| In this study, a high performance liquid chromatographic method for the simultaneous determination of environmental estrogens including bishphenol A (BPA) , 4-nonylphenol(NP), 17α-ethinylestradiol(EE2) and endogenic estrogens including 17α—estradiol(E2- α), 17β—estradioI(E2- β) and estriol(E3) in human urine and animal serum samples was developed and evaluated. After derivatization of the six target compounds with p— nitrobenzoyl chloride at room temperature, 0.40ml acetonitrile-water(30:70) was added and vortex agitated for 1 min. The supernatant (20 μ l) was injected into the chromatographic system. The method used gradient elution for the baseline separation of the six estrogens in twenty minutes. The chromatographic conditions, including column temperature and fluorescent detection wavelength were investigated. The optimized chromatographic conditions were: analytical column, Phenomenex C18, 5μm, 250×4.6mm; column temperature, 25℃ ; mobile phase, acetonitrile-water; flow rate, 1ml/min; gradient elution from 0 min at 30% acetonitrile-water to 12min at 70% acetonitrile-water, finally at 12.3min changed into 100% acetonitrile until 20min when returned to 30% of acetonitrile-water. Two wavelength shifts were adopted to achieve most sensitive exciting wavelength andcompromising emission wavelength for lower resolution paired peaks. We set the excitation wavelength and emission wavelength at 282nm and 315nm respectively at the beginning, and switched to 228nm and 316nm at 8.5min ultimately.The detection limit of the method was 2.7ug/L for bisphenol A and 17P-estradiol, 2.9ug/L for 4-nonylphenol, 4.6ug/L for 17a-estradiol and 17a-ethinylestradiol and 8.3ug/L for estriol respectively. The relative standard deviations ranged from 1.30% to 4.88%.Methanol (0.50ml) and concentrated hydrochloric acid (0.05ml) were added to 0.50 ml of urine samples to release estrogens from their conjugates, and then enriched and cleaned-up by ENV-18 Cl 8 SPE column. The estrogens on column was eluted with dichloromethane, and the eluent was evaporated to dryness under nitrogen stream. The residue was allowed to react with/?—nitrobenzoyl chloride at 25°C for 30min. The recoveries for the spiked urine samples ranged from 75.4% to 102.5%.The protein in serum sample was deposited with 2.0ml acetonitrile and then suitable amount of water, lmol/L hydrochloric acid and methanol were added to the samples, followed by extraction with 8.0ml ether for three times. The extract was evaporated to dryness under nitrogen flow. The derivatization procedure was same as that for urine samples. The recoveriesfor the spiked serum samples ranged from 72. 6% to 98. 6%.The method was applied to determining biological samples including20 urine samples and 10 serum samples. For the urine samples, five estrogens have been determined by high percentage except 17a-ethinylestradiol. Concentrations(mg/L) of 6 estrogens in urine samples were: E3: 0.004—1.81;BPA: 0.001—3.95; E2-P :0.001 — 10.64; E2-a: 0.002—0.147;EE2: 0.002 —1.31 ;NP; 0.002—2.26. For serum samples, BPA and NP have also been determined by high percentage.And the concentration(mg/L) in serum were: E3: 0.004—0.076;BPA: 0.001 — 1.03 ;E2- P :0.001—0.102;NP: 0.002 — 0.239; E2-a and EE2 had not been detected in 10 serum samples.It was concluded that this method was rapid, accurate, sensitive and economic for the determining trace estrogens in urine sample and serum sample. |
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