Determination of free fatty acids in o/w injectable emulsions by HPLC with fluorescence detection using 4-(N,N-dimethylaminosulfonyl)-7-N- piperazino-2,1,3-benzoxadiazole

Abstract

HPLC using the fluorescent reagent, 4-(N,N-dimethylaminosulfonyl)-7-N- piperazino-2,1,3-benzoxadiazole (DBD-PZ) was applied to the determination of free fatty acids in an o/w injectable emulsion. This fluorescence HPLC method was highly sensitive and the free fatty acids could be directly derivatized without any previous preparation such as solvent extraction and elimination of water. In addition, the derivatized fatty acids could be adequately separated from interfering peaks, i.e., reagent and by-product peaks. The detection limit of this HPLC method was ca. 5 fmol in the case of C(18:0) fatty acid. The mean recovery of 2.5 nmol of each fatty acid (C(8:0), C(14:0), C(16:0), C(18:0)) added to the o/w injectable emulsion was 101.6% (n = 3). This fluorescence HPLC method with DBD-PZ was applied to commercial nonaqueous injection preparations to study their stability, and each free fatty acid in these preparations could be determined successfully. These results indicate that the proposed fluorescence HPLC method can be adequately used for the determination of free fatty acids in an o/w injectable emulsion.