Quantitation of trans-fatty acids in human blood via isotope dilution-gas chromatography-negative chemical ionization-mass spectrometry

Abstract

Trans-fatty acids (TFA) are geometric isomers of naturally occurring cis-fatty acids. High dietary TFA intake has been associated with risk factors for cardiovascular disease. However, little is known about TFA levels in humans. To address this data need, we developed and validated a new isotope dilution-gas chromatography-negative chemical ionization-mass spectrometry (ID-GC-NCI-MS) method for quantitation of 27 fatty acids (FA) including 4 major TFA in human plasma, serum, and red blood cells (RBC) from 66 donors. Quantitation was performed with 18 isotope labeled internal standards and results are presented in µM and % of total FA. This method has high sensitivity and specificity due to use of pentafluorobenzyl-bromide derivatization combined with NCI-MS and a 200 m column to optimize positional and geometric FA isomer separation. The four major TFA, palmitelaidic acid, elaidic acid, trans-vaccenic acid, and linoelaidic acid, were detected in all samples, with median total TFA concentrations of 17.7 µM in plasma, 19.6 µM in serum, and 21.5 µM in RBC. The % of total FA for the TFA was 0.20% in plasma, 0.20% in serum, and 0.30% in RBC. Patterns for % FA are similar to those reported in other studies. We developed a highly specific, ID-GC-NCI-MS method to quantitate TFA and other FA in humans.

Total Fatty Acid Analysis of Human Blood Samples in One Minute by High-Resolution Mass Spectrometry

“…The rationale for this design was that negative ion mode analysis of deprotonated fatty acids (i.e., [M-H] − ) is highly sensitive, and that quantitative analysis of intact lipids by direct infusion FTMS can be performed using only a few minutes of analysis per sample [25,26,27]. Through meticulous method optimization we devised a method that uses only 1 µL of human plasma or serum, instead of 10–50 µL plasma as required by gold-standard GC-MS-based routines [13,24]. For total FA analysis, the 1 µL of plasma or serum is spiked with a defined amount of internal standard (e.g., PE 15:0/18:1(+ 2 H 7 )), followed by evaporating the sample to dryness, hydrolyzing intact lipids using sulfuric acid (H 2 SO 4 ) in acetonitrile/H 2 O (9:1, v/v), extracting the free fatty acids with hexane, and analyzing (2% of) the final total FA extract by 1 min of automated direct infusion-high resolution FTMS analysis.…”

“…Total FA analysis is typically carried out using gas-chromatography (GC)-based methods. The underlying methodology is straightforward and involves using either acid- or base-catalyzed reactions to convert FA chains of intact lipids and non-esterified fatty acids into volatile FA methyl ester (FAME) species that can be detected using GC coupled to a flame ionization detector (GC-FID) [10,11] or a mass spectrometer (GC-MS) [10,11,12,13]. By using different chromatographic parameters (incl.…”

“…column materials, gradients) and detection principles, it is possible to monitor FA analytes at different levels of structural resolution, which typically scales with increased analysis time and technical diligence of the operator [13,14]. As such, total FA analysis ranges from the identification of the total number of acyl carbon atoms and double bonds (e.g., FA 18:1), requiring about 15 min of analysis per sample, to the separation of distinct FA isomers having different positions and configurations of double bonds (e.g., FA 18:1(9Z) and FA 18:1(11E)), requiring up to 125 min of analysis [13]. Thus, the downside of increasing structural resolution is a significant increase in analysis time.…”

“…Thus, the downside of increasing structural resolution is a significant increase in analysis time. Another potential downside of GC-based approaches is that accurate quantification requires multiple isotope-labeled internal standards (isotope dilution method) as well as generation of multiple calibration curves [13,14]. Notably, in clinical settings, and in other application areas, one needs to strike a unique balance between the analysis time, the depth of structural resolution, and the required sample throughput.…”

Global Surveillance oftrans-Fatty Acids

“…Such data are not currently available in many countries. The Centers for Disease Control and Prevention (CDC) pioneered a new technique for measuring TFAs in blood that was used on a subsample of the National Health and Nutrition Examination Survey in 1999–2000 and 2009–2010, allowing the assessment of change in TFA intake over time (15,22).…”