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

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. 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. Fatty acid biosynthesis and degradation are of increasing interest in a context in which obesity affects a growing proportion of the population of the United States and is associated with higher risks of medical problems (1Bergen W.G. Mersmann H.J. Comparative aspects of lipid metabolism: impact on contemporary research and use of animal models..J. Nutr. 2005; 135: 2499-2502Crossref PubMed Scopus (202) Google Scholar, 2Center for Disease Control and Prevention-U.S. Department of Health and Human Services, National Center for Health Statistics. Accessed May, 2006 at www.cdc.gov/nchs/fastats/overwt.htm.Google Scholar). 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 (3Seppanen-Laakso T. Laakso I. Hiltunen L.R. Analysis of fatty acids by gas chromatography, and its relevance to research on health and nutrition..Anal. Chim. Acta. 2002; 465: 39-62Crossref Scopus (170) Google Scholar). However, the presence of an important in vivo endogenous pool disables short-term in vivo studies of fatty acid biosynthesis and metabolism. 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 (4Pawlosky R.J. Sprecher H.W. Salem Jr., N. High sensitivity negative ion GC-MS method for detection of desaturated and chain-elongated products of deuterated linoleic and linolenic acids..J. Lipid Res. 1992; 33: 1711-1717Abstract Full Text PDF PubMed Google Scholar, 5Pawlosky R. Barnes A. Salem Jr., N. Essential fatty acid metabolism in the feline: relationship between liver and brain production of long-chain polyunsaturated fatty acids..J. Lipid Res. 1994; 35: 2032-2040Abstract Full Text PDF PubMed Google Scholar, 6Lin Y.H. Pawlosky R.J. Salem Jr., N. Simultaneous quantitative determination of deuterium- and carbon-13 labeled essential fatty acids in rat plasma..J. Lipid Res. 2005; 46: 1974-1982Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 7Hilkert A.W. Douthitt C.B. Schluter H.J. Brand W.A. Isotope ratio monitoring gas chromatography/mass spectrometry of D/H by high temperature conversion isotope ratio mass spectrometry..Rapid Commun. Mass Spectrom. 1999; 13: 1226-1230Crossref PubMed Scopus (273) Google Scholar, 8Brenna J.T. Use of stable isotopes to study fatty acid and lipoprotein metabolism in man..Prostaglandins Leukot. Essent. Fatty Acids. 1997; 57: 467-472Abstract Full Text PDF PubMed Scopus (25) Google Scholar). 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 (4Pawlosky R.J. Sprecher H.W. Salem Jr., N. High sensitivity negative ion GC-MS method for detection of desaturated and chain-elongated products of deuterated linoleic and linolenic acids..J. Lipid Res. 1992; 33: 1711-1717Abstract Full Text PDF PubMed Google Scholar, 5Pawlosky R. Barnes A. Salem Jr., N. Essential fatty acid metabolism in the feline: relationship between liver and brain production of long-chain polyunsaturated fatty acids..J. Lipid Res. 1994; 35: 2032-2040Abstract Full Text PDF PubMed Google Scholar, 6Lin Y.H. Pawlosky R.J. Salem Jr., N. Simultaneous quantitative determination of deuterium- and carbon-13 labeled essential fatty acids in rat plasma..J. Lipid Res. 2005; 46: 1974-1982Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 8Brenna J.T. Use of stable isotopes to study fatty acid and lipoprotein metabolism in man..Prostaglandins Leukot. Essent. Fatty Acids. 1997; 57: 467-472Abstract Full Text PDF PubMed Scopus (25) Google Scholar). The use of GC for fatty acid analysis is generally associated with time-consuming sample preparation resulting from the liquid-liquid extraction and chemical derivatization steps (4Pawlosky R.J. Sprecher H.W. Salem Jr., N. High sensitivity negative ion GC-MS method for detection of desaturated and chain-elongated products of deuterated linoleic and linolenic acids..J. Lipid Res. 1992; 33: 1711-1717Abstract Full Text PDF PubMed Google Scholar, 5Pawlosky R. Barnes A. Salem Jr., N. Essential fatty acid metabolism in the feline: relationship between liver and brain production of long-chain polyunsaturated fatty acids..J. Lipid Res. 1994; 35: 2032-2040Abstract Full Text PDF PubMed Google Scholar, 6Lin Y.H. Pawlosky R.J. Salem Jr., N. Simultaneous quantitative determination of deuterium- and carbon-13 labeled essential fatty acids in rat plasma..J. Lipid Res. 2005; 46: 1974-1982Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 8Brenna J.T. 