Long chain fatty acids analysis of intertidal biofilm by direct injection liquid chromatography time of flight mass spectrometry

Abstract

The critical importance of mono- and polyunsaturated fatty acids (FAs) in a variety of biological functions, including animal nutrition and as an environmental stress monitor, is well recognized. However, while methods exist for monitoring of fatty acids, few are specific either to the profile of a microphytobenthos matrix or practical in application to multiple, diverse intertidal biofilm sample sets. In the current study, a sensitive liquid chromatography (LC) quadrupole time of flight mass spectrometry (QTOF) method was developed for the quantitative analysis of 31 FAs specific to intertidal biofilm, a thin mucilaginous layer of microalgae, bacteria, and other organisms on the surface of coastal mudflats, which provide a rich source of FAs for migratory birds. Preliminary screening of diverse biofilm samples collected from shorebird feeding grounds highlighted eight saturated (SFA), seven monounsaturated (MUFA), and sixteen polyunsaturated FAs (PUFA) that were selected for analysis. Improved method detection limits in the range 0.3 ngmL^-1 to 2.6 ngmL^-1 were achieved, excepting for stearic acid at 10.6 ngmL^-1. These excellent results were obtained without use of complex sample extraction and clean-up procedures undertaken by other published methods. An alkaline matrix of dilute aqueous ammonium hydroxide with methanol was shown to be selective for extraction and stability of the more hydrophilic fatty acid components. The direct injection method showed excellent precision and accuracy both during validation and application to hundreds of real-world intertidal biofilm samples from the Fraser River estuary (British Columbia, Canada) and other areas of the region frequented by shoreline birds.