Simple HPLC–fluorescence determination of eugenol in clove oil after pre-column derivatization with 4-(N-chloroformylmethyl-Nmethylamino)-7-nitro-2,1,3-benzoxadiazole

Abstract

Eugenol is a phenolic flavor constituent of various plants, including clove and cinnamon. Eugenol content in clove oil has been analyzed by HPLC–UV after pre-column derivatization with 4-fluoro-7-nitro- 2,1,3-benzoxadiazole (NBD-F). Here, we present a simple, more sensitive HPLC–fluorescence method for determination of eugenol in clove oil by pre-column derivatization with 4-(N-chloroformylmethyl-N-methylamino)-7-nitro-2,1,3-benzoxadiazole (NBD-COCl). The retention time of NBD-CO-eugenol was 14.9 min. A standard curve, obtained after derivatization with NBD-COCl in borate buffer (pH 9.0) at room temperature for 1 min, was linear in the range of 0.025 to 0.4 μg/mL with r 2 value of 0.9992. The limit of detection was 0.006 μg/mL (signal-to-noise ratio of 3: 1). This is 6.7-fold lower in terms of concentration and about 45-fold lower in terms of absolute amount, compared with the previous method using NBD-F. The coefficients of variation were less than 9.4%. The content of eugenol in clove oil (1.00 g) was 0.73 ± 0.05 g (range 0.65 to 0.79 g). Recovery tests were satisfactory (86 ± 5%; range 80 to 92%).