Rapid measurement of deuterium-labeled long-chain fatty acids in plasma by HPLC-ESI-MS

Abstract

Imbalanced fatty acid metabolism contributes significantly to the increased incidence of metabolic disorders. Isotope-labeled fatty acids (2H, 13C) provide efficient means to trace fatty acid metabolism in vivo. This study reports a new and rapid method for the quantification of deuterium-labeled fatty acids in plasma by HPLC-MS. The sample preparation protocol developed required only hydrolysis, neutralization, and quenching steps followed by high-performance liquid chromatography-electrospray ionization-mass spectrometry analysis in negative ion mode using single ion monitoring. Deuterium-labeled stearic acid (d7-C18:0) was synthesized to reduce matrix interference observed with d5 analog, which improved the limit of detection (LOD) significantly, depending on the products analyzed. Linearity > 0.999 between the LOD (100 nM) and 30 μM, accuracy > 90%, precision > 88%, and adequate recovery in the dynamic range were obtained for d7-C18:0 and d7-oleic acid (C18:1). Upon oral dosing of d7-C18:0 in rats, the parent compound and its desaturation and β-oxidation products, d7-C18:1 and d7-C16:0, were circulating with a maximal concentration ranging from 0.6 to 2.2 μM, with significant levels of d7-fatty acids detected for up to 72 h.

Keywords

• deuterium-labeled stearic acid
• deuterium-labeled oleic acid
• deuterium-labeled palmitic acid
• high-performance liquid chromatography-electrospray ionization-mass spectrometry
• electrospray ionization negative mode
• quantification of plasma levels