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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 deuterium-labeled fatty acids in plasma involving a simple sample preparation procedure that would significantly increase the sample preparation throughput without compromising repeatability, accuracy, and robustness. C16:0, C16:1, C18:0, C18:1, and tricosanoic acid were obtained from Sigma-Aldrich (Milwaukee, WI) and were used without any further purification (99% purity). d5-C18:0 was purchased from C/D/N Isotopes (Pointe-Claire, Canada), whereas d7-C18:0 and d7-C18:1 were synthesized in-house (S. N. Crane, K. Bateman, S. Gagne, and J-F. Levesque, unpublished data). Methanol and acetone (optima grade) were obtained from Fisher Scientific, and NH4OH was from American Chemical, Ltd. (Montreal, Canada). Water was deionized using Milli-Q Plus from Millipore. NaOH (10 N) was obtained from J. T. Baker (Phillipsburg), and formic acid (99%) was purchased from Acros Organics. The HPLC-ESI-MS/MS system consisted of a Waters 2790 Alliance HT chromatograph coupled to a Quattro Ultima triple quadrupole mass spectrometer (Manchester, England) equipped with an electrospray source. The analytical column used was the X-Terra® MS C8 from Waters which is stable at high mass of the endogenous and deuterated a of from Waters coupled to a Waters liquid chromatograph was were by using a from The phase was of NH4OH and NH4OH to the negative ionization of the acid of fatty acids. HPLC separation was using a from to in The was for followed by a period at A. The was and the column was at The was from the mass spectrometer the and time to the The was The Quattro was in negative the was at the at the at the temperature at the temperature at the at the at and were in single ion monitoring mode The of the was using of and a on of the mass with a the of system was in negative the was at the at the at the ion at the at the temperature at the temperature at the at the at and were using a time of and a range of was the mass of the of fatty acids and single ion monitoring. in a new single ion monitoring. and were by Canada), by and and by samples were from in and plasma was by at for at the rat study of were and sample was obtained by d7-C18:0 was at or 30 in at and plasma samples were at and 72 samples were The were to and was at all the samples were at and were on at of plasma was in and of NaOH of the tricosanoic was The sample was and at for h. the was at temperature for and of of formic acid and of acetone were in with sample at The was at for and of the was in a and by HPLC-MS. The samples were using the protocol that of a of d7-C18:0 and d7-C18:1 was to the of plasma for concentration between the limit of detection (LOD) and 30 The used for fatty acid was by for The for compounds were on the mass of a a on the endogenous The sensitivity of the method was by a of d7-C18:0 and d7-C18:1 in rat which was as The sensitivity is reported on a ratio of for deuterium-labeled The dynamic the repeatability, the and the recovery were using the The was from at in the dynamic range on a single The precision was from over the dynamic range and for by the on the but on The recovery was by a concentration in rat plasma and using a The recovery was at and for reported in the for fatty acid analysis long sample liquid-liquid hydrolysis, and chemical derivatization followed by GC-MS analysis (4Pawlosky R.J. Sprecher H.W. Salem Jr., N. High sensitivity negative ion GC-MS method for detection of desaturated and chain-elongated products of deuterated linoleic and linolenic acids..J. Lipid Res. 1992; 33: 1711-1717Abstract Full Text PDF PubMed Google Scholar, 5Pawlosky R. Barnes A. Salem Jr., N. Essential fatty acid metabolism in the feline: relationship between liver and brain production of long-chain polyunsaturated fatty acids..J. Lipid Res. 1994; 35: 2032-2040Abstract Full Text PDF PubMed Google Scholar, 6Lin Y.H. Pawlosky R.J. Salem Jr., N. Simultaneous quantitative determination of deuterium- and carbon-13 labeled essential fatty acids in rat plasma..J. Lipid Res. 2005; 46: 1974-1982Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). This paper a rapid and new method developed for the of natural and deuterium-labeled fatty acids in plasma by The approach consists of the of fatty acids the and other followed by the of the (4Pawlosky R.J. Sprecher H.W. Salem Jr., N. High sensitivity negative ion GC-MS method for detection of desaturated and chain-elongated products of deuterated linoleic and linolenic acids..J. Lipid Res. 1992; 33: 1711-1717Abstract Full Text PDF PubMed Google Scholar, 5Pawlosky R. Barnes A. Salem Jr., N. Essential fatty acid metabolism in the feline: relationship between liver and brain production of long-chain polyunsaturated fatty acids..J. Lipid Res. 1994; 35: 2032-2040Abstract Full Text PDF PubMed Google Scholar, 6Lin Y.H. Pawlosky R.J. Salem Jr., N. Simultaneous quantitative determination of deuterium- and carbon-13 labeled essential fatty acids in rat plasma..J. Lipid Res. 2005; 46: 1974-1982Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), and a rapid and simple using samples are and are by This protocol significantly the sample preparation time required to fatty acids by steps involved in derivation and liquid-liquid extraction (4Pawlosky R.J. Sprecher H.W. Salem Jr., N. High sensitivity negative ion GC-MS method for detection of