A-245 Development and validation of fatty acid analysis in whole blood by GC-FID

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Abstract Background Fatty acids are essential for cellular function, energy metabolism, and overall health. They serve as key components of cell membranes, energy sources, and precursors for bioactive molecules that regulate inflammation, immune responses, and cardiovascular function. Omega-3 and omega-6 fatty acids are particularly important for brain development, cognitive function, and cardiovascular health. Whole blood analysis of fatty acids provides a more stable representation of long-term dietary intake compared to plasma or serum. It helps assess metabolic disorders, cardiovascular risk, and inflammatory conditions while guiding personalized dietary and therapeutic interventions. Methods This protocol is designed to assess and validate the analytical performance of a laboratory-developed test for whole blood fatty acid profiling using GC-FID on the Agilent 8890 and to compare its performance to the existing GC-FID method on the Agilent 6890. The validation process consists of four key components: precision, method comparison, accuracy/traceability, and carryover assessment. Twenty-five fatty acid concentrations are reported as relative percentage weight with two decimal places, and the acceptable specimen type for this analysis is EDTA whole blood. The %W of the calibration standard is reviewed to ensure accuracy. All FAMEs (fatty acid methyl esters), except for the methyl ester of DPA n-6 (3.89%W), should have a %W of 4.00 ± 0.01. The % weight of 25 assayed fatty acids is used in the calculation of ratios, sums, index, and score values. A report template is used for data analysis, containing formulas that automatically calculate these values. Results Quantitative analysis was performed using a 25 FAMEs mix. Fatty acid standards were identified based on retention times and compared with chromatographic profiles from the certificate of analysis of the standard mix. All compounds were detected within 15 minutes of GC-FID analysis, with proper peak separation. Precision was expressed as the coefficient of variation (CV%). Within-run CV ranged from 0.1% to 5.1%, while total precision ranged from 0.6% to 14.2%. For accuracy, a strong correlation with the NIST standard was observed (y = 1.024x - 0.135). Additionally, comparison of 50 patient samples with the previous GC method showed good agreement, with a correlation equation of y = 0.9909x + 0.0948 and an R² value of 0.992. Conclusion In conclusion, this method identified and quantified fatty acids, omega-3, and omega-6 accurately and precisely and can be used effectively for routine FAME analysis in whole blood.

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