GC Determination of Long and Very Long Chain Fatty Acids (C24:0-C36:0) that Compose D003 in 5-mg Film-coated Tablets by a Fast Methylation Method

A fast gas chromatographic methodology for determining 5-mg of D003 (active ingredient consisting of a mixture of free saturated long and very long chain fatty acids, from C-24:0 to C-36:0, with antioxidant, antiosteoporotic, antiplatelet and cholesterol-lowering effects) in film-coated tablets was validated. This methodology applied first a hot-extraction of the fatty acids from tablets with chloroform. Then, it involved simultaneously the steps of methylation with a 2% sulphuric acid-methanol and the extraction of the formed esters with n-hexane. The gas chromatographic analysis was carried out using a DB-5 wide-bore column and nonadecanoic acid as internal standard. While 90 min had been proved to be necessary to reach the maximum efficiency for D003 methylation reaction in a previous GC method validated with the same aim, only 10 min were enough by the current method. Validation assays, applied to latter method, proved: good linearity (correlation coefficient higher than 0.9991, RSD of slope and RSD of response factors were 0.99% and 1.42%, respectively, and no bias) and accuracy (average recovery from 98.7 to 101.1%) over a range 38-150% of the nominal concentration. Good specificity was also proved, even for samples subject to stress conditions. Intra-day and inter-day precision at the nominal concentration were characterized by RSDs of 1.06% and 0.8%, respectively, and non-difference in the quantitative results by the current method with the previous method was demonstrated too. With regards to previous method, the current method was found more suitable for quality control and stability studies of these film-coated tablets.