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 ( 2 H, 13 C) 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 mM, accuracy . 90%, precision . 88%, and adequate recovery in the dynamic range were obtained for d7-

Rapid measurement of plasma free fatty acid concentration and isotopic enrichment using LC/MS

“…We found only one publication describing the use of HPLC/MS to measure fatty acid enrichment ( 20 ). The authors describe an LC/MS in negative mode using SIM approach to measure deuterium-labeled fatty acids.…”

Derivatization-based High-throughput Bioanalysis by LC-MS

“…[34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51] However, the sensitive determination of negatively charged molecules, such as carboxylic acids, is not always promising in spite of the highly sensitive MS detection. 52,53 In such a case, the derivatization technique, which generally uses a basic reagent possessing amine(s), is effective for increasing the detection sensitivity and separation efficiency of reversed-phase chromatography, 54 because the resulting derivatives are easily charged as a cation by acidic mobile-phase containing formic and acetic acids. Although the use of positively charged reagents is furthermore effective for sensitive detection, the peak-to-peak separation of the derivatives is not always easy because of their high hydrophilicities.…”

Preparation of deuterium‐labeled monounsaturated and saturated fatty acids for use as stable isotope metabolic tracers

“…5 Protection of the alcohol in 8 as a TBS-ether in 9 was followed by removal of the acetylenic hydrogen with n-BuLi to generate the corresponding lithium acetylide, that was coupled to 3 RhCl, PhH, >99%; (e) TBAF, THF, 94%; (f) 48% HBr, conc H 2 SO 4 , 1008C, 89%; (g) NaI, acetone, reflux, 89%; (h) 1,3-diaminopropane, NaH, 708C, 88%; (i) TBSCI, DMAP, Et 3 N, CH 2 Cl 2 , 75%; (j) n-BuLi, THF, hexanes, HMPA, -788-08C, 86% for X ¼ I; (k) 1 atm H 2 , Lindlar catalyst, quinoline, hexanes, >99%; (l) TBAF, THF, 97%; (m) Jones oxidation (71% for 12, 65% for 13; 93% for 15); (n) 40 PSI H 2 , 10% Pd/C, EtOAc, >99%; (o) TBAF, THF, 78%; and (p) LiAIH 4 , diglyme, 1608C, 90% iodide 7 to give 10 in 86% yield. 6 The coupling reaction could also be conducted with the corresponding bromide 6, albeit in a more modest yield (59%).…”

“…2b We have recently reported a highthroughput LC-MS method for the quantitative analysis of deuterium labeled fatty acids in biological samples. 3 The requirement for a minimum incorporation of seven deuterium (d 7 ) atoms in these tracers arose out of the necessity to optimally discriminate the tracer from naturally occurring fatty acids present in biological samples. In order to support our studies in the area of metabolic syndrome-associated dyslipidaemia, we required access to a variety of d 7 -labeled saturated and monounsaturated fatty acids for use as standards in an array of in vivo and ex vivo experiments.…”