Studies On The Determination Of Testosterone Propionate Residue In Aquatic Products

As a synthetic testosterone derivative, testosterone propionate is a steroid proteinanabolic hormone. It has been widely used to improve feed conversion efficiency andpromote growth rates in aquiculture. Because testosterone propionate residues inaquatic products is harmful to human health, and even has potential carcinogeniceffect.So the use of testosterone propionate in food-producing animals has beenrestricted and prohibited in many countries. Therefore, it is essential and important toestablish a reliable analytical method to control the illegal use of steroids and monitorthe residual contents in aquatic products. The aim of this paper is to develop a gaschromatography-mass spectrometry （GC/MS） and Liquid chromatography tandemmass spectrometry （HPLC-MS/MS） method for the determination of testosteronepropionate residues in aquatic products.1. A gas chromatography-mass spectrometry （GC/MS） method was developedfor the identification and quantitative determination of testosterone propionateresidues in the aquatic muscle tissues using internal standard nandrolone-D₃. Thecracking process of testosterone propionate trimethylsilylation derivative product inthe mass spectrometry is analyzed for the first time. Homogenized tissue samplesadded with nandrolone-D₃and Saturated sodium chloride solution were extracted bytert-butyl ether under ultrasonic and oscillation. Clean-up was carried out withfreezing, followed by Silica-solid phase extraction （SPE）. To enhance the detectionsensitivity, a derivation of the drug was performed prior to GC/MS analysis withN-methyl-N-（trimethylsilyl）-trifluoroacetamide/iodotrimethylsilane/dithioeryth-ritol（MSTFA-TMIS-DTE）. The quantification analysis using isotope-labeled internalstandard was based on the ratio of peak area of testosterone propionate derivative topeak area of internal standard Nandrolone-D₃derivative in the selected ionmonitoring （SIM） mode with electron impact （EI） source. The linearity ranged from0.005to0.2mg/L, the regression equation was calculated to be Y=26.81X-0.005. Thelimit of quantitation （LOQ） was2.0μg/kg. At1.0,2.0,10.0μg/kg spiked levels, themean recoveries were74.1%～99.5%, and the relative standard deviations were3.0%～8.3%. The spiked recovery experiments in the eel, soft-shelled turtle weredemonstrated that the developed method could be applied for high-fat fishes. Such aresult was further proved by the analysis of various real samples from local markets,and fit well with the inter-laboratory validation.2. A liquid chromatography tandem mass spectrometry （HPLC-MS/MS） methodwas established for the identification and quantitative determination of testosteronepropionate residues in the aquatic muscle tissues using internal standardnandrolone-D₃. The pretreatment of samples （including extraction and purification）and the conditions of chromatography and mass spectrometry were investigated andoptimized. The method of liquid chromatography tandem time of flight mass spectrometry was developed for definitive detection of testosterone propionatethrough optimizing the detection parameters. Homogenized tissue samples added withnandrolone-D₃and were extracted by tert-butyl ether under ultrasonic and oscillation.After centrifugation, the supernatant was evaporated to dryness, reconstituted withacetonitrile and0.1%formic acid （9:1）. The complex solution was centrifugatedunder high speed after frozen in－80℃for30min. The supernatant was filtered bymembrane and then detected by liquid chromatography tandem mass spectrometry（HPLC-MS/MS）. Quantification using isotope-labeled internal standard was based onthe ratio of peak area of testosterone propionate to peak area of internal standardNandrolone-D₃in the multiple reaction ion monitoring （MRM） mode withElectrospray ionization （ESI） source. The linearity range was demonstrated to be from0.5ng/mL to100ng/mL, the regression equation was calculated to be Y=0.9964X-0.0356, and the correlation coefficient was calculated as0.9989. The lowest limit ofquantification （LOQ） was calculated to be0.5μg/kg. At the spiking levels of0.5μg/kg,1.0μg/kg and2.0μg/kg, the average recovery was in the range of89.1%～104.5%,and the relative standard deviation was in the range of2.3%～5.5%. All these resultsallowed us to suggest the developed method as an effective technique forconfirmatory analysis of propionate residue in aquatic products.