User interviews December 2020
Our field of activity is contract research and testing for life sciences industries including pharma, biotech, nutraceuticals, environmental sciences, food&agri testing, and diagnostics.
For LabSolutions™ LCMS Software
Of the short-chain fatty acids produced by intestinal bacteria, acetic acid, propionic acid, and butyric acid are well known, and it has been reported that there are some connections between them and lifestyle-related diseases such as obesity and diabetes. Generally speaking, short-chain fatty acids are highly volatile and highly hydrophilic. This makes it dificult to perform LCMS analysis using a conventional reverse phase system. For that reason, this method package targets short-chain fatty acids (C2 to C5) that have been derivatized using 3-nitrophenylhydrazine (3-NPH). Through making settings for MRM transitions, it can be used for the simultaneous analysis (of 22 components), including organic acids related to the central metabolic pathways.
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When making MRM transition settings, selectivity can be enhanced by specifying characteristic product ions for 3-nitrophenylhydrazine derivatives. Note that some organic acids have a carbonyl group derived from ketones, so 3-nitrophenylhydrazine derivatives that reacted with carboxylic acid and the carbonyl group are targeted for MRM transition.
The entire protocol necessary for pretreatment, including the derivatization step using 3-nitrophenylhydrazine, is contained in the Instruction Manual for this method package. By following these procedures, an analyst should be able to perform the entire process, from derivatization to data acquisition and data analysis, immediately after the package is introduced.
For the derivatization using 3-nitrophenylhydrazine, carbodiimide is used as a condensing agent, and reaction proceeds under mild conditions. Moreover, in order to efficiently carry out reactions with not only short-chain fatty acids, but with organic acids as well, this method package includes derivatization using 3-NPH instead of the more commonly used 2-NPH. Also, 3-NPH derivatizations after reactions are stable, and quantitative analysis of the short-chain fatty acids or organic acids in the sample can be performed through reaction with an appropriate amount of derivatization reagent. Additionally, as reactions take place in water solutions using this derivatization reagent, the sample will not be dried and volatile components will not be lost.
Derivatization Reaction with 3-nitrophenylhydrazine (3-NPH)
The following example shows an analysis using the LCMS-8060 of a standard mixed solution of short-chain fatty acids/organic acids. An MRM chromatogram of short-chain fatty acids/organic acids derivatized using 3-NPH is shown. As can be seen, sufficient separations of the components, such as the structural isomers butyric acid, isobutyric acid, fumaric acid, maleic acid, citric acid, and isocitric acid were obtained. This method package includes MRM transitions that were formulated beforehand in order to achieve optimization for 3-NPH derivatizations for short-chain fatty acids/organic acids. As a result, short-chain fatty acids (C2 to C5) and organic acids representative of central metabolic pathways, which are difficult to analyze with LCMS using a conventional reversed phase system, can be analyzed simultaneously.
Remarks and Precautions
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User interviews December 2020
Our field of activity is contract research and testing for life sciences industries including pharma, biotech, nutraceuticals, environmental sciences, food&agri testing, and diagnostics.
LCMS Food Safety Application is now available.
Application Note: "Analysis of Pharmaceuticals' Impurity - Regulations and Analysis for Carcinogenic Substances -"
Application Note: "Various Analysis Techniques for Organic Acids and Examples of Their Application"
Shimadzu has released the Metabolites Method Package Suite.
Provides ready-to-use methods for over 1900 metabolites
This suite allows comprehensive analysis of over 1900 metabolites without the need for investigation of separation conditions, MRM optimization or parameter settings.
Direct Probe Ionization Mass Spectrometer - APPLICATION NOTEBOOK -