A Comprehensive GC-MS Sub-Microscale Assay for Fatty Acids and its Applications

Abstract

Fatty acid analysis is essential to a broad range of applications including those associated with the nascent algal biofuel and algal bioproduct industries. Current fatty acid profiling methods require lengthy, sequential extraction and transesterification steps necessitating significant quantities of analyte. We report the development of a rapid, microscale, single-step, in situ protocol for GC-MS lipid analysis that requires only 250 μg dry mass per sample. We furthermore demonstrate the broad applications of this technique by profiling the fatty acids of several algal species, small aquatic organisms, insects and terrestrial plant material. When combined with fluorescent techniques utilizing the BODIPY dye family and flow cytometry, this micro-assay serves as a powerful tool for analyzing fatty acids in laboratory and field collected samples, for high-throughput screening, and for crop assessment. Additionally, the high sensitivity of the technique allows for population analyses across a wide variety of taxa.

Fig. 1

GC–MS total ion chromatogram of a representative sample total ion chromatogram of a sample taken from the Cell Cycle Experiment (D0, replicate 1) showing an absence of interfering compounds. Abundance is electron multiplier counts. The three visible internal standards, in order of elution, are: Acenaphthene d-10, Phenanthrene d-10, and Chrysene d-12

Fig. 2

A comparison of cell cycle parameters Left axis: filled diamonds culture density (Coulter Counter). Right axis: filled squares neutral lipid concentration in relative fluorescent units (RFU), measured using BODIPY 505/515 dye; filled triangles Fatty acid per cell (picograms); filled circles fatty acid per liter of culture in mg using GC–MS. Error (by 95% confidence interval) for all data points is less than ~5%. Samples were run in quadruplicate

Fig. 3

Lipid bodies in Chrysochromulina sp. Cells were maintained on a 12 h light:12 h dark photoperiod. D11.5 cells were harvested at 11.5 h into the dark period. L11.5 cells were harvested at 11.5 h into the light. Cells were stained with BODIPY 505/515 dye (green) as described in “Materials and methods”. The chloroplast auto-fluoresces red. The scale bar is 10 μm. The cell volumes (computed from mean diameter) at these points were about 23 μm³ at D11.5 and 44 μm³ at L11.5. Excitation was at 450–490 nm and emission wavelengths were imaged through a 515 nm long-pass filter (color figure online)

Fig. 4

Fatty acid profiles over the 12 h light:12 h dark photoperiod of Chrysochromulina sp. a The relationship between cell volume (computed from cell diameter) and fatty acid content. b–d Changes in fatty acid composition as measured by GC–MS. Y axis represents the percent of total fatty acids. X axis represents the hour in the light/dark cycle