Total Serum Fatty Acid Analysis by GC-MS: Assay Validation and Serum Sample Stability

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 degrees 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.