Measurement of fatty acids with UPLC.

Because not all laboratories have access to mass spectrometry, we developed an ultraperformance liquid chromatography (UPLC) UPLC approach that involves UV detection to measure palmitoleic acid by modifying our previously published HPLC approach (Miles, 1987). We measured fatty acid concentrations using an Acquity UPLC System (Waters Corporation, Milford, MA) coupled with a heated (55° C) Acquity UPLC BEH C18 column (1.7μm; 2.1 × 150 mm; Waters Corporation, Milford, MA) and a ACQUITY UPLC Tuneable UV (TUV) detector set at 254 nm. The mobile phase was 72 % acetonitrile:28% water at a flow rate of 0.6 ml/min. The internal standard [²H₃₁]palmitic acid (2μg/sample) was used to allow measurement of arachidonic, eladic, linoleic, linolenic, myristic, palmiteladic, palmitic, palmitoleic, oleic, and stearic acids. The mixed FFA concentration standard solution was newly created for the purpose of this study using pure (as measured by LC/MS), gravimetrically measured fatty acids (Sigma-Aldrich, St. Louis, MO) suspended in fatty acid free bovine serum albumin (BSA) (Sigma-Aldrich).

We also measured plasma FFA concentrations on 30 of the same plasma samples using liquid chromatography/mass spectrometry (Persson, 2010, Song, 2019) for comparison with UPLC with UV detection.