Quantification of three triterpenic acids in dried rosemary using HPLC-fluorescence detection and 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride derivatization.

Abstract

Functional triterpenic acids such as ursolic acid (UA), oleanolic acid (OA) and betulinic acid (BA) are representative ingredients in rosemary that may have health benefits. UA, OA and BA in rosemary extracts were derivatized with 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride (DIB-Cl) and detected using HPLC-fluorescence (FL). Dried rosemary (50mg) was ground, added to 3ml of ethanol, sonicated for 40min, then the sample solution was added to a mixture of 1% trimethylamine and 1mM DIB-Cl in acetonitrile. The mixture was settled for 5min at room temperature, then the DIB-triterpenic acid derivatives were separated using a Wakopak Handy ODS column (250×4.6mm, 6μm) eluted with 25mM acetate buffer (pH4.5)/methanol/acetonitrile (=8:10:82v/v/v%). The fluorescence intensity of the eluent was monitored at 365 (λex ) and 490nm (λem ) and the maximum retention time of the derivatives was 30min. Calibration curves constructed using rosemary extract spiked with standards showed good linearity (r≥0.997) in the range 2.5-100ng/ml. The detection limits at 3σ for internal BA, UA and OA peaks in rosemary extract were 0.2, 0.4 and 0.5ng/ml, respectively. This method was used to quantify BA, UA and OA in commercially available dried rosemary products.