Study On The Determination Of Environmental Estrogens By Chromatography

Objective: In order to provide sensitive and reliable technique for the study of the relationship between enviromental estrogens and diseases, analytical methods for three kinds of representative environmental estrogens such as mycotoxins (zearalenone and its metabolites, aflatoxin B₁ and ochratoxin A), persistent pesticide pollutants (hexachlorocyclohexane and dichlorodiphenyltrichloroethane) and industrial chemicals (bisphenol A and 4-nonylphenol) with chromatography were to develop.Methods:!. Zearalenone, its metabolites(a-Zearalenol and (3-Zearalenol) , and aflatoxin B₁ in corn, flour and wheat samples were extracted ultrasonically with methanol-water, cleaned up with C₁₈ cartridges and measured by reverse phase HPLC with fluorescence detector.2. Mycotoxins such as zearalenone, its metabolites(α-Zearalenol and β-Zearalenol ) ,ochratoxin A and aflatoxin B₁ in grain samples were extracted ultrasonically with liquid -liquid partition, cleaned up with C₁₈ cartridges, then determined by micellar electrokinetic capillary chromatography (MEKC).3. Hexachlorocyclohexane and dichlorodiphenyltrichloroethane in human serum were extracted ultrasonically with hexane, cleaned up with concentrated sulfuric acid, concentrated and determined by gas chromatography with ECD.4. Bisphenol A and 4-nonylphenol in human serum were extracted ultrasonically with hexane, cleaned up with Qg cartridges and determined by HPLC with fluorescence detector.Results: 1. Optimizations for pretreatment grain sample and conditions of HPLC detection for mycotoxins were done. The linear ranges were 14ng/ml¹46ng/ml > 14ng/ml²00ng/ml , 80ng/ml¹60ng/ml > 0.2ng/ml².0 jig/ml and the detection limits of the method were 1.4 ng/g> 1.4 ng/g^ 8. 0 ng/g and 0.02ng/g for zeralenone, cc-zearalenol, {J-zearalenol, aflatoxin Bi, respectively. The between-day precisions were 4.5%⁹.2%, the within-day precisions were 2."^-7.4%.The recoveries for the spiked samples ranged from 80.0% tollO.0%. The target chemicals from market and picked grain samples like ear and kernel with aim were determined and their contents in samples were compared with those obtained by AOAC, relative error were -8.33%⁵.26%.2. Optimizations for conditions of capillary electrophoresis (CE) detection for mycotoxins were done. The detection limits of the method were 3.0 ng/g> 6.0 ng^ 5.0 ng/g> 0.16 ng/g> 4.0ng/g for zeralenone, a-zearalenol, P-zearalenol, aflatoxin Bj, ochratoxin A, respectively, linear ranges were 0.03 ng/ml ²3.36fig/ml, 0.06ng/ml ³2.00fig/ml, 0.05ng/ml ²5.60jig/mi, 0.002ng/ml⁰.32ng/ml,0.04ng/ml¹6.00|j.g/ml, respectively. The within-day precisions were 0.63^-2.85%, the between-day precisions were2.68ï¿¡5.83%.The recoveries for the spiked samples ranged from 89.9%¹02.0%. The target chemicals in corn, flour and wheat samples were determined . Compared with those obtained by HPLC , the determination results reached an consistence by and large with relative error -9.09%⁷.46%.3. Four isomers of hexachlorocyclohexanes and four isomers of dichlorodiphenyltrichloroethanes in human serum were determined by GC-ECD. The within-day precisions were 2.16%⁶.83%, the between-day precisions were 2.50%⁷.40%. The recoveries for the spiked samples ranged from 89.9% to 102.0%. Under these conditions, BHC and DDT were well separated and showed good linearity in the studies ranges (0.2ng/ml⁰.50ng/ml for or BHC * y^b"C > S-BHC and p.p1 -DDS, 0.4ng/ml⁰.50ng/ml for p"-BHC and o,p'-DDD, 0.7ng/ml⁰.50M.g/ml for o,p'-DDT and 1.0ng/ml⁰.50(Ag/ml for p,p-DDT) with correlation coefficients greater than 0.99. The detection limits of the method were 0.07ng/ml⁰.52ng/ml. 67 human serum samples were determined including 52 control samples and 15 mammary cancer samples.4. Bisphenol A and 4-nonylphenol in human serum determined by HPLC with fluorescence detection. The within-day precisions were 4.01% 5.01%, the between-day precisions were 5.48%⁷.65%. Under these conditions, BPA and NP were well separated and showed good linearity in the studies ranges (l^ng/ml-^lO.O^g/ml for BPA and 2.2ng/ml¹5.0/ 98.8% and 94.7% 97.1%,respectively. 80 human serum samples were determined including 29 control samples and 51 mammary cancer samples.Conclusions: 1. Compared with the present AOAC method, the proposed method including liquid-liquid ultrasonic extraction, Qs cartridge clean-up and simultaneous detection with fluorescence detector for zeralenone, a-zearalenol, p-zearalenol, aflatoxin Bi in grain samples was more simple and could determine more analytes. This method was sensitive, accurate and easy to operate for the determining zearalenone and its metabolites in grain. To our knowledge, this is the first report of such kind in China. The results of samples by proposed method were consistent with those by AOAC, the relative error was less than 10%.2. The capillary electrophoresis method was applied for the first time to determine zearlenone, a-zearalenol, P-zearalenol, aflatoxin Bi, ochratoxin A in grain samples with satisfactory results. It was concluded that this method was cheap and rapid and used a small amount of an organic solvent. The results of samples by proposed method were consistent with those by AOAC, the relative error was less than 10%.3.The GC-ECD method was applied to determine organochloride pesticides in human serum and combined with epidemiological analysis for the first time at home. Analytes were determined in 52 control serum samples and 15 mammary cancer serum samples. The results showed that the mean concentrations of seven of eight isomers were greater in case group than those in control group and there was a significant difference for p,p'-DDT, p,p'-DDE and oj^DDT in detecable rate between case group and control group, respectively, assessed by Student's Mest with P < 0.05. This indicated that thesesubstances may be related factors of occurrence of mammary cancer.4. The HPLC-FLD method was applied to determine BPA and 4-NP in human serum and combined with epidemiological analysis for the first time . 80 serum samples(29 control serum samples and 51 mammary cancer serum samples) were determined. The results showed that the pollution levels of BPA and 4-NP were high in China with detecable rate 46.25% for BPA and 96.25% for 4-NP, respectively. Maximum concentrations, mean and detecable rates of BPA and 4-NP in case group were higher than those in control group, respectively. Statistical analysis was assessed by Student's r-test with P < 0.05 being considered significant. There were significant differences^ < 0.05) in mean between control group and case group for BPA and NP, respectively , and there was a significant difference(,P = 0.010) in detecable rate between control group and case group for NP. This may indicate that there was a positive correlation between BPA and 4-NP and occurrence of mammary cancer.In brief, the sensitive, reliable and practical methods were presented for determination of environmental estrogens. Experimental bases for further research of pollution of mycotoxins in grain and establishment of hygienic limit standards of zearalenone in corn were proposed. Reliable experimental methods for further epidemiological investigation and study the relationship between environmental estrogens and mammary cancer were presented. The significant practical meaning can exist in controlling and reducing pollution of xenoestrogens, reducing the exposure levels of environmental estrogens to people and the occurrence risk of related diseases.