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Originally published In Press as doi:10.1194/jlr.D600037-JLR200 on October 4, 2006

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Journal of Lipid Research, Vol. 48, 252-259, January 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Methods

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

Sébastien Gagné1, Sheldon Crane, Zheng Huang, Chun Sing Li, Kevin P. Bateman and Jean-François Lévesque

Merck Frosst Canada & Co., Medicinal Chemistry Department, Kirkland, Quebec, Canada H9H 3L1

Published, JLR Papers in Press, October 4, 2006.

1 To whom correspondence should be addressed. e-mail: sebastien_gagne{at}merck.com

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 ß-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.

Supplementary key words 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

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


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