabolic compounds, it can reflect the metabolic conditions and disease information of the human body. It is useful for the research and diagnosis of diseases, especially the diagnosis, treatment and Intervention has helped. However, in the field of urine metabolite detection, the existing technology has the following deficiencies: [0003] First of all, most of the existing methods detect organic acids (see Chinese patent, CN 102621262 A, 2012.08.01), which detects a relatively single type of metabolites, which cannot meet the requirements of screening multiple types of metabolites in one analysis. [0004] Secondly, the p...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/025
Inventor 张必良胡俊杰吕小侠叶奕栋
Owner GUANGZHOU RIBOBIO
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