Determination Of Residual Sex Hormones In Animal Tissue By Gas Chromatography-mass Spectrometer

This item is under the General Administration of Quality Supervision, Inspection andQuarantine of the People’s Republic of China’s command, the item unmber is2002IK042.The aim of this study was to develop an analytical method for the determination of Hexestrol,diethylstilbesterol, dienestrol, etiocholan-3α-ol-17-one, epitestosterone, estrone, estradiol,ethinylestradiol, estriol nine sex hormones in animal tissues use GC-MS.Sex hormones was extracted with acetonitrile followed by homogenizing and ultrasonicprocedure. Use n-hexane remove fatty components, followed by C18-solid phase extraction(SPE) clean up without any significant loss of growth hormones. For further clean-up,silica-and aminopropyl-SPE were used. To enhance GC-MS detection sensitivity, the sexhormones are derivatized with50μL BSTFA:TMCS(99:1) added with50μL pyridine solution.In this paper, a domestic microwave-oven was used for microwave-assistant derivatizationfollowing gas chromatographic-mass spectrometric analysis of nine hormones veterinarydrugs. Effects of irradiation time (15~120s) at the irradiation power level700W on the yieldof the derivatization were investigated, and it is compared with the conventional heatingderivatization.The derivatization under the irradiation of700W microwave for60s producedcomparable results when compared with the conventional heating process in water bath for in45min at70℃, shorten the time consumedly. Calibration curves under these derivatizationconditions were built using nine sex hormones concentrations in a range between0.0015and0.48μg/mL. The square of the regression coefficients (R~2) range from0.9961to0.9982. Theresults demonstrated that microwave-accelerated derivatization is an efficient and suitablesample preparation method for the GC-MS analysis of sex hormones.The mass selective detector was operated in the electron ionization mode using selected ionmonitoring (SIM). The GC column was DB-5MS (30m×0.25mm i.d., with0.25μm filmthickness) with dimethylpolysiloxane phase. Helium (99.999%) was used as carrier gas at1mL/min, pressure at11.6psi(1psi=6.89kPa, about79.9kPa), splitless injection volume2μL,injector temperature:280℃.The GC oven temperature was initially maintained at120℃for1 min and then programmed to250℃at a rate15℃/min, then to280℃at5℃/min andmaintained for6min. The temperature of the direct transfer line was maintained at280℃.The source and quadrupole temperatures were150℃.The application of this newly developed method was demonstrated by analyzing variousbeef, pork, and several animal internal organ samples from local markets, with externalstandards Quantitation. The method detection limits were0.1~1μg/kg for all hormones, andthe quantity limits were0.2~2μg/kg. Overall recoveries of sex hormones were higher than72%, with coefficients of variation of4.1~22%for the complete procedure. The detection limitwas below the minimum required performance limits (MRPLs) established by the EuropeanCommunity (EC).The real sample tests showed this method can be used for the sensitive andaccurate determination of multi-sex hormones residues in biological samples.