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Novel internal standard substance for detecting amino acid, organic acid or sugar phosphate metabolite in sample

A technology for metabolites and internal standards, which can be used in the field of new internal standards to solve the problems of unsatisfactory signal values ​​and low sensitivity.

Active Publication Date: 2019-02-05
EAST CHINA UNIV OF SCI & TECH
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Problems solved by technology

[0004] However, in the previous research process, the inventors found that when p-aminobenzoic acid was used as the internal standard of amino acid fermentation metabolites, organic acid fermentation metabolites, and phosphate sugar fermentation metabolites, the signal value was not ideal, as The internal standard of low concentration metabolites has low sensitivity and other issues

Method used

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  • Novel internal standard substance for detecting amino acid, organic acid or sugar phosphate metabolite in sample
  • Novel internal standard substance for detecting amino acid, organic acid or sugar phosphate metabolite in sample
  • Novel internal standard substance for detecting amino acid, organic acid or sugar phosphate metabolite in sample

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preparation example Construction

[0048] 3. Preparation of standard and internal standard

[0049] Standard mixtures of 17 kinds of amino acids, 7 kinds of organic acids and 8 kinds of phosphate sugars were prepared respectively, with the final concentration of 200 μmol / L, and stored at -80°C (Table 1).

[0050] Five internal standard solutions were prepared respectively: p-aminobenzoic acid (PABA), L-norleucine (NLE), malonic acid (PPD), adipic acid (HEX) and monobutyl phosphate (MBP). The concentration was 200 μmol / L and stored at -80°C.

[0051] Table 1. Abbreviation list of standard products

[0052]

[0053] Amino acid standard, organic acid standard, sugar phosphate standard (Sigma company); derivatizing agent TBDMS, derivatizing agent TMS, pyridine (Sigma company); methanol, ethanol (analytical grade AR, Shanghai Lingfeng Chemical Reagent Co., Ltd.); Pure water was produced by a Millipore ultrafiltration water maker.

[0054] 4. Sample handling

[0055] (1) Rapid sampling, quenching and extractio...

Embodiment 1

[0075] Embodiment 1, the selection of characteristic ion fragments

[0076] In the previous research process, the present inventors found that p-aminobenzoic acid (PABA) was used as the internal standard of fermentation metabolites, which was not ideal for the detection of low-concentration metabolites, and the signal value was not good. Therefore, the inventor attempts to search for some compounds that have not been used as internal standards in the past, and seeks compounds that are more ideal than p-aminobenzoic acid (PABA) as internal standards to replace 13 C isotopes perform qualitative or semi-quantitative analysis of metabolites, and at the same time need to integrate the corresponding peak area of ​​metabolites.

[0077]In this example, intracellular metabolites were analyzed by single ion monitoring (SIM) method to obtain higher selectivity and sensitivity. For fragment fragmentation methods, see Kiefer P, Nicolas C, Letisse F, et al. Determination of carbon labelin...

Embodiment 2

[0084] Embodiment 2, amino acid detection

[0085] The inventors investigated a large number of compounds, and selected a series of candidate compounds by comprehensively considering the molecular structures of amino acid metabolites and candidate compounds, combined with the inventor's research experience and the cost of preparation or purchase of the compounds. It is mainly used for initial screening or selection based on the following basic principles: 1) It does not exist in nature; 2) The peak time can be separated from other amino acids; 3) Similar to homologues, that is, similar in structure. After further screening, norleucine (NLE) was used as a better choice for amino acid detection compounds.

[0086] Add 100 μL of 200 μmol / L p-aminobenzoic acid (PABA) and norleucine (NLE) internal standards to 100 μL of 200 μmol / L 18 mixed amino acid standard respectively, and the sample processing procedures are consistent during the process (see Materials for details and methods...

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Abstract

The invention relates to a novel internal standard substance for detecting amino acid, organic acid or sugar phosphate metabolite in a sample. The invention discloses a novel compound suitably used asthe internal standard substance during the detection process of the amino acid, organic acid or sugar phosphate metabolite in a to-be-detected sample, and builds a detection method. By the internal standard substance and the detection method, the detection accuracy of a fermentation metabolite can be effectively improved, and the internal standard substance is simple and convenient in process andrelatively low in cost.

Description

technical field [0001] The invention belongs to the field of substance detection, and more specifically, the invention relates to a novel internal standard for detecting amino acids, organic acids or phosphate sugar metabolites in samples. Background technique [0002] In the fermentation industry, the detection of intracellular metabolites is generally required. Due to the characteristics of various types, low concentration and fast turnover of intermediate metabolites, gas chromatography-mass spectrometry (GC-MS) has been applied to the qualitative and quantitative analysis of these substances (GUOMeng-Lei, LIU Xiao-Yun, HUANG Ming-Zhi , et al. 13 C-assisted ultra-high performance liquid chromatography triple quadrupole mass spectrometry method for precise determination of intracellular metabolites in Pichia pastoris. Chinese J. Anal. Chem., 2016, 44(2):232-240). At present, the detection of intracellular metabolites mostly adopts the isotope dilution-mass spectrometry q...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88
CPCG01N30/88G01N2030/8818
Inventor 储炬刘秀王永红杭海峰田锡炜沈玉婷
Owner EAST CHINA UNIV OF SCI & TECH
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