desaturated and chain-elongated products of deuterated linoleic and linolenic acids..J. Lipid Res. 1992; 33: 1711-1717Abstract Full Text PDF PubMed Google Scholar, 5Pawlosky R. Barnes A. Salem Jr., N. Essential fatty acid metabolism in the feline: relationship between liver and brain production of long-chain polyunsaturated fatty acids..J. Lipid Res. 1994; 35: 2032-2040Abstract Full Text PDF PubMed Google Scholar, 6Lin Y.H. Pawlosky R.J. Salem Jr., N. Simultaneous quantitative determination of deuterium- and carbon-13 labeled essential fatty acids in rat plasma..J. Lipid Res. 2005; 46: 1974-1982Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). a fatty acids were using monitoring as an approach to interference from the plasma using as a compound for the T. of HPLC in with tandem MS for the quantification of in human plasma..J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. PubMed Scopus Google Scholar). in ionization of was obtained in negative electrospray ion but its an sensitivity with The maximal of the parent as reported D. Gentili A. Marchese S. Sergi M. Caporossi L. Determination of free fatty acids in chocolate by liquid chromatography with tandem mass spectrometry..Rapid Commun. Mass Spectrom. 2004; 18: 1989-1994Crossref PubMed Scopus (43) Google with a ion which with a of from the carboxylic acid This The mode was to sensitivity with the the that analytical interference in mode in The simple sample preparation procedure with the method in mode detection of the natural fatty acids C16:0, C16:1, C18:0, and C18:1, as in plasma of endogenous fatty acids were to in the high or range in rat, and plasma Y.H. Pawlosky R.J. Salem Jr., N. Simultaneous quantitative determination of deuterium- and carbon-13 labeled essential fatty acids in rat plasma..J. Lipid Res. 2005; 46: 1974-1982Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, H. J. H. P. analysis of free fatty acids in rat plasma using mass spectrometry with as PubMed Scopus Google Scholar). high levels of natural fatty acids the to use isotopically labeled fatty acids to in vivo biosynthesis and metabolism of fatty acids over a short period with minimal interference from endogenous and used for such in which and essential fatty acid were followed in rat, cat, and human (4Pawlosky R.J. Sprecher H.W. Salem Jr., N. High sensitivity negative ion GC-MS method for detection of desaturated and chain-elongated products of deuterated linoleic and linolenic acids..J. Lipid Res. 1992; 33: 1711-1717Abstract Full Text PDF PubMed Google Scholar, 5Pawlosky R. Barnes A. Salem Jr., N. Essential fatty acid metabolism in the feline: relationship between liver and brain production of long-chain polyunsaturated fatty acids..J. Lipid Res. 1994; 35: 2032-2040Abstract Full Text PDF PubMed Google Scholar, 6Lin Y.H. Pawlosky R.J. Salem Jr., N. Simultaneous quantitative determination of deuterium- and carbon-13 labeled essential fatty acids in rat plasma..J. Lipid Res. 2005; 46: 1974-1982Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 8Brenna J.T. Use of stable isotopes to study fatty acid and lipoprotein metabolism in man..Prostaglandins Leukot. Essent. Fatty Acids. 1997; 57: 467-472Abstract Full Text PDF PubMed Scopus (25) Google Scholar). was as a fatty acid to the use of this new analytical for the detection of isotopically labeled for labeled are and the presence of a or on fatty acids the of the trace by endogenous fatty However, the mass from the isotope to the natural of the endogenous natural and isotopically labeled by chromatography (4Pawlosky R.J. Sprecher H.W. Salem Jr., N. High sensitivity negative ion GC-MS method for detection of desaturated and chain-elongated products of deuterated linoleic and linolenic acids..J. Lipid Res. 1992; 33: 1711-1717Abstract Full Text PDF PubMed Google Scholar, 5Pawlosky R. Barnes A. Salem Jr., N. Essential fatty acid metabolism in the feline: relationship between liver and brain production of long-chain polyunsaturated fatty acids..J. Lipid Res. 1994; 35: 2032-2040Abstract Full Text PDF PubMed Google Scholar, 6Lin Y.H. Pawlosky R.J. Salem Jr., N. Simultaneous quantitative determination of deuterium- and carbon-13 labeled essential fatty acids in rat plasma..J. Lipid Res. 2005; 46: 1974-1982Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). the presence of isotopes on were to on the of involved in fatty acid metabolism Y.H. Pawlosky R.J. Salem Jr., N. Simultaneous quantitative determination of deuterium- and carbon-13 labeled essential fatty acids in rat plasma..J. Lipid Res. 2005; 46: 1974-1982Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, J. method for in the of an of high 2003; PubMed Scopus Google Scholar). at were to attributable to the of endogenous fatty the d5-C18:0 was considered as a tracer for in vivo studies in the presence of interference from the plasma plasma from rat, and was and with to C16:0, C16:1, C18:0, and labeled with obtained in rat plasma