Studies On The Determination Of Hormone Multi-residues In Seafood By Gas Chromatography-mass Spectrometry

Hormonal chemicals have been widely used to improve feed conversion efficiency and promote growth rates in aquiculture. However, the use of natural and synthetic steroids in food-producing animals has been restricted and prohibited in many countries because of the possible toxic or carcinogenic effects on public health. Therefore, it is essential to establish reliable analytical methods to control the illegal use of steroids and monitor the residual contents of these compounds in fishery products. The aim of this paper is to develop a gas chromatography-mass spectrometry (GC-MS) method for the determination of various hormone residues in seafood.1. A GC-MS method was developed for the identification and quantitative determination ofβ-estradiol residues in the muscle tissues of various fishes/shellfishes, using deuterated internal standard. Homogenized tissue samples added withβ-estradiol-D2 and acetate buffer (pH5.2) were extracted by acetonitrile under ultrasonication. Clean-up was carried out with hexane, followed by C18-solid phase extraction (SPE). To enhance detection sensitivity, derivatization was achieved prior to GC-MS analysis with N-methyl-N-(trimethylsilyl)-trifluoroacetamide/iodo trimethylsilane/dithioerythritol (MSTFA-TMIS-DTE). Quantitation using isotope -labelled internal standard was based on the ratio of peak area ofβ-estradiol derivative to peak area of internal standard derivative in the selected ion monitoring (SIM) mode with electron impact (EI) source. Good linearity ranged from 0.005 to 0.25μg/mL. The limit of quantitation (LOQ) was 0.2μg/kg. At 0.2, 1.0, 5.0μg/kg spiked levels, the mean recoveries were within 68.5%~100.1%, and the relative standard deviations were within 4.8%~10.1%. The application of this method was further demonstrated by analyzing various real samples from local markets and participating in interlaboratory validation.2. A multi-residue method was developed for simultaneous determination of a wide range of anabolic steroids in seafood by GC-MS. Homogenized tissue samples added withβ-estradiol-D2 and acetate buffer (pH5.2) were hydrolyzed byβ-glucuronidase. Consequently, polar extraction with acetonitrile and non-polar extraction with hexane and ethyl acetate were carried out under ultrasonication. Clean-up was performed with hexane liquid-liquid partitioning, followed by dual polymer and amino-propyl solid phase extraction cartridges. To enhance detection sensitivity, the extracts obtained were derivatized separately with MSTFA-TMIS-DTE, heptafluorobutyric anhydride (HFBA) and MSTFA, and analyzed by GC-MS. Quantitation using spiked blank meat was based on the ratio of peak area of steroid derivative to peak area of internal standard derivative in the selected ion monitoring mode with electron impact source. Good linearity of each compound was determined and acceptable correlation coefficient was over 0.98 in the range examined. The limits of quantitation were 0.1~2.0μg/kg for all growth hormones. Overall mean recoveries were 61.1%~115.3% with coefficients of variations of 1.0%~14.1% for the complete procedure. The real sample analysis showed this method could be successfully applied for the sensitive and accurate determination of anabolic steroids multi-residues in fishery products. In comparison to the human daily production of hormones and alimentary intake of these compounds from other food-producing animals and plants, the consumption safety of seafood was primarily evaluated because of the residual contents of steroids.