Development Of A High-performance Liquid Chromatography-fluorescence Derivatization Method For Detecting Alcohol Metabolites Of 1,3-butadiene

1,3-butadiene(BD)is an important petrochemical product,and a ubiquitous environmental pollutant,has been shown to cause carcinogenic effects.BD can cause human leukemia.The target organ of butadiene is the lymphatic hematopoietic system.But the carcinogenic mechanism of butadiene is still not clear.The present study considered that the carcinogenicity of butadiene is most probably mediated by its metabolic intermediates biotransformed by cytochrome P450 enzymes.BD can also be converted in vitro by myeloperoxidase(MPO)into1-chloro-3-butene-2-ol(CHB),trans-4-chloro-2-buten-1-ol(E-CBol)and cis-4-chloro-2-buten-1-ol(Z-CBol)etc.CHB has been proved to be cytotoxic,genotoxic and mutagenic.The conversion of BD by MPO may be an important potential metabolic pathway in human carcinogenic.Because this pathway is expected to occur in MPO-rich bone marrow cells and neutrophils,which are consistent with the cancer target organs caused by BD in human.However,the conversion of BD by MPO has only been proved in vitro.So the in vivo evidence of the MPO pathway is the critical issue.Detecting E-CBol,Z-CBo and CHB in the blood of experimental animals exposed to BD can be a feasible method to prove it.But,the method in the preliminary work can not detect these componds in real samples with complex matrix because of the low recovery rate in common method like exaction-concentration-GC MS.In order to improve the ability of detection,a liquid chromatography-fluorescence derivatization detection method for E-CBol,Z-CBol,CHB,BDD and EBD was established in this paper.9-Fluorenylacetic acid(FAA)is used as erivatization reagent to label metabolic products through the derivatization reaction,then detected by HPLC-FLD.The derivatization reaction products of these componds were prepared by HPLC(8 products total),and the structures of products were confirmed by NMR spectroscopy.Then two HPLC method were developed to detecting 3 metabolites E-CBol,Z-CBol and CHB.Optimized excitation and emission wavelengths of the derivatized products are 264 nm and 319 nm,respectively.Optimized derivatization reaction time is 90 min.Based on these optimized condition,we established the HPLC-fluorescence derivatization method for detecting the metabolites E-CBol,Z-CBol and CHB in PBS(phosphate buffer saline).In summary,we develop and optimize the HPLC-fluorescence derivatization method for detecting the metabolites of BD in this paper.The method has been used in PBS sample for E-CBol,Z-CBol and CHB.The standard curves show good linearity(R~2>0.99).The accuracy is among 80.7%-110.1%,.The limit of detection in PBS for E-CBol,Z-CBol and CHB is 1,0.5,0.5?M,respectively.Then this method is successfully applied to detect E-CBol,Z-CBol and CHB in blood.This paper provides a powerful tool for studying alcohol metabolites of BD.