| Ethyl carbamate was a kind of cancerogen produced in the fermented food under natural conditions. It was showed that urea was the main precursor of ethyl carbamate in rice wine.A high performance liquid chromatography method was established for the determination of urea in rice wine, and establishing the standard methods for ethyl carbamate detect. This study mainly illustrated the methods of sample-pretreate for ethyl carbamate analyzing in rice wine using the gas chromatographic/mass spectrometric system, and chosing n-propyl carbamate as the internal standard. Then the best pretreatment method was selected. Finally the content of the urea and ethyl carbamate was compared after the heat treatment. The main results were as follows:(1) The analysis method was established for tracing the content of urea in rice wine. Methods of the HPLC separation was performed on Amino Alltima Amino Column (4.6x250mm) column with a mixture acetonitrile water(95’.5, v:v)as the mobile phase, at a flow rate of0.8mL/min. Detection wavelength was210nm by multiwavelength diode array, and column temperature was38℃. The limit of detection was2mg/L. The correlation coefficient of standard curve was0.99957, the recovery rate was between93.54%and100.6%, and the standard deviation was between2.21%and3.65%when the detection range was between2mg/L and100mg/L.The key to detect urea with HPLC in rice wine was choosing the right chromatographic column. It was concluded that using SPE LC-NH could get better accuracy and higher precision.(2) The method of liquid/liquid extraction’s parameters were optimized. And the pH of the wine was natural pH, the concentration of KC1was35%, and the dosage of CH2Cl2was3×90mL. The limit of detection was5.35μg/L. Under this extraction conditions, the recovery was between74.13%and80.29%, and the relative standard deviation (RSD) in one day was between4.60%and7.77%and that in two days was between5.65%and8.08%.The liquid/liquid extraction method could be applied in the EC extraction without expensive equipment, but emulsification was serious and lead to a larger system error.(3) The parameters of the solid phase microextraction was optimized. The sample pH was9; the concentration of NaCl (pH10.0) was30%; the sample vials were shaken at60℃for30min at the speed of800rpm in balanced mixer; the85μmPA fiber was inserted into the headspace at40℃for35min, then moved the fiber from the sample vials and rhen inserted to the injection system of the GC for10min. In this conditions, the recoveries were between81.8%and90.4%, and RSD of the precision in one day was between6.39%and9.57%and that in two days was between7.64%and10.69%. The limit of detection was1.72μg/mL.The SPME was successfully applied in determination of ethyl carbamate’s content in rice wine. This extraction method was simple, rapid and environmentalfriendly for didn’t use any organic solvent. However, this method of operation long time and the method precision and accuracy were lower.(4) The optimum parameters of SPE were:the pH was9.0; the dosage of CH2Cl2was3×60mL. The recovery was between94.62%and102.11%, and the RSD measured on the same day was between2.67%and5.98%and the results on the next three day was between3.07%and6.25%. The Instrument detection limit was1.62μg/mL. SPE provided reproducibility and good recovery Moreover, comparied with the liquid/liquid extraction method and the solid phase microextraction method, the solid phase extraction method was environmental-friendly, fast and simple. In this study, the solid phase extraction method was chosen as the standard pretretement methods to extract the ethyl carbamatefrom rice wine.(5) The content of urea and ethyl carbamate in the rice wine which suffered heating treatment were determined. It was showed that urea decreased with the rising of rice wine temperature, and ethyl carbamate increased with the rising of rice wine temperature. |
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