Profiling and quantitative analysis of underivatized fatty acids in Chlorella vulgaris microalgae by liquid chromatography-high resolution mass spectrometry.

Chlorella vulgaris is a popular microalga used for biofuel production; nevertheless, it possesses a strong cell wall that hinders the extraction of molecules, especially lipids within the cell wall. For tackling this issue, we developed an efficient and cost-effective method for optimal lipid extraction. Microlaga cell disruption by acid hydrolysis was investigated comparing different temperatures and reaction times; after hydrolysis, lipids were extracted with n-hexane. The best recoveries were obtained at 140°C for 90 min. The microalgae were then analyzed by an untargeted approach based on liquid chromatography with high-resolution mass spectrometry, providing the tentative identification of 28 fatty acids. First, a relative quantification on the untargeted data was performed using peak area as a surrogate of analyte abundance. Then, a targeted quantitative method was validated for the tentatively identified fatty acids, in terms of recovery (78-100%), intra- and interday relative standard deviations (<10 and <9%, respectively) and linearity (R2  > 0.98). The most abundant fatty acids were palmitic, palmitoleic, oleic, linoleic, linolenic, and stearic acids.