Total Serum Fatty Acid Analysis by GC-MS: Assay Validation and Serum Sample Stability
Jianwei Ren, Ellen L. Mozurkewich, Ananda Sen, Anjel M. Vahratian, Thomas G. Ferreri, Alexander N. Morse and Zora Djuric
Affiliation: 1500 E. Medical Center Drive, Room 2150 Cancer Center, University of Michigan, Ann Arbor, MI 48109-5930, USA.
Analysis of n3 fatty acids in serum samples has clinical applications in supplementation trials, but the analysis
can be challenging due to low levels, stability issues and intra-individual variation. This study presents the single laboratory
validation of a gas chromatographic-mass spectral (GC-MS) assay for analysis of fatty acid methyl esters (FAME)
using sensitive single ion monitoring and provides data on fatty acid stability under different sample handling conditions.
Recovery of total fatty acids from serum with Folch extraction was optimized and parallelism tests with spiked samples
indicated that the serum matrix did not interfere with mass spectral quantitation. Precision and accuracy of the assay at the
lowest limit of quantitation and at low, medium and high levels met with accepted guidelines for single laboratory validation.
Several storage conditions that can be encountered with clinical samples also were evaluated for impact on fatty acid
levels in serum. Serum from blood that was stored refrigerated for 3 days yielded similar results as serum that was prepared
and frozen at -80°C immediately. Serum storage at room temperature for 3-24 hours and serum subjected to one
freeze/thaw cycle had minimal effects on fatty acid levels. The intra-individual variability in pregnant women was reasonably
small, with significant correlation coefficients ranging from 0.35 to 0.76 for blood drawn between 12-20 weeks
versus 34-36 weeks of gestation. These results indicate that GC-MS with single ion monitoring is valid for the analysis of
total fatty acids in clinical samples, even when blood processing cannot be performed in a timely manner.
Keywords: Fatty acids, fatty acid methyl esters, GC-MS, fish oils, pregnancy, blood levels.
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