are in are of obtained interference was detected in the trace of a and for a whereas were observed for and d5-C18:0 at the on the endogenous This interference by the use of for d7-C16:0, and the is from interference at the of all compounds in and The use of fatty acids would detection of the fatty acids of which would using a fatty acid of for the detection of was as a of high levels of whereas the d7-C18:1 trace was the of fatty acids as from plasma extraction at and the of this d7-C18:0 and d7-C18:1 were synthesized in-house and by (S. N. Crane, K. Bateman, S. Gagne, and J-F. Levesque, unpublished and by mass accuracy using an liquid of were in with the mass The analytical was for d7-C16:0, and in rat The ester of d7-fatty acid the analytical the the was previously in the of the sample preparation procedure for endogenous fatty acids in such as time and were were reported in the (4Pawlosky R.J. Sprecher H.W. Salem Jr., N. High sensitivity negative ion GC-MS method for detection of desaturated and chain-elongated products of deuterated linoleic and linolenic acids..J. Lipid Res. 1992; 33: 1711-1717Abstract Full Text PDF PubMed Google Scholar, 5Pawlosky R. Barnes A. Salem Jr., N. Essential fatty acid metabolism in the feline: relationship between liver and brain production of long-chain polyunsaturated fatty acids..J. Lipid Res. 1994; 35: 2032-2040Abstract Full Text PDF PubMed Google Scholar, 6Lin Y.H. Pawlosky R.J. Salem Jr., N. Simultaneous quantitative determination of deuterium- and carbon-13 labeled essential fatty acids in rat plasma..J. Lipid Res. 2005; 46: 1974-1982Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). all of the analytical The LOD for d7-C18:0 and d7-C18:1 was and minimal was with the acid The was between the LOD and a maximal concentration at 30 μM, and for 0.999 was > and precision > were obtained for the of mass ionization in the for d7-C18:0 and d7-C18:1 by to 30 to 30 high-performance liquid chromatography-electrospray ionization-mass limit of in a new high-performance liquid chromatography-electrospray ionization-mass limit of the recovery was for d7-C18:0 and and the obtained are in the dynamic range the recovery an for d7-fatty acid at of and an range at for d7-C18:0 and analytical were for fatty acid analysis by in other plasma A. Latorre A. Lacorte S. Barcelo D. Direct determination of resin and fatty acids in process waters of paper industries by liquid chromatography/mass spectrometry..J. Mass Spectrom. 2003; 38: 417-426Crossref PubMed Scopus (21) Google Scholar, 19Perret D. Gentili A. Marchese S. Sergi M. Caporossi L. Determination of free fatty acids in chocolate by liquid chromatography with tandem mass spectrometry..Rapid Commun. Mass Spectrom. 2004; 18: 1989-1994Crossref PubMed Scopus (43) Google Scholar). This analytical for the extraction and analysis of d7-fatty acids was used to plasma samples from an in vivo for d7-C18:0 and d7-C18:1 by in a new the of this new tracer to study fatty acid d7-C18:0 was at and the plasma samples were using the analytical L. Fatty acid and for d7-C18:0 and β-oxidation of its chain as as to or However, the plasma in the sample preparation disables the detection of d7-fatty acid plasma samples were and a is in were observed for d7-C18:0 and d7-C18:1 at all time whereas was detected only up to h. only the the LOD at all time the of d7-fatty acids a 30 of obtained for the were that were observed for d7-fatty acid of were observed for the d7-fatty acids analyzed. 72 important levels of d7-C18:0 and d7-C18:1 was still and were to and that and β-oxidation are the involved in d7-C18:0 metabolism in vivo in rats, as reported in the for the fatty acid biosynthesis L. Fatty acid and Scholar, and J. Nutr. 1995; PubMed Scopus Google Scholar). a d7-C18:0 and d7-C18:1 as previously in rat T. H. N. lipids and lipid oral in rat liver and Nutr. Sci. 2005; PubMed Scopus Google of rat dosed with 30 an improved was developed for the analysis of natural fatty acids C16:0, and and and deuterium-labeled in plasma involving only hydrolysis, neutralization, and quenching steps followed by analysis in negative ion mode using This method is rapid and and is for the detection of fatty acids from to the high new d7-fatty acid was synthesized to reduce matrix interference observed with its d5 analog, which significantly improved the LOD depending on the products analyzed. Upon the 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. This to other d7-fatty as on obtained with endogenous fatty acids. the of only d7-C18:0 and d7-C18:1 were was to the involved in d7-C18:0 and the of labeled and The the of the Department of Comparative at for with the animal The used in this was by palmitic acid palmitoleic acid stearic acid oleic acid high-performance liquid chromatography-electrospray ionization-mass spectrometry limit of detection monitoring single ion monitoring

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