A sensitive pre-column derivatization method for the analysis of free fatty acids by RP-HPLC with fluorescence detector and its application to Caragana species.

Caragana korshinskii Kom. (CK), one of afforestation tree species, is widely planted in northwest region of China. To compare the constituents as references for further utilization of CK, C. microphyll Lam. (CM) and C. jubata L. (CJ), been used as traditional Chinese medicine, were taken into consideration. To obtain more information on CK for further utilization, a sensitive and stable pre-column derivatization method for the analysis of fatty acids (FAs) was established using a novel labeling reagent 2-(5H-benzo[a]-carbazol-11(6H)-yl)ethyl hydrazine-carboxylate (BCEHC) by HPLC with fluorescence detector. The derivatives exhibit predominant fluorescence property at excitation and emission wavelengths of 330nm and 380nm, respectively. 16 derivatives of FAs including 13 saturated FAs and 3 unsaturated FAs are separated on a reversed-phase column with gradient elution within 30min. The validation of method indicated that all FAs were given excellent linear responses with good linear coefficient of correlation being equal to or greater than 0.9985. The limits of detection (LODs) at a signal-to-noise ratio of 3 varied from 63.12 to 116.21ngL. The developed method was successfully applied to determine the contents of free FAs (FFAs) in flowers, leaves and bark of CK and the samples were extracted by a green and simple method of gas purge microsyringe extraction. The results show that the contents of linoleic acid and linolenic acid are high in flowers and leaves while the bark is rich in linoleic acid. The total content of FFAs in all parts of CK is higher than that of CM. The distribution of FFAs in plants is obviously different even in the congeneric among different species.