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Method for simultaneously analyzing amino acid and organic acid metabolite spectrum

An amino acid and organic acid technology, applied in the field of chromatographic analysis, can solve the problems of complicated operation, long analysis time, low sensitivity, etc., and achieve the effects of simple operation, good selectivity and strong universality.

Inactive Publication Date: 2009-05-13
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing methods for detecting amino acids mainly use ion exchange chromatography, high performance liquid chromatography, gas chromatography, capillary electrophoresis, etc. as separation means, and use ultraviolet or fluorescence detectors as detection methods. Ultraviolet absorption, not easy to vaporize, so derivatization steps are required when using the above four methods, the operation is cumbersome, the analysis time is long or the sensitivity is low, and it is difficult to detect dozens of common amino acids and organic acids at the same time

Method used

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  • Method for simultaneously analyzing amino acid and organic acid metabolite spectrum
  • Method for simultaneously analyzing amino acid and organic acid metabolite spectrum
  • Method for simultaneously analyzing amino acid and organic acid metabolite spectrum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Sample pretreatment: Take 200 μL of mixed standard solution, add 400 μL methanol, mix on a vortex mixer for 2 minutes, centrifuge at 12000 r / min for 10 minutes, remove 500 μL supernatant, and concentrate to dryness in a centrifugal concentrator, add 200 μL of water to the residue After fully dissolving, centrifuge at 12000r / min for 10min to take the supernatant for sample analysis.

[0020] Preparation of standard product and internal standard solution: Take about 1.0-8.0 mg of each amino acid and organic acid standard product, weigh them accurately, dissolve them in water or an aqueous solution containing a certain concentration of 1% ammonia water, and make concentrations of 1.0-8.0 mg / mL respectively standard stock solution. Before use, dilute with water to make a series of standard solutions. Take about 1.0 mg of palmatine hydrochloride standard substance, accurately weigh it, dissolve it in water, and make an internal standard solution with a concentration of 0.01...

Embodiment 2

[0027] Sample pretreatment: Take 200L normal human urine, add 400L methanol, mix on a vortex mixer for 2min, centrifuge at 12000r / min for 10min, pipette 500L supernatant, concentrate to dryness in a centrifugal concentrator, add 200L water to the residue After fully dissolving, centrifuge at 12000r / min for 10min to take the supernatant for sample analysis.

[0028] Capillary electrophoresis conditions: the buffer solution contains 4.0% formic acid and 15% methanol, the separation voltage is 15kV, and the separation temperature is 25°C. Sample injection: inject the sample solution for 40s under 50mbar pressure.

[0029] Mass spectrometry conditions: the same as in Example 1.

[0030] Amino acids and organic acids in normal human urine were analyzed by capillary electrophoresis-mass spectrometry, and the results can be found in the appendix image 3 with Figure 4 . Figure 4 Middle: 1: Glycine, 2: β-Alanine, 3: Alanine, 4: β-Aminoisobutyric Acid, 5: 3-Hydroxybutyric Acid, 6...

Embodiment 3

[0032] Sample pretreatment: Take 200L of urine from breast cancer patients, add 400L methanol, mix on a vortex mixer for 5min, centrifuge at 12000r / min for 10min, pipette 500L of supernatant, concentrate to dryness in a centrifugal concentrator, add 200L of water to the residue After fully dissolving, centrifuge at 12000r / min for 10min to take the supernatant for sample analysis.

[0033] Capillary electrophoresis conditions: the buffer contains 4.0% formic acid and 2.5% methanol, the separation voltage is 18kV, and the separation temperature is 25°C. Sample injection: 14% concentrated ammonia water (v / v) was injected under 50mbar pressure for 3s, and then the sample solution was injected under 50mbar pressure for 40s.

[0034] Mass spectrometry conditions: SIM mode detection, m / z values ​​are shown in Table 1; the sheath liquid is a solution containing 80% methanol and 0.05% formic acid, the flow rate is 3L / min; the nebulizer gas pressure is 2psig; the drying gas flow rate an...

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Abstract

The invention provides a method for testing a spectrum of amino acid and organic acid in urine, wherein, a conventional protein deposition treatment is carried out on biological samples; capillary electrophoresis is adopted as a separate method; and a mass spectrometry detector which has good universality and high sensitivity is adopted as a detector. The method has the advantages of high precision and recovery, derivatization omission, simple operation, high sensitivity and good selectivity and universality and can simultaneously analyze 22 amino acids and 5 organic acids in the biological samples.

Description

technical field [0001] The invention relates to chromatographic analysis technology, and in particular provides a method for simultaneously analyzing amino acid and organic acid metabolite profiles in biological samples through capillary electrophoresis-mass spectrometry. Background technique [0002] Amino acids and organic acids are important metabolites in metabolomics research, and are closely related to the metabolism of sugar, lipid, protein and nucleic acid. Studies have shown that changes in the concentration of amino acids and organic acids in the human body are closely related to various diseases. The existing methods for detecting amino acids mainly use ion exchange chromatography, high performance liquid chromatography, gas chromatography, capillary electrophoresis, etc. as separation means, and use ultraviolet or fluorescence detectors as detection methods. Ultraviolet absorption, not easy to vaporize, so the above four methods all need derivatization steps, cum...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N33/48
Inventor 王书芳沈朋邵青刘昌孝程翼宇
Owner ZHEJIANG UNIV
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