Automated Trimethyl Sulfonium Hydroxide Derivatization Method for High-Throughput Fatty Acid Profiling by Gas Chromatography–Mass Spectrometry

Abstract

Fatty acid profiling on gas chromatography–mass spectrometry (GC–MS) platforms is typically performed offline by manually derivatizing and analyzing small batches of samples. A GC–MS system with a fully integrated robotic autosampler can significantly improve sample handling, standardize data collection, and reduce the total hands-on time required for sample analysis. In this study, we report an optimized high-throughput GC–MS-based methodology that utilizes trimethyl sulfonium hydroxide (TMSH) as a derivatization reagent to convert fatty acids into fatty acid methyl esters. An automated online derivatization method was developed, in which the robotic autosampler derivatizes each sample individually and injects it into the GC–MS system in a high-throughput manner. This study investigated the robustness of automated TMSH derivatization by comparing fatty acid standards and lipid extracts, derivatized manually in batches and online automatically from four biological matrices. Automated derivatization improved reproducibility in 19 of 33 fatty acid standards, with nearly half of the 33 confirmed fatty acids in biological samples demonstrating improved reproducibility when compared to manually derivatized samples. In summary, we show that the online TMSH-based derivatization methodology is ideal for high-throughput fatty acid analysis, allowing rapid and efficient fatty acid profiling, with reduced sample handling, faster data acquisition, and, ultimately, improved data reproducibility.

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“…The top apolar phase was transferred to a new tube and dried at 4 °C using refrigerated Labconco CentriVap Concentrator attached to − 84 °C CentriVap Cold Trap (Kansas City, MO, USA). Metabolites were derivatized by combining 30 μL of trimethyl sulfonium hydroxide (Sigma-Aldrich) (20 mg/mL) and 60ul of methyl tert-butyl ether (Sigma- Aldrich) in an automated manner as described in previous publication [ 23 ] A small volume (3 μL) of derivatized samples were injected via spitless injection into a Thermo Scientific Thermo Scientific Trace 1310 gas chromatograph loaded with a Phenomenex Zebron ZB- (Torrance, CA, USA) 1 ms fused silica capillary column (length = 30 m, I.D. = 0.25 mm, film = 0.25 μm), which was connected to a Thermo ISQ (Thermo Fisher Scientific) single quadrupole mass spectrometer.…”

“…Palmitate retention time was confirmed by standards. Total ion current peaks of different metabolites were normalized to the internal standard 3 deuterium labeled heptadecanoic acid [ 23 ].…”

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“…The processing temperatures are lower, which may reduce unwanted reactions. TMSH is commonly used for the derivatization of free fatty acids prior to GC analysis (Ishida et al, 1999;Chiu and Kuo, 2020;Gries et al, 2021). However, it has been reported that TMSH is not efficient at derivatizing some of the unsaturated fatty acids (Ostermann et al, 2014).…”