Investigation On The Form Of Environmental Hormone In Aquatic Products And Its Efficient Extraction And Detection

With the pollution of water resource being serious day by day, aquatic productsquality and safely has gained gradual attention nowadays. The environmental hormoneis one kind of high pollutant and can be accumulated in the environment. However, thedetermination of environmental hormones in aquatic products has not been reported.Therefore, it is essential to develop a method for environmental hormones analysis inaquatic products. Determination of drugs was often very difficult in complex matrixsamples, for the presences of various interferents. The key problem for accurate andeffective detection of chemical residue in complex matrix samples is sample preparation.This paper deals with a detailed review on the above questions. The fluorescence quenchingmethod was applied to study the drugs-protein interaction. Based on the studies of above, theeffect of the protein on extraction of drugs was investigated in detail. Coupled withGC–MS and HPLC, the aqueous two phase extraction technique and modifiedQuEChERS method were employed as sample pretreatment for the determination ofenvironmental hormones in aquatic products. The proposed methods have been shownto be highly practical because of their environmentally friendly, low organic solventconsumption, high reproducibility, and convenient method detection of limits.The paper can be divided into four sections:Section one: This section discussed the origin, the harmfulness and the currentdetection methods of environmental hormones. The pretreatment methods forenvironmental hormone samples were reviewed, and the content and significance ofthis paper were introduced.Section two: Samples were pretreated by2-propanol-K2HPO₄-H2O system.PAEs in aquatic products was detected by gas chromatography combined with massspectrometry detector （GC–MS）The fluorescence quenching method and GC-MS wereapplied to study the form of PAEs in protein matrix of aquatic products. Based on thestudies of above, the effect of the protein on extraction of PAEs was investigated indetail. The results showed that PAEs strongly bound with BSA by the hydrogen bond and Van der Waals force. In the φ （2-propanol）80%aqueous solution proteindenaturation could occur and the PAEs binding with the protein could be dissociatedfully. Following the PAEs was extracted by aqueous two phase system with highextraction efficiencies. Owing to the high polar of φ （2-propanol）80%aqueous solution,less fat-soluble substance was extracted and the purification steps were greatlysimplified. After standing for a moment,2.0mL of supernatant was transferred to10-mL centrifuge tubes containing anhydrous magnesium sulfate （MgSO₄） and20mgprimary secondary amine （PSA）. The were added in upper phase to eliminate themoisture and other impurities. Then, the PAEs in extract （upper phase） was directlyanalysed by GC-MS. The accuracy of this method was evaluated by measuring therecovery from spiked samples. Overall recoveries were81.9⁹2.7%with RSD valuesof1.2⁴.8%.Section three: A simple, environmentally friendly, high efficient extractionmethod for the determination of five phthalate in aquatic products by gaschromatography combined with mass spectrometry detector （GC–MS） was proposed.In this method, samples were pretreated by modified QuEChERS （QUick, Easy, CHeap,Effective, Rugged and Safe） method. An environmentally friendly extractant （ethanolaqueous solution） replaces toxic acetonitrile for extracting phthalates in theprotein-matrix samples. Fluorescence quenching spectra of BSA with phthalatesshowed that there was a high-affinity interaction between phthalates and protein, whichdecreased the extraction efficiency of phthalates in samples. Our study has found thatin the80%（volume fraction） ethanol aqueous solution, the denaturation protein wasslow but full procedure, which would cause the unfolding of protein and release ofphthalates. Meanwhile, the fat-soluble impurities are extracted less in φ（ethanol）80%aqueous solution than others. Therefore the purification steps were greatly simplified.Overall recoveries were81.8%～90.5%with limits of detection between2.5and9.6μg/L, relative standard deviation values at1.1%～4.8%. The proposed approach wassatisfactorily applied to the determination of phthalates in real aquatic products, such asfish, shrimp and oyster. Section fore: A simple, rapid, high efficient extraction method for thedetermination of nonyphenol （NP）、octylphenol （OP）、bisphenol A（BPA）、diethylstilbestrol （DES） in quatic products samples by HPLC. In this method,samples were pretreated by acetonitrile-salt-H2O two-phase system in theprotein-matrix samples. The influence factors on the extraction efficiency wereinvestigated deeply. Fluorescence quenching spectra of FSA with estrogen showed thatthere was a high-affinity interaction between estrogen and protein, which results inlower extraction efficiency of estrogen from samples. While in the80%（volumefraction） acetonitril （ACN） aqueous solution, a slow but full protein denaturation takesplace, which would cause the unfolding of protein and release of estrogen. This resultedin a high extraction efficiency of the estrogen from protein matrix samples. Meanwhile,the fat-soluble impurities are extracted less in φ （ACN）80%aqueous solution than thatof in φ （ACN）100%or in hydrophobic solvents as liquid-liquid extraction procedure.Therefore the purification steps were greatly simplified. Overall recoveries were87.5%～96.7%with limits of detection between2.53and6.15μg/L, relative standarddeviation values at1.8%～8.7%, which indicated good method precision. Theapproach developed was applied to the determination of nonyphenol（NP）、octylphenol （OP）、bisphenol A （BPA）、diethylstilbestrol （DES） in realsamples.