High-Throughput RPLC-MS/MS Quantification of Short- and Medium-Chain Fatty Acids
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Date
2025
Publication Type
Book Chapter
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yes
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Abstract
Short- and medium-chain fatty acids (SMCFA) are monocarboxylic acids with a carbon chain length of 1–12 carbon atoms. They are mainly produced in humans by the gut microbiota, play crucial metabolic roles, are vital for intestinal health, and have multifaceted impact on immune and neurological functions. Accurate detection and quantification of SMCFA in different human biofluids is achieved using 3-nitro phenylhydrazine (3-NPH) derivatization of the free fatty acids followed by reverse phase liquid chromatography (RPLC) separation and detection by tandem mass spectrometry (MS/MS). Here, we describe the simultaneous measurement of 14 SMCFA and lactate in detail. All 3-NPH-SMCFA-hydrazones are separated in less than 5 min with an 8-min total run time (injection-to-injection). Linear dynamic range over 0.1–500 μM is achieved for most SCFAs, while it is 0.05–100 μM for MCFAs. Validation of the procedure depicts good linearity (R2 > 0.98) and repeatability (CV ≤ 20%). The lower limit of detection (LLOD) is 10–30 nM. The lower limit of quantification (LLOQ) is 50–100 nM for most analytes, while it is 0.5 μM for acetate. In conclusion, the method offers several benefits compared to alternative methods regarding throughput, selectivity, sensitivity, and robustness.
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Publication status
published
Book title
Clinical Metabolomics
Journal / series
Volume
2855
Pages / Article No.
195 - 207
Publisher
Humana
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Edition / version
Methods
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Subject
Personalized medicine; Immune system; Cancer therapy; Signaling molecules; Gut microbiome; Short-chain fatty acids; Branched-short-chain fatty acid; Medium-chain fatty acids; 3-Nitro-phenylhydrazine derivatization; Targeted metabolomics
Organisational unit
02207 - Functional Genomics Center Zurich / Functional Genomics Center Zurich