Evaluation of five methods for derivatization and GC determination of a mixture of very long chain fatty acids (C24:0–C36:0)

D003 is a new active ingredient consisting of a mixture of very long chain saturated fatty acids (C24:0–C36:0) in a definite proportion, which shows antioxidant, antiosteoporotic, antiplatelet and cholesterol-lowering effects in experimental models. Five derivatization methods for determining these fatty acids by gas chromatography (GC), using diazomethane, sulphuric acid–methanol, hydrochloric acid–methanol, boron trifluoride–methanol and N-methyl-N-trimethylsilyltrifluoroacetamide were evaluated. GC analysis was carried out using a BPX-5 wide-bore column and 1-nonadecanoic acid (C19:0) as internal standard. Methods were similar on account of the fatty acid content determined (84.2–86.6%). However, whereas the hydrochloric acid–methanol method needed 90 min to complete the derivatization, the other methods only required 10 min. Considering costs, speed, safety and GC response, the method using sulphuric acid–methanol was found the most appropriate for determining these fatty acids. The validation of this method: linearity over a range 40–160%, accuracy assessed through a recovery study, precision within day and inter-day, and specificity, even for samples subject to stress conditions, proved it is suitable for quality control and stability studies of the very long chain fatty acids composing this active ingredient.