A validated method for analyzing polyunsaturated free fatty acids from dried blood spots using LC–MS/MS

Omega-3 and omega-6 polyunsaturated free fatty acids (PUFA-FFA) are precursors to potent downstream lipid mediators that are regulators of inflammation. We describe the development and validation of a novel and sensitive method for quantification of individual PUFA-FFA in a dried blood spot using liquid chromatography tandem mass spectrometry (LC-MS/MS). Lipids were extracted from dried blood spot and six individual PUFA-FFA were quantified by LC-MS/MS using stable isotope dilution analysis with deuterated internal standards. PUFA-FFA concentrations in blood samples from 30 subjects were measured using the new method and compared to the traditional approach of thin layer chromatography followed by gas chromatography with flame ionization detection (TLC-GC). Responses for each PUFA-FFA were linear throughout a range of concentrations expected in clinical samples. Intra-day and inter-day variations for all PUFA-FFA were ≤ 16%. The concentrations of all PUFA-FFA measured by LC-MS/MS were positively correlated with measures of the same PUFA-FFA obtained by a traditional TLC-GC method. This novel method for the quantification of PUFA-FFA extracted from dried blood is sensitive and precise, and accurately measures levels of biologically important PUFA-FFA in blood.