Study On The Application Of Subcritical R134a Extraction In Sex Hormone Multi-residues Analysis In Aquatic Products

Sex hormones are anabolic hormones, they have been widely used in animal breeding processas their strong protein assimilation in the last century. However, some hormones are chemicallystable, not easily to decompose, easily accumulate and remain in animal tissue. Long-term humanconsumption of animal tissue containing these hormones will lead to metabolic disorders,developmental abnormalities and even induce cancer. Therefore, since the beginning of the1980sthe EU has passed legislation to restrict or prohibit the use of hormones in animal breedingindustry. Chinese agriculture ministry also issued a series of announcements to regulate the use ofsex hormones. However, driven by the interests, phenomenon of illegally adding banned hormonedrugs in aquaculture is still exist in society today. So, continuing to strengthen determination andevaluation of sex hormones in aquatic products are still of great significance to the safety ofaquatic food.Sample pretreatment technology is the key point in sex hormone residue analysis. Simple,fast and efficient are the development trend of future for samples pretreatment technology.However, sample pretreatment methods used in our country currently mainly including traditionalsolvent extraction, liquid-liquid partition and column chromatography purification. These methodshave many drawbacks, such as low degree of automation, low efficiency of purification, slow andserious environmental pollution. Therefore, develop sample pretreatment technology whichpossess specialty of good selectivity, high sensitivity, fast, efficient and green is an important partof food safety and inspection. Subcritical R134a extraction technology was used as a new andefficient sample pretreatment method for analysing several kinds of sex hormone residues inaquatic products in this study, it was coupled to HPLC and GC-MS off-line. Extraction conditions,purification method and detection condition were optimized, the main conclusion are as follows:1. An analytical method for determination of methyltestosterone, testosterone propionate andmedroxyprogesterone was developed by using subcritical R134a extraction coupled with HPLCoff-line. Target compounds were first extracted by subcritical R134a extraction, and then theextracts were purified by freezing filtration and C18SPE column,at last the sample was analyzedby HPLC. Extraction conditions were optimized by single factor experiment, the optimumextraction conditions were as follows: extraction temperature25℃,extraction pressure4MPa andthe cosolvent amount6mL. Under the optimum extraction, purification and detection conditions,the precision and accuracy of the method were validation by analysing the spiked sample. At thespiked level of20~100μg/kg, the mean recoveries were between75.4%and94.2%, and the relative standard deviations (RSDs) ranged from5.1%to11.2%. The detection limits of themethod were between8.4~12.5μg/kg.2. An analytical method for determination of6sex hormones was developed by usingsubcritical R134a extraction coupled with GC-MS off-line. Target compounds were first extractedby subcritical R134a extraction, and then the extracts were purified by freezing filtration and C18,NH2SPE column, at last the sample was analyzed by GC-MS. Diethylstilbestrol was selected asthe representative and the extraction conditions were optimized by response surface based on thesingle factor experiment, the optimum extraction conditions were as follows: extraction pressure4MPa, extraction temperature26℃and the cosolvent amount4.7mL. Under the optimumextraction, purification and detection conditions, the precision and accuracy of the method werevalidation by analysing the spiked sample. At the spiked level of1~10μg/kg, the mean recoverieswere between70.5%and103.6%, and the relative standard deviations (RSDs) ranged from2.1%to12.1%. The detection limits of the method were between0.2~1μg/kg.3. The sex hormones studied in this work were not detected in the real sample from themarket. In order to validation the applicable of the proposed method, positive samples wereprepared by feeding fancy carp with spiked feed. Diethylstilbestrol was detected in the positivesample with a average concentration of13.8μg/kg.A new sample pretreatment method was developed for multi sex hormones residue analysisin aquatic products. The method has many advantages, such as simple, quick, efficient, goodselectivity and high sensitivity. It has broad application prospects in multi trace residue analysis.