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 microM, 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 beta-oxidation products, d7-C18:1 and d7-C16:0, were circulating with a maximal concentration ranging from 0.6 to 2.2 microM, with significant levels of d7-fatty acids detected for up to 72 h.
Highlights
Imbalanced fatty acid metabolism contributes significantly to the increased incidence of metabolic disorders
Analysis of natural fatty acids such as palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), and oleic (C18:1) acids in biological samples is commonly performed by gas chromatography coupled to flame ionization detection or electron impact/chemical ionization mass spectrometry [3]
Chemicals C16:0, C16:1, C18:0 and d7-oleic acid (C18):0, C18:1, and tricosanoic acid were obtained from Sigma-Aldrich (Milwaukee, WI) and were used without any Abbreviations: C16:0, palmitic acid; C16:1, palmitoleic acid; C18:0, stearic acid; C18:1, oleic acid; HPLC-ESI-MS, high-performance liquid chromatography-electrospray ionization-mass spectrometry; LOD, limit of detection; MRM, multiple reaction monitoring; SIM, single ion monitoring
Summary
Imbalanced fatty acid metabolism contributes significantly to the increased incidence of metabolic disorders. An alternative approach commonly used consists of dosing isotopically labeled fatty acids (2H or 13C) to generate a mass shift that allows selective detection of the tracer using either GC-MS or GC coupled to isotope ratio mass spectrometry, with minimal interference attributable to natural fatty acids [4,5,6,7,8] Tracers such as [13C]C18:0 and deuterium-labeled (d4)-linoleic acid were dosed orally, and the disappearance of parent compound and the formation of labeled metabolites were reported in rat, cat, and human over a short time period [4,5,6, 8]. The objective of this work was to find a novel and rapid analytical method using high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) for the analysis of natural and deuteriumlabeled fatty acids in plasma involving a simple sample preparation procedure that would significantly increase the sample preparation throughput without compromising repeatability, accuracy, and robustness.
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