Standardization and validation of assay of selected omega-3 and omega-6 fatty acids from phospholipid fraction of red cell membrane using gas chromatography with flame ionization detector

Abstract

Background: Docosahexaenoic acid (DHA) is an important structural component of human brain and retina. Evidence exists linking nutritional status of pregnant mothers and cognitive functions of their born infants. The DHANI (Maternal DHA Supplementation and Offspring Neurodevelopment in India) trial was implemented to evaluate the effect of maternal supplementation with DHA during pregnancy and for 6 months following delivery on motor and mental development of infants at 1 and 12 months. We describe here the standardization and validation of an assay for measurement of selected omega-3 and omega-6 fatty acids from the phospholipid fraction of red blood cells to assess their status in mothers at baseline, delivery and 6 months post-delivery and for infants in cord blood and at 1 and 12 months of age. The validated method has been used for the analysis of samples for DHANI. Methods: Lipids were extracted from a pool of red blood cells, separated using thin layer chromatography. The phospholipid fraction was esterified, and fatty acids were separated by gas chromatography using a flame ionization detector. Result: The method accuracy for DHA was between 97 - 98% and between 91 - 95% for arachidonic acid (AA) at three different concentrations. The intra-assay and inter-assay coefficient of variation for the fatty acids ranged from 1.19 to 5.7% and 0.78 to 13.0% respectively. Intraclass correlation (ICC), as a measure of reproducibility, ranged between 0.689 and 0.996. A good linearity was observed for all the fatty acids between concentrations of 0.2–4 μg/ml. Conclusion: The standardized and validated method is suitable for implementation in large epidemiological studies for evaluation of fatty acids and in nutritional trials for assessment of fatty acid content of various lipid classes.