Abstract: A new fluorescent derivatization reagent, 10-ethyl-9-oxo-9,10-dihydroacridine-2-sulfonyl chloride (EASC), was synthesized. A pre-column derivatization with EASC and reversed-phase high performance liquid chromatographic (RP-HPLC) method with fluorescence （FL） detection and mass spectrometry （MS） identification was performed for the trace analysis of oestradiol (E2) and oestriol (E3) in urine. This reagent shows higher sensitivities in ultraviolet （UV）, FL and MS detection than those of dansyl chloride (DNS-Cl), and the fluorescence intensity of EASC is 1000 times higher than that of DNS-Cl. The results of derivatization indicated that the derivatives can be obtained by the labeling reaction of EASC with estradiol (E2) and estriol (E3) in the presence of NaHCO3 buffer (pH 10.5) at 60 ℃ for 3 min. The excitation wavelength (λex) and emission wavelength (λem) were 270 nm and 430 nm, respectively. The established method exhibited excellent reproducibility and recovery. The calibration curves were linear with regression coefficients over0.9990, and the detection limits (S/N=3) were 40, 31 fmol for the studied compounds. The practical applicability of the method was demonstrated by analyzing trace of free oestradiol and oestriol in the urine of root voles.

Key words: dansyl chloride (DNS-Cl), oestradiol, oestriol, pre-column derivatization, reversed-phase high performance liquid chromatography (RP-HPLC), urine , 10-ethyl-9-oxo-9,10-dihydroacridine-2-sulfonyl chloride (EASC)