(P26-019-23) Robust and Improved GCMS Method for the Simultaneous Analysis of Diverse Fatty Acids in Large Cohorts

Objectives: The aim of this study was to optimize a gas chromatography-mass spectrometry (GCMS) methods that involve one-step plasma/RBC fatty acid methyl esterification (FAME) as a rapid and robust analytical tool for simultaneous analysis of diverse fatty acids suitable for large numbers of samples.

Methods: The current method was optimized and validated for direct analysis of fatty acids by GCMS in human plasma and RBCs (Plasma n=550, RBC, n=100). Initially, a standard of 28 FA (27 FA from Nu-Chek Prep, Inc (Part: GLC 87) and Methyl-15-Methyl hexadecanoate from Larodan AB Corp (Part:21-1615) were procured, combined, and used as a calibrator. A stock solution was prepared prior to the extraction using acetyl chloride and methanol in addition to methyl tridecanoate as an internal standard. Stock solution was added to human plasma/RBCs samples in a screw-capped glass tube and heated at 100°C on a heating block for 60 mins, then allowed to cool, vortexed, dried under nitrogen and later extracted in hexane. The extracts were processed in a Shimadzu GCMS-QP2010 SE fitted with a length of 60 m, 0.20 µm thickness and 0.25 mm diameter column. The column oven temperature was set at 180°C ramped to 220°C for 34 mins, and total flow: 34.1 mL/min for a split ratio of 50:50. The MS parameters were set to ion source and interface temp at 205°C and 245°C, detector threshold was 1000 using scan mode, event time 0.30 sec start at m/z: 45 and end at m/z: 400.

Results: The FA peaks were uniquely sharp and well separated. The data obtained from the standard consisted of 28 peaks which were integrated and identified using the NIST library were registered to create a unique compound table that was used as a reference for qualitative and quantitative analysis of FA in human plasma/RBC. The method was further successfully validated for linearity, accuracy precision, LOD and LOQ.

Conclusions: The current method was shown to be rapid and precise for analysis of fatty acids in human plasma/RBC in a large population cohort compared to the traditional Bligh & Dyer Extraction method.

Funding Sources: USDA National Institute of Food and Agriculture and New Hampshire Agricultural Experiment Station (Hatch Project #NH00688).
The first two authors equally contributed.