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Method for simultaneously determining tryptophan, kynurenine and kynurenicacid by using high performance liquid chromatography-fluorescence method

A high-performance liquid chromatography and kynurenic acid technology, applied in the field of analytical chemistry, can solve the problems of cumbersome and time-consuming experimental operations, and achieve the effects of simple operation, true and accurate determination, and rapid separation

Inactive Publication Date: 2010-07-14
THE SECOND XIANGYA HOSPITAL OF CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method of simultaneous detection of tryptophan, kynurenine and kynurenic acid reported in foreign countries requires the combined application of ultraviolet and fluorescence detectors for detection, which is cumbersome and time-consuming
At present, there is no report on the simultaneous determination of tryptophan, kynurenine and kynurenic acid by high performance liquid chromatography fluorescence method at home and abroad.

Method used

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  • Method for simultaneously determining tryptophan, kynurenine and kynurenicacid by using high performance liquid chromatography-fluorescence method
  • Method for simultaneously determining tryptophan, kynurenine and kynurenicacid by using high performance liquid chromatography-fluorescence method
  • Method for simultaneously determining tryptophan, kynurenine and kynurenicacid by using high performance liquid chromatography-fluorescence method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] 1. Establishment of chromatographic conditions and preparation of standard solutions

[0038] Preparation of standard solution

[0039] Accurately weigh Trp100.0mg, Kyn108.0mg and Kyna99.0mg, dissolve and dilute to 100ml with 0.312mol / L perchloric acid solution, mix well, and make 4900μmol / L Trp, 1960μmol / L Kyn and 104.67μmol / L respectively Standard stock solution of L Kyna, aliquoted and stored in a -20°C refrigerator for later use. Before use, take the standard stock solution and add ultrapure water to make a mixed standard solution (containing Kyn 0.98μmol / L, Kyna 26.17nmol / L and Trp 24.5μmol / L).

[0040] Second, the choice of high performance liquid chromatography conditions

[0041] 1. Selection of detection wavelength

[0042] Using the manual stop-flow technique, the maximum excitation light wavelength is obtained by scanning the excitation light spectrum (range 200-450nm) and emission light spectrum (range 300-550nm) of Kyn, Kyna and Trp respectively: λex Tr...

Embodiment 2

[0054] Sample testing

[0055] 1. Detection of mixed standard solution

[0056] Take the standard stock solution prepared above and add ultrapure water to make a mixed standard solution (containing Kyn 0.98 μmol / L, Kyna 26.17nmol / L and Trp 24.5 μmol / L) for analysis. High performance liquid chromatography conditions: what the chromatographic column selects is Hypersil C18 250mm×4.6mm i.d., 7μm chromatographic column, mobile phase: acetonitrile containing zinc acetate 0.2mol, acetic acid 8.3mmol and 2.5v / v% in every liter of mobile phase, flow rate: 1.5ml / min; injection volume: 20μl, column temperature: 25°C, adjustable wavelength setting: 0-11min excitation wavelength 365nm, emission wavelength 480nm; 11-15.5min excitation wavelength 344nm, emission wavelength 404nm; 15.5-20min excitation light wavelength 254nm, emission light wavelength 404nm ( figure 1 ).

[0057] 2. Detection of serum samples

[0058] Take 200 μl of serum samples into an eppendorf tube, add an equal amou...

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Abstract

The invention relates to a method for simultaneously determining tryptophan, kynurenine and kynurenicacid by using high performance liquid chromatography-fluorescence method, which comprises the following steps: a. Hypersil C18 chromatographic column (250x4.6mm, particle size of 7 Mum) is adopted; b. each liter mobile phase contains 0.2 mol of zinc acetate, 8.3mol of acetic acid and 2.5v / v% of acetonitrile; c. flow velocity is 1.5ml / mim, sample size is 20 Mul, column temperature is 25 DEG C; and d. adjustable wavelength is set under the following conditions: at 0-11min, excitation wavelength is 365nm, emission wavelength is 480nm; at 11-15.5 min, excitation wavelength is 344nm, emission wavelength is 404nm; and at 5-20min, excitation wavelength is 254nm, and emission wavelength is 404nm. The method has simple method, quick separation, no derivation pre-column and post-column, high sensitivity and real and accurate result, and can simultaneously determine tryptophan, kynurenine and kynurenicacid.

Description

technical field [0001] The present invention belongs to the field of analytical chemistry. It relates to a chromatographic detection method, in particular to a method for the simultaneous determination of tryptophan, kynurenine and kynurenic acid by high performance liquid chromatography fluorescence method, that is, the high performance liquid chromatography fluorescence method using on-line derivation and adjustable wavelength A method for the simultaneous determination of tryptophan, kynurenine and kynurenic acid. Background technique [0002] Tryptophan (Trp) is one of the essential amino acids for human body. The Trp ingested in the body is mainly metabolized through the kynurenine (Kyn) pathway, that is, Trp is metabolized by tryptophan 2,3-dioxygenase (L-tryptophan 2,3-dioxygenase, TDO) or indoleamine 2,3 Formylkynurenine is generated under the action of dioxygenase (indoleamine2, 3-dioxygenase, IDO), and the latter quickly generates Kyn under the action of kynureni...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/36G01N30/74
Inventor 唐爱国项忠元罗昔波肖乐东皮兰敢周前选任亚萍
Owner THE SECOND XIANGYA HOSPITAL OF CENT SOUTH UNIV
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