Method for detecting neonatal metabolites based on ultra performance liquid chromatography-tandem mass spectrometry

A technology of ultra-high performance liquid chromatography and tandem mass spectrometry, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of repeatability, accuracy, low sensitivity, low accuracy, and large sample volume, and achieve high sensitivity and high Accuracy, effect of small sample volume

Active Publication Date: 2018-10-12
SOUTHERN MEDICAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional examination of metabolite levels mainly relies on laboratory-specific examinations. One experiment can only detect one metabolite. This method requires a large amount of samples, and the cycle is long and the accuracy is low

Method used

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  • Method for detecting neonatal metabolites based on ultra performance liquid chromatography-tandem mass spectrometry
  • Method for detecting neonatal metabolites based on ultra performance liquid chromatography-tandem mass spectrometry
  • Method for detecting neonatal metabolites based on ultra performance liquid chromatography-tandem mass spectrometry

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, blood collection, transportation and preservation of dried blood sheet:

[0036] Blood samples were collected 48 hours after the newborns were fed with breast milk. Heel blood was collected with a blood collection needle and dropped on a filter paper sheet (Whatman 903), allowing the blood to naturally penetrate into an 8mm blood spot. Collect 3 blood spots for reexamination. Air-dried at room temperature, sealed in a sealed bag, and transported at room temperature. If it cannot be sent for inspection in time, it should be stored at 2-8°C for short-term storage, and -20°C for long-term storage.

Embodiment 2

[0037] Embodiment 2, preparation of related reagents:

[0038] 2.1 Dissolution of internal standard stock solution: Add 1ml of 50% methanol aqueous solution to the bottles containing NSK-A and NSK-B internal standard substances respectively for dissolution, and ultrasonicate for 20min to prepare NSK-A internal standard stock solution and NSK-B internal standard stock solution. The standard stock solution can be stored at 4°C for 1 month; return to room temperature before use, of which NSK-A and NSK-B contain 12 kinds of amino acid isotope internal standards and 8 kinds of carnitine isotope internal standards, Cambridge Isotope Laboratories (CIL ) The components of NSK-A provided are shown in Table 1, and the components of NSK-B are shown in Table 2.

[0039] 2.2 Derivatization reagents: Add acetyl chloride to n-butanol in a ratio of 1:9 in an ice-water bath and mix. The reagents are prepared and used immediately.

[0040] 2.3 Mobile phase complex solution: 80% acetonitrile aq...

Embodiment 3

[0046] Embodiment 3, pretreatment of dried blood sample

[0047] 3.1. Turn on the 96-well plate centrifuge and wait for the instrument to stabilize; turn on the nitrogen blower and incubator, and adjust the ready-to-run status; adjust the LC-MS / MS run status.

[0048] 3.2 Punching: Take the sample blood sheet and quality control blood sheet out of the -20°C refrigerator and place them at room temperature, punch holes (3.2 mm) with a puncher, and take blood samples that are evenly dispersed within the range of the dotted line (no blood samples in the center of the blood sheet) Obvious agglutinated blood clot), put into 96-well filter plate.

[0049] 3.4 Preparation of internal standard working solution: first add 11ml of methanol to the bottle, add 55μl of NSK-A internal standard stock solution and NSK-B internal standard stock solution respectively, shake for 2min after preparation, and mix well.

[0050] 3.5 Quickly add internal standard working solution, 100 μl per well, an...

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Abstract

The invention provides a method for joint detection of metabolic markers based on ultra high performance liquid chromatography-tandem mass spectrometry. Qualitative and quantitative analysis of more than 134 metabolites including amino acids and carnitines is completed only through one-time detection of only an extremely low amount, being 3 [mu]L, of a blood sample by adopting an isotope-labeled amino acid standard substance and an isotope-labeled carnitine standard substance as internal standard substances, a high-concentration quality control blood tablet and a low-concentration quality control blood sheet as positive control substances and an acetonitrile-water solution as a mobile phase through reasonably setting parent ion scanning, neutral loss scanning and multi-reaction ion monitoring scanning combined ultra performance liquid chromatograph-tandem mass spectrometer detection conditions. The method has the advantages of small sample volume, high sensitivity, high accuracy, highthroughput and high repeatability, and is suitable for large-scale analysis of clinical dried blood tablet samples.

Description

technical field [0001] The invention relates to the field of metabolite detection, in particular to a method for detecting neonatal metabolites based on ultra-high performance liquid chromatography tandem mass spectrometry technology. Background technique [0002] Many life activities in cells occur at the level of metabolites, such as cell signaling, energy transfer, intercellular communication, etc. are all regulated by metabolites. The regulation of metabolites involves many substances such as amino acids, organic acids, fatty acids, urea cycle, carbohydrates, and steroids. The variety of them makes the metabolic network intricate. Through the detection and analysis of metabolites, it can be used to find biomarkers of diseases and establish Metabolic fingerprinting. [0003] The traditional examination of metabolite levels mainly relies on laboratory-specific examinations. One experiment can only detect one metabolite. This method requires a large sample volume, and the ...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/042
Inventor 宁云山王宗珊周景权李妍
Owner SOUTHERN MEDICAL UNIVERSITY
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