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 (C(24:0)-C(36:0)) 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 (C(19:0)) 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.