LC/MS/MS Method Package for Short Chain Fatty Acids

For LabSolutions LCMS Software

Short-Chain Fatty Acids and Organic Acids Targeted for Analysis

LC/MS/MS Method Package for Short Chain Fatty Acids

The role gut microbiota plays in many metabolic disorders, such as obesity and diabetes, is gaining recognition. While many short-chain fatty acids (SCFA) are well characterized, the influence and impact of these SCFA is currently being studied. Analyzing SCFA using LCMS can be challenging due to their volatile and hydrophilic nature. This method package helps minimize method development time by providing MRM transitions for short-chain fatty acids (C2 to C5) that have been derivatized using 3-nitrophenylhydrazine (3-NPH). Analytical conditions are provided for key short-chain fatty acids and organic acids involved in the central metabolic pathways.

Short-Chain Fatty Acids
Acetic acid
Propionic acid
Butyric acid
Isobutyric acid
Valeric acid
Isovaleric acid
Organic Acids
2-oxobutyric acid 2-hydroxyglutaric acid α-ketoglutaric acid
β-hydroxybutyric acid Isocitric acid Oxaloacetic acid
Citric acid Glyoxylic acid Glycolic acid
Succinic acid Lactic acid Pyruvic acid
Fumaric acid Maleic acid Malonic acid
Malic acid

MRM Transition Settings

Selectivity is enhanced by identifying characteristic product ions for 3-nitrophenylhydrazine derivatives. 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.

Pretreatment Protocol Including Derivatization

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 the reaction proceeds under mild conditions. In order to efficiently carry out reactions with not only short-chain fatty acids, but with organic acids, this method package includes derivatization using 3-NPH instead of the more commonly used 2-NPH. 3-NPH derivatizations are stable, and quantitative analysis of the short-chain fatty acids or organic acids in the sample can be performed with the appropriate amount of derivatization reagent. Additionally, volatile compounds are not lost during sample preparation. This is because derivatization is occurring in water, and a dry down step is not required as a result. 

Derivatization reaction with 3-NPH

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. 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.

LC/MS/MS Method Package for Short Chain Fatty Acids

Remarks and Precautions

  1. LabSolutions LCMS Ver. 5.91 or later is required.
  2. For Research Use Only. Not for Use in Diagnostic Procedures.
  3. Shimadzu makes no warranty regarding the accuracy of information included in the database or the usefulness of information obtained from using the database.

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