Determination of environmental estrogens in human urine by high performance liquid chromatography after fluorescent derivatization with p-nitrobenzoyl chloride

A high performance liquid chromatographic method (HPLC) for the simultaneous determination of 4-nonylphenol, bisphenol A, 17(alpha)-ethinylestradiol and three endogenic estrogens including 17(alpha)-estradiol, 17(beta)-estradiol, estriol in urine sample, based on precolumn derivatization with p-nitrobenzoyl chloride, is presented in this paper. The estrogens mentioned above in urine were firstly hydrolyzed with 0.6 mol/l HCl, and then enriched and cleaned-up by ENV-18 C18 solid phase extraction (SPE) column. The estrogens on column were eluted with dichloromethane, and the eluent was evaporated to dryness under gentle nitrogen flow. The residue was allowed to react with p-nitrobenzoyl chloride at 25 °C for 30 min. Separation was performed on a C18 column with gradient elution using acetonitrile and water as mobile phase. A fluorescence detection system was used to detect the fluorescent derivatization products. The detection limit of the method was 2.7 (mu)g/l for bisphenol A and 17(beta)-estradiol, 2.9 (mu)g/l for 4-nonylphenol, 4.6 (mu)g/l for 17(alpha)-estradiol and 17(alpha)-ethinylestradiol and 8.3 (mu)g/l for estriol, respectively. The relative standard deviations (R.S.D.) ranged from 1.29 to 4.52% and the recoveries ranged from 85.5 to 99.9%. The method was applied to the determination of those six estrogens mentioned above in human urine samples collected from 20 healthy volunteers (aged 21–29). Bisphenol A (BPA) and 4-nonylphenol (NP) were detected with average contents of 1.22 (plus-minus) 1.38 mg/l and 0.38 (plus-minus) 0.77 mg/l in 10 male urine samples and 1.29 (plus-minus) 1.22 mg/l and 0.05 (plus-minus) 0.05 mg/l in 10 female urine samples, respectively. 17(alpha)-ethinylestradiol ((alpha)-EE2) was also detected with average contents of 0.13 (plus-minus) 0.41 mg/l and 0.06 (plus-minus) 0.15 mg/l in male and female urine samples, respectively.